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Sonstige beteiligte Institutionen
- Department of Hematology and Oncology, Sana Hospital Hof, Hof, Germany (1)
- Department of Laboratory Medicine and Medicine Huddinge, Karolinska Institutet and University Hospital, Stockholm, Sweden (1)
- Department of Medicine A, University Hospital of Münster, Münster, Germany (1)
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- Johns Hopkins School of Medicine (1)
- University of Bari Medical School, Bari, Italy (1)
The piranha enjoys notoriety due to its infamous predatory behavior but much is still not understood about its evolutionary origins and the underlying molecular mechanisms for its unusual feeding biology. We sequenced and assembled the red-bellied piranha (Pygocentrus nattereri) genome to aid future phenotypic and genetic investigations. The assembled draft genome is similar to other related fishes in repeat composition and gene count. Our evaluation of genes under positive selection suggests candidates for adaptations of piranhas’ feeding behavior in neural functions, behavior, and regulation of energy metabolism. In the fasted brain, we find genes differentially expressed that are involved in lipid metabolism and appetite regulation as well as genes that may control the aggression/boldness behavior of hungry piranhas. Our first analysis of the piranha genome offers new insight and resources for the study of piranha biology and for feeding motivation and starvation in other organisms.
BACKGROUND: The barrier to diffusion of organic solutes across the plant cuticle is composed of waxes consisting of very long-chain aliphatic (VLCA) and, to varying degrees, cyclic compounds like pentacyclic triterpenoids. The roles of both fractions in controlling cuticular penetration by organic solutes, e.g. the active ingredients (AI) of pesticides, are unknown to date. We studied thepermeabilityof isolated leaf cuticularmembranes from Garcinia xanthochymus andPrunus laurocerasus for lipophilic azoxystrobin and theobromine as model compounds for hydrophilic AIs.
RESULTS: The wax of P. laurocerasus consists of VLCA (12%) and cyclic compounds (88%), whereas VLCAs make up 97% of the wax of G. xanthochymus.We showthat treating isolated cuticles with methanol almost quantitatively releases the cyclic fraction while leaving the VLCA fraction essentially intact. All VLCAs were subsequently removed using chloroform. In both species, the permeance of the two model compounds did not change significantly after methanol treatment, whereas chloroform extraction had a large effect on organic solute permeability.
CONCLUSION: The VLCA wax fractionmakes up the permeability barrier for organic solutes, whereas cyclic compounds even in high amounts have a negligible role. This is of significance when optimizing the foliar uptake of pesticides.
Background: Non-alcoholic steatohepatitis (NASH) is a chronic liver disease with severe complications and without approved therapies. Currently, there is limited data on the overall burden of the disease for patients or on patient needs and preferences. This study investigates patient preferences in relation to potential future therapies for NASH. In addition, the factors that are relevant to patients and their importance in relation to future treatment options are explored.
Method: Telephone in-depth interviews (TDIs) preceded an online 30-min quantitative survey. The online survey included (1) multiple choice questions (MCQs) on NASH diagnosis and disease background. (2) An exercise to determine patients' satisfaction levels with information provided at diagnosis, and to explore symptomatology in detail. (3) Exercises to evaluate potential new products and product attributes, including a "drag and drop" ranking exercise, and an adaptive choice-based conjoint exercise (ACBC). (4) The EQ-5D-5L questionnaire and the Visual Analog Scale (VAS), which measures patients' health status. (5) Collection of socio-demographic data, and (6) Questions to measure patient satisfaction with the survey.
Results: There were 166 patients included in this study from Canada [n = 36], Germany [n = 50], the UK [n = 30], and USA [n = 50]. Fifty seven percent of patients [n = 94] had had a liver biopsy for confirmation of NASH. Patients were often unable to link their symptoms to NASH or other conditions. ACBC results showed that efficacy, defined as "impact on liver status" was the single most important attribute of a potential future NASH therapy. Other attributes considered to have secondary importance included impact on weight, symptom control and the presence of side effects. The EQ-5D utility score was 0.81 and VAS = 67.2.
Conclusion: "Impact on liver status" is the primary outcome sought. Patients demonstrate a general lack of understanding of their disease and appeared to be unfamiliar with longer-term consequences of NASH. It is necessary to improve patient understanding of NASH and its progressive nature, and there is a need for improving confirmatory diagnosis and monitoring.
The P429L loss of function mutation of the human glycine transporter 2 associated with hyperekplexia
(2019)
Glycine transporter 2 (GlyT2) mutations across the entire sequence have been shown to represent the presynaptic component of the neurological disease hyperekplexia. Dominant, recessive and compound heterozygous mutations have been identified, most of them leading to impaired glycine uptake. Here, we identified a novel loss of function mutation of the GlyT2 resulting from an amino acid exchange of proline 429 to leucine in a family with both parents being heterozygous carriers. A homozygous child suffered from severe neuromotor deficits. We characterised the GlyT2P429L variant at the molecular, cellular and protein level. Functionality was determined by glycine uptake assays. Homology modelling revealed that the mutation localises to α‐helix 5, presumably disrupting the integrity of this α‐helix. GlyT2P429L shows protein trafficking through various intracellular compartments to the cellular surface. However, the protein expression at the whole cell level was significantly reduced. Although present at the cellular surface, GlyT2P429L demonstrated a loss of protein function. Coexpression of the mutant with the wild‐type protein, reflecting the situation in the parents, did not affect transporter function, thus explaining their non‐symptomatic phenotype. Nevertheless, when the mutant was expressed in excess compared with the wild‐type protein, glycine uptake was significantly reduced. Thus, these data demonstrate that the proline residue at position 429 is structurally important for the correct formation of α‐helix 5. The failure in functionality of the mutated GlyT2 is most probably due to structural changes localised in close proximity to the sodium‐binding site of the transporter.
The origin of the solvent dependence of fluorescence quantum yields in dipolar merocyanine dyes
(2019)
Fluorophores with high quantum yields are desired for a variety of applications. Optimization of promising chromophores requires an understanding of the non-radiative decay channels that compete with the emission of photons. We synthesized a new derivative of the famous laser dye 4-dicyanomethylen-2-methyl-6-p-dimethylaminostyryl-4H-pyran (DCM),i.e., merocyanine 4-(dicyanomethylene)-2-tert-butyl-6-[3-(3-butyl-benzothiazol-2-ylidene)1-propenyl]-4H-pyran (DCBT). We measured fluorescence lifetimes and quantum yields in a variety of solvents and found a trend opposite to the energy gap law.This motivated a theoretical investigation into the possible non-radiative decay channels. We propose that a barrier to a conical intersection exists that is very sensitive to the solvent polarity. The conical intersection is characterized by a twisted geometry which allows a subsequent photoisomerization. Transient absorption measurements confirmed the formation of a photoisomer in unpolar solvents, while the measurements of fluorescence quantum yields at low temperature demonstrated the existence of an activation energy barrier.
There is a specialized niche for the electrohydrodynamic jetting of melts, from biomedical products to filtration and soft matter applications. The next frontier includes optics, microfluidics, flexible electronic devices, and soft network composites in biomaterial science and soft robotics. The recent emphasis on reproducibly direct‐writing continual molten jets has enabled a spectrum of contemporary microscale 3D objects to be fabricated. One strong suit of melt processing is the capacity for the jet to solidify rapidly into a fiber, thus fixing a particular structure into position. The ability to direct‐write complex and multiscaled architectures and structures has greatly contributed to a large number of recent studies, explicitly, toward fiber–hydrogel composites and fugitive inks, and has expanded into several biomedical applications such as cartilage, skin, periosteum, and cardiovascular tissue engineering. Following the footsteps of a publication that summarized melt electrowriting literature up to 2015, the most recent literature from then until now is reviewed to provide a continuous and comprehensive timeline that demonstrates the latest advances as well as new perspectives for this emerging technology.
Cushing’s disease (CD) is a rare condition caused by adrenocorticotropic hormone (ACTH)-producing adenomas of the pituitary, which lead to hypercortisolism that is associated with high morbidity and mortality. Treatment options in case of persistent or recurrent disease are limited, but new insights into the pathogenesis of CD are raising hope for new therapeutic avenues. Here, we have performed a meta-analysis of the available sequencing data in CD to create a comprehensive picture of CD’s genetics. Our analyses clearly indicate that somatic mutations in the deubiquitinases are the key drivers in CD, namely USP8 (36.5%) and USP48 (13.3%). While in USP48 only Met415 is affected by mutations, in USP8 there are 26 different mutations described. However, these different mutations are clustering in the same hotspot region (affecting in 94.5% of cases Ser718 and Pro720). In contrast, pathogenic variants classically associated with tumorigenesis in genes like TP53 and BRAF are also present in CD but with low incidence (12.5% and 7%). Importantly, several of these mutations might have therapeutic potential as there are drugs already investigated in preclinical and clinical setting for other diseases. Furthermore, network and pathway analyses of all somatic mutations in CD suggest a rather unified picture hinting towards converging oncogenic pathways.
The NEDD8-activating enzyme (NAE) inhibitor MLN4924 inhibits cullin-RING ubiquitin ligase complexes including the SKP1-cullin-F-box E3 ligase βTrCP. MLN4924 therefore inhibits also the βTrCP-dependent activation of the classical and the alternative NFĸB pathway. In this work, we found that a subgroup of multiple myeloma cell lines (e.g., RPMI-8226, MM.1S, KMS-12BM) and about half of the primary myeloma samples tested are sensitized to TNF-induced cell death by MLN4924. This correlated with MLN4924-mediated inhibition of TNF-induced activation of the classical NFκB pathway and reduced the efficacy of TNF-induced TNFR1 signaling complex formation. Interestingly, binding studies revealed a straightforward correlation between cell surface TNFR1 expression in multiple myeloma cell lines and their sensitivity for MLN4924/TNF-induced cell death. The cell surface expression levels of TNFR1 in the investigated MM cell lines largely correlated with TNFR1 mRNA expression. This suggests that the variable levels of cell surface expression of TNFR1 in myeloma cell lines are decisive for TNF/MLN4924 sensitivity. Indeed, introduction of TNFR1 into TNFR1-negative TNF/MLN4924-resistant KMS-11BM cells, was sufficient to sensitize this cell line for TNF/MLN4924-induced cell death. Thus, MLN4924 might be especially effective in myeloma patients with TNFR1+ myeloma cells and a TNFhigh tumor microenvironment.
The name is the game: nicknames as predictors of personality and mating strategy in online dating
(2019)
Objective: We investigated the communicative function of online dating nicknames. Our aim was to assess if it is possible to correctly guess personality traits of a user simply by reading his/her nickname.
Method: We had 69 nickname users (average age: 33.59 years, 36 female) complete questionnaires assessing their personality (Big 5 + narcissism) and mating strategy (short- vs. long-term). We then checked (using a total of 638 participants, average age: 26.83 years, 355 female), whether personality and mating strategy of the nickname users could be assessed correctly based only on the nickname. We also captured the motivation to contact the user behind a nickname and looked at linguistic features of the nicknames.
Results: We found that personality and mating strategy could be inferred from a nickname. Furthermore, going by trends, women were better at intersexual personality judgments, whereas men were better in intrasexual judgements. We also found several correlates of the motivation to contact the person behind the nickname. Among other factors, long nicknames seemed to deter people from contacting the nickname user.
Conclusions: Findings display that humans are capable of making accurate personality judgements in computer-mediated communication by means of even small cues like nicknames.
YAP and TAZ, downstream effectors of the Hippo pathway, are important regulators of proliferation. Here, we show that the ability of YAP to activate mitotic gene expression is dependent on the Myb-MuvB (MMB) complex, a master regulator of genes expressed in the G2/M phase of the cell cycle. By carrying out genome-wide expression and binding analyses, we found that YAP promotes binding of the MMB subunit B-MYB to the promoters of mitotic target genes. YAP binds to B-MYB and stimulates B-MYB chromatin association through distal enhancer elements that interact with MMB-regulated promoters through chromatin looping. The cooperation between YAP and B-MYB is critical for YAP-mediated entry into mitosis. Furthermore, the expression of genes coactivated by YAP and B-MYB is associated with poor survival of cancer patients. Our findings provide a molecular mechanism by which YAP and MMB regulate mitotic gene expression and suggest a link between two cancer-relevant signaling pathways.
Background:
Pedicled perforator flaps have expanded reconstructive options in extremity reconstruction. Despite preoperative mapping, intraoperative findings may require microvascular tissue transfer when no adequate perforators can be found. The free peroneal artery perforator flap may serve as a reliable back-up plan in small defects.
Methods:
In 16 patients with small soft tissue defects on the upper and lower extremities, perforator-based propeller flaps were planned. The handheld Doppler device was used to localize potential perforators for a propeller flap in close proximity to the defect. Perforators of the proximal peroneal artery were also marked to allow conversion to microvascular tissue transfer.
Results:
In 6 cases, no adequate perforators were found intraoperatively. In 4 patients, the peroneal artery perforator flap was harvested and transferred. The pedicle length did not exceed 4 cm. No flap loss occurred.
Conclusions:
When no adequate perforator capable of nourishing a propeller flap can be found intraoperatively, the free peroneal artery flap is a good option to reconstruct small soft tissue defects in the distal extremities. The short vascular pedicle is less ideal in cases with a large zone of injury requiring a more distant site of anastomosis or when recipient vessels are located in deeper tissue planes.
FinO domain proteins such as ProQ of the model pathogen Salmonella enterica have emerged as a new class of major RNA-binding proteins in bacteria. ProQ has been shown to target hundreds of transcripts, including mRNAs from many virulence regions, but its role, if any, in bacterial pathogenesis has not been studied. Here, using a Dual RNA-seq approach to profile ProQ-dependent gene expression changes as Salmonella infects human cells, we reveal dysregulation of bacterial motility, chemotaxis, and virulence genes which is accompanied by altered MAPK (mitogen-activated protein kinase) signaling in the host. Comparison with the other major RNA chaperone in Salmonella, Hfq, reinforces the notion that these two global RNA-binding proteins work in parallel to ensure full virulence. Of newly discovered infection-associated ProQ-bound small noncoding RNAs (sRNAs), we show that the 3′UTR-derived sRNA STnc540 is capable of repressing an infection-induced magnesium transporter mRNA in a ProQ-dependent manner. Together, this comprehensive study uncovers the relevance of ProQ for Salmonella pathogenesis and highlights the importance of RNA-binding proteins in regulating bacterial virulence programs.
IMPORTANCE
The protein ProQ has recently been discovered as the centerpiece of a previously overlooked “third domain” of small RNA-mediated control of gene expression in bacteria. As in vitro work continues to reveal molecular mechanisms, it is also important to understand how ProQ affects the life cycle of bacterial pathogens as these pathogens infect eukaryotic cells. Here, we have determined how ProQ shapes Salmonella virulence and how the activities of this RNA-binding protein compare with those of Hfq, another central protein in RNA-based gene regulation in this and other bacteria. To this end, we apply global transcriptomics of pathogen and host cells during infection. In doing so, we reveal ProQ-dependent transcript changes in key virulence and host immune pathways. Moreover, we differentiate the roles of ProQ from those of Hfq during infection, for both coding and noncoding transcripts, and provide an important resource for those interested in ProQ-dependent small RNAs in enteric bacteria.
Hepatic activation of protein kinase C (PKC) isoforms by diacylglycerol (DAG) promotes insulin resistance and contributes to the development of type 2 diabetes (T2D). The closely related protein kinase D (PKD) isoforms act as effectors for DAG and PKC. Here, we showed that PKD3 was the predominant PKD isoform expressed in hepatocytes and was activated by lipid overload. PKD3 suppressed the activity of downstream insulin effectors including the kinase AKT and mechanistic target of rapamycin complex 1 and 2 (mTORC1 and mTORC2). Hepatic deletion of PKD3 in mice improved insulin-induced glucose tolerance. However, increased insulin signaling in the absence of PKD3 promoted lipogenesis mediated by SREBP (sterol regulatory element-binding protein) and consequently increased triglyceride and cholesterol content in the livers of PKD3-deficient mice fed a high-fat diet. Conversely, hepatic-specific overexpression of a constitutively active PKD3 mutant suppressed insulin-induced signaling and caused insulin resistance. Our results indicate that PKD3 provides feedback on hepatic lipid production and suppresses insulin signaling. Therefore, manipulation of PKD3 activity could be used to decrease hepatic lipid content or improve hepatic insulin sensitivity.
Introduction: The long head of the biceps (LHB) is often resected in shoulder surgery. However, its contribution to inflammatory processes in the shoulder remains unclear. In the present study, inflamed and noninflamed human LHBs were comparatively characterized for features of inflammation. Materials and methods: Twenty-two resected LHB tendons were classified into inflamed (n = 11) and noninflamed (n = 11) samples. For histological examination, samples were stained with hematoxylin eosin, Azan, van Gieson, and Masson Goldner trichrome. Neuronal tissue was immunohistochemically visualized. In addition, specific inflammatory marker gene expression of primary LHB-derived cell cultures were analyzed. Results: Features of tendinopathy, such as collagen disorganization, infiltration by inflammatory cells, neovascularization, and extensive neuronal innervation were found in the tendinitis group. Compared to noninflamed samples, inflamed LHBs showed a significantly increased inflammatory marker gene expression Conclusion: Structural and biomolecular differences of both groups suggest that the LHB tendon acts as an important pain generator in the shoulder joint. These findings can, on the one hand, contribute to the understanding of the biomolecular genesis of LHB tendinitis and, on the other hand, provide possibilities for new therapeutic approaches.
Myeloma is characterized by extensive inter-patient genomic heterogeneity due to multiple different initiating events. A recent multi-region sequencing study demonstrated spatial differences, with progression events, such as TP53 mutations, frequently being restricted to focal lesions. In this review article, we describe the clinical impact of these two types of tumor heterogeneity. Target mutations are often dominant at one site but absent at other sites, which poses a significant challenge to personalized therapy in myeloma. The same holds true for high-risk subclones, which can be locally restricted, and as such not detectable at the iliac crest, which is the usual sampling site. Imaging can improve current risk classifiers and monitoring of residual disease, but does not allow for deciphering the molecular characteristics of tumor clones. In the era of novel immunotherapies, the clinical impact of heterogeneity certainly needs to be re-defined. Yet, preliminary observations indicate an ongoing impact of spatial heterogeneity on the efficacy of monoclonal antibodies. In conclusion, we recommend combining molecular tests with imaging to improve risk prediction and monitoring of residual disease. Overcoming intra-tumor heterogeneity is the prerequisite for curing myeloma. Novel immunotherapies are promising but research addressing their impact on the spatial clonal architecture is highly warranted.
Background
The aim of this analysis was to model the effect of local control (LC) on overall survival (OS) in patients treated with stereotactic body radiotherapy (SBRT) for liver or lung metastases from colorectal cancer.
Methods
The analysis is based on pooled data from two retrospective SBRT databases for pulmonary and hepatic metastases from 27 centers from Germany and Switzerland. Only patients with metastases from colorectal cancer were considered to avoid histology as a confounding factor. An illness-death model was employed to model the relationship between LC and OS.
Results
Three hundred eighty-eight patients with 500 metastatic lesions (lung n = 209, liver n = 291) were included and analyzed. Median follow-up time for local recurrence assessment was 12.1 months. Ninety-nine patients with 112 lesions experienced local failure. Seventy-one of these patients died after local failure. Median survival time was 27.9 months in all patients and 25.4 months versus 30.6 months in patients with and without local failure after SBRT. The baseline risk of death after local failure exceeds the baseline risk of death without local failure at 10 months indicating better survival with LC.
Conclusion
In CRC patients with lung or liver metastases, our findings suggest improved long-term OS by achieving metastatic disease control using SBRT in patients with a projected OS estimate of > 12 months.
Many parts of sub-Saharan Africa (SSA) are prone to land use and land cover change (LULCC). In many cases, natural systems are converted into agricultural land to feed the growing population. However, despite climate change being a major focus nowadays, the impacts of these conversions on water resources, which are essential for agricultural production, is still often neglected, jeopardizing the sustainability of the socio-ecological system. This study investigates historic land use/land cover (LULC) patterns as well as potential future LULCC and its effect on water quantities in a complex tropical catchment in Tanzania. It then compares the results using two climate change scenarios. The Land Change Modeler (LCM) is used to analyze and to project LULC patterns until 2030 and the Soil and Water Assessment Tool (SWAT) is utilized to simulate the water balance under various LULC conditions. Results show decreasing low flows by 6–8% for the LULC scenarios, whereas high flows increase by up to 84% for the combined LULC and climate change scenarios. The effect of climate change is stronger compared to the effect of LULCC, but also contains higher uncertainties. The effects of LULCC are more distinct, although crop specific effects show diverging effects on water balance components. This study develops a methodology for quantifying the impact of land use and climate change and therefore contributes to the sustainable management of the investigated catchment, as it shows the impact of environmental change on hydrological extremes (low flow and floods) and determines hot spots, which are critical for environmental development.
Phylogenetically related groups of species contain lineage-specific genes that exhibit no sequence similarity to any genes outside the lineage. We describe here that the Jekyll gene, required for sexual reproduction, exists in two much diverged allelic variants, Jek1 and Jek3. Despite low similarity, the Jek1 and Jek3 proteins share identical signal peptides, conserved cysteine positions and direct repeats. The Jek1/Jek3 sequences are located at the same chromosomal locus and inherited in a monogenic Mendelian fashion. Jek3 has a similar expression as Jek1 and complements the Jek1 function in Jek1-deficient plants. Jek1 and Jek3 allelic variants were almost equally distributed in a collection of 485 wild and domesticated barley accessions. All domesticated barleys harboring the Jek1 allele belong to single haplotype J1-H1 indicating a genetic bottleneck during domestication. Domesticated barleys harboring the Jek3 allele consisted of three haplotypes. Jekyll-like sequences were found only in species of the closely related tribes Bromeae and Triticeae but not in other Poaceae. Non-invasive magnetic resonance imaging revealed intrinsic grain structure in Triticeae and Bromeae, associated with the Jekyll function. The emergence of Jekyll suggests its role in the separation of the Bromeae and Triticeae lineages within the Poaceae and identifies the Jekyll genes as lineage-specific.
Introduction: The German PID-NET registry was founded in 2009, serving as the first national registry of patients with primary immunodeficiencies (PID) in Germany. It is part of the European Society for Immunodeficiencies (ESID) registry. The primary purpose of the registry is to gather data on the epidemiology, diagnostic delay, diagnosis, and treatment of PIDs.
Methods: Clinical and laboratory data was collected from 2,453 patients from 36 German PID centres in an online registry. Data was analysed with the software Stata® and Excel.
Results: The minimum prevalence of PID in Germany is 2.72 per 100,000 inhabitants. Among patients aged 1-25, there was a clear predominance of males. The median age of living patients ranged between 7 and 40 years, depending on the respective PID. Predominantly antibody disorders were the most prevalent group with 57% of all 2,453 PID patients (including 728 CVID patients). A gene defect was identified in 36% of patients. Familial cases were observed in 21% of patients. The age of onset for presenting symptoms ranged from birth to late adulthood (range 0-88 years). Presenting symptoms comprised infections (74%) and immune dysregulation (22%). Ninety-three patients were diagnosed without prior clinical symptoms. Regarding the general and clinical diagnostic delay, no PID had undergone a slight decrease within the last decade. However, both, SCID and hyper IgE-syndrome showed a substantial improvement in shortening the time between onset of symptoms and genetic diagnosis. Regarding treatment, 49% of all patients received immunoglobulin G (IgG) substitution (70%-subcutaneous; 29%-intravenous; 1%-unknown). Three-hundred patients underwent at least one hematopoietic stem cell transplantation (HSCT). Five patients had gene therapy.
Conclusion: The German PID-NET registry is a precious tool for physicians, researchers, the pharmaceutical industry, politicians, and ultimately the patients, for whom the outcomes will eventually lead to a more timely diagnosis and better treatment.
We have sequenced the genome of the largest freshwater fish species of the world, the arapaima. Analysis of gene family dynamics and signatures of positive selection identified genes involved in the specific adaptations and unique features of this iconic species, in particular it’s large size and fast growth. Genome sequences from both sexes combined with RAD-tag analyses from other males and females led to the isolation of male-specific scaffolds and supports an XY sex determination system in arapaima. Whole transcriptome sequencing showed that the product of the gland-like secretory organ on the head surface of males and females may not only provide nutritional fluid for sex-unbiased parental care, but that the organ itself has a more specific function in males, which engage more in parental care.
Purpose: The effective point of measurement (EPOM) of cylindrical ionization chambers differs from their geometric center. The exact shift depends on chamber construction details, above all the chamber size, and to some degree on the field-size and beam quality. It generally decreases as the chamber dimensions get smaller. In this work, effective points of measurement in small photon fields of a range of cylindrical chambers of different sizes are investigated, including small chambers that have not been studied previously.
Methods: In this investigation, effective points of measurement for different ionization chambers (Farmer type, scanning chambers, micro-ionization chambers) and solid state detectors were determined by measuring depth-ionization curves in a 6 MV beam in field sizes between 2 9 2 cm2 and 10 9 10 cm2 and comparing those curves with curves measured with plane-parallel chambers.
Results: It was possible to average the results to one shift per detector, as the results were sufficiently independent of the studied field sizes. For cylindrical ion chambers, shifts of the EPOM were determined to be between 0.49 and 0.30 times the inner chamber radius from the reference point.
Conclusions: We experimentally confirmed the previously reported decrease of the EPOM shift with decreasing detector size. Highly accurate data for a large range of detectors, including new very small ones, were determined. Thus, small chambers noticeably differ from the 0.5-times to 0.6-times the inner chamber radius recommendations in current dosimetry protocols. The detector-individual EPOMs need to be considered for measurements of depth-dose curves.
Sensory processing and attention allocation are shaped by threat, but the role of trait-anxiety in sensory processing as a function of threat predictability remains incompletely understood. Therefore, we measured steady-state visual evoked potentials (ssVEPs) as an index of sensory processing of predictable and unpredictable threat cues in 29 low (LA) and 29 high (HA) trait-anxious participants during a modified NPU-paradigm followed by an extinction phase. Three different contextual cues indicated safety (N), predictable (P) or unpredictable threat (U), while foreground cues signalled shocks in the P-condition only. All participants allocated increased attentional resources to the central P-threat cue, replicating previous findings. Importantly, LA individuals exhibited larger ssVEP amplitudes to contextual threat (U and P) than to contextual safety cues, while HA individuals did not differentiate among contextual cues in general. Further, HA exhibited higher aversive ratings of all contexts compared to LA. These results suggest that high trait-anxious individuals might be worse at discriminating contextual threat stimuli and accordingly overestimate the probability and aversiveness of unpredictable threat. These findings support the notion of aberrant sensory processing of unpredictable threat in anxiety disorders, as this processing pattern is already evident in individuals at risk of these disorders.
Background
Fabry disease is caused by a deficiency of the lysosomal enzyme α-galactosidase, resulting in progressive accumulation of globotriaosylceramide (GL-3). The disease can manifest early during childhood and adolescence. Enzyme replacement therapy (ERT) with recombinant human α-galactosidase is the first specific treatment for Fabry disease and has been available in Europe since 2001. This paper presents the findings of a systematic literature review of clinical outcomes with ERT in paediatric patients with Fabry disease.
Methods
A comprehensive systematic review of published literature on ERT in Fabry disease was conducted in January 2017. The literature analysis included all original articles reporting outcomes of ERT in paediatric patients.
Results
Treatment-related outcomes in the paediatric population were reported in six publications derived from open-label clinical trials and in 10 publications derived from observational or registry-based studies. ERT was shown to significantly reduce plasma and urine GL-3 levels in paediatric patients with Fabry disease. The effect of ERT on GL-3 clearance from renal podocytes appeared to be agalsidase dose-dependent. ERT relieved pain and improved gastrointestinal symptoms and quality of life.
Conclusions
Based on the published literature, the use of ERT in paediatric patients can significantly clear GL-3 accumulation, ameliorate the early symptoms of Fabry disease, and improve quality of life. Treatment with ERT in paediatric patients with Fabry disease may be important to prevent further disease progression and overt organ damage.
Background
Enzyme replacement therapy (ERT) with recombinant human α-galactosidase has been available for the treatment of Fabry disease since 2001 in Europe and 2003 in the USA. Treatment outcomes with ERT are dependent on baseline patient characteristics, and published data are derived from heterogeneous study populations.
Methods
We conducted a comprehensive systematic literature review of all original articles on ERT in the treatment of Fabry disease published up until January 2017. This article presents the findings in adult male patients.
Results
Clinical evidence for the efficacy of ERT in adult male patients was available from 166 publications including 36 clinical trial publications. ERT significantly decreases globotriaosylceramide levels in plasma, urine, and in different kidney, heart, and skin cell types, slows the decline in estimated glomerular filtration rate, and reduces/stabilizes left ventricular mass and cardiac wall thickness. ERT also improves nervous system, gastrointestinal, pain, and quality of life outcomes.
Conclusions
ERT is a disease-specific treatment for patients with Fabry disease that may provide clinical benefits on several outcomes and organ systems. Better outcomes may be observed when treatment is started at an early age prior to the development of organ damage such as chronic kidney disease or cardiac fibrosis. Consolidated evidence suggests a dose effect. Data described in male patients, together with female and paediatric data, informs clinical practice and therapeutic goals for individualized treatment.
Background
Heterozygous females with Fabry disease have a wide range of clinical phenotypes depending on the nature of their mutation and their X-chromosome inactivation pattern; it is therefore important to examine outcomes of enzyme replacement therapy (ERT) in the female patient population specifically. This paper presents the findings of a systematic literature review of treatment outcomes with ERT in adult female patients.
Methods
A comprehensive systematic literature review was conducted through January 2017 to retrieve published papers with original data on ERT in the treatment of Fabry disease. The review included all original articles that presented ERT outcomes data on patients with Fabry disease, irrespective of the study type.
Results
Clinical evidence for the efficacy of ERT in female patients was available from 67 publications including six clinical trial publications, and indicates significant reductions in plasma and urine globotriaosylceramide (GL-3) accumulation (in female patients with elevated pre-treatment levels) and improvements in cardiac parameters and quality of life (QoL). To date, data are insufficient to conclude on the effects of ERT on the nervous system, gastrointestinal manifestations, and pain in female patients with Fabry disease.
Conclusions
This review of available literature data demonstrates that ERT in adult female patients with Fabry disease has a beneficial effect on GL-3 levels and cardiac outcomes. The current evidence also suggests that ERT may improve QoL in this patient population, though further studies are needed to examine these results.
Central Europe experienced several droughts in the recent past, such as in the year 2018, which was characterized by extremely low rainfall rates and high temperatures, resulting in substantial agricultural yield losses. Time series of satellite earth observation data enable the characterization of past drought events over large temporal and spatial scales. Within this study, Moderate Resolution Spectroradiometer (MODIS) Enhanced Vegetation Index (EVI) (MOD13Q1) 250 m time series were investigated for the vegetation periods of 2000 to 2018. The spatial and temporal development of vegetation in 2018 was compared to other dry and hot years in Europe, like the drought year 2003. Temporal and spatial inter- and intra-annual patterns of EVI anomalies were analyzed for all of Germany and for its cropland, forest, and grassland areas individually. While vegetation development in spring 2018 was above average, the summer months of 2018 showed negative anomalies in a similar magnitude as in 2003, which was particularly apparent within grassland and cropland areas in Germany. In contrast, the year 2003 showed negative anomalies during the entire growing season. The spatial pattern of vegetation status in 2018 showed high regional variation, with north-eastern Germany mainly affected in June, north-western parts in July, and western Germany in August. The temporal pattern of satellite-derived EVI deviances within the study period 2000-2018 were in good agreement with crop yield statistics for Germany. The study shows that the EVI deviation of the summer months of 2018 were among the most extreme in the study period compared to other years. The spatial pattern and temporal development of vegetation condition between the drought years differ.
Two different chromophores, namely a dipolar and an octupolar system, were prepared and their linear and nonlinear optical properties as well as their bioimaging capabilities were compared. Both contain triphenylamine as the donor and a triarylborane as the acceptor, the latter modified with cationic trimethylammonio groups to provide solubility in aqueous media. The octupolar system exhibits a much higher two‐photon brightness, and also better cell viability and enhanced selectivity for lysosomes compared with the dipolar chromophore. Furthermore, both dyes were applied in two‐photon excited fluorescence (TPEF) live‐cell imaging.
Two different chromophores, namely a dipolar and an octupolar system, were prepared and their linear and nonlinear optical properties as well as their bioimaging capabilities were compared. Both contain triphenylamine as the donor and a triarylborane as the acceptor, the latter modified with cationic trimethylammonio groups to provide solubility in aqueous media. The octupolar system exhibits a much higher two‐photon brightness, and also better cell viability and enhanced selectivity for lysosomes compared with the dipolar chromophore. Furthermore, both dyes were applied in two‐photon excited fluorescence (TPEF) live‐cell imaging.
Besides external characteristics and reading a piece of DNA (barcode), the DNA weight per nucleus (genome size) via flow cytometry is a key value to detect species and hybrids and determine ploidy. In addition, the DNA weight appears to be related to various properties, such as the size of the cell and the nucleus, the duration of mitosis and meiosis and the generation time. Sometimes it is even possible to distinguish between groups or sections, which can lead to new classification of the genera. The variation in DNA weight is also useful to analyze biodiversity, genome evolution and relationships between related taxa. Moreover, it is important to know how large a genome is before one determines the base sequence of the DNA of a plant. Flow cytometry is also important for understanding fundamental processes in plants such as growth and development and recognizing chimeras. In the literature, DNA weight measurements are usually limited to one genus and often only locally (Siljak et al. 2010; Bai et al. 2012). In this study, however, it was decided to investigate all vascular plants from one country. This can also contribute to the protection of rare plants. This study is the first flora in the world whose weight of DNA per nucleus and peak patterns has been determined. More than 6400 plants, representing more than 2350 (sub)species (more than 90%) have been collected, thanks to the help of almost 100 volunteers of Floristisch Onderzoek Nederland (Floron). Multiple specimens of many species have therefore been measured, preferably from different populations, in some cases more than fifty. For 1370 species, these values were not previously published. Moreover, a good number of the remaining 45% are new for The Netherlands. In principle, each species has a fixed weight of DNA per nucleus. It has also been found that, especially between the genera, there are strong differences in the number of peaks that determine the DNA weight, from one to five peaks. This indicates that in a plant or organ there are sometimes nuclei with multiples of its standard DNA weight (multiple ploidy levels). It is impossible to show graphs of more than 2350 species. Therefore, we have chosen to show the peak pattern in a new way in a short formula. Within most genera there are clear differences in the DNA weights per nucleus between the species, in some other genera the DNA weight is hardly variable. Based on about twenty genera that were previously measured completely in most cases (‘t Hart et al. 2003: Veldkamp and Zonneveld 2011; Soes et al. 2012; Dirkse et al. 2014, 2015; Verloove et al. 2017; Zonneveld [et al.] 2000−2018), it can be noted that even if all species of a genus have the same number of chromosomes, there can still be a difference of up to three times in the weight of the DNA. Therefore, a twice larger DNA weight does not have to indicate four sets of chromosomes. Finally, this research has also found clues to examine further the current taxonomy of a number of species or genera.
The retinal pigment epithelium (RPE) is a unique epithelium, with major roles which are essential in the visual cycle and homeostasis of the outer retina. The RPE is a monolayer of polygonal and pigmented cells strategically placed between the neuroretina and Bruch membrane, adjacent to the fenestrated capillaries of the choriocapillaris. It shows strong apical (towards photoreceptors) to basal/basolateral (towards Bruch membrane) polarization. Multiple functions are bound to a complex structure of highly organized and polarized intracellular components: the cytoskeleton. A strong connection between the intracellular cytoskeleton and extracellular matrix is indispensable to maintaining the function of the RPE and thus, the photoreceptors. Impairments of these intracellular structures and the regular architecture they maintain often result in a disrupted cytoskeleton, which can be found in many retinal diseases, including age-related macular degeneration (AMD). This review article will give an overview of current knowledge on the molecules and proteins involved in cytoskeleton formation in cells, including RPE and how the cytoskeleton is affected under stress conditions — especially in AMD.
The cytoskeletal crosslinking protein MACF1 is dispensable for thrombus formation and hemostasis
(2019)
Coordinated reorganization of cytoskeletal structures is critical for key aspects of platelet physiology. While several studies have addressed the role of microtubules and filamentous actin in platelet production and function, the significance of their crosstalk in these processes has been poorly investigated. The microtubule-actin cross-linking factor 1 (MACF1; synonym: Actin cross-linking factor 7, ACF7) is a member of the spectraplakin family, and one of the few proteins expressed in platelets, which possess actin and microtubule binding domains thereby facilitating actin-microtubule interaction and regulation. We used megakaryocyte- and platelet-specific Macf1 knockout (Macf1fl/fl, Pf4-Cre) mice to study the role of MACF1 in platelet production and function. MACF1 deficient mice displayed comparable platelet counts to control mice. Analysis of the platelet cytoskeletal ultrastructure revealed a normal marginal band and actin network. Platelet spreading on fibrinogen was slightly delayed but platelet activation and clot traction was unaffected. Ex vivo thrombus formation and mouse tail bleeding responses were similar between control and mutant mice. These results suggest that MACF1 is dispensable for thrombopoiesis, platelet activation, thrombus formation and the hemostatic function in mice.
One of the main objectives of the ANTARES telescope is the search for point- like neutrino sources. Both the pointing accuracy and the angular resolution of the detector are important in this context and a reliableway to evaluate this performance is needed. In order to measure the pointing accuracy of the detector, one possibility is to study the shadow of the Moon, i. e. the deficit of the atmospheric muon flux from the direction of the Moon induced by the absorption of cosmic rays. Analysing the data taken between 2007 and 2016, theMoon shadow is observed with 3.5s statistical significance. The detector angular resolution for downwardgoing muons is 0.73. +/- 0.14.. The resulting pointing performance is consistent with the expectations. An independent check of the telescope pointing accuracy is realised with the data collected by a shower array detector onboard of a ship temporarily moving around the ANTARES location.
Hsp90 is a dimeric molecular chaperone that is essential for the folding and activation of hundreds of client proteins. Co-chaperone proteins regulate the ATP-driven Hsp90 client activation cycle. Aha-type co-chaperones are the most potent stimulators of the Hsp90 ATPase activity but the relationship between ATPase regulation and in vivo activity is poorly understood. We report here that the most strongly conserved region of Aha-type co-chaperones, the N terminal NxNNWHW motif, modulates the apparent affinity of Hsp90 for nucleotide substrates. The ability of yeast Aha-type co-chaperones to act in vivo is ablated when the N terminal NxNNWHW motif is removed. This work suggests that nucleotide exchange during the Hsp90 functional cycle may be more important than rate of catalysis.
Background
One of the most important steps taken by Beyond Batten Disease Foundation in our quest to cure juvenile Batten (CLN3) disease is to understand the State of the Science. We believe that a strong understanding of where we are in our experimental understanding of the CLN3 gene, its regulation, gene product, protein structure, tissue distribution, biomarker use, and pathological responses to its deficiency, lays the groundwork for determining therapeutic action plans.
Objectives
To present an unbiased comprehensive reference tool of the experimental understanding of the CLN3 gene and gene product of the same name.
Methods
BBDF compiled all of the available CLN3 gene and protein data from biological databases, repositories of federally and privately funded projects, patent and trademark offices, science and technology journals, industrial drug and pipeline reports as well as clinical trial reports and with painstaking precision, validated the information together with experts in Batten disease, lysosomal storage disease, lysosome/endosome biology.
Results
The finished product is an indexed review of the CLN3 gene and protein which is not limited in page size or number of references, references all available primary experiments, and does not draw conclusions for the reader.
Conclusions
Revisiting the experimental history of a target gene and its product ensures that inaccuracies and contradictions come to light, long-held beliefs and assumptions continue to be challenged, and information that was previously deemed inconsequential gets a second look. Compiling the information into one manuscript with all appropriate primary references provides quick clues to which studies have been completed under which conditions and what information has been reported. This compendium does not seek to replace original articles or subtopic reviews but provides an historical roadmap to completed works.
Life on earth adapted to the daily reoccurring changes in environment by evolving an endogenous circadian clock. Although the circadian clock has a crucial impact on survival and behavior of solitary bees, many aspects of solitary bee clock mechanisms remain unknown. Our study is the first to show that the circadian clock governs emergence in Osmia bicornis, a bee species which overwinters as adult inside its cocoon. Therefore, its eclosion from the pupal case is separated by an interjacent diapause from its emergence in spring. We show that this bee species synchronizes its emergence to the morning. The daily rhythms of emergence are triggered by temperature cycles but not by light cycles. In contrast to this, the bee’s daily rhythms in locomotion are synchronized by light cycles. Thus, we show that the circadian clock of O. bicornis is set by either temperature or light, depending on what activity is timed. Light is a valuable cue for setting the circadian clock when bees have left the nest. However, for pre-emerged bees, temperature is the most important cue, which may represent an evolutionary adaptation of the circadian system to the cavity-nesting life style of O. bicornis.
It is assumed that a properly timed circadian clock enhances fitness, but only few studies have truly demonstrated this in animals. We raised each of the three classical Drosophila period mutants for >50 generations in the laboratory in competition with wildtype flies. The populations were either kept under a conventional 24-h day or under cycles that matched the mutant’s natural cycle, i.e., a 19-h day in the case of pers mutants and a 29-h day for perl mutants. The arrhythmic per0 mutants were grown together with wildtype flies under constant light that renders wildtype flies similar arrhythmic as the mutants. In addition, the mutants had to compete with wildtype flies for two summers in two consecutive years under outdoor conditions. We found that wildtype flies quickly outcompeted the mutant flies under the 24-h laboratory day and under outdoor conditions, but perl mutants persisted and even outnumbered the wildtype flies under the 29-h day in the laboratory. In contrast, pers and per0 mutants did not win against wildtype flies under the 19-h day and constant light, respectively. Our results demonstrate that wildtype flies have a clear fitness advantage in terms of fertility and offspring survival over the period mutants and – as revealed for perl mutants – this advantage appears maximal when the endogenous period resonates with the period of the environment. However, the experiments indicate that perl and pers persist at low frequencies in the population even under the 24-h day. This may be a consequence of a certain mating preference of wildtype and heterozygous females for mutant males and time differences in activity patterns between wildtype and mutants.
The Best for the Most Important: Maintaining a Pristine Proteome in Stem and Progenitor Cells
(2019)
Pluripotent stem cells give rise to reproductively enabled offsprings by generating progressively lineage-restricted multipotent stem cells that would differentiate into lineage-committed stem and progenitor cells. These lineage-committed stem and progenitor cells give rise to all adult tissues and organs. Adult stem and progenitor cells are generated as part of the developmental program and play critical roles in tissue and organ maintenance and/or regeneration. The ability of pluripotent stem cells to self-renew, maintain pluripotency, and differentiate into a multicellular organism is highly dependent on sensing and integrating extracellular and extraorganismal cues. Proteins perform and integrate almost all cellular functions including signal transduction, regulation of gene expression, metabolism, and cell division and death. Therefore, maintenance of an appropriate mix of correctly folded proteins, a pristine proteome, is essential for proper stem cell function. The stem cells' proteome must be pristine because unfolded, misfolded, or otherwise damaged proteins would interfere with unlimited self-renewal, maintenance of pluripotency, differentiation into downstream lineages, and consequently with the development of properly functioning tissue and organs. Understanding how various stem cells generate and maintain a pristine proteome is therefore essential for exploiting their potential in regenerative medicine and possibly for the discovery of novel approaches for maintaining, propagating, and differentiating pluripotent, multipotent, and adult stem cells as well as induced pluripotent stem cells. In this review, we will summarize cellular networks used by various stem cells for generation and maintenance of a pristine proteome. We will also explore the coordination of these networks with one another and their integration with the gene regulatory and signaling networks.
The rhodium(I) complex [Rh(κ3-P,O,P-Xantphos)(η2-PhC≡CPh)][BArF4] (ArF = 3,5-(CF3)2C6H4) is an effective catalyst for the cis-selective hydroboration of the alkyne diphenylacetylene using the amine-borane H3B·NMe3. Detailed mechanistic studies, that include initial rate measurements, full simulation of temporal profiles for a variety of catalyst and substrate concentrations, and speciation experiments, suggest a mechanism that involves initial coordination of alkyne and a saturation kinetics regime for amine-borane binding. The solid-state molecular structure of a model complex that probes the proposed resting state is also reported, [Rh(κ3-P,O,P-Xantphos)(NCMe)(η2-PhC≡CPh)][BArF4].
By introduction of four hydroxy (HO) groups into the two perylene bisimide (PBI) bay areas, new HO‐PBI ligands were obtained which upon deprotonation can complex ZnII ions and photosensitize semiconductive zinc oxide thin films. Such coordination is beneficial for dispersing PBI photosensitizer molecules evenly into metal oxide films to fabricate organic–inorganic hybrid interlayers for organic solar cells. Supported by the photoconductive effect of the ZnO:HO‐PBI hybrid interlayers, improved electron collection and transportation is achieved in fullerene and non‐fullerene polymer solar cell devices, leading to remarkable power conversion efficiencies of up to 15.95 % for a non‐fullerene based organic solar cell.
Objectives
The long head of the biceps (LHB) is often resected in shoulder surgery and could therefore serve as a cell source for tissue engineering approaches in the shoulder. However, whether it represents a suitable cell source for regenerative approaches, both in the inflamed and non-inflamed states, remains unclear. In the present study, inflamed and native human LHBs were comparatively characterized for features of regeneration.
Methods
In total, 22 resected LHB tendons were classified into inflamed samples (n = 11) and non-inflamed samples (n = 11). Proliferation potential and specific marker gene expression of primary LHB-derived cell cultures were analyzed. Multipotentiality, including osteogenic, adipogenic, chondrogenic, and tenogenic differentiation potential of both groups were compared under respective lineage-specific culture conditions.
Results
Inflammation does not seem to affect the proliferation rate of the isolated tendon-derived stem cells (TDSCs) and the tenogenic marker gene expression. Cells from both groups showed an equivalent osteogenic, adipogenic, chondrogenic and tenogenic differentiation potential in histology and real-time polymerase chain reaction (RT-PCR) analysis.
Conclusion
These results suggest that the LHB tendon might be a suitable cell source for regenerative approaches, both in inflamed and non-inflamed states. The LHB with and without tendinitis has been characterized as a novel source of TDSCs, which might facilitate treatment of degeneration and induction of regeneration in shoulder surgery.
Background
Increasing attention is payed to the contribution of somatosensory processing in motor control. In particular, temporal somatosensory discrimination has been found to be altered differentially in common movement disorders. To date, there have only been speculations as to how impaired temporal discrimination and clinical motor signs may relate to each other. Prior to disentangling this relationship, potential confounders of temporal discrimination, in particular age and peripheral nerve conduction, should be assessed, and a quantifiable measure of proprioceptive performance should be established.
ObjectiveTo assess the influence of age and polyneuropathy (PNP) on somatosensory temporal discrimination threshold (STDT), temporal discrimination movement threshold (TDMT), and behavioral measures of proprioception of upper and lower limbs.
Methods
STDT and TDMT were assessed in 79 subjects (54 healthy, 25 with PNP; age 30–79 years). STDT was tested with surface electrodes over the thenar or dorsal foot region. TDMT was probed with needle electrodes in flexor carpi radialis (FCR) and tibialis anterior (TA) muscle. Goniometer-based devices were used to assess limb proprioception during (i) active pointing to LED markers, (ii) active movements in response to variable visual cues, and (iii) estimation of limb position following passive movements. Pointing (or estimation) error was taken as a measure of proprioceptive performance.
Results
In healthy subjects, higher age was associated with higher STDT and TDMT at upper and lower extremities, while age did not correlate with proprioceptive performance. Patients with PNP showed higher STDT and TDMT values and decreased proprioceptive performance in active pointing tasks compared to matched healthy subjects. As an additional finding, there was a significant correlation between performance in active pointing tasks and temporal discrimination thresholds.
Conclusion
Given their notable impact on measures of temporal discrimination, age and peripheral nerve conduction need to be accounted for if STDT and TDMT are applied in patients with movement disorders. As a side observation, the correlation between measures of proprioception and temporal discrimination may prompt further studies on the presumptive link between these two domains.
An intricate network of molecular and cellular actors orchestrates the delicate balance between effector immune responses and immune tolerance. The pleiotropic cytokine tumor necrosis factor-alpha (TNF) proves as a pivotal protagonist promoting but also suppressing immune responses. These opposite actions are accomplished through specialist cell types responding to TNF via TNF receptors TNFR1 and TNFR2. Recent findings highlight the importance of TNFR2 as a key regulator of activated natural FoxP3+ regulatory T cells (Tregs) in inflammatory conditions, such as acute graft-vs.-host disease (GvHD) and the tumor microenvironment. Here we review recent advances in our understanding of TNFR2 signaling in T cells and discuss how these can reconcile seemingly conflicting observations when manipulating TNF and TNFRs. As TNFR2 emerges as a new and attractive target we furthermore pinpoint strategies and potential pitfalls for therapeutic targeting of TNFR2 for cancer treatment and immune tolerance after allogeneic hematopoietic cell transplantation.
Platelet collagen interactions at sites of vascular injuries predominantly involve glycoprotein VI (GPVI) and the integrin α2β1. Both proteins are primarily expressed on platelets and megakaryocytes whereas GPVI expression is also shown on endothelial and integrin α2β1 expression on epithelial cells. We recently showed that depletion of GPVI improves stroke outcome without increasing the risk of cerebral hemorrhage. Genetic variants associated with higher platelet surface integrin α2 (ITGA2) receptor levels have frequently been found to correlate with an increased risk of ischemic stroke in patients. However until now, no preclinical stroke study has addressed whether platelet integrin α2β1 contributes to the pathophysiology of ischemia/reperfusion (I/R) injury. Focal cerebral ischemia was induced in C57BL/6 and Itga2\(^{−/−}\) mice by a 60 min transient middle cerebral artery occlusion (tMCAO). Additionally, wild-type animals were pretreated with anti-GPVI antibody (JAQ1) or Fab fragments of a function blocking antibody against integrin α2β1 (LEN/B). In anti-GPVI treated animals, intravenous (IV) recombinant tissue plasminogen activator (rt-PA) treatment was applied immediately prior to reperfusion. Stroke outcome, including infarct size and neurological scoring was determined on day 1 after tMCAO. We demonstrate that targeting the integrin α2β1 (pharmacologic; genetic) did neither reduce stroke size nor improve functional outcome on day 1 after tMCAO. In contrast, depletion of platelet GPVI prior to stroke was safe and effective, even when combined with rt-PA treatment. Our results underscore that GPVI, but not ITGA2, is a promising and safe target in the setting of ischemic stroke.
γ-aminobutyric acid type A receptors (GABA\(_A\)Rs) are the major mediators of synaptic inhibition in the brain. Aberrant GABA\(_A\)R activity or regulation is observed in various neurodevelopmental disorders, neurodegenerative diseases and mental illnesses, including epilepsy, Alzheimer’s and schizophrenia. Benzodiazepines, anesthetics and other pharmaceutics targeting these receptors find broad clinical use, but their inherent lack of receptor subtype specificity causes unavoidable side effects, raising a need for new or adjuvant medications. In this review article, we introduce a new strategy to modulate GABAeric signaling: targeting the intracellular protein interactors of GABA\(_A\)Rs. Of special interest are scaffolding, anchoring and supporting proteins that display high GABA\(_A\)R subtype specificity. Recent efforts to target gephyrin, the major intracellular integrator of GABAergic signaling, confirm that GABA\(_A\)R-associated proteins can be successfully targeted through diverse molecules, including recombinant proteins, intrabodies, peptide-based probes and small molecules. Small-molecule artemisinins and peptides derived from endogenous interactors, that specifically target the universal receptor binding site of gephyrin, acutely affect synaptic GABA\(_A\)R numbers and clustering, modifying neuronal transmission. Interference with GABA\(_A\)R trafficking provides another way to modulate inhibitory signaling. Peptides blocking the binding site of GABA\(_A\)R to AP2 increase the surface concentration of GABA\(_A\)R clusters and enhance GABAergic signaling. Engineering of gephyrin binding peptides delivered superior means to interrogate neuronal structure and function. Fluorescent peptides, designed from gephyrin binders, enable live neuronal staining and visualization of gephyrin in the post synaptic sites with submicron resolution. We anticipate that in the future, novel fluorescent probes, with improved size and binding efficiency, may find wide application in super resolution microscopy studies, enlightening the nanoscale architecture of the inhibitory synapse. Broader studies on GABA\(_A\)R accessory proteins and the identification of the exact molecular binding interfaces and affinities will advance the development of novel GABA\(_A\)R modulators and following in vivo studies will reveal their clinical potential as adjuvant or stand-alone drugs.
Targeting bromodomain-containing protein 4 (BRD4) inhibits MYC expression in colorectal cancer cells
(2019)
The transcriptional regulator BRD4 has been shown to be important for the expression of several oncogenes including MYC. Inhibiting of BRD4 has broad antiproliferative activity in different cancer cell types. The small molecule JQ1 blocks the interaction of BRD4 with acetylated histones leading to transcriptional modulation. Depleting BRD4 via engineered bifunctional small molecules named PROTACs (proteolysis targeting chimeras) represents the next-generation approach to JQ1-mediated BRD4 inhibition. PROTACs trigger BRD4 for proteasomale degradation by recruiting E3 ligases. The aim of this study was therefore to validate the importance of BRD4 as a relevant target in colorectal cancer (CRC) cells and to compare the efficacy of BRD4 inhibition with BRD4 degradation on downregulating MYC expression. JQ1 induced a downregulation of both MYC mRNA and MYC protein associated with an antiproliferative phenotype in CRC cells. dBET1 and MZ1 induced degradation of BRD4 followed by a reduction in MYC expression and CRC cell proliferation. In SW480 cells, where dBET1 failed, we found significantly lower levels of the E3 ligase cereblon, which is essential for dBET1-induced BRD4 degradation. To gain mechanistic insight into the unresponsiveness to dBET1, we generated dBET1-resistant LS174t cells and found a strong downregulation of cereblon protein. These findings suggest that inhibition of BRD4 by JQ1 and degradation of BRD4 by dBET1 and MZ1 are powerful tools for reducing MYC expression and CRC cell proliferation. In addition, downregulation of cereblon may be an important mechanism for developing dBET1 resistance, which can be evaded by incubating dBET1-resistant cells with JQ1 or MZ1.
Targeted panel sequencing in pediatric primary cardiomyopathy supports a critical role of TNNI3
(2019)
The underlying genetic mechanisms and early pathological events of children with primary cardiomyopathy (CMP) are insufficiently characterized. In this study, we aimed to characterize the mutational spectrum of primary CMP in a large cohort of patients ≤18 years referred to a tertiary center. Eighty unrelated index patients with pediatric primary CMP underwent genetic testing with a panel‐based next‐generation sequencing approach of 89 genes. At least one pathogenic or probably pathogenic variant was identified in 30/80 (38%) index patients. In all CMP subgroups, patients carried most frequently variants of interest in sarcomere genes suggesting them as a major contributor in pediatric primary CMP. In MYH7, MYBPC3, and TNNI3, we identified 18 pathogenic/probably pathogenic variants (MYH7 n = 7, MYBPC3 n = 6, TNNI3 n = 5, including one homozygous (TNNI3 c.24+2T>A) truncating variant. Protein and transcript level analysis on heart biopsies from individuals with homozygous mutation of TNNI3 revealed that the TNNI3 protein is absent and associated with upregulation of the fetal isoform TNNI1. The present study further supports the clinical importance of sarcomeric mutation—not only in adult—but also in pediatric primary CMP. TNNI3 is the third most important disease gene in this cohort and complete loss of TNNI3 leads to severe pediatric CMP.
Melt electrowriting (MEW) is an additive manufacturing technology that is recently used to fabricate voluminous scaffolds for biomedical applications. In this study, MEW is adapted for the seeding of multicellular spheroids, which permits the easy handling as a single sheet-like tissue-scaffold construct. Spheroids are made from adipose-derived stromal cells (ASCs). Poly(ε-caprolactone) is processed via MEW into scaffolds with box-structured pores, readily tailorable to spheroid size, using 13–15 µm diameter fibers. Two 7–8 µm diameter “catching fibers” near the bottom of the scaffold are threaded through each pore (360 and 380 µm) to prevent loss of spheroids during seeding. Cell viability remains high during the two week culture period, while the differentiation of ASCs into the adipogenic lineage is induced. Subsequent sectioning and staining of the spheroid-scaffold construct can be readily performed and accumulated lipid droplets are observed, while upregulation of molecular markers associated with successful differentiation is demonstrated. Tailoring MEW scaffolds with pores allows the simultaneous seeding of high numbers of spheroids at a time into a construct that can be handled in culture and may be readily transferred to other sites for use as implants or tissue models.
Background
T1 mapping sequences such as MOLLI, ShMOLLI and SASHA make use of different technical approaches, bearing strengths and weaknesses. It is well known that obtained T1 relaxation times differ between the sequence techniques as well as between different hardware. Yet, T1 quantification is a promising tool for myocardial tissue characterization, disregarding the absence of established reference values. The purpose of this study was to evaluate the feasibility of native and post-contrast T1 mapping methods as well as ECV maps and its diagnostic benefits in a clinical environment when scanning patients with various cardiac diseases at 3 T.
Methods
Native and post-contrast T1 mapping data acquired on a 3 T full-body scanner using the three pulse sequences 5(3)3 MOLLI, ShMOLLI and SASHA in 19 patients with clinical indication for contrast enhanced MRI were compared. We analyzed global and segmental T1 relaxation times as well as respective extracellular volumes and compared the emerged differences between the used pulse sequences.
Results
T1 times acquired with MOLLI and ShMOLLI exhibited systematic T1 deviation compared to SASHA. Myocardial MOLLI T1 times were 19% lower and ShMOLLI T1 times 25% lower compared to SASHA. Native blood T1 times from MOLLI were 13% lower than SASHA, while post-contrast MOLLI T1-times were only 5% lower. ECV values exhibited comparably biased estimation with MOLLI and ShMOLLI compared to SASHA in good agreement with results reported in literature. Pathology-suspect segments were clearly differentiated from remote myocardium with all three sequences.
Conclusion
Myocardial T1 mapping yields systematically biased pre- and post-contrast T1 times depending on the applied pulse sequence. Additionally calculating ECV attenuates this bias, making MOLLI, ShMOLLI and SASHA better comparable. Therefore, myocardial T1 mapping is a powerful clinical tool for classification of soft tissue abnormalities in spite of the absence of established reference values.
The massive infiltration of lymphocytes into the skin is a hallmark of numerous human skin disorders. By co-culturing murine keratinocytes with splenic T cells we demonstrate here that T cells affect and control the synthesis and secretion of chemokines by keratinocytes. While pre-activated CD8\(^+\)T cells induce the synthesis of CXCL9 and CXCL10 in keratinocytes and keep in check the synthesis of CXCL1, CXCL5, and CCL20, keratinocytes dampen the synthesis of CCL3 and CCL4 in pre-activated CD8\(^+\)T cells. One key molecule is IFN-γ that is synthesized by CD8\(^+\)T cells under the control of NFATc1 and NFATc2. CD8\(^+\)T cells deficient for both NFAT factors are unable to induce CXCL9 and CXCL10 expression. In addition, CD8\(^+\)T cells induced numerous type I IFN-inducible “defense genes” in keratinocytes encoding the PD1 and CD40 ligands, TNF-α and caspase-1. The enhanced expression of type I IFN-inducible genes resembles the gene expression pattern at the dermal/epidermal interface in lichen planus, an inflammatory T lymphocyte-driven skin disease, in which we detected the expression of CXCL10 in keratinocytes in close vicinity to the infiltration front of T cells. These data reflect the multifaceted interplay of lymphocytes with keratinocytes at the molecular level.
There is an urgent need for therapies that could reduce the disease burden of preterm hypoxic-ischemic encephalopathy. Here, we evaluate the long-term effects of multipotent adult progenitor cells (MAPC) on long-term behavioral outcomes in a preterm rat model of perinatal asphyxia. Rats of both sexes were treated with two doses of MAPCs within 24 h after the insult. Locomotor, cognitive and psychiatric impairments were evaluated starting at 1.5 (juvenile) and 6 months (adult). Hypoxia-ischemia affected locomotion, cognition, and anxiety in a sex-dependent manner, with higher vulnerability observed in males. The MAPC therapy partially attenuated deficits in object recognition memory in females of all tested ages, and in the adult males. The hypoxic insult caused delayed hyperactivity in adult males, which was corrected by MAPC therapy. These results suggest that MAPCs may have long-term benefits for neurodevelopmental outcome after preterm birth and global hypoxia-ischemia, which warrants further preclinical exploration.
Among the Microbacteriaceae the species of Subtercola and Agreia form closely associated clusters. Phylogenetic analysis demonstrated three major phylogenetic branches of these species. One of these branches contains the two psychrophilic species Subtercola frigoramans and Subtercola vilae, together with a larger number of isolates from various cold environments. Genomic evidence supports the separation of Agreia and Subtercola species. In order to gain insight into the ability of S. vilae to adapt to life in this extreme environment, we analyzed the genome with a particular focus on properties related to possible adaptation to a cold environment. General properties of the genome are presented, including carbon and energy metabolism, as well as secondary metabolite production. The repertoire of genes in the genome of S. vilae DB165\(^T\) linked to adaptations to the harsh conditions found in Llullaillaco Volcano Lake includes several mechanisms to transcribe proteins under low temperatures, such as a high number of tRNAs and cold shock proteins. In addition, S. vilae DB165\(^T\) is capable of producing a number of proteins to cope with oxidative stress, which is of particular relevance at low temperature environments, in which reactive oxygen species are more abundant. Most important, it obtains capacities to produce cryo-protectants, and to combat against ice crystal formation, it produces ice-binding proteins. Two new ice-binding proteins were identified which are unique to S. vilae DB165\(^T\). These results indicate that S. vilae has the capacity to employ different mechanisms to live under the extreme and cold conditions prevalent in Llullaillaco Volcano Lake.
Synergy of chemo- and photodynamic therapies with C\(_{60}\) Fullerene-Doxorubicin nanocomplex
(2019)
A nanosized drug complex was explored to improve the efficiency of cancer chemotherapy, complementing it with nanodelivery and photodynamic therapy. For this, nanomolar amounts of a non-covalent nanocomplex of Doxorubicin (Dox) with carbon nanoparticle C\(_{60}\) fullerene (C\(_{60}\)) were applied in 1:1 and 2:1 molar ratio, exploiting C\(_{60}\) both as a drug-carrier and as a photosensitizer. The fluorescence microscopy analysis of human leukemic CCRF-CEM cells, in vitro cancer model, treated with nanocomplexes showed Dox’s nuclear and C\(_{60}\)'s extranuclear localization. It gave an opportunity to realize a double hit strategy against cancer cells based on Dox's antiproliferative activity and C\(_{60}\)'s photoinduced pro-oxidant activity. When cells were treated with 2:1 C\(_{60}\)-Dox and irradiated at 405 nm the high cytotoxicity of photo-irradiated C\(_{60}\)-Dox enabled a nanomolar concentration of Dox and C\(_{60}\) to efficiently kill cancer cells in vitro. The high pro-oxidant and pro-apoptotic efficiency decreased IC\(_{50}\) 16, 9 and 7 × 10\(^3\)-fold, if compared with the action of Dox, non-irradiated nanocomplex, and C\(_{60}\)'s photodynamic effect, correspondingly. Hereafter, a strong synergy of therapy arising from the combination of C\(_{60}\)-mediated Dox delivery and C\(_{60}\) photoexcitation was revealed. Our data indicate that a combination of chemo- and photodynamic therapies with C\(_{60}\)-Dox nanoformulation provides a promising synergetic approach for cancer treatment.
Symptomatic vs. asymptomatic 20–40% internal carotid artery stenosis: Does the plaque size matter?
(2019)
Background: Around 9–15% of ischemic strokes are related to internal carotid artery (ICA)-stenosis ≥50%. However, the extent to which ICA-stenosis <50% causes ischemic cerebrovascular events is uncertain. We examined the relation between plaque cross-sectional area and length and the risk of ischemic stroke or TIA among patients with ICA-stenosis of 20–40%.
Methods: We retrospectively identified patients admitted to the Department of Neurology, University Hospital of Würzburg, from January 2011 until September 2016 with ischemic stroke or TIA and concomitant ICA-stenosis of 20–40%, either symptomatic or asymptomatic. Plaque length and cross-sectional area were assessed on ultrasound scans.
Results: We identified 41 patients with ischemic stroke or TIA and ICA-stenosis of 20–40%; 14 symptomatic and 27 asymptomatic. The plaque cross-sectional area was significantly larger among symptomatic than asymptomatic ICA-stenosis; median values (IQR) were 0.45 (0.21–0.69) cm2 and 0.27 (0.21–0.38) cm2, p = 0.03, respectively. A plaque cross-sectional area ≥0.36 cm2 had a sensitivity of 71% and a specificity of 76% for symptomatic compared with asymptomatic ICA-stenosis. In a sex-adjusted multivariate logistic regression, a plaque cross-sectional area ≥0.36 cm2 and a plaque length ≥1.65 cm were associated with an OR (95% CI) of 5.54 (1.2–25.6), p = 0.028 and 1.78 (0.36–8.73), p = 0.48, respectively, for symptomatic ICA-stenosis.
Conclusion: Large plaques might increase the risk of ischemic stroke or TIA among patients with low-grade ICA-stenosis of 20–40%. Sufficiently powered prospective longitudinal cohort studies are needed to definitively test the stroke risk stratification value of carotid plaque length and cross-sectional area in the setting of current optimal medical treatment.
We report on a 14 months old toddler who sustained a traumatic hemipelvectomy by being crushed between a car and a stone wall. After stabilization in the resuscitation room he was treated operatively by laparotomy, osteosynthesis of the pelvic ring, reconstruction of the both external iliac vessels and the urethra and reposition of the testicles. After 66 days he was discharged into rehabilitation. Implants were removed after eight months. 20 months after the injury, the leg was plegic, initial radiological signs of femoral head necrosis showed up but the infant was able to walk with an orthesis and a walker.
Up to our knowledge, this is the youngest patient described in the literature with a survived traumatic hemipelvectomy and salvaged limb.
Knowledge on trauma survival time prior to death following a lethal traumatic brain injury (TBI) may be essential for legal purposes. Immunohistochemistry studies might allow to narrow down this survival interval. The biomarkers interleukin-6 (IL-6) and glial fibrillary acidic protein (GFAP) are well known in the clinical setting for their usability in TBI prediction. Here, both proteins were chosen in forensics to determine whether neuronal or glial expression in various brain regions may be associated with the cause of death and the survival time prior to death following TBI. IL-6 positive neurons, glial cells and GFAP positive astrocytes all concordantly increase with longer trauma survival time, with statistically significant changes being evident from three days post-TBI (p < 0.05) in the pericontusional zone, irrespective of its definite cortical localization. IL-6 staining in neurons increases significantly in the cerebellum after trauma, whereas increasing GFAP positivity is also detected in the cortex contralateral to the focal lesion. These systematic chronological changes in biomarkers of pericontusional neurons and glial cells allow for an estimation of trauma survival time. Higher numbers of IL-6 and GFAP-stained cells above threshold values in the pericontusional zone substantiate the existence of fatal traumatic changes in the brain with reasonable certainty.
Background
Epidural catheters are state of the art for postoperative analgesic in abdominal surgery. Due to neurolysis it can lead to postoperative urinary tract retention (POUR), which leads to prolonged bladder catheterization, which has an increased risk for urinary tract infections (UTI). Our aim was to identify the current perioperative management of urinary catheters and, second, to identify the optimal time of suprapubic bladder catheter removal in regard to the removal of the epidural catheter.
Methods
We sent a questionnaire to 102 German hospitals and analyzed the 83 received answers to evaluate the current handling of bladder drainage and epidural catheters. Then, we conducted a retrospective study including 501 patients, who received an epidural and suprapubic catheter after abdominal surgery at the University Hospital Würzburg. We divided the patients into three groups according to the point in time of suprapubic bladder drainage removal in regard to the removal of the epidural catheter and analyzed the onset of a UTI.
Results
Our survey showed that in almost all hospitals (98.8%), patients received an epidural catheter and a bladder drainage after abdominal surgery. The point in time of urinary catheter removal was equally distributed between before, simultaneously and after the removal of the epidural catheter (respectively: ~28–29%). The retrospective study showed a catheter-associated UTI in 6.7%. Women were affected significantly more often than men (10,7% versus 2,5%, p<0.001). There was a non-significant trend to more UTIs when the suprapubic catheter was removed after the epidural catheter (before: 5.7%, after: 8.4%).
Conclusion
The point in time of suprapubic bladder drainage removal in relation to the removal of the epidural catheter does not seem to correlate with the rate of UTIs. The current handling in Germany is inhomogeneous, so further studies to standardize treatment are recommended.
Superconductivity from the condensation of topological defects in a quantum spin-Hall insulator
(2019)
The discovery of quantum spin-Hall (QSH) insulators has brought topology to the forefront of condensed matter physics. While a QSH state from spin-orbit coupling can be fully understood in terms of band theory, fascinating many-body effects are expected if it instead results from spontaneous symmetry breaking. Here, we introduce a model of interacting Dirac fermions where a QSH state is dynamically generated. Our tuning parameter further allows us to destabilize the QSH state in favour of a superconducting state through proliferation of charge-2e topological defects. This route to superconductivity put forward by Grover and Senthil is an instance of a deconfined quantum critical point (DQCP). Our model offers the possibility to study DQCPs without a second length scale associated with the reduced symmetry between field theory and lattice realization and, by construction, is amenable to large-scale fermion quantum Monte Carlo simulations.
Immunotherapy with chimeric antigen receptor-engineered T-cells (CAR-T) is under investigation in multiple myeloma. There are reports of myeloma remission after CD19 CAR-T therapy, although CD19 is hardly detectable on myeloma cells by flow cytometry (FC). We apply single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM), and demonstrate CD19 expression on a fraction of myeloma cells (10.3–80%) in 10 out of 14 patients (density: 13–5,000 molecules per cell). In contrast, FC detects CD19 in only 2 of these 10 patients, on a smaller fraction of cells. Treatment with CD19 CAR-T in vitro results in elimination of CD19-positive myeloma cells, including those with <100 CD19 molecules per cell. Similar data are obtained by dSTORM analyses of CD20 expression on myeloma cells and CD20 CAR-T. These data establish a sensitivity threshold for CAR-T and illustrate how super-resolution microscopy can guide patient selection in immunotherapy to exploit ultra-low density antigens.
Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for epidemic meningitis and sepsis worldwide. A critical step in the development of meningitis is the interaction of bacteria with cells forming the blood-cerebrospinal fluid barrier, which requires tight adhesion of the pathogen to highly specialized brain endothelial cells. Two endothelial receptors, CD147 and the β2-adrenergic receptor, have been found to be sequentially recruited by meningococci involving the interaction with type IV pilus. Despite the identification of cellular key players in bacterial adhesion the detailed mechanism of invasion is still poorly understood. Here, we investigated cellular dynamics and mobility of the type IV pilus receptor CD147 upon treatment with pili enriched fractions and specific antibodies directed against two extracellular Ig-like domains in living human brain microvascular endothelial cells. Modulation of CD147 mobility after ligand binding revealed by single-molecule tracking experiments demonstrates receptor activation and indicates plasma membrane rearrangements. Exploiting the binding of Shiga (STxB) and Cholera toxin B (CTxB) subunits to the two native plasma membrane sphingolipids globotriaosylceramide (Gb3) and raft-associated monosialotetrahexosylganglioside GM1, respectively, we investigated their involvement in bacterial invasion by super-resolution microscopy. Structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) unraveled accumulation and coating of meningococci with GM1 upon cellular uptake. Blocking of CTxB binding sites did not impair bacterial adhesion but dramatically reduced bacterial invasion efficiency. In addition, cell cycle arrest in G1 phase induced by serum starvation led to an overall increase of GM1 molecules in the plasma membrane and consequently also in bacterial invasion efficiency. Our results will help to understand downstream signaling events after initial type IV pilus-host cell interactions and thus have general impact on the development of new therapeutics targeting key molecules involved in infection.
A new series of [Pd2(L)4] cages based on photochromic dithienylethene (DTE) ligands allowed us to gain insight into the successive photoswitching of multiple DTE moieties in a confined metallo-supramolecular assembly. Three new X-ray structures of [Pd2(o-L4)4], [Pd2(o-L1)2(c-L1)2] and [Pd2(c-L1)4] (o-L and c-L = open and closed forms of DTE ligands, respectively) were obtained. The structures deliver snapshots of three different combinations of DTE photoisomeric states within the cage, facilitating a comparison of the all-open with the all-closed, and most notably, an intermediate form where open and closed switches co-exist in the same cage. Moreover, a series of spherical anionic borate clusters was introduced in order to study their roles in the light-controllable host–guest chemistry. The binding guests show higher affinities with the flexible open cage [Pd2(o-L1)4] than with the rigid closed cage [Pd2(c-L1)4]. For the [B12F12]2− guest, thermodynamic data obtained from NMR experiments was compared to results from isothermal titration calorimetry (ITC).
Structure-fluorescence activation relationships of a large Stokes shift fluorogenic RNA aptamer
(2019)
The Chili RNA aptamer is a 52 nt long fluorogen-activating RNA aptamer (FLAP) that confers fluorescence to structurally diverse derivatives of fluorescent protein chromophores. A key feature of Chili is the formation of highly stable complexes with different ligands, which exhibit bright, highly Stokes-shifted fluorescence emission. In this work, we have analyzed the interactions between the Chili RNA and a family of conditionally fluorescent ligands using a variety of spectroscopic, calorimetric and biochemical techniques to reveal key structure - fluorescence activation relationships (SFARs). The ligands under investigation form two categories with emission maxima of ~540 nm or ~590 nm, respectively, and bind with affinities in the nanomolar to low-micromolar range. Isothermal titration calorimetry was used to elucidate the enthalpic and entropic contributions to binding affinity for a cationic ligand that is unique to the Chili aptamer. In addition to fluorescence activation, ligand binding was also observed by NMR spectroscopy, revealing characteristic signals for the formation of a G-quadruplex only upon ligand binding. These data shed light on the molecular features required and responsible for the large Stokes shift and the strong fluorescence enhancement of red and green emitting RNA-chromophore complexes.
γ-Aminobutyric acid type A receptors (GABAARs) mediate the majority of fast synaptic inhibition in the central nervous system (CNS). GABAARs belong to the Cys-loop superfamily of pentameric ligand-gated ion channels (pLGIC) and are assembled from 19 different subunits. As dysfunctional GABAergic neurotransmission manifests itself in neurodevelopmental disorders including epilepsy and anxiety, GABAARs are key drug targets. The majority of synaptic GABAARs are anchored at the inhibitory postsynaptic membrane by the principal scaffolding protein gephyrin, which acts as the central organizer in maintaining the architecture of the inhibitory postsynaptic density (iPSD). This interaction is mediated by the long intracellular loop located in between transmembrane helices 3 and 4 (M3–M4 loop) of the receptors and a universal receptor-binding pocket residing in the C-terminal domain of gephyrin. In 2014, the crystal structure of the β3-homopentameric GABAAR provided crucial information regarding the architecture of the receptor; however, an understanding of the structure and assembly of heteropentameric receptors at the atomic level was lacking. This review article will highlight recent advances in understanding the structure of heteropentameric synaptic GABAARs and how these structures have provided fundamental insights into the assembly of these multi-subunit receptors as well as their modulation by diverse ligands including the physiological agonist GABA. We will further discuss the role of gephyrin in the anchoring of synaptic GABAARs and glycine receptors (GlyRs), which are crucial for maintaining the architecture of the iPSD. Finally, we will also summarize how anti-malarial artemisinin drugs modulate gephyrin-mediated inhibitory neurotransmission.
Structure of heteropentameric GABA\(_A\) receptors and receptor-anchoring properties of gephyrin
(2019)
γ-Aminobutyric acid type A receptors (GABA\(_A\)Rs) mediate the majority of fast synaptic inhibition in the central nervous system (CNS). GABA\(_A\)Rs belong to the Cys-loop superfamily of pentameric ligand-gated ion channels (pLGIC) and are assembled from 19 different subunits. As dysfunctional GABAergic neurotransmission manifests itself in neurodevelopmental disorders including epilepsy and anxiety, GABA\(_A\)Rs are key drug targets. The majority of synaptic GABA\(_A\)Rs are anchored at the inhibitory postsynaptic membrane by the principal scaffolding protein gephyrin, which acts as the central organizer in maintaining the architecture of the inhibitory postsynaptic density (iPSD). This interaction is mediated by the long intracellular loop located in between transmembrane helices 3 and 4 (M3–M4 loop) of the receptors and a universal receptor-binding pocket residing in the C-terminal domain of gephyrin. In 2014, the crystal structure of the β3-homopentameric GABA\(_A\)R provided crucial information regarding the architecture of the receptor; however, an understanding of the structure and assembly of heteropentameric receptors at the atomic level was lacking. This review article will highlight recent advances in understanding the structure of heteropentameric synaptic GABA\(_A\)Rs and how these structures have provided fundamental insights into the assembly of these multi-subunit receptors as well as their modulation by diverse ligands including the physiological agonist GABA. We will further discuss the role of gephyrin in the anchoring of synaptic GABA\(_A\)Rs and glycine receptors (GlyRs), which are crucial for maintaining the architecture of the iPSD. Finally, we will also summarize how anti-malarial artemisinin drugs modulate gephyrin-mediated inhibitory neurotransmission.
Background
Atrial fibrillation (AF) is present in 15–20% of patients with acute ischemic stroke. Oral anticoagulation reduces the risk of AF-related recurrent stroke but clinical guideline recommendations are rather vague regarding its use in the acute phase of stroke. We aimed to assess the current clinical practice of medical stroke prevention in AF patients during the acute phase of ischemic stroke.
Methods
In April 2017, a standardized anonymous questionnaire was sent to clinical leads of all 298 certified stroke units in Germany.
Results
Overall, 154 stroke unit leads participated (response rate 52%). Anticoagulation in the acute phase of stroke is considered feasible in more than 90% of AF patients with ischemic stroke. Clinicians assume that about two thirds of all AF patients (range 20–100%) are discharged on oral anticoagulation. According to local preferences, acetylsalicylic acid is given orally in the majority of patients with delayed initiation of oral anticoagulation. A non-vitamin K-dependent oral anticoagulant (NOAC) is more often prescribed than a vitamin K-dependent oral anticoagulant (VKA). VKA is more often chosen in patients with previous VKA intake than in VKA naive patients. In the minority of patients, stroke unit leads discuss the prescription of a specific oral anticoagulant with the treating general practitioner. Adherence to medical stroke prevention after hospital discharge is not assessed on a regular basis in any patient by the majority of participating stroke centers.
Conclusions
Early secondary stroke prevention in AF patients in German stroke units is based on OAC use but prescription modalities vary in clinical practice.
Bacterial meningitis is a serious life threatening infection of the CNS. To cause meningitis, blood–borne bacteria need to interact with and penetrate brain endothelial cells (BECs) that comprise the blood–brain barrier. BECs help maintain brain homeostasis and they possess an array of efflux transporters, such as P-glycoprotein (P-gp), that function to efflux potentially harmful compounds from the CNS back into the circulation. Oftentimes, efflux also serves to limit the brain uptake of therapeutic drugs, representing a major hurdle for CNS drug delivery. During meningitis, BEC barrier integrity is compromised; however, little is known about efflux transport perturbations during infection. Thus, understanding the impact of bacterial infection on P-gp function would be important for potential routes of therapeutic intervention. To this end, the meningeal bacterial pathogen, Streptococcus agalactiae, was found to inhibit P-gp activity in human induced pluripotent stem cell-derived BECs, and live bacteria were required for the observed inhibition. This observation was correlated to decreased P-gp expression both in vitro and during infection in vivo using a mouse model of bacterial meningitis. Given the impact of bacterial interactions on P-gp function, it will be important to incorporate these findings into analyses of drug delivery paradigms for bacterial infections of the CNS.
Mutual coupling and injection locking of semiconductor lasers is of great interest in non-linear dynamics and its applications for instance in secure data communication and photonic reservoir computing. Despite its importance, it has hardly been studied in microlasers operating at mu W light levels. In this context, vertically emitting quantum dot micropillar lasers are of high interest. Usually, their light emission is bimodal, and the gain competition of the associated linearly polarized fundamental emission modes results in complex switching dynamics. We report on selective optical injection into either one of the two fundamental mode components of a bimodal micropillar laser. Both modes can lock to the master laser and influence the non-injected mode by reducing the available gain. We demonstrate that the switching dynamics can be tailored externally via optical injection in very good agreement with our theory based on semi-classical rate equations. (C) 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
The steric and electronic properties of aryl substituents in monoaryl borohydrides (Li[ArBH\(_3\)]) and dihydroboranes were systematically varied and their reactions with [Ru(PCy\(_3\))\(_2\)HCl(H\(_2\))] (Cy: cyclohexyl) were studied, resulting in bis(σ)‐borane or terminal borylene complexes of ruthenium. These variations allowed for the investigation of the factors involved in the activation of dihydroboranes in the synthesis of terminal borylene complexes. The complexes were studied by multinuclear NMR spectroscopy, mass spectrometry, X‐ray diffraction analysis, and density functional theory (DFT) calculations. The experimental and computational results suggest that the ortho‐substitution of the aryl groups is necessary for the formation of terminal borylene complexes.
Stereospecific Synthesis and Photophysical Properties of Propeller-Shaped C\(_{90}\)H\(_{48}\) PAH
(2019)
Herein, we have synthesized an enantiomerically pure propeller‐shaped PAH, C\(_{90}\)H\(_{48}\), possessing three [7]helicene and three [5]helicene subunits. This compound can be obtained in gram quantities in a straightforward manner. The photophysical and chiroptical properties were investigated using UV/Vis absorption and emission, optical rotation and circular dichroism spectroscopy, supported by DFT calculations. The nonlinear optical properties were investigated by two‐photon absorption measurements using linearly and circularly polarized light. The extremely twisted structure and packing of the homochiral compound were investigated by single‐crystal X‐ray diffraction analysis.
Background and Purpose: Internal carotid artery stenosis (ICAS)≥70% is a leading cause of ischemic cerebrovascular events (ICVEs). However, a considerable percentage of stroke survivors with symptomatic ICAS (sICAS) have <70% stenosis with a vulnerable plaque. Whether the length of ICAS is associated with high risk of ICVEs is poorly investigated. Our main aim was to investigate the relation between the length of ICAS and the development of ICVEs.
Methods: In a retrospective cross-sectional study, we identified 95 arteries with sICAS and another 64 with asymptomatic internal carotid artery stenosis (aICAS) among 121 patients with ICVEs. The degree and length of ICAS as well as plaque echolucency were assessed on ultrasound scans.
Results: A statistically significant inverse correlation between the ultrasound-measured length and degree of ICAS was detected for sICAS≥70% (Spearman correlation coefficient ρ = –0.57, p < 0.001, n = 51) but neither for sICAS<70% (ρ = 0.15, p = 0.45, n = 27) nor for aICAS (ρ = 0.07, p = 0.64, n = 54). The median (IQR) length for sICAS<70% and ≥70% was 17 (15–20) and 15 (12–19) mm (p = 0.06), respectively, while that for sICAS<90% and sICAS 90% was 18 (15–21) and 13 (10–16) mm, respectively (p < 0.001). Among patients with ICAS <70%, a cut-off length of ≥16 mm was found for sICAS rather than aICAS with a sensitivity and specificity of 74.1% and 51.1%, respectively. Irrespective of the stenotic degree, plaques of the sICAS compared to aICAS were significantly more often echolucent (43.2 vs. 24.6%, p = 0.02).
Conclusion: We found a statistically insignificant tendency for the ultrasound-measured length of sICAS<70% to be longer than that of sICAS≥70%. Moreover, the ultrasound-measured length of sICAS<90% was significantly longer than that of sICAS 90%. Among patients with sICAS≥70%, the degree and length of stenosis were inversely correlated. Larger studies are needed before a clinical implication can be drawn from these results.
Purpose:
To quantify the contribution of penumbra in the improvement of healthy tissue sparing at reduced source‐to‐axis distance (SAD) for simple spherical target and different prescription isodoses (PI).
Method:
A TPS‐independent method was used to estimate three‐dimensional (3D) dose distribution for stereotactic treatment of spherical targets of 0.5 cm radius based on single beam two‐dimensional (2D) film dosimetry measurements. 1 cm target constitutes the worst case for the conformation with standard Multi‐Leaf Collimator (MLC) with 0.5 cm leaf width. The measured 2D transverse dose cross‐sections and the profiles in leaf and jaw directions were used to calculate radial dose distribution from isotropic beam arrangement, for both quadratic and circular beam openings, respectively. The results were compared for standard (100 cm) and reduced SAD 70 and 55 cm for different PI.
Results:
For practical reduction of SAD using quadratic openings, the improvement of healthy tissue sparing (HTS) at distances up to 3 times the PTV radius was at least 6%–12%; gradient indices (GI) were reduced by 3–39% for PI between 40% and 90%. Except for PI of 80% and 90%, quadratic apertures at SAD 70 cm improved the HTS by up to 20% compared to circular openings at 100 cm or were at least equivalent; GI were 3%–33% lower for reduced SAD in the PI range 40%–70%. For PI = 80% and 90% the results depend on the circular collimator model.
Conclusion:
Stereotactic treatments of spherical targets delivered at reduced SAD of 70 or 55 cm using MLC spare healthy tissue around the target at least as good as treatments at SAD 100 cm using circular collimators. The steeper beam penumbra at reduced SAD seems to be as important as perfect target conformity. The authors argue therefore that the beam penumbra width should be addressed in the stereotactic studies.
Even today, the automatic digitisation of scanned documents in general, but especially the automatic optical music recognition (OMR) of historical manuscripts, still remains an enormous challenge, since both handwritten musical symbols and text have to be identified. This paper focuses on the Medieval so-called square notation developed in the 11th–12th century, which is already composed of staff lines, staves, clefs, accidentals, and neumes that are roughly spoken connected single notes. The aim is to develop an algorithm that captures both the neumes, and in particular its melody, which can be used to reconstruct the original writing. Our pipeline is similar to the standard OMR approach and comprises a novel staff line and symbol detection algorithm based on deep Fully Convolutional Networks (FCN), which perform pixel-based predictions for either staff lines or symbols and their respective types. Then, the staff line detection combines the extracted lines to staves and yields an F\(_1\) -score of over 99% for both detecting lines and complete staves. For the music symbol detection, we choose a novel approach that skips the step to identify neumes and instead directly predicts note components (NCs) and their respective affiliation to a neume. Furthermore, the algorithm detects clefs and accidentals. Our algorithm predicts the symbol sequence of a staff with a diplomatic symbol accuracy rate (dSAR) of about 87%, which includes symbol type and location. If only the NCs without their respective connection to a neume, all clefs and accidentals are of interest, the algorithm reaches an harmonic symbol accuracy rate (hSAR) of approximately 90%. In general, the algorithm recognises a symbol in the manuscript with an F\(_1\) -score of over 96%.
ST18 Enhances PV-IgG-Induced Loss of Keratinocyte Cohesion in Parallel to Increased ERK Activation
(2019)
Pemphigus is an autoimmune blistering disease targeting the desmosomal proteins desmoglein (Dsg) 1 and Dsg3. Recently, a genetic variant of the Suppression of tumorigenicity 18 (ST18) promoter was reported to cause ST18 up-regulation, associated with pemphigus vulgaris (PV)-IgG-mediated increase in cytokine secretion and more prominent loss of keratinocyte cohesion. Here we tested the effects of PV-IgG and the pathogenic pemphigus mouse anti-Dsg3 antibody AK23 on cytokine secretion and ERK activity in human keratinocytes dependent on ST18 expression. Without ST18 overexpression, both PV-IgG and AK23 induced loss of keratinocyte cohesion which was accompanied by prominent fragmentation of Dsg3 immunostaining along cell borders. In contrast, release of pro-inflammatory cytokines such as IL-1 alpha, IL-6, TNF alpha, and IFN-gamma was not altered significantly in both HaCaT and primary NHEK cells. These experiments indicate that cytokine expression is not strictly required for loss of keratinocyte cohesion. Upon ST18 overexpression, fragmentation of cell monolayers increased significantly in response to autoantibody incubation. Furthermore, production of IL-1 alpha and IL-6 was enhanced in some experiments but not in others whereas release of TNF-alpha dropped significantly upon PV-IgG application in both EV- and ST18-transfected HaCaT cells. Additionally, in NHEK, application of PV-IgG but not of AK23 significantly increased ERK activity. In contrast, ST18 overexpression in HaCaT cells augmented ERK activation in response to both c-IgG and AK23 but not PV-IgG. Because inhibition of ERK by U0126 abolished PV-IgG- and AK23-induced loss of cell cohesion in ST18-expressing cells, we conclude that autoantibody-induced ERK activation was relevant in this scenario. In summary, similar to the situation in PV patients carrying ST18 polymorphism, overexpression of ST18 enhanced keratinocyte susceptibility to autoantibody-induced loss of cell adhesion, which may be caused in part by enhanced ERK signaling.
Spread and clinical severity of respiratory syncytial virus A genotype ON1 in Germany, 2011–2017
(2019)
Background
The Respiratory Syncytial Virus (RSV) A genotype ON1, which was first detected in Ontario (Canada) in 2010/11, appeared in Germany in 2011/12. Preliminary observations suggested a higher clinical severity in children infected with this new genotype. We investigated spread and disease severity of RSV-A ON1 in pediatric in- and outpatient settings.
Methods
During 2010/11 to 2016/17, clinical characteristics and respiratory samples from children with acute respiratory tract infections (RTI) were obtained from ongoing surveillance studies in 33 pediatric practices (PP), one pediatric hospital ward (PW) and 23 pediatric intensive care units (PICU) in Germany. RSV was detected in the respiratory samples by PCR; genotypes were identified by sequencing. Within each setting, clinical severity markers were compared between RSV-A ON1 and RSV-A non-ON1 genotypes.
Results
A total of 603 children with RSV-RTI were included (132 children in PP, 288 in PW, and 183 in PICU). Of these children, 341 (56.6%) were infected with RSV-A, 235 (39.0%) with RSV-B, and one child (0.2%) with both RSV-A and RSV-B; in 26 (4.3%) children, the subtype could not be identified. In the 341 RSV-A positive samples, genotype ON1 was detected in 247 (72.4%), NA1 in 92 (26.9%), and GA5 in 2 children (0.6%). RSV-A ON1, rarely observed in 2011/12, was the predominant RSV-A genotype in all settings by 2012/13 and remained predominant until 2016/17. Children in PP or PW infected with RSV-A ON1 did not show a more severe clinical course of disease compared with RSV-A non-ON1 infections. In the PICU group, hospital stay was one day longer (median 8 days, inter-quartile range (IQR) 7–12 vs. 7 days, IQR 5–9; p = 0.02) and duration of oxygen treatment two days longer (median 6 days, IQR 4–9 vs. 4 days, IQR 2–6; p = 0.03) for children infected with RSV-A ON1.
Conclusions
In children, RSV-A ON1 largely replaced RSV-A non-ON1 genotypes within two seasons and remained the predominant RSV-A genotype in Germany during subsequent seasons. A higher clinical severity of RSV-A ON1 was observed within the group of children receiving PICU treatment, whereas in other settings clinical severity of RSV-A ON1 and non-ON1 genotypes was largely similar.
The helical distribution of the electronic density in chiral molecules, such as DNA and bacteriorhodopsin, has been suggested to induce a spin–orbit coupling interaction that may lead to the so-called chirality-induced spin selectivity (CISS) effect. Key ingredients for the theoretical modelling are, in this context, the helically shaped potential of the molecule and, concomitantly, a Rashba-like spin–orbit coupling due to the appearance of a magnetic field in the electron reference frame. Symmetries of these models clearly play a crucial role in explaining the observed effect, but a thorough analysis has been largely ignored in the literature. In this work, we present a study of these symmetries and how they can be exploited to enhance chiral-induced spin selectivity in helical molecular systems.
Spin echo based cardiac diffusion imaging at 7T: An ex vivo study of the porcine heart at 7T and 3T
(2019)
Purpose of this work was to assess feasibility of cardiac diffusion tensor imaging (cDTI) at 7 T in a set of healthy, unfixed, porcine hearts using various parallel imaging acceleration factors and to compare SNR and derived cDTI metrics to a reference measured at 3 T. Magnetic resonance imaging was performed on 7T and 3T whole body systems using a spin echo diffusion encoding sequence with echo planar imaging readout. Five reference (b = 0 s/mm\(^2\)) images and 30 diffusion directions (b = 700 s/mm\(^2\)) were acquired at both 7 T and 3 T using a GRAPPA acceleration factor R = 1. Scans at 7 T were repeated using R = 2, R = 3, and R = 4. SNR evaluation was based on 30 reference (b = 0 s/mm\(^2\)) images of 30 slices of the left ventricle and cardiac DTI metrics were compared within AHA segmentation. The number of hearts scanned at 7 T and 3 T was n = 11. No statistically significant differences were found for evaluated helix angle, secondary eigenvector angle, fractional anisotropy and apparent diffusion coefficient at the different field strengths, given sufficiently high SNR and geometrically undistorted images. R≥3 was needed to reduce susceptibility induced geometric distortions to an acceptable amount. On average SNR in myocardium of the left ventricle was increased from 29±3 to 44±6 in the reference image (b = 0 s/mm\(^2\)) when switching from 3 T to 7 T. Our study demonstrates that high resolution, ex vivo cDTI is feasible at 7 T using commercial hardware.
Sphingolipids are major components of cellular membranes, and at steady-state level, their metabolic fluxes are tightly controlled. On challenge by external signals, they undergo rapid turnover, which substantially affects the biophysical properties of membrane lipid and protein compartments and, consequently, signaling and morphodynamics. In T cells, external cues translate into formation of membrane microdomains where proximal signaling platforms essential for metabolic reprograming and cytoskeletal reorganization are organized. This review will focus on sphingomyelinases, which mediate sphingomyelin breakdown and ensuing ceramide release that have been implicated in T-cell viability and function. Acting at the sphingomyelin pool at the extrafacial or cytosolic leaflet of cellular membranes, acid and neutral sphingomyelinases organize ceramide-enriched membrane microdomains that regulate T-cell homeostatic activity and, upon stimulation, compartmentalize receptors, membrane proximal signaling complexes, and cytoskeletal dynamics as essential for initiating T-cell motility and interaction with endothelia and antigen-presenting cells. Prominent examples to be discussed in this review include death receptor family members, integrins, CD3, and CD28 and their associated signalosomes. Progress made with regard to experimental tools has greatly aided our understanding of the role of bioactive sphingolipids in T-cell biology at a molecular level and of targets explored by a model pathogen (measles virus) to specifically interfere with their physiological activity.
In chemical education, it is often a challenge to understand the basic principles of spectroscopic techniques due to missing connections to the real world. Therefore, the present contribution offers context-based applications of UV/Vis spectroscopy for analytics of food colorings with which learners can improve their skills regarding this method. The spectroscopic determination of food colorings seems to be a promising approach due to the long tradition and omnipresence of dyes in supermarket products. The therefor-required spectral data for commonly used dyes are provided for educational usage. Qualitative and quantitative analytics of food colorings in four different lemonades and chocolate beans have been used to introduce learners to important analytical techniques like sample preparation or elimination of confounding factors. These analytics also display the limitations of the method in the visible range of light in the case of tartrazine and curcumin. By applying Lambert-Beer-Bouguer’s Law in different variations, typical calculations of concentrations can be studied in quantitative analyses. The studied food samples demonstrate the different usage of food colorings depending on the country of sale. Finally, a 3D-printable low-cost photometer suitable for the discussed quantitative analytics in educational contexts is presented.
Bone Morphogenetic Proteins (BMPs) together with the Growth and Differentiation Factors (GDFs) form the largest subgroup of the Transforming Growth Factor (TGF)β family and represent secreted growth factors, which play an essential role in many aspects of cell communication in higher organisms. As morphogens they exert crucial functions during embryonal development, but are also involved in tissue homeostasis and regeneration in the adult organism. Their involvement in maintenance and repair processes of various tissues and organs made these growth factors highly interesting targets for novel pharmaceutical applications in regenerative medicine. A hallmark of the TGFβ protein family is that all of the more than 30 growth factors identified to date signal by binding and hetero-oligomerization of a very limited set of transmembrane serine-threonine kinase receptors, which can be classified into two subgroups termed type I and type II. Only seven type I and five type II receptors exist for all 30plus TGFβ members suggesting a pronounced ligand-receptor promiscuity. Indeed, many TGFβ ligands can bind the same type I or type II receptor and a particular receptor of either subtype can usually interact with and bind various TGFβ ligands. The possible consequence of this ligand-receptor promiscuity is further aggravated by the finding that canonical TGFβ signaling of all family members seemingly results in the activation of just two distinct signaling pathways, that is either SMAD2/3 or SMAD1/5/8 activation. While this would implicate that different ligands can assemble seemingly identical receptor complexes that activate just either one of two distinct pathways, in vitro and in vivo analyses show that the different TGFβ members exert quite distinct biological functions with high specificity. This discrepancy indicates that our current view of TGFβ signaling initiation just by hetero-oligomerization of two receptor subtypes and transduction via two main pathways in an on-off switch manner is too simplified. Hence, the signals generated by the various TGFβ members are either quantitatively interpreted using the subtle differences in their receptor-binding properties leading to ligand-specific modulation of the downstream signaling cascade or additional components participating in the signaling activation complex allow diversification of the encoded signal in a ligand-dependent manner at all cellular levels. In this review we focus on signal specification of TGFβ members, particularly of BMPs and GDFs addressing the role of binding affinities, specificities, and kinetics of individual ligand-receptor interactions for the assembly of specific receptor complexes with potentially distinct signaling properties.
The East Himalaya is one of the world’s most biodiverse ecosystems. However, very little is known about the abundance and distribution of many plant and animal taxa in this region. Bumble bees are a group of cold-adapted and high elevation insects that fulfil an important ecological and economical function as pollinators of wild and agricultural flowering plants and crops. The Himalayan mountain range provides ample suitable habitats for bumble bees. Systematic study of Himalayan bumble bees began a few decades ago and the main focus has centred on the western region, while the eastern part of the mountain range has received little attention and only a few species have been verified. During a three-year survey, more than 700 bumble bee specimens of 21 species were collected in Arunachal Pradesh, the largest of the north-eastern states of India. The material included a range of species that were previously known from a limited number of collected specimens, which highlights the unique character of the East Himalayan ecosystem. Our results are an important first step towards a future assessment of species distribution, threat, and conservation. Clear elevation patterns of species diversity were observed, which raise important questions about the functional adaptations that allow bumble bees to thrive in this particularly moist region in the East Himalaya.
Investigating approach-avoidance behavior regarding affective stimuli is important in broadening the understanding of one of the most common psychiatric disorders, social anxiety disorder. Many studies in this field rely on approach-avoidance tasks, which mainly assess hand movements, or interpersonal distance measures, which return inconsistent results and lack ecological validity. Therefore, the present study introduces a virtual reality task, looking at avoidance parameters (movement time and speed, distance to social stimulus, gaze behavior) during whole-body movements. These complex movements represent the most ecologically valid form of approach and avoidance behavior. These are at the core of complex and natural social behavior. With this newly developed task, the present study examined whether high socially anxious individuals differ in avoidance behavior when bypassing another person, here virtual humans with neutral and angry facial expressions. Results showed that virtual bystanders displaying angry facial expressions were generally avoided by all participants. In addition, high socially anxious participants generally displayed enhanced avoidance behavior towards virtual people, but no specifically exaggerated avoidance behavior towards virtual people with a negative facial expression. The newly developed virtual reality task proved to be an ecological valid tool for research on complex approach-avoidance behavior in social situations. The first results revealed that whole body approach-avoidance behavior relative to passive bystanders is modulated by their emotional facial expressions and that social anxiety generally amplifies such avoidance.
Background
Skeletal muscle function dysfunction has been reported in patients with cystic fibrosis (CF). Studies so far showed inconclusive data whether reduced exercise capacity is related to intrinsic muscle dysfunction in CF.
Methods
Twenty patients with CF and 23 age-matched controls completed an incremental cardiopulmonary cycling test. Further, a Wingate anaerobic test to assess muscle power was performed. In addition, all participants completed an incremental knee-extension test with 31P magnetic resonance spectroscopy to assess muscle metabolism (inorganic phosphate (Pi) and phosphocreatinine (PCr) as well as intracellular pH). In the MRI, muscle cross-sectional area of the M. quadriceps (qCSA) was also measured. A subgroup of 15 participants (5 CF, 10 control) additionally completed a continuous high-intensity, high-frequency knee-extension exercise task during 31P magnetic resonance spectroscopy to assess muscle metabolism.
Results
Patients with CF showed a reduced exercise capacity in the incremental cardiopulmonary cycling test (VO2peak: CF 77.8 ± 16.2%predicted (36.5 ± 7.4 ml/qCSA/min), control 100.6 ± 18.8%predicted (49.1 ± 11.4 ml/qCSA/min); p < 0.001), and deficits in anaerobic capacity reflected by the Wingate test (peak power: CF 537 ± 180 W, control 727 ± 186 W; mean power: CF 378 ± 127 W, control 486 ± 126 W; power drop CF 12 ± 5 W, control 8 ± 4 W. all: p < 0.001). In the knee-extension task, patients with CF achieved a significantly lower workload (p < 0.05). However, in a linear model analysing maximal work load of the incremental knee-extension task and results of the Wingate test, respectively, only muscle size and height, but not disease status (CF or not) contributed to explaining variance. In line with this finding, no differences were found in muscle metabolism reflected by intracellular pH and the ratio of Pi/PCr at submaximal stages and peak exercise measured through MRI spectroscopy.
Conclusions
The lower absolute muscle power in patients with CF compared to controls is exclusively explained by the reduced muscle size in this study. No evidence was found for an intrinsic skeletal muscle dysfunction due to primary alterations of muscle metabolism.
Introduction: Acute stroke care delivered by interdisciplinary teams is time-sensitive. Simulation-based team training is a promising tool to improve team performance in medical operations. It has the potential to improve process times, team communication, patient safety, and staff satisfaction. We aim to assess whether a multi-level approach consisting of a stringent workflow revision based on peer-to-peer review and 2–3 one-day in situ simulation trainings can improve acute stroke care processing times in high volume neurocenters within a 6 months period.
Methods and Analysis: The trial is being carried out in a pre-test-post-test design at 7 tertiary care university hospital neurocenters in Germany. The intervention is directed at the interdisciplinary multiprofessional stroke teams. Before and after the intervention, process times of all direct-to-center stroke patients receiving IV thrombolysis (IVT) and/or endovascular therapy (EVT) will be recorded. The primary outcome measure will be the “door-to-needle” time of all consecutive stroke patients directly admitted to the neurocenters who receive IVT. Secondary outcome measures will be intervention-related process times of the fraction of patients undergoing EVT and effects on team communication, perceived patient safety, and staff satisfaction via a staff questionnaire.
Interventions: We are applying a multi-level intervention in cooperation with three “STREAM multipliers” from each center. First step is a central meeting of the multipliers at the sponsor's institution with the purposes of algorithm review in a peer-to-peer process that is recorded in a protocol and an introduction to the principles of simulation training and debriefing as well as crew resource management and team communication. Thereafter, the multipliers cooperate with the stroke team trainers from the sponsor's institution to plan and execute 2–3 one-day simulation courses in situ in the emergency department and CT room of the trial centers whereupon they receive teaching materials to perpetuate the trainings.
Clinical Trial Registration: STREAM is a registered trial at https://clinicaltrials.gov/ct2/show/NCT03228251.
Projected climate changes for the 21st century may cause great uncertainties on the hydrology of a river basin. This study explored the impacts of climate change on the water balance and hydrological regime of the Jhelum River Basin using the Soil and Water Assessment Tool (SWAT). Two downscaling methods (SDSM, Statistical Downscaling Model and LARS-WG, Long Ashton Research Station Weather Generator), three Global Circulation Models (GCMs), and two representative concentration pathways (RCP4.5 and RCP8.5) for three future periods (2030s, 2050s, and 2090s) were used to assess the climate change impacts on flow regimes. The results exhibited that both downscaling methods suggested an increase in annual streamflow over the river basin. There is generally an increasing trend of winter and autumn discharge, whereas it is complicated for summer and spring to conclude if the trend is increasing or decreasing depending on the downscaling methods. Therefore, the uncertainty associated with the downscaling of climate simulation needs to consider, for the best estimate, the impact of climate change, with its uncertainty, on a particular basin. The study also resulted that water yield and evapotranspiration in the eastern part of the basin (sub-basins at high elevation) would be most affected by climate change. The outcomes of this study would be useful for providing guidance in water management and planning for the river basin under climate change.
Transposable elements are endogenous DNA sequences able to integrate into and multiply within genomes. They constitute a major source of genetic innovations, as they can not only rearrange genomes but also spread ready-to-use regulatory sequences able to modify host gene expression, and even can give birth to new host genes. As their evolutionary success depends on their vertical transmission, transposable elements are intrinsically linked to reproduction. In organisms with sexual reproduction, this implies that transposable elements have to manifest their transpositional activity in germ cells or their progenitors. The control of sexual development and function can be very versatile, and several studies have demonstrated the implication of transposable elements in the evolution of sex. In this review, we report the functional and evolutionary relationships between transposable elements and sexual reproduction in animals. In particular, we highlight how transposable elements can influence expression of sexual development genes, and how, reciprocally, they are tightly controlled in gonads. We also review how transposable elements contribute to the organization, expression and evolution of sexual development genes and sex chromosomes. This underscores the intricate co-evolution between host functions and transposable elements, which regularly shift from a parasitic to a domesticated status useful to the host.
Brain serotonin (5-hydroxytryptamine, 5-HT) system dysfunction is implicated in exaggerated fear responses triggering various anxiety-, stress-, and trauma-related disorders. However, the underlying mechanisms are not well understood. Here, we investigated the impact of constitutively inactivated 5-HT synthesis on context-dependent fear learning and extinction using tryptophan hydroxylase 2 (Tph2) knockout mice. Fear conditioning and context-dependent fear memory extinction paradigms were combined with c-Fos imaging and electrophysiological recordings in the dorsal hippocampus (dHip). Tph2 mutant mice, completely devoid of 5-HT synthesis in brain, displayed accelerated fear memory formation and increased locomotor responses to foot shock. Furthermore, recall of context-dependent fear memory was increased. The behavioral responses were associated with increased c-Fos expression in the dHip and resistance to foot shock-induced impairment of hippocampal long-term potentiation (LTP). In conclusion, increased context-dependent fear memory resulting from brain 5-HT deficiency involves dysfunction of the hippocampal circuitry controlling contextual representation of fear-related behavioral responses.
Background
The honeybee (Apis mellifera) represents a model organism for social insects displaying behavioral plasticity. This is reflected by an age-dependent task allocation. The most protruding tasks are performed by young nurse bees and older forager bees that take care of the brood inside the hive and collect food from outside the hive, respectively. The molecular mechanism leading to the transition from nurse bees to foragers is currently under intense research. Circular RNAs, however, were not considered in this context so far. As of today, this group of non-coding RNAs was only known to exist in two other insects, Drosophila melanogaster and Bombyx mori. Here we complement the state of circular RNA research with the first characterization in a social insect.
Results
We identified numerous circular RNAs in the brain of A. mellifera nurse bees and forager bees using RNA-Seq with exonuclease enrichment. Presence and circularity were verified for the most abundant representatives. Back-splicing in honeybee occurs further towards the end of transcripts and in transcripts with a high number of exons. The occurrence of circularized exons is correlated with length and CpG-content of their flanking introns. The latter coincides with increased DNA-methylation in the respective loci. For two prominent circular RNAs the abundance in worker bee brains was quantified in TaqMan assays. In line with previous findings of circular RNAs in Drosophila, circAmrsmep2 accumulates with increasing age of the insect. In contrast, the levels of circAmrad appear age-independent and correlate with the bee's task. Its parental gene is related to amnesia-resistant memory.
Conclusions
We provide the first characterization of circRNAs in a social insect. Many of the RNAs identified here show homologies to circular RNAs found in Drosophila and Bombyx, indicating that circular RNAs are a common feature among insects. We find that exon circularization is correlated to DNA-methylation at the flanking introns. The levels of circAmrad suggest a task-dependent abundance that is decoupled from age. Moreover, a GO term analysis shows an enrichment of task-related functions. We conclude that circular RNAs could be relevant for task allocation in honeybee and should be investigated further in this context.
KM3NeT will be a network of deep-sea neutrino telescopes in the Mediterranean Sea. The KM3NeT/ARCA detector, to be installed at the Capo Passero site (Italy), is optimised for the detection of high-energy neutrinos of cosmic origin. Thanks to its geographical location on the Northern hemisphere, KM3NeT/ARCA can observe upgoing neutrinos from most of the Galactic Plane, including the Galactic Centre. Given its effective area and excellent pointing resolution, KM3NeT/ARCA will measure or significantly constrain the neutrino flux from potential astrophysical neutrino sources. At the same time, it will test flux predictions based on gamma-ray measurements and the assumption that the gamma-ray flux is of hadronic origin. Assuming this scenario, discovery potentials and sensitivities for a selected list of Galactic sources and to generic point sources with an E-2 spectrum are presented. These spectra are assumed to be time independent. The results indicate that an observation with 3 sigma significance is possible in about six years of operation for the most intense sources, such as Supernovae Remnants RX J1713.7-3946 and Vela Jr. If no signal will be found during this time, the fraction of the gamma-ray flux coming from hadronic processes can be constrained to be below 50% for these two objects. (C) 2019 The Authors. Published by Elsevier B.V.
Unprecedented urbanization in particular in countries of the global south result in informal urban development processes, especially in mega cities. With an estimated 1 billion slum dwellers globally, the United Nations have made the fight against poverty the number one sustainable development goal. To provide better infrastructure and thus a better life to slum dwellers, detailed information on the spatial location and size of slums is of crucial importance. In the past, remote sensing has proven to be an extremely valuable and effective tool for mapping slums. The nature of used mapping approaches by machine learning, however, made it necessary to invest a lot of effort in training the models. Recent advances in deep learning allow for transferring trained fully convolutional networks (FCN) from one data set to another. Thus, in our study we aim at analyzing transfer learning capabilities of FCNs to slum mapping in various satellite images. A model trained on very high resolution optical satellite imagery from QuickBird is transferred to Sentinel-2 and TerraSAR-X data. While free-of-charge Sentinel-2 data is widely available, its comparably lower resolution makes slum mapping a challenging task. TerraSAR-X data on the other hand, has a higher resolution and is considered a powerful data source for intra-urban structure analysis. Due to the different image characteristics of SAR compared to optical data, however, transferring the model could not improve the performance of semantic segmentation but we observe very high accuracies for mapped slums in the optical data: QuickBird image obtains 86–88% (positive prediction value and sensitivity) and a significant increase for Sentinel-2 applying transfer learning can be observed (from 38 to 55% and from 79 to 85% for PPV and sensitivity, respectively). Using transfer learning proofs extremely valuable in retrieving information on small-scaled urban structures such as slum patches even in satellite images of decametric resolution.
Background
The guideline recommendation to not measure carotid intima-media thickness (CIMT) for cardiovascular risk prediction is based on the assessment of just one single carotid segment. We evaluated whether there is a segment-specific association between different measurement locations of CIMT and cardiovascular risk factors.
Methods
Subjects from the population-based STAAB cohort study comprising subjects aged 30 to 79 years of the general population from Würzburg, Germany, were investigated. CIMT was measured on the far wall of both sides in three different predefined locations: common carotid artery (CCA), bulb, and internal carotid artery (ICA). Diabetes, dyslipidemia, hypertension, smoking, and obesity were considered as risk factors. In multivariable logistic regression analysis, odds ratios of risk factors per location were estimated for the endpoint of individual age- and sex-adjusted 75th percentile of CIMT.
Results
2492 subjects were included in the analysis. Segment-specific CIMT was highest in the bulb, followed by CCA, and lowest in the ICA. Dyslipidemia, hypertension, and smoking were associated with CIMT, but not diabetes and obesity. We observed no relevant segment-specific association between the three different locations and risk factors, except for a possible interaction between smoking and ICA.
Conclusions
As no segment-specific association between cardiovascular risk factors and CIMT became evident, one simple measurement of one location may suffice to assess the cardiovascular risk of an individual.
Seasonal Occurrence and Carbapenem Susceptibility of Bovine Acinetobacter baumannii in Germany
(2019)
Acinetobacter baumannii is one of the leading causes of nosocomial infections in humans. To investigate its prevalence, distribution of sequence types (STs), and antimicrobial resistance in cattle, we sampled 422 cattle, including 280 dairy cows, 59 beef cattle, and 83 calves over a 14-month period. Metadata, such as the previous use of antimicrobial agents and feeding, were collected to identify putative determining factors. Bacterial isolates were identified via MALDI-TOF/MS and PCR, antimicrobial susceptibility was evaluated via VITEK2 and antibiotic gradient tests, resistance genes were identified by PCR. Overall, 15.6% of the cattle harbored A. baumannii, predominantly in the nose (60.3% of the A. baumannii isolates). It was more frequent in dairy cows (21.1%) than in beef cattle (6.8%) and calves (2.4%). A seasonal occurrence was shown with a peak between May and August. The rate of occurrence of A. baumannii was correlated with a history of use of 3rd generation cephalosporins in the last 6 months prior to sampling Multilocus sequence typing (Pasteur scheme) revealed 83 STs among 126 unique isolates. Nine of the bovine STs have previously been implicated in human infections. Besides known intrinsic resistance of the species, the isolates did not show additional resistance to the antimicrobial substances tested, including carbapenems. Our data suggest that cattle are not a reservoir for nosocomial A. baumannii but carry a highly diverse population of this species. Nevertheless, some STs seem to be able to colonize both cattle and humans.
Astrophysical sources of gravitational waves, such as binary neutron star and black hole mergers or core-collapse supernovae, can drive relativistic outflows, giving rise to non-thermal high-energy emission. High-energy neutrinos are signatures of such outflows. The detection of gravitational waves and high-energy neutrinos from common sources could help establish the connection between the dynamics of the progenitor and the properties of the outflow. We searched for associated emission of gravitational waves and high-energy neutrinos from astrophysical transients with minimal assumptions using data from Advanced LIGO from its first observing run O1, and data from the Antares and IceCube neutrino observatories from the same time period. We focused on candidate events whose astrophysical origins could not be determined from a single messenger. We found no significant coincident candidate, which we used to constrain the rate density of astrophysical sources dependent on their gravitational-wave and neutrino emission processes.
This Letter presents a search for new light resonances decaying to pairs of quarks and produced in association with a high-p(T) photon or jet. The dataset consists of proton-proton collisions with an integrated luminosity of 36.1 fb(-1) at a centre-of-mass energy of root s = 13 TeV recorded by the ATLAS detector at the Large Hadron Collider. Resonance candidates are identified as massive large-radius jets with substructure consistent with a particle decaying into a quark pair. The mass spectrum of the candidates is examined for local excesses above background. No evidence of a new resonance is observed in the data, which are used to exclude the production of a lepto-phobic axial-vector Z' boson. (C) 2018 The Author(s). Published by Elsevier B.V.
A search for high-mass dielectron and dimuon resonances in the mass range of 250 GeV to 6TeV is presented. The data were recorded by the ATLAS experiment in proton-proton collisions at a centre-ofmass energy of root s = 13 TeV during Run 2 of the Large Hadron Collider and correspond to an integrated luminosity of 139 fb(-1). A functional form is fitted to the dilepton invariant-mass distribution to model the contribution from background processes, and a generic signal shape is used to determine the significance of observed deviations from this background estimate. No significant deviation is observed and upper limits are placed at the 95% confidence level on the fiducial cross-section times branching ratio for various resonance width hypotheses. The derived limits are shown to be applicable to spin-0, spin-1 and spin-2 signal hypotheses. For a set of benchmark models, the limits are converted into lower limits on the resonance mass and reach 4.5 TeV for the E-6-motivated Z(psi)' boson. Also presented are limits on Heavy Vector Triplet model couplings. (C) 2019 The Author. Published by Elsevier B.V.
A search for new particles decaying into a pair of top quarks is performed using proton-proton collision data recorded with the ATLAS detector at the Large Hadron Collider at a center-of-mass energy of root s = 13 TeV corresponding to an integrated luminosity of 36.1 fb(-1). Events consistent with top-quark pair production and the fully hadronic decay mode of the top quarks are selected by requiring multiple high transverse momentum jets including those containing b-hadrons. Two analysis techniques, exploiting dedicated top-quark pair reconstruction in different kinematic regimes, are used to optimize the search sensitivity to new hypothetical particles over a wide mass range. The invariant mass distribution of the two reconstructed top-quark candidates is examined for resonant production of new particles with various spins and decay widths. No significant deviation from the Standard Model prediction is observed and limits are set on the production cross-section times branching fraction for new hypothetical Z' bosons, dark-matter mediators, Kaluza-Klein gravitons and Kaluza-Klein gluons. By comparing with the predicted production cross sections, the Z' boson in the topcolor-assisted-technicolor model is excluded for masses up to 3.1-3.6 TeV, the dark-matter mediators in a simplified framework are excluded in the mass ranges from 0.8 to 0.9 TeV and from 2.0 to 2.2 TeV, and the Kaluza-Klein gluon is excluded for masses up to 3.4 TeV, depending on the decay widths of the particles.
This paper reports on a search for electroweak diboson (WW/WZ/ZZ) production in association with a high-mass dijet system, using data from proton-proton collisions at a center-of-mass energy of N root s = 13 TeV. The data, corresponding to an integrated luminosity of 35.5 fb(-1), were recorded with the ATLAS detector in 2015 and 2016 at the Large Hadron Collider. The search is performed in final states in which one boson decays leptonically, and the other boson decays hadronically. The hadronically decaying W/Z boson is reconstructed as either two small-radius jets or one large-radius jet using jet substructure techniques. The electroweak production of WW/WZ/ZZ in association with two jets is measured with an observed (expected) significance of 2.7 (2.5) standard deviations, and the fiducial cross section is measured to be 45.1 +/- 8.6(stat.)(-14.6)(+15.9)(syst.) fb.
Narrow resonances decaying into WW, WZ or ZZ boson pairs are searched for in 139 fb(-1) of proton-proton collision data at a centre-of-mass energy of root s = 13TeV recorded with the ATLAS detector at the Large Hadron Collider from 2015 to 2018. The diboson system is reconstructed using pairs of high transverse momentum, large-radius jets. These jets are built from a combination of calorimeter- and tracker-inputs compatible with the hadronic decay of a boosted W or Z boson, using jet mass and substructure properties. The search is performed for diboson resonances with masses greater than 1.3TeV. No significant deviations from the background expectations are observed. Exclusion limits at the 95% confidence level are set on the production cross-section times branching ratio into dibosons for resonances in a range of theories beyond the Standard Model, with the highest excluded mass of a new gauge boson at 3.8TeV in the context of mass-degenerate resonances that couple predominantly to gauge bosons.
A search for a heavy charged-boson resonance decaying into a charged lepton (electron or muon) and a neutrino is reported. A data sample of 139 fb(-1) of proton-proton collisions at root s = 13 TeV collected with the ATLAS detector at the LHC during 2015-2018 is used in the search. The observed transverse mass distribution computed from the lepton and missing transverse momenta is consistent with the distribution expected from the Standard Model, and upper limits on the cross section for pp -> W'-> lv are extracted (l = e or mu). These vary between 1.3 pb and 0.05 tb depending on the resonance mass in the range between 0.15 and 7.0 TeV at 95% confidence level for the electron and muon channels combined. Gauge bosons with a mass below 6.0 and 5.1 TeV are excluded in the electron and muon channels, respectively, in a model with a resonance that has couplings to fermions identical to those of the Standard Model W boson. Cross-section limits are also provided for resonances with several fixed Gamma/m values in the range between 1% and 15%. Model-independent limits are derived in single-bin signal regions defined by a varying minimum transverse mass threshold. The resulting visible cross-section upper limits range between 4.6 (15) ph and 22 (22) ab as the threshold increases from 130 (110) GeV to 5.1 (5.1) TeV in the electron (muon) channel.
Polyphenols exert beneficial effects in type 2 diabetes mellitus (T2DM). However, their mechanism of action remains largely unknown. Endothelial Akt-kinase plays a key role in the pathogenesis of cardiovascular complications in T2DM and therefore the modulation of its activity is of interest. This work aimed to characterize effects of structurally different polyphenols on Akt-phosphorylation (pAkt) in endothelial cells (Ea.hy926) and to describe structure-activity features. A comprehensive screening via ELISA quantified the effects of 44 polyphenols (10 µM) on pAkt Ser473. The most pronounced inhibitors were luteolin (44 ± 18%), quercetin (36 ± 8%), urolithin A (35 ± 12%), apigenin, fisetin, and resveratrol; (p < 0.01). The results were confirmed by Western blotting and complemented with corresponding experiments in HUVEC cells. A strong positive and statistically significant correlation between the mean inhibitory effects of the tested polyphenols on both Akt-residues Ser473 and Thr308 (r = 0.9478, p = 0.0003) was determined by immunoblotting. Interestingly, the structural characteristics favoring pAkt inhibition partially differed from structural features enhancing the compounds’ antioxidant activity. The present study is the first to quantitatively compare the influence of polyphenols from nine different structural subclasses on pAkt in endothelial cells. These effects might be advantageous in certain T2DM-complications involving over-activation of the Akt-pathway. The suggested molecular mode of action of polyphenols involving Akt-inhibition contributes to understanding their effects on the cellular level.
In recent years, covalent organic frameworks (COFs) have attracted considerable attention due to their crystalline and porous nature, which positions them as intriguing candidates for diverse applications such as catalysis, sensing, or optoelectronics. The incorporation of dyes or semiconducting moieties into a rigid two-dimensional COF can offer emergent features such as enhanced light harvesting or charge transport. However, this approach can be challenging when dealing with dye molecules that exhibit a large aromatic backbone, since the steric demand of solubilizing side chains also needs to be integrated into the framework. Here, we report the successful synthesis of DPP2-HHTP-COF consisting of diketopyrrolopyrrole (DPP) diboronic acid and hexahydroxytriphenylene (HHTP) building blocks. The well-known boronate ester coupling motif guides the formation of a planar and rigid backbone and long-range molecular DPP stacks, resulting in a highly crystalline and porous material. DPP2-HHTP-COF exhibits excellent optical properties including strong absorption over the visible spectral range, broad emission into the NIR and a singlet lifetime of over 5 ns attributed to the formation of molecular stacks with J-type interactions between the DPP subcomponents in the COF. Electrical measurements of crystalline DPP2-HHTP-COF pellets revealed conductivity values of up to 10(-6) S cm(-1).
With the technological advances of the last decade, it is now feasible to analyze microbiome samples, such as human stool specimens, using multi-omic techniques. Given the inherent sample complexity, there exists a need for sample methods which preserve as much information as possible about the biological system at the time of sampling. Here, we analyzed human stool samples preserved and stored using different methods, applying metagenomics as well as metaproteomics. Our results demonstrate that sample preservation and storage have a significant effect on the taxonomic composition of identified proteins. The overall identification rates, as well as the proportion of proteins from Actinobacteria were much higher when samples were flash frozen. Preservation in RNAlater overall led to fewer protein identifications and a considerable increase in the share of Bacteroidetes, as well as Proteobacteria. Additionally, a decrease in the share of metabolism-related proteins and an increase of the relative amount of proteins involved in the processing of genetic information was observed for RNAlater-stored samples. This suggests that great care should be taken in choosing methods for the preservation and storage of microbiome samples, as well as in comparing the results of analyses using different sampling and storage methods. Flash freezing and subsequent storage at −80 °C should be chosen wherever possible.