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Several oncolytic viruses (OVs) including various human and canine adenoviruses, canine distemper virus, herpes-simplex virus, reovirus, and members of the poxvirus family, such as vaccinia virus and myxoma virus, have been successfully tested for canine cancer therapy in preclinical and clinical settings. The success of the cancer virotherapy is dependent on the ability of oncolytic viruses to overcome the attacks of the host immune system, to preferentially infect and lyse cancer cells, and to initiate tumor-specific immunity. To date, several different strategies have been developed to overcome the antiviral host defense barriers. In our study, we used canine adipose-derived mesenchymal stem cells (cAdMSCs) as a “Trojan horse” for the delivery of oncolytic vaccinia virus Copenhagen strain to achieve maximum oncolysis against canine soft tissue sarcoma (CSTS) tumors. A single systemic administration of vaccinia virus-loaded cAdMSCs was found to be safe and led to the significant reduction and substantial inhibition of tumor growth in a CSTS xenograft mouse model. This is the first example that vaccinia virus-loaded cAdMSCs could serve as a therapeutic agent against CSTS tumors.
Head and neck squamous cell carcinoma (HNSCC) is known to overexpress a variety of receptor tyrosine kinases, such as the HGF receptor Met. Like other malignancies, HNSCC involves a mutual interaction between the tumor cells and surrounding tissues and cells. We hypothesized that activation of HGF/Met signaling in HNSCC influences glucose metabolism and therefore substantially changes the tumor microenvironment. To determine the effect of HGF, we submitted three established HNSCC cell lines to mRNA sequencing. Dynamic changes in glucose metabolism were measured in real time by an extracellular flux analyzer. As expected, the cell lines exhibited different levels of Met and responded differently to HGF stimulation. As confirmed by mRNA sequencing, the level of Met expression was associated with the number of upregulated HGF-dependent genes. Overall, Met stimulation by HGF leads to increased glycolysis, presumably mediated by higher expression of three key enzymes of glycolysis. These effects appear to be stronger in Met\(^{high}\)-expressing HNSCC cells. Collectively, our data support the hypothesized role of HGF/Met signaling in metabolic reprogramming of HNSCC.
Specialization of plant-pollinator interactions increases with temperature at Mt. Kilimanjaro
(2020)
Aim: Species differ in their degree of specialization when interacting with other species, with significant consequences for the function and robustness of ecosystems. In order to better estimate such consequences, we need to improve our understanding of the spatial patterns and drivers of specialization in interaction networks.
Methods: Here, we used the extensive environmental gradient of Mt. Kilimanjaro (Tanzania, East Africa) to study patterns and drivers of specialization, and robustness of plant–pollinator interactions against simulated species extinction with standardized sampling methods. We studied specialization, network robustness and other network indices of 67 quantitative plant–pollinator networks consisting of 268 observational hours and 4,380 plant–pollinator interactions along a 3.4 km elevational gradient. Using path analysis, we tested whether resource availability, pollinator richness, visitation rates, temperature, and/or area explain average specialization in pollinator communities. We further linked pollinator specialization to different pollinator taxa, and species traits, that is, proboscis length, body size, and species elevational ranges.
Results: We found that specialization decreased with increasing elevation at different levels of biological organization. Among all variables, mean annual temperature was the best predictor of average specialization in pollinator communities. Specialization differed between pollinator taxa, but was not related to pollinator traits. Network robustness against simulated species extinctions of both plants and pollinators was lowest in the most specialized interaction networks, that is, in the lowlands.
Conclusions: Our study uncovers patterns in plant–pollinator specialization along elevational gradients. Mean annual temperature was closely linked to pollinator specialization. Energetic constraints, caused by short activity timeframes in cold highlands, may force ectothermic species to broaden their dietary spectrum. Alternatively or in addition, accelerated evolutionary rates might facilitate the establishment of specialization under warm climates. Despite the mechanisms behind the patterns have yet to be fully resolved, our data suggest that temperature shifts in the course of climate change may destabilize pollination networks by affecting network architecture.
Neutral sphingomyelinase-2 (NSM2) is a member of a superfamily of enzymes responsible for conversion of sphingomyelin into phosphocholine and ceramide at the cytosolic leaflet of the plasma membrane. Upon specific ablation of NSM2, T cells proved to be hyper-responsive to CD3/CD28 co-stimulation, indicating that the enzyme acts to dampen early overshooting activation of these cells. It remained unclear whether hyper-reactivity of NSM2-deficient T cells is supported by a deregulated metabolic activity in these cells. Here, we demonstrate that ablation of NSM2 activity affects metabolism of the quiescent CD4\(^+\) T cells which accumulate ATP in mitochondria and increase basal glycolytic activity. This supports enhanced production of total ATP and metabolic switch early after TCR/CD28 stimulation. Most interestingly, increased metabolic activity in resting NSM2-deficient T cells does not support sustained response upon stimulation. While elevated under steady-state conditions in NSM2-deficient CD4\(^+\) T cells, the mTORC1 pathway regulating mitochondria size, oxidative phosphorylation, and ATP production is impaired after 24 h of stimulation. Taken together, the absence of NSM2 promotes a hyperactive metabolic state in unstimulated CD4\(^+\) T cells yet fails to support sustained T cell responses upon antigenic stimulation.
Folliculin Controls the Intracellular Survival and Trans-Epithelial Passage of Neisseria gonorrhoeae
(2020)
Neisseria gonorrhoeae, a Gram-negative obligate human pathogenic bacterium, infects human epithelial cells and causes sexually transmitted diseases. Emerging multi-antibiotic resistant gonococci and increasing numbers of infections complicate the treatment of infected patients. Here, we used an shRNA library screen and next-generation sequencing to identify factors involved in epithelial cell infection. Folliculin (FLCN), a 64 kDa protein with a tumor repressor function was identified as a novel host factor important for N. gonorrhoeae survival after uptake. We further determined that FLCN did not affect N. gonorrhoeae adherence and invasion but was essential for its survival in the cells by modulating autophagy. In addition, FLCN was also required to maintain cell to cell contacts in the epithelial layer. In an infection model with polarized cells, FLCN inhibited the polarized localization of E-cadherin and the transcytosis of gonococci across polarized epithelial cells. In conclusion, we demonstrate here the connection between FLCN and bacterial infection and in particular the role of FLCN in the intracellular survival and transcytosis of gonococci across polarized epithelial cell layers.
Comparison of the central human and mouse platelet signaling cascade by systems biological analysis
(2020)
Background
Understanding the molecular mechanisms of platelet activation and aggregation is of high interest for basic and clinical hemostasis and thrombosis research. The central platelet protein interaction network is involved in major responses to exogenous factors. This is defined by systemsbiological pathway analysis as the central regulating signaling cascade of platelets (CC).
Results
The CC is systematically compared here between mouse and human and major differences were found. Genetic differences were analysed comparing orthologous human and mouse genes. We next analyzed different expression levels of mRNAs. Considering 4 mouse and 7 human high-quality proteome data sets, we identified then those major mRNA expression differences (81%) which were supported by proteome data. CC is conserved regarding genetic completeness, but we observed major differences in mRNA and protein levels between both species. Looking at central interactors, human PLCB2, MMP9, BDNF, ITPR3 and SLC25A6 (always Entrez notation) show absence in all murine datasets. CC interactors GNG12, PRKCE and ADCY9 occur only in mice. Looking at the common proteins, TLN1, CALM3, PRKCB, APP, SOD2 and TIMP1 are higher abundant in human, whereas RASGRP2, ITGB2, MYL9, EIF4EBP1, ADAM17, ARRB2, CD9 and ZYX are higher abundant in mouse. Pivotal kinase SRC shows different regulation on mRNA and protein level as well as ADP receptor P2RY12.
Conclusions
Our results highlight species-specific differences in platelet signaling and points of specific fine-tuning in human platelets as well as murine-specific signaling differences.
Objective
The biological interpretation of gene expression measurements is a challenging task. While ordination methods are routinely used to identify clusters of samples or co-expressed genes, these methods do not take sample or gene annotations into account. We aim to provide a tool that allows users of all backgrounds to assess and visualize the intrinsic correlation structure of complex annotated gene expression data and discover the covariates that jointly affect expression patterns.
Results
The Bioconductor package covRNA provides a convenient and fast interface for testing and visualizing complex relationships between sample and gene covariates mediated by gene expression data in an entirely unsupervised setting. The relationships between sample and gene covariates are tested by statistical permutation tests and visualized by ordination. The methods are inspired by the fourthcorner and RLQ analyses used in ecological research for the analysis of species abundance data, that we modified to make them suitable for the distributional characteristics of both, RNA-Seq read counts and microarray intensities, and to provide a high-performance parallelized implementation for the analysis of large-scale gene expression data on multi-core computational systems. CovRNA provides additional modules for unsupervised gene filtering and plotting functions to ensure a smooth and coherent analysis workflow.
The opportunistic human pathogen Staphylococcus aureus causes serious infectious diseases that range from superficial skin and soft tissue infections to necrotizing pneumonia and sepsis. While classically regarded as an extracellular pathogen, S. aureus is able to invade and survive within human cells. Host cell exit is associated with cell death, tissue destruction, and the spread of infection. The exact molecular mechanism employed by S. aureus to escape the host cell is still unclear. In this study, we performed a genome-wide small hairpin RNA (shRNA) screen and identified the calcium signaling pathway as being involved in intracellular infection. S. aureus induced a massive cytosolic Ca\(^{2+}\) increase in epithelial host cells after invasion and intracellular replication of the pathogen. This was paralleled by a decrease in endoplasmic reticulum Ca\(^{2+}\) concentration. Additionally, calcium ions from the extracellular space contributed to the cytosolic Ca2+ increase. As a consequence, we observed that the cytoplasmic Ca\(^{2+}\) rise led to an increase in mitochondrial Ca\(^{2+}\) concentration, the activation of calpains and caspases, and eventually to cell lysis of S. aureus-infected cells. Our study therefore suggests that intracellular S. aureus disturbs the host cell Ca\(^{2+}\) homeostasis and induces cytoplasmic Ca\(^{2+}\) overload, which results in both apoptotic and necrotic cell death in parallel or succession.
IMPORTANCE Despite being regarded as an extracellular bacterium, the pathogen Staphylococcus aureus can invade and survive within human cells. The intracellular niche is considered a hideout from the host immune system and antibiotic treatment and allows bacterial proliferation. Subsequently, the intracellular bacterium induces host cell death, which may facilitate the spread of infection and tissue destruction. So far, host cell factors exploited by intracellular S. aureus to promote cell death are only poorly characterized. We performed a genome-wide screen and found the calcium signaling pathway to play a role in S. aureus invasion and cytotoxicity. The intracellular bacterium induces a cytoplasmic and mitochondrial Ca\(^{2+}\) overload, which results in host cell death. Thus, this study first showed how an intracellular bacterium perturbs the host cell Ca\(^{2+}\) homeostasis."
Metabolism and signaling of cytokinins was first established in plants, followed by cytokinin discoveries in all kingdoms of life. However, understanding of their role in mammalian cells is still scarce. Kinetin is a cytokinin that mitigates the effects of oxidative stress in mammalian cells. The effective concentrations of exogenously applied kinetin in invoking various cellular responses are not well standardized. Likewise, the metabolism of kinetin and its cellular targets within the mammalian cells are still not well studied. Applying vitality tests as well as comet assays under normal and hyper-oxidative states, our analysis suggests that kinetin concentrations of 500 nM and above cause cytotoxicity as well as genotoxicity in various cell types. However, concentrations below 100 nM do not cause any toxicity, rather in this range kinetin counteracts oxidative burst and cytotoxicity. We focus here on these effects. To get insights into the cellular targets of kinetin mediating these pro-survival functions and protective effects we applied structural and computational approaches on two previously testified targets for these effects. Our analysis deciphers vital residues in adenine phosphoribosyltransferase (APRT) and adenosine receptor (A2A-R) that facilitate the binding of kinetin to these two important human cellular proteins. We finally discuss how the therapeutic potential of kinetin against oxidative stress helps in various pathophysiological conditions.
MORN (Membrane Occupation and Recognition Nexus) repeat proteins have a wide taxonomic distribution, being found in both prokaryotes and eukaryotes. Despite this ubiquity, they remain poorly characterised at both a structural and a functional level compared to other common repeats. In functional terms, they are often assumed to be lipid-binding modules that mediate membrane targeting. We addressed this putative activity by focusing on a protein composed solely of MORN repeats-Trypanosoma brucei MORN1. Surprisingly, no evidence for binding to membranes or lipid vesicles by TbMORN1 could be obtained either in vivo or in vitro. Conversely, TbMORN1 did interact with individual phospholipids. High- and low-resolution structures of the MORN1 protein from Trypanosoma brucei and homologous proteins from the parasites Toxoplasma gondii and Plasmodium falciparum were obtained using a combination of macromolecular crystallography, small-angle X-ray scattering, and electron microscopy. This enabled a first structure-based definition of the MORN repeat itself. Furthermore, all three structures dimerised via their C-termini in an antiparallel configuration. The dimers could form extended or V-shaped quaternary structures depending on the presence of specific interface residues. This work provides a new perspective on MORN repeats, showing that they are protein-protein interaction modules capable of mediating both dimerisation and oligomerisation.