title: Fasting reduces inhibitory and attentional control of food-related cues

abstract: The metabolic and cognitive systems interact to create the motivational drive that occasionally leads to disrupted consummatory eating behaviors. In this study, we investigated whether stimulus-specific alterations of the inhibitory function are present following a period of food deprivation. Twenty-six participants with normal weight performed the Stop Signal Task (SST) and the Go/No-go (GNG) task to measure response inhibition to food images containing high or low caloric content after following -or not- a 12-hour fasting period. Response inhibition performance in the SST did not exhibit significant differences when considering neither fasting, stimulus type nor food caloric content. We instead found a higher percentage of commission errors in the No-go trials of the GNG task in the fasting session, and specially with high-caloric food items. In contrast, the accuracy in the Go trials was similar between conditions. A mixed logistic regression model confirmed the remarkable impact of fasting on the performance of response inhibition. Overall, our findings support an interpretation of the motivational drive to eat strongly associated with aspects of the inhibitory function underlying high attentional control, rather than to a proper response inhibition per se.

category: neuroscience


title: Improving the understanding of cytoneme-mediated morphogen gradients by in silico modeling

abstract: Morphogen gradients are crucial for the development of organisms. The biochemical properties of many morphogens prevent their extracellular free diffusion, indicating the need of an active mechanism for transport. The involvement of filopodial structures (cytonemes) has been proposed for morphogen signaling. Here, we describe an in silico model based on the main general features of cytoneme-meditated gradient formation and its implementation into Cytomorph, an open software tool. We have tested the spatial and temporal adaptability of our model quantifying Hedgehog (Hh) gradient formation in two Drosophila tissues. Cytomorph is able to reproduce the gradient and explain the different scaling between the two epithelia. After experimental validation, we studied the predicted impact of a range of features such as length, size, density, dynamics and contact behavior of cytonemes on Hh morphogen distribution. Our results illustrate Cytomorph as an adaptive tool to test different morphogens gradients and to generate hypotheses that are difficult to study experimentally.

category: genetics


category: developmental biology


title: Beta bursting in the retrosplenial cortex is a neurophysiological correlate of environmental novelty which is disrupted in a mouse model of Alzheimer's disease.

abstract: The retrosplenial cortex (RSC) plays a significant role in spatial learning and memory, and is functionally disrupted in the early stages of Alzheimers disease. In order to investigate neurophysiological correlates of spatial learning and memory in this region we employed in vivo electrophysiology in awake, behaving mice, comparing neural activity between wild-type and J20 mice, a mouse model of Alzheimers disease-associated amyloidopathy. To determine the response of the RSC to environmental novelty local field potentials were recorded while mice explored novel and familiar recording arenas. In familiar environments we detected short, phasic bursts of beta (20-30 Hz) oscillations (beta bursts) which arose at a low but steady rate. Exposure to a novel environment rapidly initiated a dramatic increase in the rate, size and duration of beta bursts. Additionally, theta-beta cross-frequency coupling was significantly higher during novelty, and spiking of neurons in the RSC was significantly enhanced during beta bursts. Finally, aberrant beta bursting was seen in J20 mice, including increased beta bursting during novelty and familiarity, yet a loss of coupling between beta bursts and spiking activity. These findings, support the concept that beta bursting may be responsible for the activation and reactivation of neuronal ensembles underpinning the formation and maintenance of cortical representations, and that disruptions to this activity in J20 mice may underlie cognitive impairments seen in these animals.


title: Perivascular cells support folliculogenesis in the developing ovary

abstract: Granulosa cells, supporting cells of the ovary, are essential for ovarian differentiation by providing a nurturing environment for oogenesis. Sufficient numbers of granulosa cells are vital for establishment of follicles and the oocyte reserve; therefore, identifying the cellular source from which granulosa cells are derived is critical for understanding basic ovarian biology. One cell type that has received little attention in this field is the perivascular cell. Here we use lineage tracing and organ culture techniques in mice to identify ovarian Nestin+ perivascular cells as multipotent progenitors that contribute to granulosa, thecal, and pericyte lineages. Maintenance of these progenitors was dependent on vascular-mesenchymal Notch signaling. Ablation of postnatal Nestin+ cells resulted in a disruption of granulosa cell specification and an increased incidence of polyovular ovarian follicles, thus uncovering key roles for vasculature in ovarian differentiation. These findings may provide new insights into the origins of female gonad dysgenesis and infertility.


title: Non-breeding waterbirds benefit from protected areas when adjusting their distribution to climate warming

abstract: Climate warming is driving changes in species distributions, although many species show a so-called climatic debt, where their range shifts lag behind the fast shift in temperature isoclines. Protected areas (PAs) may impact the rate of distribution changes both positively and negatively. At the cold edges of species distributions, PAs can facilitate species distribution changes by increasing the colonization required for distribution change. At the warm edges, PAs can mitigate the loss of species, by reducing the local extinction of vulnerable species. To assess the importance of PAs to affect species distribution change, we evaluated the changes in a non-breeding waterbird community as a response to temperature increase and PA status, using changes of species occurrence in the Western-Palearctic over 25 years (97 species, 7,071 sites, 39 countries, 1993- 2017). We used a community temperature index (CTI) framework based on species thermal affinities to investigate the species turn-over induced by temperature increase. In addition, we measured whether the thermal community adjustment was led by cold-dwelling species extinction and/or warm-dwelling species colonization, by modelling the change in standard deviation of the CTI (CTIsd). Using linear mixed-effects models, we investigated whether communities within PAs had lower climatic debt and different patterns of community change regarding the local PA surface. Thanks to the combined use of the CTI and CTIsd, we found that communities inside PAs had more species, higher colonization, lower extinction and the climatic debt was 16% lower than outside PAs. The results suggest the importance of PAs to facilitate warm-dwelling species colonization and attenuate cold-dwelling species extinction. The community adjustment was however not sufficiently fast to keep pace with the strong temperature increase in central and northeastern Western-Palearctic regions. Our study underlines the potential of the combined CTI and CTIsd metrics to understand the colonization-extinction patterns driven by climate warming.

category: ecology


title: Markov models provide many solutions for appropriate modeling of inapplicable morphological characters in phylogenetics

abstract: This paper describes new Markov models for phylogenetic inference with inapplicable (anatomically dependent) morphological characters. It shows that the new solutions fall into two categories depending on the type of character hierarchy and require use of different Markov models. A combination of the new models and previous solutions is capable to model all general types of dependencies that may exists between morphological traits. The performance of the new models is assessed using theory and simulations. Bayesian phylogenetic inference with the new models and their construction for a hierarchy of custom complexity is demonstrated using RevBayes and the R package rphenoscate respectively.

category: evolutionary biology


title: New phylogenetic Markov models for inapplicable morphological characters


title: Rephine.r: a pipeline for correcting gene calls and clusters to improve phage pangenomes and phylogenies

abstract:
BackgroundA pangenome is the collection of all genes found in a set of related genomes. For microbes, these genomes are often different strains of the same species, and the pangenome offers a means to compare gene content variation with differences in phenotypes, ecology, and phylogenetic relatedness. Though most frequently applied to bacteria, there is growing interest in adapting pangenome analysis to bacteriophages. However, working with phage genomes presents new challenges. First, most phage families are under-sampled, and homologous genes in related viruses can be difficult to identify. Second, homing endonucleases and intron-like sequences may be present, resulting in fragmented gene calls. Each of these issues can reduce the accuracy of standard pangenome analysis tools.

MethodsWe developed an R pipeline called Rephine.r that takes as input the gene clusters produced by an initial pangenomics workflow. Rephine.r then proceeds in two primary steps. First, it identifies three common causes of fragmented gene calls: 1) indels creating early stop codons and new start codons; 2) interruption by a selfish genetic element; and 3) splitting at the ends of the reported genome. Fragmented genes are then fused to create new sequence alignments. In tandem, Rephine.r searches for distant homologs separated into different gene families using Hidden Markov Models. Significant hits are used to merge families into larger clusters. A final round of fragment identification is then run, and results may be used to infer single-copy core genomes and phylogenetic trees.

ResultsWe applied Rephine.r to three well-studied phage groups: the Tevenvirinae (e.g. T4), the Studiervirinae (e.g. T7), and the Pbunaviruses (e.g. PB1). In each case, Rephine.r recovered additional members of the single-copy core genome and increased the overall bootstrap support of the phylogeny. The Rephine.r pipeline is provided through GitHub (https://www.github.com/coevoeco/Rephine.r) as a single script for automated analysis and with utility functions and a walkthrough for researchers with specific use cases for each type of correction.

category: bioinformatics


title: Secondary Structure Prediction for RNA Sequences Including N6-methyladenosine

abstract: There is increasing interest in the roles played by covalently modified nucleotides in mRNAs and non-coding RNAs. New high-throughput sequencing technologies localize these modifications to exact nucleotide positions. There has been, however, and inability to account for these modifications in secondary structure prediction because of a lack of software tools for handling modifications and a lack of thermodynamic parameters for modifications. Here, we report that we solved these issues for N6-methyladenosine (m6A), for the first time allowing secondary structure prediction for a nucleotide alphabet of A, C, G, U, and m6A. We revised the RNAstructure software package to work with any user-defined alphabet of nucleotides. We also developed a set of nearest neighbor parameters for helices and loops containing m6A, using a set of 45 optical melting experiments. Interestingly, N6-methylation decreases the folding stability of structures with adenosines in the middle of a helix, has little effect on the folding stability of adenosines at the ends of helices, and stabilizes the folding stability for structures with unpaired adenosines stacked on the end of a helix. The parameters were tested against an additional two melting experiments, including a consensus sequence for methylation and an m6A dangling end. The utility of the new software was tested using predictions of the structure of a molecular switch in the MALAT1 lncRNA, for which a conformation change is triggered by methylation. Additionally, human transcriptome-wide calculations for the effect of N6-methylation on the probability of an adenosine being buried in a helix compare favorably with PARS structure mapping data. Now users of RNAstructure are able to develop hypothesis for structure-function relationships for RNAs with m6A, including conformational switching triggered by methylation.


title: Dedifferentiation alters chondrocyte nuclear mechanics during in vitro culture and expansion

abstract:
Dedifferentiation of chondrocytes during in vitro passaging before implantation, and post implantation in vivo, is a critical limitation in cartilage tissue engineering. Several biophysical features define the dedifferentiated state including a flattened cell morphology and increased stress fiber formation. However, how dedifferentiation influences nuclear mechanics, and the possible long-term implications of this state, are unknown. In this study, we investigated how chondrocyte dedifferentiation affects the mechanics of the chromatin architecture inside the cell nucleus and the gene expression of the structural proteins located at the nuclear envelope. Through an experimental model of cell stretching and a detailed spatial intranuclear strain quantification, we identified that strain is amplified and distribution of strain within the chromatin is altered under tensile loading in the dedifferentiated state. Further, using a confocal microscopy image-based finite element model and simulation of cell stretching, we found that the cell shape is the primary determinant of the strain amplification inside the chondrocyte nucleus in the dedifferentiated state. Additionally, we found that nuclear envelope proteins have lower gene expression in the dedifferentiated state suggesting a weaker nuclear envelope which can further intensify the intranuclear strain amplification. Our results indicate that dedifferentiation and altered nuclear strain could promote gene expression changes at the nuclear envelope, thus promoting further deviation from chondrocyte phenotype. This study highlights the role of cell shape on nuclear mechanics and lays the groundwork to design biophysical strategies for the maintenance and enhancement of the chondrocyte phenotype during expansion with a goal of successful cartilage tissue engineering.

SIGNIFICANCEChondrocytes dedifferentiate into a fibroblast-like phenotype in a non-native biophysical environment. Using high resolution microscopy, intranuclear strain analysis, finite element method based computational modeling, and molecular biology techniques, we investigated how mechanical force causes abnormal intranuclear strain distribution in chondrocytes during the dedifferentiation process. Overall, our results suggest that the altered cell geometry aided by an altered or weakened nuclear envelope structure are responsible for abnormal intranuclear strain during chondrocyte dedifferentiation that can further deviate chondrocytes to a more dedifferentiated state.

category: biophysics


title: De novo identification of universal cell mechanics regulators

abstract: Mechanical proprieties determine many cellular functions, such as cell fate specification, migration, or circulation through vasculature. Identifying factors governing cell mechanical phenotype is therefore a subject of great interest. Here we present a mechanomics approach for establishing links between mechanical phenotype changes and the genes involved in driving them. We employ a machine learning-based discriminative network analysis method termed PC-corr to associate cell mechanical states, measured by real-time deformability cytometry (RT-DC), with large-scale transcriptome datasets ranging from stem cell development to cancer progression, and originating from different murine and human tissues. By intersecting the discriminative networks inferred from two selected datasets, we identify a conserved module of five genes with putative roles in the regulation of cell mechanics. We validate the power of the individual genes to discriminate between soft and stiff cell states in silico, and demonstrate experimentally that the top scoring gene, CAV1, changes the mechanical phenotype of cells when silenced or overexpressed. The data-driven approach presented here has the power of de novo identification of genes involved in cell mechanics regulation and paves the way towards engineering cell mechanical properties on demand to explore their impact on physiological and pathological cell functions.


title: Quantifying chromosomal instability from intratumoral karyotype diversity using agent-based modeling and Bayesian inference

abstract: Chromosomal instability (CIN) -- persistent chromosome gain or loss through abnormal karyokinesis -- is a hallmark of cancer that drives aneuploidy. Intrinsic chromosome mis-segregation rates, a measure of CIN, can inform prognosis and are a likely biomarker for response to anti-microtubule agents. However, existing methodologies to measure this rate are labor intensive, indirect, and confounded by karyotype selection reducing observable diversity. We developed a framework to simulate and measure CIN, accounting for karyotype selection, and recapitulated karyotype-level clonality in simulated populations. We leveraged approximate Bayesian computation using phylogenetic topology and diversity to infer mis-segregation rates and karyotype selection from single-cell DNA sequencing data. Experimental validation of this approach revealed extensive chromosome mis-segregation rates caused by the chemotherapy paclitaxel (17.5{+/-}0.14/division). Extending this approach to clinical samples revealed the inferred rates fell within direct observations of cancer cell lines. This work provides the necessary framework to quantify CIN in human tumors and develop it as a predictive biomarker.

category: cancer biology


title: The Lack of Nrf2 Causes Hepatocyte Dedifferentiation and Reduced Albumin Production in an Experimental Extrahepatic Cholestasis Model

abstract:
The transcription factor Nrf2 modulates the initiation and progression of a number of diseases including liver disorders. The aim of this study was to evaluate whether Nrf2 mediates hepatic adaptive responses to cholestasis. Wild-type and Nrf2-null mice were subjected to bile duct ligation (BDL) or a sham operation. Various assessments were performed at different days after surgery. Significant genotype-dependent changes in liver size, biliary ductular reaction, hepatocyte proliferation, and fibrotic response were not observed. However, as cholestasis progressed to Day 15 post-BDL, hepatocytes in the wild-type mice exhibited a tendency to dedifferentiate, indicated by the very weak expression of hepatic progenitor markers: CD133 and fibroblast growth factor-inducible 14 (Fn14). During the same period, Nrf2 deficiency augmented this tendency, manifested by higher CD133 expression, earlier, stronger, and continuous induction of Fn14 expression, and markedly reduced albumin production. Remarkably, as cholestasis advanced to the late stage (40 days after BDL), hepatocytes in the wild-type mice exhibited a Fn14+ phenotype and strikingly upregulated the expression of deleted in malignant brain tumor 1 (DMBT1), a protein essential for epithelial differentiation during development. In contrast, at this stage, hepatocytes in the Nrf2-null mice entirely inhibited the upregulation of DMBT1 expression, displayed a strong CD133+/Fn14+ phenotype indicative of severe dedifferentiation, and persistently reduced albumin production. Collectively, our studies revealed that Nrf2 maintains hepatocytes in the differentiated state potentially via the increased activity of the Nrf2/DMBT1 pathway during cholestasis. These findings enable us to gain novel insight into how hepatocytes respond to cholestasis.

New and NoteworthyWe found that, when hepatocytes are exposed to cholestasis, they exhibit a tendency of dedifferentiation. In this case, Nrf2 is highly activated to markedly up-regulate the expression of epithelial differentiation gene DMBT1, which potentially prevent hepatocytes from dedifferentiation. Our findings revealed a plastic property of hepatocytes in response to cholestasis and demonstrated a novel Nrf2/DMBT1 pathway likely controlling this property of hepatocytes.

category: pathology


title: Characterization of methylation signatures in spontaneous preterm birth

abstract: Preterm birth is a global public health crisis which results in significant neonatal and maternal mortality. Yet little is known regarding the molecular mechanisms of idiopathic spontaneous preterm birth, and we have few diagnostic markers for adequate assessment of placental development and function. Previous studies of placental pathology and our transcriptomics studies suggest a role for placental maturity in idiopathic spontaneous preterm birth. It is known that placental DNA methylation changes over gestation. We hypothesized that if placental hypermaturity is present in our samples, we would observe a unique idiopathic spontaneous preterm birth DNA methylation profile potentially driving the gene expression differences we previously identified in our placental samples. Our results indicate the idiopathic spontaneous preterm birth DNA methylation pattern mimics the term birth methylation pattern suggesting hypermaturity. Only seven significant differentially methylated regions fitting the idiopathic spontaneous preterm birth specific (relative to the controls) profile were identified, indicating unusually high similarity in DNA methylation between idiopathic spontaneous preterm birth and term birth samples. We identified an additional 1,718 significantly methylated regions in our gestational age matched controls were the idiopathic spontaneous preterm birth DNA methylation pattern mimics the term birth methylation pattern, again indicating a striking level of similarity between the idiopathic spontaneous preterm birth and term birth samples. Pathway analysis of these regions revealed differences in genes within the WNT and Cadherin signaling pathways, both of which are essential in placental development and maturation. Taken together, these data demonstrate that the idiopathic spontaneous preterm birth samples are molecularly more mature than expected given their respective gestational age which likely impacts birth timing.

category: physiology


title: Characterization of methylation profiles in spontaneous preterm birth placental villous tissue


title: Redundancy between spectral and higher-order texture statistics for natural image segmentation

abstract: Visual texture, defined by local image statistics, provides important information to the human visual system for perceptual segmentation. Second-order or spectral statistics (equivalent to the Fourier power spectrum) are a well-studied segmentation cue. However, the role of higher-order statistics (HOS) in segmentation remains unclear, particularly for natural images. Recent experiments indicate that, in peripheral vision, the HOS of the widely adopted Portilla-Simoncelli texture model are a weak segmentation cue compared to spectral statistics, despite the fact that both are necessary to explain other perceptual phenomena and to support high-quality texture synthesis. Here we test whether this discrepancy reflects a property of natural image statistics. First, we observe that differences in spectral statistics across segments of natural images are redundant with differences in HOS. Second, using linear and nonlinear classifiers, we show that each set of statistics individually affords high performance in natural scenes and texture segmentation tasks, but combining spectral statistics and HOS produces relatively small improvements. Third, we find that HOS improve segmentation for a subset of images, although these images are difficult to identify. We also find that different subsets of HOS improve segmentation to a different extent, in agreement with previous physiological and perceptual work. These results show that the HOS add modestly to spectral statistics for natural image segmentation. We speculate that tuning to natural image statistics under resource constraints could explain the weak contribution of HOS to perceptual segmentation in human peripheral vision.


title: Affordable and Effective Optokinetic Response Methods to Assess Visual Acuity and Contrast Sensitivity in Larval to Juvenile Zebrafish

abstract:
BackgroundThe optokinetic response (OKR) is an effective behavioural assay to investigate functional vision in zebrafish. The rapid and widespread use of gene editing, drug screening and environmental modulation technologies have resulted in a broader need for visual neuroscience researchers to access affordable and more sensitive OKR, contrast sensitivity (CS) and visual acuity (VA) assays. Here, we demonstrate how 2D- and 3D-printed, striped patterns or drums coupled with a motorised base and microscope provide a simple, cost-effective but efficient means to assay OKR, CS and VA in larval-juvenile zebrafish.

ResultsIn wild-type, 5 days post-fertilisation (dpf) zebrafish, the 2D or 3D drums printed with the standard OKR stimulus of 0.02 cycles per degree (cpd), 100% black-white contrast evoked equivalent responses of 24.2 or 21.8 saccades per minute, respectively. Furthermore, although the OKR number was significantly reduced compared to the 0.02 cpd drum (p<0.0001), the 2D and 3D drums evoked respectively equivalent responses with the 0.06 and 0.2 cpd drums. Notably, standard OKR responses varied with time of day; peak responses of 29.8 saccades per minute occurred in the early afternoon with significantly reduced responses occurring in the early morning or late afternoon, (18.5 and 18.4 saccades per minute, respectively). A customised series of 2D printed drums enabled analysis of visual acuity and contrast sensitivity in 5-21 dpf zebrafish. The saccadic frequency in visual acuity and contrast sensitivity assays, was inversely proportional to age, spatial frequency and contrast of the stimulus.

ConclusionsOKR, VA and CS of zebrafish larvae can be efficiently measured using 2D- or 3D-printed striped drums. For data consistency the luminance of the OKR light source, the time of day when the analysis performed, and the order of presentation of VA and CS drums must be considered. These simple methods allow effective and more sensitive analysis of functional vision in zebrafish.


title: The effects of epigallocatechin gallate and caffeine on risky decision-making

abstract: Epigallocatechin-3-gallate (EGCG) and caffeine are the two primary compounds found in green tea. While EGCG has anxiolytic and anti-inflammatory effects, its acute effects on cognition are not well understood. Furthermore, despite widespread green tea consumption, little is known about how EGCG and caffeine co-administration impact behavior. Here, we investigated the effects of multiple doses of either EGCG or caffeine on a rat model of risk-taking. This was assessed using the risky decision-making task (RDT), in which rats choose between a small, safe reward and a large reward with escalating risk of mild footshock. Rats were tested in RDT after acute systemic administration of EGCG, caffeine, or joint EGCG and caffeine. EGCG caused a dose dependent reduction in risk-taking without affecting reward discrimination or task engagement. Caffeine did not impact risk taking, but elevated locomotor activity and reduced task engagement at high doses. Finally, exposure to both EGCG and caffeine had no effect on risk-taking, suggesting that low-dose caffeine is sufficient to mask the risk-aversion caused by EGCG. These data suggest EGCG as a potential therapeutic treatment for psychological disorders that induce compulsive risky decision-making.


title: Mechanical control of innate immune responses against viral infection revealed in a human Lung Alveolus Chip

abstract: Mechanical forces associated with breathing play a fundamental role in lung development and disease but the molecular pathways remain largely unknown. Here, we used a mechanically actuatable Human Lung Alveolus Chip that recapitulates human lung alveolar type I and type II cell differentiation, alveolar-capillary interface formation, and genome-wide gene expression profiles characteristic of the distal lung to investigate the role of physical forces associated with cyclic breathing motions in lung innate immune responses to viral infection. When the mechanically active Alveolus Chips are infected with the influenza H3N2 virus, a cascade of host responses is elicited on-chip, including increased production of cytokines and expression of inflammation-associated genes in pulmonary epithelial and endothelial cells, resulting in enhanced recruitment of circulating immune cells as occurs during viral infection in vivo. Surprisingly, studies carried out in parallel with static chips revealed that physiological breathing motions suppress viral replication by activating protective innate immune responses in epithelial and endothelial cells. This is mediated at least in part through upregulation of S100 calcium-binding protein A7 (S100A7), which binds to the Receptor for Advanced Glycation End Products (RAGE), an inflammatory mediator that is most highly expressed in the lung alveolus in vivo. This mechano-immunological control mechanism is further supported by the finding that existing RAGE inhibitor drugs can suppress the production of inflammatory cytokines in response to influenza virus infection in this model. S100A7-RAGE interactions and modulation of mechanical ventilation parameters could therefore serve as new targets for therapeutic intervention in patients infected with influenza and other potential pandemic viruses that cause life-threatening lung inflammation.

category: immunology


title: Sequential single cell transcriptional and protein marker profiling reveals TIGIT as a marker of CD19 CAR-T cell dysfunction in patients with non-Hodgkin lymphoma

abstract: Chimeric antigen receptor T cell (CAR-T cell) therapy is known to produce durable remissions in the treatment of CD19+ relapsed/refractory B cell malignancies. Nonetheless, a significant portion of patients receiving the therapy experience poor outcomes in the acute response for unknown reasons. Given the decreased expansion and persistence of CD8 CAR-T cells in poor outcome groups, this failure may be attributed to CAR-T cell dysfunction. However, a comparison of the post-infusion transcriptional profiles and phenotypes between CAR-T cells of poor and favorable response groups has not been performed. Here, we employed single cell RNA sequencing and protein surface marker profiling of serial CAR-T cell blood samples from patients with CD19+ relapsed/refractory non-Hodgkins lymphoma (NHL) to reveal CAR-T cell evolution, identify biomarkers of response, and test for evidence of exhaustion in CAR-T cells of poor responders. At the transcriptional and protein levels, we note the evolution of a majority of CAR-T cells toward a non-proliferative and highly-differentiated state. In poor outcome patients, we observed a more marked enrichment of an exhaustion profile as compared to favorable outcome patients. Lastly, we identified the checkpoint receptor TIGIT (T cell immunoreceptor with Ig and ITIM domains) as a novel prognostic biomarker and potential driver of CAR-T cell exhaustion. Altogether, we provide evidence of CAR-T cell dysfunction marked by TIGIT expression driving poor response in NHL patients.


title: Trypanosoma brucei Tim50 Plays a Critical Role in Cell Cycle Regulation and Parasite Infectivity

abstract:
Tim50 is a receptor subunit of the preprotein-translocase of the mitochondrial inner membrane, TIM23. Trypanosoma brucei, the infective agent for African trypanosomiasis, possesses a homologue of Tim50 (TbTim50) with a pair of characteristic DXDX(T/V) phosphatase signature motifs. Here, we demonstrated that besides its protein phosphatase activity, the recombinant TbTim50 binds and hydrolyzes phosphatidic acid in a concentration-dependent manner. In silico structural homology models identify the putative binding interfaces that may accommodate different phospho-substrates. Interestingly, TbTim50 depletion in the bloodstream form (BF) of T. brucei reduced cardiolipin (CL) levels and decreased mitochondrial membrane potential ({Delta}{Psi}). TbTim50 knockdown (KD) also reduced the population of G2 phase and increased G1 phase; thus, BF cell growth was reduced. Confocal and electron microscopy revealed a defect in regulation of kinetoplast (kDNA) replication due to TbTim50 KD. Depletion of TbTim50 increased the levels of AMPK phosphorylation, and parasite morphology was changed to stumpy-like with upregulation of few stumpy marker gene expressions. Importantly, we observed that TbTim50-depleted parasites were unable to establish infection in mice and rats. Proteomics analysis showed reductions of the translation factors, flagellar transport proteins, and many proteasomal subunits, including the mitochondrial HslVU that is known to play a role in kDNA replication. Reduction of the level of HslV in TbTim50 KD cells was further validated by immunoblot analysis. Altogether, our results showed that TbTim50 is essential for mitochondrial function, regulation of kDNA replication, and cell cycle in the BF. Therefore, TbTim50 is an important target for structure-based drug design to combat African trypanosomiasis.

ImportanceAfrican trypanosomiasis, a neglected tropical disease caused by parasitic protozoan Trypanosoma brucei, is transmitted by the tsetse fly prevalent in sub-Saharan Africa. During its digenetic life cycle, T. brucei undergoes multiple developmental changes to adapt in different environments. T. brucei BF, dwelling in mammalian blood, generates ATP from glycolysis and hydrolyzes ATP in mitochondria for inner membrane potential. We found that TbTim50, a HAD-family phosphatase, is critical for T. brucei BF survival in vitro and in vivo. Depletion of TbTim50 in BF reduced CL levels and mitochondrial {Delta}{Psi} and caused a detrimental effect on many cellular functions. Cells accumulated in G1-S phase, and kinetoplast was over-replicated due to depletion of mitochondrial proteasomes, HslVU, a master-regulator of kDNA replication. Cell growth inhibition was accompanied by changes in morphology, AMPK phosphorylation, and upregulation of stumpy-specific gene expression. TbTim50 is essential for T. brucei survival and an important T. brucei therapeutic target.

category: cell biology


title: EVALUATION OF TAURINE'S NEUROPROTECTIVE EFFECTS ON SH-SY5Y CELLS UNDER OXIDATIVE STRESS

abstract:
Alzheimers disease (AD) is a type of dementia that affects millions of people. Although there is no cure, several study strategies seek to elucidate the mechanisms of the disease. Recent studies address the benefits of taurine. Thus, the present study aims to analyze the neuroprotective effect of taurine on human neuroblastoma, using an in vitro experimental model of oxidative stress induced by hydrocortisone in the SH-SY5Y cell line as a characteristic model of AD. The violet crystal assay was used for cell viability and the evaluation of cell morphology was performed by scanning electron microscopy (SEM). After pretreatment with taurine, the SH-SY5Y cell showed an improvement in cell viability in the face of oxidative stress and improved cell morphology. Thus, the treatment presented a neuroprotective effect.

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title: Seed banks alter metacommunity diversity: the interactive effects of competition, germination, and survival

abstract: Dispersal and dormancy are two common strategies allowing for species persistence and the maintenance of ecological diversity in variable environments. However, theory and empirical tests of spatial diversity patterns tend to examine either mechanism in isolation. Here, we developed a stochastic, spatially explicit metacommunity model incorporating seed banks with varying germination and survival rates. We found that dormancy and dispersal had interactive, nonlinear effects on the maintenance and distribution of metacommunity diversity, where scale-dependent effects of seed banks were modified by local competitive interactions and dispersal. The interplay between seed germination and survival regulated the benefits of seed banks for diversity. Our study shows that the role of seed banks depends critically on spatial processes, and that classic predictions for how dispersal affects metacommunity diversity can be strongly influenced by dormancy. Together, these results highlight the need to consider both temporal and spatial storage when predicting multi-scale patterns of diversity.


title: Fitness estimation for viral variants in the context of cellular coinfection

abstract: Animal models are frequently used to characterize the within-host dynamics of emerging zoonotic viruses. More recent studies have also deep-sequenced longitudinal viral samples originating from experimental challenges to gain a better understanding of how these viruses may evolve in vivo and between transmission events. These studies have often identified nucleotide variants that can replicate more efficiently within hosts and also transmit more effectively between hosts. Quantifying the degree to which a mutation impacts viral fitness within a host can improve identification of variants that are of particular epidemiological concern and our ability to anticipate viral adaptation at the population level. While methods have been developed to quantify the fitness effects of mutations using observed changes in allele frequencies over the course of a hosts infection, none of the existing methods account for the possibility of cellular coinfection. Here, we develop mathematical models to project variant allele frequency changes in the context of cellular coinfection and, further, integrate these models with statistical inference approaches to demonstrate how variant fitness can be estimated alongside cellular multiplicity of infection. We apply our approaches to empirical longitudinally-sampled H5N1 sequence data from ferrets. Our results indicate that previous studies may have significantly underestimated the within-host fitness advantage of viral variants. These findings underscore the importance of considering the process of cellular coinfection when studying within-host viral evolutionary dynamics.


title: Mapping and modeling the genomic basis of differential RNA isoform expression at single-cell resolution with LR-Split-seq

abstract: Alternative RNA isoforms are defined by promoter choice, alternative splicing, and polyA site selection. Although differential isoform expression is known to play a large regulatory role in eukaryotes, it has proved challenging to study with standard short-read RNA-seq because of the uncertainties it leaves about the full-length structure and precise termini of transcripts. The rise in throughput and quality of long-read sequencing now makes it possible, in principle, to unambiguously identify most transcript isoforms from beginning to end. However, its application to single-cell RNA-seq has been limited by throughput and expense. Here, we develop and characterize long-read Split-seq (LR-Split-seq), which uses a combinatorial barcoding-based method for sequencing single cells and nuclei with long reads. We show that LR-Split-seq can associate isoforms with cell types with relative economy and design flexibility. We characterize LR-Split-seq for whole cells and nuclei by using the well-studied mouse C2C12 system in which mononucleated myoblast cells differentiate and fuse into multinucleated myotubes. We show that the overall results are reproducible when comparing long- and short-read data from the same cell or nucleus. We find substantial evidence of differential isoform expression during differentiation including alternative transcription start site (TSS) usage. We integrate the resulting isoform expression dynamics with snATAC-seq chromatin accessibility to validate TSS-driven isoform choices. LR-Split-seq provides an affordable method for identifying cluster-specific isoforms in single cells that can be further quantified with companion deep short-read scRNA-seq from the same cell populations.

category: genomics


title: Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages

abstract: CRISPR-Cas9 screening libraries have arisen as a powerful tool to identify both protein coding (pc) and non-coding genes playing a role along different processes. In particular, the usage of a nuclease active Cas9 coupled to a single gRNA has proven to efficiently impair the expression of pc-genes by generating deleterious frameshifts. Here, we first demonstrate that the usage of a second gRNA targeting the same gene synergistically enhances the capacity of the CRISPR-Cas9 system to knock out pc-genes. We next take advantage of our paired-guide (pgRNA) system to design a library to simultaneously target 874 pc-genes and 166 lncRNAs which are known to change expression during the transdifferentiation from pre-B cells to macrophages. We show that this system is able to identify known players in this process, and also predicts 26 potential novel ones, of which we select four for deeper characterization. Two of these, FURIN and NFE2, code for proteins related to cell differentiation and macrophage function; the other two, LINC02432 and MIR3945HG, are lncRNAs associated with cancerous and infectious diseases, respectively. The CRISPR-Cas9 coupled to pgRNAs system is, therefore, a suitable tool to target simultaneously pc-genes and lncRNAs for genomic perturbation assays.


title: Relative Mutant N501Y SARS-CoV-2 Spike Protein RBD Inhibition of Anti-Spike Protein IgG and ACE-2 Binding to Spike Protein Species

abstract: In the SARS-CoV-2 coronavirus pandemic of 2019 (COVID-19), it has become evident that the ACE-2 receptor-binding domain (RBD) of the viral spike protein (SP) is the target of neutralizing antibodies that comprise a critical element of protective immunity to the virus. The most definitive confirmation of this contention is that the two mRNA COVID-19 vaccines in general use, which elicit antibodies specific for the RBD, exhibit approximately 95% protective efficacy against COVID-19. A potential challenge to vaccine efficacy is the emergence of SARS-CoV-2 variants possessing multiple mutations affecting amino acid residues in the RBD. Of concern are variants that arose in the United Kingdom, Brazil and South Africa. One of the variants, designated B.1.351, has shown a higher transmissibility due to greater affinity for the ACE-2 receptor and decreased neutralization by convalescent plasma, therapeutic monoclonal antibodies, and post-vaccination plasma. Common to several of the variants is the N501Y mutation in the RBD, which may be responsible for at least part of the observed variant properties. To test this hypothesis, we measured the ability of the Y501 RBD to inhibit binding of the wild type RBD and full SP (S1 + S2) to the ACE-2 protein and a human monoclonal IgG antibody elicited to the wild type RBD, relative to the wild type RBD in two enzyme-linked immunosorbent assays (ELISAs). We found no significant difference in the IC50 of the two RBD species inhibition of ACE-2 binding, but unexpectedly found that the IC50 of the wild type RBD inhibition of antibody binding was nearly twice that of the Y501 RBD, reflecting a lower affinity. These results suggest that the individual N501Y mutation does not contribute to altered viral properties by itself, but may contribute to a collective conformational shift produced by multiple mutations.

category: biochemistry


title: Changes in Cellular Crosstalk between Skeletal Muscle Myoblasts and Bone Osteoblasts with Aging

abstract: Musculoskeletal function declines with aging, resulting in an increased incidence of trips and falls. Both bone and muscle experience age-related losses in tissue mass that alter their mechanical interactions in a well characterized manner, but changes in the biochemical interactions between bone and muscle with aging are not well understood. Of note, insulin-like growth factor 1 (IGF-1), a potent growth factor for bone and muscle, can be negatively altered with aging and may help explain losses in these tissues. We recently developed a co-culture system for simultaneous growth of bone mesenchymal stem cells (MSCs) and muscle satellite cells (SCs) to investigate the biochemical crosstalk between the two cell types. Here, we utilized an aging rat model to study cellular changes between young and old rat MSCs and SCs, in particular whether 1) young MSCs and SCs have increased proliferation and differentiation compared to old MSCs and SCs; 2) young cells have increased IGF-1 and collagen expression as a measure of crosstalk compared to old cells; and 3) young cells can mitigate the aging phenotype of old cells in co-culture. Rat MSCs and SCs were either mono- or co-cultured in Transwell(R) plates, grown to confluence, and allowed to differentiate for 14 days. Across the 14 days, cell proliferation was measured, with differentiation and crosstalk measurements evaluated at 14 days. The results suggest that in both young and old, proliferation is greater in mono-cultures compared to co-cultures, yet age and cell type did not have a significant effect. Differentiation did not differ between young and old cells, yet MSCs and SCs demonstrated the greatest amount of differentiation in co-culture. Finally, age, cell type, and culture type did not have a significant effect on collagen or IGF-1 expression. These results suggest co-culture may have a controlling effect, with the two cell types acting together to promote differentiation more than in mono-cultures, yet this response was not altered by age. In general, results for old cells had higher variability, suggesting a wider variety in the aging phenotypes demonstrated in these animals. This study was the first to use this rat aging model to investigate changes between bone and skeletal muscle cells, however further investigations are required to determine what signaling changes occur in response to age. Determining these signaling changes could lead to new targets for mitigating the progression of aging.

category: bioengineering


title: Particle Dynamics and Bioaerosol Viability of Aerosolized BCG Vaccine Using Jet and Vibrating Mesh Clinical Nebulizers

abstract:
BackgroundBacillus Calmette-Guerin (BCG) is a vaccine used to protect against tuberculosis primarily in infants to stop early infection in areas of the world where the disease is endemic. Normally administered as a percutaneous injection, BCG is a live, significantly attenuated bacteria that is now being investigated for its potential within an inhalable vaccine formulation. This work investigates the feasibility and performance of four jet and ultrasonic nebulizers aerosolizing BCG and the resulting particle characteristics and residual viability of the bacteria post-aerosolization.

MethodsA jet nebulizer (Collison) outfitted either with a 3- or 6-jet head, was compared to two clinical nebulizers, the vibrating mesh Omron MicroAir and Aerogen Solo devices. Particle characteristics, including aerodynamic particle sizing, was performed on all devices within a common aerosol chamber configuration and comparable BCG innocula concentrations. Integrated aerosol samples were collected for each generator and assayed for bacterial viability using conventional microbiological technique.

Results and ConclusionsA batch lot of BCG (Danish) was grown to titer and used in all generator assessments. Aerosol particles within the respirable range were generated from all nebulizers at four different concentrations of BCG. The jet nebulizers produced a uniformly smaller particle size than the ultrasonic devices, although particle concentrations by mass were similar across all devices tested with the exception of the Aerogen Solo, which resulted in a very low concentration of BCG aerosols. The resulting measured viable BCG aerosol concentration fraction produced by each device approximated one another; however, a measurable decrease of efficiency and overall viability reduction in the jet nebulizer was observed in higher BCG inoculum starting concentrations, whereas the vibrating mesh nebulizer returned a remarkably stable viable aerosol fraction irrespective of inoculum concentration.


title: Modeling breast cancer by grafting patient tumor samples in the avian embryo: an in vivo platform for therapy evaluation coupled to large scale molecular analyses

abstract: Lack of preclinical patient-derived xenograft (PDX) cancer models in which to conduct large scale molecular studies seriously impairs the development of effective personalized therapies. We report here on an in vivo concept consisting of implanting human tumor cells in targeted tissues of an avian embryo, delivering therapeutics, evaluating their efficacy by measuring tumors using light sheet confocal microscopy, and conducting large scale RNAseq analysis to characterize therapeutic-induced changes in gene expression. The model was established to recapitulate triple negative breast cancer (TNBC) and validated using TNBC standards of care (SOCs) and an investigational therapeutic agent.


title: Determining clinically relevant features in cytometry data using persistent homology

abstract:
Cytometry experiments yield high-dimensional point cloud data that is difficult to interpret manually. Boolean gating techniques coupled with comparisons of relative abundances of cellular subsets is the current standard for cytometry data analysis. However, this approach is unable to capture more subtle topological features hidden in data, especially if those features are further masked by data transforms or significant batch effects or donor-to-donor variations in clinical data. We present that persistent homology, a mathematical structure that summarizes the topological features, can distinguish different sources of data, such as from groups of healthy donors or patients, effectively. Analysis of publicly available cytometry data describing non-naive CD8+ T cells in COVID-19 patients and healthy controls shows that systematic structural differences exist between single cell protein expressions in COVID-19 patients and healthy controls.

Our method identifies proteins of interest by a decision-tree based classifier and passes them to a kernel-density estimator (KDE) for sampling points from the density distribution. We then compute persistence diagrams from these sampled points. The resulting persistence diagrams identify regions in cytometry datasets of varying density and identify protruded structures such as  elbows. We compute Wasserstein distances between these persistence diagrams for random pairs of healthy controls and COVID-19 patients and find that systematic structural differences exist between COVID-19 patients and healthy controls in the expression data for T-bet, Eomes, and Ki-67. Further analysis shows that expression of T-bet and Eomes are significantly downregulated in COVID-19 patient non-naive CD8+ T cells compared to healthy controls. This counter-intuitive finding may indicate that canonical effector CD8+ T cells are less prevalent in COVID-19 patients than healthy controls. This method is applicable to any cytometry dataset for discovering novel insights through topological data analysis which may be difficult to ascertain otherwise with a standard gating strategy or in the presence of large batch effects.

Author summaryIdentifying differences between cytometry data seen as a point cloud can be complicated by random variations in data collection and data sources. We apply persistent homology used in topological data analysis to describe the shape and structure of the data representing immune cells in healthy donors and COVID-19 patients. By looking at how the shape and structure differ between healthy donors and COVID-19 patients, we are able to definitively conclude how these groups differ despite random variations in the data. Furthermore, these results are novel in their ability to capture shape and structure of cytometry data, something not described by other analyses.

category: systems biology


title: Sensory evidence accumulation using optic flow in a naturalistic navigation task

abstract: Sensory evidence accumulation is considered a hallmark of decision-making in noisy environments. Integration of sensory inputs has been traditionally studied using passive stimuli, segregating perception from action. Lessons learned from this approach, however, may not generalize to ethological behaviors like navigation, where there is an active interplay between perception and action. We designed a sensory-based sequential decision task in virtual reality in which humans and monkeys navigated to a memorized location by integrating optic flow generated by their own joystick movements. A major challenge in such closed-loop tasks is that subjects actions will determine future sensory input, causing ambiguity about whether they rely on sensory input rather than expectations based solely on a learned model of the dynamics. To test whether subjects performed sensory integration, we used three independent experimental manipulations: unpredictable optic flow perturbations, which pushed subjects off their trajectory; gain manipulation of the joystick controller, which changed the consequences of actions; and manipulation of the optic flow density, which changed the reliability of sensory evidence. Our results suggest that both macaques and humans relied heavily on optic flow, thereby demonstrating a critical role for sensory evidence accumulation during naturalistic action-perception closed-loop tasks.


title: Eye movements reveal spatiotemporal dynamics of active sensing and planning in navigation

abstract: Goal-oriented navigation is widely understood to depend upon internal maps. Although this may be the case in many settings, humans tend to rely on vision in complex, unfamiliar environments. To study the nature of gaze during visually-guided navigation, we tasked humans to navigate to transiently visible goals in virtual mazes of varying levels of difficulty, observing that they took near-optimal trajectories in all arenas. By analyzing participants eye movements, we gained insights into how they performed visually-informed planning. The spatial distribution of gaze revealed that environmental complexity mediated a striking trade-off in the extent to which attention was directed towards two complimentary aspects of the world model: the reward location and task-relevant transitions. The temporal evolution of gaze revealed rapid, sequential prospection of the future path, evocative of neural replay. These findings suggest that the spatiotemporal characteristics of gaze during navigation are significantly shaped by the unique cognitive computations underlying real-world, sequential decision making.


title: Eye movements reveal spatiotemporal dynamics of visually-informed planning in navigation


title: Vascular and perivascular cell profiling reveals the molecular and cellular bases of blood-brain barrier heterogeneity

abstract:
SUMMARY

The blood-brain barrier (BBB) is critical for protecting the brain and maintaining neuronal homeostasis. Although the BBB is a unique feature of the central nervous system (CNS) vasculature, not all brain regions have the same degree of impermeability. Differences in BBB permeability are important for controlling the local extracellular environment of specific brain regions to regulate the function and plasticity of particular neural circuits. However, how BBB heterogeneity occurs is poorly understood. Here, we demonstrate how regional specialization of the BBB is achieved. With unbiased cell profiling in small, defined brain regions, we compare the median eminence, which has a naturally leaky BBB, with the cortex, which has an impermeable BBB. We identify hundreds of molecular differences in endothelial cells (ECs) and demonstrate the existence of differences in perivascular astrocytes and pericytes in these regions, finding 3 previously unknown subtypes of astrocytes and several key differences in pericytes. By serial electron microscopy reconstruction and a novel, aqueous-based tissue clearing imaging method, we further reveal previously unknown anatomical specializations of these perivascular cells and their unique physical interactions with neighboring ECs. Finally, we identify ligand-receptor pairs between ECs and perivascular cells that may regulate regional BBB integrity in ECs. Using a bioinformatic approach we identified 26 and 26 ligand-receptor pairs underlying EC-pericyte and EC-astrocyte interactions, respectively. Our results demonstrate that differences in ECs, together with region-specific physical and molecular interactions with local perivascular cells, contribute to BBB functional heterogeneity. These regional cell inventories serve as a platform for further investigation of the dynamic and heterogeneous nature of the BBB in other brain regions. Identification of local BBB specializations provides insight into the function of different brain regions and will permit the development of region-specific drug delivery in the CNS.


title: Long-lasting humoral immunity in Covid-19 infected patients at a University Hospital Clinic in Ostergotland County Council during 2020-2021.

abstract: Longitudinal serum samples and nasopharyngeal/nasal swab samples were collected from forty-eight individuals (median age 66yrs) with Covid-19 PCR-positive test results at Linkoping University Hospital. Samples were collected from initial visit and for 6 months follow up. Presence of serum IgG and IgA against SARS-CoV-2 antigens (S1-spike, nucleocapsid and NSP3) were analyzed. Nasal swabs were tested for presence of IgA against the outer envelope S1 spike protein. Ninety-two percent of participants were seropositive against SARS-CoV-2 recombinant proteins at day 28 from study entry and all (100%) were seropositive from samples collected at 2 months or later. The most common antibody responses (both serum IgG, mainly IgG1 and IgA) were detected against the S1-spike protein and the nucleoprotein. In samples collected from nasal tissues considerably lower frequencies of IgA-positive reactivities were detected. Sixteen to 18 percent of study participants showed detectable IgA levels in nasal samples, except at day 60 when 36% of tested individuals showed presence of IgA against the S1-spike protein. The study suggests that the absolute majority of studied naturally infected Covid-19 patient in the Linkoping, Ostergotland health region develop over 6 months lasting detectable levels of serum IgG and IgA responses towards the SARS-CoV-2 S1-spike protein as well as against the nucleoprotein, but not against the non-structural protein 3.


title: Nucleocapsid vaccine elicits spike-independent SARS-CoV-2 protective immunity

abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Neutralizing antibodies target the receptor binding domain of the spike (S) protein, a focus of successful vaccine efforts. Concerns have arisen that S-specific vaccine immunity may fail to neutralize emerging variants. We show that vaccination with HAd5 expressing the nucleocapsid (N) protein can establish protective immunity, defined by reduced weight loss and viral load, in both Syrian hamsters and k18-hACE2 mice. Challenge of vaccinated mice was associated with rapid N-specific T cell recall responses in the respiratory mucosa. This study supports the rationale for including additional viral antigens, even if they are not a target of neutralizing antibodies, to broaden epitope coverage and immune effector mechanisms.


title: Inositol lipid synthesis is widespread in host-associated Bacteroidetes

abstract: Ubiquitous in eukaryotes, inositol lipids have finely tuned roles in cellular signaling and membrane homeostasis. In Bacteria, however, inositol lipid production is rare. Recently, the prominent human gut bacterium Bacteroides thetaiotaomicron (BT) was reported to produce inositol lipids, including inositol sphingolipids, but the pathways remain ambiguous and their prevalence unclear. Here, we investigated the gene cluster responsible for inositol lipid synthesis in BT using a novel strain with inducible control of sphingolipid synthesis. We characterized the biosynthetic pathway from myo-inositol-phosphate (MIP) synthesis to phosphoinositol-dihydroceramide, including structural and kinetic studies of the enzyme MIP synthase (MIPS). We determined the crystal structure of recombinant BT MIPS with bound NAD cofactor at 2.0 [A] resolution, and identified the first reported phosphatase for the conversion of bacterially-derived phosphatidylinositol phosphate (PIP) to phosphatidylinositol (PI). Transcriptomic analysis indicated inositol production is nonessential but its loss alters BT capsule expression. Bioinformatic and lipidomic comparisons of Bacteroidetes species revealed a novel second putative pathway for bacterial PI synthesis without a PIP intermediate. Our results indicate that inositol sphingolipid production, via one of the two pathways, is widespread in host-associated Bacteroidetes, and may be implicated in host interactions both indirectly via the capsule and directly through inositol lipid provisioning.

category: microbiology


title: Generation of a Sleeping Beauty transposon-based cellular system for rapid and sensitive identification of SARS-CoV-2 host dependency and restriction factors

abstract:
The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. The common methods to monitor and quantitate SARS-CoV-2 infectivity in cell culture are so far time-consuming and labor-intensive. Using the Sleeping Beauty transposase system, we generated a robust and versatile reporter cell system that allows SARS-CoV-2 infection experiments compatible for high-throughput and live cell imaging. The reporter cell is based on lung derived A549 cells, which show a profound interferon response and convenient cell culture characteristics. ACE2 and TMPRSS2 were introduced for constitutive expression in A549 cells. Subclones with varying levels of ACE2/TMPRSS2 were screened for optimal SARS-CoV2 susceptibility. Furthermore, extensive evaluation demonstrated that SARS-CoV-2 infected reporter cells were distinguishable from mock-infected cells and already showed approximately 12 h post infection a clear signal to noise ratio in terms of cell roughness, fluorescence and a profound visible cytopathic effect. Moreover, due to the high transfection efficiency and proliferation capacity, Sleeping Beauty transposase-based overexpression cell lines with a second inducible fluorescence reporter cassette (eGFP) can be generated in a very short time, enabling the investigation of host and restriction factors in a doxycycline-inducible manner. Thus, the novel reporter cell line allows rapid and sensitive detection of SARS-CoV-2 infection and the screening for host factors essential for viral replication.

Highlights- Sleeping Beauty transposon-based cellular system was used to generate a highly susceptible cell line for monitoring SARS-CoV-2 infection
- The versatile reporter cell line A549-AT is suitable for rapid and sensitive high-throughput assays
- Additional gene specific expression cassettes allow the identification of SARS-CoV-2 host dependency and restriction factors


title: Iota-carrageenan prevents the replication of SARS-CoV-2 on an in vitro respiratory epithelium model

abstract: There are, except for remdesivir, no approved antivirals for the treatment or prevention of SARS-CoV-2 infections. Iota-carrageenan formulated into a nasal spray has already been proven safe and effective in viral respiratory infections. We explored this antiviral activity in Calu-3, a human respiratory model cell line. A formula of iota-carrageenan and sodium chloride, as a nasal spray, already approved for human use, effectively inhibited SARS-CoV-2 infection in vitro, providing a more substantial reference for further clinical studies or developments.


title: Testing the Tea Bag Index as a potential indicator for assessing litter decomposition in aquatic ecosystems

abstract: The Tea Bag Index (TBI) approach is a standardized method for assessing litter decomposition in terrestrial ecosystems. This method allows determination of the stabilized portion of the hydrolysable fraction during the decomposition process, and derivation of a decomposition constant (k) using single measurements of the mass-loss ratios of green and rooibos teas. Although this method is being applied to aquatic systems, it has not been validated in these environments, where initial leaching tends to be higher than in terrestrial ecosystems. Here, we first validated a critical assumption of the TBI method that green tea decomposition plateaus during the standard incubation period of 90 days, and then tested the accuracy of a TBI-based asymptote model using a second model obtained from fitting actual decomposition data. Validation data were obtained by incubating tea bags in water samples taken from a stream, a pond, and the ocean in Kumamoto, Japan. We found that green tea decomposition did not plateau during the 90-day period, contradicting a key assumption of the TBI method. Moreover, the TBI-based asymptote models disagreed with actual decomposition data. Subtracting the leachable fraction from the initial tea mass improved the TBI-based model, but discrepancies with the actual decomposition data remained. Thus, we conclude that the TBI approach, which was developed for a terrestrial environment, is not appropriate for aquatic ecosystems. However, the use of tea bags as a standard material in assessments of aquatic litter decomposition remains beneficial.


title: The parallel origins of vertebrate venoms

abstract: Evolution can occur with surprising predictability when organisms face similar ecological challenges. How and why traits arise repeatedly remains a central question in evolutionary biology, but the complexity of most traits makes it challenging to answer. Reptiles and mammals independently evolved oral venoms consisting of proteinaceous cocktails that allow mapping between genotype and phenotype. Although biochemically similar toxins can occur as major venom components across many taxa, whether these toxins evolved via convergent or parallel processes remains unknown. Most notable among them are kallikrein-like serine proteases that form the core of most vertebrate oral venoms. We used a combination of comparative genomics and phylogenetics to investigate whether serine protease recruitment into the venom occurred independently or in parallel in mammalian and reptilian lineages. Using syntenic relationships between genes flanking known toxins, we traced the origin of kallikreins to a single locus containing one or more nearby paralogous kallikrein-like clusters. Independently, phylogenetic analysis of vertebrate serine proteases revealed that kallikrein-like toxins in mammals and reptiles are homologous. Given the shared regulatory and genetic machinery, these findings suggest a unified model underlying vertebrate venom evolution. Namely, the common tetrapod ancestor possessed salivary genes that were biochemically suitable for envenomation. We term such genes  toxipotent - in the case of salivary kallikreins they already had potent vasodilatory activity that was weaponized by venomous lineages. Furthermore, the ubiquitous distribution of kallikreins across vertebrates suggests that the evolution of envenomation may be more common than previously recognized, blurring the line between venomous and non-venomous animals.


title: Ancient gene duplications, rather than polyploidization, facilitate diversification of petal pigmentation patterns in Clarkia gracilis (Onagraceae)

abstract: It has been suggested that gene duplication and polyploidization create opportunities for the evolution of novel characters. However, the connections between the effects of polyploidization and morphological novelties have rarely been examined. In this study, we investigated whether petal pigmentation patterning in an allotetraploid Clarkia gracilis has evolved as a result of polyploidization. C. gracilis is thought to be derived through a recent polyploidization event with two diploid species, C. amoena huntiana and an extinct species that is closely related to C. lassenensis. We reconstructed phylogenetic relationships of the R2R3-MYBs (the regulators of petal pigmentation) from two subspecies of C. gracilis and the two purported progenitors, C. a. huntiana and C. lassenensis. The gene tree reveals that these R2R3-MYB genes have arisen through duplications that occurred before the divergence of the two progenitor species, i.e., before polyploidization. After polyploidization and subsequent gene loss, only one of the two orthologous copies inherited from the progenitors was retained in the polyploid, turning it to diploid inheritance. We examined evolutionary changes in these R2R3-MYBs and in their expression, which reveals that the changes affecting patterning (including expression domain contraction, loss-of-function mutation, cis-regulatory mutation) occurred after polyploidization within the C. gracilis lineages. Our results thus suggest that polyploidization itself is not necessary in producing novel petal color patterns. By contrast, duplications of R2R3-MYB genes in the common ancestor of the two progenitors have apparently facilitated diversification of petal pigmentation patterns.


title: Force-dependent interactions between talin and full-length vinculin

abstract: Talin and vinculin are part of a multi-component system involved in mechanosensing in cell-matrix adhesions. Both exist in auto-inhibited forms, and activation of vinculin requires binding to mechanically activated talin, yet how forces affect talins interaction with vinculin has not been investigated. Here by quantifying the force-dependent talin-vinculin interactions and kinetics using single-molecule analysis, we show that mechanical exposure of a single vinculin binding site (VBS) in talin is sufficient to relieve the autoinhibition of vinculin resulting in high-affinity binding. We provide evidence that the vinculin undergoes dynamic fluctuations between an auto-inhibited closed conformation and an open conformation that is stabilized upon binding to the VBS. Furthermore, we discover an additional level of regulation in which the mechanically exposed VBS binds vinculin significantly more tightly than the isolated VBS alone. Molecular dynamics simulations reveal the basis of this new regulatory mechanism, identifying a sensitive force-dependent change in the conformation of an exposed VBS that modulates binding. Together, these results provide a comprehensive understanding of how the interplay between force and autoinhibition provides exquisite complexity within this major mechanosensing axis.


title: Stress-responsive Long Non-coding RNA, hsrω, is a genetic modifier of JNK-dependent Intrinsic Tumor Suppression in Drosophila

abstract:
Cells incurring oncogenic hits are often eliminated by cell death via built-in anti-cancer defense mechanisms, broadly termed as intrinsic tumor suppression (ITS). Identification of genetic modifiers of ITS-induced cell death would provide better understanding of inherent tumor-resistance and/or susceptibility. Using a Drosophila model of loss of a tumor suppressor-mediated epithelial tumorigenesis, here we show that perturbations in levels of stress-responsive nuclear long non-coding RNA (lncRNA) hsr{omega} gene, promote epithelial tumorigenesis. Thus, while somatic clones with loss of a tumor suppressor, Lgl, are eliminated by JNK-induced cell death, lgl mutant somatic clones induced either in an hsr{omega} loss-of-function heterozygous genetic background, or upon cell autonomous up- or down-regulation of hsr{omega} in lgl somatic clones, override the JNK-mediated cell death and progress to full blown tumors. These tumors display deregulation of Hippo pathway as seen from a gain of downstream target of inhibition, Diap1, an inhibitor of cell death. We finally show that downregulation in sat III non-coding RNA, a functional analog of hsr{omega} in humans, increases sensitivity of cancer cells to cytotoxic stress-induced cell death. lncRNA hsr{omega}, therefore, constitutes a novel genetic modifier of ITS in Drosophila and of stress-induced cell death in human cancers.

SummaryA long non-coding RNA, hsr{omega}, is a novel regulator of JNK-mediated intrinsic tumor suppression in Drosophila.

HighlightsO_LIlgl clones induced in hsr{omega} heterozygous loss-of-function genetic background escape intrinsic tumor suppression (ITS).
C_LIO_LIPerturbation of hsr{omega} in lgl mutant clones, too, leads to their escape from ITS.
C_LIO_LIhsr{omega} homeostasis required for JNK-dependent ITS.
C_LIO_LIHuman sat III, a functional analog of hsr{omega}, confers stress-resistant to human cancer cells.
C_LI


title: THC modifies the impact of heroin delivered by vapor inhalation in rats

abstract:
Opioids are effective medications, but they have several key limitations including the development of tolerance, establishment of dependence, diversion for non-medical use and the development of addiction. Therefore, any drugs which act in an additive or synergistic fashion with opioids to address medical applications have the potential to reduce opioid-related harms. This study was conducted to determine if heroin and {Delta}9-tetrahydrocannabinol (THC) interact in an additive or independent manner to alter nociception, body temperature and spontaneous locomotor activity when inhaled or injected.

Groups of male and female rats implanted with radiotelemetry transmitters were exposed to vapor for assessment of effects on temperature and activity. Heroin (50 mg/mL in the propylene glycol; PG) inhalation increased temperature and activity whereas THC (50 mg/mL) inhalation decreased temperature and activity. Effects of combined inhalation were in opposition, and additional experiments found the same outcome for the injection of heroin (0.5 mg/kg, s.c.) and THC (10 mg/kg, i.p.) alone and in combination. In contrast, the co-administration of Heroin and THC by either inhalation or injection produced additive effects on thermal nociception assessed with a warm water tail-withdrawal assay in male and female Sprague-Dawley and Wistar rats.

The conclusion of this study is that additive effects of THC with heroin on a medical endpoint such as analgesia may not generalize to other behavioral or physiological effects, which may be a positive outcome for unwanted side effects.

category: pharmacology and toxicology


title: Tryptophan derivatives regulate the seed germination and radicle growth of a root parasitic plant, Orobanche minor

abstract: Root parasitic plant germination is induced by the host-derived chemical, strigolactone (SL). We found that a major microbial culture broth component, tryptone, inhibits the SL-inducible germination of a root parasitic plant, Orobanche minor. O_SCPLOWLC_SCPLOW-tryptophan (1a, O_SCPLOWLC_SCPLOW-Trp) was isolated as the active compound from tryptone. We further found that O_SCPLOWLC_SCPLOW-Trp related compounds (1b-11), such as a major plant hormone auxin (8, indole-3-acetic acid; IAA), also inhibit the germination and post-radicle growth of O. minor. We designed a hybrid chemical (13), in which IAA is attached to a part of SL, and found that this synthetic analog induced the germination of O. minor, and also inhibited post-radicle growth. Moreover, we found that N-acetyl Trp (9) showed germination stimulating activity, and introduction of a substitution at C-5 position incresed its activity (12a-12f). Our data, in particular, the discovery of a structurally hybrid compound that has two activities that induce spontaneous germination and inhibit subsequent radical growth, would provide new types of germination regulators for root parasitic plants.

category: plant biology


title: Integrated spatial genomics in tissues reveals invariant and cell type dependent nuclear architecture

abstract: Nuclear architecture in tissues can arise from cell-type specific organization of nuclear bodies, chromatin states and chromosome structures. However, the lack of genome-wide measurements to interrelate such modalities within single cells limits our overall understanding of nuclear architecture. Here, we demonstrate integrated spatial genomics in the mouse brain cortex, imaging thousands of genomic loci along with RNAs and subnuclear markers simultaneously in individual cells. We revealed chromatin fixed points, combined with cell-type specific organization of nuclear bodies, arrange the interchromosomal organization and radial positioning of chromosomes in diverse cell types. At the sub-megabase level, we uncovered a collection of single-cell chromosome domain structures, including those for the active and inactive X chromosomes. These results advance our understanding of single-cell nuclear architecture in complex tissues.


title: Understanding double descent through the lens of principal component regression

abstract: A number of recent papers have studied the double-descent phenomenon: as the number of parameters in a supervised learning model increasingly exceeds that of data points ("second-descent"), the empirical risk curve has been observed to not overfit, instead decreasing monotonically, sometimes to a level even better than the best "first-descent" model (using a subset of features not exceeding the number of data points). Understanding exactly when this happens and why it happens is an important theoretical problem. Focusing on the over-parameterized linear regression setting, a commonly chosen case study in the double-descent literature, we present two theoretical results: 1) final second-descent (regression using all of the predictor variables) and principal component (PC) regression without dimensionality reduction are equivalent; 2) the PCR risk curve can be expected to lower bound not only all linearly transformed first-descent models, but also all linearly transformed second-descent models (including the elimination of features as a special case); 3) if the smallest singular value of the design matrix is "large enough" (we will define mathematically), final second-descent can be expected to outperform any first-descent or second-descent model. These insights have important ramifications for a type of semi-supervised learning problem, a scenario which can explain why a face representation trained on unlabeled faces from one race would be better for later supervised-learning tasks on the same race of faces than for faces from another race - this can both provide a scientific explanation for the other-race effect seen in humans and give hints for how to mitigate similar issues in the domain of ethical AI.

category: neuroscience


title: Engagement of the contralateral limb can enhance the facilitation of motor output by loud acoustic stimuli

abstract: When intense sound is presented during light muscle contraction, inhibition of the corticospinal tract is observed. During action preparation, this effect is reversed, with sound resulting in excitation of the corticospinal tract. We investigated how the combined maintenance of a muscle contraction during preparation for a ballistic action impacts the magnitude of the facilitation of motor output by a loud acoustic stimulus (LAS) - a phenomenon known as the StartReact effect. Participants executed ballistic wrist flexion movements and a LAS was presented simultaneously with the imperative signal in a subset of trials. We examined whether the force level or muscle used to maintain a contraction during preparation for the ballistic response impacted reaction time and/or the force of movements triggered by the LAS. These contractions were sustained either ipsilaterally or contralaterally to the ballistic response. The magnitude of facilitation by the LAS was greatest when low force flexion contractions were maintained in the limb contralateral to the ballistic response during preparation. There was little change in facilitation when contractions recruited the contralateral extensor muscle, or when they were sustained in the same limb that executed the ballistic response. We conclude that a larger network of neurons which may be engaged by a contralateral sustained contraction prior to initiation may be recruited by the LAS, further contributing to the motor output of the response. These findings may be particularly applicable in stroke rehabilitation where engagement of the contralesional side may increase the benefits of a LAS to the functional recovery of movement.


title: Stress granules are shock absorbers that prevent excessive innate immune responses to dsRNA

abstract: Proper defense against microbial infection depends on the controlled activation of the immune system. This is particularly important for the innate immune receptors that recognize viral double-stranded RNA (dsRNA) and initiate antiviral immune responses with the potential of triggering systemic inflammation and immunopathology. How the functions of the dsRNA receptors and their downstream effector molecules are coordinately regulated to avoid excessive immune response is poorly understood. We here demonstrate that stress granules (SGs), biomolecular condensates that form in response to various stresses including viral dsRNA1, 2, play key roles in regulating dsRNA-triggered immune response. Upon dsRNA stimulation, SGs recruit many innate immune molecules, including RIG-I-like receptors (RLRs), protein kinase R (PKR) and oligoadenylate synthases (OASes), target these molecules and dsRNA for autophagy and limit their functions through sequestration. In the absence of SGs, dsRNA stimulation results in hyperactivation of inflammatory signaling pathways, global translational arrest and bulk RNA degradation, altogether compromising the cellular capacity to restore homeostasis and triggering cell death. In contrast to most dsRNA-induced immune signaling pathways that are hyperactivated in the absence of SGs, a sub-branch of the RLR pathway (IRF3-dependent type I interferon signaling) shows time-dependent changes, where the initial spike in signaling is followed by a significant drop due to increased caspase-dependent negative feedback regulation. This highlights the role of SGs in regulating the delicate balance between the type I interferon pathway and cell death. Altogether, our data suggest that cells utilize SGs as shock absorbers to moderate antiviral innate immune response, thereby allowing cells to guard against its own immune system as well as viruses.


title: A Pan-Cancer Census of Dominant Tumor Immune Archetypes

abstract:
Cancers display significant heterogeneity with respect to tissue of origin, driver mutations and other features of the surrounding tissue. It is likely that persistent tumors differentially engage inherent patterns-here  Archetypes-of the immune system, to both benefit from a tumor immune microenvironment (TIME) and to disengage tumor-targeting. To discover dominant immune system archetypes, the Immunoprofiler Initiative (IPI) processed 364 individual tumors across 12 cancer types using standardized protocols. Computational clustering of flow cytometry and transcriptomic data obtained from cell sub compartments uncovered archetypes that exist across indications. These Immune composition-based archetypes differentiate tumors based upon unique immune and tumor gene-expression patterns. Archetypes discovered this way also tie closely to well-established classifications of tumor biology. The IPI resource provides a template for understanding cancer immunity as a collection of dominant patterns of immune infiltration and provides a rational path forward to learn how to modulate these patterns to improve therapy.

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title: A Scenic River in Rural Ohio Shows Elevated Antibiotic Resistance Genes, Including Acinetobacter Tetracycline and Macrolide Resistance, Downstream of Wastewater Treatment Plant Effluent

abstract:
The entry of antibiotic resistance genes (ARGs) into aquatic systems has been documented for large municipal wastewater treatment plants, but there is less study of the impact of smaller plants that are situated on small rural rivers. We sampled water metagenomes for ARG and taxa composition from the Kokosing River, a small rural river in Knox County, Ohio, which has been designated an Ohio State Scenic River for retention of natural character. Samples were obtained 1.0 km upstream, 120 m downstream, and 6.4 km downstream from the effluent release of the Mount Vernon wastewater treatment plant (WWTP). ARGS were identified in metagenomes using ShortBRED markers from the CARD database screened against UniPROT. Through all seasons, the metagenome just downstream of the WWTP effluent showed a substantial elevation of at least 15 different ARGs, including 6 ARGs commonly associated with Acinetobacter baumannii such as msrE, mphE (macrolide resistance) and tet(39) (tetracycline resistance). The ARGs most prevalent near the effluent pipe persisted 6.4 km downriver. Using MetaPhlAn2 clade-specific marker genes, the taxa distribution near the effluent showed elevation of reads annotated as Acinetobacter species as well as gut-associated taxa, Bacteroides and Firmicutes. The ARG levels and taxa prevalence showed little dependence on seasonal chlorination of the effluent. Nitrogen and phosphorus were elevated near the effluent pipe but had no consistent correlation with ARG levels. We show that in a rural river microbiome, year-round wastewater effluent substantially elevates ARGs including those associated with multidrug-resistant A. baumanii.

IMPORTANCEAntibiotic resistance is a growing problem worldwide, with frequent transmission between pathogens and environmental organisms. Rural rivers can support high levels of recreational use by people unaware of inputs from treated wastewater, while WWTPs can generate a small but significant portion of flow volume into a river surrounded by forest and agriculture. There is little information on the rural impacts of WWTP effluent on the delivery and transport of antibiotic resistance genes. In our study, the river water proximal to wastewater effluent shows evidence for the influx of multidrug-resistant Acinetobacter baumanii, an opportunistic pathogen of concern for hospitals but also widespread in natural environments. Our work highlights the importance of wastewater effluent in management of environmental antibiotic resistance, even in high quality, rural river systems.


title: An Ohio State Scenic River Shows Elevated Antibiotic Resistance Genes, Including Acinetobacter Tetracycline and Macrolide Resistance, Downstream of Wastewater Treatment Plant Effluent


title: Activation of ACC synthase 2/6 increases stomatal density and cluster on the Arabidopsis leaf epidermis during drought

abstract:
It is known that the transcription factor SPEECHLESS (SPCH) drives entry of epidermal cells into stomatal lineage, and that the activation of subtilisin-like protease SDD1 reduces stomatal density and cluster on the epidermis. However, there is still a big gap in our understanding of the relationship between stomatal development and the establishment of stomatal density and pattern, especially during drought. Interestingly, 1-aminocyclopropane-1-carboxylic acid (ACC) not only promotes stomatal development, but also is involved in the establishment of stomatal density and pattern. ACC generation comes from the activity of ACC synthase (ACS), while ACS activity could be mediated by drought. This work showed that the Arabidopsis SPCH activated ACS2/6 expression and ACC-dependent stomatal generation with an increase of stomatal density and cluster under drought conditions; and the possible mechanisms were that ACC-induced Ca2+ shortage in stomatal lineage reduced the inhibition of the transcription factor GT-2 Like 1 (GTL1) on SDD1 expression. These suggest that ACS2/6-dependent ACC accumulation integrated stomatal development with the establishment of stomatal density and pattern by mediating Ca2+ levels in stomatal lineage cells on the leaf epidermis, and this integration is directly related to the growth or survival of plants under escalated drought stress.

HighlightACC synthase ACS2/6 activation integrated stomatal individual development with space setting between stomata by mediating Ca2+ levels in stomatal lineage on the leaf epidermis in response to drought.


title: Gambogic acid and gambogenic acid induce a thiol-dependent heat shock response and disrupt the interaction between HSP90 and HSF1 or HSF2

abstract: Cancer cells rely on heat shock proteins (HSPs) for growth and survival. Especially HSP90 has multiple client proteins and plays a critical role in malignant transformation, and therefore different types of HSP90 inhibitors are being developed. The bioactive natural compound gambogic acid (GB) is a prenylated xanthone with antitumor activity and it has been proposed to function as an HSP90 inhibitor. However, there are contradicting reports whether GB induces a heat shock response (HSR), which is cytoprotective for cancer cells and therefore a potentially problematic feature for an anticancer drug. In this study, we show that GB and a structurally related compound, called gambogenic acid (GBA), induce a robust HSR, in a thiol-dependent manner. Using heat shock factor 1 (HSF1) or HSF2 knockout cells, we show that the GB or GBA-induced HSR is HSF1-dependent. Intriguingly, using closed form ATP-bound HSP90-mutants that can be co-precipitated with HSF1, a known facilitator of cancer, we show that also endogenous HSF2 binds to the HSP90-HSF1 complex. GB and GBA treatment disrupt the interaction between HSP90 and HSF1 and HSF2. Our study implies that these compounds should be used cautiously if developed for cancer therapies, since GB and its derivative GBA are strong inducers of the HSR, in multiple cell types, by involving the dissociation of a HSP90-HSF1-HSF2 complex.


title: Cdc42 activity in Sertoli cells is essential for maintenance of spermatogenesis

abstract: Sertoli cells are highly polarized testicular supporting cells that simultaneously nurture progressively maturing germ cells. Proper localization of polarity protein complexes within Sertoli cells, including those responsible for blood-testis barrier formation, are vital for successful spermatogenesis. However, the mechanisms and developmental timing that underlie the establishment of polarity are poorly understood. To investigate this aspect of testicular function, we conditionally deleted Cdc42, encoding a Rho GTPase involved in regulating cell polarity, specifically in Sertoli cells. Cdc42 deletion disrupted adult Sertoli cell maturation and localization of polarity proteins, but did not affect fetal and early postnatal testicular development, nor the onset of the first wave of spermatogenesis. By early adulthood, however, conditional knockout males exhibited a loss of spermatogenic cells, resulting in a complete lack of sperm. These findings demonstrate that Cdc42 plays an essential role in establishing adult Sertoli cell polarity and, thus, maintaining steady-state spermatogenesis and healthy sperm production.

category: developmental biology


title: Stingless bee floral visitation in the global tropics and subtropics

abstract: Bees play a key role in maintaining healthy terrestrial ecosystems by pollinating plants. Stingless bees (Apidae: Meliponini) are a diverse clade of social bees (>500 species) with a pantropical distribution spanning South and Central America, Africa, India and Austral-Asia. They are garnering increasing attention as commercially-beneficial pollinators of some crops, yet their contribution to the pollination of native plants in the tropics and subtropics remains poorly understood. Here we conduct for the first time a global review of the plants visited by stingless bees. We compile a database of reported associations (flower visits) between stingless bees and plants, from studies that have made either direct observations of foraging bees or analysed the pollen stored in nests. Worldwide, we find stingless bees have been reported to visit the flowers of plants from at least 215 different families and 1434 genera, with frequently reported interactions for many of the tropics most species-diverse plant families including Fabaceae, Asteraceae, Rubiaceae, Poaceae, Euphorbiaceae, Myrtaceae, Malvaceae, Arecaceae, Solanaceae, and Anacardiaceae. The stingless bee fauna of each of three major biogeographic regions (Neotropical, Afrotropical and Indo-Malayan-Australasian) were frequent visitors of many of the same plant families, however we detected differences in the proportional use of plant families by the stingless bees of the Indo-Malayan-Australasian and Neotropical regions, likely reflecting differences in the available flora of those regions. Stingless bees in all regions visit a range of exotic species in their preferred plant families (crops, ornamental plants and weeds), in addition to native plants. Although most reports of floral visitation on wild plants do not confirm effective pollen transfer, it is likely that stingless bees make at least some contribution to pollination for the majority of plants they visit. In all, our database supports the view that stingless bees play an important role in the ecosystems of the global tropics and subtropics as pollinators of an exceptionally large and diverse number of plants. This database also highlights important gaps in our knowledge of stingless bee resource use and should benefit future efforts to understand stingless bee-plant interactions.

category: ecology


title: Robust and annotation-free analysis of isoform variation using short-read scRNA-seq data

abstract: Although alternative splicing is a fundamental and pervasive aspect of gene expression in higher eukaryotes, it is often omitted from single-cell studies due to quantification challenges inherent to commonly used short-read sequencing technologies. Here, we undertake the analysis of alternative splicing across numerous diverse murine cell types from two large-scale single-cell datasets--the Tabula Muris and BRAIN Initiative Cell Census Network--while accounting for understudied technical artifacts and unannotated isoforms. We find strong and general cell-type-specific alternative splicing, complementary to total gene expression but of similar discriminatory value, and identify a large volume of novel isoforms. We specifically highlight splicing variation across different cell types in primary motor cortex neurons, bone marrow B cells, and various epithelial cells; and show that the implicated transcripts include many genes which do not display total expression differences. To elucidate the regulation of alternative splicing, we build a custom predictive model based on splicing factor activity, recovering several known interactions while generating new hypotheses, including potential regulatory roles for novel alternative splicing events in critical genes including Khdrbs3 and Rbfox1. We make our results available using public interactive browsers to spur further exploration by the community.


title: Robust and annotation-free analysis of alternative splicing using short-read scRNA-seq data


title: Robust and annotation-free analysis of alternative splicing across diverse cell types in mice


title: Stochastic Neural Networks for Automatic Cell Tracking in Microscopy Image Sequences of Bacterial Colonies

abstract: We describe an automated analysis method to quantify the detailed growth dynamics of a population of bacilliform bacteria. We propose an innovative approach to frame-sequence tracking of deformable-cell motion by the automated minimization of a new, specific cost functional. This minimization is implemented by dedicated Boltzmann machines (stochastic recurrent neural networks). Automated detection of cell divisions is handled similarly by successive minimizations of two cost functions, alternating the identification of children pairs and parent identification. We validate this automatic cell tracking algorithm using recordings of simulated cell colonies that closely mimic the growth dynamics of E. coli in microfluidic traps. On a batch of 1100 image frames, cell registration accuracies per frame ranged from 94.5% to 100%, with a high average. Our initial tests using experimental image sequences of E. coli colonies also yield convincing results, with a registration accuracy ranging from 90% to 100%.

category: bioinformatics