title: Nutrient profiling reveals extracellular uridine as a fuel for pancreatic cancer through uridine phosphorylase 1

abstract: Pancreatic ductal adenocarcinoma (PDA) is a lethal disease characterized by high invasiveness, therapeutic resistance, and metabolic aberrations. Although altered metabolism drives PDA growth and survival, the complete spectrum of metabolites used as nutrients by PDA remains largely unknown. Here, we aimed to determine novel nutrients utilized by PDA. We assessed how >175 metabolites impacted metabolic activity in 19 PDA cell lines under nutrient-restricted conditions. This analysis identified uridine as a novel metabolite driver of PDA survival in glucose-deprived conditions. Uridine utilization strongly correlated with expression of the enzyme uridine phosphorylase 1 (UPP1). Metabolomics profiling, notably 13C-stable isotope tracing, revealed that uridine-derived ribose is the relevant component supporting redox balance, survival, and proliferation in glucose-deprived PDA cells. We demonstrate that UPP1 catabolizes uridine, shunting its ribose component into central carbon metabolism to support glycolysis, the tricarboxylic acid (TCA) cycle and nucleotide biosynthesis. Compared to non-tumoral tissues, we show that PDA tumors express high UPP1, which correlated with poor overall survival in multiple patient cohorts. Further, uridine is enriched in the pancreatic tumor microenvironment, and we demonstrate that this may be provided in part by tumor associated macrophages. Finally, we found that inhibition of UPP1 restricted the ability of PDA cells to use uridine, and that UPP1 knockout impairs tumor growth in vivo. Our data identifies uridine catabolism as a critical aspect of compensatory metabolism in nutrient-deprived PDA cells, suggesting a novel metabolic axis for PDA therapy.


title: Conservation and diversity in transcriptional responses among host plants forming distinct arbuscular mycorrhizal morphotypes

abstract:
O_LIThe morphotype of arbuscular mycorrhizal (AM) roots is distinct mostly depending on AM host species: Arum, Paris, and Intermediate types. We previously reported that gibberellin (GA) promotes the establishment of Paris-type AM symbiosis in Eustoma grandiflorum despite its negative effects on Arum-type AM symbiosis in model plants. However, the molecular mechanisms underlying the differential effects of GA on different morphotypes, including Intermediate-type AM symbiosis, remain elusive.
C_LIO_LIComparative transcriptomics revealed that several symbiosis-related genes were transcriptionally promoted upon AM fungal colonization in Lotus japonicus (Arum-type), Daucus carota (Intermediate-type), and E. grandiflorum (Paris-type). Interestingly, upon GA treatment, the fungal colonization levels and expression of symbiosis-related genes were suppressed in L. japonicus and D. carota but were promoted in E. grandiflorum.
C_LIO_LIExogenous GA transcriptionally inhibited the biosynthetic process of a host-derived signal molecule involved in AM symbiosis, strigolactone, in L. japonicus and E. grandiflorum. Additionally, disaccharides mainly metabolized in AM roots would be different between L. japonicus and D. carota/E. grandiflorum.
C_LIO_LIThis study uncovered the conserved transcriptional responses during mycorrhization and diverse responses to GA in AM roots with distinct morphotypes among phylogenetically distant host plants.
C_LI

category: plant biology


title: Rat anterior cingulate cortex continuously signals decision variables in a patchforaging task

abstract: In patch foraging tasks, animals must decide whether to remain with a depleting resource or to leave it in search of a potentially better source of reward. In such tasks, animals consistently follow the general predictions of optimal foraging theory (the Marginal Value Theorem; MVT): to leave a patch when the reward rate in the current patch depletes to the average reward rate across patches. Prior studies implicate an important role for the anterior cingulate cortex (ACC) in foraging decisions based on MVT: within single trials, ACC activity increases immediately preceding foraging decisions, and across trials, these dynamics are modulated as the value of staying in the patch depletes to the average reward rate. Here, we test whether these activity patterns reflect dynamic encoding of decision-variables and whether these signals are directly involved in decision-making. We developed a leaky accumulator model based on the MVT that generates estimates of decision variables within and across trials, and tested model predictions against ACC activity recorded from rats performing a patch foraging task. Model predicted changes in MVT decision variables closely matched rat ACC activity. Next, we pharmacologically inactivated ACC to test the contribution of these signals to decision-making. ACC inactivation had a profound effect on rats foraging decisions and response times yet rats still followed the MVT decision rule. These findings suggest that the ACC encodes foraging-related variables for reasons unrelated to patch-leaving decisions.

category: neuroscience


abstract:
Reading creates accessibility to the outer world information and is an integral skill for academic achievement. It involves visual perception, reasoning at symbolic level (text reading) and phonological processing. But does perception, visuospatial processing at pre-symbolic level affect reading skill? To understand and untangle the mechanism fifty children with reading problem (developmental dyslexia, DDC) and twentyfive age-matched typical readers (healthy controls, HC) were studied. In DDC, the variable-performance of non-symbolic visual search (pictorial-level) was associated with bilateral brain activity (functional MRI) of ventral stream (inferior occipital and fusiform) and greater involvement of frontal-prefrontal regions, suggesting the modified dorsal route-gating for figure-ground filtering. Performance variability in picture-concept reasoning (71.9% and significant group mean difference) and visuospatial-organization (51.3%) of DDC children indicate attentional or executive differences at nonsymbolic level. In symbolic discrimination performance, no significant difference observed for single alphabet or number (digit) reading but significant differences at word level for visual and phonological performance (considering orthographic differences), attributes to distractor-inhibition problems. Nonsymbolic to symbolic coding/decoding speed and writing error (copying) differences are suggestive of execution variability rather than motor coordination in DDC. In HC, the top-down dorsal route gating and hypoactivity induced in right frontal-anterior cingulate cortices, play a major role in reading by allocating spatial attention and inhibiting distractors. It has been elaborated in the "neurobiological model".

How perception and attention participate in reading skill?O_ST_ABSResearch HighlightsC_ST_ABSO_LIVisuospatial attention, executive control (nonsymbolic level) and visual discrimination (symbolic level) performance contribute to reading.
C_LIO_LIIn children with reading problems (dyslexia) pictorial visual processing task (fMRI) recruited higher activation in right hemispheric frontal, inferior parietal and anterior cingulate regions.
C_LIO_LIBilateral activation of occipital-striate, extra-striate, fusiform for nonsymbolic is associated with significantly more errors at bigger-symbol-string, in dyslexia.
C_LIO_LIAuditory discrimination differences in shallow-deep orthography.
C_LIO_LICorrelation of non-symbolic executive and symbolic performances.
C_LIO_LIRole of the prelexical visual perception and dorsal-route gating in reading skill explained as neurobiological model.
C_LI


title: Estimated connectivity networks outperform observed connectivity networks when classifying people with multiple sclerosis into disability groups

abstract:
BackgroundMultiple Sclerosis (MS), a neurodegenerative and neuroinflammatory disease, causing lesions that disrupt the brains anatomical and physiological connectivity networks, resulting in cognitive, visual and/or motor disabilities. Advanced imaging techniques like diffusion and functional MRI allow measurement of the brains structural connectivity (SC) and functional connectivity (FC) networks, and can enable a better understanding of how their disruptions cause disability in people with MS (pwMS). However, advanced MRI techniques are used mainly for research purposes as they are expensive, time-consuming and require high-level expertise to acquire and process. As an alternative, the Network Modification (NeMo) Tool can be used to estimate SC and FC using lesion masks derived from pwMS and a reference set of controls connectivity networks.

ObjectiveHere, we test the hypothesis that estimated SC and FC (eSC and eFC) from the NeMo Tool, based only on an individuals lesion masks, can be used to classify pwMS into disability categories just as well as SC and FC extracted from advanced MRI directly in pwMS. We also aim to find the connections most important for differentiating between no disability vs evidence of disability groups.

Materials and MethodsOne hundred pwMS (age:45.5 {+/-} 11.4 years, 66% female, disease duration: 12.97 8.07 years) were included in this study. Expanded Disability Status Scale (EDSS) was used to assess disability, 67 pwMS had no disability (EDSS < 2). Observed SC and FC were extracted from diffusion and functional MRI directly in pwMS, respectively. The NeMo Tool was used to estimate the remaining structural connectome (eSC), by removing streamlines in a reference set of tractograms that intersected the lesion mask. The NeMo Tools eSC was used then as input to a deep neural network to estimate the corresponding FC (eFC). Logistic regression with ridge regularization was used to classify pwMS into disability categories (no disability vs evidence of disability), based on demographics/clinical information (sex, age, race, disease duration, clinical phenotype, and spinal lesion burden) and either pairwise entries or regional summaries from one of the following matrices: SC, FC, eSC, and eFC. The area under the ROC curve (AUC) was used to assess the classification performance. Both univariate statistics and parameter coefficients from the classification models were used to identify features important to differentiating between the groups.

ResultsThe regional eSC and eFC models outperformed their observed FC and SC counterparts (p-value<0.05), while the pairwise eSC and SC performed similarly (p=0.10). Regional eSC and eFC models had higher AUC (0.66-0.68) than the pairwise models (0.60-0.65), with regional eFC having highest classification accuracy across all models. Ridge regression coefficients for the regional eFC and regional observed FC models were significantly correlated (Pearsons r = 0.52, p-value < 10e-7). Decreased estimated SC node strength in default mode and ventral attention networks and increased eFC node strength in visual networks was associated with evidence of disability.

DiscussionHere, for the first time, we use clinically-acquired lesion masks to estimate both structural and functional connectomes in patient populations to better understand brain lesion-dysfunction mapping in pwMS. Models based on the NeMo Tools estimates of SC and FC better classified pwMS by disability level than SC and FC observed directly in the individual using advanced MRI. This work provides a viable alternative to performing high-cost, advanced MRI in patient populations, bringing the connectome one step closer to the clinic.

HighlightsO_LIWe compared the accuracy of models based on observed functional connectivity (FC) and structural connectivity (SC) networks extracted from advanced MRI and estimated FC and SC networks derived using only lesion masks from conventional MRI in classifying people with multiple sclerosis (pwMS) into disability groups.
C_LIO_LIEstimated SC and FC generally outperformed observed SC and FC in classifying pwMS into no disability vs evidence of disability groups, with regional estimated SC and FC having the best performance.
C_LIO_LIIncreased estimated FC node strength of regions in the visual network was associated with disability.
C_LIO_LIDecreased estimated SC node strength of regions in the default mode and ventral attention networks was associated with disability.
C_LIO_LIDespite their varied sources of origin, feature weights for the regional estimated FC and the regional observed FC classification models was significantly correlated (Pearsons r = 0.52, p-value < 10e-7).
C_LI


title: A personalized anti-cancer vaccine for melanoma based on an approved vaccine against measles, mumps, and rubella

abstract: Common vaccines for infectious diseases have been repurposed as cancer immunotherapies. Intratumoural administration of these repurposed vaccines can induce immune cell infiltration into the treated tumour. Here, we have used an approved trivalent live attenuated measles, mumps, and rubella (MMR) vaccine in our previously developed PeptiENV anti-cancer vaccine platform. Intratumoural administration of this novel MMR-containing PeptiENV anti-cancer vaccine significantly increased both intratumoural as well as systemic tumour-specific T cell responses. In addition, PeptiENV therapy, in combination with immune checkpoint inhibitor therapy, significantly improved tumour growth control and survival as well as increased the number of mice responsive to immune checkpoint inhibitor therapy.

category: immunology


title: Cool temperatures limit Zika virus genome replication

abstract:
Zika virus is a mosquito-borne flavivirus known to cause severe birth defects and neuroimmunological disorders. We have previously demonstrated that mosquito transmission of Zika virus decreases with temperature. While transmission was optimized at 29{degrees}C, it was limited at cool temperatures (< 22{degrees}C) due to poor virus establishment in the mosquitoes. Temperature is one of the strongest drivers of vector-borne disease transmission due to its profound effect on ectothermic mosquito vectors, viruses, and their interaction. Although there is substantial evidence of temperature effects on arbovirus replication and dissemination inside mosquitoes, little is known about whether temperature affects virus replication directly or indirectly through mosquito physiology. In order to determine the mechanisms behind temperature-induced changes in Zika virus transmission potential, we investigated different steps of the virus replication cycle in mosquito cells (C6/36) at optimal (28{degrees}C) and cool (20{degrees}C) temperatures. We found that cool temperature did not alter Zika virus entry or translation but reduced the amount of double-stranded RNA replication intermediates. If replication complexes were first formed at 28{degrees}C and the cells were subsequently shifted to 20{degrees}C, the late steps in the virus replication cycle were efficiently completed. These data suggest that cool temperature decreases the efficiency of Zika virus genome replication in mosquito cells. This phenotype was observed in the Asian-lineage of Zika virus, while the African-lineage Zika virus was less restrictive at 20{degrees}C.

ImportanceWith half of the human population at risk, arboviral diseases represent a substantial global health burden. Zika virus, previously known to cause sporadic infections in humans, emerged in the Americas in 2015 and quickly spread worldwide. There was an urgent need to better understand the disease pathogenesis, develop therapeutics and vaccines, as well as to understand, predict, and control virus transmission. In order to efficiently predict the seasonality and geography for Zika virus transmission, we need a deeper understanding of the host-pathogen interactions and how they can be altered by environmental factors such as temperature. Identifying the step in the virus replication cycle that is inhibited in cool conditions can have implications in modeling the temperature suitability for arbovirus transmission as global environmental patterns change. Understanding the link between pathogen replication and environmental conditions can potentially be exploited to develop new vector control strategies in the future.

category: microbiology


title: The NF-κB pathway regulates heterochromatin at intronic young LINE-1 elements and hematopoietic stem cell gene expression during irradiation stress

abstract: Ionizing radiations (IR) alter hematopoietic stem cell (HSC) function on the long-term, but the mechanisms underlying these effects are still poorly understood. We recently showed that IR induces the derepression of L1Md, the mouse young subfamilies of LINE-1/L1 retroelements. L1 contribute to gene regulatory networks. However, how L1Md are derepressed and impact HSC gene expression are not known. Here we show that IR triggers genome-wide H3K9me3 decrease that occurs mainly at L1Md. Loss of H3K9me3 at intronic L1Md harboring NF-{kappa}B binding sites motifs but not at promoters is associated with the repression of HSC specific genes. This is correlated with reduced NFKB1 repressor expression. TNF--treatment before IR rescued all these effects and prevented IR-induced HSC loss of function in vivo. This TNF-/NF-{kappa}B/H3K9me3/L1Md axis might be important to maintain of HSCs while allowing expression of immune genes during myeloid regeneration or damage-induced bone marrow ablation.

category: cell biology


title: Adaptive reprogramming during early seed germination requires temporarily enhanced fermentation a critical role for alternative oxidase (AOX) regulation that concerns also microbiota effectiveness

abstract:
Plants respond to environmental cues via adaptive cell reprogramming that can affect whole plant and ecosystem functionality. Microbiota constitutes part of plants inner and outer environment. This Umwelt underlies steady dynamics, due to complex local and global biotic and abiotic changes. Hence, adaptive plant holobiont responses are crucial for continuous metabolic adjustment at systems levels. Plants require oxygen-dependent respiration for energy-dependent adaptive morphology, such as, germination, root and shoot growth, formation of adventitious, clonal and reproductive organs, fruits and seeds. Fermentative paths can help in acclimation and, to our view the role of alternative oxidase (AOX) in coordinating complex metabolic and physiologic adjustments is underestimated.

Cellular level of sucrose is an important sensor of environmental stress. We explored the role of exogenous sucrose and its interplay with AOX during early seed germination. We found that sucrose-dependent initiation of fermentation during the first 12 hours after imbibition (HAI) was beneficial to germination. However, parallel enhanced AOX expression was essential to control negative effects by prolonged sucrose treatment. Early down-regulated AOX activity until 12 HAI improved germination efficiency in the absence of sucrose, but suppressed early germination in its presence. Our results also suggest that seeds-inoculated arbuscular mycorrhizal fungi can buffer sucrose stress during germination to restore normal respiration more efficiently.

Following this approach, we propose a simple method to identify organic seeds and low-cost on-farm perspectives for early selection on disease tolerance, predicting plant holobiont behavior and improving germination. Furthermore, our research strengthens the view that AOX can serve as powerful functional marker source for seed hologenomes.


title: The impact of housing conditions on porcine adult stem cell populations differ between adipose tissue and skeletal muscle

abstract:
BackgroundIn pigs, the ratio between lean mass and fat mass in the carcass determines production efficiency and is strongly influenced by the number and size of cells in tissues. During growth, the increase in the number of cells results from the recruitment of different populations of multipotent mesenchymal stromal/stem cells (MSCs) residing in the tissues. We hypothesized that the impact of hygiene of housing conditions during growth on the proportions of MSCs in adipose tissue and skeletal muscle may differ between pigs with different residual feed intake (RFI), a measure of feed efficiency.

MethodsAt the age of 12 weeks, Large White pigs from two lines divergently selected for low and high RFI were housed in two contrasting hygiene conditions (good vs poor). After six weeks, pigs were slaughtered (n = 30; 5-9/group). Samples of subcutaneous adipose tissue and longissimus skeletal muscle were collected, and cells from the stromal vascular fraction (SVF), which includes mesenchymal stromal/stem cell populations, were isolated from each tissue. Adipose and muscle cell populations from the SVF were phenotyped by flow cytometry using antibodies that targeted different cell surface markers (CD45 to separate hematopoietic cells from MSCs; CD34, CD38, CD56 and CD140a to identify MSC populations with adipogenic and/or myogenic potential).

ResultsAdipose tissue and muscle shared some common MSC populations although MSC diversity was higher in muscle than in adipose tissue. In muscle, the CD45-CD56+CD34-CD140a+ and CD45-CD56+CD34+CD140a+ cell populations were abundant. Of these two cell populations, only the proportions of CD45-CD56+CD34+CD140a+ cells increased (P < 0.05) in pigs housed in poor hygiene as compared with pigs in good hygiene conditions. For the CD45-CD56-CD34- cell population, present in low proportion, there was an interaction between hygiene condition and genetic line (P < 0.05) with a decrease in low RFI pigs housed in poor hygiene conditions. In adipose tissue, the two abundant MSC populations were CD45-CD56-CD34- and CD45-CD56+CD34-. The proportion of CD45-CD56-CD34- cells increased (P < 0.05) whereas the proportion of CD45-CD56+CD34- tended to decrease (P < 0.1) in pigs housed in poor conditions. This study shows that the proportions of some MSC populations were affected by hygiene of housing conditions in a tissue-dependent manner in pigs of both RFI lines. It suggests that MSCs may play a significant role in adipose tissue and skeletal muscle homeostasis and may influence later growth and body composition in growing animals.


title: The impact of housing conditions on mesenchymal stromal/stem cell populations differ between adipose tissue and skeletal muscle


title: Inhibition of aryl hydrocarbon receptor signaling promotes the terminal differentiation of human erythrocytes

abstract: The aryl hydrocarbon receptor (AHR) plays an important role during mammalian embryo development. Inhibition of AHR signaling promotes the development of hematopoietic stem/progenitor cells. AHR also regulates the functional maturation of blood cells, such as T cells and megakaryocytes. However, little is known about the role of AHR modulation during the development of erythroid cells. In this study, we used the AHR antagonist StemRegenin 1 (SR1) and the AHR agonist 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) during different stages of human erythropoiesis to elucidate the function of AHR. We found that antagonizing AHR signaling improved the production of human embryonic stem cell (hESC)-derived erythrocytes and enhanced erythroid terminal differentiation. RNA-sequencing showed that SR1 treatment of proerythroblasts upregulated the expression of erythrocyte differentiation-related genes and downregulated actin organization-associated genes. We found that SR1 promoted F-actin remodeling in terminally differentiated erythrocytes, favoring the maturation of the cytoskeleton and enucleation. We demonstrated that the effects of AHR inhibition on erythroid maturation resulted from an increase in F-actin remodeling. Our findings help uncover the mechanism for AHR-mediated human erythroid cell differentiation. We also provide a new approach toward the large-scale production of functionally mature hPSC-derived erythrocytes for use in translational applications.


title: Ergonomic risk factors and Work-related Musculoskeletal Disorders among Fireworks workers in West Bengal, India: A cross-sectional study

abstract: Fireworks industries are very old, unorganized cottage industries in West Bengal mainly confined in South 24 Parganas. The present investigation was intended to investigate the prevalence of work-related musculoskeletal disorders among the workers and to identify the causative factors behind it. In this present study, 152 male fireworks workers from different age groups and 100 control subjects were investigated. Modified Nordic questionnaires were used to identify the region-wise disorders. The Hand Grip strength of both groups was also estimated. Among the fireworks workers posture related musculoskeletal disorders were severely observed in the lower back which was aggravated with the advancement of age and working experience. Pain and stiffness were also reported in the neck, upper back, wrist, elbow, knee and ankle. A lower backrest with support at the lumber region is strongly recommended.

category: physiology


title: Aberrant hippocampal transmission and behavior in mice with a stargazin mutation linked to intellectual disability

abstract: Mutations linked to neurodevelopmental disorders, such as intellectual disability (ID), are frequently found in genes that encode for proteins of the excitatory synapse. Transmembrane AMPA receptor regulatory proteins (TARPs) are AMPA receptor auxiliary proteins that regulate crucial aspects of receptor function. Here, we investigate an ID-associated mutant form of the TARP family member stargazin. Molecular dynamics analyses showed that the stargazin V143L variant weakens the overall interface of the AMPAR:stargazin complex and hinders the stability of the complex. Knock-in mice for the V143L stargazin mutation manifest cognitive and social deficits and hippocampal synaptic transmission defects. In the hippocampus of stargazin V143L mice, CA1 neurons show impaired spine maturation in basal dendrites, and synaptic ultrastructural alterations. These data demonstrate a causal role for mutated stargazin in the pathogenesis of ID and highlight its role in the development and function of hippocampal synapses.


title: Peer presence and familiarity as key factors to reduce cocaine intake: an effect mediated by the Subthalamic Nucleus

abstract: Stimulant use, including cocaine, often occurs in a social context whose influence is important to understand to decrease intake and reduce associated harms. Given the regulatory role of the subthalamic nucleus (STN) on cocaine intake and emotions, we investigate its role on such influence of social context on cocaine intake. We explored the influence of peer presence and familiarity on the frequency of self-administered cocaine and its neurobiological basis. We first compared cocaine intake in various conditions (alone or with peers with different characteristics: observing or self-administering, familiar or not, cocaine-naive or not, dominant or subordinate) in rats (n=90). The risk of drug consumption was reduced when a peer was present, observing or self-administering as well, and further diminished when the peer was unfamiliar (vs familiar). The presence of a cocaine-naive peer further decreased cocaine consumption. The presence of a non-familiar and drug-naive peer represents thus key conditions to diminish cocaine intake. We tested the effects of STN lesions in these various conditions and also conducted social experiments to validate the role of STN in social cognition. The STN lesion by itself reduced cocaine intake to the level reached in presence of a stranger naive peer and affected social cognition, positioning the STN as one neurobiological substrate of social influence on drug intake. Finally, with a translational research approach, we compared the drug intake in these conditions in human drug users (n=77). This human study confirmed the beneficial effect of social presence, especially of strangers. Our results indirectly support the use of social interventions and harm reduction strategies, in particular supervised consumption rooms for stimulant users.


title: Mapping Potential Antigenic Drift Sites (PADS) on SARS-CoV-2 Spike in Continuous Epitope-Paratope Space

abstract:
SARS-CoV-2 mutations with antigenic effects pose a risk to immunity developed through vaccination and natural infection. While vaccine updates for current variants of concern (VOCs) are underway, it is likewise important to prepare for further antigenic mutations as the virus navigates the heterogeneous global landscape of host immunity. Toward this end, a wealth of data and tools exist that can augment existing genetic surveillance of VOC evolution. In this study, we integrate published datasets describing genetic, structural, and functional constraints on mutation along with computational analyses of antibody-spike co-crystal structures to identify a set of potential antigenic drift sites (PADS) within the receptor binding domain (RBD) and N-terminal domain (NTD) of SARS-CoV-2 spike protein. Further, we project the PADS set into a continuous epitope-paratope space to facilitate interpretation of the degree to which newly observed mutations might be antigenically synergistic with existing VOC mutations, and this representation suggests that functionally convergent and synergistic antigenic mutations are accruing across VOC NTDs. The PADS set and synergy visualization serve as a reference as new mutations are detected on VOCs, enable proactive investigation of potentially synergistic mutations, and offer guidance to antibody and vaccine design efforts.

Graphical Abstract

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title: Gut bacterial aggregates as living gels

abstract: The spatial organization of gut microbiota influences both microbial abundances and host-microbe interactions, but the underlying rules relating bacterial dynamics to large-scale structure remain unclear. To this end we studied experimentally and theoretically the formation of three-dimensional bacterial clusters, a key parameter controlling susceptibility to intestinal transport and access to the epithelium. Inspired by models of structure formation in soft materials, we sought to understand how the distribution of gut bacterial cluster sizes emerges from bacterial-scale kinetics. Analyzing imaging-derived data on cluster sizes for eight different bacterial strains in the larval zebrafish gut, we find a common family of size distributions that decay approximately as power laws with exponents close to -2, becoming shallower for large clusters in a strain-dependent manner. We show that this type of distribution arises naturally from a Yule-Simons-type process in which bacteria grow within clusters and can escape from them, coupled to an aggregation process that tends to condense the system toward a single massive cluster, reminiscent of gel formation. Together, these results point to the existence of general, biophysical principles governing the spatial organization of the gut microbiome that may be useful for inferring fast-timescale dynamics that are experimentally inaccessible.


title: The transcription factor Roc1 is a regulator of cellulose degradation in the wood-decaying mushroom Schizophyllum commune

abstract: Wood-decaying fungi of the class Agaricomycetes (phylum Basidiomycota) are saprotrophs that break down lignocellulose and play an important role in the nutrient recycling. They secrete a wide range of extracellular plant cell wall degrading enzymes that break down cellulose, hemicellulose and lignin, the main building blocks of plant biomass. Although the production of these enzymes is regulated mainly at the transcriptional level, no activating regulators have been identified in any wood-decaying fungus in the class Agaricomycetes. We studied the regulation of cellulase expression in the wood-decaying fungus Schizophyllum commune. Comparative genomics and transcriptomics on two wild isolates revealed a Zn2Cys6-type transcription factor gene (roc1) that was highly up-regulated during growth on cellulose, when compared to glucose. It is only conserved in the class Agaricomycetes. A roc1 knockout strain showed an inability to grow on medium with cellulose as sole carbon source, and growth on cellobiose and xylan (other components of wood) was inhibited. Growth on non-wood-related carbon sources was not inhibited. Cellulase activity was reduced in the growth medium of the {Delta}roc1 strain. ChIP-Seq identified 1474 binding sites of the Roc1 transcription factor. Promoters of genes involved in lignocellulose degradation were enriched with these binding sites, especially those of LPMO (lytic polysaccharide monooxygenase) CAZymes, indicating that Roc1 directly regulates these genes. A GC-rich motif was identified as the binding site of Roc1, which was confirmed by a functional promoter analysis. Together, Roc1 is a key regulator of cellulose degradation and the first identified in wood-decaying fungi in the phylum Basidiomycota.


title: Seed microbiota revealed by a large-scale meta-analysis including 50 plant species

abstract: Seed microbiota constitutes a primary inoculum for plants that is gaining attention due to its role for plant health and productivity. Here, we performed a meta-analysis on 63 seed microbiota studies covering 50 plant species to synthesize knowledge on the diversity of this habitat. Seed microbiota are diverse and extremely variable, with taxa richness varying from one to thousands of taxa. Hence, seed microbiota presents a variable (i.e flexible) microbial fraction but we also identified a stable (i.e. core) fraction across samples. Around 30 bacterial and fungal taxa are present in most plant species and in samples from all over the world. Core taxa, such as Pantoea agglomerans, Pseudomonas viridiflava, P. fluorescens, Cladosporium perangustum and Alternaria sp., are dominant seed taxa. The characterization of the core and flexible seed microbiota provided here will help uncover seed microbiota roles for plant health and design effective microbiome engineering.


title: The expression of virulence increases outer-membrane permeability and sensitivity to envelope stress in Salmonella Typhimurium

abstract: Environmental cues modulate the expression of virulence in bacterial pathogens. However, while cues that upregulate virulence are often intuitive and mechanistically well understood, this is less so for cues that downregulate virulence. In this study, we noticed that upregulation of the HilD virulence regulon in Salmonella Typhimurium (S.Tm) sensitized cells to membrane stress mediated by cholate, Tris/EDTA or heat. Further monitoring of membrane status and stress resistance of S.Tm cells in relation to virulence expression, revealed that co-expressed virulence factors embedded in the envelope (including the Type Three Secretion System 1 and the flagella) increased permeability, and stress sensitivity of the membrane. Importantly, pretreating the bacteria by sublethal stress inhibited virulence expression and restored stress resistance. As such, these results demonstrate a trade-off between virulence and stress resistance, which explains the downregulation of virulence expression in response to harsh environments in S.Tm.


title: FXa cleaves the SARS-CoV-2 spike protein and blocks cell entry to protect against infection with inferior effects in B.1.1.7 variant

abstract: The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human natural defense mechanisms against SARS-CoV-2 are largely unknown. Serine proteases (SPs) including furin and TMPRSS2 cleave SARS-CoV-2 spike protein, facilitating viral entry. Here, we show that FXa, a SP for blood coagulation, is upregulated in COVID-19 patients compared to non-COVID-19 donors and exerts anti-viral activity. Mechanistically, FXa cleaves the SARS-CoV-2 spike protein, which prevents its binding to ACE2, and thus blocks viral entry. Furthermore, the variant B.1.1.7 with several mutations is dramatically resistant to the anti-viral effect of FXa compared to wild-type SARA-CoV-2 in vivo and in vitro. The anti-coagulant rivaroxaban directly inhibits FXa and facilitates viral entry, whereas the indirect inhibitor fondaparinux does not. In a lethal humanized hACE2 mouse model of SARS-CoV-2, FXa prolonged survival while combination with rivaroxaban but not fondaparinux abrogated this protection. These preclinical results identify a previously unknown SP function and associated anti-viral host defense mechanism and suggest caution in considering direct inhibitors for prevention or treatment of thrombotic complications in COVID-19 patients.


title: In depth analysis of Cyprus-specific mutations of SARS-CoV-2 strains using computational approaches

abstract: This study aims to characterize SARS-CoV-2 mutations which are primarily prevalent in the Cypriot population. Moreover, using computational approaches, we assess whether these mutations are associated with changes in viral virulence. We utilize genetic data from 144 sequences of SARS-CoV-2 strains from the Cypriot population obtained between March 2020 and January 2021, as well as all data available from GISAID. We combine this with countries regional information, such as deaths and cases per million, as well as COVID-19-related public health austerity measure response times. Initial indications of selective advantage of Cyprus-specific mutations are obtained by mutation tracking analysis. This entails calculating specific mutation frequencies within the Cypriot population and comparing these with their prevalence world-wide throughout the course of the pandemic. We further make use of linear regression models to extrapolate additional information that may be missed through standard statistical analysis. We report a single mutation found in the ORF1ab gene (S6059F) that appears to be significantly enriched within the Cypriot population. We further analyse this mutation using regression models to investigate possible associations with increased deaths and cases per million. Moreover, protein structure prediction tools show that the mutation infers a conformational change to the protein that significantly alters its structure when compared to the reference protein. Investigating Cyprus-specific mutations for SARS-CoV-2 can not only lead to important findings from which to battle the pandemic on a national level, but also provide insights into viral virulence worldwide.

category: bioinformatics


title: Bacterioplankton Diversity and Distribution in Relation to Phytoplankton Community Structure in the Ross Sea surface waters

abstract: Primary productivity in the Ross Sea region is characterized by intense phytoplankton blooms whose temporal and spatial distribution are driven by changes in environmental conditions as well as interactions with the bacterioplankton community. Exchange of exudates, metabolism by-products and cofactors between the phytoplankton and the bacterioplankton communities drive a series of complex interactions affecting the micronutrient availability and co-limitation, as well as nutrient uptakes in Antarctic waters. Yet, the number of studies reporting the simultaneous diversity of the phytoplankton and bacterioplankton in Antarctic waters are limited. Here we report data on the bacterial diversity in relation to phytoplankton community in the surface waters of the Ross Sea during the austral summer 2017. Our results show partially overlapping bacterioplankton communities between the stations located in the Terra Nova Bay coastal waters and the Ross Sea open waters, suggesting that the two communities are subjected to different drivers. We show that the rate of diversity change between the two locations is influenced by both abiotic (salinity and the nitrogen to phosphorus ratio) and biotic (phytoplankton community structure) factors. Our data provides new insight into the coexistence of the bacterioplankton and phytoplankton in Antarctic waters.


title: The wall-less bacterium Spiroplasma poulsonii builds a polymeric cytoskeleton composed of interacting MreB isoforms

abstract:
A rigid cell wall defines the morphology of most bacteria. MreB, a bacterial homologue of actin, plays a major role in coordinating cell wall biogenesis and defining a cells shape. In contrast with most bacteria, the Mollicutes family is devoid of cell wall. As a consequence, many Mollicutes have undefined morphologies. Spiroplasma species are an exception as they robustly grow with a characteristic helical shape, but how they maintain their morphology remains unclear. Paradoxal to their lack of cell wall, the genome of Spiroplasma contains five homologues of MreB (SpMreBs). Since MreB is a homolog of actin and that short MreB filaments participate in its function, we hypothesize that SpMreBs form a polymeric cytoskeleton. Here, we investigate the function of SpMreB in forming a polymeric cytoskeleton by focusing on the Drosophila endosymbiont Spiroplasma poulsonii. We found that in vivo, Spiroplasma maintain a high concentration of all five MreB isoforms. By leveraging a heterologous expression system that bypasses the poor genetic tractability of Spiroplasma, we found that strong intracellular levels of SpMreb systematically produced polymeric filaments of various morphologies. Using co-immunoprecipitation and co-expression of fluorescent fusions, we characterized an interaction network between isoforms that regulate the filaments formation. Our results point to a sub-functionalization of each isoform which, when all combined in vivo, form a complex inner polymeric network that shapes the cell in a wall-independent manner. Our work therefore supports the hypothesis where MreB mechanically supports the cell membrane, thus forming a cytoskeleton.

Significance statementBacteria shape is determined by their cell wall. The actin homologue MreB essentially determines shape by organizing cell wall synthesis at the subcellular level. Despite their lack of cell wall, Spiroplasma robustly grow into long helical bacteria. Surprisingly, its genome retains five copies of mreB while it lost genes encoding canonical MreB interactors. We sought to delineate the exact function of Spiroplasma MreBs (SpMreBs). We leveraged in vivo data along with functional studies to systematically investigate MreB polymerization behavior. We uncovered that SpMreBs build into filaments, which structure it determined by a complex interaction network between isoforms. Our results support the hypothesis that MreB can mechanically support the membrane of Spiroplasma, hence acting as a load-bearing cytoskeletal protein.


title: The LysR-type transcriptional regulator BsrA (PA2121) controls vital metabolic pathways in Pseudomonas aeruginosa

abstract:
Pseudomonas aeruginosa, a facultative human pathogen causing nosocomial infections, has complex regulatory systems involving many transcriptional regulators. LTTR (LysR-Type Transcriptional Regulator) family proteins are involved in the regulation of various processes including stress responses, motility, virulence and amino acid metabolism. The aim of this study was to characterize the LysR-type protein BsrA (PA2121), previously described as a negative regulator of biofilm formation in P. aeruginosa. Genome wide identification of BsrA binding sites using ChIP-seq revealed 765 BsrA-bound regions in the P. aeruginosa PAO1161 genome, including 367 sites in intergenic regions. The motif T-N11-A was identified within sequences bound by BsrA. Transcriptomic analysis showed altered expression of 157 genes in response to BsrA excess, of which 35 had a BsrA binding site within their promoter regions, suggesting a direct influence of BsrA on the transcription of these genes. BsrA-repressed loci included genes encoding proteins engaged in key metabolic pathways such as the tricarboxylic acid cycle. The panel of loci possibly directly activated by BsrA, included genes involved in pili/fimbriae assembly as well as secretion and transport systems. In addition, DNA pull-down and regulatory analyses showed the involvement of PA2551, PA3398 and PA5189 in regulation of bsrA expression, indicating that this gene is part of an intricate regulatory network. Taken together, these findings reveal the existence of a BsrA regulon, which performs important functions in P. aeruginosa.

IMPORTANCEThis study shows that BsrA, a LysR-type transcriptional regulator from P. aeruginosa, previously identified as a repressor of biofilm synthesis, is part of an intricate global regulatory network. BsrA acts directly and/or indirectly as the repressor and/or activator of genes from vital metabolic pathways (e.g. pyruvate, acetate, tricarboxylic acid cycle), and is involved in control of transport functions and the formation of surface appendages. Expression of the bsrA gene is increased in the presence of antibiotics, which suggests its induction in response to stress, possibly reflecting the need to redirect metabolism under stressful conditions. This is particularly relevant for the treatment of infections caused by P. aeruginosa. In summary, the findings of this study demonstrate that the BsrA regulator performs important roles in carbon metabolism, biofilm formation and antibiotic resistance in P. aeruginosa.

category: molecular biology


title: ROS/RNS balancing, aerobic fermentation regulation and cell cycle control a complex early trait ('CoV-MAC-TED') for combating SARS-CoV-2-induced cell reprogramming

abstract:
In a perspective entitled  From plant survival under severe stress to anti-viral human defense we raised and justified the hypothesis that transcript level profiles of justified target genes established from in vitro somatic embryogenesis (SE) induction in plants as a reference compared to virus-induced profiles can identify differential virus signatures that link to harmful reprogramming. A standard profile of selected genes named  ReprogVirus was proposed for in vitro-scanning of early virus-induced reprogramming in critical primary infected cells/tissues as target trait. For data collection, the  ReprogVirus platform was initiated. This initiative aims to identify in a common effort across scientific boundaries critical virus footprints from diverse virus origins and variants as a basis for anti-viral strategy design. This approach is open for validation and extension.

In the present study, we initiated validation by experimental transcriptome data available in public domain combined with advancing plant wet lab research. We compared plant-adapted transcriptomes according to  RegroVirus complemented by alternative oxidase (AOX) genes during de novo programming under SE-inducing conditions with in vitro corona virus-induced transcriptome profiles. This approach enabled identifying a major complex trait for early de novo programming during SARS-CoV-2 infection, called  CoV-MAC-TED. It consists of unbalanced ROS/RNS levels, which are connected to increased aerobic fermentation that links to alpha-tubulin-based cell restructuration and progression of cell cycle.

We conclude that anti-viral/anti-SARS-CoV-2 strategies need to rigorously target  CoV-MAC-TED in primary infected nose and mouth cells through prophylactic and very early therapeutic strategies. We also discuss potential strategies in the view of the beneficial role of AOX for resilient behavior in plants.

Furthermore, following the general observation that ROS/RNS equilibration/redox homeostasis is of utmost importance at the very beginning of viral infection, we highlight that  de-stressing disease and social handling should be seen as essential part of anti-viral/anti-SARS-CoV-2 strategies.


title: ER-lysosome lipid transfer protein VPS13C/PARK23 prevents aberrant mtDNA-dependent STING signaling

abstract: Mutations in VPS13C cause early onset, autosomal recessive Parkinsons Disease (PD). We have established that VPS13C encodes a lipid transfer protein localized to contact sites between the endoplasmic reticulum (ER) and late endosomes/lysosomes. In the current study, we demonstrate that depleting VPS13C in HeLa cells causes an accumulation of lysosomes with an altered lipid profile, including an accumulation of di-22:6-BMP, a biomarker of the PD-associated leucine-rich repeat kinase 2 (LRRK2) G2019S mutation. In addition, the DNA-sensing cGAS/STING pathway, which was recently implicated in PD pathogenesis, is activated in these cells. This activation results from a combination of elevated mitochondrial DNA in the cytosol and a defect in the degradation of activated STING, a lysosome-dependent process. These results suggest a link between ER-lysosome lipid transfer and innate immune activation and place VPS13C in pathways relevant to PD pathogenesis.


title: Microfluidic deep mutational scanning of the human executioner caspases reveals differences in structure and regulation

abstract: The human caspase family comprises 12 cysteine proteases that are centrally involved in cell death and inflammation responses. The members of this family have conserved sequences and structures, highly similar enzymatic activities and substrate preferences, and overlapping physiological roles. In this paper, we present a deep mutational scan of the executioner caspases CASP3 and CASP7 to dissect differences in their structure, function, and regulation. Our approach leverages high-throughput microfluidic screening to analyze hundreds of thousands of caspase variants in tightly controlled in vitro reactions. The resulting data provides a large-scale and unbiased view of the impact of amino acid substitutions on the proteolytic activity of CASP3 and CASP7. We use this data to pinpoint key functional differences between CASP3 and CASP7, including a secondary internal cleavage site, CASP7 Q196 that is not present in CASP3. Our results will open avenues for inquiry in caspase function and regulation that could potentially inform the development of future caspasespecific therapeutics.

category: biochemistry


title: Single domain shark VNAR antibodies neutralize SARS-CoV-2 infection in vitro

abstract: Single domain shark VNAR antibodies can offer a viable alternative to conventional Ig-based monoclonal antibodies in treating COVID-19 disease during the current pandemic. Here we report the identification of neutralizing single domain VNAR antibodies selected against the SARS-CoV-2 spike protein derived from the Wuhan variant using phage display. We identified 56 unique binding clones that exhibited high affinity and specificity to the spike protein. Of those, 10 showed an ability to block both the spike protein receptor binding domain from the Wuhan variant and the N501Y mutant from interacting with recombinant ACE2 receptor in vitro. In addition, 3 antibody clones retained in vitro blocking activity when the E484K spike protein mutant was used. The inhibitory property of the VNAR antibodies was further confirmed for all 10 antibody clones using ACE2 expressing cells with spike protein from the Wuhan variant. The viral neutralizing potential of the VNAR clones was also confirmed for the 10 antibodies tested using live Wuhan variant virus in in vitro cell infectivity assays. Single domain VNAR antibodies due to their low complexity, small size, unique epitope recognition and formatting flexibility should be a useful adjunct to existing antibody approaches to treat COVID-19.

category: bioengineering


title: Predicted Coronavirus Nsp5 Protease Cleavage Sites in the Human Proteome: A Resource for SARS-CoV-2 Research

abstract:
BackgroundThe coronavirus nonstructural protein 5 (Nsp5) is a cysteine protease required for processing the viral polyprotein and is therefore crucial for viral replication. Nsp5 from several coronaviruses have also been found to cleave host proteins, disrupting molecular pathways involved in innate immunity. Nsp5 from the recently emerged SARS-CoV-2 virus interacts with and can cleave human proteins, which may be relevant to the pathogenesis of COVID-19. Based on the continuing global pandemic, and emerging understanding of coronavirus Nsp5-human protein interactions, we set out to predict what human proteins are cleaved by the coronavirus Nsp5 protease using a bioinformatics approach.

ResultsUsing a previously developed neural network trained on coronavirus Nsp5 cleavage sites (NetCorona), we made predictions of Nsp5 cleavage sites in all human proteins. Structures of human proteins in the Protein Data Bank containing a predicted Nsp5 cleavage site were then examined, generating a list of 92 human proteins with a highly predicted and accessible cleavage site. Of those, 48 are expected to be found in the same cellular compartment as Nsp5. Analysis of this targeted list of proteins revealed molecular pathways susceptible to Nsp5 cleavage and therefore relevant to coronavirus infection, including pathways involved in mRNA processing, cytokine response, cytoskeleton organization, and apoptosis.

ConclusionsThis study combines predictions of Nsp5 cleavage sites in human proteins with protein structure information and protein network analysis. We predicted cleavage sites in proteins recently shown to be cleaved in vitro by SARS-CoV-2 Nsp5, and we discuss how other potentially cleaved proteins may be relevant to coronavirus mediated immune dysregulation. The data presented here will assist in the design of more targeted experiments, to determine the role of coronavirus Nsp5 cleavage of host proteins, which is relevant to understanding the molecular pathology of SARS-CoV-2 infection.


title: Locally secreted BiTEs complement CAR T cells by enhancing solid tumor killing

abstract: Bispecific T-cell engagers (BiTEs) are bispecific antibodies that redirect T cells to target antigen-expressing tumors. BiTEs can be secreted by T cells through genetic engineering and perform anti-tumor activity. We hypothesized that BiTE-secreting T cells could be a valuable T cell-directed therapy in solid tumors, with distinct properties in mono- or multi-valent strategies incorporating chimeric antigen receptor (CAR) T cells. Glioblastomas represent a good model for solid tumor heterogeneity and represent a significant therapeutic challenge. We detected expression of tumor-associated epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), and interleukin-13 receptor alpha 2 (IL13R2) on glioma tissues and glioma cancer stem cells. These antigens formed the basis of a multivalent approach, using a conformation-specific tumor-related EGFR targeting antibody (806) and Hu08, an IL13R2-targeting antibody, as the scFvs to generate new BiTE molecules. Compared with 806CAR T cells and Hu08CAR T cells, BiTE T cells demonstrated prominent activation, cytokine production, and cytotoxicity in response to target-positive gliomas. Superior response activity was also demonstrated in BiTE secreting bivalent targeting T cells compared with bivalent targeting CAR T cells, which significantly delayed tumor growth in a glioma mouse model. In summary, BiTEs secreted by mono- or multi- valent targeting T cells have potent anti-tumor activity in vitro and in vivo with significant sensitivity and specificity, demonstrating a promising strategy in solid tumor therapy.

category: cancer biology


title: Targeting of Protein Kinase CK2 Elicits Antiviral Activity on Bovine Coronavirus Infection

abstract: Coronaviruses constitute a global threat to human population since three highly pathogenic coronaviruses (SARS-CoV, MERS-CoV and SARS-CoV-2) have crossed species to cause severe human respiratory disease. Considering the worldwide emergency status due to the current COVID-19 pandemic, effective pan-coronavirus antiviral drugs are required to tackle the ongoing as well as future (re)emerging virus outbreaks. Protein kinase CK2 has been deemed a promising therapeutic target in COVID-19 supported by its in vitro pharmacologic inhibition and molecular studies on SARS-CoV-2 infected cells. CIGB-325 is a first-in-class synthetic peptide impairing the CK2-mediated signaling whose safety and clinical benefit have been evidenced in Covid-19 and cancer patients after intravenous administration. Here, we explored the putative antiviral effect of CIGB-325 over MDBK cells infected by bovine coronavirus (BCoV) Mebus. Importantly, CIGB-325 inhibited both the cytopathic effect and the number of plaques forming units with a half-inhibitory concentrations IC50 = 3.5 M and 17.7 M, respectively. Accordingly, viral protein accumulation at the cytoplasm was clearly reduced by treating BCoV-infected cells with CIGB-325 over time, as determined by immunocytochemistry. Of note, data from pull-down assay followed by western blot and/or mass spectrometry identification revealed physical interaction of CIGB-325 with nucleocapsid (N) protein and a bona fide cellular CK2 substrates. Functional enrichment and network analysis from the CIGB-325 interacting proteins indicated cytoskeleton reorganization and protein folding as the most represented biological processes disturbed by this anti-CK2 peptide. Altogether, our findings not only unveil the direct antiviral activity of CIGB-325 on coronavirus infection but also provide molecular clues underlying such effect. Also, our data reinforce the scientific rationality behind the pharmacologic inhibition of CK2 to treat coronavirus infections.

category: pharmacology and toxicology


title: The Ebola virus matrix protein clusters phosphatidylserine, a critical step in viral budding

abstract: Phosphatidylserine (PS) has been shown to be a critical lipid factor in the assembly and spread of numerous lipid enveloped viruses. Here, we describe the ability of the Ebola virus (EBOV) matrix protein eVP40 to induce clustering of PS and promote viral budding in vitro, as well as the ability of an FDA approved drug, fendiline, to reduce PS clustering subsequently reducing virus budding and entry. To gain mechanistic insight into fendiline inhibition of EBOV replication, multiple in vitro assays were employed including imaging, viral budding and viral entry assays. Fendiline reduced the PS content in mammalian cells and PS in the plasma membrane, reducing the ability of VP40 to form new virus particles. Further, particles that do form from fendiline treated cells have altered particle morphology and decreased infectivity capacity. These complementary studies reveal the mechanism by which filovirus matrix proteins cluster PS to enhance viral assembly, budding, and spread from the host cell while also laying the groundwork for fundamental drug targeting strategies.


title: Shrimp parvovirus circular DNA fragments arise from both endogenous viral elements (EVE) and the infecting virus

abstract: Some insects use endogenous reverse transcriptase (RT) to make variable viral copy DNA (vcDNA) fragments from viral RNA in linear (lvcDNA) and circular (cvcDNA) forms. The latter form is easy to extract selectively. The vcDNA produces small interfering RNA (siRNA) variants that inhibit viral replication via the RNA interference (RNAi) pathway. The vcDNA is also autonomously inserted into the host genome as endogenous viral elements (EVE) that can also result in RNAi. We hypothesized that similar mechanisms occurred in shrimp. We used the insect methods to extract circular viral copy DNA (cvcDNA) from the giant tiger shrimp (Penaeus monodon) infected with a virus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV). Simultaneous injection of the extracted cvcDNA plus IHHNV into whiteleg shrimp (Penaeus vannamei) resulted in a significant reduction in IHHNV replication when compared to shrimp injected with IHHNV only. Next generation sequencing (NGS) revealed that the extract contained a mixture of two general IHHNV-cvcDNA types. One showed 98 to 99% sequence identity to GenBank record AF218266 from an extant type of infectious IHHNV. The other type showed 98% sequence identity to GenBank record DQ228358, an EVE formerly called non-infectious IHHNV. The startling discovery that EVE could also give rise to cvcDNA revealed that cvcDNA provided an easy means to identify and characterize EVE in shrimp and perhaps other organisms. These studies open the way for identification, characterization and use of protective cvcDNA as a potential shrimp vaccine and as a tool to identify, characterize and select naturally protective EVE to improve shrimp tolerance to homologous viruses in breeding programs.


title: Analysis of subcellular transcriptomes by RNA proximity labeling with Halo-seq

abstract: Thousands of RNA species display nonuniform distribution within cells. However, quantification of the spatial patterns adopted by individual RNAs remains difficult, in part by a lack of quantitative tools for subcellular transcriptome analysis. In this study, we describe an RNA proximity labeling method that facilitates the quantification of subcellular RNA populations with high spatial specificity. This method, termed Halo-seq, pairs a light-activatable, radical generating small molecule with highly efficient Click chemistry to efficiently label and purify spatially defined RNA samples. We compared Halo-seq with previously reported similar methods and found that Halo-seq displayed a higher efficiency of RNA labeling, indicating that it is well suited to the investigation of small, precisely localized RNA populations. We then used Halo-seq to quantify nuclear, nucleolar, and cytoplasmic transcriptomes, characterize their dynamic nature following perturbation, and identify RNA sequence features associated with their composition. Specifically, we found that RNAs containing AU-rich elements are relatively enriched in the nucleus. This enrichment becomes stronger upon treatment with the nuclear export inhibitor leptomycin B, both expanding the role of HuR in RNA export and generating a comprehensive set of transcripts whose export from the nucleus depends on HuR.


title: A programmable pAgo nuclease with RNA target preference from the psychrotolerant bacteria Mucilaginibacter paludis

abstract: Argonaute (Ago) proteins are programmable nuclease found in both eukaryotes and prokaryotes. Prokaryotic Argonaute proteins (pAgos) share a high degree of structural homology with eukaryotic Argonaute proteins (eAgos) and eAgos are considered to evolve from pAgos. However, the majority of studied pAgos prefer to cleave DNA targets, and eAgos exclusively cleave RNA targets. Here, we characterize a novel pAgo, MbpAgo, from psychrotolerant bacteria Mucilaginibacter paludis that can be programmed with DNA guides and prefers to cleave RNA targets rather than DNA targets. MbpAgo can be active at a wide range of temperatures (4-65{degrees}C). In comparison with previously studied pAgos, MbpAgo is able to utilize 16-nt long 5phosphorylated and 5hydroxylated DNA guides for efficient and precise cleavage and displays no obvious preference for the 5end nucleotide of a guide. Furthermore, the cleavage efficiency can be regulated by mismatches in the central and 3supplementary regions of the guide. MbpAgo can efficiently cleave highly-structured RNA targets using both 5phosphorylated and 5hydroxylated DNA guides in the presence of Mg2+ or Mn2+. In conclusion, we have demonstrated that MbpAgo is a unique programmable nuclease that has a strong preference for RNA targets, with great potential applications in the field of nucleic acid biotechnology.


title: Cortical waves mediate the cellular response to electric fields

abstract: Electrotaxis, the directional migration of cells in a constant electric field, is important in regeneration, development, and wound healing. Electrotaxis has a slower response and a smaller dynamic range than guidance by other cues, suggesting that the mechanism of electrotaxis share both similarities and differences with chemical-gradient-sensing pathways. We examined a mechanism centered on the excitable system consisting of cortical waves of biochemical signals coupled to cytoskeletal reorganization, which has been implicated in random cell motility. We use electro-fused giant Dictyostelium discoideum cells to decouple waves from cell motion and employ nanotopographic surfaces to limit wave dimensions and lifetimes. We demonstrate that wave propagation in these cells is guided by electric fields. The wave area and lifetime gradually increase in the first 10 minutes after an electric field is turned on, leading to more abundant and wider protrusions in the cell region nearest the cathode. The wave directions display "U-turn" behavior upon field reversal, and this switch occurs more quickly on nanotopography. Our results suggest that electric fields guide cells by controlling waves of signal transduction and cytoskeletal activity, which underlie cellular protrusions. Whereas surface receptor occupancy triggers both rapid activation and slower polarization of signaling pathways, electric fields appear to act primarily on polarization, explaining why cells respond to electric fields more slowly than to other guidance cues.


title: Cell-to-cell variability in inducible Caspase9-mediated cell death

abstract: iCasp9 suicide gene has been widely used as a promising killing strategy in various cell therapies. However, different cells show significant heterogeneity in response to apoptosis inducer, posing challenges in clinical applications of killing strategy. The cause of the heterogeneity remains elusive so far. Here, by simultaneously monitoring the dynamics of iCasp9 dimerization, Caspase3 activation and cell fate in single cells, we found that the heterogeneity was mainly due to cell-to-cell variability in initial iCasp9 expression and XIAP/Caspase3 ratio. Moreover, multiple-round drugging cannot increase the killing efficiency. Instead, it will place selective pressure on protein levels, especially on the level of initial iCasp9, leading to drug resistance. We further show this resistance can be largely eliminated by combinatorial drugging with XIAP inhibitor at the end, but not at the beginning, of the multiple-round treatments. Our results unveil the source of cell fate heterogeneity and drug resistance in iCasp9-mediated cell death, which may enlighten better therapeutic strategies for optimized killing.


title: Ssdp influences Wg expression and embryonic somatic muscle identity in Drosophila melanogaster

abstract: The somatic muscles of the Drosophila embryo and larvae share structural and functional similarities with vertebrate skeletal muscles and serve as a powerful model for studying muscle development. Here we show that the evolutionarily conserved Ssdp protein is required for the correct patterning of somatic muscles. Ssdp is part of the conserved Chi/LDB-Ssdp (ChiLS) complex that is a core component of the conserved Wg/Wnt enhanceosome, which responds to Wg signals to regulate gene transcription. Ssdp shows isoform specific expression in developing somatic muscles and its loss of function leads to an aberrant somatic muscle pattern due to a deregulated muscle identity program. Ssdp mutant embryos fail to maintain adequate expression levels of muscle identity transcription factors and this results in aberrant muscle morphology, innervation, attachment and fusion. We also show that the epidermal expression of Wg is downregulated in Ssdp mutants and that Ssdp interacts with Wg to regulate the properties of a subset of ventral muscles. Thus, our data unveil the dual contribution of Ssdp contribution to muscle diversification by regulating the expression of muscle-intrinsic identity genes and by interacting with the extrinsic factor, Wg. The knowledge gained here about Ssdp and its interaction with Wg could be relevant to vertebrate muscle development.

category: developmental biology


title: The Streptochaeta genome and the evolution of the grasses

abstract: In this work, we sequenced and annotated the genome of Streptochaeta angustifolia, one of two genera in the grass subfamily Anomochlooideae, a lineage sister to all other grasses. The final assembly size is over 99% of the estimated genome size, capturing most of the gene space. Streptochaeta is similar to other grasses in the structure of its fruit (a caryopsis or grain) but has peculiar flowers and inflorescences that are distinct from those in the outgroups and in other grasses. To provide tools for investigations of floral structure, we analyzed two large families of transcription factors, AP2-like and R2R3 MYBs, that are known to control floral and spikelet development in rice and maize among other grasses. Many of these are also regulated by small RNAs. Structure of the gene trees showed that the well documented whole genome duplication at the origin of the grasses ({rho}) occurred before the divergence of the Anomochlooideae lineage from the lineage leading to the rest of the grasses (the spikelet clade) and thus that the common ancestor of all grasses probably had two copies of the developmental genes. However, Streptochaeta (and by inference other members of Anomochlooideae) has lost one copy of many genes. The peculiar floral morphology of Streptochaeta may thus have derived from an ancestral plant that was morphologically similar to the spikelet-bearing grasses. We further identify 114 loci producing microRNAs and 89 loci generating phased, secondary siRNAs, classes of small RNAs known to be influential in transcriptional and post-transcriptional regulation of several plant functions.

category: evolutionary biology


title: Convergent loss of the necroptosis pathway in disparate mammalian lineages shapes virus countermeasures

abstract: Programmed cell death is a vital process in the life cycle of an organism. Necroptosis, an evolutionary restricted form of programmed necrosis, contributes to the innate immune response by killing pathogen-infected cells. This virus-host interaction pathway is organized around two key components: the receptor-interacting protein kinase 3 (RIPK3), which recruits and phosphorylates the mixed lineage kinase-like protein (MLKL), thus inducing cellular plasma membrane rupture and cell death. Critically, the presence of necroptotic inhibitors in viral genomes validates necroptosis as an important host defense mechanism. Here, we show, counterintuitively, that in different mammalian lineages of mammalian, central components of the necroptotic pathway, such as RIPK3 and MLKL genes, are deleted or display inactivating mutations. Frameshifts or premature stop codons are observed in all the studied species of cetaceans and leporids. In carnivores genomes, the MLKL gene is deleted, while in a small number of species from afrotheria and rodentia premature stop codons are observed in RIPK3 and/or MLKL. Interestingly, we also found a strong correlation between the disruption of necroptosis in leporids and cetaceans and the absence of the C-terminal domain of E3-like homologs (responsible for necroptosis inhibition) in their naturally infecting poxviruses. Overall, our study provides the first comprehensive picture of the molecular evolution of necroptosis in mammals. The loss of necroptosis multiple times during mammalian evolution highlights the importance of gene/pathway loss for species adaptation and suggests that necroptosis is not required for normal mammalian development. Moreover, this study highlights a co-evolutionary relationship between poxviruses and their hosts, emphasizing the role of host adaptation in shaping virus evolution.


title: Host phenology can select for multiple stable parasite virulence strategies

abstract: Host phenology is an important driver of parasite transmission dynamics and evolution. Prior research has demonstrated that host phenology can drive monocyclic, obligate-killer parasites to evolve an intermediate virulence strategy where all parasites kill their host just before the season ends to limit the death of parasite progeny in the environment. The impact of host seasonality on parasites that are not constrained to a monocyclic life-cycle, however, cannot be inferred from these results. Here we present a mathematical model that demonstrates that many, but not all, seasonal host activity patterns support multiple evolutionarily stable parasite strategies (ESS), although these strategies cannot coexist in the same system. The specific monocyclic and polycyclic parasite evolutionarily stable strategies in each phenological pattern are interspersed with less-fit mono- and polycyclic strategies (evolutionary repellors). The ESS that dominates each system at equilibrium is a function of the strategy of the parasite introduced into the system. The results demonstrate that host phenology can, in theory, maintain diverse parasite strategies among isolated geographic locations.


title: Parallel and non-parallel divergence within polymorphic populations of brook stickleback, Culaea inconstans (Actinopterygii: Gasterosteidae)

abstract: Studying parallel evolution allows us to draw conclusions about the repeatability of adaptive evolution. Whereas populations likely experience similar selective pressures in similar environments, it is not clear if this will always result in parallel divergence of ecologically relevant traits. Our study investigates the extent of parallelism associated with the evolution of pelvic spine reduction in brook stickleback populations. We find that populations with parallel divergence in pelvic spine morphology do not exhibit parallel divergence in head and body morphology but do exhibit parallel divergence in diet. In addition, we compare these patterns associated with pelvic reduction in brook stickleback to well-studied patterns of divergence between spined and unspined threespine stickleback. Whereas spine reduction is associated with littoral habitats and a benthic diet in threespine stickleback, spine reduction in brook stickleback is associated with a planktonic diet. Hence, we find that pelvic spine divergence is associated with largely non-parallel ecological consequences across species.


title: Population structure analysis and laboratory monitoring of Shigella with a standardised core-genome multilocus sequence typing scheme: a validation study

abstract: The laboratory surveillance of bacillary dysentery is based on a standardised Shigella typing scheme that classifies Shigella strains into four serogroups and more than 50 serotypes on the basis of biochemical tests and lipopolysaccharide O-antigen serotyping. Real-time genomic surveillance of Shigella infections has been implemented in several countries, but without the use of a standardised typing scheme. We studied over 4,000 reference strains and clinical isolates of Shigella, covering all serotypes, with both the current serotyping scheme and the standardised EnteroBase core-genome multilocus sequence typing scheme (cgMLST). The Shigella genomes were grouped into eight phylogenetically distinct clusters, within the E. coli species. The cgMLST hierarchical clustering (HC) analysis at different levels of resolution (HC2000 to HC400) recognised the natural groupings for Shigella. By contrast, the serotyping scheme was affected by horizontal gene transfer, leading to a conflation of genetically unrelated Shigella strains and a separation of genetically related strains. The use of this cgMLST scheme will enhance the laboratory surveillance of Shigella infections.

category: genomics


title: Population structure analysis and laboratory monitoring of Shigella with a standardised core-genome multilocus sequence typing scheme


title: An Efficient and Cost-effective Purification Methodology for Cas9 Nuclease

abstract: With an ever-increasing demand for laboratory-grade Cas9 proteins by many groups advancing the use of CRISPR technology, a more efficient and scalable process for generating the proteins, coupled with rapid purification methods is in urgent demand. Here, we introduce a modified methodology for rapid purification of active SaCas9 protein within 24 hours. The product has over 90% protein purity. The simplicity and cost-effectiveness of such methodology will enable general labs to produce a sizable amount of Cas9 proteins, further accelerating the advancement of CRISPR/Cas9-based research.


title: Online μSEC2-nRPLC-MS for improved sensitivity of intact protein detection of IEF separated non-human primate cerebrospinal fluid proteins

abstract: Proteoform-resolved information, obtained by top-down (TD) "intact protein" proteomics, is expected to contribute substantially to the understanding of molecular pathogenic mechanisms and in turn, identify novel therapeutic and diagnostic targets. However, the robustness of mass spectrometry (MS) analysis of intact proteins in complex biological samples is hindered by high dynamic range in protein concentration and mass, protein instability, and buffer complexity. Here, we describe an evolutionary step for intact protein investigations through the online implementation of tandem microflow size exclusion chromatography with nanoflow reversed-phase liquid chromatography and MS (SEC2-nRPLC-MS). Online serial high-/low-pass SEC filtration overcomes the aforementioned hurdles to intact proteomic analysis through automated sample desalting/cleanup and enrichment of target mass ranges (5-155 kDa) prior to nRPLC-MS. The coupling of SEC to nRPLC is achieved through a novel injection volume control (IVC) strategy of inserting protein trap columns pre- and post-SEC columns to enable injection of dilute samples in high volumes without loss of sensitivity or resolution. Critical characteristics of the approach are tested via rigorous investigations on samples of varied complexity and chemical background. Application of the platform to cerebrospinal fluid (CSF) pre-fractionated by OFFGEL isoelectric focusing drastically increases the number of intact mass tags (IMTs) detected within the target mass range (5-30 kDa) in comparison to one-dimensional nRPLC-MS with approximately 100x less CSF than previous OFFGEL studies. Furthermore, the modular design of the SEC2-nRPLC-MS platform is robust and promises significant flexibility for large-scale TDMS analysis of diverse samples either directly or in concert with other multidimensional fractionation steps.


title: Optimization of Phosphate Limited Autoinduction Broth for 2-Stage Heterologous Protein Expression in E. coli

abstract: Autoinducible, 2-stage protein expression leveraging phosphate inducible promoters has been recently shown to enable not only high protein titers but also consistent performance across scales from screening systems (microtiter plates) to instrumented bioreactors. However, to date small scale production using microtiter plates and shake flasks rely on a complex autoinduction broth (AB) that requires making numerous media components, not all amenable to autoclaving. In this report, we develop a simpler media formulation (AB-2) with just a few autoclavable components. We show that AB-2 is robust to small changes in its composition and performs equally, if not better, than AB across different scales. AB-2 will facilitate adoption of phosphate limited 2-stage protein expression protocols.


title: Removing Thermostat Distortions of Protein Dynamics in Constant-Temperature Molecular Dynamics Simulations

abstract:
Molecular dynamics simulations are widely used to determine equilibrium and dynamic properties of proteins. Nearly all simulations nowadays are carried out at constant temperature, with a Langevin thermostat among the most widely used. Thermostats distort protein dynamics, but whether or how such distortions can be corrected has long been an open question. Here we show that constant-temperature simulations with a Langevin thermostat dilate protein dynamics and present a correction scheme to remove the dynamic distortions. Specifically, ns-scale time constants for overall rotation are dilated significantly but sub-ns time constants for internal motions are dilated modestly, while all motional amplitudes are unaffected. The correction scheme involves contraction of the time constants, with the contraction factor a linear function of the time constant to be corrected. The corrected dynamics of eight proteins are validated by NMR data for rotational diffusion and for backbone amide and side-chain methyl relaxation. The present work demonstrates that, even for complex systems like proteins with dynamics spanning multiple timescales, one can predict how thermostats distort protein dynamics and remove such distortions. The correction scheme will have wide applications, facilitating force-field parameterization and propelling simulations to be on par with NMR and other experimental techniques in determining dynamic properties of proteins.

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category: biophysics


title: A mechano-osmotic feedback couples cell volume to the rate of cell deformation

abstract: Mechanics has been a central focus of physical biology in the past decade. In comparison, the osmotic and electric properties of cells are less understood. Here we show that a parameter central to both the physics and the physiology of the cell, its volume, depends on a mechano-osmotic coupling. We found that cells change their volume depending on the rate at which they change shape, when they spread, migrate or are externally deformed. Cells undergo slow deformation at constant volume, while fast deformation leads to volume loss. We propose a mechano-sensitive pump and leak model to explain this phenomenon. Our model and experiments suggest that volume modulation depends on the state of the actin cortex and the coupling of ion fluxes to membrane tension. This mechano-osmotic coupling defines a membrane tension homeostasis module constantly at work in cells, causing volume fluctuations associated with fast cell shape changes, with potential consequences on cellular physiology.


title: TissueGrinder, a novel technology for rapid generation of patient-derived single cell suspensions from solid tumors by mechanical tissue dissociation

abstract:
IntroductionRecent advances hold promise of making personalized medicine a step closer to implementation in clinical settings. However, traditional sample preparation methods are not robust and reproducible. In this study, the TissueGrinder, a novel mechanical semi-automated benchtop device, which can isolate cells from tissue in a very fast and enzyme-free way is tested for cell isolation from surgically resected tumor tissues.

Methods33 surgically resected tumor tissues from various but mainly pancreatic, liver or colorectal origins were processed by both novel TissueGrinder and explant method. An optimized processing program for tumors from pancreatic, liver or colorectal cancer was developed. The viability and morphological characteristics of the isolated cells were evaluated microscopically. Expression of pancreatic cancer markers was evaluated in cells isolated from pancreatic tumors. Finally, the effect of mechanical stress on the cells was evaluated by assessing apoptosis markers via western blotting.

ResultsTissueGinder was more efficient in isolating cells from tumor tissue with a success rate of 75% when compared to explant method 45% in terms of cell outgrowth six weeks after processing. Cells isolated with TissueGinder had a higher abundance and were more heterogeneous in composition as compared to explant method. Mechanical processing of the cells with TissueGrinder does not lead to apoptosis but causes slight stress to the cells.

DiscussionOur results show that TissueGrinder can process solid tumor tissues more rapidly and efficiently and with higher success rate compared to the conventionally used explant method. The results of the study suggest that the TissueGrinder might be a suitable method for obtaining cells, which is important for its application in individualized therapy. Due to the great variance in different tumor entities and the associated individual tissue characteristics, a further development of the dissociation protocol for other types of tumors and normal will be targeted.


title: Vitamin D modulation of mitochondrial oxidative metabolism and mTOR enforces stress tolerance and anti-cancer responses

abstract: The relationship between vitamin D and reactive oxygen species (ROS), two integral signaling and damaging molecules of the cell, is poorly understood. This is striking, given that both factors are involved in cancer cell regulation and metabolism. Mitochondria (mt) dysfunction is one of the main drivers of cancer, producing higher cellular energy and ROS that can enhance oxidative stress and stress tolerance responses. To study the effects of vitamin D on metabolic and mt dysfunction, we used the vitamin D receptor (VDR)-sensitive MG-63 osteosarcoma cell model. Using biochemical approaches, active vitamin D (1,25-dihydroxyvitamin D, 1,25(OH)2D3) decreased mt ROS levels, membrane potential ({Delta}{Psi}mt), biogenesis, and translation, while enforcing endoplasmic reticulum/mitohormetic stress tolerance responses. Using a mitochondria-focused transcriptomic approach, gene set enrichment and pathway analyses show that 1,25(OH)2D3 lowered mt fusion/fission and oxidative phosphorylation (OXPHOS). By contrast, mitophagy, ROS defense, and epigenetic gene regulation were enhanced after 1,25(OH)2D3 treatment, as well as key metabolic enzymes that regulate fluxes of substrates for cellular architecture and a shift toward non-oxidative energy metabolism. ATACseq revealed putative oxi-sensitive and tumor-suppressing transcription factors that may regulate important mt functional genes such as the mTORC1 inhibitor, DDIT4/REDD1. DDIT4/REDD1 was predominantly localized to the outer mt membrane in untreated MG-63 cells yet sequestered in the cytoplasm after 1,25(OH)2D3 and rotenone treatments, suggesting a level of control by membrane depolarization to facilitate its cytoplasmic mTORC1 inhibitory function. The results show that vitamin D activates distinct adaptive metabolic responses involving mitochondria to regain redox balance and control the growth of cancer cells.


title: Metabolomics of lung microdissections reveals region- and sex-specific metabolic effects of acute naphthalene exposure in mice

abstract: Naphthalene is a ubiquitous environmental contaminant produced by combustion of fossil fuels and is a primary constituent of both mainstream and side stream tobacco smoke. Naphthalene elicits region-specific toxicity in airway club cells through cytochrome P450 (P450)-mediated bioactivation, resulting in depletion of glutathione and subsequent cytotoxicity. While effects of naphthalene in mice have been extensively studied, few experiments have characterized global metabolomic changes in the lung. In individual lung regions, we found metabolomic changes in microdissected mouse lung conducting airways and parenchyma obtained from animals sacrificed 2, 6, and 24 hours following naphthalene treatment. Data on 577 unique identified metabolites were acquired by accurate mass spectrometry-based assays focusing on lipidomics and non-targeted metabolomics of hydrophilic compounds. Statistical analyses revealed distinct metabolite profiles between the two major lung regions. In addition, the number and magnitude of statistically significant exposure-induced changes in metabolite abundance were different between lung airways and parenchyma for unsaturated lysophosphatidylcholines (LPCs), dipeptides, purines, pyrimidines, and amino acids. Importantly, temporal changes were found to be highly distinct for male and female mice, with males exhibiting predominant treatment-specific changes only at two hours post-exposure. In females, metabolomic changes persisted until six hours post-naphthalene treatment, which may explain the previously characterized higher susceptibility of female mice to naphthalene toxicity. In both males and females, treatment-specific changes corresponding to lung remodeling, oxidative stress response, and DNA damage were observed, which may provide insights into potential mechanisms contributing to the previously reported effects of naphthalene exposure in the lung.


title: The Innate Immune Response to Invasive Pulmonary Aspergillosis: A Systems Modeling Approach

abstract: Aspergillus species are ubiquitous environmental molds, the spores of which are inhaled daily by most humans. Immunocompromised hosts can develop an invasive infection resulting in high mortality. There is, therefore, a pressing need for host-centric therapeutics for this infection. To address this need, we created a multi-scale computational model of the infection, focused on its interaction with the innate immune system and iron, a critical nutrient for the pathogen. The model, parameterized using published data, was found to recapitulate a wide range of biological features and was experimentally validated in vivo. Conidial swelling was identified as critical in fungal strains with high growth, whereas the siderophore secretion rate is an essential prerequisite for establishment of the infection in low-growth strains. In immunocompetent hosts, high growth, high swelling probability, and impaired leukocyte activation lead to a high conidial germination rate. Similarly, in neutropenic mice, high fungal growth was achieved through synergy between high growth rate, high swelling probability, slow leukocyte activation, and high siderophore secretion. In summary, the model reveals a small set of parameters related to fungal growth, iron acquisition, and leukocyte activation as key determinants of the fate of the infection.

category: systems biology


title: Multiscale mechanistic modelling of the host defense in invasiveaspergillosis reveals leukocyte activation and iron acquisition as driversof infection outcome


title: Nanoscale organization and calcium influx in dendritic spines guarantee ER store refilling without depletion

abstract: Dendritic spines are critical components of the neuronal synapse as they receive and transform the synaptic input into a succession of biochemical events regulated by calcium signaling. The spine apparatus (SA), an extension of smooth endoplasmic reticulum (ER), regulates slow and fast calcium dynamics in spines. Calcium release events from SA result in a rapid depletion of calcium ion reservoir, yet the next cycle of signaling requires replenishment of SA calcium stores. How dendritic spines achieve this without triggering calcium release remains unclear. Using computational modeling, calcium and STED super-resolution imaging, we showed that the refilling of calcium-deprived SA involves store-operated calcium entry during spontaneous calcium transients in spine heads. We identified two main conditions that guarantee SA replenishment without depletion: (1) a small amplitude and slow timescale for calcium influx, and (2) a close proximity between SA and plasma membranes. Thereby, molecular nano-organization creates the conditions for a clear separation between SA replenishment and depletion. We further conclude that the nanoscale organization of SA receptors underlies the specificity of calcium dynamics patterns during the induction of long-term synaptic changes.


title: Shotgun Immunoproteomic Approach for the Discovery of Linear B Cell Epitopes in Biothreat Agents Francisella tularensis and Burkholderia pseudomallei

abstract: Peptide-based subunit vaccines are coming to the forefront of current vaccine approaches, with safety and cost-effective production among their top advantages. Peptide vaccine formulations consist of multiple synthetic linear epitopes that together trigger desired immune responses that can result in robust immune memory. The advantages of peptide epitopes are their simple structure, ease of synthesis, and ability to stimulate immune responses by means that do not require complex 3D conformation. Identification of linear epitopes is currently an inefficient process that requires thorough characterization of previously identified full-length protein antigens, or laborious techniques involving genetic manipulation of organisms. In this study, we apply a newly developed generalizable screening method that enables efficient identification of B cell epitopes in the proteomes of pathogenic bacteria. As a test case, we used this method to identify epitopes in the proteome of Francisella tularensis (Ft), a Select Agent with a well-characterized immunoproteome. Our screen identified many peptides that map to known antigens, including verified and predicted outer membrane proteins and extracellular proteins, validating the utility of this approach. We then used the method to identify seroreactive peptides in the less characterized immunoproteome of Select Agent Burkholderia pseudomallei (Bp). This screen revealed known Bp antigens as well as proteins that have not been previously identified as antigens. The present workflow is easily adaptable to detecting peptide targets relevant to the immune systems of other mammalian species, including humans (depending upon the availability of convalescent sera from patients), and could aid in accelerating the discovery of B cell epitopes and development of vaccines to counter emerging biological threats.


title: Context-aware genomic surveillance reveals hidden transmission of a carbapenemase-producing Klebsiella pneumoniae

abstract: Genomic surveillance can inform effective public health responses to pathogen outbreaks. However, integration of non-local data is rarely done. We investigate two large hospital outbreaks of a carbapenemase-carrying Klebsiella pneumoniae strain in Germany and show the value of contextual data. By screening more than ten thousand genomes, 500 thousand metagenomes, and two culture collections using in silico and in vitro methods, we identify a total of 415 closely related genomes reported in 28 studies. We identify the relationship between the two outbreaks through time-dated phylogeny, including their respective origin. One of the outbreaks presents extensive hidden transmission, with descendant isolates only identified in other studies. We then leverage the genome collection from this meta-analysis to identify genes under positive selection. We thereby identify an inner membrane transporter (ynjC) with a putative role in colistin resistance. Contextual data from other sources can thus enhance local genomic surveillance at multiple levels and should be integrated by default when available.


title: BROADLY NEUTRALIZING ANTI- HIV-1 ANTIBODIES DO NOT INHIBIT HIV-1 ENV-MEDIATED CELL-CELL FUSION

abstract: PG9, PG16, PGT121, and PGT145 antibodies were identified from culture media of activated memory B-cells of an infected donor and shown to neutralize many HIV-1 strains. Since HIV-1 spreads via both free virions and cell-cell fusion, we examined the effect of the antibodies on HIV-1 Env-mediated cell-cell fusion. Clone69TRevEnv cells that express Env in the absence of tetracycline were labeled with Calcein-AM Green, and incubated with CD4+ SupT1 cells labeled with CellTrace Calcein Red-Orange, with or without antibodies. Monoclonal antibodies PG9, PG16, 2G12, PGT121, and PGT145 (at up to 50 g/mL) had little or no inhibitory effect on fusion between HIV-Env and SupT1 cells. By contrast, Hippeastrum hybrid agglutinin completely inhibited fusion. Our results indicate that transmission of the virus or viral genetic material would not be inhibited by these broadly neutralizing antibodies. Thus, antibodies generated by HIV-1 vaccines should be screened for their inhibitory effect on Env-mediated cell-cell fusion.


title: Vestibular and auditory hair cell regeneration following targeted ablation of hair cells with diphtheria toxin in zebrafish

abstract: Millions of Americans experience hearing or balance disorders due to loss of hair cells in the inner ear. The hair cells are mechanosensory receptors used in the auditory and vestibular organs of all vertebrates as well as the lateral line systems of aquatic vertebrates. In zebrafish and other non-mammalian vertebrates, hair cells turn over during homeostasis and regenerate completely after being destroyed or damaged by acoustic or chemical exposure. However in mammals, destroying or damaging hair cells results in permanent impairments to hearing or balance. We sought an improved method for studying hair cell damage and regeneration in adult aquatic vertebrates by generating a transgenic zebrafish with the capacity for targeted and inducible hair cell ablation in vivo. This model expresses the human diphtheria toxin receptor (hDTR) gene under the control of the myo6b promoter, resulting in hDTR expressed only in hair cells. Cell ablation is achieved by an intraperitoneal injection of diphtheria toxin (DT) in adult zebrafish or DT dissolved in the water for larvae. In the lateral line of 5 dpf zebrafish, ablation of hair cells by DT treatment occurred within 2 days in a dose-dependent manner. Similarly, in adult utricles and saccules, a single intraperitoneal injection of 0.05 ng DT caused complete loss of hair cells in the utricle and saccule by 5 days post-injection. Full hair cell regeneration was observed for the lateral line and the inner ear tissues. This study introduces a new method for efficient conditional hair cell ablation in adult zebrafish inner ear sensory epithelia (utricles and saccules) and demonstrates that zebrafish hair cells will regenerate in vivo after this treatment.