title: Olfactory Sensitivity Aligns with Caste Revealing Multipotential Minors and Specialized Soldier Majors in Camponotus floridanus Ants

abstract:
Camponotus floridanus ant colonies are comprised of a single reproductive queen and thousands of sterile female offspring that consist of two morphologically distinct castes: smaller minors and larger majors. Minors perform most of the tasks within the colony, including brood care and food collection, whereas majors have fewer clear roles and have been hypothesized to act as a specialized solider caste associated with colony defense. The allocation of workers to these different tasks depends on the detection and processing of local information including pheromones and other chemical blends such as cuticular hydrocarbons. We examined the electrophysiological responses to general odorants, cuticular extracts, and a trail pheromone in adult minor and major C. floridanus workers, revealing that the repertoire of social behaviors is positively correlated with olfactory sensitivity. Minors in particular display primarily excitatory responses to olfactory stimuli, whereas major workers respond primarily with inhibitory signals. The notable exception to this paradigm is that both minors and majors display robust, dose-dependent excitatory responses to conspecific, non-nestmate cuticular extracts. Moreover, while both minors and majors actively aggress non-nestmate foes, majors display significantly enhanced capabilities to rapidly subdue and kill opponents. Overall, our data suggest that C. floridanus majors do indeed represent a physiologically and behaviorally specialized soldier caste and support a model in which caste-specific olfactory sensitivity plays an important role in task allocation and the regulation of social behavior in ant colonies.

Significance StatementThe detection and odor coding of chemical cues are essential components of the collective behavior observed in eusocial ants. To better understand the interdependent relationship between olfactory sensitivity and the allocation of worker castes to the various tasks critical for the success of the colony, a series of behavioral assessments and an electrophysiological survey of the antennae comprising general odorants, cuticular extracts, and a trail pheromone were undertaken. These studies reveal the behavioral repertoire of minors and majors aligned with profound shifts in olfactory sensitivity and odor coding. Our data support the hypothesis that minors are multipotential workers with broad excitatory sensitivity, and majors are dedicated soldiers with a highly specialized olfactory system for the detection of non-nestmate foes.


title: Efflux-linked Accelerated Evolution of Antibiotic Resistance at a Population Edge

abstract: Efflux is a common mechanism of resistance to antibiotics. We show that efflux itself promotes accumulation of antibiotic-resistance mutations (ARM). This phenomenon was initially discovered in a bacterial swarm where the linked phenotypes of high efflux and high mutation frequencies spatially segregated to the swarm edge, driven there by motility. We uncovered and validated a global regulatory network that connects high efflux to downregulation of specific DNA repair pathways even in non-swarming states. The efflux-DNA repair link was corroborated in a clinical  resistome database: genomes with mutations that increase efflux exhibit a significant increase in ARMs. Accordingly, efflux inhibitors decreased evolvability to antibiotic resistance. Swarms also revealed how bacterial populations serve as reservoir of ARMs even in absence of antibiotic selection pressure. High efflux at the edge births mutants that, despite compromised fitness, survive there because of reduced competition. This finding is relevant to biofilms where efflux activity is high.

category: microbiology


title: Bacterial threat assessment of bacteriophage infection is mediated by intracellular polyamine accumulation and Gac/Rsm signaling

abstract:
When eukaryotic cells are killed by pathogenic microorganisms, damage-associated and pathogen-associated signals are generated that alert other cells of nearby danger. Bacteria can detect the death of their kin; however, how bacteria make threat assessments of cellular injury is largely unexplored. Here we show that polyamines released by lysed bacteria serve as damage-associated molecules in Pseudomonas aeruginosa. In response to exogenous polyamines, Gac/Rsm and cyclic-di-GMP signaling is activated and intracellular polyamine levels increase. In the absence of a threat, polyamines are catabolized, and intracellular polyamines return to basal levels, but cells infected by bacteriophage increase and maintain intracellular polyamine levels, which inhibits phage replication. Phage species not inhibited by polyamines did not trigger polyamine accumulation by P. aeruginosa, suggesting polyamine accumulation and metabolism are targets in the phage-host arms-race. Our results suggest that like eukaryotic cells, bacteria can differentiate damage-associated and pathogen-associated signals to make threat assessments of cellular injury.

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title: Allopeptimicins: unique antibacterial metabolites generated by hybrid PKS-NRPS, with original self-defense mechanism in Actinoallomurus

abstract: In the search for structurally novel metabolites with antibacterial activity, innovative approaches must be implemented to increase the probability of discovering novel chemistry from microbial sources. Here we report on the application of metabolomic tools to the genus Actinoallomurus, a poorly explored member of the Actinobacteria. From examining extracts derived from 88 isolates belonging to this genus, we identified a family of cyclodepsipeptides acylated with a C20 polyketide chain, which we named allopeptimicins. These molecules possess unusual structural features, including several double bonds in the amino-polyketide chain and four non-proteinogenic amino acids in the octapeptide. Remarkably, allopeptimicins are produced as a complex of active and inactive congeners, the latter carrying a sulfate group on the polyketide amine. This modification is also a mechanism of self-protection in the producer strain. The structural uniqueness of allopeptimicins is reflected in a biosynthetic gene cluster showing a mosaic structure, with dedicated gene cassettes devoted to formation of specialized precursors and modular assembly lines related to those from different pathways.


title: Overexpression of the WAPO-A1 gene increases the number of spikelets per spike in bread wheat

abstract: Two homoeologous QTLs for number of spikelets per spike (SPS) were mapped on chromosomes 7AL and 7BL using two wheat MAGIC populations. Sets of lines contrasting for the QTL on 7AL were developed which allowed for the validation and fine mapping of the 7AL QTL and for the identification of a previously described candidate gene, WHEAT ORTHOLOG OF APO1 (WAPO1). Using transgenic overexpression in both a low and a high SPS line, we provide a functional validation for the role of this gene in determining SPS also in hexaploid wheat. We show that the expression levels of this gene positively correlate with SPS in multiple MAGIC founder lines under field conditions as well as in transgenic lines grown in the greenhouse. This work highlights the potential use of WAPO1 in hexaploid wheat for further yield increases. The impact of WAPO1 and SPS on yield depends on other genetic and environmental factors, hence, will require a finely balanced expression level to avoid the development of detrimental pleiotropic phenotypes.

category: plant biology


title: CryoGrid-PIXUL-RNA: High throughput RNA isolation platform for tissue transcript analysis

abstract:
Disease molecular complexity requires high throughput workflows to map disease pathways through analysis of vast tissue repositories. Great progress has been made in life sciences analytical technologies. To match the high throughput of these advanced analytical platforms, we have previously developed a multipurpose microplate sonicator, PIXUL, that can be used in multiple workflows to extract analytes from cultured cells and tissue fragments for various downstream molecular assays. And yet, the sample preparation devices, such as PIXUL, along with the downstream analytical capabilities have not been fully exploited to interrogate tissues because storing and sampling of such specimens remain, in comparison, inefficient. To mitigate this bottleneck, we have developed a low-cost user-friendly system, the CryoGrid, that consists of CryoBlock, thermometer/thermocouple, and QR coded CryoTrays to freeze and store frozen tissue fragments, and hand-held CryoCore tool for tissue sampling supported by iPad and Google apps to display tissues, direct coring and share metadata.

RNA is one of the most studied analytes. There is a decades-long history of developing methods to isolate and analyze RNA. Still, the throughput of sampling and RNA extraction from tissues has not matched that of the high throughput transcriptome analytical platforms. To address this need, we have integrated the CryoGrid system with PIXUL-based methods to isolate RNA for gene-specific qPCR and genome-wide transcript analyses. TRIzol is commonly used to isolate RNA but it is labor-intensive, hazardous, requires fume-hoods, and is an expensive reagent. We developed a PIXUL-based TRIzol-free RNA isolation fast protocol that uses a buffer containing proteinase K (PK). Virtually every disease (and often therapeutic agents toxicity) is a systemic syndrome but often only one organ is examined. CryoGrid-PIXUL, integrated with either TRIzol or PK buffer RNA isolation protocols, yielded similar RNA profiles in a multiorgan (brain, heart, kidney and liver) mouse model of sepsis. Thus, RNA isolation using the CryoGrid-PIXUL system combined with the PK buffer offers an inexpensive user-friendly workflow to study transcriptional responses in tissues in health and disease as well as in therapeutic interventions.

category: molecular biology


title: Spatial-CITE-seq: spatially resolved high-plex protein and whole transcriptome co-mapping

abstract: We present spatial-CITE-seq for high-plex protein and whole transcriptome co-mapping, which was firstly demonstrated for profiling 189 proteins and transcriptome in multiple mouse tissue types. It was then applied to human tissues to measure 273 proteins and transcriptome that revealed spatially distinct germinal center reaction in tonsil and early immune activation in skin at the COVID-19 mRNA vaccine injection site. Spatial-CITE-seq may find a range of applications in biomedical research.

category: genomics


title: Late maturation of executive control promotes conceptual development

abstract: Control processes are critical for the generation of task-appropriate behaviour across cognitive domains, yet children have a long developmental period with reduced executive control. Traditionally, this is viewed as a negative but necessary consequence of the time taken to learn control processes and develop the prefrontal cortex. Here, we exploit a recent computational model of controlled semantic cognition to formally test an alternative (yet perhaps complementary) view that a developmental period without control promotes conceptual knowledge acquisition. Our simulations show that maturational delay and anatomical connectivity conspire to promote conceptual learning. Learning conceptual structure necessitates a connection from control to more peripheral regions rather than the deep multimodal hub. Delayed control speeds conceptual learning without compromising conceptual representations, particularly when control connects to intermediate layers. These results are counterintuitive as delayed control onset can produce overall faster mastery of controlled behaviours. To assess whether delayed semantic control is also observed over development, we conducted a meta-analysis of the classic triadic matching task where participants decide which of two options best matches a third. Matching can be based on taxonomic or thematic relations, and when these conflict, participants must use task context to determine which relation to choose--that is, they must exert semantic control. Children aged 2 have the representations to identify both relations when distractors are unrelated, but when relations conflict, they cannot use context to guide their choices. Context-sensitivity develops later than conceptual structure and shows large increases around 6, a critical time for executive function across domains.

category: neuroscience


title: Explaining flexible continuous speech comprehension from individual motor rhythms

abstract: When speech is too fast, the tracking of the acoustic signal along the auditory pathway deteriorates, leading to suboptimal speech segmentation and decoding of speech information. Thus, speech comprehension is limited by the temporal constraints of the auditory system. Previous research suggests that individual differences in auditory-motor coupling strength might shape such temporal constraints. In two behavioral experiments, we characterize individual differences in the comprehension of naturalistic speech as function of the individual coupling strength between the auditory and motor systems, and the preferred frequencies of the systems. As expected, speech comprehension declined at higher rates. Importantly, both, higher auditory-motor coupling and higher spontaneous speech motor production rates were related to better speech-comprehension performance. Furthermore, performance increased with higher linguistic sentence predictability (using a recurrent neural network) -particularly at higher speech rates and for individuals with high auditory-motor coupling strength- and with participants higher working memory capacity (Digit Span). These findings support the notion of an individual preferred auditory regime that allows for optimal auditory (speech) processing. Crucially, we provide evidence for a model that assigns a central role to motor-system-dependent individual flexibility in continuous speech comprehension.


title: Inter-Individual Variability in Dorsal Stream Dynamics During Word Production

abstract: The current standard model of language production involves a sensorimotor dorsal stream connecting areas in the temporo-parietal junction with those in the inferior frontal gyrus and lateral premotor cortex. These regions have been linked to various aspects of word production such as phonological processing or articulatory programming, primarily through neuropsychological and functional imaging group studies. Most if not all the theoretical descriptions of this model imply that the same network should be identifiable across individual speakers. We tested this hypothesis by quantifying the variability of activation observed across individuals within each dorsal stream anatomical region. This estimate was based on electrical activity recorded directly from the cerebral cortex with millisecond accuracy in awake epileptic patients clinically implanted with intracerebral depth electrodes for pre-surgical diagnosis. Each regions activity was quantified using two different metrics, intra-cerebral evoked related potentials and high gamma activity, at the level of the group, the individual, and the recording contact. Using picture naming task, the two metrics show simultaneous activation of parietal and frontal regions in line with models that posit interactive processing during word retrieval. They also reveal different levels of variability across brain regions and patients except in auditory and motor regions. The independence and non-uniformity of cortical activity according to the two metrics push the current model towards sub-second and sub-region explorations focused on individualized language speech production. Several hypotheses are considered for this within-region heterogeneity.


title: Robustness of the ferret model for influenza risk assessment studies: a cross-laboratory exercise

abstract: Ferrets represent the preferred animal model for assessing the transmission potential of newly emerged zoonotic influenza viruses. However, heterogeneity among established experimental protocols and facilities across different laboratories may lead to variable results, complicating interpretation of transmission experimental data. Between 2018-2020, a global exercise was conducted by 11 participating laboratories to assess the range of variation in ferret transmission experiments using two common stock H1N1 influenza viruses that possess different transmission characteristics in ferrets. Inoculation route, dose, and volume were standardized, and all participating laboratories followed the same experimental conditions for respiratory droplet transmission, including a strict 1:1 donor:contact ratio. Additional host and environmental parameters likely to affect influenza transmission kinetics were monitored throughout. Overall transmission outcomes for both viruses across 11 laboratories were concordant, suggesting the robustness of the ferret model for zoonotic influenza risk assessment. To attain high confidence in identifying zoonotic influenza viruses with moderate-to-high or low transmissibility, our analyses support that as few as three but as many as five laboratories, respectively, would need to independently perform viral transmission experiments with concordant results. This exercise facilitates the development of a more homogenous protocol for ferret transmission experiments that are employed for the purposes of risk assessment.


title: Engineering a more specific E. coli glyoxylate/hydroxypyruvate reductase for coupled steady state kinetics assays

abstract: The E. coli glyoxylate reductase/hydroxypyruvate reductase A (EcGhrA) was investigated as a coupling enzyme to monitor the transamination of 2-ketoarginine and glycine by the L-enduracididine biosynthetic enzyme MppQ. Surprisingly, 2-ketoarginine proved to be an efficient substrate for EcGhrA. Since the promiscuity of EcGhrA prevented its use as a coupling enzyme to monitor the aminotransferase activity of MppQ, we set about engineering a more specific variant. X-ray crystal structures of EcGhrA were determined in the unliganded state, as well as with glyoxylate and 2-ketoarginine bound. The electron density maps of EcGhrA with 2-ketoarginine bound showed weak electron density for the side chain of this substrate, complicating the choice of active site residues to target for site-directed mutagenesis. The structure of the complex did, however, suggest that the side chain of W45 could interact with the guanidinium group of 2-ketoarginine. We therefore generated the EcGhrAW45F variant and tested it for activity with 2-ketoarginine, glyoxylate, oxaloacetate, -ketoglutarate, -oxofuranacetic acid, phenyl pyruvate, 3-mercaptopyruvate and 2-ketobutyric acid. The W45F variant exhibited a [~]10-fold decrease in the specificity constant (kcat/KM) for 2-ketoarginine, while the reaction with glyoxylate was not significantly impaired. The reactions of the W45F variant with the alternative substrates oxaloacetate and -ketoglutarate were also impaired. Thus, the W45F variant is a less promiscuous enzyme than the wild-type. This engineered EcGhrAW45F variant could be generally useful as a coupling system for enzymes that produce glyoxylate, such as 4-hydroxy-2-oxoglutarate aldolase or isocitrate lyase.

category: biochemistry


title: The optimal strategy of incompatible insect technique (IIT) using Wolbachia to control Malaria

abstract: For decades, techniques to control vector population with low environmental impact have been widely explored in both field and theoretical studies. The incompatible insect method (IIT) using Wolbachia, based on cytoplasmic incompatibility, is a technique that Wolbachia-infected male mosquitoes are incapable of producing viable offspring after mating with wildtype female mosquitoes. While the IIT method experimentally ensured its effectiveness in several field works, the failure of female mosquito population control owing to the accidental contamination of Wolbachia-infected female mosquitoes has been a concern and an obstacle in implementing the IIT method in nature. In this study, we developed a population-based IIT mathematical model using cytoplasmic incompatibility and evaluated the effectiveness of the IIT method in scenarios where contamination was present or absent. In addition, by extending the model to assess the disease infection status of the human population with malaria, we evaluated the optimal release strategy and cost for successful disease control. Our study proves that IIT could be a promising method to control mosquito-borne diseases without perfect eradication of vector mosquito population regardless of contamination.

category: pathology


title: Building molecular band-pass filters via molecular sequestration

abstract: Engineered genetic circuits with tailored functions that mimic how cells process information in changing environments (e.g. cell fate decision, chemotaxis, immune response) have great applications in biomedicine and synthetic biology. Although there is a lot of progress toward the design of gene circuits yielding desired steady states (e.g. logic-based networks), building synthetic circuits for dynamic signal processing (e.g. filters, frequency modulation, and controllers) is still challenging. Here, we provide a model-based approach to build gene networks that can operate as band-pass filters by taking advantage of molecular sequestration. By suitably approximating the dynamics of molecular sequestration, we analyze an Incoherent Feed-Forward Loop (IFFL) and a Negative Feedback (NF) circuit and illustrate how they can achieve band-pass filter behavior. Computational analysis shows that a circuit that incorporates both IFFL and NF motifs improves the filter performance. Our approach facilitates the design of sequestration-based filters, and may support the synthesis of molecular controllers with desired specifications.

category: synthetic biology


title: Modulation of C5a-C5aR1 signaling alters the dynamics of AD progression

abstract:
BackgroundThe complement system is part of the innate immune system that clears pathogens and cellular debris. In the healthy brain, complement influences neurodevelopment and neurogenesis, synaptic pruning, clearance of neuronal blebs, recruitment of phagocytes, and protects from pathogens. However, excessive downstream complement activation that leads to generation of C5a, and C5a engagement with its receptor C5aR1, instigates a feed-forward loop of inflammation, injury, and neuronal death, making C5aR1 a potential therapeutic target for neuroinflammatory disorders. C5aR1 ablation in the Arctic (Arc) model of Alzheimers disease protects against cognitive decline and neuronal injury without altering amyloid plaque accumulation.

MethodsTo elucidate the effects of C5a-C5aR1 signaling on AD pathology, we crossed Arc mice with a C5a overexpressing mouse (ArcC5a+) and tested hippocampal memory. RNA-seq was performed on hippocampus and cortex from Arc, ArcC5aR1KO, and ArcC5a+ mice at 2.7-10 months and age-matched controls to assess mechanisms involved in each system. Immunohistochemistry was used to probe for protein markers of microglia and astrocytes activation states.

ResultsArcC5a+ mice had accelerated cognitive decline compared to Arc. Deletion of C5ar1 delayed or prevented the expression of some, but not all, AD-associated genes in the hippocampus and a subset of pan-reactive and A1 reactive astrocyte genes, indicating a separation between genes induced by amyloid plaques alone and those influenced by C5a-C5aR1 signaling.. Biological processes associated with AD and AD mouse models, including inflammatory signaling, microglial cell activation, and astrocyte migration, were delayed in the ArcC5aR1KO hippocampus. Interestingly, C5a overexpression also delayed the increase of some AD-, complement-, and astrocyte-associated genes, suggesting the possible involvement of neuroprotective C5aR2. However, these pathways were enhanced in older ArcC5a+ mice compared to Arc. Immunohistochemistry confirmed that C5a-C5aR1 modulation in Arc mice delayed the increase in CD11c-positive microglia, while not affecting other pan-reactive microglial or astrocyte markers.

ConclusionC5a-C5aR1 signaling in AD largely exerts its effects by enhancing microglial activation pathways that accelerate disease progression. While C5a may have neuroprotective effects via C5aR2, engagement of C5a with C5aR1 is detrimental in AD models. These data support specific pharmacological inhibition of C5aR1 as a potential therapeutic strategy to treat AD.


title: Pleiotrophin signals through ALK receptor to enhance growth of neurons in the presence of inhibitory CSPGs

abstract:
Chondroitin sulfate proteoglycans (CSPGs), one of the major extracellular matrix components of the glial scar that surrounds central nervous system (CNS) injuries, are known to inhibit the regeneration of neurons. This study investigated whether pleiotrophin (PTN), a growth factor upregulated during early CNS development, can overcome the inhibition mediated by CSPGs and promote the neurite outgrowth of neurons in vitro. The data showed that a CSPG matrix inhibited the outgrowth of neurites in primary cortical neuron cultures compared to a control matrix. PTN elicited a dose dependent increase in the neurite outgrowth even in the presence of the growth inhibitory CSPG matrix, with optimal growth at 15 ng mL-1 of PTN (114.8% of neuronal outgrowth relative to laminin control). The growth promoting effect of PTN was blocked by inhibition of the receptor anaplastic lymphoma kinase (ALK) by alectinib in a dose dependent manner. Neurite outgrowth in the presence of this CSPG matrix was induced by activation of the protein kinase B (AKT) pathway, a key downstream mediator of ALK activation. This study identified PTN as a dose-dependent regulator of neurite outgrowth in primary cortical neurons cultured in the presence of a CSPG matrix, and identified ALK activation as a key driver of PTN-induced growth.

Summary statementFunction in the central nervous system (CNS) is attributed to the complex interactions of neurons and glia. These cells are anchored in extracellular matrix (ECM) which constitutes about 10% - 20% of brain volume. Cells in the brain produce different components of the ECM in brain including chondroitin sulfate proteoglycans (CSPGs). After a nervous system injury, glial cells produce excess CSPGs that restrict the regeneration of neurons, thus limiting functional recovery. This study examines the role of the endogenous growth factor pleiotrophin (PTN) in driving the growth of neurons even in the presence of inhibitory CSPGs, and anaplastic lymphoma kinase (ALK) receptor as a key mediator by which PTN potentiates growth.


title: The WalRK Two-component system is Essential for Proper Cell Envelope Biogenesis in Clostridioides difficile

abstract:
The WalR-WalK two-component regulatory system (TCS) is found in all Firmicutes, where it regulates the expression of multiple genes required for remodeling the cell envelope during growth and division. Unlike most TCSs, WalRK is essential for viability, so it has attracted interest as a potential antibiotic target. Here we used overexpression of WalR and CRISPR interference to investigate the Wal system of Clostridioides difficile, a major cause of hospital-associated diarrhea in high-income countries. We confirmed the wal operon is essential and identified morphological defects and cell lysis as the major terminal phenotypes of altered wal expression. We also used RNA-seq to identify over 150 genes whose expression changes in response to WalR levels. This gene set is enriched in cell envelope genes and includes several predicted PG hydrolases and proteins that could regulate PG hydrolase activity. A distinct feature of the C. difficile cell envelope is the presence of an S-layer and we found WalR affects expression of several genes which encode S-layer proteins. An unexpected finding was that some Wal-associated phenotypic defects were inverted in comparison to what has been reported in other Firmicutes. For example, down-regulation of Wal signaling caused C. difficile cells to become longer rather than shorter, as in Bacillus subtilis. Likewise, down-regulation of Wal rendered C. difficile more sensitive to vancomycin, whereas reduced Wal activity is linked to increased vancomycin resistance in Staphylococcus aureus.

ImportanceThe WalRK two-component system (TCS) is essential for coordinating synthesis and turnover of peptidoglycan in Firmicutes. Here we investigate the WalRK TCS in Clostridioides difficile, an important bacterial pathogen with an atypical cell envelope. We confirmed that WalRK is essential and regulates cell envelope biogenesis, although several of the phenotypic changes we observed were opposite to what has been reported in other Firmicutes. We also identified over 150 genes whose expression is controlled either directly or indirectly by WalR. Overall, our findings provide a foundation for future investigations of an important regulatory system and potential antibiotic target in C. difficile.


title: Robust Variation in Infant Gut Microbiome Assembly Across a Spectrum of Lifestyles

abstract:
Infant microbiome assembly is intensely studied in infants from industrialized nations, but little is known about this process in populations living non-industrialized lifestyles. In this study we deeply sequenced infant stool samples from the Hadza hunter-gatherers of Tanzania and analyzed them in a global meta-analysis. Infant microbiomes develop along lifestyle-associated trajectories, with over twenty percent of genomes detected in the Hadza infant gut representing phylogenetically diverse novel species. Industrialized infants, even those who are breastfed, have microbiomes characterized by a paucity of Bifidobacterium infantis and gene cassettes involved in human milk utilization. Strains within lifestyle-associated taxonomic groups are shared between mother-infant dyads, consistent with early-life inheritance of lifestyle-shaped microbiomes. The population-specific differences in infant microbiome composition and function underscore the importance of studying microbiomes from people outside of wealthy, industrialized nations.

Recognition of work on indigenous communitiesResearch involving indigenous communities is needed for a variety of reasons including to ensure that scientific discoveries and understanding appropriately represent all populations and do not only benefit those living in industrialized nations. Special considerations must be made to ensure that this research is conducted ethically and in a non-exploitative manner. In this study we performed deep metagenomic sequencing on fecal samples that were collected from Hadza hunter-gatherers in 2013/2014 and were analyzed in previous publications using different methods (1, 2). A material transfer agreement with the National Institute for Medical Research in Tanzania ensures that stool samples collected are used solely for academic purposes, permission for the study was obtained from the National Institute of Medical Research (MR/53i 100/83, NIMR/HQ/R.8a/Vol.IX/1542) and the Tanzania Commission for Science and Technology, and verbal consent was obtained from the Hadza after the studys intent and scope was described with the help of a translator. The publications that first described these samples included several scientists and Tanzanian field-guides as co-authors for the critical roles they played in sample collection, but as no new samples were collected in this study, only scientists who contributed to the analyses described here were included as co-authors in this publication. It is currently not possible for us to travel to Tanzania and present our results to the Hadza people, however we intend to do so once the conditions of the COVID-19 pandemic allow it.


title: Novel Requirement for Staphylococcal Cell Wall-Anchored Protein SasD in Pulmonary Infection

abstract:
Staphylococcus aureus can complicate preceding viral infections, including influenza virus. A bacterial infection combined with a preceeding viral infection, known as super-infection, leads to worse outcomes compared to single infection. Most of the super-infection literature focuses on the changes in immune responses to bacteria between homeostatic and virally infected lungs. However, it is unclear how much of an influence bacterial virulence factors have in super-infection. Staphylococcal species express a broad range of cell wall-anchored proteins (CWAs) that have roles in host adhesion, nutrient acquisition, and immune evasion. We screened the importance of these CWAs using mutants lacking individual CWAs in vivo in both bacterial pneumonia and influenza super-infection. In bacterial pneumonia, lacking individual CWAs led to varying decreases in bacterial burden, lung damage, and immune infiltration into the lung. However, the presence of a preceding influenza infection partially abrogated the requirement for CWAs. In the screen, we found that the uncharacterized CWA S. aureus surface protein D (SasD) induced changes in both inflammatory and homeostatic lung markers. We further characterized a SasD mutant (sasD A50.1) in the context of pneumonia. Mice infected with sasD A50.1 had decreased bacterial burden, inflammatory responses, and mortalty compared to wildtype S. aureus. Mice also had reduced levels of IL-1{beta} compared with wildtype, likely derived from macrophages. Reductions in IL-1{beta} transcript levels as well as increased macrophage viability implicate altered macrophage cell death pathways. These data identify a novel virulence factor for S. aureus that influences inflammatory signaling within the lung.

ImportanceStaphylococcus aureus is a common commensal bacteria that can cause severe infections, such as pneumonia. In the lung, viral infections increase the risk of staphylococcal pneumonia, leading to combined infections known as super-infections. The most common virus associated with S. aureus pneumonia is influenza, and super-infections lead to worse patient outcomes compared to either infection alone. While there is much known about how the immune system differs between healthy and virally infected lungs, the role of bacterial virulence factors in super-infection is less understood. The significance of our research is identifying new bacterial virulence factors that play a role in the initiation of infection and lung injury, which could lead to future therapies to prevent pulmonary single or super-infection with S. aureus.


title: Activation of the integrative and conjugative element Tn916 causes growth arrest and death of host bacteria

abstract: Integrative and conjugative elements (ICEs) serve as major drivers of bacterial evolution. These elements often confer some benefit to a host cell, including antibiotic resistance, metabolic capabilities, or pathogenic determinants. ICEs can also have negative impacts on their host cells. Here, we investigated the effects of the ICE (conjugative transposon) Tn916 on host cells. Because Tn916 is active in a relatively small subpopulation of host cells, we developed a fluorescent reporter system for monitoring activation of Tn916 in single cells. We found that when active in Bacillus subtilis and its natural host Enterococcus faecalis, Tn916 inhibited cell division and most cells died. We also observed these phenotypes on the population level in B. subtilis utilizing a modified version of Tn916 that can be activated in the majority of cells. We identified two genes (orf17 and orf16) in Tn916 that were sufficient to cause host growth defects and identified a single gene, yqaR, that is found in a defective phage (skin) in the B. subtilis chromosome that is required for this phenotype. However, these three genes are only partially responsible for the growth defect caused by Tn916, indicating that Tn916 possesses multiple mechanisms to affect growth and viability of host cells. These results highlight the complex relationships that conjugative elements have with their host cells and the interplay between mobile genetic elements.


title: Extensive heterogeneity of the HIV-1 infected CD4+ T-cell reservoir revealed by single-cell viral ASAPseq

abstract:
Defining the long-lived HIV reservoir during antiretroviral therapy remains a challenge for HIV cure research. To determine if unifying cell surface or genomic features can be identified in unperturbed HIV-1 infected CD4+ T-cells, we developed single-cell viral ASAPseq, which leverages scATACseq and surface marker profiling to define HIV-infected cells via the presence of integrated proviral DNA. Here, we employed this strategy to define infected CD4+ T-cells both in vitro and ex vivo from blood and lymph nodes of, respectively, treated and untreated people living with HIV. In each setting, we identified HIV-infected cells with both novel and known surface markers associated with provirus. We also uncovered epigenetic profiles and transcription factor motifs enriched in HIV-infected cells that more deeply define the reservoir. Our data highlight the broad heterogeneity of the HIV reservoir and create a foundation for a multiomic atlas to develop targeted elimination strategies.

Graphical Abstract

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title: The fluorescence-activating and absorption-shifting tag (FAST) enables live-cell fluorescence imaging of Methanococcus maripaludis

abstract:
Live-cell fluorescence imaging in methanogenic archaea has been limited due to the strictly anoxic conditions required for growth and issues with autofluorescence associated with electron carriers in central metabolism. Here, we show that the fluorescence-activating and absorption-shifting tag (FAST) when complexed with the fluorogenic ligand 4-hydroxy-3-methylbenzylidene-rhodanine (HMBR) overcomes these issues and displays robust fluorescence in Methanococcus maripaludis. We also describe a mechanism to visualize cells under anoxic conditions using a fluorescence microscope. Derivatives of FAST were successfully applied for protein abundance analysis, subcellular localization, and determination of protein-protein interactions. FAST fusions to both formate dehydrogenase (Fdh) and F420-reducing hydrogenase (Fru) displayed increased fluorescence in cells grown on formate containing medium, consistent with previous studies suggesting increased abundance of these proteins in the absence of H2. Additionally, FAST fusions to both Fru and the ATPase associated with the archaellum (FlaI) showed membrane localization in single cells observed using anoxic fluorescence microscopy. Finally, a split reporter translationally fused to the alpha and beta subunits of Fdh reconstituted a functionally fluorescent molecule in vivo via bimolecular fluorescence complementation. Together, these observations demonstrate the utility of FAST as a tool for studying members of the methanogenic archaea.

ImportanceMethanogenic archaea are important members of anaerobic microbial communities where they catalyze essential reactions in the degradation of organic matter. Developing additional tools for studying the cell biology of these organisms is essential to understanding them at a mechanistic level. Here, we show that FAST, in combination with the fluorogenic ligand HMBR, can be used to monitor protein dynamics in live cells of M. maripaludis. Application of FAST holds promise for future studies focused on the metabolism and physiology of methanogenic archaea.


title: Control of pancreatic islet function and glucose homeostasis by a novel microexon program misregulated in type 2 diabetes

abstract: Pancreatic islets control glucose homeostasis by the balanced secretion of insulin and other hormones, and its abnormal function causes diabetes or hypoglycemia. Here, we uncover a conserved program of alternative microexons included in mRNAs of islet cells, particularly in genes involved in vesicle transport and exocytosis. Islet microexons (IsletMICs) are regulated by the RNA binding protein SRRM3 and represent a subset of the larger neural program that are particularly sensitive to the levels of this regulator. Both SRRM3 and IsletMICs are induced by elevated glucose levels, and depletion of SRRM3 in beta cell lines and mouse islets, or repression of particular IsletMICs using antisense oligonucleotides, leads to inappropriate insulin secretion. Consistently, SRRM3 mutant mice display defects in islet cell identity and function, leading to hyperinsulinemic hypoglycemia. Importantly, human genetic variants that influence SRRM3 expression and IsletMIC inclusion in islets are associated with fasting glucose variation and type 2 diabetes risk.


title: Mmp14-controlled extracellular matrix homeostasis is required for circadian rhythm

abstract: The circadian clock regulates extracellular matrix (ECM) homeostasis but whether altered ECM homeostasis regulates the clock was unknown. Thus, we generated mice with a Col1a2-Cre-ERT2 conditional deletion of matrix metalloproteinase-14 (Mmp14) to disrupt matrix homeostasis in postnatal mice. We show here that tamoxifen-treated Col1a2-Cre-ERT2::Mmp14F/F mice developed a severe tendon matrix phenotype with dorsiflexion of hind limbs, tissue thickening, and accumulation of hydroxylysine aldehyde (pyridinoline) crosslinked narrow-diameter collagen fibrils typically seen accumulating during embryonic development. Proteomic analysis of tamoxifen-treated postnatal mice showed [~]16% of detected proteins lost (176 proteins) or reversed (19 proteins) time-dependent rhythmicity. Ontological analysis identified  cytoskeleton as an affected system. Subsequent studies in CRISPR-Cas9 Mmp14 null cells showed decreased F-/G-actin ratio, decreased cell spreading, elevated levels of pyridinoline crosslink factor Plod2, nuclear translocation of transcription factor Pitx2, and loss of circadian rhythm. Thus, our study shows a bidirectional dependence of collagen-ECM homeostasis and the circadian clock.

category: cell biology


title: Xport-A functions as a chaperone by stabilizing the first 5 transmembrane domains of Rhodopsin-1

abstract: Rhodopsin-1 (Rh1), the main photo-sensitive protein of Drosophila, is a seven transmembrane domain protein, which is inserted co-translationally in the endoplasmic reticulum (ER) membrane. Maturation of Rh1 occurs in the ER, where various chaperones interact with Rh1 to aid in its folding and subsequent transport in the secretory pathway. Xport-A has been shown to be a chaperone/ transport factor for Rh1, but the exact molecular mechanism for Xport-A activity upon Rh1 is not known. Here, based on computational predictions, we propose a model where Xport-A functions as a chaperone in the biosynthesis of Rh1 by stabilizing the first 5 transmembrane domains of Rh1, but not the full length Rh1 protein.


title: Unifying genetic association tests via regression: Prospective and retrospective, parametric and non-parametric, and genotype- and allele-based tests

abstract:
Genetic association analysis, evaluating the relationship between genetic markers and complex and heritable traits, is the basis of genome-wide association studies. In response, many association tests have been developed, and they are generally classified as prospective vs. retrospective, parametric vs. non-parametric, and genotype- vs. allele-based association tests. While method classification is useful, it is confusing and challenging for practitioners to decide on the  optimal test to use for their data. Although there are known differences between some of the popular association tests, we provide new results that show the analytical connections between the different tests for both population- and family-based study designs.

ResumeInserer votre resume ici. We will supply a French abstract for those authors who cant prepare it themselves.


title: Appia: a web interface for chromatography

abstract: Chromatography is used ubiquitously in biology and chemistry to prepare, purify, and assess a wide variety of samples. Software written by chromatography instrument manufacturers is proprietary, which limits its use to licensed computers, and complex, which increases the burden of cognitive load on the user. Here we present Appia, a free, open-source chromatography processing and visualization package focused on making analysis, collaboration, and publication quick, easy, and aesthetically appealing.


title: Cotranslational folding and assembly of the dimeric E. coli inner membrane protein EmrE

abstract:
In recent years, it has become clear that many homo- and heterodimeric cytoplasmic proteins in both prokaryotic and eukaryotic cells start to dimerize cotranslationally, i.e., while at least one of the two chains is still attached to the ribosome. Whether this is possible also for integral membrane proteins is unknown, however. Here, we apply Force Profile Analysis (FPA) - a method where a translational arrest peptide (AP) engineered into the polypeptide chain is used to detect force generated on the nascent chain during membrane insertion - to demonstrate cotranslational interactions between a fully membrane-inserted monomer and a nascent, ribosome-tethered monomer of the E. coli inner membrane protein EmrE. Similar cotranslational interactions are also seen when the two monomers are fused into a single polypeptide. Further, we uncover an apparent intrachain interaction between E14 in TMH1 and S64 in TMH3 that forms at a precise nascent chain length during cotranslational membrane insertion of an EmrE monomer. Like soluble proteins, inner membrane proteins can thus both start to fold and start to dimerize during the cotranslational membrane-insertion process.

Significance statementMany water-soluble proteins are known to fold and even dimerize cotranslationally, i.e., when still attached to the ribosome. Here, we show that an E. coli inner membrane protein can also start to fold and dimerize cotranslationally, establishing the generality of these cotranslational maturation processes.


title: Highly multiplexed targeted plasma proteomics quantifies several hundred blood proteins in serum from colorectal carcinoma patients

abstract:
The rapid analysis of human serum and plasma can provide deep insights into changes of the blood proteome in response to different patient treatments or diseases. Targeted proteomics techniques, like SRM and PRM, can be utilized to monitor proteins at high sensitivitym but so far were limited to smaller protein panels, which can be monitored in one experiment. The recently, on a Bruker tims-TOF pro mass spectrometer, developed parallel reaction monitoring-parallel accumulation - serial fragmentation (prm-PASEF) method expands the standard PRM method by using ion-mobility. The use of ion mobility as a fourth separation dimension increases the proteome coverage while reducing the length of the necessary chromatogeaphic separation. By combining an isotope-labeled reference standard, which covers 579 plasma proteins, we were able to quantify 565 proteins in plasma using prm-PASEF, with the least abundant protein being quantified at 7 amol. We continued the analysis by combining the isotype-labeled reference standard with dia-PASEF, which allowed the quantification of 549 proteins. Both methods were used to analyze 20 patient plasma samples from a colorectal cancer (CRC) cohort. The analysis identified 16 differentially regulated proteins between the CRC patient and control individual plasma samples. 15 of the 16 proteins showed a high correlation to the mRNA expression in CRC tumor samples, showing the technique s potential for the rapid identification of potential biomarkers in larger cohorts, abolishing the need for preselection of potential biomarker proteins.

Graphical abstract

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title: Clinically-relevant treatment of PDX models reveals patterns of neuroblastoma chemoresistance

abstract:
Chemotherapy resistance and relapses are common in high-risk neuroblastoma (NB), an aggressive pediatric solid tumor of the sympathetic nervous system. Here, we developed a clinically-relevant in vivo treatment protocol mimicking the first line five-chemotherapy treatment regimen of high-risk NB and applied this protocol to mice with MYCN-amplified NB patient-derived xenografts (PDXs). Genomic and transcriptomic analyses were used to reveal the genetic and non-genetic mechanisms involved in NB chemoresistance. We observed convergent and parallel evolution of key NB genetic aberrations over time. Intrinsic resistance to chemotherapy was associated with high genetic diversity and an embryonic phenotype. Relapsed NB PDX tumors with acquired resistance showed an immature mesenchymal-like phenotype resembling multipotent Schwann cell precursors that are found in the adrenal gland. NBs with a successful treatment response presented a lineage-committed adrenergic phenotype similar to normal neuroblasts, reduced cell cycle gene expression, and negative regulation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascade. NB organoids established from relapsed PDX tumors retained drug resistance, tumorigenicity, and transcriptional cell states ex vivo. This work sheds light on mechanisms involved in NB chemotherapy response in vivo and ex vivo using a clinically-relevant protocol, and emphasizes the importance of transcriptional cell states in treatment response. Detailed characterization of resistance mechanisms is essential for the development of novel treatment strategies in non-responsive or relapsed high-risk NB.

One Sentence SummaryCOJEC chemotherapy treatment of neuroblastoma PDX models uncovers patterns of transcriptional plasticity and chemoresistance.

category: cancer biology


title: Oncogenic mutations in the DNA-binding domain of FOXO1 disrupt folding: quantitative insights from experiments and molecular simulations

abstract: FOXO1, a member of the family of winged-helix motif Forkhead box (FOX) transcription factors, is the most abundantly expressed FOXO member in mature B-cells. Sequencing of diffuse large B-cell lymphoma (DLBCL) tumors and cell lines identified specific mutations in the forkhead domain linked to loss of function. Differential scanning calorimetry and thermal shift assays were used to characterize how eight of these mutations affect the stability of the FOX domain. Mutations L183P and L183R were found to be particularly destabilizing. Electrophoresis mobility shift assays show these same mutations also disrupt FOXO1 binding to their canonical DNA sequences, suggesting the loss of function is due to destabilization of the folded structure. Computational modeling of the effects of mutations on FOXO1 folding was performed using alchemical FEP, and a Markov model of the entire folding reaction was constructed from massively parallel molecular simulations, which predicts folding pathways involving the late folding of helix 3. Although FEP is able to qualitatively predict the destabilization from L183 mutations, we find that a simple hydrophobic transfer model, combined with estimates of unfolded-state solvent accessible surface areas from molecular simulation, is able to more accurately predict changes in folding free energies due to mutations. These results suggest that atomic detail provided from simulation is important for accurate prediction of mutational effects on folding stability. Corresponding disease-associated mutations in other FOX family members support further experimental and computational studies of the folding mechanism of FOX domains.


title: Microgravity enhances the phenotype of Arabidopsis zigzag-1 and reduces the Wortmannin-induced vacuole fusion in root cells

abstract: The spaceflight environment of the International Space Station poses a multitude of stresses on plant growth including reduced gravity. Plants exposed to microgravity and other conditions on the ISS display root skewing, changes in gene expression and protein abundance that may result in changes in cell wall composition, antioxidant accumulation and modification of growth anisotropy. Systematic studies that address the effects of microgravity on cellular organelles are lacking but altered numbers and sizes of vacuoles have been detected in previous flights. The prominent size of plant vacuoles makes them ideal models to study organelle dynamics in space. Here, we used Arabidopsis zigzag1 (zig-1) as a sensitized genotype to study the effect of microgravity on plant vacuole fusion. Wortmannin was used to induce vacuole fusion in seedlings and a formaldehyde-based fixation protocol was developed to visualize plant vacuole morphology after sample return, using confocal microscopy. Our results indicate that microgravity enhances the zig-1 phenotype by reducing hypocotyl growth and vacuole fusion in some cells. This study demonstrates the feasibility of chemical inhibitor treatments for plant cell biology experiments in space.


title: Computational design and construction of an Escherichia coli strain engineered to produce a non-standard amino acid

abstract: Introducing heterologous pathways into host cells constitutes a promising strategy for synthesizing nonstandard amino acids (nsAAs) to enable the production of proteins with expanded chemistries. However, this strategy has proven challenging as the expression of heterologous pathways can disrupt cellular homeostasis of the host cell. Here, we sought to optimize the heterologous production of the nsAA para-aminophenylalanine (pAF) in Escherichia coli. First, we incorporated a heterologous pAF biosynthesis pathway into a genome-scale model of E. coli metabolism, and computationally identified metabolic interventions in the hosts native metabolism to improve pAF production. Next, we explored different ways of imposing these flux interventions experimentally and found that the upregulation of flux in chorismate biosynthesis pathway through the elimination of feedback inhibition mechanisms could significantly raise pAF titers ([~]20 fold) while maintaining a reasonable pAF yield-growth rate trade-off. Overall, this study provides a promising strategy for the biosynthesis of nsAAs in engineered cells.

category: systems biology


title: Reduced cutaneous CD200:CD200R1 signalling in psoriasis enhances neutrophil recruitment to skin

abstract:
The skin immune system is tightly regulated to prevent inappropriate inflammation in response to harmless environmental substances. This regulation is actively maintained by mechanisms including cytokines and cell surface receptors and its loss results in inflammatory disease. In the case of psoriasis, inappropriate immune activation leads to IL-17-driven chronic inflammation, but molecular mechanisms underlying this loss of regulation are not well understood.

We reveal that immunoglobulin superfamily member CD200, and signalling via its receptor, CD200R1 are reduced in non-lesional psoriasis skin. To examine the consequences of this, CD200R1 was blocked in a mouse model of psoriasis demonstrating that the receptor limits psoriasis-like inflammation. Specifically, CD200R1 blockade enhances acanthosis, CCL20 production and neutrophil recruitment but surprisingly, macrophage function and IL-17 production were not affected, and neutrophil reactive oxygen species production was reduced.

Collectively, our data show that CD200R1 affects neutrophil function and limits inflammatory responses in healthy skin by restricting neutrophil recruitment. However, the CD200 pathway is reduced in psoriasis, resulting in a loss of immune control, and increased neutrophil recruitment in mouse models. In conclusion, we highlight a pathway that might be targeted to dampen inflammation in patients with psoriasis.

category: immunology


title: IMC10 and LMF1 mediate membrane contact between the mitochondrion and the inner membrane complex in Toxoplasma gondii

abstract:
The single mitochondrion of Toxoplasma gondii is highly dynamic, being predominantly in a peripherally distributed lasso-shape in intracellular parasites and collapsed in extracellular ones. The peripheral positioning of the mitochondrion is associated with apparent contacts between the mitochondrion membrane and the parasite pellicle. The outer mitochondrial membrane-associated protein LMF1 is critical for the correct positioning of the mitochondrion, and in its absence, intracellular parasites fail to form the lasso-shaped mitochondrion. To identify other proteins that participate in tethering the parasites mitochondrion to the pellicle, we performed a yeast two-hybrid screen for LMF1 interactors. We identified 70 putative interactors, six of which are known to localize to the apical end of the parasite, two to the mitochondrial membrane, and three localize to the inner membrane complex (IMC), a component of the parasite pellicle. Using reciprocal immunoprecipitation and proximity ligation assays, we confirmed the interaction of LMF1 with the pellicle protein IMC10, with a hypothetical protein known to be part of the conoid, and with an ATPase-Guanylyl Cyclase. Conditional knockdown of IMC10 does not affect parasite viability but severely affects mitochondrial morphology in intracellular parasites and mitochondrial distribution to the daughter cells during division. In effect, IMC10 knockdown phenocopies disruption of LMF1, suggesting that these two proteins define a novel membrane tether between Toxoplasmas mitochondrion and the inner membrane complex.

IMPORTANCE

Toxoplasma gondii is an opportunistic parasite that can cause life-threatening disease in immunocompromised patients and those infected congenitally. As current therapies against this parasite can be poorly tolerated and are not effective against the latent stage of the parasite, there is an urgent need to identify new drug targets. The single mitochondrion of this parasite is a validated drug target, but little is known about the machinery that controls its division and structure, information that would be critical for a thorough exploration of the mitochondrion as a drug target. We have identified parasite-specific proteins that are essential to maintain the normal structure of the mitochondrion. We have discovered a complex of two proteins that tether the mitochondrion to the periphery of the parasite. Loss of this connection results in changes in mitochondrial morphology and cell division defects. Our results provide important insight into the molecular mechanisms regulating Toxoplasma mitochondrial morphology.


title: Artificial selection methods from evolutionary computing show promise for directed evolution of microbes

abstract: Directed microbial evolution harnesses evolutionary processes in the laboratory to construct microorganisms with enhanced or novel functional traits. Attempting to direct evolutionary processes for applied goals is fundamental to evolutionary computation, which harnesses the principles of Darwinian evolution as a general purpose search engine for solutions to challenging computational problems. Despite their overlapping approaches, artificial selection methods from evolutionary computing are not commonly applied to living systems in the laboratory. In this work, we ask if parent selection algorithms--procedures for choosing promising progenitors--from evolutionary computation might be useful for directing the evolution of microbial populations when selecting for multiple functional traits. To do so, we introduce an agent-based model of directed microbial evolution, which we used to evaluate how well three selection algorithms from evolutionary computing (tournament selection, lexicase selection, and non-dominated elite selection) performed relative to methods commonly used in the laboratory (elite and top-10% selection). We found that multi-objective selection techniques from evolutionary computing (lexicase and non-dominated elite) generally outperformed the commonly used directed evolution approaches when selecting for multiple traits of interest. Our results motivate ongoing work transferring these multi-objective selection procedures into the laboratory. Additionally, our findings suggest that more sophisticated artificial selection methods from evolutionary computation should also be evaluated for use in directed microbial evolution.

category: evolutionary biology


title: Approximations to the expectations and variances of ratios of tree properties under the coalescent

abstract: Properties of gene genealogies such as tree height (H), total branch length (L), total lengths of external (E) and internal (I) branches, mean length of basal branches (B), and the underlying coalescence times (T) can be used to study population-genetic processes and to develop statistical tests of population-genetic models. Uses of tree features in statistical tests often rely on predictions that depend on pairwise relationships among such features. For genealogies under the coalescent, we provide exact expressions for Taylor approximations to expected values and variances of ratios Xn/Yn, for all 15 pairs among the variables {Hn, Ln, En, In, Bn, Tk}, considering n leaves and 2 [≤] k [≤] n. For expected values of the ratios, the approximations match closely with empirical simulation-based values. The approximations to the variances are not as accurate, but they generally match simulations in their trends as n increases. Although En has expectation 2 and Hn has expectation 2 in the limit as n [->] {infty}, the approximation to the limiting expectation for En/Hn is not 1, instead equaling{pi} 2/3 - 2 {approx} 1.28987. The new approximations augment fundamental results in coalescent theory on the shapes of genealogical trees.

category: genetics


title: DeepAcr: Predicting Anti-CRISPR with Deep Learning

abstract: As an important group of proteins discovered in phages, anti-CRISPR inhibits the activity of the immune system of bacteria (i.e., CRISPR-Cas), showing great potential for gene editing and phage therapy. However, the prediction and discovery of anti-CRISPR are challenging for its high variability and fast evolution. Existing biological studies often depend on known CRISPR and anti-CRISPR pairs, which may not be practical considering the huge number of pairs in reality. Computational methods usually struggle with prediction performance. To tackle these issues, we propose a novel deep learning method for anti-CRISPR analysis (DeepAcr), which achieves impressive performance. On both the cross-fold and cross-dataset validation, our method outperforms the previous state-of-the-art methods significantly. Impressively, DeepAcr improves the prediction performance by at least 40% regarding the F1 score for the cross-dataset test. Moreover, DeepAcr is the first computational method to predict the detailed anti-CRISPR classes, which may help illustrate the anti-CRISPR mechanism. Taking advantage of a Transformer protein language model pre-trained on 250 million protein sequences, DeepAcr overcomes the data scarcity problem. Extensive experiments and analysis suggest that Transformer model feature, evolutionary feature, and local structure feature complement each other, which indicates the critical properties of anti-CRISPR proteins. Combined with AlphaFold prediction, further motif analysis and docking experiments demonstrate that DeepAcr captures the evolutionarily conserved pattern and the interaction between anti-CRISPR and the target implicitly. With the impressive prediction capability, DeepAcr can serve as a valuable tool for anti-CRISPR study and new anti-CRISPR discovery.

category: bioinformatics


title: TP53 somatic mutations in Asian breast cancer are associated with subtype-specific effects

abstract: Recent genomics studies of breast cancer in Asian cohorts have found a higher prevalence of TP53 mutations in Asian breast cancer patients relative to Caucasian patients. However, the effect of TP53 mutations on Asian breast tumours has not been comprehensively studied. Here, we report an analysis of 492 breast cancer samples from the Malaysian Breast Cancer (MyBrCa) cohort where we examined the impact of TP53 somatic mutations in relation to PAM50 subtypes by comparing whole exome and transcriptome data from tumours with mutant and wild type TP53. We found that the magnitude of impact of TP53 somatic mutations appears to vary between different subtypes. TP53 somatic mutations were associated with larger and more consistent differences in HR deficiency scores as well as transcriptional alterations in the luminal A and luminal B subtypes compared to the basal-like and Her2-enriched subtypes. The only pathways that were consistently dysregulated when comparing tumours with mutant and wild type TP53 across the different subtypes were the mTORC1 signaling and glycolysis pathways. These results suggest that therapies that target TP53 or other downstream pathways may be more effective against luminal A and B tumours in the Asian population.


title: Dual pathway architecture underlying vocal learning in songbirds

abstract: Song acquisition and production in songbirds is governed by a dedicated neural circuitry that involves two parallel pathways: a motor pathway for the production and a basal ganglia (BG) pathway for the acquisition. Juveniles learn by imitating adult vocalizations and proceed by trial and error, errors being conveyed by a dopaminergic signal. The complex nature of the relationship between neural control and syrinx musculature makes song learning a complicated problem to solve. Reinforcement learning (RL) has been widely hypothesized to underlie such sensorimotor learning even though this can lead to sub-optimal solutions under uneven contours in continuous action spaces. In this article, we propose to re-interpret the role of a dual pathway architecture, underlying avian vocal learning, that helps overcome these limitations. We posit that the BG pathway conducts exploration by inducing large daily shifts in the vocal production while the motor pathway gradually consolidates this exploration. This process can be understood as a modified form of a simulated annealing process. Simulations on Gaussian performance landscapes and a syrinx-based performance landscape are demonstrated and compared with standard approaches. Taking behavioral constraints into account (60 days of learning, 1000 trials per day), the model allows to reach the global optimum in complex landscapes and thus provides a sound insight into the role of the dual pathway architecture underlying vocal learning.


title: A retrospective and stepwise learning strategy revealed by neuronal activity in the basal forebrain

abstract: Associative learning is a fundamental cognitive capacity that allows animals and humans to learn the predictive relationship between behavioral events and rewarding outcomes. While the process of learning is commonly conceptualized as a prospective strategy (learning how behavioral events predict future rewards), here we provide behavioral and neurophysiological evidence to show that animals may instead employ a retrospective and stepwise learning strategy (learning how the reward is predicted by preceding behavioral events). In rats learning a new association in which the reward was paired with a sequence of behavioral events, learning started from the event closest to the reward and sequentially incorporated earlier events into animals internal model. The learning of each behavioral event as a new reward predictor was accompanied by the emergence of basal forebrain (BF) neuronal responses toward that event. BF activities quantitatively conveyed a reward prediction error signal associated with the behavioral event, and promoted reward-seeking behavioral sequences containing the newly learned event. As the internal model incorporated more behavioral events as reward predictors, non-rewarded behavioral sequences that were once compatible with the internal model during early stages of learning became incompatible and were sequentially eliminated. Together, these results demonstrate how the retrospective and stepwise learning strategy can effectively establish animals internal model during the learning process and lead to the sequential refinement of reward-seeking behaviors. These results also highlight the functional significance of BF neuronal activities, which provided unique insights into the covert dynamics of the learning process in single trials.


title: FCHO controls AP2's critical endocytic roles through a PtdIns4,5P2 membrane-dependent switch

abstract:
Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo-adaptor AP2 requires membrane-localised FCHO. Here, live-cell eTIRF-SIM shows that FCHO marks sites of clathrin- coated pit (CCP) initiation, which mature into uniform sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Eps15 ring. We dissect the network of interactions between the FCHO interdomain-linker and AP2, which concentrates, orients, tethers and partially destabilizes closed AP2 at the plasma membrane. AP2s subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with PtdIns4,5P2, clathrin, cargo and CME accessory factors. FCHO can now relocate toward a CCPs outer edge to engage and activate further AP2s to drive CCP growth/maturation.

125 character summaryFCHO primes AP2 for CCV incorporation, a process that triggers FCHO release to enable activation/recruitment of further AP2s


title: Reduced mitochondria provide an essential function for the cytosolic methionine cycle

abstract: It has been long hypothesised that mitochondrial reduction is intrinsically related to the remodelling of Fe-S clusters assembly. Yet as our knowledge of divergent free-living protists broadens, so does the spectrum of variability within the range of mitochondrial-related organelles (MROs) fundamental functions. We resolved to high precision the MRO proteome of Paratrimastix pyriformis using Localisation of Organelle Proteins by Isotope Tagging (LOPIT) and demonstrate its role in the synthesis of folate derivates bearing one-carbon (1C) units, its link to the glycine cleavage system (GCS) and their only conceivable role as suppliers for the cytosolic methionine cycle, involved in recycling of S-adenosine methionine. This observation provides congruity to the presence of GCS in MROs of free-living anaerobes and its absence in endobionts, which typically lose the methionine cycle and, in the case of oxymonads, also mitochondria.


title: A Locally Activatable Sensor for Robust Quantification of Organellar Glutathione

abstract: Glutathione (GSH) is the main determinant of intracellular redox potential and participates in multiple cellular signaling pathways. Achieving a detailed understanding of intracellular GSH trafficking and regulation depends on the development of tools to map GSH compartmentalization and intra-organelle fluctuations. Herein, we present a new GSH sensing platform, TRaQ-G, for live-cell imaging. This small-molecule/protein hybrid sensor possesses a unique reactivity turn-on mechanism that ensures that the small molecule is only sensitive to GSH in the desired location. Furthermore, TRaQ-G can be fused to a fluorescent protein of choice to give a ratiometric response. Using TRaQ-G-mGold, we demonstrated that the nuclear and cytosolic GSH pools are independently regulated during cell proliferation. We also used this sensor, in combination with roGFP, to quantify redox potential and GSH concentration simultaneously in the endoplasmic reticulum. Finally, by exchanging the fluorescent protein, we created a near-infrared, targetable and quantitative GSH sensor.


title: Death-seq identifies regulators of cell death and senolytic therapies

abstract: Selectively ablating senescent cells ("senolysis") is an evolving therapeutic approach for age-related diseases. Current senolytics are limited to local administration by potency and side effects. While genetic screens could identify senolytics, current screens are underpowered for identifying genes that regulate cell death due to limitations in screen methodology. Here, we establish Death-seq, a positive selection CRISPR screen optimized to identify enhancers and mechanisms of cell death. Our screens identified synergistic enhancers of cell death induced by the known senolytic ABT-263, a BH3 mimetic. SMAC mimetics, enhancers in our screens, synergize with ABT-199, another BH3 mimetic that is not senolytic alone, clearing senescent cells in models of age-related disease while sparing human platelets, avoiding the thrombocytopenia associated with ABT-263. Death-seq enables the systematic screening of cell death pathways to uncover molecular mechanisms of regulated cell death subroutines and identify drug targets for diverse pathological states such as senescence, cancer, and neurodegeneration.


title: Pathogenic variants of sphingomyelin synthase SMS2 disrupt lipid landscapes in the secretory pathway

abstract: Sphingomyelin is a dominant sphingolipid in mammalian cells. Its production in the trans-Golgi traps cholesterol synthesized in the ER to promote formation of a sphingomyelin/sterol gradient along the secretory pathway. This gradient marks a fundamental transition in physical membrane properties that help specify organelle identify and function. We previously identified mutations in sphingomyelin synthase SMS2 that cause osteoporosis and skeletal dysplasia. Here we show that SMS2 variants linked to the most severe bone phenotypes retain full enzymatic activity but fail to leave the ER owing to a defective autonomous ER export signal. Cells harboring pathogenic SMS2 variants accumulate sphingomyelin in the ER and display a disrupted transbilayer sphingomyelin asymmetry. These aberrant sphingomyelin distributions also occur in patient-derived fibroblasts and are accompanied by imbalances in cholesterol organization, glycerophospholipid profiles and lipid order in the secretory pathway. We postulate that pathogenic SMS2 variants undermine the capacity of osteogenic cells to uphold nonrandom lipid distributions that are critical for their bone forming activity.


title: A dual mechanism of APC/C inhibition by MAP kinases

abstract: Mitotic anaphase onset is a key cellular process that is tightly regulated by multiple kinases. The involvement of mitogen-activated protein kinases (MAPKs) in this process has been established long ago in Xenopus egg extracts. However, despite its importance, it is still unclear which MAPK(s) is actually involved, this impedes the further understanding of the regulatory cascade. In this study, we first demonstrated that the involvement of MAPKs in mitotic anaphase onset regulation is evolutionarily conserved in the fission yeast (Schizosaccharomyces pombe). Then, we found that two of the three fission yeast MAPK signaling pathways act in concert to restrain anaphase-promoting complex/cyclosome (APC/C) activity upon activation of the spindle assembly checkpoint (SAC). The first pathway involves the phosphorylation of Mad2, a component of the core mitotic check complex (MCC), by MAPK Sty1, which enhances the tight binding of MCC to APC/C. The second pathway involves MAPK Pmk1 phosphorylation of Slp1Cdc20, the fission yeast homologue of Cdc20 and the co-activator of APC/C, which promotes the degradation of Slp1Cdc20. Both phosphorylation events are required to sustain mitotic arrest in response to spindle defects. These results clarified a detailed regulation cascade of the ubiquitous MAPK signaling in spindle checkpoint activation, APC/C inhibition and anaphase entry, which is vital for accurate chromosome segregation and cell viability.


title: Surface curvature and basal hydraulic stress induce spatial bias in cell extrusion

abstract: Cell extrusion is a coordinated biological process by which epithelial cells are removed from the epithelium without compromising the tissues natural barrier function [1-3]. Cell extrusions not only occur when injuries or genetic mutations arise [4, 5], they also serve to maintain homeostatic cell numbers [6] and have been found to drive morphogenesis [1]. However, the effects of unique environmental settings--e.g., the complex geometric landscape and varying solute gradients in intestinal epithelia [2,7]-- on cell extrusions are less explored. Here we show, using epithelial monolayers cultured on periodic wave substrates, that surface curvature can induce a spatial bias in cell extrusions, with cells more frequently extruding from the negatively curved valleys. Surprisingly, increasing media osmolarity significantly reduced this bias, and also overall extrusion numbers. Furthermore, basal fluid spaces were found smaller under monolayers in hyper-osmotic media compared to iso-osmotic conditions, and similarly smaller on the positively curved hills as compared to valleys. The latter corresponded with the measured inward pointing normal forces on the hills. Remarkably, higher focal adhesion kinase (FAK) and Akt activation were observed in hyper-osmotically treated monolayers. Likewise, higher FAK activation was also observed in wave-hill residing cells. Our results show that surface curvature can play a modulating role in osmotically induced hydraulic stress levels, which act to disrupt focal adhesions and affect cell extrusions via FAK and the downstream Akt survival pathway. These findings highlight the fact that multiple environmental cues can synergistically influence cell extrusions. And an awareness of this will benefit our understanding of spatiotemporal preferences in cell extrusions seen in homeostasis, disease and development [8].


title: Dietary αKG inhibits SARS CoV-2 infection and rescues inflamed lungs to restore normal O2 saturation in animals

abstract: Our recent works described the rescue effect of -ketoglutarate (KG, a metabolite of Krebs cycle) on thrombosis and inflammation in animals. KG augments activity of prolyl hydroxylase 2 (PHD2), which in turn degrades proline residues of substrates like phosphorylated Akt (pAkt) and hypoxia inducible factor (HIF). Here we describe the inhibitory effect of octyl KG on pAkt as well as on HIF1/HIF2, and in turn decreasing SARS CoV-2 replication in Vero E6 cells. KG failed to inhibit the viral replication and Akt phosphorylation in PHD2-knockdown U937 cells transiently expressing ACE2. Contrastingly, triciribine (TCN, an Akt-inhibitor) inhibited viral replication alongside a downmodulation of pAkt in PHD2-KD cells. Dietary KG significantly inhibited viral infection and rescued hamsters from thrombus formation and inflammation in lungs, the known causes of acute respiratory distress syndrome (ARDS) in COVID-19. KG supplementation also reduced the apoptotic death of lung tissues in infected animals, alongside a downmodulation of pAkt and HIF2. KG supplementation neither affected IgG levels against SARS CoV-2 RBD protein nor altered the neutralization antibody response against SARS CoV-2. It did not interfere with the percentage of interferon-{gamma} positive (IFN{gamma}+) CD4+ and IFN{gamma}+CD8+ T cells in infected animals. The extended work in balb/c mice transiently expressing ACE2 showed a similar effect of KG in reducing accumulation of inflammatory immune cells and cytokines, including IL6, IL1{beta} and TNF, in lungs as well as in circulation of infected animals. Pro-thrombotic markers like platelet microparticles and platelet-leukocyte aggregates were reduced significantly in infected mice after KG supplementation. Importantly, KG supplementation restored the O2 saturation (SpO2) in circulation of SARS CoV-2 infected hamsters and mice, suggesting a potential therapeutic role of this metabolite in COVID-19 treatment.


title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

abstract: Methylation of RNA at the N6 position of adenosine (m6A) is one of the most common RNA modifications, impacting RNA stability as well as its transport and translation. Previous studies uncovered RNA destabilization in models of amyotrophic lateral sclerosis (ALS), in association with accumulation of the RNA-binding protein TDP43, which is itself mislocalized from the nucleus in >95% of those with ALS. Here, we show that TDP43 recognizes m6A-modified RNA, and that RNA methylation is critical for both TDP43 binding and autoregulation. We also observed extensive hypermethylation of coding and non-coding transcripts in ALS spinal cord, many of which overlap with methylated TDP43 target RNAs. Emphasizing the importance of m6A for TDP43 binding and function, we identified several m6A factors that enhance or suppress TDP43-mediated toxicity via a single-cell CRISPR/Cas9 candidate-based screen in primary neurons. The most promising genetic modifier among these--the canonical m6A reader YTHDF2-- accumulated within spinal motor neurons in ALS postmortem sections, and its knockdown prolonged the survival of human neurons carrying ALS-associated mutations. Collectively, these data show that m6A modifications modulate RNA binding by TDP43, and that m6A is pivotal for TDP43-related neurodegeneration in ALS.


title: Conformational Flexibility in Neutralization of SARS-CoV-2 by Naturally Elicited Anti-SARS-CoV-2 Antibodies

abstract: As new variants of SARS-CoV-2 continue to emerge, it is important to assess the neutralizing capabilities of naturally elicited antibodies against SARS-CoV-2. In the present study, we evaluated the activity of nine anti-SARS-CoV-2 monoclonal antibodies (mAbs), previously isolated from convalescent donors infected with the Wuhan-Hu-1 strain, against the SARS-CoV-2 variants of concern (VOC) Alpha, Beta, Gamma, Delta and Omicron. By testing an array of mutated spike receptor binding domain (RBD) proteins, cell-expressed spike proteins from VOCs, and neutralization of SARS-CoV-2 VOCs as pseudoviuses, or as the authentic viruses in culture, we show that mAbs directed against the ACE2 binding site (ACE2bs) are far more sensitive to viral evolution compared to anti-RBD non-ACE2bs mAbs, two of which kept their potency against all VOCs tested. At the second part of our study, we reveal the neutralization mechanisms at high molecular resolution of two anti-SARS-CoV-2 neutralizing mAbs by structural characterization. We solved the structures of the Delta-neutralizing ACE2bs mAb TAU-2303 with the SARS-CoV-2 spike trimer and RBD at 4.5 [A] and 2.42 [A], respectively, revealing a similar mode of binding to that between the RBD and the ACE2 receptor. Furthermore, we provide five additional structures (at resolutions of 5.54 [A], 7.76 [A], 6.47 [A], 3.45 [A], and 7.32 [A]) of a second antibody, non-ACE2bs mAb TAU-2212, complexed with the SARS-CoV-2 spike trimer. TAU-2212 binds an exclusively quaternary epitope, and exhibits a unique, flexible mode of neutralization that involves transitioning between five different conformations, with both arms of the antibody recruited for cross linking intra- and inter-spike RBD subunits. Our study provides new mechanistic insights about how antibodies neutralize SARS-CoV-2 and its emerging variants and provides insight about the likelihood of reinfections.


title: SARS-CoV-2 spike S375F mutation characterizes the Omicron BA.1 variant

abstract:
Recent studies have revealed the unique virological characteristics of Omicron, the newest SARS-CoV-2 variant of concern, such as pronounced resistance to vaccine-induced neutralizing antibodies, less efficient cleavage of the spike protein, and poor fusogenicity. However, it remains unclear which mutation(s) in the spike protein determine the virological characteristics of Omicron. Here, we show that the representative characteristics of the Omicron spike are determined by its receptor-binding domain. Interestingly, the molecular phylogenetic analysis revealed that the acquisition of the spike S375F mutation was closely associated with the explosive spread of Omicron in the human population. We further elucidate that the F375 residue forms an interprotomer pi-pi interaction with the H505 residue in another protomer in the spike trimer, which confers the attenuated spike cleavage efficiency and fusogenicity of Omicron. Our data shed light on the evolutionary events underlying Omicron emergence at the molecular level.

HighlightsO_LIOmicron spike receptor binding domain determines virological characteristics
C_LIO_LISpike S375F mutation results in the poor spike cleavage and fusogenicity in Omicron
C_LIO_LIAcquisition of the spike S375F mutation triggered the explosive spread of Omicron
C_LIO_LIF375-H505-mediated {pi}-{pi} interaction in the spike determines the phenotype of Omicron
C_LI


title: Compound interaction screen on a photoactivatable cellulose membrane (CISCM) identifies drug targets

abstract: Identifying the protein targets of drugs is an important but tedious process. Existing proteomic approaches enable unbiased target identification but lack the throughput needed to screen larger compound libraries. Here, we present a compound interaction screen on a photoactivatable cellulose membrane (CISCM) that enables target identification of several drugs in parallel. To this end, we use diazirine-based undirected photoaffinity labeling (PAL) to immobilize compounds on cellulose membranes. Functionalized membranes are then incubated with protein extract and specific targets are identified via quantitative affinity purification and mass spectrometry. CISCM reliably identifies known targets of natural products in less than three hours of analysis time per compound. In summary, we show that combining undirected photoimmobilization of compounds on cellulose with quantitative interaction proteomics provides an efficient means to identify the targets of natural products.


title: Kinetic and Redox Characterization of KRAS G12C Inhibition

abstract: The development of mutant-selective inhibitors for the KRASG12C allele has generated considerable excitement. These KRASG12C inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRASG12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain, motivating the present studies. Measurement of the C12 thiol pKa by NMR and an independent biochemical assay found a depressed pKa (relative to free cysteine) of 7.6 consistent with its susceptibility to chemical ligation. Using a novel and validated fluorescent KRASY137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRASG12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that ARS-853 addition at 5{degrees}C elicited both a rapid first phase (attributed to binding, yielding a Kd of 36.0 {+/-} 0.7 M), and a second, slower pH-dependent phase taken to represent covalent ligation. Consistent with the lower pKa of the C12 thiol, we found that reversible and irreversible oxidation of KRASG12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRASG12C thiol to sulfinic acid alters RAS conformation and dynamics to be more similar to KRASG12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRASG12C drug discovery efforts as well as identifying the occurrence of G12C oxidation with currently unknown biological ramifications.


title: Molecular basis of neurodevelopmental disorder-causing mutation in nonsense-mediated mRNA decay factor UPF3B

abstract: UPF3B is a key nonsense-mediated mRNA decay (NMD) factor required for surveillance of mRNA and regulation of eukaryotic gene expression. Mutations in UPF3B cause intellectual disability. The underlying molecular mechanisms remain unexplored as the mutations lie in an uncharacterized region of UPF3B. Here, we show that UPF3B shares structural and functional homology to the Drosophila Behavior/Human Splicing protein family comprising an RNA-recognition motif-like domain (RRM-L), a NONA/paraspeckle-like domain (NOPS-L), and extended -helical domains essential for ribosome- and RNA-binding and RNA-induced oligomerization. A co-crystal structure of UPF3B with the third middle domain of eukaryotic initiation factor 4G (MIF4GIII) of UPF2 reveals an unexpectedly intimate binding interface. UPF3Bs disease-causing mutation Y160D located in the NOPS-L domain reduces the UPF2 binding affinity ~40-fold compared to wildtype UPF3B. UPF3Bs paralogue UPF3A, an NMD antagonist which is upregulated in patients with the UPF3B-Y160D mutation, binds UPF2 with ~10-fold higher affinity than UPF3B, leading to impaired NMD activity and upregulation of mRNAs involved in neurodevelopment.


title: SEACells: Inference of transcriptional and epigenomic cellular states from single-cell genomics data

abstract: Metacells are cell groupings derived from single-cell sequencing data that represent highly granular, distinct cell states. Here, we present single-cell aggregation of cell-states (SEACells), an algorithm for identifying metacells; overcoming the sparsity of single-cell data, while retaining heterogeneity obscured by traditional cell clustering. SEACells outperforms existing algorithms in identifying accurate, compact, and well-separated metacells in both RNA and ATAC modalities across datasets with discrete cell types and continuous trajectories. We demonstrate the use of SEACells to improve gene-peak associations, compute ATAC gene scores and measure gene accessibility in each metacell. Metacell-level analysis scales to large datasets and are particularly well suited for patient cohorts, including facilitation of data integration. We use our metacells to reveal expression dynamics and gradual reconfiguration of the chromatin landscape during hematopoietic differentiation, and to uniquely identify CD4 T cell differentiation and activation states associated with disease onset and severity in a COVID-19 patient cohort.


title: A co-operative knock-on mechanism underpins Ca2+-selective cation permeation in TRPV channels

abstract: The selective exchange of ions across cellular membranes is a vital biological process. Ca2+-mediated signalling is implicated in a broad array of physiological processes in cells, whilst elevated intracellular concentrations of Ca2+ are cytotoxic. Due to the significance of this cation, strict Ca2+ concentration gradients are maintained across the plasma and organelle membranes. Therefore, Ca2+ signalling relies on permeation through selective ion channels that control the flux of Ca2+ ions. A key family of Ca2+-permeable membrane channels are the polymodal signal-detecting Transient Receptor Potential (TRP) ion channels. TRP channels are activated by a wide variety of cues including temperature, small molecules, transmembrane voltage and mechanical stimuli. Whilst most members of this family permeate a broad range of cations non-selectively, TRPV5 and TRPV6 are unique due to their strong Ca2+-selectivity. Here, we address the question of how some members of the TRPV subfamily show a high degree of Ca2+-selectivity whilst others conduct a wider spectrum of cations. We present results from all-atom molecular dynamics simulations of ion permeation through two Ca2+-selective and two non-selective TRPV channels. Using a new method to quantify permeation co-operativity based on mutual information, we show that Ca2+-selective TRPV channel permeation occurs by a three binding site knock-on mechanism, whereas a two binding site knock-on mechanism is observed in non-selective TRPV channels. Each of the ion binding sites involved displayed greater affinity for Ca2+ over Na+. As such, our results suggest that coupling to an extra binding site in the Ca2+-selective TRPV channels underpins their increased selectivity for Ca2+ over Na+ ions. Furthermore, analysis of all available TRPV channel structures shows that the selectivity filter entrance region is wider for the non-selective TRPV channels, slightly destabilising ion binding at this site, which is likely to underlie mechanistic decoupling.

category: biophysics


title: Farnesyltransferase inhibition overcomes the adaptive resistance to osimertinib in EGFR-mutant NSCLC

abstract: Drug-tolerant "dormant" cells (DTC) have emerged as one of the major non-genetic mechanisms driving resistance to targeted therapy in lung cancer, although the sequence of events leading to entry and exit from dormancy remain poorly described. Here, we performed real-time monitoring of the cell cycle dynamics during the adaptive response to Epidermal Growth Factor Receptor tyrosine kinase inhibitors (EGFR-TKi) in a panel of EGFR-mutated lung cancer cell lines. We identified a rare population of S/G2 cycling cells (referred to as early escapers) that emerged in the first hours of treatment amongst stably arrested and progressively dying G1 cells. We determined that early escapers evolved from a non-proliferative differentiated alveolar type 1 (AT1) phenotype which was invariably associated with cytoskeletal remodeling through Rho/ROCK pathway activation. Using a panel of Rho-pathway inhibitors, we found that the farnesyltransferase inhibitor tipifarnib induced a complete clearance of EGFR-TKi-induced DTC thus fully preventing relapse in vitro. Using a xenograft model and a PDX model of EGFRL858R/T790M lung cancer, co-treatment with tipifarnib prevented relapse to osimertinib for up to 6 months with no evidence of toxicity. Among the farnesylated proteins regulated during osimertinib treatment, concomitant inhibition of RHOE and LaminB1 was sufficient to recapitulated FTIs effect. Osimertinib and tipifarnib co-treatment completely suppressed the emergence of AT1 phenotype, prevented mitosis of S/G2-treated cells and increased the apoptotic response through activation of ATF4-CHOP-dependent Integrated Stress Response (ISR) pathway. Our data strongly support the use of tipifarnib in combination with osimertinib in patients to effectively and durably prevent relapse.


title: NSMCE2, a Novel Super-Enhancer Regulated Gene, is Linked to Poor Prognosis and Therapy Resistance in Breast Cancer

abstract: In this study, we identified two novel super-enhancer associated genes: NSMCE2 and MAL2, highly upregulated in breast tumors, for which high RNA levels significantly and specifically correlate with breast cancer patients poor prognosis. To approach this, we took advantage of existing datasets containing super-enhancers associated genes identified in primary breast tumors and public databases comprising gene expression, genomic and clinical outcomes for patients diagnosed with breast cancer. Through in-vitro pharmacological super-enhancer disruption assays in breast cancer cells we confirmed that super-enhancers are involved in NSMCE2 and MAL2 transcript upregulation and through bioinformatics we found that high levels of NSMCE2 strongly associate with poor response to chemotherapy. This was observed especially for patients diagnosed with aggressive triple negative and HER2 positive tumor types. Finally, we showed that treating breast cancer cells with chemotherapeutic agents while simultaneously decreasing NSMCE2 gene expression by super-enhancer blockade or by directly silencing it, reduces cell viability thus increasing the effectiveness of chemotherapy. Our results indicate that moderating the transcript levels of the novel identified super-enhancer associated gene NSMCE2 could improve patients response to standard chemotherapy and, consequently, may improve disease outcome. In summary by mining existing public breast cancer datasets, our work demonstrates that searching for super-enhancer regulated genes and their association to patients survival and response to treatment, could be an effective method for identifying a signature of tumor specific -not frequently mutated, but super-enhancer dysregulated genes. Our approach offers a new avenue to identify novel biomarkers of poor prognosis and potential pharmacological targets for improving cancer treatment.


title: Modulation of Receptor-like Trans-Membrane Kinase 1 nuclear localisation by DA1 peptidases in Arabidopsis.

abstract: The activities of proteins can depend on their cleavage, which influences their cellular location, stability and functions. The cleavage of intracellular domains of Receptor -Like Kinases (RLK) has an important functional role in the transduction of signals from the cell surface to the nucleus in many organisms. However, the peptidases that catalyse protein cleavage during signal transduction remain poorly understood despite their crucial roles in diverse signalling processes. Here we report in the flowering plant Arabidopsis thaliana that members of the DA1 family of ubiquitin-regulated Zn metallopeptidases cleave the cytoplasmic kinase domain of TransMembrane Kinase 1 (TMK1), releasing it for nuclear localisation where it represses auxin-responsive cell growth during apical hook formation by phosphorylation and stabilisation of the transcriptional repressors IAA32 and IAA34. Mutations in DA1 family members exhibited reduced apical hook formation, and DA1 family mediated cleavage of TMK1 was promoted by auxin treatment. Expression of the DA1 family - generated intracellular kinase domain of TMK1 by an auxin-responsive promoter fully restored apical hook formation in a tmk1 mutant, establishing the function of DA1 family peptidase activities in TMK1-mediated differential cell growth and apical hook formation. DA1 family peptidase activity therefore modulates TMK1 kinase activity between a membrane location where it stimulates acid cell growth and initiates an auxin-dependent kinase cascade controlling cell proliferation in lateral roots and a nuclear localisation where it represses auxin mediated gene expression and growth.