GFAP splice variants fine-tune glioma cell invasion and tumour dynamics by modulating migration persistence Glioma is the most common form of malignant primary brain tumours in adults. Their highly invasive nature makes the disease incurable to date, emphasizing the importance of better understanding the mechanisms driving glioma invasion. Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is characteristic for astrocyte- and neural stem cell-derived gliomas. Glioma malignancy is associated with changes in GFAP alternative splicing, as the canonical isoform GFAP is downregulated in higher-grade tumours, leading to increased dominance of the GFAP{delta} isoform in the network. In this study, we used intravital imaging and an ex vivo brain slice invasion model. We show that the GFAP{delta} and GFAP isoforms differentially regulate the tumour dynamics of glioma cells. Depletion of either isoform increases the migratory capacity of glioma cells. Remarkably, GFAP{delta}-depleted cells migrate randomly through the brain tissue, whereas GFAP-depleted cells show a directionally persistent invasion into the brain parenchyma. This study shows that distinct compositions of the GFAP-network lead to specific migratory dynamics and behaviours of gliomas.
Dynamic Phosphorylation of RGS Provides Spatial Regulation of MAP Kinase And Promotes Completion of Cytokinesis during the Yeast Pheromone Response Yeast use a G-protein coupled receptor (GPCR) signaling pathway to detect mating pheromone, arrest in G1, and direct polarized growth towards the potential mating partner. The primary negative regulator of this pathway is the regulator of G-protein signaling (RGS), Sst2, which induces G GTPase activity and subsequent inactivation of all downstream signaling including a MAPK cascade. The MAPK Fus3 phosphorylates the RGS in response to pheromone, but the role of this modification is unknown. We set out to examine the role of RGS phosphorylation during the pheromone response. We found that RGS phosphorylation peaks early in the pheromone response and diminishes RGS localization to the polarity site and focuses MAPK complexes there. At later time points, RGS is predominantly unphosphorylated, which promotes RGS localization to the polar cap and broadens the distribution of MAPK complexes relative to the Cdc42 polarity machinery. Surprisingly, we found that phosphorylation of the RGS is required for the completion of cytokinesis prior to pheromone induced growth. The completion of cytokinesis in the presence of pheromone is promoted by the formin Bnr1 and the kelch-repeat protein, Kel1, both proteins previously found to interact with the RGS.
Herbivore life histories are altered by drought stress in their hosts plants Drought associated with climate change can stress plants, altering their interactions with phytophagous arthropods. Dryness not only impacts cultivated plants but also their parasites, which in some cases are favoured by drought. Herbivorous arthropods feeding on drought-stressed plants typically produce bigger offspring and develop faster. However, how much responses to abiotic factors differ among populations of a species remains poorly documented. Here, we evaluate variability among populations of a major agricultural pest, the two spotted spider mite, Tetranychus urticae, in response to drought-stress. We compare key life-history parameters of twelve populations that originate from climates ranging from wet and cool Atlantic locations to medium to dry hot Mediterranean locations. We evaluated how plant drought stress affects four life history traits: development time, fecundity, sex-ratio and emigration rate in an experiment comparing well-watered and drought-stressed bean plants. Mites feeding on drought-stressed plants developed faster and attempted to leave leaves less often, and young females were more fecund. The mites from wet temperate climates exhibited greater plasticity between the two water regimes than mites originating from dryer and hot climates. Thus, climate in the area of origin influences mite response to drought. Differed cell division angle, position of cell proliferative area, and localization of ANGUSTIFOLIA3 in lateral organs Leaf meristem is a cell proliferative zone present in the lateral organ primordia. In this study, we investigated how the proliferative zone affects the final morphology of the lateral organs. We examined how cell proliferative zones differ in the primordia of planar floral organs and polar auxin transport inhibitor (PATI)-treated leaves from normal foliage leaf primordia of Arabidopsis thaliana with a focus on the spatial accumulation pattern of ANGUSTIFOLIA3 (AN3), a key element for leaf meristem positioning. We found that organ shape changes by PATI treatment were correlated to the angle of the cell division plane relative to the leaf primordia axis in the leaf meristem (cell division angle), but not with leaf-meristem positioning, size of the leaf meristem, or the localization pattern of AN3 protein. In contrast, different shapes between sepals and petals compared with foliage leaves were associated with both altered meristem position associated with altered AN3 expression patterns and different distributions of cell division angles. These results suggest that lateral organ shapes are regulated via two aspects: position of meristem and cell division angles

Summary statementDifferent lateral organs with different morphology possess different properties of meristems; cell division angles, position of cell proliferative area and AN3 localization patterns. Desalination and temperature increase will shift seasonal grazing patterns of invasive Gammarus tigrinus on charophytes Charophytes are a refuge for zooplankton and stabilize sediments, but they are also a food source for various animal species (water birds, fishes, invertebrates). Especially the introduction of new species, like Gammarus tigrinus, into the Baltic Sea led to yet not understood changes in the food web. Furthermore, future projections point to increased water temperatures at lowered salinity levels affecting species capacity to acclimatize to changing abiotic factors. In this study we investigated the influence of temperature and salinity on the grazing pressure of Gammarus tigrinus on two charophyte species: Chara aspera and Chara tomentosa. The grazing experiments were conducted in a full factorial design with the factors salinity (3 - 13 g kg-1), temperature (5 - 30 {degrees}C), and charophyte species. Grazing rates were determined as mass deviation within 48 hours considering biomass changes in the presence and absence of gammarids. Grazing rate were further used to calculate charophyte losses in two coastal lagoons with different salinity concentrations for recent and future time periods. The potential grazing peak of about 24 {degrees}C is not yet reached in these coastal waters but may be reached in the near future as shown by our future projection results. However, the temperature increase, and desalination will cause a shift in seasonal individual grazing patterns from summer to spring and autumn. Desalination and temperature increase can lead to a shift in optimal habitats for G. tigrinus in the future. Characterization of regulatory transcriptional mechanisms in hepatocyte lipotoxicity Non-alcoholic fatty liver disease is a continuum of disorders among which non-alcoholic steatohepatitis (NASH) is particularly associated with a negative prognosis. Hepatocyte lipotoxicity is one of the main pathogenic factors of liver fibrosis and NASH. However, the molecular mechanisms regulating this process are poorly understood. The main aim of this study was to dissect transcriptional mechanisms regulated by lipotoxicity in hepatocytes. We achieved this aim by combining transcriptomic, proteomic and chromatin accessibility analyses from human liver and mouse hepatocytes. This integrative approach revealed several transcription factor networks deregulated by NASH and lipotoxicity. To validate these predictions, genetic deletion of the transcription factors MAFK and TCF4 was performed, resulting in hepatocytes that were better protected against saturated fatty acid oversupply. MAFK- and TCF4-regulated gene expression profiles suggest a mitigating effect against cell stress, while promoting cell survival and growth. Moreover, in the context of lipotoxicity, some MAFK and TCF4 target genes were to the corresponding differentially regulated transcripts in human liver fibrosis. Collectively, our findings comprehensively profile the transcriptional response to lipotoxicity in hepatocytes, revealing new molecular insights and providing a valuable resource for future endeavours to tackle the molecular mechanisms of NASH. "Neighbourhood watch" model: embryonic epiblast cells assess positional information in relation to their neighbours In many developing and regenerating systems, tissue pattern is established through gradients of informative morphogens, but we know little about how cells interpret these. Using experimental manipulation of early chick embryos including misexpression of an inducer (VG1 or ACTIVIN) and an inhibitor (BMP4), we test two alternative models for their ability to explain how the site of primitive streak formation is positioned relative to the rest of the embryo. In one model, cells read morphogen concentrations cell-autonomously. In the other, cells sense changes in morphogen status relative to their neighbourhood. We find that only the latter model can account for the experimental results, including some counter-intuitive predictions. This mechanism (which we name "neighbourhood watch" model) illuminates the classic "French Flag Problem" and how positional information is interpreted by a sheet of cells in a large developing system.

Summary statementIn a large developing system, the chick embryo before gastrulation, cells interpret gradients of positional signals relative to their neighbours to position the primitive streak, establishing bilateral symmetry. Macroscopic control of synchronous electrical signaling with chemically-excited gene expression Excitable cells can convert electrical signals into chemical outputs to facilitate the active transport of information across larger distances. This electrical-to-chemical conversion requires a tightly regulated expression of ion channels. Alterations of ion channel expression provide landmarks of numerous pathological diseases, such as cardiac arrhythmia, epilepsy, or cancer. Although the activity of ion channels can be locally regulated by external light or chemical stimulus, it remains challenging to coordinate the expression of ion channels on extended spatial-temporal scales in a non-invasive manner. Here, we have engineered yeast S. cerevisiae to read and convert local chemical concentrations into a dynamic electrical field distributed across cell populations. The core mechanism encodes a chemically-excitable dual-feedback gene circuit that precisely tunes the expression domain of potassium channels, globally coordinating cyclic firing of the plasma membrane potential (PMP). We demonstrate that this mechanism leverages an engineered constitutively open bacterial potassium channel KcsA to directly couple chemical stimuli with ion flux through gene expression and it can interface with the host ion channels through the pulsatile production of toxins. Our study provides a robust synthetic transcriptional toolbox underlying the conversion of local chemical environments into spatiotemporally organized electrical impulses for various cellular engineering, synthetic biology, and potential therapeutic applications. Laminar compartmentalization of attention modulation in area V4 aligns with the demands of visual processing hierarchy in the cortex Contrast is a key feature of the visual scene that aids object recognition. Attention has been shown to selectively enhance the responses to low contrast stimuli in visual area V4, a critical hub that sends projections both up and down the visual hierarchy. Veridical encoding of contrast information is a key computation in early visual areas, while later stages encode higher level features that benefit from improved sensitivity to low contrast. How area V4 meets these distinct information processing demands in the attentive state is not known. We found that attentional modulation of contrast responses in area V4 is cortical layer and cell-class specific. Putative excitatory neurons in the superficial output layers that project to higher areas show enhanced boosting of low contrast information. On the other hand, putative excitatory neurons of deep output layers that project to early visual areas exhibit contrast-independent scaling. Computational modeling revealed that such layer-wise differences may result from variations in spatial integration extent of inhibitory neurons. These findings reveal that the nature of interactions between attention and contrast in V4 is highly compartmentalized, in alignment with the demands of the visual processing hierarchy. Molecular basis for gating of cardiac ryanodine receptor: underlying mechanisms for gain- and loss-of function mutations Cardiac ryanodine receptor (RyR2) is a large Ca2+ release channel in the sarcoplasmic reticulum and indispensable for excitation-contraction coupling in the heart. RyR2 is activated by Ca2+ and RyR2 mutations are implicated in severe arrhythmogenic diseases. Yet, the structural basis underlying channel opening and how mutations affect the channel remain unknown. Here, we addressed gating mechanism of RyR2 by combining high-resolution structures determined by cryo-electron microscopy with quantitative functional analysis of channels carrying various mutations in specific residues. We demonstrated two fundamental mechanisms for channel gating: interactions close to the channel pore stabilize the channel to prevent hyperactivity and a series of interactions in the surrounding regions is necessary for channel opening upon Ca2+ binding. Mutations at the residues involved in the former and the latter mechanisms cause gain-of-function and loss-of-function, respectively. Our results reveal gating mechanisms of the RyR2 channel and alterations by pathogenic mutations at the atomic level. The transposable element-rich genome of the cereal pest Sitophilus oryzae BackgroundThe rice weevil Sitophilus oryzae is one of the most important agricultural pests, causing extensive damage to cereal in fields and to stored grains. S. oryzae has an intracellular symbiotic relationship (endosymbiosis) with the Gram-negative bacterium Sodalis pierantonius and is a valuable model to decipher host-symbiont molecular interactions.

ResultsWe sequenced the Sitophilus oryzae genome using a combination of short and long reads to produce the best assembly for a Curculionidae species to date. We show that S. oryzae has undergone successive bursts of transposable element (TE) amplification, representing 72% of the genome. In addition, we show that many TE families are transcriptionally active, and changes in their expression are associated with insect endosymbiotic state. S. oryzae has undergone a high gene expansion rate, when compared to other beetles. Reconstruction of host-symbiont metabolic networks revealed that, despite its recent association with cereal weevils (30 Kyear), S. pierantonius relies on the host for several amino acids and nucleotides to survive and to produce vitamins and essential amino-acids required for insect development and cuticle biosynthesis.

ConclusionsHere we present the genome of an agricultural pest beetle, which may act as a foundation for pest control. In addition, S. oryzae may be a useful model for endosymbiosis, and studying TE evolution and regulation, along with the impact of TEs on eukaryotic genomes. Naming Human Diseases: Ethical Principles of Curating Exclusive Substitute for Inopportune Nosology BackgroundIn the medical sphere, understanding naming conventions strengthen the integrity of naming human diseases remains nominal rather than substantial yet. Since the current nosology-based standard for human diseases could not offer a one-size-fits-all corrective mechanism, many idiomatic but flawed names frequently appear in scientific literature and news outlets at the cost of sociocultural impacts.

ObjectiveWe attempt to examine the ethical oversights of current naming practices and propose heuristic rationales and approaches to determine a pithy name instead of an inopportune nosology.

MethodsFirst, we examined the compiled global online news volumes and emotional tones on some inopportune nosology like German measles, Middle Eastern Respiratory Syndrome, Spanish flu, Hong Kong flu, and Huntingtons disease in the wake of COVID-19. Second, we prototypically scrutinize the lexical dynamics and pathological differentials of German measles and common synonyms by leveraging the capacity of the Google Books Ngram Corpus. Third, we demonstrated the empirical approaches to curate an exclusive substitute for an anachronistic nosology German measles based on deep learning models and post-hoc explanations.

ResultsThe infodemiological study shows that the public informed the offensive names with extremely negative tones in textual and visual narratives. The findings of the historiographical study indicate that many synonyms of German measles did not survive, while German measles became an anachronistic usage, and rubella has taken the dominant place since 1994. The PubMedBERT model could identify rubella as a potential substitution for German measles with the highest semantic similarity. The results of the semantic drift experiments further indicate that rubella tends to survive during the ebb and flow of semantic drift.

ConclusionsOur findings indicate that the nosological evolution of anachronistic names could result in sociocultural impacts without a corrective mechanism. To mitigate such impacts, we introduce some ethical principles for formulating an improved naming scheme. Based on deep learning models and post-hoc explanations, our illustrated experiments could provide hallmark references to the remedial mechanism of naming practices and pertinent credit allocations. Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin inflammation ObjectiveUltraviolet B (UVB) is an important trigger of skin inflammation and lupus with leukocyte recruitment to inflamed skin. We recently reported the involvement of neutrophil NETosis in UVB-induced skin inflammation, and that NETotic nuclear envelope rupture is driven by PKC-mediated nuclear lamin B disassembly. To address the role of Actin cytoskeleton in NETosis, we investigated the effects of Rho kinase (ROCK) and its downstream actomyosin cytoskeletal networks on PKC nuclear translocation and NET formation, as well as their involvement in UVB-induced skin inflammation.

MethodsWe studied the dynamic changes of ROCK and actomyosin cytoskeletal networks during NETosis induction and their involvement in PKC nuclear translocation. Using mice with hematopoietic-specific ROCK1 deficiency, we investigated the effects of ROCK1 deficiency on NETosis, and its involvement in UVB-induced skin inflammation.

ResultsOur time course studies demonstrated the dynamic changes of actin polymerization and ROCK activation, support the role of actin cytoskeleton in nuclear translocation of cytosolic PKC in early stage of NETosis induction. Inhibition of actin polymerization or key molecules of the ROCK/MLCK/myosin pathway decreased PKC nuclear translocation and NET formation. Genetic deficiency of ROCK1, inhibited NETosis ex vivo and in vivo, decreased extracellular display of NET-associated IL-17A, TNF, IFN{gamma}, and IFN in inflamed skin, which were correlated with the ameliorated skin inflammation in UVB-irradiated mice with hematopoietic-specific ROCK1 deficiency.

ConclusionsROCK regulated NETosis through modulation of PKC nuclear translocation via actomyosin cytoskeletal networks in neutrophils. ROCK1 deficiency ameliorated UVB- induced skin inflammation by attenuation of NETosis and NET-associated cytokines. Structural effects driven by rare point mutations in amylin hormone, the type II diabetes-associated peptide BackgroundAmylin is a 37-amino-acid peptide hormone co-secreted with insulin, which participates in glucose homeostasis. This hormone is able to aggregate in a {beta}-sheet conformation and deposit in islet amyloids, a hallmark in type II diabetes. Since amylin is a gene-encoded hormone, this peptide has variants caused by point mutations that can impact its functions.

MethodsHere, we analyzed the structural effects caused by S20G and G33R point mutations which, according to the 1000 Genomes Project, have frequency in East Asian and European populations, respectively. The analyses were performed by means of aggrescan server, SNP functional effect predictors, and molecular dynamics.

ResultsWe found that both mutations have aggregation potential and cause changes in the monomeric forms when compared with wild-type amylin. Furthermore, comparative analyses with pramlintide, an amylin drug analogue, allowed us to infer that second -helix maintenance may be related to the aggregation potential.

ConclusionsThe S20G mutation has been described as pathologically related, which is in agreement with our findings. In addition, our data suggest that the G33R mutation might have a deleterious effect. The data presented here also provide new therapy opportunities, whether for creating more effective drugs for diabetes or implementing specific treatment for patients with these mutations.

General SignificanceOur data could help to better understand the impact of mutations on the wild-type amylin sequence, as a starting point for the evaluation and characterization of other variations. Moreover, these findings could improve the health of patients with type II diabetes. Targeted Gene Editing in Plants using CRISPR-Cas9, Single-Stranded DNA Oligonucleotides, and Protoplast Regeneration Versatile genome editing can be facilitated by the insertion of DNA sequences into specific locations. Current protocols involving clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins rely on low efficiency homology-directed repair or non-homologous end joining with modified double-stranded DNA oligonucleotides as donors. Our simple protocol eliminates the need for expensive equipment, chemical and enzymatic donor DNA modification or plasmid construction by using polyethylene glycol-calcium to deliver non-modified single-stranded DNA oligonucleotides and CRISPR-Cas9 ribonucleoprotein into protoplasts. Plants regenerated via edited protoplasts achieved targeted insertion frequencies of up to 50.0% in Nicotiana benthamiana and 13.6% in rapid cycling Brassica oleracea without antibiotic selection. Using a 60-nt donor containing 27 nt in each homologous arm, 6 of 22 regenerated N. benthamiana plants showed targeted insertions, and one contained a precise insertion of a 6-bp HindIII site. The inserted sequences were transmitted to the next generation and invite the possibility of future exploration of versatile genome editing by targeted DNA insertion in plants. Extreme parallel evolution of flagellar motility facilitated by silent mutations Mutational hotspots can determine evolutionary outcomes and make evolution repeatable. Hotspots are products of multiple evolutionary forces including mutation rate heterogeneity, but this variable is often hard to identify. In this work we reveal that a powerfully deterministic genetic hotspot can be built and broken by a handful of silent mutations. We observed this when studying homologous immotile variants of the bacteria Pseudomonas fluorescens, AR2 and Pf0-2x. AR2 resurrects motility through highly repeatable de novo mutation of the same nucleotide in >95% lines in minimal media (ntrB A289C). Pf0-2x, however, evolves via a number of mutations meaning the two strains diverge significantly during adaptation. We determined that this evolutionary disparity was owed to just 6 synonymous variations within the ntrB locus, which we demonstrated by swapping the sites and observing that we were able to both break (>95% to 0% in AR2) and build (0% to 80% in Pf0-2x) a powerfully deterministic mutational hotspot. Our work reveals a fundamental role for silent genetic variation in determining adaptive outcomes. Oxytocin promotes social grooming in bonobos Oxytocin has attracted research attention due to its role in promoting social bonding. One notable recent hypothesis is the biobehavioral feedback loop, which posits that the oxytocin system has evolved to support the formation and maintenance of social bonds through a positive feedback loop, where oxytocin promotes social behaviours which then cause oxytocin release themselves. In the two Pan species, humans closest relatives, oxytocin is known to be released following key behaviours related to social bonding, such as social grooming in chimpanzees and female-female sexual behaviour in bonobos. However, no experimental evidence has demonstrated that oxytocin promotes such socio-positive behaviours. To test this, we administered nebulized oxytocin or saline placebo to a group of female bonobos and subsequently observed the change in their gross behavior during free interaction. We found that bonobos groomed other group members significantly more frequently in the oxytocin compared to placebo condition. Other behavioural measures did not largely differ between conditions, except for a nonsignificant trend for reduction in abnormal regurgitation/reingestion behaviour. Overall, we found that oxytocin promoted socio-positive interaction in bonobos, providing support for the biobehavioural feedback loop hypothesis of oxytocin in bonobo social evolution. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Differences in gait stability and acceleration characteristics between healthy young and older females Our aim was to evaluate differences in gait acceleration intensity, variability and stability of feet and trunk between older females and young females using inertial sensors. Twenty older females (OF; mean age 68.4, SD 4.1 years) and eighteen young females (YF; mean age 22.3, SD 1.7 years) were asked to walk straight for 100 meters at their preferred speed, while wearing inertial sensors on heels and lower back. We calculated spatiotemporal measures, foot and trunk acceleration characteristics and their variability, as well as trunk stability using the local divergence exponent (LDE). Two-way analysis of variance (including the factors foot and age), Students t-test, and Mann-Whitney U test were used to compare statistical differences of measures between groups. Cohens d effects were calculated for each variable. Foot maximum vertical acceleration and amplitude, trunk-foot vertical acceleration attenuation, as well as their variability were significantly smaller in OF than in YF. In contrast, trunk mediolateral acceleration amplitude, maximum vertical acceleration, and amplitude, as well as their variability were significantly larger in OF than in YF. Moreover, OF showed lower stability (i.e., higher LDE values) in mediolateral acceleration, mediolateral and vertical angular velocity of the trunk. Even though we measured healthy older females, these participants showed lower vertical foot accelerations with higher vertical trunk acceleration, lower trunk-foot vertical acceleration attenuation, less gait stability, and more variability of the trunk, and hence, were more likely to fall. These findings suggest that instrumented gait measurements may help for early detection of changes or impairments in gait performance, even before this can be observed by clinical eye or gait speed. Amoxicillin-resistant Streptococcus pneumoniae can be resensitized by targeting the mevalonate pathway as indicated by sCRilecs-seq Antibiotic resistance in the important opportunistic human pathogen Streptococcus pneumoniae is on the rise. This is particularly problematic in the case of the {beta}-lactam antibiotic amoxicillin, which is the first-line therapy. It is therefore crucial to uncover targets that would kill or resensitize amoxicillin-resistant pneumococci. To do so, we developed a genome-wide, single-cell based, gene silencing screen using CRISPR interference called sCRilecs-seq (subsets of CRISPR interference libraries extracted by fluorescence activated cell sorting coupled to next generation sequencing). Since amoxicillin affects growth and division, sCRilecs-seq was used to identify targets that are responsible for maintaining proper cell size. Our screen revealed that downregulation of the mevalonate pathway leads to extensive cell elongation. Further investigation into this phenotype indicates that it is caused by insufficient transport of cell wall precursors across the cell membrane due to a limitation in the production of undecaprenyl phosphate (Und-P), the lipid carrier responsible for this process. The data suggest that whereas peptidoglycan synthesis continues even with reduced Und-P levels, cell constriction is specifically halted. We successfully exploited this knowledge to create a combination treatment strategy where the FDA-approved drug clomiphene, an inhibitor of Und-P synthesis, is paired up with amoxicillin. Our results show that clomiphene potentiates the antimicrobial activity of amoxicillin and that combination therapy resensitizes amoxicillin-resistant S. pneumoniae. These findings could provide a starting point to develop a solution for the increasing amount of hard-to-treat amoxicillin-resistant pneumococcal infections. Define protein variant functions with high-complexity mutagenesis libraries and enhanced mutation detection software ASMv1.0 Open reading frame (ORF) variant libraries have advanced our ability to query the functions of a large number of variants of a protein simultaneously in a single experiment. Variant libraries targeting full-length ORFs typically consists of all possible single-amino-acid substitutions and a stop codon at each amino-acid position. Because a variant differs from the template ORF by a single codon variation, variant quantification presents the most profound challenge to this technology. Efforts such as dividing a library into sub-libraries for direct sequencing, or tag-directed subassembly are practical only for small ORFs. Our approach, however, features an enhanced variant-calling tool that processes massive shotgun sequencing data, allowing one to screen a single variant library for ORFs sized up to [~]3600 bases. The variant-calling tool detects variants reliably, and also presents variant annotations in datafiles enabling analyses that have reshaped our strategies governing library design, screen deconvolution, sequencing and sequencing data analysis. P2X2 receptor subunit interfaces are missense variant hotspots where mutations tend to increase apparent ATP affinity Background and PurposeP2X receptors (P2XRs) are trimeric ligand-gated ion channels (LGICs) that open a cation-selective pore in response to ATP binding to their large extracellular domain (ECD). The seven known P2XR subtypes can assemble as homo- or heterotrimeric complexes and contribute to numerous physiological functions, including nociception, inflammation and hearing. The overall structure of P2XRs is well established, but little is known about the spectrum and prevalence of human genetic variations and the functional implications in specific domains.

Experimental ApproachHere we examine the impact of P2X2 receptor (P2X2R) inter-subunit interface missense variants identified in the human population or through structural predictions. We test both single and double mutants through electrophysiological and biochemical approaches.

Key resultsWe demonstrate that predicted ECD inter-subunit interfaces display a higher-than-expected density of missense variations and that the majority of mutations that disrupt putative inter-subunit interactions result in channels with higher apparent ATP affinity. Lastly, we show that double mutants at the subunit interface show significant energetic coupling, especially if located in close proximity.

Conclusions and ImplicationsWe provide the first structural mapping of the mutational burden across the human population in a LGIC and show that the density of missense mutations is constrained between protein domains, indicating evolutionary selection at the domain level. Our data may indicate that, unlike other LGICs, P2X2Rs have evolved an intrinsically high threshold for activation, possibly to allow for additional modulation or as a cellular protection mechanism against overstimulation.

Bullet point summary What is already known: O_LIP2X2 receptors are ATP-activated ion channels implicated in hearing and nociceptice pathways
C_LI
 What this study adds: O_LIA structural mapping of missense variants observed in the human population
C_LIO_LIWe identify the intersubunit-interface as a variant hotspot and decipher functional impact of mutations
C_LI
 Clinical significance: O_LIThe development of both inhibitors and activators of P2X2 receptor function may be required
C_LI Establishing combination PAC-1 and TRAIL regimens for treating ovarian cancer based on patient-specific pharmacokinetic profiles using in silico clinical trials Ovarian cancer is commonly diagnosed in its late stages, and new treatment modalities are needed to improve patient outcomes and survival. We have recently established the synergistic effects of combination tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and procaspase activating compound (PAC-1) therapies in granulosa cell tumours (GCT) of the ovary, a rare form of ovarian cancer, using a mathematical model of the effects of both drugs in a GCT cell line. Here, to understand the mechanisms of combined TRAIL and PAC-1 therapy, study the viability of this treatment strategy, and accelerate preclinical translation, we leveraged our mathematical model in combination with population pharmacokinetics (PopPK) models of both TRAIL and PAC-1 to expand a realistic heterogeneous cohort of virtual patients and optimize treatment schedules. Using this approach, we investigated treatment responses in this virtual cohort and determined optimal therapeutic schedules based on patient-specific pharmacokinetic characteristics. Our results showed that schedules with high initial doses of PAC-1 were required for therapeutic efficacy. Further analysis of individualized regimens revealed two distinct groups of virtual patients within our cohort: one with high PAC-1 elimination, and one with normal PAC-1 elimination. In the high elimination group, high weekly doses of both PAC-1 and TRAIL were necessary for therapeutic efficacy, however virtual patients in this group were predicted to have a worse prognosis when compared to those in the normal elimination group. Thus, PAC-1 pharmacokinetic characteristics, particularly clearance, can be used to identify patients most likely to respond to combined PAC-1 and TRAIL therapy. This work underlines the importance of quantitative approaches in preclinical oncology. C. elegans PEZO-1 is a Mechanosensitive Ion Channel Involved in Food Sensation PIEZO channels are force sensors essential for physiological processes, including baroreception and proprioception. The Caenorhabditis elegans genome encodes an ortholog gene of the Piezo family, pezo-1, which is expressed in several tissues, including the pharynx. This myogenic pump is an essential component of the C. elegans alimentary canal, whose contraction and relaxation are modulated by mechanical stimulation elicited by food content. Whether pezo-1 encodes a mechanosensitive ion channel and contributes to pharyngeal function remain unknown. Here, we leverage genome editing, genetics, microfluidics, and electropharyngeogram recording to establish that pezo-1 is expressed in the pharynx, including in a proprioceptive-like neuron, and regulates pharyngeal function. Knockout (KO) and gain-of-function (GOF) mutants reveal that pezo-1 is involved in fine-tuning pharyngeal pumping frequency, sensing osmolarity, and food mechanical properties. Using pressure-clamp experiments in primary C. elegans embryo cultures, we determine that pezo-1 KO cells do not display mechanosensitive currents, whereas cells expressing wild-type or GOF PEZO-1 exhibit mechanosensitivity. Moreover, infecting the Spodoptera frugiperda cell line with a baculovirus containing the G-isoform of pezo-1 (among the longest isoforms) demonstrates that pezo-1 encodes a mechanosensitive channel. Our findings reveal that pezo-1 is a mechanosensitive ion channel that regulates food sensation in worms. Omicron-specific mRNA vaccination alone and as a heterologous booster against SARS-CoV-2 The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has high transmissibility and recently swept the globe. Due to the extensive number of mutations, this variant has high level of immune evasion, which drastically reduced the efficacy of existing antibodies and vaccines. Thus, it is important to test an Omicron-specific vaccine, evaluate its immune response against Omicron and other variants, and compare its immunogenicity as boosters with existing vaccine designed against the reference wildtype virus (WT). Here, we generated an Omicron-specific lipid nanoparticle (LNP) mRNA vaccine candidate, and tested its activity in animals, both alone and as a heterologous booster to existing WT mRNA vaccine. Our Omicron-specific LNP-mRNA vaccine elicited strong and specific antibody response in vaccination-naive mice. Mice that received two-dose WT LNP-mRNA, the one mimicking the commonly used Pfizer/Moderna mRNA vaccine, showed a >40-fold reduction in neutralization potency against Omicron variant than that against WT two weeks post second dose, which further reduced to background level >3 months post second dose. As a booster shot for two-dose WT mRNA vaccinated mice, a single dose of either a homologous booster with WT LNP-mRNA or a heterologous booster with Omicron LNP-mRNA restored the waning antibody response against Omicron, with over 40-fold increase at two weeks post injection as compared to right before booster. Interestingly, the heterologous Omicron LNP-mRNA booster elicited neutralizing titers 10-20 fold higher than the homologous WT booster against the Omicron variant, with comparable titers against the Delta variant. All three types of vaccination, including Omicron mRNA alone, WT mRNA homologous booster, and Omicron heterologous booster, elicited broad binding antibody responses against SARS-CoV-2 WA-1, Beta, and Delta variants, as well as other Betacoronavirus species such as SARS-CoV, but not Middle East respiratory syndrome coronavirus (MERS-CoV). These data provided direct proof-of-concept assessments of an Omicron-specific mRNA vaccination in vivo, both alone and as a heterologous booster to the existing widely-used WT mRNA vaccine form. Medium-throughput zebrafish optogenetic platform identifies deficits in subsequent neural activity following brief early exposure to cannabidiol and Δ-9-tetrahydrocannabinol In the light of legislative changes and the widespread use of cannabis as a recreational and medicinal drug, delayed effects of cannabis upon brief exposure during embryonic development are of high interest as early pregnancies often go undetected. Here, zebrafish embryos were exposed to cannabidiol (CBD) and {Delta}-9-tetrahydrocannabinol l (THC) until the end of gastrulation (1-10 hours post-fertilization) and analyzed later in development (4-5 days post-fertilization). In order to measure neural activity, we implemented CaMPARI (Calcium-Modulated Photoactivatable Ratiometric Integrator) and optimized the protocol for a 96-well format complemented by locomotor analysis. Our results revealed that neural activity was decreased by CBD more than THC. At higher doses, both cannabinoids could dramatically reduce neural activity and locomotor activity. Interestingly, the decrease was more pronounced when CBD and THC were combined. At the receptor level, CBD-mediated reduction of locomotor activity was partially prevented using cannabinoid type 1 and 2 receptor inhibitors. Overall, we report that CBD toxicity occurs via two cannabinoid receptors and is synergistically enhanced by THC exposure to negatively impact neural activity late in larval development. Future studies are warranted to reveal other cannabinoids and receptors involved in this pathway to understand the subsequent health implications of cannabis consumption on fetal development.