title: Joint effects of genes underlying a temperature specialization tradeoff in yeast

abstract:
A central goal of evolutionary genetics is to understand, at the molecular level, how organisms adapt to their environments. For a given trait, the answer often involves the acquisition of variants at unlinked sites across the genome. Genomic methods have achieved landmark successes in pinpointing adaptive loci. To figure out how a suite of adaptive alleles work together, and to what extent they can reconstitute the phenotype of interest, requires their transfer into an exogenous background. We studied the joint effect of adaptive, gain-of-function thermotolerance alleles at eight unlinked genes from Saccharomyces cerevisiae, when introduced into a thermosensitive sister species, S. paradoxus. Although the loci damped each others beneficial impact (that is, they were subject to negative epistasis), most boosted high-temperature growth alone and in combination, and none was deleterious. The complete set of eight genes was sufficient to confer [~]15% of the S. cerevisiae phenotype in the S. paradoxus background. The same loci also contributed to a heretofore unknown advantage in cold growth by S. paradoxus. Together, our data establish temperature resistance in yeasts as a model case of a genetically complex evolutionary tradeoff, which can be partly reconstituted from the sequential assembly of unlinked underlying loci.

Author summaryOrganisms adapt to threats in the environment by acquiring DNA sequence variants that tweak traits to improve fitness. Experimental studies of this process have proven to be a particular challenge when they involve manipulation of a suite of genes, all on different chromosomes. We set out to understand how so many loci could work together to confer a trait. We used as a model system eight genes that govern the ability of the unicellular yeast Saccharomyces cerevisiae to grow at high temperature. We introduced these variant loci stepwise into a non-thermotolerant sister species, and found that the more S. cerevisiae alleles we added, the better the phenotype. We saw no evidence for toxic interactions between the genes as they were combined. We also used the eight-fold transgenic to dissect the biological mechanism of thermotolerance. And we discovered a tradeoff: the same alleles that boosted growth at high temperature eroded the organisms ability to deal with cold conditions. These results serve as a case study of modular construction of a trait from nature, by assembling the genes together in one genome.


title: Sex-specific transcript diversity is regulated by a maternal pioneer factor in early Drosophila embryos

abstract: Maternally deposited factors play a crucial role in initiating zygotic transcription. However, the mechanisms by which maternal factors regulate early zygotic transcript diversity through alternative splicing remain unclear. Furthermore, how early in development widespread sex-specific transcript diversity occurs is not known. We show that widespread sex-specific transcript diversity occurs much earlier than previously thought and present a new pipeline called time2splice to quantify splicing changes over time. Using the powerful Drosophila model, we define several mechanisms by which a maternal factor regulates sex-specific zygotic transcriptome diversity: 1) GA-binding pioneer factor CLAMP (Chromatin linked adapter for MSL proteins) binds to the gene bodies of sex-specifically spliced genes to alter the recruitment of spliceosome components; 2) CLAMP modulates chromatin accessibility of an alternatively-spliced exon within sxl, the master regulator of sex determination; 3) CLAMP regulates the distribution of the spliceosome component Maleless (MLE) to prevent aberrant sex-specific splicing. Overall, we provide key insight into how maternal factors influence sex-specific transcript diversity.

category: genomics


title: Deficiency of MicroRNA-181a Results in Transcriptome-Wide Cell Specific Changes in the Kidney that Lead to Elevated Blood Pressure and Salt Sensitivity

abstract: MicroRNA miR-181a is down-regulated in the kidneys of hypertensive patients and hypertensive mice. In vitro, miR-181a is a posttranslational inhibitor of renin expression, but pleiotropic mechanisms by which miR-181a may influence blood pressure (BP) are unknown. Here we determined whether deletion of miR-181a/b-1 in vivo changes BP and the molecular mechanisms involved at the single-cell level. We developed a knockout mouse model lacking miR-181a/b-1 genes using CRISPR/Cas9 technology. Radio-telemetry probes were implanted in twelve-week-old C57BL/6J wild-type and miR-181a/b-1 knockout mice. Systolic and diastolic BP were 4-5mmHg higher in knockout compared with wild-type mice over 24-hours (P<0.01). Compared with wild-type mice, renal renin was higher in the juxtaglomerular cells of knockout mice. BP was similar in wild-type mice on a high (3.1%) versus low (0.3%) sodium diet (+0.4{+/-}0.8mmHg) but knockout mice showed salt sensitivity (+3.3{+/-}0.8mmHg, P<0.001). Since microRNAs can target several mRNAs simultaneously, we performed single-nuclei RNA-sequencing in 6,699 renal cells. We identified 12 distinct types of renal cells, all of which had genes that were dysregulated. This included genes involved in renal fibrosis and inflammation such as Stat4, Col4a1, Cd81, Flt3l, Cxcl16, Smad4. We observed up-regulation of pathways related to the immune system, inflammatory response, reactive oxygen species and nerve development, consistent with higher tyrosine hydroxylase. In conclusion, downregulation of the miR-181a gene led to increased BP and salt sensitivity in mice. This is likely due to an increase in renin expression in juxtaglomerular cells, as well as microRNA-driven pleiotropic effects impacting renal pathways associated with hypertension.


title: Identification and assessment of cardiolipin interactions with E. coli inner membrane proteins

abstract: Integral membrane proteins are localised and/or regulated by lipids present in the surrounding bilayer. Whilst bacteria such as E. coli have relatively simple membranes when compared to eukaryotic cells, there is ample evidence that many bacterial proteins bind to specific lipids, especially the anionic lipid cardiolipin. Here, we apply molecular dynamics simulations to assess lipid binding to 42 different E. coli inner membrane proteins. Our data reveals a strong asymmetry between the membrane leaflets, with a marked increase of anionic lipid binding to the inner leaflet regions of membrane proteins, particularly for cardiolipin. From our simulations we identify over 700 independent cardiolipin binding sites, allowing us to identify the molecular basis of a prototypical cardiolipin binding site, which we validate against structures of bacterial proteins bound to cardiolipin. This allows us to construct a set of metrics for defining a high affinity cardiolipin binding site on (bacterial) membrane proteins, paving the way for a heuristic approach to defining more complex protein-lipid interactions.

category: biochemistry


title: Variations in phenolic levels in grapevine buds at eco-dormancy after chemically-induced stress conditions

abstract:
Viticulture is one of the briskest economic activities worldwide. A major obstacle impeding such grape yields to satisfy the demand for increased production is the insufficient period of chilling temperatures which, due to climate change, is becoming briefer. The shorter period of cold leads to poor budbreak which, in turn, results in reduced yields. To combat this issue, agronomists have resorted to treatments with chemical regulators to meet the chilling requirement for bud dormancy release several fruit-bearing plants have, including grapevines.

This study aimed at identifying and quantifying during eco-dormancy the individual polyphenolic compounds, and their possible variations, in the latent buds of the  Prime(C) and  Ralli table grape cultivars. The study induced chemical stress by means of four different solutions, at three discrete dates per year, for three consecutive years. Phenolics in the latent buds of the studied varieties were analyzed via HPLC. Their quantitative analysis showed variations both between the varieties and between the samples of those varieties collected after the eight treatments. The analysis indicated that the concentration of phenolics continued progressing during the annual growth cycle of the grapevine, with strong positive correlations being detected between catechin, epicatechin, chlorogenic acid, o-coumaric, piceid, procyanidins B1-B2, rutin, {varepsilon}-viniferin. Luteolin registered a positive correlation with quercetin, resveratrol, o-coumaric, but not with the remaining polyphenols. The results confirmed that (a) the concentration of phenolics of the latent buds is affected by chemically-induced stress conditions, (b) depending on the date of application, significant changes appear in the variations of those phenolics.

One sentence summaryChanges in the levels of polyphenol compounds of the grapevines latent buds hints, for the first time, how much stress the buds actually undergo under chemically-induced stress conditions


title: Validating the automation of different measures of high temperature tolerance of small terrestrial insects

abstract: Accurately phenotyping numerous test subjects is essential for most experimental research. Collecting such data can be tedious or time-consuming, and can be biased or limited by manual observations. The thermal tolerance of small ectotherms is a good example of this type of phenotypic data, and it is widely used to investigate thermal adaptation, acclimation capacity and climate change resilience of small ectotherms. Here, we present the results of automatically generated thermal tolerance data using motion tracking on video recordings using two Drosophila species and temperature acclimation to create variation in thermal tolerances and two different heat tolerance assays. We find similar effect sizes of acclimation and hardening responses between manual and automated approaches, but different absolute tolerance estimates. This discrepancy likely reflects both technical differences and the behavioral cessation of movement rather than physiological failure measured in other assays. We conclude that both methods generate biological meaningful results, which reflect different aspects of the thermal biology, find no evidence of inflated variance in the manually scored assays, but find that automation can increase throughput without compromising quality. Further we show that the method can be applied to a wide range of arthropod taxa. We suggest that our automated method is a useful example of through-put phenotyping, and suggest this approach might be applied to other tedious laboratory traits, such as desiccation or starvation tolerance, with similar benefits to through-put. However, the interpretation and potential comparison to results using different methodology rely on thorough validation of the assay and the involved biological mechanism.

category: physiology


title: Medium optimization for biomass production of three peat moss (Sphagnum L.) species using fractional factorial design and response surface methodology

abstract: Peat moss (Sphagnum) biomass is a promising bioresource to substitute peat in growing media with a renewable material. For sustainable production on a large scale, the productivity of Sphagnum mosses has to be increased by optimizing culture conditions. Optimization was achieved using fractional factorial design and response surface methodology based on central composite design to determine concentrations of eight factors leading to highest biomass yield. We improved a standard Sphagnum medium by reducing the concentrations of NH4NO3, KH2PO4, KCl, MgSO4, Ca(NO3)2, FeSO4 and a microelement solution up to 50 %. Together with a reduced sucrose concentration for Sphagnum fuscum, while it remained unchanged for Sphagnum palustre and Sphagnum squarrosum, moss productivities were enhanced for all tested species in shake flasks. Further upscaling to 5 L photobioreactors increased the biomass yield up to nearly 50-fold for S. fuscum, 40-fold for S. palustre and 25-fold for S. squarrosum in 24 days.

category: plant biology


title: Abi3 regulates microglial ramification and dynamic tissue surveillance in vivo.

abstract: A rare coding variant of Abelson-interactor gene family member 3 (Abi3) is associated with increased risk of late-onset Alzheimers Disease (AD). Although Abi3 is recognised as a core microglial gene, its role in microglia is largely unknown. Here we demonstrate that Abi3 is crucial for normal microglial morphology, distribution, and homeostatic tissue surveillance activity in vivo.

category: neuroscience


title: Anti-fibrotic activity of an antimicrobial peptide in a Drosophila model.

abstract: Fibrotic lesions accompany several pathological conditions including tumors. We show that expression of a dominant-active form of the Ras oncogene in Drosophila salivary glands (SGs) leads to redistribution of components of the basement membrane (BM) and fibrotic lesions. Similar to several types of mammalian fibrosis, the disturbed BM attracts clot components including insect transglutaminase and phenoloxidase. SG epithelial cells show reduced apico-basal polarity accompanied by a loss of secretory activity. Both the fibrotic lesions and the reduced cell polarity are alleviated by ectopic expression of the antimicrobial peptide Drosomycin (Drs), which also restores secretory activity of the SGs. In addition to ECM components, both Drs and F-actin localize to fibrotic lesions.

category: immunology


title: Versatile genetic toolbox for Prevotella copri enables studying polysaccharide utilization systems

abstract: Prevotella copri is a prevalent inhabitant of the human gut and has been associated with plant-rich diet consumption and diverse health states. The underlying genetic basis of these associations remains enigmatic due to the lack of genetic tools. Here, we developed a novel versatile genetic toolbox for rapid and efficient genetic insertion and allelic exchange applicable to P. copri strains from multiple clades. Enabled by the genetic platform, we systematically investigated the specificity of polysaccharide utilization loci (PULs), and identified four highly conserved PULs for utilizing arabinan, pectic galactan, arabinoxylan and inulin, respectively. Further genetic and functional analysis of arabinan utilization systems illustrate that P. copri has evolved two distinct types of arabinan-processing PULs (PULAra) and that the type-II PULAra is significantly enriched in individuals consuming a vegan diet compared to other diets. In summary, this genetic toolbox will enable functional genetic studies for P. copri in the future.

category: microbiology


title: Genomic Evidence of Environmental and Resident Salmonella Senftenberg and Montevideo Contamination in the Pistachio Supply-chain

abstract:
Pistachios have been implicated in two salmonellosis outbreaks and multiple recalls in the U.S. This study performed a retrospective data analysis of Salmonella associated with pistachios and a storage study to evaluate the survivability of Salmonella on inoculated inshell pistachios to further understand the genetics and microbiological dynamics of this commodity-pathogen pair. The retrospective data analysis on isolates associated with pistachios was performed from both short-read and long-read sequencing technologies. The sequence data were analyzed using the FDAs Center for Food Safety and Applied Nutrition Single Nucleotide Polymorphism (SNP) analysis and Whole Genome Multi-locus Sequence Typing (wgMLST) pipeline. The storage study evaluated the survival of five strains of Salmonella on pistachios, both in a cocktail as well as individually. Our results demonstrate: i) evidence of persistent Salmonella Senftenberg and Salmonella Montevideo strains in pistachio environments, some of which may be due to clonal resident strains and some of which may be due to preharvest contamination; ii) presence of the Copper Homeostasis and Silver Resistance Island (CHASRI) in Salmonella Senftenberg and Montevideo strains in the pistachio supply chain; and iii) different serovars of Salmonella enterica, including Salmonella Senftenberg and Salmonella Montevideo, are able to survive in pistachios over an extended period of time.

ImportancePistachios have been linked to multistate outbreaks caused by Salmonella serovar Senftenberg (2013, 2016) and serovar Montevideo (2016). This comprehensive study of whole-genome-sequence (WGS) data from Senftenberg and Montevideo isolates associated with pistachio outbreaks, recalls, and investigations over a nine-year period (2009-2018) was conducted to provide insights into evolutionary relationships and persistence. Multiple analytical approaches were applied including MLST, wgMLST and phylogenetic SNP analysis. The CHASRI was identified in all isolates associated with the sequence type 14 (ST14) and ST316, suggesting an adaptation in response to extrinsic pressures, such as in the farm environment. There is evidence of persistent Salmonella Senftenberg and Montevideo strains in pistachio pre-harvest and post-harvest environments. Utilizing metagenomic sequencing during an inoculated storage study of pistachios allowed for determination of surviving serovars over an extended period of time. Defining the mechanisms of persistence of these strains is of high importance to public health.


title: Inhibition of select actinobacteria by the organophosphate pesticide chlorpyrifos

abstract:
Organophosphorus compounds have an extensive history as both agricultural pesticides as well as chemical nerve agents. Decades of research have demonstrated numerous links between these chemicals and their direct and indirect effects on humans and other organisms. The inhibitory effects of organophosphate pesticides (OPPs) on metazoan physiology, are well-characterized; however, the effects of organophosphorus compounds on soil microbes - essential contributors to key agricultural processes - are poorly understood. Chlorpyrifos (CPF) is an OPP that is used globally for crop protection. Studies of CPF application to soils have shown transient effects on soil microbial communities with conflicting data. Here, we directly test the effect of CPF on a panel of 196 actinobacteria strains, examining the effects of CPF on their growth and in vitro phenotypes on solid media. Strains were grown and replica-plated onto media containing CPF or a vehicle control and grown at 28{degrees}C. CPF dramatically inhibited the growth of most strains and/or altered colony morphologies, with 13 strains completely inhibited by CPF. In disk diffusion assays with CPF, its degradation product 3,5,6-trichloropyridinol (TCP), malathion, parathion, monocrotophos and mevinphos, only CPF exhibited direct antimicrobial activity suggesting that the observed effects were due to CPF itself.

IMPORTANCEChlorpyrifos is a globally used pesticide with documented neurological effects on non-target organisms in the environment. Finding that chlorpyrifos can inhibit the growth of some soil microbes in vitro may have implications for the composition, stability, and health of the soil microbiome. Due to the importance of soil microbes to numerous biogeochemical processes in agricultural systems, additional investigations into the non-target effects of CPF on soil microbes are clearly needed.


title: Common dandelion (Taraxacum officinale) efficiently blocks the interaction between ACE2 cell surface receptor and SARS-CoV-2 spike protein D614, mutants D614G, N501Y, K417N and E484K in vitro

abstract:
On 11th March 2020, coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, was declared as a global pandemic by the World Health Organization (WHO). To date, there are rapidly spreading new "variants of concern" of SARS-CoV-2, the United Kingdom (B.1.1.7), the South African (B.1.351) or Brasilian (P.1) variant. All of them contain multiple mutations in the ACE2 receptor recognition site of the spike protein, compared to the original Wuhan sequence, which is of great concern, because of their potential for immune escape. Here we report on the efficacy of common dandelion (Taraxacum officinale) to block protein-protein interaction of spike S1 to the human ACE2 cell surface receptor. This could be shown for the original spike D614, but also for its mutant forms (D614G, N501Y, and mix of K417N, E484K, N501Y) in human HEK293-hACE2 kidney and A549-hACE2-TMPRSS2 lung cells. High molecular weight compounds in the water-based extract account for this effect. Infection of the lung cells using SARS-CoV-2 spike pseudotyped lentivirus particles was efficiently prevented by the extract and so was virus-triggered pro-inflammatory interleukin 6 secretion. Modern herbal monographs consider the usage of this medicinal plant as safe. Thus, the in vitro results reported here should encourage further research on the clinical relevance and applicability of the extract as prevention strategy for SARS-CoV-2 infection.

Significance statementSARS-CoV-2 is steadily mutating during continuous transmission among humans. This might eventually lead the virus into evading existing therapeutic and prophylactic approaches aimed at the viral spike. We found effective inhibition of protein-protein interaction between the human virus cell entry receptor ACE2 and SARS-CoV-2 spike, including five relevant mutations, by water-based common dandelion (Taraxacum officinale) extracts. This was shown in vitro using human kidney (HEK293) and lung (A549) cells, overexpressing the ACE2 and ACE2/TMPRSS2 protein, respectively. Infection of the lung cells using SARS-CoV-2 pseudotyped lentivirus was efficiently prevented by the extract. The results deserve more in-depth analysis of dandelions effectiveness in SARS-CoV-2 prevention and now require confirmatory clinical evidence.


title: In-vivo protein nitration and de-nitration facilitate Vibrio cholerae cell survival under anaerobic condition: Consequences of Nitrite induced protein nitration

abstract: Protein tyrosine nitration (PTN), a highly selective post translational modification, occurs in both prokaryotic and eukaryotic cells under nitrosative stress1. It is reported that the activities of many proteins are altered due to PTN2. PTN is found to be associated with many pathophysiological conditions like neurodegenerative and cardiac diseases etc.3. However, its physiological function is not yet clear. Like all other gut pathogens Vibrio cholerae also faces nitrosative stress in the gut environment which makes its proteome more vulnerable to PTN. Here, we report for the first time in-vivo PTN in V. cholerae. We show that in-vivo protein nitration is nitrite dependent and nitration-denitration phenomenon actually facilitates V. cholerae cell survival in anaerobic or hypoxic condition. In our study, we found that the extent of in-vivo nitration is negatively correlated with the intracellular nitrite content and maximum nitration occurs during log phase of V. cholerae. Most interestingly, a significant denitration was associated with increase in intracellular nitrate content during anaerobic incubation of aerobically grown late log phase cultures. In-vivo nitration could provide an avenue for toxic nitrite storage and nitrosative stress tolerance mechanism in many gut pathogens, whereas denitration could supply nitrate for cell survival in anaerobic nitrate deficient environment.


title: Differential CTCF binding in motor neurons and lymphocytes

abstract: Transcriptional regulation of protein-coding genes is a primary control mechanism of cellular function. Similarities in regulation of expression for select genes between lymphocytes and neurons have led to proposals that such genes may be useful biomarkers for some neurological disorders that can be monitored via patient lymphocyte populations. Examination of shared molecular mechanisms underlying neurogenesis and lymphocyte differentiation may give help to identify relevant pathways and suggest additional biomarkers in lymphocytes that are relevant to neurological disorders. In this study, we analysed similarities and conserved regions in several genes regulated by CCCTC-binding factor (CTCF) during lymphocyte and neuronal developmental stages. We performed epigenetic analysis of CTCF binding Trak1, Gabpa, Gabpb1, Gabpb2, Gfi1, Gfi1b gene loci at T and B lymphocytes at different developmental stages, as well as in neural progenitor cells and motor neurons. Common and shared CTCF binding events at Trak1 gene suggest additional transcriptional regulatory factors that control Trak1 gene expression levels differ in neurons and lymphocytes. Gabpb1 gene includes a common CTCF binding site shared with neurons and lymphocytes. Correlation of CTCF binding analysis and gene expression profile suggests that CTCF binding plays a role in epigenetic regulation of Gabpb1 gene. In contrast, while Gfi1a gene is phylogenetically well-conserved and CTCF sites are occupied in lymphocytes, there are no CTCF binding occupied in neurons and neural progenitor cells. Low expression levels of Gfi1s in neurons indicate that regulation of this gene is CTCF-independent in neurons. Although Gfi1b is highly homologous to Gfi1, differences in expression levels suggest that Gfi1b is critical for both lymphogenesis and neurogenesis. Neurons and lymphocytes have multiple common CTCF binding sites in the Gfi1b gene, although lineage specific transcriptional regulators that play a role in their different expression levels still need to be identified. The partial overlap in CTCF regulatory sites for some genes in neurons and lymphocytes suggest that there may be markers that can exhibit parallel changes in these cells and serve as biomarkers.

category: developmental biology


title: Blunted Fas signaling favors RIPK1-driven neutrophil necroptosis in critically ill COVID-19 patients

abstract: Critically ill COVID-19 patients are characterized by a severely dysregulated cytokine profile and elevated neutrophil counts, which are thought to contribute to disease severity. However, to date it remains unclear how neutrophils contribute to pathophysiology during COVID-19. Here, we assessed the impact of the dysregulated cytokine profile on the tightly regulated cell death program of neutrophils. We show that in a subpopulation of neutrophils, canonical apoptosis was skewed towards rapidly occurring necroptosis. This phenotype was characterized by abrogated caspase-8 activity and increased RIPK1 levels, favoring execution of necroptosis via the RIPK1-RIPK3-MLKL axis, as further confirmed in COVID-19 biopsies. Moreover, reduction of sFas-L levels in COVID-19 patients and hence decreased signaling to Fas directly increased RIPK1 levels and correlated with disease severity. Our results suggest an important role for Fas signaling in the regulation of cell death program ambiguity via the ripoptosome in neutrophils during COVID-19 and a potential therapeutic target to curb inflammation and thus influence disease severity and outcome.


title: Pronounced genetic separation among varieties of the Primula cusickiana species complex, a Great Basin endemic

abstract: Distinguishing between unique species and populations with strong genetic structure is a common challenge in population genetics, especially in fragmented habitats where allopatric speciation may be widespread and distinct groups may be morphologically similar. Such is often the case with species complexes across sky island environments. In these scenarios, biogeography may help to explain the relations between species complex members, and RADseq methods are commonly used to compare closely related species across thousands of genetic loci. Here we use RADseq to clarify the relations between geographically distinct but morphologically similar varieties of the Primula cusickiana species complex, and to contextualize past findings of strong genetic structure among populations within varieties. Our genomic analyses demonstrate pronounced separation between isolated populations of this Great Basin endemic, indicating that the current varietal classification of complex members is inaccurate and emphasizing their conservation importance. We discuss how these results correspond to recent biogeographical models used to describe the distribution of other sky island taxa in western North America. Our findings also fit into a wider trend observed for alpine Primula species complexes, and we consider how heterostylous breeding systems may be contributing to frequent diversification via allopatric speciation in this genus.

category: evolutionary biology


title: Genetic Susceptibility to Enteric Fever in Experimentally Challenged Human Volunteers

abstract:
BackgroundInfection with Salmonella enterica serovars Typhi and Paratyphi A cause an estimated 14 million cases of enteric fever annually. Here the controlled nature of challenge studies is exploited to identify genetic variants associated with enteric fever susceptibility.

MethodsHuman challenge participants were genotyped by Illumina OmniExpress-24 BeadChip array (n=176) and/or transcriptionally profiled by RNA-sequencing (n=178).

ResultsTwo SNPs within CAPN14 and MIATNB were identified with p<10-5 for association with development of symptoms or bacteraemia following oral S. Typhi or S. Paratyphi A challenge. Imputation of classical human leukocyte antigen (HLA) types from genomic and transcriptomic data identified HLA-B*27:05, previously associated with non-typhoidal Salmonella-induced reactive arthritis, as the HLA type most strongly associated with enteric fever susceptibility (p=0.012). Genes related to the unfolded protein response and heat shock were over-represented in HLA-B*27:05+ participants following challenge (p=0.01). Furthermore, intracellular replication of S. Typhi is higher in C1R cells transfected with HLA-B*27:05 (p=0.02).

ConclusionThese data suggest that activation of the unfolded protein response by HLA-B*27:05 misfolding may create an intracellular environment conducive to S. Typhi replication, increasing susceptibility to enteric fever.

category: genetics


title: Insecticide resistance characteristic of Anopheles vector species successfully controlled by deployment of pyrethroid and PBO long lasting insecticidal treated nets (LLINs) in Tanzania

abstract: Long lasting insecticidal nets (LLINs) are a proven tool to reduce malaria transmission, but in Africa efficacy is being reduced by pyrethroid resistance in the major vectors. A cluster randomized trial in Muleba district, Tanzania demonstrated that permethrin LLINs co-treated with piperonyl butoxide (PBO), a synergist that can block pyrethroid-metabolizing enzymes in the mosquito, had much greater efficacy than pyrethroid-only nets. Insecticide resistance profiles and underlying mechanisms were investigated in Anopheles gambiae and An. funestus from Muleba during the trial. Diagnostic dose bioassays using permethrin, together with intensity assays, suggest pyrethroid resistance that is both strong and very common, but not extreme. Transcriptomic analysis found multiple P450 genes over expressed including CYP6M2, CYP6Z3, CYP6P3, CYP6P4, CYP6AA1 and CYP9K1 in An. gambiae and CYP6N1, CYP6M7, CYP6M1 and CYP6Z3 in An. funestus. Indeed, very similar suites of P450 enzymes commonly associated with resistant populations elsewhere in Africa were detected as over expressed suggesting a convergence of mechanisms across Sub-Saharan African malaria vectors. The findings give insight into factors that may correlate with pyrethroid PBO LLIN success, broadly supporting model predictions, but revision to guidelines previously issued by the World Health Organization is warranted.

category: molecular biology


title: DHA but not EPA induces the trans-differentiation of C2C12 cells into white-like adipocytes phenotype

abstract: Muscle derived stem cells (MDSCs) and myoblast play an important role in myotube regeneration when muscle tissue is injured. However, these cells can be induced to differentiate into adipocytes once exposed to PPAR{gamma} activator like EPA and DHA that are highly suggested during pregnancy. The objective of this study aims at determining the identity of trans-differentiated cells by exploring the effect of EPA and DHA on C2C12 undergoing differentiation into brown and white adipocytes. DHA but not EPA committed C2C12 cells reprograming into white like adipocyte phenotype. Also, DHA promoted the expression of lipolysis regulating genes but had no effect on genes regulating {beta}-oxidation referring to its implication in lipid re-esterification. Furthermore, DHA impaired C2C12 cells differentiation into brown adipocytes through reducing the thermogenic capacity and mitochondrial biogenesis of derived cells independent of UCP1. Accordingly, DHA treated groups showed an increased accumulation of lipid droplets and suppressed mitochondrial maximal respiration and spare respiratory capacity. EPA, on the other hand, reduced myogenesis regulating genes, but no significant differences were observed in the expression of adipogenesis key genes. Likewise, EPA suppressed the expression of WAT signature genes indicating that EPA and DHA have an independent role on white adipogeneis. Unlike DHA treatment, EPA supplementation had no effect on the differential of C2C12 cells into brown adipocytes. In conclusion, DHA is a potent adipogenic and lipogenic factor that can change the metabolic profile of muscle cells by increasing myocellular fat.

category: cell biology


title: Exploring an experiment-split method to estimate the generalization ability in new data: DeepKme as an example

abstract: A Large number of predictors have been built based on different data sets for predicting different post-translational modification sites. However, limited to our knowledge, most of them gave an overfitting estimation of their generalization ability in new data because of the intrinsic trait--not considering the experimental sources of the new data--of the cross-validation method. Thus, we proposed and explored a new method--the experiment-split method--imitating the blinded assessment to deal with the overfitting problem in the new data. The experiment-split method logically split the training and test data based on the datas different experimental sources, and the new data can be regarded as the data from different experimental sources. To specifically illustrate the experiment-split method, we combined an actual application, DeepKme--a predictor built by us for the lysine methylation sites, to demonstrate how it be used in the true scenarios. We compared the cross-validation method with the experiment-split method. The result suggested the experiment-split method could effectively relieve the overfitting compared with the cross-validation method and may be widely used in the field of identification participated by multiple experiments. We believe DeepKme would facilitate the related researchers deep thought of the experiment-split method and the overfitting phenomenon, and of course, advance the study of the lysine methylation and similar fields.

category: bioinformatics


title: Revealing the transcription factor regulatory context of human specific cortical development using single-cell multi-omics

abstract: Human behaviors are at least partially driven by genomic regions that influence human-specific neurodevelopment. This includes genomic regions undergoing human specific sequence acceleration (Human Accelerated Regions or HARs) and regions showing human-specific enhancer activity (Human Gained Enhancers or HGEs) not present in other primates. However, prior studies on HAR/HGE activities involved mixtures of brain cell types and focused only on putative downstream target genes. Here, we directly measured cell type specific HAR/HGE activity in the developing fetal human brain using two independent single-cell chromatin accessibility datasets with matching single-cell gene expression data. Transcription factor (TF) motif analyses identified upstream TFs binding to HARs/HGEs and identified LHX2, a key regulator of forebrain development, as an active HGE regulator in neuronal progenitors. We integrated our TF motif analyses with published chromatin interaction maps to build detailed regulatory networks where TFs are linked to downstream genes via HARs/HGEs. Through these networks, we identified a potential regulatory role for NFIC in human neuronal progenitor networks via modulating the Notch signaling and cell adhesion pathways. Therefore, by using a single cell multi-omics approach, we were able to capture both the upstream and downstream regulatory context of HARs/HGEs, which may provide a more comprehensive picture of the roles HARs/HGEs play amongst diverse fetal cell types of the developing human brain.


title: Invariant Representation of Physical Stability in the Human Brain

abstract: Successful engagement with the world requires the ability to predict what will happen next. Here we investigate how the brain makes the most basic prediction about the physical world: whether the situation in front of us is stable, and hence likely to stay the same, or unstable, and hence likely to change in the immediate future. Specifically, we ask if judgements of stability can be supported by the kinds of representations that have proven to be highly effective at visual object recognition in both machines and brains, or instead if the ability to determine the physical stability of natural scenes may require generative algorithms that simulate the physics of the world. To find out, we measured responses in both convolutional neural networks (CNNs) and the brain (using fMRI) to natural images of physically stable versus unstable scenarios. We find no evidence for generalizable representations of physical stability in either standard CNNs trained on visual object and scene classification (ImageNet), or in the human ventral visual pathway, which has long been implicated in the same process. However, in fronto-parietal regions previously implicated in intuitive physical reasoning we find both scenario-invariant representations of physical stability, and higher univariate responses to unstable than stable scenes. These results demonstrate abstract representations of physical stability in the dorsal but not ventral pathway, consistent with the hypothesis that the computations underlying stability entail not just pattern classification but forward physical simulation.


title: Identifying disease-critical cell types and cellular processes across the human body by integration of single-cell profiles and human genetics

abstract: Genome-wide association studies (GWAS) provide a powerful means to identify loci and genes contributing to disease, but in many cases the related cell types/states through which genes confer disease risk remain unknown. Deciphering such relationships is important for identifying pathogenic processes and developing therapeutics. Here, we introduce sc-linker, a framework for integrating single-cell RNA-seq (scRNA-seq), epigenomic maps and GWAS summary statistics to infer the underlying cell types and processes by which genetic variants influence disease. We analyzed 1.6 million scRNA-seq profiles from 209 individuals spanning 11 tissue types and 6 disease conditions, and constructed gene programs capturing cell types, disease progression, and cellular processes both within and across cell types. We evaluated these gene programs for disease enrichment by transforming them to SNP annotations with tissue-specific epigenomic maps and computing enrichment scores across 60 diseases and complex traits (average N=297K). Cell type, disease progression, and cellular process programs captured distinct heritability signals even within the same cell type, as we show in multiple complex diseases that affect the brain (Alzheimers disease, multiple sclerosis), colon (ulcerative colitis) and lung (asthma, idiopathic pulmonary fibrosis, severe COVID-19). The inferred disease enrichments recapitulated known biology and highlighted novel cell-disease relationships, including GABAergic neurons in major depressive disorder (MDD), a disease progression M cell program in ulcerative colitis, and a disease-specific complement cascade process in multiple sclerosis. In autoimmune disease, both healthy and disease progression immune cell type programs were associated, whereas for epithelial cells, disease progression programs were most prominent, perhaps suggesting a role in disease progression over initiation. Our framework provides a powerful approach for identifying the cell types and cellular processes by which genetic variants influence disease.


title: The responsiveness of gait and balance outcomes to disease progression in Friedreich ataxia

abstract:
ObjectiveTo identify gait and balance measures that are responsive to change during the timeline of a clinical trial in Friedreich ataxia (FRDA) we administered a battery of potential measures three times over a 12-month period.

MethodsSixty-one ambulant individuals with FRDA underwent assessment of gait and balance at baseline, six months and 12 months. Outcomes included: GAITRite(R) spatiotemporal gait parameters; Biodex Balance System Postural Stability Test (PST) and Limits of Stability; Berg Balance Scale (BBS); Timed 25 Foot Walk Test; Dynamic Gait Index (DGI); SenseWear MF Armband step and energy activity; and the Friedreich Ataxia Rating Scale Upright Stability Subscale (FARS USS). The standardised response mean (SRM) or correlation coefficients were reported as effect size indices for comparison of internal responsiveness. Internal responsiveness was also analysed in subgroups.

ResultsSenseWear Armband daily step count had the largest effect size of all the variables over six months (SRM=-0.615), while the PST medial-lateral index had the largest effect size (SRM=0.829) over 12 months. The FARS USS (SRM=0.824) and BBS (SRM=-0.720) were the only outcomes able to detect change over 12 months in all subgroups. The DGI was the most responsive outcome in children, detecting a mean change of -2.59 (95% CI -3.52 to -1.66, p<0.001, SRM=-1.429).

ConclusionsThe FARS USS and BBS are highly responsive and can detect change in a wide range of ambulant individuals with FRDA. However, therapeutic effects in children may be best measured by the DGI.


title: Specific protein-membrane interactions promote the export of metallo-β-lactamases via outer membrane vesicles

abstract: Outer membrane vesicles (OMVs) act as carriers of resistance determinants such as metallo-{beta}-lactamases. The metallo-{beta}-lactamase NDM-1 is present in OMVs produced by Gram-negative bacteria since it is a lipidated, membrane-anchored protein. The soluble domain of NDM-1 also forms electrostatic interactions with the membrane. Herein, we show that these interactions promote its export into OMVs produced by Escherichia coli. We report that favorable electrostatic protein-membrane interactions are also at work in the soluble enzyme IMP-1, while being absent in VIM-2. These interactions correlate with an enhanced secretion into OMVs of IMP-1 compared to VIM-2. Disruption of these interactions in NDM-1 and IMP-1 impairs export into vesicles, confirming their role in defining the protein cargo in OMVs. These results also indicate that export of metallo-{beta}-lactamases into vesicles in their active form is a common phenomenon that involves cargo selection based on molecular features.


title: Gene network simulations provide testable predictions for the molecular domestication syndrome

abstract: The domestication of plant species lead to repeatable morphological evolution, often referred to as the phenotypic domestication syndrome. Domestication is also associated with important genomic changes, such as the loss of genetic diversity compared to adequately large wild populations, and modifications of gene expression patterns. Here, we explored theoretically the effect of a domestication-like scenario on the evolution of gene regulatory networks. We ran population genetics simulations in which individuals were featured by their genotype (an interaction matrix encoding a gene regulatory network) and their gene expressions, representing the phenotypic level. Our domestication scenario included a population bottleneck and a selection switch mimicking human-mediated directional and canalizing selection, i.e., change in the optimal gene expression level and selection towards more stable expression across environments. We showed that domestication profoundly alters genetic architectures. Based on four examples of plant domestication scenarios, our simulations predict (i) a drop in neutral allelic diversity, (ii) a change in gene expression variance that depends upon the domestication scenario, (iii) transient maladaptive plasticity, (iv) a deep rewiring of the gene regulatory networks, with a trend towards gain of regulatory interactions, and (v) a global increase in the genetic correlations among gene expressions, with a loss of modularity in the resulting coexpression patterns and in the underlying networks. We provide empirically testable predictions on the differences of genetic architectures between wild and domesticated forms. The characterization of such systematic evolutionary changes in the genetic architecture of traits contributes to define a molecular domestication syndrome.


title: Influence of the TorD signal peptide chaperone on Tat-dependent protein translocation

abstract: The twin-arginine translocation (Tat) pathway transports folded proteins across energetic membranes. Numerous Tat substrates contain co-factors that are inserted before transport with the assistance of redox enzyme maturation proteins (REMPs), which bind to the signal peptide of precursor proteins. How signal peptides are transferred from a REMP to a binding site on the Tat receptor complex remains unknown. Since the signal peptide mediates both interactions, possibilities include: i) a coordinated hand-off mechanism; or ii) a diffusional search after REMP dissociation. We investigated the binding interaction between substrates containing the TorA signal peptide (spTorA) and its cognate REMP, TorD, and the effect of TorD on the in vitro transport of such substrates. We found that Escherichia coli TorD is predominantly a monomer at low micromolar concentrations (dimerization KD> 50 {micro}M), and this monomer binds reversibly to spTorA (KD{approx} 1 {micro}M). While TorD binds to membranes (KD{approx} 100 nM), it has no apparent affinity for Tat translocons and it inhibits binding of a precursor substrate to the membrane. TorD has a minimal effect on substrate transport by the Tat system, being mildly inhibitory at high concentrations. These data are consistent with a model in which the REMP-bound signal peptide is shielded from recognition by the Tat translocon, and spontaneous dissociation of the REMP allows the substrate to engage the Tat machinery. Thus, the REMP does not assist with targeting to the Tat translocon, but rather temporarily shields the signal peptide.


title: A versatile CRISPR toolbox to study neurological CaV3.2 channelopathies by promoter-mediated transcription control

abstract: Precise genome editing in combination with viral delivery systems provides a valuable tool for neuroscience research. Traditionally, the role of genes in neuronal circuits has been addressed by overexpression or knock-out/knock-down systems. However, those techniques do not manipulate the endogenous loci and therefore have limitations. Those constraints include that many genes exhibit extensive alternative splicing, which can be regulated by neuronal activity. This complexity cannot be easily reproduced by overexpression of one protein variant. The CRISPR activation and interference/inhibition systems (CRISPRa/i) directed to promoter sequences can modulate the expression of selected target genes in a highly specific manner. This strategy could be particularly useful for the overexpression of large proteins and for alternatively spliced genes, e.g. for studying large ion channels known to be affected in ion channelopathies in a variety of neurological diseases. Here, we demonstrate the feasibility of a newly developed CRISPRa/i toolbox to manipulate the promoter activity of the Cacna1h gene. Impaired, function of the low-voltage-activated T-Type calcium channel CaV3.2 is involved in genetic/mutational as well as acquired/transcriptional channelopathies that emerge with epileptic seizures. We show CRISPR-induced activation and inhibition of the Cacna1h locus in NS20Y cells and primary cortical neurons, as well as activation in mouse organotypic slice cultures. In future applications, the system offers the intriguing perspective to study functional effects of gain-of-function or loss-of-function variations in the Cacna1h gene in more detail. A better understanding of CaV3.2 channelopathies might result in a major advancement in the pharmacotherapy of CaV3.2 channelopathy diseases.


title: RIM-Binding Protein 2 organizes Ca2+ channel topography and regulates release probability and vesicle replenishment at a fast central synapse

abstract:
RIM-Binding Protein 2 (RIM-BP2) is a multi-domain protein of the presynaptic active zone (AZ). By binding to Rab-interacting protein (RIM), bassoon and voltage-gated Ca{superscript 2} channels (CaV), it is considered to be a central organizer of the topography of CaV and release sites of synaptic vesicles (SVs) at the AZ. Here, we investigated the role of RIM-BP2 at the endbulb of Held synapse of auditory nerve fibers with bushy cells of the cochlear nucleus, a fast relay of the auditory pathway with high release probability. Disruption of RIM-BP2 lowered release probability altering short-term plasticity and reduced evoked excitatory postsynaptic currents (EPSCs). Analysis of SV pool dynamics during high frequency train stimulation indicated a reduction of SVs with high release probability but an overall normal size of the readily releasable SV pool (RRP). The Ca2+-dependent fast component of SV replenishment after RRP depletion was slowed. Ultrastructural analysis by super-resolution light and electron microscopy revealed an impaired topography of presynaptic CaV and a reduction of docked and membrane-proximal SVs at the AZ. We conclude that RIM-BP2 organizes the topography of CaV, and promotes SV tethering and docking. This way RIM-BP2 is critical for establishing a high initial release probability as required to reliably signal sound onset information that we found to be degraded in bushy cells of RIM-BP2-deficient mice in vivo.

Significance StatementRIM-binding proteins (RIM-BPs) are key organizers of the active zone (AZ). Using a multidisciplinary approach to the calyceal endbulb of Held synapse that transmit auditory information at rates of up to hundreds of Hertz with sub-millisecond precision we demonstrate a requirement for RIM-BP2 for normal auditory signaling. Endbulb synapses lacking RIM-BP2 show a reduced release probability despite normal whole-terminal Ca2+ influx and abundance of the key priming protein Munc13-1, a reduced rate of SV replenishment, as well as an altered topography of CaV2.1 Ca2+ channels, and fewer docked and membrane proximal synaptic vesicles. This hampers transmission of sound onset information likely affecting downstream neural computations such as of sound localization.


title: Two-pore channel 2 is a key regulator of adipocyte differentiation via the cAMP signaling pathway with calpain as downstream effector

abstract: We investigated whether the endolysosomal two-pore channel TPC2 is a mediator of adipocyte differentiation. We show that Tpcn2 mRNA is expressed transiently during induction of C3H10T1/2 mesenchymal stem cells to differentiate into adipocytes, and that this expression is triggered by cAMP. This is the first demonstration of a cell signaling pathway that can regulate TPC gene expression. We also identified an important functional role for TPC2 in adipocyte differentiation. First, ectopic TPC2 expression in C3H10T1/2 cells partially rescued the block to adipocyte differentiation caused by cAMP absence. Second, inhibition of endogenous TPC2 expression in primary preadipocytes substantially reduced their ability to differentiate into adipocytes. Finally, genetic variation at the Tpcn2 locus is associated with increased upper-body fat distribution in women concomitant with reduced Tpcn2 expression in abdominal adipose tissue. Our findings implicate TPC2 as an important mediator of adipogenesis and may aid identification of new drug targets for treatment of obesity.


title: Talin mechanosensitivity is modulated by a direct interaction with cyclin-dependent kinase-1

abstract:
Talin is a mechanosensitive component of adhesion complexes that directly couples integrins to the actin cytoskeleton. In response to force, talin undergoes switch-like behaviour of its multiple rod domains that modulate interactions with its binding partners. Cyclin-dependent kinase-1 (CDK1) is a key regulator of the cell cycle, exerting its effects through synchronised phosphorylation of a large number of protein targets. CDK1 activity also maintains adhesion during interphase, and its inhibition is a prerequisite for the tightly choreographed changes in cell shape and adhesiveness that are required for successful completion of mitosis. Using a combination of biochemical, structural and cell biological approaches, we demonstrate a direct interaction between talin and CDK1 that occurs at sites of integrin-mediated adhesion. Mutagenesis demonstrated that CDK1 contains a functional talin-binding LD motif, and the binding site within talin was pinpointed to helical bundle R8 through the use of recombinant fragments. Talin also contains a consensus CDK1 phosphorylation motif centred on S1589; a site that was phosphorylated by CDK1 in vitro. A phosphomimetic mutant of this site within talin lowered the binding affinity of KANK and weakened the mechanical response of the region, potentially altering downstream mechanotransduction pathways. The direct binding of the master cell cycle regulator, CDK1, to the primary integrin effector, talin, therefore provides a primordial solution for coupling the cell proliferation and cell adhesion machineries, and thereby enables microenvironmental control of cell division in multicellular organisms.

SummaryThe direct binding of the master cell cycle regulator, CDK1, to the primary integrin effector, talin, provides a primordial solution for coupling the cell proliferation and cell adhesion machineries, and thereby enables microenvironmental control of cell division.


title: Drosophila Male Accessory Gland Displays Developmental Ground Plan of a Primitive Prostate

abstract: Conservation of developmental genetic toolkits of functionally comparable organs from disparate phyla reveals their deep homology, which may help overcome the challenges of their confounding categorization as either homologous or analogous organs. A male accessory sexual organ in mammals, prostate, for instance, is anatomically disparate from its phylogenetically distant counterpart--the male accessory gland (MAG)--in insects like Drosophila. By examining a select set of toolkit gene expression patterns, here we show that Drosophila MAG displays deep homology with the mammalian prostate. Like mammalian prostate, MAG morphogenesis is marked by recruitment of fibroblast growth factor receptor, FGFR, a tubulogenesis toolkit signaling pathway, starting early during its adepithelial genesis. Specialization of the individual domains of the developing MAG tube on the other hand is marked by expression of a posterior Hox gene transcription factor, Abd-B, while Hh-Dpp signaling marks its growth. Drosophila MAG thus reveals developmental design of unitary bud-derived tube--a ground plan that appears to have been reiteratively co-opted during evolutionary diversification of male accessory sexual organs across distant phylogeny.


title: Viscoelasticity and cell jamming state transition

abstract: Although collective cell migration (CCM) is a highly coordinated migratory mode, perturbations in the form of jamming state transitions and vice versa often occur even in 2D. These perturbations are involved in various biological processes, such as embryogenesis, wound healing and cancer invasion. CCM induces accumulation of cell residual stress which has a feedback impact to cell packing density. Density-mediated change of cell mobility influences the state of viscoelasticity of multicellular systems and on that base the jamming state transition. Although a good comprehension of how cells collectively migrate by following molecular rules has been generated, the impact of cellular rearrangements on cell viscoelasticity remains less understood. Thus, considering the density driven evolution of viscoelasticity caused by reduction of cell mobility could result in a powerful tool in order to address the contribution of cell jamming state transition in CCM and help to understand this important but still controversial topic. In addition, five viscoelastic states gained within three regimes: (1) convective regime, (2) conductive regime, and (3) damped-conductive regime was discussed based on the modeling consideration with special emphasis of jamming and unjamming states.


title: Nestmate recognition of early brood in ants

abstract: Brood is critically important in social insect colonies. It carries the colony fitness through delivering future reproductive adults as well as workers that will increase the colonys workforce. Adoption of non-nestmate brood can be a mean to increase colonys workforce but entails the risk of rearing unrelated sexuals or social parasites. For early brood (eggs and L1 larvae), this balance is less positive as young brood need a substantial amount of resource before becoming workers. Thus, it appears beneficial for ant workers to discriminate between nestmate and alien brood using the chemical cues displayed at the broods surface. However, the chemical signature of ant early brood stages and its use by workers remains understudied. To fill this gap, we investigated the chemical basis of early brood nestmate and cross-species recognition in six Formicoid ants. We also tested the discrimination behaviour of workers in brood retrieval trials. We observed clear species-level cues and discrimination against heterospecific brood. We also found that eggs and most young larvae display a colony signature but that only some species discriminate against non-nestmate eggs and L1 larvae. Interestingly, these species appear to also be those belonging to genera subject to brood parasitism.

category: animal behavior and cognition


title: Visualising formation of the ribosomal active site in mitochondria

abstract: Ribosome assembly is an essential and complex process that is regulated at each step by specific biogenesis factors. Using cryo-electron microscopy, we identify and order major steps in the formation of the highly conserved peptidyl transferase centre (PTC) and tRNA binding sites in the large subunit of the human mitochondrial ribosome (mitoribosome). The conserved GTPase GTPBP7 regulates the folding and incorporation of core 16S ribosomal RNA (rRNA) helices and the ribosomal protein bL36m, and ensures that the PTC base U3039 has been 2'-O-methylated. Additionally, GTPBP7 binds the RNA methyltransferase NSUN4 and MTERF4, which facilitate earlier steps by sequestering H68-71 of the 16S rRNA and allowing biogenesis factors to access the maturing PTC. Consistent with the central role of NSUN4*MTERF4 and GTPBP7 during mitoribosome biogenesis, in vivo mutagenesis designed to disrupt binding of their Caenorhabditis elegans orthologs to the large subunit potently activates mitochondrial stress responses and results in severely reduced viability, developmental delays and sterility. Next-generation RNA sequencing reveals widespread gene expression changes in these mutant animals that are indicative of mitochondrial stress response activation. We also answer the long-standing question of why NSUN4 but not its enzymatic activity, is indispensable for mitochondrial protein synthesis in metazoans.


title: N-myristoyltransferase inhibition is synthetic lethal in MYC-deregulated cancers

abstract:
Human N-myristoyltransferases (NMTs) catalyze N-terminal protein myristoylation, a modification regulating membrane trafficking and interactions of >100 proteins. NMT is a promising target in cancer, but a mechanistic rationale for targeted therapy remains poorly defined. Here, large-scale cancer cell line screens against a panel of NMT inhibitors (NMTi) were combined with systems-level analyses to reveal that NMTi is synthetic lethal with deregulated MYC. Synthetic lethality is mediated by post-transcriptional failure in mitochondrial respiratory complex I protein synthesis concurrent with loss of myristoylation and degradation of complex I assembly factor NDUFAF4, followed by mitochondrial dysfunction specifically in MYC-deregulated cancer cells. NMTi eliminated MYC-deregulated tumors in vivo without overt toxicity, providing a new paradigm in which targeting a constitutive co-translational protein modification is synthetically lethal in MYC-deregulated cancers.

One-sentence summaryN-myristoyltransferase inhibition leads to post-transcriptional complex I failure and cell death in MYC-deregulated cancers

category: cancer biology


title: Testing the inferred transcription rates of a dynamic, gene network model in absolute units

abstract: The circadian clock coordinates plant physiology and development. Mathematical clock models have provided a rigorous framework to understand how the observed rhythms emerge from disparate, molecular processes. However, models of the plant clock have largely been built and tested against RNA timeseries data in arbitrary, relative units. This limits model transferability, refinement from biochemical data and applications in synthetic biology. Here, we incorporate absolute mass units into a detailed, gene circuit model of the clock in Arabidopsis thaliana. We re-interpret the established P2011 model, highlighting a transcriptional activator that overlaps the function of REVEILLE 8/LHY-CCA1-LIKE 5, and refactor dynamic equations for the Evening Complex. The U2020 model incorporates the repressive regulation of PRR genes, a key feature of the most detailed clock model F2014, without greatly increasing model complexity. We tested the experimental error distributions of qRT-PCR data calibrated for units of RNA transcripts/cell and of circadian period estimates, in order to link the models to data more appropriately. U2019 and U2020 models were constrained using these data types, recreating previously-described circadian behaviours with RNA metabolic processes in absolute units. To test their inferred rates, we estimated a distribution of observed, transcriptome-wide transcription rates (Plant Empirical Transcription Rates, PETR) in units of transcripts/cell/hour. The PETR distribution and the equivalent degradation rates indicated that the models predicted rates are biologically plausible, with individual exceptions. In addition to updated, explanatory models of the plant clock, this validation process represents an advance in biochemical realism for models of plant gene regulation.

category: systems biology


title: Unbiased transcriptomic analysis of chondrocyte differentiation in a high-density cell culture model

abstract: In the developing vertebrate skeleton, cartilage is an important precursor to the formation of bones. Cartilage is produced by chondrocytes, which derive from embryonic mesoderm and undergo a stereotypical program of differentiation and maturation. Here we modeled this process in vitro, using primary fetal mouse rib chondrocytes in a high-density cell culture model of cartilage differentiation, and performed genome-wide gene expression profiling over the course of culture. The overarching goal of this study was to characterize the molecular pathways involved in cartilage differentiation and maturation. Our results also enable a comprehensive appraisal of distinctions between common in vitro models for cartilage differentiation, and of differences in their molecular resemblance to cartilage formation in vivo.


title: Bacterial hopanoids: a newly identified potent inducer of monocyte to macrophage differentiation

abstract: Monocyte to macrophage differentiation is an extremely essential cellular and immunological process aimed to combat the assault of foreign pathogens as well as this process has immense importance in the maintenance of physiological homeostasis. Differentiated macrophage, serving as specialized phagocytes is an indispensable component of innate immunity. Besides this, being a well-documented antigen-presenting cell (APC) macrophages also function as a key regulator of both humoral and cell-mediated immunity: the two integral components of adaptive immunity. Bacterial hopanoids are a primitive analogous of eukaryotic sterols that are established as a prominent immunomodulator. In this study, we have demonstrated the isolation protocol of neutral lipid fractions from different Gram-negative bacteria, and adopting several analytical approaches we proposed that the extracted lipid fractions may contain hopanoid as an active component. We have delved deeper into the biological effect of these probable hopanoids like compounds. Here, by considering the several structural and functional attributes like altered phenotype, expression of macrophage-specific markers, increased intracellular organelles, acquirement of enhanced phagocytic and inflammatory property, induction of autophagy, etc we have established hopanoids as a potential inducer of monocyte to macrophage differentiation. Thus, our study has unraveled a new immunomodulator present in Gram-negative bacteria and would undoubtedly help to understand the intricacies of host-pathogen interaction in a better and conclusive manner.


title: HLA Type and Chronic Viral Infection Impact Peripheral T-cell Receptor Sharing Between Unrelated Individuals

abstract: The human adaptive immune system must generate extraordinary diversity to be able to respond to all possible pathogens. The T-cell repertoire derives this high diversity through somatic recombination of the T-cell receptor (TCR) locus, a random process that results in repertoires that are largely private to each individual. However, certain factors such as low junctional diversity, thymic selection, and T-cell proliferation upon antigen exposure can affect TCR sharing among individuals. By immunosequencing the TCR{beta} variable region of 426 healthy individuals, we find that fewer than 1% of TCR{beta} clones are shared between individuals on average, consistent with largely private TCR{beta} repertoires. However, we detect a significant correlation between increased HLA allele sharing and increased number of shared TCR{beta} clones, with each additional shared HLA allele contributing to an increase in [~]0.01% of the total TCR{beta} clones being shared, supporting a key role for HLA type in shaping the immune repertoire. Surprisingly, we find that shared antigen exposure to CMV leads to fewer shared TCR{beta} clones, even after controlling for HLA, indicative of a largely private response to major viral antigenic exposure. Consistent with this hypothesis, we find that increased age is correlated with decreased overall TCR{beta} clone sharing, indicating that the pattern of private TCR{beta} clonal expansion is a general feature of the T-cell response to other infectious antigens. All of these factors contribute to shaping the TCR{beta} repertoire, and understanding their interplay has important implications for the use of T cells for therapeutics and diagnostics.


title: CCL28 modulates neutrophil responses and impacts the trajectory of mucosal infections

abstract: The chemokine CCL28 is highly expressed in mucosal tissues, but its role during infection is not well understood. Here we show that CCL28 promotes neutrophil accumulation to the gut of mice infected with Salmonella and to the lung of mice infected with Acinetobacter. Neutrophils isolated from the infected mucosa expressed the CCL28 receptors CCR3 and, to a lesser extent, CCR10, on their surface. The functional consequences of CCL28 deficiency were different between the two infections, as Ccl28-/- mice were highly susceptible to Salmonella gut infection, but highly resistant to otherwise lethal Acinetobacter lung infection. In vitro, unstimulated neutrophils harbored pre-formed intracellular CCR3 that was rapidly mobilized to the cell surface following phagocytosis or inflammatory stimuli. Moreover, CCL28 stimulation enhanced neutrophil antimicrobial activity, production of reactive oxygen species, and formation of extracellular traps, all processes that were largely dependent on CCR3. Consistent with the different outcomes in the two infection models, neutrophil stimulation with CCL28 boosted the killing of Salmonella but not of Acinetobacter. CCL28 thus plays a critical role in the immune response to mucosal pathogens by increasing neutrophil accumulation and activation, which can enhance pathogen clearance but also exacerbate disease depending on the mucosal site and the infectious agent.


title: CCL28 modulates neutrophil responses during infection with mucosal pathogens


title: The Card19 locus of murine chromosome 13 regulates terminal cell lysis downstream of caspase activation and Gasdermin-D cleavage

abstract:
Cell death plays a critical role in inflammatory responses. During pyroptosis, inflammatory caspases cleave Gasdermin D (GSDMD) to release an N-terminal fragment that generates plasma membrane pores that mediate cell lysis and IL-1 cytokine release. Terminal cell lysis and IL-1{beta} release following caspase activation can be uncoupled in certain cell types or in response to particular stimuli, a state termed hyperactivation. However, the factors and mechanisms that regulate terminal cell lysis downstream of GSDMD cleavage remain poorly understood. In the course of studies to define regulation of pyroptosis during Yersinia infection, we identified a line of Card19-deficient mice (Card19lxcn) whose macrophages were protected from cell lysis and showed reduced apoptosis and pyroptosis, yet had wild-type levels of caspase activation, IL-1 secretion, and GSDMD cleavage. Unexpectedly, CARD19, a mitochondrial CARD-containing protein, was not directly responsible for this, as two independently-generated CRISPR/Cas9 Card19 knockout mice showed no defect in macrophage cell lysis, and expression of CARD19 in Card19lxcn macrophages did not restore cell lysis. Card19 is located on chromosome 13, adjacent to Ninj1, which was recently reported to regulate cell lysis downstream of GSDMD activation. Intriguingly, RNA-seq and western blotting revealed that Card19lxcn BMDMs are hypomorphic for NINJ1 expression, and reconstitution of Ninj1 in Card19lxcn immortalized BMDMs restored cell lysis. Card19lxcn mice exhibited significantly increased susceptibility to Yersinia infection, demonstrating that cell lysis itself plays a key role in protection against bacterial infection. Our findings identify genetic targeting of Card19 being responsible for off-target effects on the adjacent Ninj1 gene, thereby disrupting the ability of macrophages to undergo plasma membrane rupture downstream of gasdermin cleavage and impacting host survival and bacterial control during Yersinia infection.

Author SummaryProgrammed cell death is critical for regulating tissue homeostasis and host defense against infection. Pyroptosis is an inflammatory form of programmed cell death that couples cell lysis with release of inflammatory cytokines. Cell lysis is triggered by activation of particular intracellular pore forming proteins, but how regulation of cell lysis occurs is not well understood. Genetic targeting of Card19 on chromosome 13 resulted in decreased expression of the adjacent gene, Ninj1 which was recently found to regulate terminal lysis events in response to cell death-inducing stimuli. We found that macrophages from Card19-deficient mice were resistant to multiple forms of cell death in response to a variety of inflammatory stimuli, including canonical and non-canonical inflammasome activation, as well as triggers of cell-extrinsic apoptosis. Notably, Card19-deficient mice were more susceptible to Yersinia infection, indicating that cell lysis contributes to control of bacterial infections. Our data provide new insight into the impact of terminal cell lysis on control of bacterial infection and highlight the role of additional factors that regulate lytic cell death downstream of gasdermin cleavage.


title: Genetic targeting of Card19 is linked to disrupted Ninj1 expression, impaired cell lysis, and increased susceptibility to Yersinia infection


title: Systemic and mucosal mobilization of granulocyte subsets during lentivirus infection

abstract: Granulocytes mediate broad immunoprotection through phagocytosis, extracellular traps, release of cytotoxic granules, antibody effector functions and recruitment of other immune cells against pathogens. However, descriptions of granulocytes in HIV infection and mucosal tissues are limited. Our goal was to characterize granulocyte subsets in systemic, mucosal and lymphoid tissues during lentivirus infection using the rhesus macaque (RM) model. Mononuclear cells from jejunum, colon, cervix, vagina, lymph nodes, spleen, liver, and whole blood from naive, and chronically SHIVsf162p3-infected RM were analyzed by microscopy and polychromatic flow cytometry. Granulocytes were identified using phenotypes designed specifically for RM: eosinophils - CD45+CD66+CD49d+; neutrophils - CD45+CD66+CD14+; and basophils - CD45+CD123+FcR{varepsilon}+. Nuclear visualization with DAPI staining and surface marker images by ImageStream (cytometry/microscopy) further confirmed granulocytic phenotypes. Flow cytometric data showed that all RM granulocytes expressed CD32 (FcR{gamma}II) but did not express CD16 (FcR{gamma}III). Additionally, constitutive expression of CD64 (FcR{gamma}I) on neutrophils and FcR{varepsilon} on basophils, indicates the differential expression of Fc receptors on granulocyte subsets. Granulocytic subsets in naive whole blood ranged 25.4-81.5% neutrophils, 0.59-13.3% eosinophils and 0.059-1.8% basophils. Interestingly, elevated frequencies of circulating neutrophils, colorectal neutrophils, and colorectal eosinophils were all observed in chronic lentivirus disease. Conversely, circulating basophils, jejunal eosinophils, vaginal neutrophils, and vaginal eosinophils of SHIVsf162p3-infected RM declined in frequency. Overall, our data suggest modulation of granulocytes in chronic lentivirus infection, most notably in the gastrointestinal mucosae where significant inflammation and disruption occurs in lentivirus-induced disease. Furthermore, granulocytes may migrate to inflamed tissues during infection and could serve as targets of immunotherapeutic intervention.


title: Life history strategies explain bacterial activity in the soil carbon cycle

abstract: Soil microorganisms determine the fate of soil organic matter (SOM), and their activities comprise a major component of the global carbon (C) cycle. We employed a multi-substrate DNA-stable isotope probing experiment to track bacterial assimilation of C derived from distinct sources that varied in bioavailability. This approach allowed us to measure microbial contributions to SOM processing by measuring the C assimilation dynamics of diverse microorganisms as they interact within soil. We identified and tracked 1,286 bacterial taxa that assimilated 13C in an agricultural soil over a period of 48 days. Overall 13C-assimilation dynamics of bacterial taxa, defined by the source and timing of the 13C they assimilated, exhibited low phylogenetic conservation. We identified bacterial guilds comprised of taxa that had similar 13C assimilation dynamics. We show that C source bioavailability explained significant variation in both C mineralization dynamics and guild structure. In addition, guild structure explained significant variation in bacterial growth dynamics. We demonstrate that the observed guild structure is consistent with predictions made by bacterial life history theory. We also demonstrate that the guild structure explains significant variation in the biogeographical distribution of bacteria at continental and global scales. We interpret these findings in the context of bacterial life history strategies and their relationship to terrestrial C-cycling.


title: Multi-substrate DNA stable isotope probing reveals guild structure in bacterially mediated soil carbon cycling


title: Deciphering Bedaquiline and Clofazimine Resistance in Tuberculosis: An Evolutionary Medicine Approach

abstract: Bedaquiline (BDQ) and clofazimine (CFZ) are core drugs for treatment of multidrug resistant tuberculosis (MDR-TB), however, our understanding of the resistance mechanisms for these drugs is sparse which is hampering rapid molecular diagnostics. To address this, we employed a unique approach using experimental evolution, protein modelling, genome sequencing, and minimum inhibitory concentration data combined with genomes from a global strain collection of over 14,151 Mycobacterium tuberculosis complex isolates and an extensive literature review. Overall, 230 genomic variants causing elevated BDQ and/or CFZ MICs could be discerned, with 201 (87.4%) variants affecting the transcriptional repressor (Rv0678) of an efflux system (mmpS5-mmpL5). Structural modelling of Rv0678 suggests four major mechanisms that confer resistance: impairment of DNA binding, reduction in protein stability, disruption of protein dimerization, and alteration in affinity for its fatty acid ligand. These modelling and experimental techniques will improve personalized medicine in an impending drug resistant era.


title: Revisiting the source of wilt symptoms: X-ray microcomputed tomography provides direct evidence that Ralstonia biomass clogs xylem vessels.

abstract: Plant pathogenic Ralstonia cause wilt diseases by colonizing xylem vessels and disrupting water transport. Due to the abundance of Ralstonia cells in vessels, the dogma is that bacterial biomass clogs vessels and reduces the flow of xylem sap. However, the physiological mechanism of xylem disruption during bacterial wilt disease is untested. Using a tomato and Ralstonia pseudosolanacearum GMI1000 model, we visualized and quantified the spatiotemporal dynamics of xylem disruption during bacterial wilt disease. First, we measured stomatal conductance of leaflets on mock-inoculated and wilt-symptomatic plants. Wilted leaflets had reduced stomatal conductance, as did turgid leaflets located on the same petiole as wilted leaflets. Next, we used X-ray microcomputed tomography (X-ray microCT) and light microscopy to differentiate between mechanisms of xylem disruption: blockage by bacterial biomass, blockage by vascular tyloses, or sap displacement by gas embolisms. We imaged stems on plants with intact roots and leaves to quantify embolized vessels. Embolized vessels were rare, but there was a slight trend of increased vessel embolisms in infected plants with low bacterial population sizes. To test the hypothesis that vessels are clogged during bacterial wilt, we imaged excised stems after allowing the sap to evaporate during a brief dehydration. Most xylem vessels in mock-infected plants emptied their contents after excision, but non-conductive clogged vessels were abundant in infected plants by 2 days post infection. At wilt onset when bacterial populations exceeded 5x108 cfu/g stem tissue, approximately half of the xylem vessels were clogged with electron-dense bacterial biomass. We found no evidence of tyloses in the X-ray microCT reconstructions or light microscopy on the preserved stems. Bacterial blockage of vessels appears to be the principal cause of vascular disruption during Ralstonia wilt.


title: Transcriptome-wide spatial RNA profiling maps the cellular architecture of the developing human neocortex

abstract: Spatial genomic technologies can map gene expression in tissues, but provide limited potential for transcriptome-wide discovery approaches and application to fixed tissue samples. Here, we introduce the GeoMX Whole Transcriptome Atlas (WTA), a new technology for transcriptome-wide spatial profiling of tissues with cellular resolution. WTA significantly expands the Digital Spatial Profiling approach to enable in situ hybridisation against 18,190 genes at high-throughput using a sequencing readout. We applied WTA to generate the first spatial transcriptomic map of the fetal human neocortex, validating transcriptome-wide spatial profiling on formalin-fixed tissue material and demonstrating the spatial enrichment of autism gene expression in deep cortical layers. To demonstrate the value of WTA for cell atlasing, we integrated single-cell RNA-sequencing (scRNA-seq) and WTA data to spatially map dozens of neural cell types and showed that WTA can be used to directly measure cell type specific transcriptomes in situ. Moreover, we developed computational tools for background correction of WTA data and accurate integration with scRNA-seq. Our results present WTA as a versatile transcriptome-wide discovery tool for cell atlasing and fixed tissue spatial transcriptomics.


title: Molecular basis for recognition of the Group A Carbohydrate backbone by the PlyC streptococcal bacteriophage endolysin

abstract: Endolysins are peptidoglycan (PG) hydrolases that function as part of the bacteriophage (phage) lytic system to release progeny phage at the end of a replication cycle. Notably, endolysins alone can produce lysis without phage infection, which offers an attractive alternative to traditional antibiotics. Endolysins from phage that infect Gram-positive bacterial hosts contain at least one enzymatically active domain (EAD) responsible for hydrolysis of PG bonds and a cell wall binding domain (CBD) that binds a cell wall epitope, such as a surface carbohydrate, providing some degree of specificity for the endolysin. Whilst the EADs typically cluster into conserved mechanistic classes with well-defined active sites, relatively little is known about the nature of the CBDs and only a few binding epitopes for CBDs have been elucidated. The major cell wall components of many streptococci are the polysaccharides that contain the polyrhamnose (pRha) backbone modified with species-specific and serotype-specific glycosyl side chains. In this report, using molecular genetics, microscopy, flow cytometry and lytic activity assays, we demonstrate the interaction of PlyCB, the CBD subunit of the streptococcal PlyC endolysin, with the pRha backbone of the cell wall polysaccharides, Group A Carbohydrate (GAC) and serotype c-specific carbohydrate (SCC) expressed by the Group A Streptococcus and Streptococcus mutans, respectively.


title: A multiplex microchamber diffusion assay for the antibody-based detection of microRNAs on randomly ordered microbeads

abstract:
BackgroundMicroRNAs (miRNAs) are small, conserved, noncoding RNAs regulating gene expression that functions in RNA silencing and post-transcriptional regulation of gene expression. Altered miRNA profiles have been implicated in many human diseases, and due to their circulating abilities, they have excited great interest in their use as clinical biomarkers. The development of innovative methods for miRNA detection has become of high scientific and clinical interest.

MethodsWe developed a diffusion-driven microbead assay and combined it with an antibody-based miRNA detection. The diffusion process was carried out in two different approaches a) co-diffusion of miRNA and antibodies (termed diffusion approach I, DAI) and b) diffusion of miRNA in an antibody-saturated environment (DAII). In both approaches, neutravidin-coated microbeads were loaded with specific biotinylated DNA capture probes, which targets either miR-21-5p, miR-30a-3p or miR-93-5p. The miRNAs were time- and dose-dependently detected in a diffusion microchamber by primary anti-DNA:RNA hybrid and fluorescence-labeled secondary antibodies using our in-house developed inverse fluorescence microscope imaging platform VideoScan.

ResultsOur assay offers the advantage that several target molecules can be detected simultaneously and in real-time in one reaction environment (multiplex), without any amplification steps. We recorded the diffusion process over a period of 24 h and found that the reaction was almost completed after 2 h. The specificity of the assay was 96.7 % for DAI and 92.3 % for DAII. The detection limits were in a concentration range of 0.03-0.43 nM for DAI and 0.14-1.09 nM for DAII, depending on the miRNA.

ConclusionThe miRNAs are successively exposed to the capture probe-loaded randomly ordered microbeads (p value of CSR 0.23-0.96), which leads to microbeads that become saturated with the target molecules first in front rows. Non-bonded miRNAs continue to diffuse further and can therefore subsequently bind to the microbeads with free binding sites. Our detection principle differs from other microbead assays, in which all microbeads are simultaneously mixed with the sample solution, so that all target molecules bind equally distributed to the microbeads, resulting in an averaged signal intensity.


title: The middle lipin (M-Lip) domain is a new dimeric protein fold that binds membranes

abstract: Phospholipid synthesis and fat storage as triglycerides is regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized and novel protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in full-length lipin 1 influences PAP activity, membrane binding, subcellular localization, oligomerization, and adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a new dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.


title: DNA Supercoiling Drives a Transition between Collective Modes of Gene Synthesis

abstract: Recent experiments showed that multiple copies of the molecular machine RNA polymerase (RNAP) can efficiently synthesize mRNA collectively in the active state of the promoter. However, environmentally-induced promoter repression results in long-distance antagonistic interactions that drastically reduce the speed of RNAPs and cause a quick arrest of mRNA synthesis. The mechanism underlying this transition between cooperative and antagonistic dynamics remains poorly understood. In this Letter, we introduce a continuum deterministic model for the translocation of RNAPs, where the speed of an RNAP is coupled to the local DNA supercoiling as well as the density of RNAPs on the gene. We assume that torsional stress experienced by individual RNAPs is exacerbated by high RNAP density on the gene and that transcription factors act as physical barriers to the diffusion of DNA supercoils. We show that this minimal model exhibits two transcription modes mediated by the torsional stress: a fluid mode when the promoter is active and a torsionally stressed mode when the promoter is repressed, in quantitative agreement with experimentally observed dynamics of co-transcribing RNAPs. Our work provides an important step towards understanding the collective dynamics of molecular machines involved in gene expression.

category: biophysics


title: An atlas of inter- and intra-tumor heterogeneityof apoptosis competency in colorectal cancertissue at single cell resolution

abstract: Cancer cells ability to inhibit apoptosis is key to malignant transformation and limits response to therapy. Here, we performed multiplexed immunofluorescence analysis on tissue microarrays with 373 cores from 168 patients, segmentation of 2.4 million individual cells and quantification of 20 cell lineage and apoptosis proteins. Ordinary differential equation-based modelling of apoptosis sensitivity at single cell resolution was conducted and an atlas of inter- and intra-tumor heterogeneity in apoptosis susceptibility generated. We identified an enrichment for BCL2 in immune, and BAK, SMAC and XIAP in cancer cells. ODE-based modelling at single cell resolution identified an enhanced sensitivity of cancer cells to mitochondrial permeabilization and executioner caspase activation compared to immune and stromal cells, with significant inter- and intra-tumor heterogeneity. However, we did not find increased spatial heterogeneity of apoptosis signaling in cancer cells, suggesting that such heterogeneity is an intrinsic, non-genomic property not increased by the process of malignant transformation.


title: A bidirectional corticoamygdala circuit for the encoding and retrieval of detailed reward memories

abstract: Adaptive reward-related decision making often requires accurate and detailed representation of potential available rewards. Environmental reward-predictive stimuli can facilitate these representations, allowing one to infer which specific rewards might be available and choose accordingly. This process relies on encoded relationships between the cues and the sensory-specific details of the reward they predict. Here we interrogated the function of the basolateral amygdala (BLA) and its interaction with the lateral orbitofrontal cortex (lOFC) in the ability to learn such stimulus-outcome associations and use these memories to guide decision making. Using optical recording and inhibition approaches, Pavlovian cue-reward conditioning, and the outcome-selective Pavlovian-to-instrumental transfer (PIT) test in male rats, we found that the BLA is robustly activated at the time of stimulus-outcome learning and that this activity is necessary for sensory-specific stimulus-outcome memories to be encoded, so they can subsequently influence reward choices. Direct input from the lOFC was found to support the BLA in this function. Based on prior work, activity in BLA projections back to the lOFC was known to support the use of stimulus-outcome memories to influence decision making. By multiplexing optogenetic and chemogenetic inhibition we performed a serial circuit disconnection and found that the lOFC[->]BLA and BLA[->]lOFC pathways form a functional circuit regulating the encoding (lOFC[->]BLA) and subsequent use (BLA[->]lOFC) of the stimulus-dependent, sensory-specific reward memories that are critical for adaptive, appetitive decision making.


title: Plasticity in gustatory and nociceptive neurons controls decision making in C. elegans salt navigation

abstract: A conventional understanding of perception assigns sensory organs the role of capturing the environment. Better sensors result in more accurate encoding of stimuli, allowing for cognitive processing downstream. Here we show that plasticity in sensory neurons mediates a behavioral switch in C. elegans between attraction to NaCl in naive animals and avoidance of NaCl in preconditioned animals, called gustatory plasticity. Ca2+ imaging in ASE and ASH NaCl sensing neurons reveals multiple cell-autonomous and distributed circuit adaptation mechanisms. A computational model quantitatively accounts for observed behaviors and reveals roles for sensory neurons in the control and modulation of motor behaviors, decision making and navigational strategy. Sensory adaptation dynamically alters the encoding of the environment. Rather than encoding the stimulus directly, therefore, we propose that these C. elegans sensors dynamically encode a context-dependent value of the stimulus. Our results demonstrate how adaptive sensory computation can directly control an animals behavioral state.


title: The Human Milk Oligosaccharide 3-Sialyllactose Promotes Inflammation Resolution and Reduces Atherosclerosis Development in Mice.

abstract: Macrophages contribute to the induction and resolution of inflammation and play a central role in the chronic low-grade inflammation in cardiovascular diseases caused by atherosclerosis. Human milk oligosaccharides (HMOs) are complex unconjugated glycans unique to human milk that benefit infant health and act as innate immune modulators. Here, we identify the HMO 3sialyllactose (3SL) as a natural inhibitor of TLR4-induced low-grade inflammation in macrophages and endothelium. Transcriptome analysis in macrophages revealed that 3SL attenuates a selected set of inflammatory gene expression and promotes activity of LXR and SREBP. These acute anti-inflammatory effects of 3SL were associated with reduced histone H3K27 acetylation at a subset of LPS-inducible enhancers distinguished by preferential enrichment for CTCF, IRF2, BCL6, and other transcription factor recognition motifs. In a murine atherosclerosis model, both subcutaneous and oral administration of 3SL significantly reduced atherosclerosis development and the associated inflammation. This study provides evidence that 3SL attenuates inflammation by a transcriptional mechanism to reduce atherosclerosis development in the context of cardiovascular disease.


title: GABAergic signaling in human and murine NK cells upon challenge with Toxoplasma gondii

abstract:
Protective cytotoxic and proinflammatory cytokine responses by natural killer (NK) cells impact the outcome of infections by Toxoplasma gondii, a common parasite in humans and other vertebrates. However, T. gondii can also sequester within NK cells and downmodulate their effector functions. Recently, the implication of {gamma}-aminobutyric acid (GABA) signaling in infection and inflammation-related responses of mononuclear phagocytes and T cells has become evident. Yet, the role of GABAergic signaling in NK cells has remained unknown. Here, we report that human and murine NK cells synthesize and secrete GABA in response to infection challenge. Parasitized NK cells secreted GABA while activation stimuli, such as IL-12/IL-18 or parasite lysates, failed to induce GABA secretion. GABA secretion by NK cells was associated to a transcriptional upregulation of GABA synthesis enzymes (GAD65/67) and was abrogated by GAD-inhibition. Further, NK cells expressed GABA-A receptor subunits and GABA signaling regulators, with transcriptional modulations taking place upon challenge with T. gondii. Exogenous GABA and GABA-containing supernatants from parasitized dendritic cells (DCs) impacted NK cell function by reducing the degranulation and cytotoxicity of NK cells. Conversely, GABA-containing supernatants from NK cells enhanced the migratory responses of parasitized DCs. This enhanced DC migration was abolished by GABA-A receptor antagonism or GAD-inhibition and was reconstituted by exogenous GABA. Jointly, the data show that NK cells are GABAergic cells and that GABA hampers NK cell cytotoxicity in vitro. We hypothesize that GABA secreted by parasitized immune cells modulates the immune responses to T. gondii infection.

Summary sentenceIn response to infection challenge, NK cells synthesize and secrete{gamma} -aminobutyric acid (GABA), which impacts NK cell and dendritic cell functions via GABA-A receptors.


title: The within-host evolutionary dynamics of seasonal and pandemic human influenza A viruses in young children

abstract: The evolution of influenza viruses is fundamentally shaped by within-host processes. However, the within-host evolutionary dynamics of influenza viruses remain incompletely understood, in part because most studies have focused on within-host virus diversity of infections in otherwise healthy adults based on single timepoint data. Here, we analysed the within-host evolution of 82 longitudinally-sampled individuals, mostly young children, infected with A/H3N2 or A/H1N1pdm09 viruses between 2007 and 2009. For A/H1N1pdm09 infections during the 2009 pandemic, nonsynonymous changes were common early in infection but decreased or remained constant throughout infection. For A/H3N2 viruses, early infection was dominated by purifying selection. However, as infections progressed, nonsynonymous variants increased in frequencies even though within-host virus titres decreased, leading to the maintenance of virus diversity via mutation-selection balance. Our findings suggest that this maintenance of genetic diversity in these children combined with their longer duration of infection may provide important opportunities for within-host virus evolution.


title: Within-host evolutionary dynamics of seasonal and pandemic human influenza A viruses in young children