title: A comparative meta-analysis of membraneless organelle associated proteins with age related proteome of C. elegans

abstract:
Proteome imbalance can lead to protein misfolding and aggregation which is associated with pathologies. Protein aggregation can also be an active, organized process and can be exploited by cells as a survival strategy. In adverse conditions, it is beneficial to deposit the proteins in a condensate rather degrading and resynthesizing. Membraneless organelles (MLOs) are biological condensates formed through liquid-liquid phase separation (LLPS), involving cellular components such as nucleic acids and proteins. LLPS is a regulated process, which when perturbed, can undergo a transition from a physiological liquid condensate to pathological solid-like protein aggregates.

To understand how the MLO-associated proteins (MLO-APs) behave during aging, we performed a comparative meta-analysis with age related proteome of C. elegans. We found that the MLO-APs are highly abundant throughout the lifespan. Interestingly, they are aggregating more in long-lived mutant worms compared to the age matched wildtype worms. GO term analysis revealed that the cell cycle and embryonic development are among the top enriched processes in addition to RNP components in insoluble proteome. Considering antagonistic pleotropic nature of these developmental genes and post mitotic status of C. elegans, we assume that these proteins phase transit during post development. As the organism ages, these MLO-APs either mature to become more insoluble or dissolve in uncontrolled manner. However, in the long-lived daf-2 mutant worms, the MLOs may attain protective states due to extended availability and association of molecular chaperones.


title: Extent of Molecular Chaperone Association Might Determine Fates of Membraneless Organelles during Aging in C. elegans

category: biochemistry


title: vRhyme enables binning of viral genomes from metagenomes

abstract: Genome binning has been essential for characterization of bacteria, archaea, and even eukaryotes from metagenomes. Yet, no approach exists for viruses. We developed vRhyme, a fast and precise software for construction of viral metagenome-assembled genomes (vMAGs). vRhyme utilizes single- or multi-sample coverage effect size comparisons between scaffolds and employs supervised machine learning to identity nucleotide feature similarities, which are compiled into iterations of weighted networks and refined bins. Using simulated viromes, we displayed superior performance of vRhyme compared to available binning tools in constructing more complete and uncontaminated vMAGs. When applied to 10,601 viral scaffolds from human skin, vRhyme advanced our understanding of resident viruses, highlighted by identification of a Herelleviridae vMAG comprised of 22 scaffolds, and another vMAG encoding a nitrate reductase metabolic gene, representing near-complete genomes post-binning. vRhyme will enable a convention of binning uncultivated viral genomes and has the potential to transform metagenome-based viral ecology.

category: bioinformatics


title: Dissecting the stability determinants of a challenging de novo protein fold using massively parallel design and experimentation

abstract:
Designing entirely new protein structures remains challenging because we do not fully understand the biophysical determinants of folding stability. Yet some protein folds are easier to design than others. Previous work identified the 43-residue {beta}{beta} fold as especially challenging: the best designs had only a 2% success rate, compared to 39-87% success for other simple folds (1). This suggested the {beta}{beta} fold would be a useful model system for gaining a deeper understanding of folding stability determinants and for testing new protein design methods. Here, we designed over ten thousand new {beta}{beta} proteins and found over three thousand of them to fold into stable structures using a high-throughput protease-based assay. Nuclear magnetic resonance, hydrogen-deuterium exchange, circular dichroism, deep mutational scanning, and scrambled sequence control experiments indicated that our stable designs fold into their designed {beta}{beta} structures with exceptional stability for their small size. Our large dataset enabled us to quantify the influence of universal stability determinants including nonpolar burial, helix capping, and buried unsatisfied polar atoms, as well as stability determinants unique to the {beta}{beta} topology. Our work demonstrates how large-scale design and test cycles can solve challenging design problems while illuminating the biophysical determinants of folding.

SignificanceMost computationally designed proteins fail to fold into their designed structures. This low success rate is a major obstacle to expanding the applications of protein design. In previous work, we discovered a small protein fold that was paradoxically challenging to design (only a 2% success rate) even though the fold itself is very simple. Here, we used a recently developed high-throughput approach to comprehensively examine the design rules for this simple fold. By designing over ten thousand proteins and experimentally measuring their folding stability, we discovered the key biophysical properties that determine the stability of these designs. Our results illustrate general lessons for protein design and also demonstrate how high-throughput stability studies can quantify the importance of different biophysical forces.

category: synthetic biology


title: Overlapping stimulons arising in response to divergent stresses in Escherichia coli

abstract: Cellular responses to stress can cause a similar change in some facets of fitness even if the stresses are different. Lactose as a sole carbon source for Escherichia coli is an established example: too little causes starvation while excessive lactose import causes toxicity as a side-effect. In an E. coli strain that is robust to osmotic and ionic differences in growth media, B REL606, the rate of antibiotic-tolerant persister formation is elevated in both starvation-inducing and toxicity-inducing concentrations of lactose in comparison to less stressful intermediate concentrations. Such similarities between starvation and toxification raise the question of how much the global stress response stimulon differs between them. We hypothesized that a common stress response is conserved between the two conditions, but that a previously shown threshold driving growth rate heterogeneity in a lactose-toxifying medium would reveal that the growing fraction of cells in that medium to be missing key stress responses that curb growth. To test this, we performed RNA-seq in three representative conditions for differential expression analysis. In comparison to nominally unstressed cultures, both stress conditions showed global shifts in gene expression, with informative similarities and differences. Functional analysis of pathways, gene ontology terms, and clusters of orthogonal groups revealed signatures of overflow metabolism, membrane component shifts, and altered cytosolic and periplasmic contents in toxified cultures. Starving cultures showed an increased tendency toward stringent response-like regulatory signatures. Along with other emerging evidence, our results show multiple possible pathways to stress responses, persistence, and possibly other phenotypes. These results suggest a set of overlapping responses that drives emergence of stress-tolerant phenotypes in diverse conditions.

category: systems biology


title: CD4 T cells are rapidly depleted from tuberculosis granulomas following acute SIV co-infection

abstract: The HIV-mediated decline in circulating CD4 T cells correlates with increased the risk of active tuberculosis (TB)1-4. However, HIV/Mycobacterium tuberculosis (Mtb) co-infected individuals also have an increased incidence of TB prior to loss of CD4 T cells in blood3,5, raising the possibility that HIV co-infection leads to disruption of CD4 T cell responses at the site of lung infection before they are observed systemically. Here we used a rhesus macaque model of SIV/Mtb co-infection to study the early effects of acute SIV infection on CD4 T cells in pulmonary Mtb granulomas. Two weeks after SIV co-infection CD4 T cells were dramatically depleted from granulomas, before significant bacterial outgrowth, disease reactivation as measured by PET-CT imaging, or CD4 T cell loss in blood, airways, and lymph nodes. Mtb-specific CD4 T cells, CCR5-expressing, in granulomas were preferentially depleted by SIV infection. Moreover, CD4 T cells were preferentially depleted from the granuloma core and lymphocyte cuff relative to B cell-rich regions, and live imaging of granuloma explants showed that SIV co-infection reduced T cell motility. Thus, Mtb-specific CD4 T cells in pulmonary granulomas may be decimated before many patients even experience the first symptoms of acute HIV infection.

category: immunology


title: Mathematical modeling suggests cooperation of plant-infecting viruses

abstract: Viruses are major pathogens of agricultural crops. Viral infections often start after the virus enters the outer layer of a tissue or surface and many successful viruses, after local replication in the infected tissue, are able to spread systemically. Quantitative details of virus dynamics in plants, however, have been poorly understood, in part, because of the lack of experimental methods allowing to accurately measure the degree of infection in individual plant tissues. Recently, by using flow cytometry and two different flourescently-labeled strains of the Tobacco etch virus (TEV), Venus and BFP, kinetics of viral infection of individual cells in leaves of Nicotiana tabacum plants was followed over time [1]. A simple mathematical model, assuming that viral spread occurs from lower to upper leaves, was fitted to these data. While the the original model could accurately describe the kinetics of viral spread locally and systemically, we also found that many alternative versions of the model, for example, if viral spread starts at upper leaves and progresses to lower leaves or when virus dissemination is stopped due to an immune response, provided fits of the data with reasonable quality, and yet with different parameter estimates. These results strongly suggest that experimental measurements of the virus infection in individual leaves may not be sufficient to identify the pathways of viral dissemination between different leaves and reasons for viral control; we propose experiments that may allow discrimination between the alternatives. By analyzing the kinetics of coinfection of individual cells by Venus and BFP strains of TEV we found a strong deviation from the random infection model, suggesting cooperation between the two strains when infecting plant cells. Importantly, we showed that many mathematical models on the kinetics of coinfection of cells with two strains could not adequately describe the data, and the best fit model needed to assume i) different susceptibility of uninfected cells to infection by two viruses locally in the leaf vs. systemically from other leaves, and ii) decrease in the infection rate depending on the fraction of uninfected cells which could be due to a systemic immune response. Our results thus demonstrate the difficulty in reaching definite conclusions from extensive and yet limited experimental data and provide evidence of potential cooperation between different viral variants infecting individual cells in plants.

category: microbiology


title: The acquisition of clinically relevant amoxicillin resistance in Streptococcus pneumoniae requires ordered horizontal gene transfer of four loci

abstract: Understanding how antimicrobial resistance spreads is critical for optimal application of new treatments. In the naturally competent human pathogen Streptococcus pneumoniae, resistance to {beta}-lactam antibiotics is mediated by recombination events in genes encoding the target proteins, resulting in reduced drug binding affinity. However, for the front-line antibiotic amoxicillin, the exact mechanism of resistance still needs to be elucidated. Through successive rounds of transformation with genomic DNA from a clinically resistant isolate, we followed amoxicillin resistance development. Using whole genome sequencing, we showed that multiple recombination events occurred at different loci during one round of transformation. We found examples of non-contiguous recombination, and demonstrated that this could occur either through multiple D-loop formation from one donor DNA molecule, or by the integration of multiple DNA fragments. We also show that the final minimum inhibitory concentration (MIC) differs depending on recipient genome, explained by differences in the extent of recombination at key loci. Finally, through back transformations of mutant alleles and fluorescently labelled penicillin (bocillin-FL) binding assays, we confirm that pbp1a, pbp2b, pbp2x, and murM are the main resistance determinants for amoxicillin resistance, and that the order of allele uptake is important for successful resistance evolution. We conclude that recombination events are complex, and that this complexity contributes to the highly diverse genotypes of amoxicillin-resistant pneumococcal isolates.


title: Antibiotic Resistance Genes from Mat and Planktonic Microbiomes of Antarctic Lake Fryxell and Lake Bonney, and a Mat-forming Culture of Rhodoferax antarcticus

abstract: The Antarctic Taylor Valley Lakes Fryxell and Bonney harbor oligotrophic microbial communities that are separated geographically from other aquatic systems. Their microbiomes include planktonic as well as lift-off mat communities that float to the underside of the perennial ice cover and eventually emerge at the surface. We investigated the antibiotic resistance genes (ARGs) from metagenomes of lift-off mats emerging from ice, from filtered water samples of Lake Fryxell, and from filtered water samples of Lake Bonney. ARG sequence markers were designed by ShortBRED-Identify using the Comprehensive Antibiotic Resistance Database (CARD). The overall proportion of ARG hits in the metagenomes was found to be similar to that found in temperate-zone rural water bodies with moderate human inputs (0.0002-0.0007%). The specific ARGs found showed distinct distributions for the two lakes, and for mat versus planktonic sources. Metagenomic taxa distributions showed that mat phototrophs were mainly cyanobacteria or betaproteobacteria, whereas the water column phototrophs were mainly protists. An enrichment culture of the betaproteobacterium Rhodoferax antarcticus from a Lake Fryxell mat sample showed a mat-forming phenotype not previously reported for this species. Its genome showed no ARGs associated with betaproteobacteria, but had ARGs consistent with a minor Pseudomonas component. The Antarctic lake mats and water showed specific ARGs distinctive to the mat and water sources, but overall ARG levels were similar to those of temperate water bodies with minimal human inputs.


title: Antibiotic Resistance Genes and Taxa Analysis from Mat and Planktonic Microbiomes of Antarctic Lake Fryxell and Lake Bonney, and a Mat-forming Culture of Rhodoferax antarcticus


title: Prior exposure to microcystin alters host gut resistome and is associated with dysregulated immune homeostasis in translatable mouse models

abstract: The increased propensity of harmful algal blooms (HABs) and exposure from HABs-cyanotoxin causes human toxicity. It has been associated with the progression of several diseases that encompass the liver, kidneys, and immune system. Recently, a strong association of cyano-HAB toxicity with the altered host gut microbiome has been shown. We tested the hypothesis that prior exposure to cyanotoxin microcystin may alter the microbiome and induce microbiome-host-resistome crosstalk. Using both wild-type and humanized mice, we show that the mice exposed to microcystin had an altered microbiome signature that harbored antimicrobial resistance genes. Host resistome phenotypes such as mefA, msrD, mel, ant6, and tet40 increased in diversity and relative abundance following microcystin exposure. Interestingly, the increased abundance of these genes was traced to resistance to common antibiotics such as tetracycline, macrolides, glycopeptide, and aminoglycosides, crucial for modern-day treatment for several diseases. Increased abundance of these genes was positively associated with increased expression of PD1, a T-cell homeostasis marker, and pleiotropic inflammatory cytokine IL-6 with a concomitant negative association with immunosurveillance markers IL7 and TLR2. Microcystin exposure also caused decreased TLR2, TLR4, and REG3G expressions, increased immunosenescence, and higher systemic levels of IL-6 in both wild-type and humanized mice. In conclusion, the results show a first-ever characterization of the host resistome of microcystin exposure and its connection to host immune status and antibiotic resistance. The results may be crucial for understanding the ability of exposed subjects to fight future bacterial infections and the progression of the debilitating disease in hospital settings.


title: A tale of two plasmids: contributions of plasmid associated phenotypes to epidemiological success among Shigella

abstract: Dissemination of antimicrobial resistance (AMR) genes by horizontal gene transfer (HGT) mediated through plasmids is a major global concern. Genomic epidemiology studies have shown varying success of different AMR plasmids during outbreaks, but the underlying reasons for these differences are unclear. Here, we investigated two Shigella plasmids (pKSR100 and pAPR100) that circulated in the same transmission network but had starkly contrasting epidemiological outcomes to identify plasmid features that may have contributed to the differences. We used plasmid comparative genomics to reveal divergence between the two plasmids in genes encoding AMR, SOS response alleviation, and conjugation. Experimental analyses revealed that these genomic differences corresponded with reduced conjugation rates for the epidemiologically successful pKSR100, but more extensive AMR, reduced fitness costs, and a reduced SOS response in the presence of antimicrobials, compared with the less successful pAPR100. The discrepant phenotypes between the two plasmids are consistent with the hypothesis that plasmid associated phenotypes contribute to determining the epidemiological outcome of AMR HGT and suggest that phenotypes relevant in responding to antimicrobial pressure and fitness impact may be more important than those around conjugation in this setting. Plasmid phenotypes could thus be valuable tools in conjunction with genomic epidemiology for predicting AMR dissemination.


title: Rational Protein Engineering of Bacterial N-Demethylases to Create Biocatalysts for the Production of Methylxanthines

abstract: Methylxanthines have a rich history as therapeutics and pharmaceuticals. However, natural dimethyl- and monomethylxanthines are difficult to produce synthetically, which has limited further exploration of these compounds in medicinal applications. A biosynthetic method for production of methylxanthines from whole cell biocatalysts is an attractive alternative. The bacterium Pseudomonas putida CBB5 contains a set of five enzymes, NdmABCDE, which are responsible for methylxanthine metabolism via N-demethylation to xanthine. The recent elucidation of the crystal structures of NdmA and NdmB, which remove the N1- and N3-methyl groups of caffeine, respectively, has opened new avenues to create biocatalysts for methylxanthine production. We have created a set of fifteen N-demethylase mutants and expressed them in E. coli BL21(DE3) as whole cell biocatalysts. The activity of each mutant was characterized for their affinity towards caffeine, theobromine, and theophylline. Two mutant enzymes in particular, labeled NdmA3 and NdmA4, both exhibited selectivity towards the N3-methyl group instead of the N1-methyl group. We also discovered that specific point mutations in NdmD resulted in the ability to tune the rate of the N-demethylase reaction. These new enzymes provide the capability of producing high-value methylxanthines, such as paraxanthine and 1-methylxanthine, through a biocatalytic route.


title: Escherichia coli BarA-UvrY regulates the pks island and kills Staphylococci via the genotoxin colibactin

abstract: Wound infections are often polymicrobial in nature and are associated with poor disease prognoses. Escherichia coli and Staphylococcus aureus are among the top five most cultured pathogens from wound infections. However, little is known about the polymicrobial interactions between E. coli and S. aureus during wound infections. In this study, we show that E. coli kills S. aureus both in vitro and in a mouse excisional wound model via the genotoxin, colibactin. We also show that the BarA-UvrY two component system (TCS) is a novel regulator of the pks island, which acts through the carbon storage global regulatory (Csr) system. Together, our data demonstrate the role of colibactin in inter-species competition and show that it is regulated by BarA-UvrY TCS, a previously uncharacterized regulator of the pks island.


title: Physico-chemical principles of HDL-small RNA binding interactions.

abstract: Extracellular small RNAs (sRNA) are abundant in many biofluids, but little is known about their mechanisms of transport and stability in RNase-rich environments. We previously reported that high-density lipoproteins (HDL) of mice were enriched with multiple classes of sRNA derived from the endogenous transcriptome, but also exogenous organisms. Here, we show that human HDL transports tRNA-derived sRNAs (tDRs) from host and non-host species which were found to be altered in human atherosclerosis. We hypothesized that HDL binds to tDRs through apolipoprotein A-I (apoA-I) and these interactions are conferred by RNA-specific features. We tested this using microscale thermophoresis and electrophoretic mobility shift assays and found that HDL bind tDRs and other single-stranded sRNAs with strong affinity, but not doublestranded RNA or DNA. Natural and synthetic RNA modifications influenced tDR binding to HDL. Reconstituted HDL bound tDRs only in the presence of apoA-I and purified apoA-I alone was sufficient for binding sRNA. Conversely, phosphatidylcholine vesicles did not bind tDRs. In summary, HDL preferentially binds to single-stranded sRNAs likely through non-ionic interactions with apoA-I. These studies highlight binding properties that likely enable extracellular RNA communication and provide a foundation for future studies to manipulate HDL-sRNA for therapeutic approaches to prevent or treat disease.

category: molecular biology


title: KDM6B sensitizes PARthanatos via suppression of O6-methylguanine DNA methyltransferase repair and sustained checkpoint response

abstract: Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA damage sensor and contributes to both DNA repair and cell death processes. However, how PARP-1 signaling is regulated to switch its function from DNA repair to cell death remains largely unknown. Here, we found that PARP-1 plays a central role in alkylating agent-induced PARthanatic cancer cell death. Lysine demethylase 6B (KDM6B) was identified as a key cell death effector in PARthanatos. Knockout of KDM6B or loss of KDM6B demethylase activity conferred cancer cells resistance to PARthanatic cell death in response to alkylating agents. Mechanistically, KDM6B knockout suppressed methylation at the promoter of O6-methylguanine-DNA methyltransferase (MGMT) to enhance MGMT expression and its direct DNA repair function, thereby inhibiting DNA damage-evoked PARP-1 hyperactivation and subsequent cell death. Moreover, KDM6B knockout triggered sustained Chk1 phosphorylation and activated a second repair machinery to fix DNA damage evading from MGMT repair. Inhibition of MGMT or checkpoint response re-sensitized KDM6B deficient cells to PARthanatos induced by alkylating agents. These findings provide new molecular insights into epigenetic regulation of PARP-1 signaling mediating DNA repair or cell death and identify KDM6B as a biomarker for prediction of cancer cell vulnerability to alkylating agent treatment.


title: Differential regulation of SHH signaling and the developmental control of species-specific jaw size through neural crest-mediated Gas1 expression

abstract:
Developmental control of jaw size is crucial to prevent disease and facilitate evolution. We have shown that species-specific differences in jaw size are established by neural crest mesenchyme (NCM), which are the jaw progenitors that migrate into the mandibular primordia. NCM relies on multiple signaling molecules including Sonic Hedgehog (SHH) to mediate interactions with mandibular epithelium that facilitate outgrowth of the jaws. SHH signaling is known to promote outgrowth and so we tested if differential regulation of the SHH pathway can account for species-specific variation in mandibular primordia size. We analyze gene expression of SHH pathway members in duck, chick, and quail, and find higher transcriptional activation in the larger mandibular primordia of duck relative to those of chick and quail. We generate quail-duck chimeras and demonstrate that such activation is NCM-mediated. Gain- and loss-of-function experiments reveal a species-specific response to SHH signaling, with the target Gas1 being most sensitive to manipulations. Gas1 overexpression and knockdown in NCM alters cell number and/or mandibular primordia size. Our work suggests that NCM-mediated changes in SHH signaling may modulate jaw size during development, disease, and evolution.

Summary StatementWe have determined that Gas1, which is a component of the SHH signaling pathway, plays a key role in the development and evolution of jaw size.

category: developmental biology


title: RNA viral communities are structured by host plant phylogeny in oak and conifer leaves

abstract: Wild plants can suffer devastating diseases, experience asymptomatic, persistent infections, and serve as reservoirs for viruses of agricultural crops, yet we have a limited understanding of the natural plant virosphere. To access representatives of locally and globally distinct wild plants and investigate their viral diversity, we extracted and sequenced dsRNA from leaves from 16 healthy oak and conifer trees in the UC Davis Arboretum (Davis, California). From de novo assemblies, we recovered 389 RNA-dependent RNA polymerase (RdRp) gene sequences from 384 putative viral species, and a further 580 putative viral contigs were identified with virus prediction software followed by manual confirmation of virus annotation. Based on similarity to known viruses, most recovered viruses were predicted to infect plants or fungi, with the highest diversity and abundance observed in the Totiviridae and Mitoviridae families. Phyllosphere viral community composition differed significantly by host plant phylogeny, suggesting the potential for host-specific viromes. The phyllosphere viral community of one oak tree differed substantially from other oak viral communities and contained a greater proportion of putative mycoviral sequences, potentially due to the trees more advanced senescence at the time of sampling. These results suggest that oaks and conifers harbor a vast diversity of viruses with as-yet unknown roles in plant health and phyllosphere microbial ecology.

category: ecology


title: Adaptive tail-length evolution in deer mice is associated with differential Hoxd13 expression in early development

abstract:
Variation in the size and number of axial segments underlies much of the diversity in animal body plans. Here, we investigate the evolutionary, genetic, and developmental mechanisms driving tail-length differences between forest and prairie ecotypes of deer mice (Peromyscus maniculatus). We first show that long-tailed forest mice perform better in an arboreal locomotion assay, consistent with tails being important for balance during climbing. The long tails of these forest mice consist of both longer and more caudal vertebrae than prairie mice. Using quantitative genetics, we identify six genomic regions that contribute to differences in total tail length, three of which associate with vertebra length and the other three with vertebra number. For all six loci, the forest allele increases tail length, consistent with the cumulative effect of natural selection. Two of the genomic regions associated with variation in vertebra number contain Hox gene clusters. Of those, we find an allele-specific decrease in Hoxd13 expression in the embryonic tail bud of long-tailed forest mice, consistent with its role in axial elongation. Additionally, we find that forest embryos have more presomitic mesoderm than prairie embryos, and that this correlates with an increase in the number of neuromesodermal progenitors (NMPs), which are modulated by Hox13 paralogs. Together, these results suggest a role for Hoxd13 in the development of natural variation in adaptive morphology on a microevolutionary timescale.

HIGHLIGHTSO_LIIn deer mice, the long-tailed forest ecotype outperforms the short-tailed prairie ecotype in climbing, consistent with the tails role in balance.
C_LIO_LILong tails are due to mutations on distinct chromosomes that affect either length or number of caudal vertebrae.
C_LIO_LIQTL mapping identifies Hox clusters, one gene of which - Hoxd13 - shows low allele-specific expression in the embryonic tail bud of forest mice.
C_LIO_LIForest mouse embryos have a larger presomitic mesoderm (PSM), likely mediated by a larger progenitor population (NMPs) and lower Hoxd13 levels.
C_LI

category: genetics


title: Single cell mRNA signals reveal a distinct developmental state of KMT2A-rearranged infant B-cell acute lymphoblastic leukemia

abstract: Infant B-cell acute lymphoblastic leukemia (B-ALL) has not followed the increasing trend towards cure seen in other childhood B-ALLs. The prognosis for infants with KMT2A gene fusions is especially poor, and the origins of this aggressive leukemia remain unknown. Here, we investigated the developmental state of KMT2A-rearranged infant B-ALL within hematopoietic hierarchies of human fetal bone marrow, using bulk mRNA meta-analysis of childhood leukemia and examination of single lymphoblast transcriptomes. KMT2A-rearranged infant B-ALL was uniquely dominated by an early lymphocyte precursor (ELP) state. Direct comparison of infant lymphoblasts with ELP cells distilled the core oncogenic transcriptome of cancer cells which harboured potentially targetable hybrid myeloid-lymphoid features. Overall our quantitative molecular analyses demonstrate a distinct developmental state of KMT2A-rearranged infant B-ALL.

category: genomics


title: Tissue- and ethnicity-independent hypervariable DNA methylation states show evidence of establishment in the early human embryo

abstract: We analysed DNA methylation data from 30 datasets comprising 3,474 individuals, 19 tissues and 8 ethnicities at CpGs covered by the Illumina450K array. We identified 4,143 hypervariable CpGs ("hvCpGs") with methylation in the top 5% most variable sites across multiple tissues and ethnicities. hvCpG methylation was influenced but not determined by genetic variation, and was not linked to probe reliability, epigenetic drift, age, sex or cell heterogeneity effects. hvCpG methylation tended to covary across tissues derived from different germ-layers and hvCpGs were enriched for associations with periconceptional environment, proximity to ERV1 and ERVK retrovirus elements and parent-of-origin-specific methylation. They also showed distinctive methylation signatures in monozygotic twins. Together, these properties position hvCpGs as strong candidates for studying how stochastic and/or environmentally influenced DNA methylation states which are established in the early embryo and maintained stably thereafter can influence life-long health and disease.


title: Cryo-EM structure of the human CST·Polα/Primase complex in a recruitment state

abstract: The CST*Pol/Primase complex is essential for telomere overhang maintenance and additionally functions to counteract resection at double-strand breaks. We report a 4.6-[A] resolution cryo-EM structure of CST*Pol/Primase, captured prior to catalysis in a recruitment state, which provides insights into the architecture and stoichiometry of the fill-in machinery. Our model informs on human disease mutations that cause Coats plus syndrome.


title: 2FAST2Q: A general-purpose sequence search and counting program for FASTQ files

abstract: The increasingly widespread use of next generation sequencing protocols has brought the need for the development of user-friendly raw data processing tools. Here, we present 2FAST2Q, a versatile and intuitive standalone program capable of extracting and counting feature occurrences in FASTQ files. 2FAST2Q can be used in any experimental setup that requires feature extraction from raw reads, being able to quickly handle mismatch alignments, nucleotide wise Phred score filtering, custom read trimming, and sequence searching within a single program. Using published CRISPRi datasets in which Escherichia coli and Mycobacterium tuberculosis gene essentiality, as well as host-cell sensitivity towards SARS-CoV2 infectivity were tested, we demonstrate that 2FAST2Q efficiently recapitulates the output in read counts per provided feature as with traditional pipelines. Moreover, we show how different FASTQ read filtering parameters impact downstream analysis, and suggest a default usage protocol. 2FAST2Q has a familiar user interface and uses a custom sequence mismatch search algorithm, taking advantage of Pythons numba module JIT runtime speeds. It is thus easier to use and faster than currently available tools, efficiently processing large CRISPRi-Seq or random-barcode sequencing datasets on any up-to-date laptop. 2FAST2Q is available as an executable file for all current operating systems without installation and as a Python3 module on the PyPI repository (available at https://veeninglab.com/2fast2q). We expect that 2FAST2Q will not only be useful for people working in microbiology but also for other fields in which amplicon sequencing data is generated.


title: Understanding the interaction of 14-3-3 proteins with hDMX and hDM2: a structural and biophysical study

abstract: p53 plays a critical role in regulating diverse biological processes: DNA repair, cell cycle arrest, apoptosis, and senescence. The p53 pathway has therefore served as the focus for drug-discovery efforts. p53 is negatively regulated by hDMX and hDM2; prior studies have identified 14-3-3 proteins as hDMX and hDM2 client proteins. 14-3-3 proteins are adaptor proteins that modulate localisation, degradation and interactions of their targets in response to phosphorylation. Thus 14-3-3 proteins may indirectly modulate the interaction between hDMX or hDM2 and p53 and represent potential targets for modulation of the p53 pathway. In this manuscript we report on the biophysical and structural characterization of peptide/protein interactions that are representative of the interaction between 14-3-3 and hDMX or hDM2. The data establish that proximal phosphosites spaced [~]20-25 residues apart in both hDMX and hDM2 co-operate to facilitate high-affinity 14-3-3 binding and provide structural insight that can be utilized in future stabilizer/inhibitor discovery efforts.

category: biophysics


title: Capturing the start point of the virus-cell interaction with high-speed 3D single-virus tracking

abstract:
The early stages of the virus-cell interaction have long evaded observation by existing microscopy methods due to the rapid diffusion of virions in the extracellular space and the large 3D cellular structures involved. Here we present an active-feedback single-virus tracking method with simultaneous volumetric imaging of the live cell environment to address this knowledge gap to present unprecedented detail to the extracellular phase of the infectious cycle. We report previously unobserved phenomena in the early stages of the virus-cell interaction, including skimming contact events at the millisecond timescale, orders of magnitude change in diffusion coefficient upon binding, and cylindrical and linear diffusion modes along filopodia. Finally, we demonstrate how this new method can move single-virus tracking from simple monolayer culture towards more tissue-like conditions by tracking single virions in tightly packed epithelial cells. This multi-resolution method presents new opportunities for capturing fast, 3D processes in biological systems.

One-Sentence SummaryActive-feedback 3D single-virus tracking enables an unprecedented look at the early stages of virus-cell interactions.


title: Intra-colony channel morphology in Escherichia coli biofilms is governed by nutrient availability and substrate stiffness

abstract: Nutrient-transporting channels have been recently discovered in mature Escherichia coli biofilms, however the relationship between intra-colony channel structure and the surrounding environmental conditions is poorly understood. Using a combination of fluorescence mesoscopy and a purpose-designed open-source quantitative image analysis pipeline, we show that growth substrate composition and nutrient availability have a profound effect on the morphology of intra-colony channels in mature E. coli biofilms. Under all nutrient conditions, intra-colony channel width was observed to increase non-linearly with radial distance from the centre of the biofilm. Notably, the channels were around 50% wider at the centre of carbon-limited biofilms compared to nitrogen-limited biofilms. Channel density also differed in colonies grown on rich and minimal media, with the former creating a network of tightly packed channels and the latter leading to well-separated, wider channels with defined edges. Our approach paves the way for measurement of internal patterns in a wide range of biofilms, offering the potential for new insights into infection and pathogenicity.


title: Dynamic localisation of DamX regulates bacterial filamentation and division during UPEC dispersal from host cells

abstract: Uropathogenic Escherichia coli (UPEC) cells can grow into highly filamentous forms during infection of bladder epithelial cells, but this process is poorly understood. Herein we found that some UPEC filaments released from infected bladder cells in vitro grew very rapidly and by more than 100 {micro}m before initiating division, whereas others did not survive, suggesting that filamentation is a stress response that promotes dispersal. The DamX bifunctional division protein, which is essential for UPEC filamentation, was initially non-localized but then assembled at multiple division sites in the filaments prior to division. DamX rings maintained consistent thickness during constriction and remained at the septum until after membrane fusion was completed, like in rod cell division. Our findings suggest a mechanism involving regulated dissipation of DamX, leading to division arrest and filamentation, followed by its reassembly into division rings to promote UPEC dispersal and survival during infection.


title: Comprehensive transcriptional profiling and mouse phenotyping reveals dispensable role for adipose tissue selective long noncoding RNA Gm15551

abstract: 1Cold and nutrient activated brown adipose tissue (BAT) is capable of increasing systemic energy expenditure via uncoupled respiration and secretion of endocrine factors thereby protecting mice against diet-induced obesity and improving insulin response and glucose tolerance in men. Long non-coding RNAs (lncRNAs) have recently been identified as fine tuning regulators of cellular function. While certain lncRNAs have been functionally characterised in adipose tissue, their overall contribution in the activation of BAT remains elusive. We identified lncRNAs correlating to inter-scapular brown adipose tissue (iBAT) function in high fat diet (HFD) and cold stressed mice. We focused on Gm15551 which has an adipose tissue specific expression profile, is highly upregulated during adipogenesis and downregulated by {beta}-adrenergic activation in mature adipocytes. Albeit we performed comprehensive transcriptional and adipocyte physiology profiling in vitro and in vivo, we could not detect an effect of gain or loss of function of Gm15551.


title: Synuclein alpha accumulation mediates podocyte injury in Fabry nephropathy

abstract: Current therapies for Fabry disease are based on reversing intra-cellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosome dysfunction. However, their effect in the reversal of endorgan damage, like kidney injury and chronic kidney disease remains unclear. First, ultrastructural analysis of serial human kidney biopsies showed that longterm use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/CAS9-mediated -Galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified alpha-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.


title: Ataxin-2, Twenty-four and Dicer-2 are components of a non-canonical cytoplasmic polyadenylation complex

abstract: Cytoplasmic polyadenylation is a mechanism to promote mRNA translation in a wide variety of biological contexts. A canonical complex centered around the conserved RNA-binding protein family CPEB has been shown to be responsible for this process. We have previously reported evidence for an alternative non-canonical, CPEB-independent complex in Drosophila, of which the RNA-interference factor Dicer-2 is a component. Here, we investigate Dicer-2 mRNA targets and protein co-factors in cytoplasmic polyadenylation. Using RIP-Seq analysis we identify hundreds of novel Dicer-2 target transcripts, [~]50% of which were previously found as targets of the cytoplasmic poly(A) polymerase Wispy, suggesting widespread roles of Dicer-2 in cytoplasmic polyadenylation. Large-scale immunoprecipitation revealed Ataxin-2 and Twenty-four among the high-confidence interactors of Dicer-2. Functional analysis indicate that both factors form an RNA-independent complex with Dicer-2, and are required for cytoplasmic polyadenylation of Dicer-2 targets. Our results reveal the composition of a novel cytoplasmic polyadenylation complex that operates during Drosophila early embryogenesis.


title: Translational readthrough as a potential therapeutic for AIPL1-associated Leber Congenital Amaurosis in a patient-derived iPSC-retinal organoid model

abstract: Leber Congenital Amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterised by severe sight impairment in infancy and rapidly progressive degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cGMP phosphodiesterase (PDE6) compared to control or CRISPR corrected organoids. Moreover, increased levels of cGMP were detected. The translational readthrough inducing drug (TRID) PTC124 was investigated as a potential therapeutic. LCA4 retinal organoids exhibited rescue of AIPL1 and PDE6; however, the level of full-length, functional AIPL1 induced through PTC124 treatment was insufficient to reduce cGMP levels and fully rescue the LCA4 phenotype. LCA4 retinal organoids are a valuable platform for the in vitro investigation of the molecular mechanisms that drive photoreceptor loss and for the evaluation of novel therapeutics.

category: cell biology


title: Dynamic cell cycle-dependent phosphorylation modulates CENP-L-CENP-N centromere recruitment

abstract: The kinetochore is a macromolecular structure that is required to ensure proper chromosome segregation during each cell division. The kinetochore is assembled upon a platform of the 16-subunit Constitutive Centromere Associated Network (CCAN), which is present at centromeres throughout the cell cycle. The nature and regulation of CCAN assembly, interactions, and dynamics required to facilitate changing centromere properties and requirements remain to be fully elucidated. The CENP-LN CCAN sub-complex displays a unique cell cycle-dependent localization behavior, peaking in S phase. Here, we demonstrate that phosphorylation of CENP-L and CENP-N controls CENP-LN complex formation and localization in a cell cycle-dependent manner. Mimicking constitutive phosphorylation of either CENP-L or CENP-N or simultaneously preventing phosphorylation of both proteins prevents CENP-LN localization and disrupts chromosome segregation. Together, our work suggests that cycles of phosphorylation and dephosphorylation are critical for CENP-LN complex recruitment and dynamics at centromeres to enable cell cycle-dependent CCAN reorganization.


title: The E3 ubiquitin ligase HECTD1 contributes to cell cycle progression through an effect on mitosis

abstract: Mechanistic studies of how protein ubiquitylation regulates the cell cycle, in particular during mitosis, has provided unique insights which have contributed to the emergence of the  Ubiquitin code. In contrast to RING E3 ubiquitin ligases such as the APC/c ligase complex, the contribution of other E3 ligase families during cell cycle progression remains less well understood. Similarly, the contribution of ubiquitin chain types beyond homotypic K48 chains in S-phase or branched K11/K48 chains assembled by APC/c during mitosis, also remains to be fully determined. Our recent findings that HECTD1 ubiquitin ligase activity assembles branched K29/K48 ubiquitin linkages prompted us to evaluate its function during the cell cycle. We used transient knockdown and genetic knockout to show that HECTD1 depletion in HEK293T and HeLa cells decreases cell proliferation and we established that this is mediated through loss of its ubiquitin ligase activity. Interestingly, we found that HECTD1 depletion increases the proportion of cells with aligned chromosomes (Prometa/Metaphase). We confirmed this molecularly using phospho-Histone H3 (Ser28) as a marker of mitosis. Time-lapse microscopy of NEBD to anaphase onset established that HECTD1-depleted cells take on average longer to go through mitosis. To explore the mechanisms involved, we used proteomics to explore the endogenous HECTD1 interactome in mitosis and validated the Mitosis Checkpoint Complex protein BUB3 as a novel HECTD1 interactor. In line with this, we found that HECTD1 depletion reduces the activity of the Spindle Assembly Checkpoint. Overall, our data suggests a novel role for HECTD1 ubiquitin ligase activity in mitosis.


title: Determination of Slow-binding HDAC Inhibitor Potency and Subclass Selectivity

abstract:
Histone deacetylases (HDACs) 1-3 regulate chromatin structure and gene expression. These three enzymes are targets for cancer chemotherapy and are studied for the treatment of immune disorders and neurodegeneration, but there is a lack of selective pharmacological tool compounds to unravel their individual roles. Potent inhibitors of HDACs 1-3 often display slow-binding kinetics, which causes a delay in inhibitor-enzyme equilibration and may affect assay readout. Here, we compare the potency and selectivity of slow-binding inhibitors measured by discontinuous and continuous assays. We find that entinostat, a clinical candidate, inhibits HDACs 1-3 by a two-step, slow-binding mechanism with lower potencies than previously reported. In addition, we show that RGFP966, commercialized as HDAC3-selective probe, is a slow-binding inhibitor with inhibitor constants of 57 nM, 31 nM, and 13 nM against HDACs 1-3, respectively. These data highlight a need for thorough kinetic investigation in the development of selective HDAC probes.

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title: Structural basis for the assembly and electron transport mechanisms of the dimeric photosynthetic RC-LH1 supercomplex

abstract: The reaction center (RC) and light-harvesting complex 1 (LH1) form a RC-LH1 core supercomplex that is vital for the primary reactions of photosynthesis in purple photosynthetic bacteria. Some species possess the dimeric RC-LH1 complex with an additional polypeptide PufX, representing the largest photosynthetic complex in anoxygenic phototrophs. However, the details of the architecture and assembly mechanism of the RC-LH1 dimer are unclear. Here we report seven cryo-electron microscopy (cryo-EM) structures of RC-LH1 supercomplexes from Rhodobacter sphaeroides. Our structures reveal that two PufX polypeptides are positioned in the center of the S-shaped RC-LH1 dimer, interlocking association between the components and mediating RC-LH1 dimerization. Moreover, we identify a new transmembrane peptide, designated PufY, which is located between the RC and LH1 subunits near the LH1 opening. PufY binds a quinone molecule and prevents LH1 subunits from completely encircling the RC, creating a channel for quinone/quinol exchange. Genetic mutagenesis, cryo-EM structures, and computational simulations enable a mechanistic understanding of the assembly and electron transport pathways of the RC-LH1 dimer and elucidate the roles of individual components in ensuring the structural and functional integrity of the photosynthetic supercomplex.


title: Music-selective cortex is sensitive to structure in both pitch and time

abstract: Converging evidence suggests that neural populations within human non-primary auditory cortex respond selectively to music. These neural populations respond strongly to a wide range of music stimuli, and weakly to other natural sounds and to synthetic control stimuli matched to music in many acoustic properties, suggesting that they are driven by high-level musical features. What are these features? Here we used fMRI to test the extent to which musical structure in pitch and time contribute to music-selective neural responses. We used voxel decomposition to derive music-selective response components in each of 15 participants individually, and then measured the response of these components to synthetic music clips in which we selectively disrupted musical structure by scrambling either the note pitches and/or onset times. Both types of scrambling produced lower responses compared to when melodic or rhythmic structure was intact. This effect was much stronger in the music-selective component than in the other response components, even those with substantial spatial overlap with the music component. We further found no evidence for any cortical regions sensitive to pitch but not time structure, or vice versa. Our results suggest that the processing of melody and rhythm are intertwined within auditory cortex.

category: neuroscience


title: Functional organization of social perception in the human brain

abstract: Humans can readily perceive a multitude of features from social interactions, but the phenomenological and neural basis of social perception has yet to be solved. Short film clips with rich social content were shown to 97 healthy participants while their haemodynamic brain activity was measured with fMRI. The stimulus clips were annotated for 112 social features yielding the initial stimulus model. Cluster analysis revealed that 13 dimensions were sufficient for describing the social perceptual space. Univariate GLM using these dimensions as predictors was used to map regional neural response profiles to different social features. Multivariate pattern analysis was then utilized to establish the regional specificity of the responses. The results revealed a posterior-anterior gradient in the processing of social information in the brain. Occipital and temporal regions responded to most social dimensions and the classifier revealed that these responses were dimension specific; in contrast Heschl gyri and parietal areas were also broadly tuned to different social signals yet the responses were domain-general and did not differentiate between dimensions. Altogether these results highlight the distributed nature of social processing in the brain as well as the specific contributions of feature-specific versus domain-general social perceptual processes.


title: Help or flight: Neural defensive circuits promote helping under threat in humans

abstract: Empathy for others distress has long been considered the driving force of helping. However, when deciding to help others in danger, one must consider not only their distress, but also the risk to oneself. Whereas the role of self-defence in helping has been overlooked in human research, studies in other animals indicate defensive responses are necessary for the protection of conspecifics. In this pre-registered study (N=49), we demonstrate that human defensive neural circuits are implicated in helping others under threat. Participants underwent fMRI scanning while deciding whether to help another participant avoid aversive electrical shocks, at the risk of also being shocked. We found that higher engagement of neural circuits that coordinate fast escape from self-directed danger (including the insula, PAG and ACC) facilitated decisions to help others. Importantly, using Representational Similarity Analysis, we found that the strength with which the amygdala and insula uniquely represented the threat to oneself (and not the others distress) predicted helping. Our findings indicate that in humans, as other mammals, defensive mechanisms play a greater role in helping behaviour than previously understood.


title: Neural defensive circuits underlie helping under threat in humans


title: Functional connectivity and information pathways in the human entorhinal-hippocampal circuitry

abstract: AO_SCPLOWBSTRACTC_SCPLOWCortical processing streams for item and contextual information come together in the entorhinal-hippocampal circuitry. Various evidence suggest that information-specific pathways organize the cortical - entorhinal interaction and the circuitrys inner communication along the transversal axis. Here, we leveraged ultra-high field functional imaging and advance Maass, Berron et al. (2015) who report two functional routes segregating the entorhinal cortex (EC) and subiculum. Our data show specific scene processing in the functionally connected posterior-medial EC and distal subiculum. The regions of another route, that connects the anterior-lateral EC and a newly identified retrosplenial-based anterior-medial EC subregion with the CA1/subiculum border, process object and scene information similarly. Our results support topographical information flow in human entorhinal-hippocampal subregions with organized convergence of cortical processing streams and a unique route for contextual information. They characterize the functional organization of the circuitry and underpin its central role in memory function and pathological decline.


title: Planning the potential future during multi-item visual working memory

abstract: Working memory allows us to retain visual information to guide upcoming future behavior. In line with this future-oriented purpose of working memory, recent studies have shown that action planning occurs during encoding and retention of a single visual item, for which the upcoming action is certain. We asked whether and how this extends to multi-item visual working memory, when visual representations serve the potential future. Human participants performed a visual working memory task with a memory-load manipulation (one/two/four items), and a delayed orientation-reproduction report (of one item). We measured EEG to track 15-25 Hz beta activity in electrodes contralateral to the required response hand - a canonical marker of action planning. We show an attenuation of beta activity, not only in load one (with one certain future action), but also in load two (with two potential future actions), compared to load four (with low prospective-action certainty). Moreover, in load two, potential action planning occurs regardless whether both visual items afford similar or dissimilar manual responses; and it predicts the speed of ensuing memory-guided behavior. This shows that potential action planning occurs during multi-item visual working memory, and brings the perspective that working memory helps us prepare for the potential future.


title: Characterization of sequence determinants of enhancer function using natural genetic variation

abstract: Sequence variation in enhancers, a class of cis-regulatory elements that control cell type-specific gene transcription, contributes significantly to phenotypic variation within human populations. Enhancers are short DNA sequences ([~]200 bp) composed of multiple binding sites (4-10 bp) for transcription factors (TFs). The transcriptional regulatory activity of an enhancer is encoded by the type, number, and distribution of TF binding sites that it contains. However, the sequence determinants of TF binding to enhancers and the relationship between TF binding and enhancer activity are complex, and thus it remains difficult to predict the effect of any given sequence variant on enhancer function. Here, we generate allele-specific maps of TF binding and enhancer activity in fibroblasts from a panel of F1 hybrid mice that have a high frequency of sequence variants. We identified thousands of enhancers that exhibit differences in TF binding and/or activity between alleles and use these data to define features of sequence variants that are most likely to impact enhancer function. Our data demonstrate a critical role for AP-1 TFs at many fibroblast enhancers, reveal a hierarchical relationship between AP-1 and TEAD TF binding at enhancers, and delineate the nature of sequence variants that contribute to AP-1 TF binding. These data represent one of the most comprehensive assessments to date of the impact of sequence variation on enhancer function in chromatin, with implications for identifying functional cis-regulatory variation in human populations.


title: Song Richness Index: A measure to understand the diversity and repetition of notes in a birdsong

abstract: Birdsong plays an important role in mate attraction and territorial defense. Many birds, especially Passerines, produce varying sequences of multiple notes resulting in complex songs. Studying the diversity of notes within these songs can give insights into an individuals reproductive fitness. We first looked at the previously described and commonly used diversity measures to understand the possible case-specific limitations. We then developed a new diversity measure-Song Richness Index (SRI). We compared SRI with three measures of diversity using all possible combinations of notes to understand the case-specific advantages and limitations of all approaches. Simulating all possible combinations gave us insights into how each diversity measure works in a real scenario. SRI showed an advantage over conventional measures of diversity like Note Diversity Index (NDI), Shannons Equitability (SH), and Simpsons Diversity (SI), especially in the cases where songs are made up of only one type of repetitive note.

category: animal behavior and cognition


title: Integration of public DNA methylation and expression networks via eQTMs improves prediction of functional gene-gene associations

abstract:
Gene co-expression networks can be used to infer functional relationships between genes, but they do not work well for all genes. We investigated whether DNA methylation can provide complementary information for such genes. We first carried out an eQTM meta-analysis of 3,574 gene expression and methylation samples from blood, brain and nasal epithelial brushed cells to identify links between methylated CpG sites and genes. This revealed 6,067 significant eQTM genes, and we observed that histone modification information is predictive of both eQTM direction and presence, enabling us to link many CpG sites to genes. We then generated a co-methylation network - MethylationNetwork - using 27,720 publicly available methylation profiles and integrated it with a public RNA-seq co-expression dataset of 31,499 samples. Here, we observed that MethylationNetwork can identify experimentally validated interacting pairs of genes that could not be identified in the RNA-seq datasets. We then developed a novel integration pipeline based on CCA and used the integrated methylation and gene networks to predict gene pairs reported in the STRING database. The integrated network showed significantly improved prediction performance compared to using a DNA co-methylation or a gene co-expression network alone. This is the first study to integrate data from two -omics layers from unmatched public samples across different tissues and diseases, and our results highlight the issues and potential of integrating public datasets from multiple molecular phenotypes. The eQTMs we identified can be used as an annotation resource for epigenome-wide association, and we believe that our integration pipeline can be used as a framework for future -omics integration analyses of public datasets.

We provide supporting materials and results, including the harmonized DNA methylation data from multiple tissues and diseases in https://data.harmjanwestra.nl/comethylation/, the discovered and predicted eQTMs, the corresponding CCA components and the trained prediction models in a Zenodo repository (https://zenodo.org/record/4666994). We provide notebooks to facilitate use of the proposed pipeline in a GitHub repository (https://github.com/molgenis/methylationnetwork).


title: Systematic analysis of low-affinity transcription factor binding site clusters in vitro and in vivo establishes their functional relevance

abstract: Transcription factor binding to a single binding site and its functional consequence in a promoter context are beginning to be relatively well understood. However, binding to clusters of sites has yet to be characterized in depth, and the functional relevance of binding site clusters remains uncertain. We employed a high-throughput biochemical method to characterize transcription factor binding to clusters varying across a range of affinities and configurations. We found that transcription factors can bind concurrently to overlapping sites, challenging the notion of binding exclusivity. Furthermore, compared to an individual high-affinity binding site, small clusters with binding sites an order of magnitude lower in affinity give rise to higher mean occupancies at physiologically-relevant transcription factor concentrations in vitro. To assess whether the observed in vitro occupancies translate to transcriptional activation in vivo, we tested low-affinity binding site clusters by inserting them into a synthetic minimal CYC1 and the native PHO5 S. cerevisiae promoter. In the minCYC1 promoter, clusters of low-affinity binding sites can generate transcriptional output comparable to a promoter containing three consensus binding sites. In the PHO5 promoter, replacing the native Pho4 binding sites with clusters of low-affinity binding sites recovered activation of these promoters as well. This systematic characterization demonstrates that clusters of low-affinity binding sites achieve substantial occupancies, and that this occupancy can drive expression in eukaryotic promoters.


title: Visual speech is processed differently in auditory and visual cortex: evidence from MEG and partial coherence analysis

abstract:
Speech perception in noisy environments is enhanced by seeing facial movements of communication partners. However, the neural mechanisms by which audio and visual speech are combined are not fully understood. We explore MEG phase locking to auditory and visual signals in MEG recordings from 14 human participants (6 females, 8 males) that reported words from single spoken sentences. We manipulated the acoustic clarity and visual speech signals such that critical speech information is present in auditory, visual or both modalities. MEG coherence analysis revealed that both auditory and visual speech envelopes (auditory amplitude modulations and lip aperture changes) were phase-locked to 2-6Hz brain responses in auditory and visual cortex, consistent with entrainment to syllable-rate components. Partial coherence analysis was used to separate neural responses to correlated audio-visual signals and showed non-zero phase locking to auditory envelope in occipital cortex during audio-visual (AV) speech. Furthermore, phase-locking to auditory signals in visual cortex was enhanced for AV speech compared to audio-only (AO) speech that was matched for intelligibility. Conversely, auditory regions of the superior temporal gyrus (STG) did not show above-chance partial coherence with visual speech signals during AV conditions, but did show partial coherence in VO conditions. Hence, visual speech enabled stronger phase locking to auditory signals in visual areas, whereas phase-locking of visual speech in auditory regions only occurred during silent lip-reading. Differences in these cross-modal interactions between auditory and visual speech signals are interpreted in line with cross-modal predictive mechanisms during speech perception.

Significance StatementVerbal communication in noisy environments is challenging, especially for hearing-impaired individuals. Seeing facial movements of communication partners improves speech perception when auditory signals are degraded or absent. The neural mechanisms supporting lip-reading or audio-visual benefit are not fully understood. Using MEG recordings and partial coherence analysis we show that speech information is used differently in brain regions that respond to auditory and visual speech. While visual areas use visual speech to improve phase-locking to auditory speech signals, auditory areas do not show phase-locking to visual speech unless auditory speech is absent and visual speech is used to substitute for missing auditory signals. These findings highlight brain processes that combine visual and auditory signals to support speech understanding.


title: Differential auditory and visual phase-locking are observed during audio-visual benefit and silent lip-reading for speech perception


title: Speech-driven Facial Animations Improve Speech-in-Noise Comprehension of Humans

abstract: Understanding speech becomes a demanding task when the environment is noisy. Comprehension of speech in noise can be substantially improved by looking at the speakers face, and this audiovisual benefit is even more pronounced in people with hearing impairment. Recent advances in AI have allowed to synthesize photorealistic talking faces from a speech recording and a still image of a persons face in an end-to-end manner. However, it has remained unknown whether such facial animations improve speech-in-noise comprehension. Here we consider facial animations produced by a recently introduced generative adversarial network (GAN), and show that humans cannot distinguish between the synthesized and the natural videos. Importantly, we then show that the end- to-end synthesized videos significantly aid humans in understanding speech in noise, although the natural facial motions yield a yet higher audiovisual benefit. We further find that an audiovisual speech recognizer benefits from the synthesized facial animations as well. Our results suggest that synthesizing facial motions from speech can be used to aid speech comprehension in difficult listening environments.


title: An improved organ explant culture method reveals stem cell lineage dynamics in the adult Drosophila intestine

abstract: In recent years, live-imaging techniques have been developed for the adult midgut of Drosophila melanogaster that allow temporal characterization of key processes involved in stem cell and tissue homeostasis. However, current organ culture techniques are limited to imaging sessions of [≤]16 hours, an interval too short to track dynamic processes such as damage responses and regeneration, which can unfold over several days. Therefore, we developed a new organ explant culture protocol capable of sustaining midguts ex vivo for up to 3 days. This was made possible by the formulation of a culture medium specifically designed for adult Drosophila tissues with an increased Na+/K+ ratio and trehalose concentration, and by placing midguts at an air-liquid interface for enhanced oxygenation. We show that midgut progenitor cells can respond to gut epithelium damage ex vivo, proliferating and differentiating to replace lost cells, but are quiescent in healthy intestines. Using ex vivo gene induction to promote stem cell proliferation, we demonstrate that intestinal stem lineages can be traced through multiple cell divisions using live imaging. Both asymmetric and symmetric divisions can be identified in the reconstructed lineages. We find that daughter cells of asymmetric divisions remain in close proximity of each other, while the progeny of symmetric divisions actively move apart, with implications for cell differentiation and tissue organization. We show that the same culture set-up is useful for imaging adult renal tubules and ovaries for up to 72 hours. By enabling both long-term imaging and real-time ex vivo gene manipulation, our simple culture protocol provides a powerful tool for studies of epithelial biology and cell lineage behavior.


title: Different temporal requirements for the LRR transmembrane receptors Tartan and Toll-2 in the formation of contractile interfaces at compartmental boundaries

abstract: Compartmental boundaries physically separate groups of epithelial cells, a property fundamental for the organization of the body plan in both insects and vertebrates. In many examples, this physical separation has been shown to be the consequence of a regulated increase in contractility of the actomyosin cortex at boundary cell-cell interfaces, a property important in developmental morphogenesis beyond compartmental boundary formation. In this study, we took an unbiased screening approach to identify cell surface receptors required for actomyosin enrichment and polarisation at parasegmental boundaries (PSBs) in early Drosophila embryos, leading us to uncover different temporal requirements for two LRR receptors, Tartan and Toll-2. First, we find that Tartan is required during germband extension for actomyosin enrichment at PSBs, confirming an earlier report. Next, by following in real time the dynamics of loss of boundary straightness in tartan mutant embryos compared to wildtype and ftz mutant embryos, we show that Tartan is not required beyond germband extension. At this stage, actomyosin enrichment at PSBs becomes regulated by Wingless signalling. We find that Wingless signalling regulates Toll-2 expression and we show that Toll-2 is required for planar polarization of actomyosin after the completion of germ-band extension. Thus the formation of contractile interfaces at PSBs depends on a dynamic set of LRR receptors cues. Our study also suggests that the number of receptor cues is small and that the receptors are interchangeable.


title: Spastic paraplegia protein ortholog, reticulon-like 1, governs presynaptic ER organization and Ca2+ dynamics

abstract: Neuronal endoplasmic reticulum (ER) appears continuous throughout the cell. Its shape and continuity are influenced by ER-shaping proteins, mutations in which can cause axon degeneration in Hereditary Spastic Paraplegia (HSP). While HSP is thought of as an axon degeneration disease, the susceptibility of distal axons suggests a "dying back" pathology, in which presynaptic terminals could also be affected. We therefore asked how loss of Rtnl1, a Drosophila ortholog of the human HSP gene RTN2 (SPG12), which encodes an ER-shaping protein, affected ER organization and the function of presynaptic terminals. Loss of Rtnl1 depleted ER membrane markers at larval presynaptic motor terminals, and appeared to deplete mainly narrow tubular ER while leaving cisternae largely unaffected, thus suggesting little change in Ca2+ storage capacity at rest. Nevertheless, these changes in presynaptic ER architecture were accompanied by major reductions in activity-evoked Ca2+ fluxes in the cytosol, ER lumen, and mitochondria, as well as by reduced evoked and spontaneous neurotransmission. Our results provide a unique model to explore the roles of presynaptic tubular ER, and show the importance of ER architecture in regulating presynaptic physiology and synaptic function. Altered presynaptic Ca2+ physiology is therefore a potential factor in the pathological changes found in HSP.


title: Chromosome remodelling by SMC/Condensin in B. subtilis is regulated by Soj/ParA during growth and sporulation

abstract: SMC complexes, loaded at ParB-parS sites, are key mediators of chromosome organization in bacteria. ParA/Soj proteins interact with ParB/Spo0J in a pathway involving ATP-dependent dimerization and DNA binding, leading to chromosome segregation and SMC loading. In Bacillus subtilis, ParA/Soj also regulates DNA replication initiation, and along with ParB/Spo0J is involved in cell cycle changes during endospore formation. The first morphological stage in sporulation is the formation of an elongated chromosome structure called an axial filament. We now show that a major redistribution of SMC complexes drives axial filament formation, in a process regulated by ParA/Soj. Unexpectedly, this regulation is dependent on monomeric forms of ParA/Soj that cannot bind DNA or hydrolyse ATP. These results reveal a new role for ParA/Soj proteins in the regulation of SMC dynamics in bacteria, and yet further complexity in the web of interactions involving chromosome replication, segregation, and organization, controlled by ParAB and SMC.


title: Proximal binding pocket Arg717 substitutions in Escherichia coli AcrB cause clinically relevant divergencies in resistance profiles

abstract:
Multidrug resistance (MDR) in bacteria can be caused by the over-expression of multidrug efflux pumps belonging to the Resistance-Nodulation-Division (RND) superfamily of proteins. These intrinsic or acquired pumps can export a wide range of antibiotics. Recently, amino acid substitutions within these pumps have been observed in resistant clinical strains. Among others, two of these worrying gain-of-function mutations are R717L and R717Q in the proximal binding pocket of efflux pump AcrB (AcrB-Sa) found in azithromycin-resistant Salmonella enterica spp. We investigated the ramifications of these (and other) mutations in phylogenetically closely related AcrB from Escherichia coli (AcrB-Ec). We found that AcrB-Ec harboring Arg717 substitutions were significantly more effective in exporting all tested macrolides, with an up to 8-fold increase in the minimum inhibitory concentration (MIC) values (from 16 to 128 g/mL for azithromycin). Interestingly, gain-of-function was also seen for fluoroquinolones (2-fold higher MICs), while there was a consistent loss-of-function for the export of novobiocin and {beta}-lactam cloxacillin (2-fold lower MICs), pointing to a protein adaptation, which simultaneously partly compromised the efflux ability of other compounds with different molecular properties. Disk diffusion susceptibility testing corroborated the findings, as the R717Q and R717L mutant strains had significantly smaller inhibition zones for macrolides and fluoroquinolones and a larger inhibition zone for novobiocin, compared to the wild type. The spread and independent emergences of these potent efflux pump mutations highlight the necessity of control of, and adjustments to, treatments with antibiotics and the need for novel antibiotics and efflux pump inhibitors.

ImportanceThe over-expression of multidrug efflux pumps is a major cause of multidrug-resistant bacteria. The emergence of gain-of-function mutations in these pumps is additionally worrying, as these render last-resort antibiotic treatment options ineffective. The Arg717-mutations in related Salmonella Typhi and Paratyphi A AcrB caused azithromycin-resistant strains, with resistance levels past breakpoints. This is worrying, as azithromycin is a last-resort antibiotic after fluoroquinolones to treat typhoid and paratyphoid fever. Our findings that the same substitutions in closely related Escherichia coli AcrB cause an increase in MICs for both fluoroquinolones and macrolides is worrying as it shows the effect of one amino acid substitution on a multitude of treatment options. Additionally, the finding that the substitutions negatively impact the export of other drugs, such as {beta}-lactam cloxacillin, suggests that treatment with multiple antibiotics may mitigate resistance and improve treatment. Our findings also imply the urge to develop novel antibiotics and inhibitors.


title: Kinesin-II motors differentially impact biogenesis of distinct extracellular vesicle subpopulations shed from C. elegans sensory cilia

abstract: Extracellular vesicles (EVs) are bioactive lipid-bilayer enclosed particles released from nearly all cells. One specialized site for EV shedding is the primary cilium, a conserved signaling organelle. The mechanisms underlying cargo enrichment and biogenesis of heterogeneous EVs shed from cilia are unclear. Here we discover the conserved ion channel CLHM-1 as a new ciliary EV cargo. Using super-resolution microscopy, we imaged EVs released into the environment from sensory neuron cilia of C. elegans expressing fluorescently-tagged CLHM-1 and TRP polycystin-2 channel PKD-2 EV cargoes at endogenous levels. We find that these proteins are enriched in distinct EV subpopulations that are differentially shed in response to availability of hermaphrodite mating partners. Both CLHM-1 and PKD-2 localize to the ciliary base and middle segment of the cilium proper, but PKD-2 alone is present in the cilium distal tip and EVs shed from this site. CLHM-1 EVs released into the environment bud from a secondary site, the periciliary membrane compartment at the ciliary base. We show that individual heterotrimeric and homomeric kinesin-II motors have discrete impacts on the colocalization of PKD-2 and CLHM-1 in both cilia and EVs. Total loss of kinesin-II activity significantly decreases shedding of PKD-2 but not CLHM-1 EVs. Our data demonstrate that anterograde kinesin-II-dependent intraflagellar transport is required for selective enrichment of specific protein cargoes into heterogeneous EVs with different signaling potentials.


title: Epithelial cells of the intestine acquire cell-intrinsic inflammation signatures during ageing

abstract: During ageing, cell-intrinsic and extrinsic factors lead to the decline of tissue function and organismal health. Disentangling these factors is important for developing effective strategies to prolong organismal healthspan. Here, we addressed this question in the mouse intestinal epithelium, which forms a dynamic interface with its microenvironment and receives extrinsic signals affecting its homeostasis and tissue ageing. We systematically compared transcriptional profiles of young and aged epithelial cells in vivo and ex vivo in cultured intestinal organoids. We found that all cell types of the aged epithelium exhibit an inflammation phenotype, which is marked by MHC class II upregulation and most pronounced in enterocytes. This was accompanied by elevated levels of the immune tolerance markers PD-1 and PD-L1 in the aged tissue microenvironment, indicating dysregulation of immunological homeostasis. Intestinal organoids from aged mice still showed an inflammation signature after weeks in culture, which was concurrent with increased chromatin accessibility of inflammation-associated loci. Our results reveal a cell-intrinsic, persistent inflammation phenotype in aged epithelial cells, which might contribute to systemic inflammation observed during ageing.


title: Microcin MccI47 selectively inhibits enteric bacteria and reduces carbapenem-resistant Klebsiella pneumoniae colonization in vivo when administered via an engineered live biotherapeutic

abstract:
BackgroundThe gastrointestinal (GI) tract is the reservoir for multidrug-resistant (MDR) pathogens, specifically carbapenem-resistant (CR) Klebsiella pneumoniae and other Enterobacteriaceae, which often lead to the spread of antimicrobial resistance genes, severe extraintestinal infections, and lethal outcomes. Selective GI decolonization has been proposed as a new strategy for preventing transmission to other body sites and minimizing spreading to susceptible individuals.

ResultsHere, we purify the to-date uncharacterized class IIb microcin I47 (MccI47) and demonstrate potent inhibition of numerous Enterobacteriaceae, including multidrug-resistant clinical isolates, in vitro at concentrations resembling those of commonly prescribed antibiotics. We then genetically modify the probiotic bacterium Escherichia coli Nissle 1917 (EcN) to produce MccI47 from a stable multicopy plasmid by using MccI47 toxin production in a counterselection mechanism to engineer one of the native EcN plasmids, which renders provisions for inducible expression and plasmid selection unnecessary. We then test the clinical relevance of the MccI47-producing engineered EcN in a murine CR K. pneumoniae colonization model and demonstrate significant MccI47-dependent reduction of CR K. pneumoniae abundance after seven days of daily oral live biotherapeutic administration without disruption of the resident microbiota.

ConclusionsThis study provides the first demonstration of MccI47 as a potent antimicrobial against certain Enterobacteriaceae, and its ability to significantly reduce the abundance of CR K. pneumoniae in a preclinical animal model, when delivered from an engineered live biotherapeutic product. This study serves as the foundational step towards the use of engineered live biotherapeutic products aimed at the selective removal of MDR pathogens from the GI tract

category: bioengineering


title: Histone demethylase UTX regulates glioblastoma progression through affecting periostin expression

abstract:
The histone H3K27 demethylase UTX participates in regulating multiple cancer types. However, less is known about the UTX function in glioblastoma (GBM). This study aims to define the effect of UTX on GBM. GEPIA2 database analysis showed that UTX expression was significantly increased in GBM and inversely correlated with survival. Knockdown UTX inhibited GBM cell proliferation, migration, and invasion while promoting apoptosis. Moreover, knockdown UTX also hampered tumor growth in the heterotopic xenograft model. RNA-seq combined with qRT-PCR and ChIP-qPCR were used to identify the target genes. The results showed that the UTX-mediated genes were strongly associated with tumor progression and the extracellular environment. Protein-protein interaction analysis suggested that periostin (POSTN) interacted with most of the other UTX-mediated genes. POSTN supplement abolished the effect of UTX knockdown in GBM cells. Furthermore, silencing UTX exhibited similar antitumor effect in patient-derived glioblastoma stem-like cells, while UTX functions were partially restored after exposing POSTN. Our findings may reveal a new insight into the onset of gliomagenesis and progression, providing a promising therapeutic strategy for GBM treatment.

Bullet PointsUTX correlates with survival in glioblastoma. Silencing UTX decreased the levels of H3K27 methylation in the POSTN gene, thereby suppressing POSTN expression. UTX-mediated POSTN expression is crucial for glioblastoma cell growth and tumorigenesis.

O_LIUTX expression is increased in GBM and negatively correlated with survival.
C_LIO_LIUTX knockdown influences proliferation and apoptosis in both GBM cells and GSCs.
C_LIO_LIThe antitumor effect of UTX knockdown is achieved by suppressing POSTN expression.
C_LI

category: cancer biology


title: REPTOR/CREBRF encode key regulators of muscle energy metabolism

abstract: Metabolic flexibility of muscle tissue describes the capacity to use glucose or lipids as energy substrates and its disruption is associated with metabolic dysfunction. Cancer-induced cachexia is a metabolic syndrome linked with muscle wasting, changes in muscle energy metabolism and lower life expectancy in cancer patients. The molecular mechanisms driving metabolic changes in muscle, however, are poorly characterized. Here, using a Drosophila model of systemic metabolic dysfunction triggered by yorkie-induced gut tumors, we identify the transcription factor REPTOR as a key regulator of energy metabolism in muscle. We show that REPTOR is upregulated in muscles of adult flies with gut yorkie-tumors, where it is necessary to modulate glucose metabolism. REPTOR expression in muscles is induced by ImpL2, a tumor-derived insulin binding protein that reduces systemic insulin signaling, or by nutritional restriction. Further, in vitro and in vivo studies indicate that high activity of REPTOR is sufficient to increase glucose content, transcriptionally repress phosphofructokinase and increase mitochondrial respiration. Consistent with the fly studies, higher levels of CREBRF, the mammalian ortholog of REPTOR, reduce glycolysis in mouse myotubes while promoting an oxidative phenotype. Altogether, our results implicate REPTOR/CREBRF as key regulators of muscle metabolism and metabolic flexibility that share a conserved function as repressors of glycolysis and promoters of oxidative phosphorylation.

category: physiology


title: A nutriepigenetic pathway links nutrient information to sensory plasticity

abstract: Diet composition has a profound influence on brain physiology and behavior, but the mechanisms through which nutrient information is transmuted into neural changes remain elusive. Here we uncover how the metabolic enzyme O-GlcNAc Transferase (OGT) transforms information about the dietary environment into taste adaptations. We show that in the fly D. melanogaster, OGT decorates the chromatin of the sweet taste neurons and provides the nutrient context to drive changes in chromatin accessibility in response to high dietary sugar. Specifically, we found that OGT cooperates with the epigenetic silencer Polycomb Repressive Complex 2.1 (PRC2.1) to promote nutrient-sensitive variations in chromatin openness; these chromatin dynamics result in changes in gene expression and taste plasticity that are dependent on the catalytic activity of OGT. Parallel nutrigenomic signatures were also observed in the lingual epithelium of rats exposed to high dietary sugar, suggesting that this conserved metabolic-epigenetic pathway may also underlie diet-dependent taste changes in mammals. Together our findings reveal a novel role for nutriepigenetic signaling in the brain: amplifying nutrient perturbations into robust changes in chromatin accessibility and transcriptional output that shape neural and behavioral plasticity.


title: Genotype and Th2 cells control monocyte to tissue resident macrophage differentiation during nematode infection of the pleural cavity

abstract:
The recent revolution in tissue-resident macrophage biology has resulted largely from murine studies performed in the C57BL/6 strain. Here, we provide a comprehensive analysis of immune cells in the pleural cavity using both C57BL/6 and BALB/c mice. Unlike C57BL/6 mice, naive tissue-resident Large Cavity Macrophages (LCM) of BALB/c mice failed to fully implement the tissue residency program. Following infection with a pleural-dwelling nematode these pre-existing differences were accentuated with LCM expansion occurring in C57BL/6 but not BALB/c mice. While infection drove monocyte recruitment in both strains, only in C57BL/6 mice were monocytes able to efficiently integrate into the resident pool. Monocyte to macrophage conversion required both T cells and IL-4R signalling. Host genetics are therefore a key influence on tissue resident macrophage biology, and during nematode infection Th2 cells control the differentiation pathway of tissue resident macrophages.

Graphical Abstract

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title: Phospho-proteomics reveals that RSK signaling is required for proliferation of natural killer cells stimulated with IL-2 or IL-15

abstract: Natural killer (NK) cells are cytotoxic lymphocytes that play a critical role in the innate immune system. Although cytokine signaling is crucial for the development, expansion, and cytotoxicity of NK cells, the signaling pathways stimulated by cytokines are not well understood. Here, we sought to compare the early signaling dynamics induced by the cytokines interleukin (IL)-2 and IL-15 using liquid chromatography-mass spectrometry (LC-MS)-based phospho-proteomics. Following stimulation of the immortalized NK cell line NK-92 with IL-2 or IL-15 for 5, 10, 15, or 30 minutes, we identified 8,692 phospho-peptides from 3,023 proteins. Comparing the kinetic profiles of 3,619 fully quantified phospho-peptides, we found that IL-2 and IL-15 induced highly similar signaling in NK-92 cells. Among the IL-2/IL-15-regulated phospho-sites were both well-known signaling events like the JAK/STAT pathway and novel signaling events with potential functional significance including LCP1 Ser5, PAK2 Ser141, and STK17B Ser12. Using bioinformatic approaches, we sought to identify kinases regulated by IL-2/IL-15 stimulation and found that the p90 ribosomal S6 kinase (p90RSK) family was activated by both cytokines. Using pharmacological inhibitors, we then discovered that RSK signaling is required for IL-2 and IL-15-induced proliferation in NK-92 cells. Taken together, our analysis represents the first phospho-proteomic characterization of cytokine signaling in NK cells and increases our understanding of how cytokine signaling regulates NK cell function.


title: DOK1 and DOK2 regulate CD8+ T cell signaling and memory formation without affecting tumor cell killing

abstract:
Targeting intracellular inhibiting proteins is a promising strategy to improve CD8+ T cell anti-tumor efficacy. DOK1 and DOK2 are CD8+ T cell inhibitory proteins that are targeted in this study in order to improve the activation and cytotoxic capacities of these cells. To evaluate the role of DOK-1 and DOK-2 depletion in physiology and effector function of T CD8+ lymphocyte and in cancer progression, a transgenic T cell receptor mouse model specific to melanoma antigen hgp100 (pmel-1 TCR Tg) was established.

Depletion of both Dok1 and Dok2 did not affect the development, proliferation, mortality, activation and cytotoxic function of naive CD8+ T cells. However, after an in vitro pre-stimulation Dok1/Dok2 DKO CD8+ T cells had higher percentage of effector memory T cells and showed an increase in levels of pAKT and pERK upon TCR stimulation. Despite this improved TCR signaling, pre-stimulated Dok1/Dok2 DKO CD8+ T cells did not show any increase in their activation or cytotoxicity capacities against melanoma cell line expressing hgp100 in vitro.

Altogether we demonstrate here a novel aspect of the negative regulation by DOK1 and DOK2 proteins in CD8+ T cells. In conclusion, DOK1 and DOK2 have an inhibitory role following long term T cell stimulations.


title: Dynamics of DNA replication during male gametogenesis in the malaria parasite Plasmodium falciparum

abstract: Malaria parasites undergo a single phase of sexual reproduction in their complex lifecycle, during which they cycle between mosquito and vertebrate hosts. Sexual reproduction occurs only at the point when parasites move into the mosquito host. It involves specialised, sexually committed cells called gametocytes, which develop very rapidly into mature gametes and then mate inside the mosquito midgut. The gamete development process is unique, involving unprecedentedly fast replication and cell division to produce male gametes. A single male gametocyte replicates its [~]23Mb genome three times over to produce 8 genomes, segregates these into newly-assembled flagellated gamete cells and releases them to seek out female gametes, all within [~]15 minutes. Here, for the first time, we use fluorescent labelling of de novo DNA synthesis to follow this process at the whole-cell and single-molecule levels, yielding several novel observations. Firstly, we confirm that no DNA replication occurs before gametogenesis is triggered, although the origin recognition complex protein Orc1 is abundant even in immature gametocytes. Secondly, between repeated rounds of DNA replication there is no detectable karyokinesis - in contrast to the repeated replicative rounds that occur in asexual schizonts. Thirdly, cytokinesis is clearly uncoupled from DNA replication, and can occur even if replication fails, implying a lack of cell cycle checkpoints. Finally the single-molecule dynamics of DNA replication are entirely different from those in asexual schizonts.


title: Metabolic Processing of Selenium-based Bioisostere of meso-diaminopimelic Acid in Live Bacteria

abstract: The bacterial cell wall supports cell shape and prevents lysis due to internal turgor pressure. A primary component of all known bacterial cell walls is the peptidoglycan (PG) layer, which is comprised of repeating units of sugars connected to short and unusual peptides. The various steps within PG biosynthesis are often the target of antibiotics as they are essential for cellular growth and survival. Synthetic mimics of PG have proven to be indispensable tools to study bacterial cell growth and remodeling. Yet, a common component of PG, meso-diaminopimelic acid (m-DAP) at the third position of the stem peptide, remains challenging to build synthetically and is not commercially available. Here, we describe the synthesis and metabolic processing of a selenium-based bioisostere of a m-DAP analogue, selenolanthionine. We show that selenolanthionine is installed within the PG of live bacteria by the native cell wall crosslinking machinery in several mycobacteria species. We envision that this probe will supplement the current methods available for investigating PG crosslinking in m-DAP containing organisms.


title: Unconstrained coevolution of bacterial size and the latent period of plastic phage

abstract: Viruses play critical roles in the dynamics of microbial communities. Lytic viruses, for example, kill significant proportions of autotrophic and heterotrophic microbes. The dynamic interplay between viruses and microbes results from an overlap of physiological, ecological, and evolutionary responses: environmental changes trigger host physiological changes, affecting the ecological interactions of host and virus and, ultimately, the evolutionary pressures influencing the two populations. Recent theoretical work studied how the dependence of viral traits on host physiology (viral plasticity) affects the evolutionarily stable host cell size and viral infection time emerging from coevolution. Here, we broaden the scope of the framework to consider any coevolutionary outcome, including potential evolutionary collapses of the system. We used the case study of Escherichia coli and T-like viruses under chemo-stat conditions, but the framework can be adapted to any microbe-virus system. Oligotrophic conditions led to smaller, lower-quality but more abundant hosts, and infections that were longer but produced a reduced viral offspring. Conversely, eutrophic conditions resulted in fewer but larger higher-quality hosts, and shorter but more productive infections. The virus influenced host evolution decreasing host radius more noticeably for low than for high dilution rates, and for high than for low nutrient input concentration. For low dilution rates, the emergent infection time minimized host need/use, but higher dilution led to an opportunistic strategy that shortened the duration of infections. System collapses driven by evolution resulted from host failure to adapt quickly enough to the evolving virus. Our results contribute to understanding the eco-evolutionary dynamics of microbes and virus, and to improving the predictability of current models for host-virus interactions. The large quantitative and qualitative differences observed with respect to a classic description (in which viral traits are assumed to be constant) highlights the importance of including viral plasticity in theories describing short- and long-term host-virus dynamics.