title: Evidence-based calibration of computational tools for missense variant pathogenicity classification and ClinGen recommendations for clinical use of PP3/BP4 criteria

abstract: Recommendations from the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) for interpreting sequence variants specify the use of computational predictors as Supporting level of evidence for pathogenicity or benignity using criteria PP3 and BP4, respectively. However, score intervals defined by tool developers, and ACMG/AMP recommendations that require the consensus of multiple predictors, lack quantitative support. Previously, we described a probabilistic framework that quantified the strengths of evidence (Supporting, Moderate, Strong, Very Strong) within ACMG/AMP recommendations. We have extended this framework to computational predictors and introduce a new standard that converts a tools scores to PP3 and BP4 evidence strengths. Our approach is based on estimating the local positive predictive value and can calibrate any computational tool or other continuous-scale evidence on any variant type. We estimate thresholds (score intervals) corresponding to each strength of evidence for pathogenicity and benignity for thirteen missense variant interpretation tools, using carefully assembled independent data sets. Most tools achieved Supporting evidence level for both pathogenic and benign classification using newly established thresholds. Multiple tools reached score thresholds justifying Moderate and several reached Strong evidence levels. One tool reached Very Strong evidence level for benign classification on some variants. Based on these findings, we provide recommendations for evidence-based revisions of the PP3 and BP4 ACMG/AMP criteria using individual tools and future assessment of computational methods for clinical interpretation.

category: bioinformatics


title: Fate mapping melanoma persister cells through regression and into recurrent disease in adult zebrafish

abstract:
Melanoma heterogeneity and plasticity underlie therapy resistance. Some tumour cells possess innate resistance while others reprogramme during drug exposure and survive to form persister cells, a source of potential cancer cells for recurrent disease. Tracing individual melanoma cell populations through tumour regression and recurrent disease remains largely unexplored, in part, because complex animal models are required for live imaging of cell populations over time. Here, we apply tamoxifen-inducible creERt2/loxP lineage tracing to a zebrafish model of MITF-dependent melanoma regression and recurrence to image and trace cell populations in vivo through disease stages. Using this strategy, we showed that melanoma persister cells at the minimal residual disease site originate from the primary tumour. Next, we fate mapped rare MITF-independent persister cells and demonstrate that these cells directly contribute to progressive disease. Multiplex immunohistochemistry confirmed MITF-independent persister cells give rise to Mitfa+ cells in recurrent disease. Taken together, our work reveals a direct contribution of melanoma persister cell populations to recurrent disease, and provides a resource for lineage tracing methodology in adult zebrafish cancer models.

Summary statementWe fate map melanoma cells from the primary tumour into a persister cell state and show that persister cells directly contribute to recurrent disease.

category: cancer biology


title: POU2AF2/C11orf53 functions as a co-activator of POU2F3 by maintaining chromatin accessibility and enhancer activity

abstract: Small cell lung cancer (SCLC), accounting for around 13% of all lung cancers, often results in rapid tumor growth, early metastasis, and acquired therapeutic resistance. The POU class 2 homeobox 3 (POU2F3) is a master regulator of tuft cell identity and defines a subtype of SCLC tumors (SCLC-P subtype) that lack the expression of neuroendocrine (NE) markers. Here, we have identified a previously uncharacterized protein, C11orf53, which is co-expressed with POU2F3 in both SCLC cell lines and patient samples, defining the same SCLC-P subtype. C11orf53 is an essential gene for the cell survivability of SCLC-P subtype, based on genome-wide CRISPR screening and genetic depletion of C11orf53 or POU2F3, inducing apoptosis in different SCLC-P subtype cell lines. Furthermore, our biochemical and genome-wide studies have demonstrated that C11orf53 is a novel chromatin-bound protein that occupies broad active enhancer domains and regulates gene expression at active enhancers. Mechanistically, C11orf53 directly interacts with POU2F3 at the POU domain, and is recruited to chromatin by POU2F3. Consequently, depletion of C11orf53 dramatically reduced enhancer H3K27ac levels and chromatin accessibility, resulting in a reduction of the expression of POU2F3-dependent tuft cell markers. Based on the expression pattern and molecular function of C11orf53, we have renamed it as "POU Class 2 Homeobox Associating Factor 2" (POU2AF2). In summary, our study has identified a new co-activator of POU2 family transcription factors, and sheds light on the therapeutic potential of targeting POU2AF2/POU2F3 heterodimer in human SCLC.


title: Distribution and frequency of salivary gland tumours: an international multicenter study

abstract:
BackgroundSalivary gland tumours (SGT) are a relatively rare group of neoplasms with a wide range of histopathological appearance and clinical features. To date, most of the epidemiological studies on salivary gland tumours are limited for a variety of reason including being out of date, extrapolated from either a single centre or country studies, or investigating either major or minor glands only.

MethodsThis study aimed to mitigate these shortcomings by analysing epidemiological data including demographic, anatomical location and histological diagnoses of SGT from multiple centres across the world. The analysed data included age, gender, location and histological diagnosis from fifteen centres covering the majority of the world health organisation (WHO) geographical regions between 2006 and 2019.

ResultsA total of 5798 cases were analysed including 65% benign and 35% malignant tumours. A slight female predilection (54%) and peak incidence between the fourth and seventh decade for both benign and malignant tumours was observed. The majority (69%) of the SGT presented in major and 31% in the minor glands. The parotid gland was the most common location (70%) for benign and minor glands (46%) for malignant tumours. Pleomorphic adenoma (70%), and Warthins tumour (17%), were the most common benign tumours whereas mucoepidermoid carcinoma (25%) and adenoid cystic carcinoma (16%) were the most frequent malignant tumours.

ConclusionsThis multicentre investigation presents the largest cohort study to date analysing salivary gland tumour data from tertiary centres scattered across the globe. These findings should serve as a baseline for future studies evaluating the epidemiological landscape of these tumours.

category: pathology


title: Spatio-temporal modeling reveals a layer of tunable control circuits for the distribution of cytokines in tissues

abstract: Cytokines are diffusible mediators of cell-cell communication among immune cells with critical regulatory functions for cell differentiation and proliferation. Previous studies have revealed considerable spatial inhomogeneities in the distribution of cytokine molecules in tissues, potentially shaping the efficacy and range of paracrine cytokine signals. How such cytokine gradients emerge and are controlled within cell populations is incompletely understood. In this work, we employed a spatial reaction-diffusion model to systematically investigate the formation and influence of spatial cytokine gradients. We found the fraction of cytokine secreting cells to be the main source of spatial inhomogeneity and subsequent activation. Positive feedback from local cytokine levels upon cytokine receptor expression leads to further increased spatial cytokine inhomogeneities. By exploring the effect of co-clustering cytokine secreting cells and cells with large amounts of receptor expression, as in the presence of regulatory T cells in the vicinity of antigen-presenting cells, we found that such constrained tissue architecture can have profound effects on the range of paracrine cytokine signals.

category: systems biology


title: Behavioral and dopaminergic signatures of resilience

abstract: Chronic stress can have lasting adverse consequences in some individuals, yet others are resilient to the same stressor1,2. While previous work found differences in the intrinsic properties of mesolimbic dopamine (DA) neurons in susceptible and resilient individuals after stress was over;3-10 the causal links between DA activity during stress, dynamic stress-evoked behavior, and individual differences in susceptibility and resilience are not known. Here, we record behavior and neural activity in DA projections to the nucleus accumbens (NAc, signals reward11-14) and to the tail striatum (TS, signals threat15-18) during a multiday chronic social defeat paradigm and discover behavioral and neural signatures of resilience. Using supervised and unsupervised behavioral quantification, we find that resilient and susceptible individuals employ different behavioral strategies during stress. In addition, NAc-DA (but not TS-DA) activity is higher in the proximity of the aggressor in resilient mice, consistent with a greater subjective value of the aggressor. Moreover, NAc-DA tends to be elevated at the onset of fighting back in resilient mice and at the offset of attacks in susceptible mice. To test whether DA activation during defeat can generate resilience, and if its timing with respect to behavior is critical, we performed optogenetic stimulation of NAc-DA in open-loop (randomly timed) during defeat or timed to specific behaviors using real-time pose-tracking and behavioral classification. We find that both open-loop DA activation and fighting-back-timed activation promote resilience, in both cases reorganizing behavior during defeat toward resilience-associated patterns. Attack offset-timed activation promotes avoidance during defeat but does not promote susceptibility afterwards. Together, these data suggest a model whereby, during stress, DA in the NAc can increase resilience primarily by elevating the subjective value of the stressor rather than by reinforcing particular stress-responsive behaviors.

category: neuroscience


title: Maternal separation induces autism spectrum disorder in young rhesus monkeys

abstract: Autism spectrum disorder (ASD) is a class of severe neurodevelopmental disorders with a high incidence in young children, and its pathogenesis remains elusive. There is no effective treatment, and ASD children usually have a hard time in integrating into society and leading a normal life, which places a heavy burden on the families and society. Studies have shown that in addition to genetic factors, environmental factors are another important risk contributing to the pathogenesis of ASD. Early environmental adversity, which can lead to abnormal brain development and affect cognition and behavior, greatly increases the incidence of a variety of brain developmental diseases including ASD. However, studies on this aspect are inadequate at present, and no clear conclusions can be drawn. We explored whether early adversity could trigger ASD core clinical symptoms in macaques by modeling early adversity through maternal separation. In this study, we conducted a rigorous behavioral analysis of 12 male macaques (1.5-2 years old) that underwent maternal separation and 9 male normal macaques of the same age that had been mother raised, and found that maternal separation could induce a small number of the young individuals to develop three core symptoms of ASD, including social impairment, stereotyped behaviors, and restricted interest simultaneously. According to DSM-V and ASD clinical diagnostic criteria, these individuals should be ASD macaques for having all the three core ASD symptoms at the same time., For the first time, this study reveals that early environmental adversity can lead to ASD pathogenesis in monkeys, and provides a new approach for future ASD studies and modeling ASD monkeys.


title: What happens after an error?

abstract:
A ubiquitous finding in cognitive science across a wide variety of tasks is that humans tend to slow down after making an error. The dominant account of this post error slowing is that people engage in adaptive control and are simply more cautious after an error. However, this explanation is challenged by the fact that, although people are slower, accuracy typically does not improve following an error. Errors negatively impact cognitive processing, but characterizing the nature of error-based impairments has been a challenge in standard paradigms. Here, we adopt a recently developed experimental approach to uncover the time course of stimulus-response processing following an error by exerting tight control over the timing of responses. This method allows us to apply a computational model of response preparation that allows us to estimate the latency of cognitive processing underlying responses. In a four alternative forced-choice task with arbitrary stimulus-response mappings, we find that human participants are less accurate after an error even when given up to two seconds to make a response. Our modeling results ruled out the possibility that errors lead to a subsequent slowing of the cognitive processing underlying responses. Instead, we found that the "efficacy" of cognitive processing in producing an intended response is impaired following errors as people commit more perseverative slips of action regardless of when a response is made. These results suggest that prior observations of post-error slowing may be an adaptive response to impaired cognitive processing rather than a strategic shift in the speed-accuracy tradeoff.

Significance StatementWhat happens after we make a mistake? It has long been established that human behavior changes after committing an error, but it has been surprisingly hard to establish how our mental processing is affected. By forcing people to respond at predetermined times, we uncover the time-course of how we go from a stimulus to a response. We find that errors do not affect the timing or the variability of stimulus-response processing. Instead, errors lead people to be more likely to immediately slip up and repeat their past mistakes, even when given ample time to recover. Our results that even though people may slow down to take their time to get it right, they are fundamentally less effective after making an error.


title: Antimicrobial peptide secretion protects endosymbionts from bacteriome autoimmunity in insects

abstract: Many insects house symbiotic intracellular bacteria (endosymbionts) that provide them with essential nutrients, thus promoting usage of nutrient-poor habitats. Endosymbiont seclusion within host specialized cells, called bacteriocytes, often organized in a dedicated organ, the bacteriome, is crucial in protecting them from host immune defenses while avoiding chronic host immune activation. Previous evidence obtained in the cereal weevil Sitophilus oryzae has shown that bacteriome immunity is activated against invading pathogens, suggesting endosymbionts might be targeted and impacted by immune effectors during an immune challenge. To pinpoint any molecular determinants associated with such challenges, we conducted a dual transcriptomic analysis of S. oryzaes bacteriome subjected to immunogenic peptidoglycan fragments. We show that upon immune challenge the bacteriome actively participates in the innate immune response via a burst of antimicrobial peptides (AMPs). Surprisingly, endosymbionts do not undergo any transcriptomic changes, indicating that this potential threat goes unnoticed. Immunohistochemistry of selected AMPs show that these are secreted outside the bacteriome, excluding direct contact with the endosymbionts. This work demonstrates that endosymbiont protection during an immune challenge is mainly achieved by efficient confinement within bacteriomes, which provides physical separation between host systemic response and endosymbionts.

category: immunology


title: Disruption of the plastid-hosted iron-sulfur cluster biogenesis pathway in Toxoplasma gondii has pleiotropic effects irreversibly impacting parasite viability

abstract: Like many other apicomplexan parasites, Toxoplasma gondii contains a plastid harbouring key metabolic pathways, including the SUF pathway that is involved in the biosynthesis of iron-sulfur clusters. These cofactors are key for a variety of proteins involved in important metabolic reactions, potentially including plastidic pathways for the synthesis of isoprenoid and fatty acids. It was shown previously that impairing the NFS2 cysteine desulfurase, involved in the first step of the SUF pathway, leads to an irreversible killing of intracellular parasites. However, the metabolic impact of disrupting the pathway remained unexplored. We have generated another mutant of the pathway, deficient for the SUFC ATPase, and we have investigated in details the phenotypic consequences of TgNFS2 and TgSUFC depletion on parasite homeostasis. Our analysis confirms that Toxoplasma SUF mutants are severely and irreversibly impacted in growth: cell division and membrane homeostasis are particularly affected. Lipidomic analysis suggests a defect in apicoplast-generated fatty acids, along with a simultaneous increase in scavenging of host-derived lipids. However, addition of exogenous lipids did not allow full restauration of growth, suggesting other more important cellular functions were impacted in addition to fatty acid synthesis. For instance, we have shown that the SUF pathway is also key for generating isoprenoid-derived precursors necessary for the proper targeting of GPI-anchored proteins as well as for the parasite gliding motility. Thus, plastid-generated iron-sulfur clusters support the functions of proteins involved in several vital downstream cellular pathways, which implies the SUF machinery may be explored for discovering new potential anti-Toxoplasma targets.

category: microbiology


title: Disrupting the plastid-hosted iron-sulfur cluster biogenesis pathway in Toxoplasma gondii has pleiotropic effects irreversibly impacting parasite viability


title: Transcriptional analysis identifies overlapping and tissue-distinct profiles between Kaposi sarcoma tumors of the skin and gastrointestinal tract

abstract:
Kaposi sarcoma (KS), caused by Kaposi sarcoma herpesvirus (KSHV), is a multicentric tumor characterized by abnormal vasculature and proliferation of KSHV-infected spindle cells. KS commonly involves the skin but in severe cases KS can also involve the gastrointestinal tract (GI). Here, we sought to compare the cellular and KSHV gene expression signatures of skin and GI KS lesions. Skin and GI KS were compared to normal matched samples using bulk RNA sequencing.Twenty-two paired samples of KS and normal tissue were obtained (skin (10 pairs) and GI (12 pairs)) from 19 patients with KS of whom 17 had concurrent HIV infection. Seven paired samples were from patients who had received prior KS therapy. Three patients provided both skin and GI samples at the same timepoint. These analyses identified 370 differentially expressed genes unique to cutaneous KS and 58 DEGs unique to GI KS compared to normal skin or GI tissues. Twenty-six differentially expressed genes overlapped between skin and GI KS, which included FLT4, which encodes for a VEGF-C and VEGF-D receptor, and STC1. KSHV infection of primary lymphatic endothelial cells (LECs) resulted in increased angiogenesis, and repression of STC1 or FLT4 inhibited angiogenesis. The analyses of KSHV expression from KS lesions identified certain lytic genes, specifically ORF75, that were consistently expressed, and these expression patterns differed from laboratory infection of LECs with KSHV and KSHV gene expression in PEL cell lines. This study demonstrates that complex patterns of gene expression are found in KS tissue that differ from the canonical latent/lytic programs seen in KSHV cell lines and also demonstrates differences in viral gene and clinically relevant host gene expression in skin and GI KS that may offer insights into the pathogenesis of these forms of KS.

One sentence summaryKaposi sarcoma that manifests in the skin and gastrointestinal tracts differ by human and viral gene expression.


title: A role of Lck annular lipids in the steady upkeep of active Lck in T cells

abstract: Theoretical work suggests that collective spatiotemporal behaviour of integral membrane proteins (IMPs) can be modulated by annular lipids sheathing their hydrophobic moiety. Here, we present evidence for this prediction in a natural membrane by investigating the mechanism that maintains steady amount of active isoform of Lck kinase (LckA) by Lck trans-autophosphorylation offset by the phosphatase CD45. We gauged experimental suitability by quantitation of CD45 and LckA subcellular localisation, LckA generation as a function of Lck and pharmacological perturbation. Steady LckA was challenged by swapping Lck membrane anchor with structurally divergent ones expected to substantially modify Lck annular lipids, such as that of Src or the transmembrane domains of LAT, CD4, palmitoylation-defective CD4 and CD45, respectively. The data showed only small alteration of LckA, except for CD45 hydrophobic anchor that thwarted LckA, due to excessive lateral proximity to CD45. The data are best explained by annular lipids facilitating or penalising IMPs lateral proximity, hence modulating IMPs protein-protein functional interactions. Our findings can contribute to improve the understanding of biomembranes organisation.

category: cell biology


title: The motif composition of variable-number tandem repeats impacts gene expression

abstract: Understanding the impact of DNA variation on human traits is a fundamental question in human genetics. Variable number tandem repeats (VNTRs) make up roughly 3% of the human genome but are often excluded from association analysis due to poor read mappability or divergent repeat content. While methods exist to estimate VNTR length from short-read data, it is known that VNTRs vary in both length and repeat (motif) composition. Here, we use a repeat-pangenome graph (RPGG) constructed on 35 haplotype-resolved assemblies to detect variation in both VNTR length and repeat composition. We align population scale data from the Genotype-Tissue Expression (GTEx) Consortium to examine how variations in sequence composition may be linked to expression, including cases independent of overall VNTR length. We find that 20,834 VNTRs are associated with nearby gene expression through motif variations, of which only 5.1% associations are accessible from length. Fine-mapping identifies 273 genes to be likely driven by variation in certain VNTR motifs and not overall length. To demonstrate the utility of association using VNTR motifs, we examine the intronic VNTR of CACNA1C, which has been reported to be associated with schizophrenia risk through motif variation. We show that in healthy populations a previously identified schizophrenia risk motif is associated with decreased expression of CACNA1C, and detect an additional novel motif with similar effect.

category: genomics


title: Functional analysis of the N-terminal region of Vibrio FlhG, a MinD-type ATPase in flagellar number control

abstract: GTPase FlhF and ATPase FlhG are two key factors involved in regulating the flagellum number in Vibrio alginolyticus. FlhG is a paralog of the Escherichia coli cell division regulator MinD, which has a longer N-terminal region. Deleting the N-terminal region of FlhG results in cells with multiple flagella. The Q9A mutation in the DQAxxLR motif of the N-terminal region prevents it from activating the GTPase activity of FlhF in vitro and results in a multi-flagellation phenotype. The mutant FlhG protein was remarkably reduced compared to that of the wild-type protein in vivo. When the mutant FlhG was expressed at the same level as the wild-type FlhG, the number of flagella was restored to the wild-type level. Once synthesized in Vibrio cells, the N-terminal region mutation in FlhG seems not to affect the protein stability. We speculated that the flhG translation efficiency is decreased by N-terminal mutation. Our results suggest that the N-terminal region of FlhG controls the number of flagella by adjusting the FlhF activity and the amount of FlhG in vivo. We speculate that the regulation by FlhG, achieved through transcription by the master regulator FlaK, is affected by the mutations, resulting in reduced flagellar formation by FlhF.

category: biochemistry


title: Studies of a siderophore-producing cyclization domain: A refined proposal of substrate binding

abstract: Nonribosomal peptide synthetase (NRPS) heterocyclization (Cy) domains generate biologically important ox-/thiazoline modifications in natural products, including in production of compounds targeting disease or siderophores that are important for bacterial pathogenicity. Cy domains share the NRPS condensation domain fold but catalyze consecutive condensation and cyclodehydration reactions via an unknown mechanism. To further understanding of Cy domain catalysis, we report the crystal structure of the second Cy domain (Cy2) of yersiniabactin synthetase from the causative agent of the plague, Yersinia pestis. We find the high-resolution structure of Cy2 adopts a conformation enabling exploration of binding the extended, thiazoline-containing cyclodehydration intermediate for catalysis and the acceptor carrier protein to which it is tethered. We also report complementary electrostatic interfaces between Cy2 and its donor carrier protein that mediate donor binding. Lastly, we explore domain flexibility through the normal mode approximation and identify small-molecule fragment binding sites to inform antibiotic design targeting Cy function. Our results suggest how carrier protein binding may influence global conformation, with consequences for active site catalytic states and inhibitor development.


title: Harnessing the power of technical and natural variation in 116 yeast datasets to benchmark long read assembly pipelines

abstract: With increases in throughput and reductions in cost, long read sequencing has become the standard for most genome assembly projects and has opened up new avenues for large-scale genomic research. While more amenable to assembly than short-read sequence data, long-read datasets tend to have higher error rates. To address this problem numerous tools have been developed to correct reads before assembly and polish assembled contigs. Although, numerous studies have been conducted to assess or benchmark these tools, few capture the real variance in long read sequence data that might affect tool performance much less full pipeline performance. To address these shortcomings, we compiled a dataset containing long-read sequences of 116 different strains of brewers yeast, S. cerevisiae, gathered largely from public databases and evaluated different assembly-related tools as well as their interactions. We found that pre-assembly short-read error correction of long reads combined with post-assembly short-read polishing provided the best assemblies. We also found that correction/polishing steps with uncorrected long reads often lead to degradation of assembly quality. Finally, we show which tools and pipelines work best with different types of input data.


title: Ratiometric Sensing of Redox Environments Inside Individual Carboxysomes Trapped in Solution

abstract:
Diffusion of biological nanoparticles in solution impedes our ability to continuously monitor individuals and measure their physical and chemical properties. To overcome this, we previously developed the Interferometric Scattering Anti-Brownian ELectrokinetic (ISABEL) trap, which uses scattering to localize a particle and applies electrokinetic forces which counteract Brownian motion, thus enabling extended observation. Here, we present an improved ISABEL trap that incorporates a near-infrared scatter illumination beam and rapidly interleaves 405 and 488 nm fluorescence excitation reporter beams. With the ISABEL trap, we monitor the internal redox environment of individual carboxysomes labeled with the ratiometric redox reporter roGFP2. Carboxysomes widely vary in scattering contrast (reporting on size) and redox-dependent ratiometric fluorescence. Further, we used redox sensing to explore the chemical kinetics within intact carboxysomes, where bulk measurements may contain unwanted contributions from aggregates or interfering fluorescent proteins. Overall, we demonstrate the ISABEL traps ability to sensitively monitor nanoscale biological objects, enabling new experiments on these systems.

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category: biophysics


title: Dynamic control of metabolic zonation and liver repair by endothelial cell Wnt2 and Wnt9b revealed by single cell spatial transcriptomics using Molecular Cartography

abstract: The conclusive identity of Wnt proteins regulating liver zonation (LZ) and regeneration (LR) remains unclear despite an undisputed role of {beta}-catenin. Using single-cell analysis of liver cells from various species, a conserved Wnt2 and Wnt9b expression in endothelial cells (ECs) in zone 3 shown to be the major Wnt cell source, was identified. Conditional EC-elimination of Wnt2 and Wnt9b led to perturbation of LZ with not only loss of {beta}-catenin targets in zone 3, but also re-appearance of zone 1 genes in zone 3, unraveling dynamicity as revealed by single-cell spatial transcriptomics using Molecular Cartography. Defective LR observed in the knockouts phenocopied other models of defective hepatic Wnt signaling. Administration of a tetravalent antibody to activate Wnt signaling rescued LZ and LR in the knockouts. Molecular Cartography on the livers of the agonist-treated animal revealed changes in LZ. Administration of the agonist also promoted LR in acetaminophen overdose acute liver failure (ALF) fulfilling an unmet clinical need. Overall, we report an unequivocal role of EC-Wnt2 and Wnt9b in LZ and LR and show the role of Wnt activators as regenerative therapy for ALF.


title: If this title is funny, will you cite me? Citation impacts of humour and other features of article titles in ecology and evolution

abstract: Titles of scientific papers pay a key role in their discovery, and "good" titles engage and recruit readers. A particularly interesting aspect of title construction is the use of humour, but little is known about whether funny titles boost or limit readership and citation of papers. We used a panel of volunteer scorers to assess title humour for 2,439 papers in ecology and evolution, and measured associations between humour scores and subsequent citation (both self-citation and citation by others). Papers with funnier titles were cited less often, but this appears to result from a confound with paper importance. Self-citation data suggest that authors give funnier titles to papers they consider less important. After correction for this confound, papers with funny titles have significantly higher citation rates, suggesting that humour recruits readers. We also examined associations between citation rates and several other features of titles. Inclusion of acronyms and taxonomic names was associated with lower citation rates, while assertive-statement phrasing and presence of colons, question marks, and political regions were associated with somewhat higher citation rates. Title length had no effect on citation. Our results suggest that scientists can use creativity with titles without having their work condemned to obscurity.

category: scientific communication and education


title: Strategies to Identify and Edit Improvements in Synthetic Genome Segments Episomally.

abstract: Genome engineering projects often utilize bacterial artificial chromosomes (BACs) to carry multi-kilobase DNA segments on low copy number vectors. However, DNA synthesis and amplification have the potential to impose mutations on the contained DNA segments that can in turn reduce or prevent viability of the final strain. Here, we describe improvements to a multiplex automated genome engineering (MAGE) protocol to improve recombineering frequency and multiplexability. This protocol was applied to  recoding an Escherichia coli strain to swap out seven codons to synonymous alternatives genome-wide. Ten 44,402 to 47,179 bp de novo synthesized BAC-contained DNA segments from the recoded strain were unable to complement deletion of the corresponding 33 to 61 wild type genes using a single antibiotic resistance marker. Next-Generation Sequencing was used to identify 1-7 non-recoding mutations in essential genes per segment, and MAGE in turn proved a useful strategy to repair these mutations on the BAC-contained recoded segment when both the recoded and wild type copies of the mutated genes had to exist by necessity during the repair process. This strategy could be adapted to mutation identification and repair for other large-scale genome engineering projects, or for incorporation of small genetic engineering sites for quick protocol adjustments.

category: synthetic biology


title: Alpha-1 Antitrypsin Limits Neutrophil Extracellular Trap Disruption of Airway Epithelial Barrier Function

abstract:
Neutrophil extracellular traps (NETs) contribute to lung injury in cystic fibrosis and asthma, but the mechanisms are poorly understood. We sought to understand the impact of NETs on barrier function in human airway epithelia. To test this, we exposed primary human bronchial epithelial cells (HBE) and a human airway epithelial cell line (wtCFBE41o-) to isolated human NETs in their apical compartment for 2-18 hours. We demonstrate that NETs disrupt airway epithelial barrier function by decreasing transepithelial electrical resistance (TEER) and increasing paracellular flux. This is in part due to NET-induced airway epithelial cell death via apoptosis. NETs selectively impact the expression of the tight junction genes claudins 4, 8 and 11. Intact epithelia exposed to NETs demonstrate visible gaps in E-cadherin staining, a decrease in full-length E-cadherin protein and the appearance of cleaved E-cadherin peptides. DNase did not alter NET-driven changes in HBE TEER or E-cadherin. Pretreatment of NETs with alpha-1 antitrypsin (A1AT) inhibits NET serine protease activity, limits E-cadherin cleavage, and preserves epithelial integrity. In conclusion, NETs disrupt human airway epithelial barrier function through bronchial cell death and degradation of E-cadherin, the latter of which limited by exogenous A1AT.

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title: Dependence on a variable residue limits the breadth of an HIV MPER neutralizing antibody, despite convergent evolution with broadly neutralizing antibodies

abstract:
Broadly neutralizing antibodies (bNAbs) that target the membrane-proximal external region (MPER) of HIV gp41 envelope, such as 4E10 and VRC42.01, can neutralize >95% of viruses. These two MPER-directed monoclonal antibodies share germline antibody genes (IGHV1-69 and IGKV3-20) and form a bNAb epitope class. Furthermore, convergent evolution within these two lineages towards a 111.2GW111.3 motif in the CDRH3 is known to enhance neutralization potency. We have previously isolated an MPER neutralizing antibody, CAP206-CH12, that uses these same germline heavy and light chain genes but lacks breadth (neutralizing only 6% of heterologous viruses). Longitudinal sequencing of the CAP206-CH12 lineage over three years revealed similar convergent evolution towards 111.2GW111.3 among some lineage members. Mutagenesis of CAP206-CH12 from 111.2GL111.3 to 111.2GW111.3 modestly improved neutralization breadth (by 9%) and potency (3-fold), but did not reach the levels of breadth and potency of VRC42.01 and 4E10. To explore the lack of potency/breadth, viral mutagenesis was performed to map the CAP206-CH12 epitope. This indicated that CAP206-CH12 is dependent on D674, a highly variable residue at the solvent-exposed elbow of MPER. In contrast, VRC42.01 and 4E10 were dependent on highly conserved residues (W672, F673, T676, and W680) facing the hydrophobic patch of the MPER. Therefore, while CAP206-CH12, VRC42.01, and 4E10 share germline genes and show some evidence of convergent evolution, their dependence on different amino acids, which impacts orientation of binding to the MPER, result in differences in breadth and potency. These data have implications for the design of HIV vaccines directed at the MPER epitope.

Author SummaryGermline-targeting immunogens are a promising HIV vaccine design strategy. This approach is reliant on the identification of broadly neutralizing antibody (bNAb) classes, which use the same germline antibody genes to target the same viral epitopes. Here, we compare three HIV Envelope MPER-directed antibodies (4E10, VRC42.01 and CAP206-CH12) that despite having shared antibody genes, show distinct neutralization profiles. We show that CAP206-CH12 targets a single highly variable residue in the MPER, which results in low neutralization breadth. In contrast, the 4E10 and VRC42.01 mAbs are dependent on highly conserved residues in the MPER, resulting in exceptional neutralization breadth. Our data suggest that while shared germline genes within bNAb epitope classes are required, in some cases these are not sufficient to produce neutralization breadth, and MPER immunogens will need to trigger responses to conserved sites.


title: Meta-analysis of the autism gut microbiome identifies factors influencing study discrepancies and machine learning classification

abstract: Autism Spectrum Disorder (ASD) is a severe neurodevelopmental disorder and accumulating evidence has suggested that dysbiosis of the gut microbiome plays an essential role. However, a body of research has investigated the ASD gut microbiome without consensus as to whether or how the ASD microbiome differs from neurotypical children. Here, we evaluate the underlying factors leading to study discrepancies by performing a meta-analysis on reprocessed 16S ribosomal RNA gene amplicon (16S) sequencing data. We compile a total of 1,740 samples across 13 carefully selected published studies together with samples from the American Gut Project, and analyze the data in aggregate and from a per-study perspective. We observed increased Bifidobacterium, Actinobacteria, and Prevotella among ASD individuals across cohorts. We further identified associations to Desulfovibrionales, Deltaproteobacteria and Prevotella that were dependent upon which 16S variable regions were sequenced. Utilizing machine learning (ML), we obtained increased accuracy in ASD classification using data collected from certain territories, on younger subjects, on unrelated age-matched rather than related controls, on samples with increased sequencing depth and when accounting for sex differences. Our work provides compelling evidence that the gut microbiome is altered in ASD patients, and highlights novel factors that are important considerations for future studies.


title: Activation of the chemokine receptor CCR1 and preferential recruitment of Gαi suppress RSV replication: implications for developing novel RSV treatment strategies

abstract: Respiratory syncytial virus (RSV) is a major pathogen that can cause acute respiratory infectious diseases of the upper and lower respiratory tract, especially in children, elderly individuals and immunocompromised people. Generally, following viral infection, respiratory epithelial cells secrete cytokines and chemokines to recruit immune cells and initiate innate and/or adaptive immune responses. However, whether chemokines affect viral replication in non-immune cells is rarely clear. In this study, we detect that chemokine CCL5 was highly expressed, while expression of its receptor, CCR1, was downregulated in respiratory epithelial cells after RSV infection. When we overexpressed CCR1 on respiratory epithelial cells in vivo/in vitro, viral load was significantly suppressed, which can be restored by the neutralizing antibody for CCR1. Interestingly, the antiviral effect of CCR1 was not related to type I interferon (IFN-I), apoptosis induction or viral adhesion or entry inhibition; in contrast, it was related to the preferential recruitment and activation of adaptor Gi, which promoted inositol 1,4,5-triphosphate receptor type 3 (ITPR3) expression, leading to inhibited STAT3 phosphorylation, explicitly, phosphorylated (p)-STAT3 was verified to be among the important factors regulating the activity of HSP90, which has been previously reported to be a chaperone of RSV RNA polymerase. In summary, we are the first to reveal that CCR1 on the surface of non-immune cells regulates RSV replication through a previously unknown mechanism that does not involve IFN-I induction.


title: A South American mouse morbillivirus sheds light into a clade of rodent-borne morbilliviruses

abstract: Morbilliviruses are negative sense single stranded monosegmented RNA viruses in the family Paramyxoviridae (order Mononegavirales). Morbilliviruses infect diverse mammals including humans, dogs, cats, small ruminants, seals, and cetaceans, which serve as natural hosts. Here, I report the identification and characterization of novel viruses associated with South American long-haired and olive field mice. The divergent viruses dubbed Raton olivaceo morbillivirus (RoMV) detected in renal samples from mice collected from Chile and Argentina are characterized by an unusually large genome including long intergenic regions and the presence of an accessory protein between the F and H genes redounding in a genome architecture consisting in 3-N-P/V/C-M-F-hp-H-L-5. Structural and functional annotation, genetic distance and evolutionary insights suggest that RoMV is a member of a novel species within genus Morbillivirus tentatively dubbed South American mouse morbillivirus. Phylogenetic analysis suggests that this mouse morbillivirus is closely related to, and clusters into a monophyletic group of novel rodent-borne morbilliviruses. This subclade of divergent viruses expands the host range, redefines the genomic organization and provides insights on the evolutionary history of genus Morbillivirus.


title: The phospholipase VlsPLA2 from the plant pathogen Verticillium longisporum is a virulence factor targeting host nuclei and suppressing PTI-related hypersensitive response

abstract: Verticillium longisporum is an amphidiploid fungal species infecting plants mainly in Brassicaceae family. It is one of the most important pathogens in rapeseed (Brassica napus) cultivation, causing severe annual yield losses worldwide. Pathogens secrete a plethora of small proteins, termed effectors, to evade plant immunity and to establish a successful infection. Genome analysis of the V. longisporum stain VL1, isolated from Swedish infested soils where rapeseed has intensively been cultivated, showed that it contains more than 80 candidate effector genes. Among them, the VlsPLA2 gene was highly induced during B. napus infection. Protein expression in E. coli cells, showed that it is an active A2 phospholipase. Further characterization showed that it has a catalytic dyad HD crucial for its enzymatic function, a signal peptide at the N-terminus, and two active NLS, one monopartite and one bipartite, with the latter one to be more crucial for its nuclear localization. Transient expression in Nicotiana benthamiana plants showed it to be localized to host nuclei 48hpi, while it is translocated to chloroplasts 72hpi. However, the enzymatically inactive VlsPLA2 protein did not enter the nucleus or localized to chloroplasts even 48hpi. Our data from B. napus plants showed that V. longisporum mutants overexpressing VlsPLA2 were more virulent as compared to strain that expressed an enzymatically inactive one. Finally, this effector is a negative regulator of hypersensitive response (HR) triggered by the Cf4/Avr4 complex, but not by Pseudomonas syringae pv. tomato DC3000 (PsPto). Also, it was able to induce genes that encode pathogen related (PR) proteins in this host. In conclusion, these data show that VlsPLA2 is a virulence factor targeting host nuclei and suppresses Pattern Triggered immunity (PTI)-related HR, possibly through interference in signal transduction pathways, altering gene expression.


title: Molecular determinants of the Bacillus subtilis chromosome origin basal unwinding system

abstract: Genome duplication is essential for cell proliferation and DNA synthesis is generally initiated by dedicated replication proteins at specific loci termed origins. During DNA replication initiation in bacteria, the ubiquitous DnaA protein engages both double-strand DNA (dsDNA) and single-stranded DNA (ssDNA) at the chromosome origin (oriC) to promote DNA duplex unwinding. While the molecular basis for DnaA binding to a specific dsDNA element ("DnaA-box") has been established, the mechanism for DnaA binding to a specific ssDNA motif ("DnaA-trio") is unclear. Here we define specific steps of DnaA-trio engagement by Bacillus subtilis DnaA. Single-molecule total internal reflection fluorescence microscopy indicates that DnaA proteins are loaded onto DnaA-trios using DnaA-boxes located on a shared DNA polymer. Chemical modification of either the phosphodiester backbone or the nucleobases revealed that three DnaA-trio repeats proximal to DnaA-boxes are necessary and sufficient to promote DnaA-dependent strand separation, and that the amino group from the central nucleobase of the DnaA-trio is critical for this reaction. Finally, based on electrophoretic mobility shift assays, we propose that during replication initiation DnaA progresses from DnaA-boxes to nucleobase recognition at DnaA-trios before engaging the phosphodiester backbone and destabilizing the DNA duplex. These results provide new molecular insight into DnaA-dependent Bacterial Unwinding System (BUS) activity at a bacterial chromosome origin.


title: The genome wide analysis deciphered virulence factors and secondary metabolites of Chryseobacterium and description of Chryseobacterium indicum sp. nov.

abstract: This study describes a novel bacterium, Chryseobacterium strain PS-8, isolated from the skin of freshwater pufferfish (Tetraodon cutcutia). Cells are Gram-negative. The genome size is 4.72 Mb, and the G+C content is 36.4%. The in silico DDH homology, ANI, and AAI were below the cutoff value, 70% and 95% to 96%, respectively, as required for novel species delineation. The phylogenomic analysis using core and non-recombinant core genes, the strain PS-8 showed clustering with Chryseobacterium gambrini DSM 18014T. Generally, Chryseobacterium species are considered opportunistic pathogens. Prediction of the virulence genes revealed genes for adherence, biofilm and stability (glf, lpxD, clpE, cps0, clpE, IlpA), proliferation (tufA, cap8E, galE, kfic, bioB, clpP, kdtB, csgD, carB), resistance to immune response (htpB, katA, wbtl, sodB, kpsF), and host-defense evasion system (kpst, clpE, kfic, ybtQ, cap8G, clpP). The cladogram based upon the virulence genes showed a similar phylogeny amongst the Chryseobacterium species. Additionally, secondary metabolites producing gene clusters were identified, including microviridin, resorcinol and polyene, terpene, etc. Our study showed that strain PS-8 constitute a novel species for which Chryseobacterium indicum sp. nov. is proposed. The type strain is PS-8T (= TBRC 15233T = NBRC 115235T).


title: SUMO monoclonal antibodies vary in sensitivity, specificity, and ability to detect SUMO conjugate types.

abstract: Monoclonal antibodies (MAb) to members of the Small Ubiquitin-like modifier (SUMO) family are essential tools in the study of cellular SUMOylation. However, many reagents are poorly validated, and the choice of which antibody to use for which detection format is without an evidence base. Here we test twenty-four anti-SUMO monoclonal antibodies for sensitivity and specificity to SUMO1-4 in monomeric and polymeric states in dot-blots, immunoblots, immunofluorescence and immunoprecipitation. We find substantial variability between SUMO MAbs for different conjugation states, detection of increased SUMOylation in response to thirteen different stress agents and as enrichment reagents for analysis of SUMOylated RanGAP1 or KAP1. All four anti-SUMO4 monoclonal antibodies tested cross-reacted with SUMO2/3, and several SUMO2/3 monoclonal antibodies cross-reacted with SUMO4. ~10% of tested monoclonal antibodies produced specific results across multiple applications.

category: molecular biology


title: Coenzyme Q regulates UCP1 expression and thermogenesis through the integrated stress responses

abstract: Coenzyme Q (CoQ) is an essential component of mitochondrial respiration1 and required for thermogenic activity in brown adipose tissues2 (BAT). CoQ deficiency leads to a wide range of pathological manifestations3 but mechanistic consequences of CoQ deficiency in specific tissues such as BAT remain poorly understood. Here we show that pharmacological or genetic CoQ deficiency (50-75% reduction) in BAT leads to accumulation of cytosolic mitochondrial RNAs (mtRNAs) and activation of the eIF2 kinase PKR resulting in the induction of the integrated stress response (ISR) and suppression of UCP1 expression in an ATF4-dependent fashion. Surprisingly, despite diminished UCP1 levels, BAT CoQ deficiency increases whole-body metabolic rates at room temperature and thermoneutrality resulting in decreased weight gain on high fat diets (HFD). This mitohormesis like effect of BAT CoQ insufficiency is dependent on the ATF4-FGF21 axis in BAT revealing an unexpected role for CoQ in the modulation of whole-body energy expenditure with wide-ranging implications for primary and secondary CoQ deficiencies.


title: Modeling the Evolution of Rates of Continuous Trait Evolution

abstract: AO_SCPLOWBSTRACTC_SCPLOWRates of phenotypic evolution vary markedly across the tree of life, from the accelerated evolution apparent in adaptive radiations to the remarkable evolutionary stasis exhibited by so-called "living fossils". Such rate variation has important consequences for large-scale evolutionary dynamics, generating vast disparities in phenotypic diversity across space, time, and taxa. Despite this, most methods for estimating trait evolution rates assume rates vary deterministically with respect to some variable of interest or change infrequently during a clades history. These assumptions may cause underfitting of trait evolution models and mislead hypothesis testing. Here, we develop a new trait evolution model that allows rates to vary gradually and stochastically across a clade. Further, we extend this model to accommodate generally decreasing or increasing rates over time, allowing for flexible modeling of "early/late bursts" of trait evolution. We implement a Bayesian method, termed "evolving rates" (evorates for short), to efficiently fit this model to comparative data. Through simulation, we demonstrate that evorates can reliably infer both how and in which lineages trait evolution rates varied during a clades history. We apply this method to body size evolution in cetaceans, recovering substantial support for an overall slowdown in body size evolution over time with recent bursts among some oceanic dolphins and relative stasis among beaked whales of the genus Mesoplodon. These results unify and expand on previous research, demonstrating the empirical utility of evorates.

category: evolutionary biology


title: Increased male investment in sperm competition results in offspring of lower quality

abstract: Male animals often show higher mutation rates than their female conspecifics. A hypothesis for this male-bias is that competition over fertilization of female gametes leads to sexual selection for increased male germline replication at the expense of maintenance and repair, resulting in a trade-off between male success in sperm competition and offspring quality. Here we test this hypothesis using experimental evolution lines of the seed beetle Callosobruchus maculatus, maintained for >50 generations under three alternative mating regimes: natural and sexual selection (N+S-lines), natural selection only (N-lines) or sexual selection only (S-lines). Previous findings suggest that S-males reduce germline maintenance when engaging in reproduction compared to N- and N+S-males. Here, we first show that S-males are superior in sperm competition compared to both N- and N+S-males, suggesting that the removal of trade-offs between naturally and sexually selected male fitness components has resulted in the evolution of increased post-copulatory reproductive success. We then show that S-males produce progeny of lower quality if engaging in socio-sexual interactions with conspecifics prior to being challenged with a dose of irradiation introducing DNA-damage in their germline. We identify 18 candidate genes that showed differential expression in response to the induced germline damage. These genes also showed significant expression changes across socio-sexual treatments of fathers and predicted the reduction in quality of their offspring. Moreover, sex differences in expression of the same 18 genes indicate a substantially higher female investment in germline maintenance. Our findings provide evidence for a trade-off between male success in sperm competition and germline maintenance, suggesting that sex-differences in the relative strengths of sexual and natural selection are causally linked to male-mutation bias.


title: The genetic and biochemical determinants of mRNA degradation rates in mammals

abstract:
BackgroundDegradation rate is a fundamental aspect of mRNA metabolism, and the factors governing it remain poorly characterized. Understanding the genetic and biochemical determinants of mRNA half-life would enable a more precise identification of variants that perturb gene expression through post-transcriptional gene regulatory mechanisms.

ResultsHere, we establish a compendium of 54 human and 27 mouse transcriptome-wide mRNA decay rate datasets. A meta-analysis of these data identified a prevalence of technical noise and measurement bias, induced partially by the underlying experimental strategy. Correcting for these biases allowed us to derive more precise, consensus measurements of half-life which exhibit enhanced consistency between species. We trained substantially improved statistical models based upon genetic and biochemical features to better predict half-life and characterize the factors molding it. Our state-of-the-art model, Saluki, is a hybrid convolutional and recurrent deep neural network which relies only upon an mRNA sequence annotated with coding frame and splice sites to predict half-life (r=0.77). Saluki predicts the impact of RNA sequences and genetic mutations therein on mRNA stability, in agreement with functional measurements derived from massively parallel reporter assays.

ConclusionsOur work produces a more robust "ground truth" with regards to transcriptome-wide mRNA half-lives in mammalian cells. Using these consolidated measurements, we trained a model that is over 50% more accurate in predicting half-life from sequence than existing models. Our best model, Saluki, succinctly captures many of the known determinants of mRNA half-life and can be rapidly deployed to predict the functional consequences of arbitrary mutations in the transcriptome.


title: Programmed-DNA Elimination in the free-living nematodes Mesorhabditis

abstract:
In most species, elaborate programs exist to protect chromatin and maintain its integrity over cell cycles and generations. However some species systematically undergo excision and elimination of portions of their genome in somatic cells in a process called programmed-DNA elimination (PDE). PDE involves the elimination of mainly repeated elements but also protein-coding genes. PDE has been described in approximately 100 species from very distinct phyla, and more extensively in the parasitic nematodes Ascaris and in the unicellular Ciliates. In Ciliates, where PDE is pervasive, the underlying mechanisms have been studied and involve small RNA-guided heterochromatinization. In striking contrast, chromatin recognition and excision mechanisms remain mysterious in Metazoans, because the study species are not amenable to functional approaches. Above all, the function of such a mechanism, which has emerged repeatedly throughout evolution, is unknown. Answering these questions will provide significant insights into our understanding of chromatin regulation and genome stability.

We fortuitously discovered the phenomenon of PDE in all species of the free-living nematode genus Mesorhabditis. Mesorhabditis, which belong to the same family as C. elegans, have a small [~]150 Mb genome and offer many experimental advantages to start elucidating the elimination mechanisms in Metazoans. In this first study, we have used a combination of cytological observation and genomic approaches to describe PDE in Mesorhabditis. We found that the dynamics of chromosome fragmentation and loss are very similar to those described in Ascaris. Elimination occurs once in development, at the third embryonic cell division in all 5 presomatic blastomeres. Similar to other species, Mesorhabditis eliminate repeated elements but also about a hundred unique sequences. Most of the eliminated unique sequences are either pseudogenes or poorly conserved protein-coding genes. Our results raise the possibility that PDE has not been selected for a gene regulatory function in Mesorhabditis but rather mainly is a mechanism to irreversibly silence repeated elements in the soma.


title: One-Pot Exosome Proteomics Enabled by a Photocleavable Surfactant

abstract: Exosomes are small extracellular vesicles (EVs) secreted by all cells and found in biological fluids, which can serve as minimally invasive liquid biopsies with high therapeutic and diagnostic potential. Mass spectrometry (MS)-based proteomics is a powerful technique to profile and quantify the protein content of exosomes but the current methods require laborious and time-consuming multi-step sample preparation that significantly limit throughput. Herein, we report a one-pot exosome proteomics method enabled by a photocleavable surfactant, Azo, for rapid and effective exosomal lysis, protein extraction, and digestion. We have applied this method to exosomes derived from isolated mammary fibroblasts and confidently identified 3,466 proteins and quantified 2,288 proteins using reversed-phase liquid chromatography coupled to trapped ion mobility spectrometry (TIMS) quadrupole time-of-flight mass spectrometer. 3,166 (91%) of the identified proteins are annotated in the exosome/EVs databases, ExoCarta and Vesiclepedia, including important exosomal markers, CD63, PDCD6IP, and SDCBP. This method is fast, simple, and highly effective at extracting exosomal proteins with high reproducibility for deep exosomal proteome coverage. We envision this method could be generally applicable for exosome proteomics applications in biomedical research, therapeutic interventions, and clinical diagnostics.


title: Wochenende - modular and flexible alignment-based shotgun metagenome analysis

abstract: Shotgun metagenome analysis provides a robust and verifiable method for comprehensive microbiome analysis of fungal, viral, archaeal and bacterial taxonomy, particularly with regard to visualization of read mapping location, normalization options, growth dynamics and functional gene repertoires. Current read classification tools use non-standard output formats, or do not fully show information on mapping location. As reference datasets are not perfect, portrayal of mapping information is critical for judging results effectively. Our alignment-based pipeline, Wochenende, incorporates flexible quality control, trimming, mapping, various filters and normalization. We observe stringent filtering of mismatches and use of mapping quality sharply reduces the number of false positives. Further modules allow genomic visualization, as well as integration and subsequent plotting of pipeline results. Our novel normalization approach additionally allows calculation of absolute abundance profiles by comparison with reads assigned to the human host genome. Wochenende has the ability to find and filter alignments to all kingdoms of life using both short and long reads, and requires only good quality reference genomes. Wochenende is available at https://github.com/MHH-RCUG/Wochenende.


title: Universal dynamics of biological pattern formation in spatio-temporal morphogen variations

abstract: In biological pattern-forming systems, generic features have been previously identified that promote robustness with respect to variation between systems in components such as protein production rates and gene-regulatory network architecture. By contrast, less is understood about how individual systems respond to spatio-temporal changes in morphogen concentrations over timescales faster than or similar to cell or tissue growth. Here, we formulate and apply a general theory of biological pattern formation in spatio-temporally varying concentrations of "pre-pattern" morphogens, which determine system parameters. Our analytical results reveal universal dynamical regimes of biological pattern-forming systems, and quantify how the dynamics in each regime depend on the mathematical properties of the relevant gene-regulatory network. By applying our theory, we predict that two paradigmatic pattern-forming systems are robust with respect to spatio-temporal morphogen variations that happen faster than growth timescales. More broadly, we predict that the dynamics of pattern-forming systems with spatio-temporally varying parameters can be classified based on the bifurcations in their governing equations.


title: AlphaFold encodes the principles to identify high affinity peptide binders

abstract: Machine learning has revolutionized structural biology by solving the problem of predicting structures from sequence information. The community is pushing the limits of interpretability and application of these algorithms beyond their original objective. Already, AlphaFolds ability to predict bound conformations for complexes has surpassed the performance of docking methods, especially for protein-peptide binding. A key question is the ability of these methods to differentiate binding affinities between several peptides that bind the same receptor. We show a novel application of AlphaFold for competitive binding of different peptides to the same receptor. For systems in which the individual structures of the peptides are well predicted, predictions in which both peptides are introduced capture the stronger binder in the bound state, and the other peptide in the unbound form. The speed and robustness of the method will be a game changer to screen large libraries of peptide sequences to prioritize for detailed experimental characterization.


title: Vimentin intermediate filament assembly is a reversible process

abstract:
Networks of intermediate filaments (IF) need to constantly reorganize to fulfill their functions at different locations within the cell. IF assembly results from end-to-end annealing, which is commonly assumed to be irreversible. By contrast, the mechanisms involved in IF disassembly are far less understood. IF fragmentation has however been observed in many cell types, and it has been suggested that it could be associated with post-translational modifications. In this article, we investigate the contribution of filament fragmentation in the assembly dynamics of type III vimentin IF using a combination of in vitro reconstitution, fluorescence imaging, and theoretical modeling. We first show that vimentin assembly at low concentrations results in an equilibrium between filament annealing and fragmentation at time [≥] 24 h. At higher concentration, entanglements kinetically trap the system out of equilibrium, and we show that this trapping is reversible upon dilution. Taking into account both fragmentation and entanglement, we estimated that the mean bond breaking time was [~]18 hours Finally, we provide direct evidence through dual color imaging that filament fragmentation and annealing coexist during assembly. By showing that IF fragmentation can occur without cofactors or post-translational-modifications, our study provides a physical understanding of the IF length regulation.

Significance StatementVimentin intermediate filaments are a key component of the cytoskeleton and are involved in many cellular functions, such as the regulation of cell shape, migration and division. These functions require cytoskeletal filaments to simultaneously assemble and disassemble throughout the life of the cell. While the mechanisms of intermediate filament assembly have been widely studied, the minimal ingredients underpinning their disassembly are not understood. Here, we demonstrate that vimentin constantly disassembles through filament breakage without the assistance of any other protein or post-translational modification, contrary to common wisdom. Our findings suggest that the dynamic cytoskeletal steady-states observed in cells could be largely shaped by simple physical effects linked to the reversible association of vimentin subunits, combined with dramatic kinetic trapping effects that hinder network reorganization as soon as the filaments become too dense and too long.


title: Proteomic identification of the interactome of stalled ribosome nascent chain complexes translating the thylakoid membrane protein D1

abstract: The synthesis of multi-span thylakoid membrane proteins initiates at ribosomes off the membrane. Subsequently, the ribosome nascent chain complexes (RNCs) are transferred to the translocase machinery in the thylakoid membrane for cotranslational protein insertion. These steps require finely tuned mechanisms for protein processing, quality control, and targeting to prevent misfolding or aggregation and to ensure efficient transfer of the nascent chain to the insertion machinery. However, little is known about the regulatory network underlying these processes. To identify factors specifically involved in the cotranslational biogenesis of the reaction center protein D1 of photosystem II we established a chloroplast-derived in vitro translation method that allows the production and affinity purification of stalled RNCs bearing nascent chains of D1 of different defined lengths. Stalled RNCs translating the soluble ribosomal subunit uS2c were affinity-purified for comparison. Quantitative tandem-mass spectrometry revealed a set of about 120 proteins specifically associated with D1 RNCs. The interactome includes proteins with broad functions in protein processing, biogenesis and metabolic pathways, such as chlorophyll biosynthesis. We identified STIC2 as a new factor specifically associated with D1 RNCs. Furthermore, our results demonstrated that the interaction of STIC2 with the thylakoid insertase Alb3 and its homologue Alb4 is mediated by the conserved motif III within the C-terminal regions of Alb3 and Alb4. Our data suggest that STIC2 is involved in cotranslational substrate delivery at the thylakoid membrane by coordinating the binding of the D1 RNCs to the insertase machinery.

category: plant biology


title: Configurable Compartmentation Enables In vitro Reconstitution of Sustained Synthetic Biology Systems

abstract: The compartmentalized and communicative nature of biological cells contributes to the complexity and endurance of living organisms. We present a general compartmentalization strategy for in vitro systems that inherits the passive transport phenomenon of biology. The strategy incorporates layered, micrometer-sized, hydrogel-based compartments featuring configurability in composition, functionality and selective permeability of biomolecules. We demonstrated the utility of the strategy by reconstituting a compartmentalized in vitro protein synthesis system which supports multiple rounds of reactions and compartmentalized living bacteria-based biosensors which sustain long-lasting functioning with markedly enhanced fitness in complex environments. The strategy should be widely applicable for constructing complex, robust and sustained in vitro synthetic molecular and cellular systems, paving the way for their practical applications.


title: Characterizing dry mass and volume changes in human multiple myeloma cells upon treatment with proteotoxic and genotoxic drugs

abstract: Multiple myeloma (MM) is a cancer of terminally differentiated plasma cells. MM remains incurable, but overall survival of patients has progressively increased over the past two decades largely due to novel agents such as proteasome inhibitors (PI) and the immunomodulatory agents. While these therapies are highly effective, MM patients can be de novo resistant and acquired resistance with prolonged treatment is inevitable. There is growing interest in early, accurate identification of responsive versus non-responsive patients, however limited sample availability and need for rapid assays are limiting factors. Here, we test dry mass and volume as label-free biomarkers to monitor early response of MM cells to treatment with bortezomib, doxorubicin, and ultraviolet light. For the dry mass measurement, we use two types of phase-sensitive optical microscopy techniques: digital holographic tomography and computationally-enhanced quantitative phase microscopy. We show that human MM cell lines (RPMI8226, MM.1S, KMS20, and AMO1) increase dry mass upon bortezomib treatment. This dry mass increase after bortezomib treatment occurs as early as 1 hour for sensitive cells and 4 hours for all tested cells. We further confirm this observation using primary multiple myeloma cells derived from patients and show that a correlation exists between increase in dry mass and sensitivity to bortezomib, supporting the use of dry mass as a biomarker. The volume measurement using Coulter counter shows a more complex behavior; RPMI8226 cells increase the volume at an early stage of apoptosis, but MM.1S cells show the volume decrease typically observed with apoptotic cells. Altogether, this cell study presents complex kinetics of dry mass and volume at an early stage of apoptosis, which may serve as a basis for the detection and treatment of MM cells.


title: A microfluidic-based model of nociceptor sensitization reveals a direct activation of sensory axons by prostaglandin E2

abstract:
Prostaglandin E2 (PGE2) is one of the major contributors to inflammatory pain hyperalgesia, however, the extent to which it modulates the activity of the nociceptive axons is incompletely understood. We used a microfluidic cell culture platform to investigate the changes in responsiveness of sensory axons following treatment with PGE2. We show that the application of PGE2 to fluidically isolated axons leads to sensitization of their responses to depolarising stimuli, and the inclusion of zatebradine, a blocker of HCN channels, blocks this enhancement. However, unexpectedly, we also found that the application of PGE2 to the axons elicited a direct and persistent spiking activity in the sensory neurons. We demonstrate that this persistent activity is due to a direct depolarization of axons by PGE2, which is inhibited by Nav1.8 sodium channel blockers but is mainly refractory to Nav1.7 channel blockade. Both the persistent activity and the membrane depolarization in the axons are abolished by the EP4 receptor inhibitor and a blocker of cAMP synthesis. Our data indicate that PGE2/EP4/cAMP pathway culminates in a sustained depolarization in the sensory axons, leading to the generation of action potentials propagating to the soma. PGE2 therefore, not only mediates nociceptor sensitization but can directly elicit discharges in nociceptive axons, hence redefining its role as a pain mediator in inflammatory conditions.

One Sentence SummaryProstaglandin E2 can depolarise nociceptive axons in the absence of any noxious stimuli leading to a sustained activation of pain sensing neurons.


title: Microglial amyloid beta clearance is driven by PIEZO1 channels

abstract:
BackgroundMicroglia are the endogenous immune cells of the brain and act as sensors of pathology to maintain brain homeostasis and eliminate potential threats. In Alzheimers disease (AD), toxic amyloid beta (A{beta}) accumulates in the brain and forms stiff plaques. In late-onset AD accounting for 95% of all cases, this is thought to be due to reduced clearance of A{beta}. Human genome-wide association studies and animal models suggest that reduced clearance results from aberrant function of microglia. While the impact of neurochemical pathways on microglia have been broadly studied, mechanical receptors regulating microglial functions remain largely unexplored.

MethodsHere we showed that a mechanotransduction ion channel, PIEZO1, is expressed and functional in human and mouse microglia. We used a small molecule agonist, Yoda1, to study how activation of PIEZO1 affects AD-related functions in human induced pluripotent stem cell (iPSC) -derived microglia-like cells (iMGL) under controlled laboratory experiments. Cell survival, metabolism, phagocytosis and lysosomal activity were assessed using real-time functional assays. To evaluate the effect of activation of PIEZO1 in vivo, 5-month-old 5xFAD male mice were infused daily with Yoda1 for two weeks through intracranial cannulas. Microglial Iba1 expression and A{beta} pathology were quantified with immunohistochemistry and confocal microscopy. Published human and mouse AD datasets were used for in-depth analysis of PIEZO1 gene expression and related pathways in microglial subpopulations.

ResultsWe show that PIEZO1 orchestrates A{beta} clearance by enhancing microglial survival, phagocytosis, and lysosomal activity. A{beta} inhibited PIEZO1-mediated calcium transients, whereas activation of PIEZO1 with a selective agonist, Yoda1, improved microglial phagocytosis resulting in A{beta} clearance both in human and mouse models of AD. Moreover, PIEZO1 expression was associated with a unique microglial transcriptional phenotype in AD as indicated by assessment of cellular metabolism, and human and mouse single cell datasets.

ConclusionThese results indicate that the compromised function of microglia in AD could be improved by controlled activation of PIEZO1 channels resulting in alleviated A{beta} burden. Pharmacological regulation of these mechanoreceptors in microglia could represent a novel therapeutic paradigm for AD.

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title: Low-Toxin Clostridioides difficile RT027 Strains Exhibit Robust Virulence

abstract: Clostridioides difficile is a leading cause of healthcare-associated infections worldwide. Currently, there is lack of consensus on a single optimal diagnostic method for C. difficile infection (CDI). Nucleic acid amplification tests (NAAT) that detect toxin genes are highly sensitive, but their specificity limitations could inflate CDI rates. Alternate multi-step diagnostic algorithms emphasize the detection of C. difficile toxins TcdA/TcdB, and are premised on the rationale that stool toxin-negative (Tox-) CDI patients have less severe disease, shorter diarrhea duration, and fewer disease complications. There have been no systematic assessments, however, of the virulence of C. difficile strains from Tox-/NAAT+ (discrepant) specimens. In our prospective analysis of 1243 C. difficile-positive patient stool specimens from three Southern Arizona hospitals, 31% were discrepant. Ribotype 027 (RT027) strains were recovered from 221 specimens; of these, 23% were discrepant. Post-culture, RT027 strains produced a range of toxin amounts including levels lower than that of the non-epidemic strain CD630. These low-toxin RT027 (LT-027) strains harbored both tcdA and tcdB genes, and their culture supernatants were cytotoxic to cultured fibroblasts. We confirmed robust colonization and virulence of a subset of LT-027 strains using multiple rodent models; lethality in animals infected with LT-027 strains was comparable to that potentiated by a high-toxin RT027 strain. Comparative genomics and proteomics analyses of several LT-027 strains identified unique genes and altered protein abundances relative to closely-related high-toxin strains. Collectively our data highlight the robust virulence and clinical relevance of low-toxin-producing, RT027 C. difficile isolates.


title: A Biological Signature for the Inhibition of Outer Membrane Lipoprotein Biogenesis

abstract:
The outer membrane (OM) of Gram-negative bacteria is an essential organelle that acts as a formidable barrier to antibiotics. Increasingly prevalent resistance to existing drugs has exacerbated the need for antibiotic discovery efforts targeting the OM. Acylated proteins, known as lipoproteins, are essential in every pathway needed to build the OM. The central role of OM lipoproteins makes their biogenesis a uniquely attractive therapeutic target, but it also complicates in vivo identification of on-pathway inhibitors, as inhibition of OM lipoprotein biogenesis broadly disrupts OM assembly. Here, we use genetics to probe the eight essential proteins involved in OM lipoprotein biogenesis. We define a biological signature consisting of three simple assays that can characteristically identify OM lipoprotein biogenesis defects in vivo. The few known chemical inhibitors of OM lipoprotein biogenesis conform to the biological signature. We also examine MAC13243, a proposed inhibitor of OM lipoprotein biogenesis, and find that it fails to conform to the biological signature. Indeed, we demonstrate that MAC13243 activity relies entirely on a target outside of the OM lipoprotein biogenesis pathway. Hence, our signature offers simple tools to easily assess whether antibiotic lead compounds target an essential pathway that is the hub of OM assembly.

IMPORTANCEGram-negative bacteria have an outer membrane, which acts as a protective barrier and excludes many antibiotics. The limited number of antibiotics active against Gram-negative bacteria, along with rising rates of antibiotic resistance, highlights the need for efficient antibiotic discovery efforts. Unfortunately, finding the target of lead compounds, especially ones targeting outer membrane construction, remains difficult. The hub of outer membrane construction is the lipoprotein biogenesis pathway. We show that defects in this pathway result in a signature cellular response that can be used to quickly and accurately validate pathway inhibitors. Indeed, we found that MAC13243, a compound previously proposed to target outer membrane lipoprotein biogenesis, does not fit the signature, and we show that it instead targets an entirely different cellular pathway. Our findings offer a streamlined approach to discovery and validation of lead antibiotics against a conserved and essential pathway in Gram-negative bacteria.


title: Development of ONT-cappable-seq to unravel the transcriptional landscape of Pseudomonas phages

abstract: RNA sequencing has become the method of choice to study the transcriptional landscape of phage-infected bacteria. However, short-read RNA sequencing approaches generally fail to capture the primary 5 and 3 boundaries of transcripts, confounding the discovery of key transcription initiation and termination events as well as operon architectures. Yet, the elucidation of these elements is crucial for the understanding of the strategy of transcription regulation during the infection process, which is currently lacking beyond a handful of model phages. To this end, we developed ONT-cappable-seq, a specialized long-read RNA sequencing technique that allows end-to-end sequencing of primary prokaryotic transcripts using the Nanopore sequencing platform. We applied ONT-cappable-seq to study transcription of Pseudomonas aeruginosa phage LUZ7, obtaining a comprehensive genome-wide map of viral transcription start sites, terminators, and complex operon structures that fine-regulate gene expression. Our work provides new insights in the RNA biology of a non-model phage, unveiling distinct promoter architectures, putative small non-coding viral RNAs, and the prominent regulatory role of terminators during infection. The robust workflow presented here offers a framework to obtain a global, yet fine-grained view of phage transcription and paves the way for standardized, in depth transcription studies for microbial viruses or bacteria in general.


title: Location of the TEMPO moiety of TEMPO-PC in phosphatidylcholine bilayers is membrane phase-dependent

abstract: The (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) moiety tethered to the headgroup of phosphatidylcholine (PC) lipid is employed in spin labeling electron paramagnetic resonance (EPR) spectroscopy to probe the water dynamics near lipid bilayer interfaces. Due to its amphiphilic character, however, TEMPO spin label could partition between aqueous and lipid phases, and may even be stabilized in the lipid phase. Accurate assessment of the TEMPO-PC configuration in bilayer membranes is essential for correctly interpreting the data from measurements. Here, we carry out all-atom molecular dynamics (MD) simulations of TEMPO-PC probe in single-component lipid bilayers at varying temperatures, using two standard MD force fields. We find that for DPPC membrane whose gel-to-fluid lipid phase transition occurs at 314 K, TEMPO is stabilized above (below) the bilayer interface if the membrane is in the gel (fluid) phase. For bilayers made of unsaturated lipids, DOPC and POPC, which adopt the fluid phase at ambient temperature, TEMPO is unequivocally stabilized inside the bilayers. Our finding of membrane phase-dependent positioning of TEMPO moiety highlights the importance of assessing the packing order and fluidity of lipids under a given measurement condition.


title: Integrated single-cell and spatial transcriptomic analyses unravel the heterogeneity of the prostate tumor microenvironment

abstract:
The treatment of primary prostate cancer delicately balances an active surveillance approach for low-risk disease with multimodal treatment including surgery, radiation therapy, and hormonal therapy for high-risk disease. Recurrence and development of metastatic disease remains a clinical problem, without a clear understanding of what drives immune escape and tumor progression. Here, we sought to comprehensively describe the tumor microenvironment of localized prostate cancer contrasting this with adjacent normal samples and healthy controls. We performed single-cell RNA sequencing and high-resolution spatial transcriptomic analysis. This revealed tumor context dependent changes in gene expression. Our data point towards an immune suppressive tumor microenvironment associated with suppressive myeloid populations and exhausted T-cells, in addition to high stromal angiogenic activity. We inferred cell-to-cell relationships at an unprecedented scale for ligand-receptor interactions within undissociated tissue sections. Our work provides a highly detailed and comprehensive resource of the prostate tumor microenvironment as well as tumor-stromal cell interactions.

HighlightsO_LICharacterization of prostate cancer by combined scRNA-seq and spatial transcriptomic analysis
C_LIO_LIPrimary prostate cancer establishes a suppressive immune microenvironment
C_LIO_LIThe prostate tumor microenvironment exhibits a high angiogenic gene expression pattern
C_LIO_LIA new computational analysis pipeline to deconvolute context-specific differential gene expression
C_LI


title: Mitochondrial Matrix Protease ClpP Agonists inhibit Cancer Stem Cell Function in Breast Cancer Cells by Disrupting Mitochondrial Homeostasis

abstract: Mitochondria are multifaceted organelles which are important for bioenergetics, biosynthesis and signaling in metazoans. Mitochondrial functions are frequently altered in cancer to promote both the energy and the necessary metabolic intermediates for biosynthesis required for tumor growth. Cancer stem cells (CSCs) contribute to chemotherapy resistance, relapse, and metastasis. Recent studies have shown that while non-stem, bulk cancer cells utilize glycolysis, breast CSCs are more dependent on oxidative phosphorylation (OxPhos) and therefore targeting mitochondria may inhibit CSC function. We previously reported that small molecule ONC201, which is an agonist for the mitochondrial caseinolytic protease (ClpP), induces mitochondrial dysfunction in breast cancer cells. In this study, we report that ClpP agonists inhibit breast cancer CSC function in vitro and in vivo. Mechanistically, we found that OxPhos inhibition downregulates multiple pathways required for CSC function, such as the mevalonate pathway, YAP, Myc, and the HIF pathway. ClpP agonists showed significantly greater inhibitory effect on CSC functions compared with other mitochondria-targeting drugs. Further studies showed that ClpP agonists deplete NAD(P)+ and NAD(P)H and induce redox imbalance, and dysregulate one-carbon metabolism and proline biosynthesis. Downregulation of these pathways by ClpP agonists further contribute to the inhibition of CSC function. In conclusion, ClpP agonists inhibit breast CSC functions by disrupting mitochondrial homeostasis in breast cancer cells and inhibiting multiple pathways critical to CSC function.


title: Hepatocyte-specific loss of LAP2α reduces hepatic steatosis in male mice by enhancing LMNA-mediated transcriptional regulation

abstract:
There is increasing evidence for the importance of the nuclear envelope in lipid metabolism, nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH). Human mutations in LMNA, encoding A-type nuclear lamins, cause early-onset insulin resistance and NASH, while hepatocyte-specific deletion of Lmna predisposes to NASH with fibrosis in male mice. Given that variants in the gene encoding LAP2, a nuclear protein that regulates LMNA, were previously identified in patients with NAFLD, we sought to determine the role of LAP2 in NAFLD using a mouse genetic model. Hepatocyte-specific Lap2-knockout (HKO) mice and littermate controls were fed normal chow or high-fat diet (HFD) for 8 weeks or 6 months. In contrast to what was observed with hepatocyte-specific Lmna deletion, male HKO mice showed no increase in hepatic steatosis or NASH compared to controls. Rather, HKO mice demonstrated reduced hepatic steatosis, particularly after long-term HFD, with decreased susceptibility to diet-induced NASH. Accordingly, whereas pro-steatotic genes Cidea, Mogat1, and Cd36 were upregulated in Lmna-KO mice, they were downregulated in HKO mice, as were pro-inflammatory and pro-fibrotic genes. These data indicate that Lap2 deletion enhances hepatoprotective LMNA-mediated regulation of gene expression in mouse hepatocytes; therefore, LAP2 might represent a potential therapeutic target in human NASH.

Brief SummaryLoss of mouse LAP2 protected against diet-induced hepatic steatosis and NASH via enhancing protective regulatory functions of LMNA.

category: physiology


title: Structural and functional insight into the plant unique multimodular triphosphosphate tunnel metalloenzymes of Arabidopsis thaliana

abstract: Triphosphate tunnel metalloenzymes (TTMs) are found in all biological kingdoms and have been characterized in microorganisms and animals. Members of the TTM family already characterized have divergent biological functions and act on a range of triphosphorylated substrates (RNA, thiamine triphosphate, inorganic polyphosphate). TTM proteins in plants have received considerably less attention and are unique in that some homologs harbor additional domains including a P-loop kinase and transmembrane domain. Here we report on structural and functional aspects of the multimodular TTM1 and TTM2 of Arabidopsis thaliana. Tissue and cellular microscopy studies show that both AtTTM1 and AtTTM2 are expressed in actively dividing (meristem) tissue and are tail-anchored proteins at the outer mitochondrial membrane - mediated by the single transmembrane domain at the C-terminus, supporting earlier studies. Crystal structures of AtTTM1 in the presence and absence of a non-hydrolyzable ATP analog reveal a catalytically incompetent TTM tunnel domain tightly interacting with the P-loop kinase domain that is locked in an inactive conformation. Structural comparison reveals that a helical hairpin may facilitate movement of the TTM domain thereby activating the kinase. Genetic studies show that AtTTM2 is important for the developmental transition from the vegetative to the reproductive phase in Arabidopsis, whereas its closest paralog AtTTM1 is not. Rational design of mutations based on the 3D structure demonstrates that both the P-loop kinase and TTM tunnel modules of AtTTM2 are required for the developmental switch.


title: Accurate Protein Domain Structure Annotation with DomainMapper

abstract:
Automated domain annotation plays a number of important roles in structural informatics and typically involves searching query sequences against Hidden Markov Model (HMM) profiles. This process can be ambiguous or inaccurate when proteins contain domains with non-contiguous residue ranges, and especially when insertional domains are hosted within them. Here we present DomainMapper, an algorithm that accurately assigns a unique domain structure annotation to any query sequence, including those with complex topologies. We validate our domain assignments using the AlphaFold database and confirm that non-contiguity is pervasive (6.5% of all domains in yeast and 2.5% in human). Using this resource, we find that certain folds have strong propensities to be non-contiguous or insertional across the Tree of Life, likely underlying evolutionary preferences for domain topology. DomainMapper is freely available and can be run as a single command line function.

HIGHLIGHTSDomainMapper generates a unique domain structure annotation, including non-contiguous and insertional domains

Automated annotations of non-contiguous domains are validated against the AlphaFold database

DomainMapper can be easily installed and used by non-experts

Certain folds have strong preferences to be non-contiguous or insertional

GRAPHICAL ABSTRACT

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category: bioinformatics


title: Fragment libraries designed to be functionally diverse recover protein binding information more efficiently than standard structurally diverse libraries

abstract: Current fragment-based drug design relies on the efficient exploration of chemical space though the use of structurally diverse libraries of small fragments. However, structurally dissimilar compounds can exploit the same interactions on a target, and thus be functionally similar. Using 3D structures of many fragments bound to multiple targets, we examined if there exists a better strategy for selecting fragments for screening libraries. We show that structurally diverse fragments can be described as functionally redundant, often making the same interactions. Ranking fragments by the number of novel interactions they made, we show that functionally diverse selections of fragments substantially increase the amount of information recovered for unseen targets compared to other methods of selection. Using these results, we design small functionally efficient libraries that are able to give significantly more information about new protein targets than similarly sized structurally diverse libraries. By covering more functional space (rather than chemical space), more diverse sets of drug leads can be generated, increasing the chances of fragment screens resulting in viable drug candidates.


title: Mechanism of homotropic cooperativity from nearest-neighbor statistical thermodynamic modelling and native mass spectrometry of ring-shaped homo-oligomeric ligand binding proteins

abstract:
Homo-oligomeric ligand-activated proteins are ubiquitous in biology. The functions of such molecules are commonly regulated by allosteric coupling between ligand binding sites. Understanding the basis for this regulation requires both quantifying the free energy {Delta}G transduced between sites, and the structural basis by which it is transduced. We consider allostery in three variants of the model ring-shaped homo-oligomeric trp RNA binding attenuation protein, TRAP. First, we developed nearest-neighbor statistical thermodynamic binding models comprising microscopic free energies for ligand binding to isolated sites {Delta}GN0, and for coupling between one or both adjacent sites, {Delta}GN1 and {Delta}GN2. Using the resulting partition function (PF) we explored the effects of these parameters on simulated population distributions for the 2N possible liganded states. We then experimentally monitored liganddependent population shifts using conventional spectroscopic and calorimetric methods, and using native mass spectrometry (MS). By resolving species with differing numbers of bound ligands by their mass, native MS revealed striking differences in their ligand-dependent population shifts. Fitting the populations to a binding polynomial derived from the PF yielded coupling free energy terms corresponding to orders of magnitude differences in cooperativity. Uniquely, this approach predicts which of the possible 2N liganded states are populated at different ligand concentrations, providing necessary insights into regulation. The combination of statistical thermodynamic modeling with native MS may provide the thermodynamic foundation for a meaningful understanding of the structure-thermodynamic linkage that drives cooperativity.

TOC Figure (draft)

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C_FIG


title: Thermodynamic coupling between neighboring binding sites in homo-oligomeric ligand sensing proteins from mass resolved ligand dependent population distributions


title: Zhx2 is a candidate gene underlying oxymorphone metabolite brain concentration associated with state-dependent oxycodone reward

abstract:
Understanding the pharmacogenomics of opioid metabolism and behavior is vital to therapeutic success as mutations can dramatically alter therapeutic efficacy and addiction liability. We found robust, sex-dependent BALB/c substrain differences in oxycodone behaviors and whole brain concentration of oxycodone metabolites. BALB/cJ females showed robust state-dependent oxycodone reward learning as measured via conditioned place preference when compared to the closely related BALB/cByJ substrain. Accordingly, BALB/cJ females also showed a robust increase in brain concentration of the inactive metabolite noroxycodone and the active metabolite oxymorphone compared to BALB/cByJ mice. Oxymorphone is a highly potent full agonist at the mu opioid receptor that could enhance drug-induced interoception and state-dependent oxycodone reward learning. Quantitative trait locus (QTL) mapping in a BALB/c F2 reduced complexity cross revealed one major QTL on chromosome 15 underlying brain oxymorphone concentration that explained 32% of the female variance. BALB/cJ and BALB/cByJ differ by fewer than 10,000 variants which can greatly facilitate candidate gene/variant identification. Hippocampal and striatal cis-expression QTL (eQTL) and exon-level eQTL analysis identified Zhx2, a candidate gene coding for a transcriptional repressor with a private BALB/cJ retroviral insertion that reduces Zhx2 expression and sex-dependent dysregulation of CYP enzymes. Whole brain proteomics corroborated the Zhx2 eQTL and identified upregulated CYP2D11 that could increase brain oxymorphone in BALB/cJ females. To summarize, Zhx2 is a highly promising candidate gene underlying brain oxycodone metabolite levels. Future studies will validate Zhx2 and its site of action using reciprocal gene editing and tissue-specific viral manipulations in BALB/c substrains.

Significance StatementOur findings show genetic variation can result in sex-specific alterations in whole brain concentrations of bioactive opioid metabolites following oxycodone administration, and reinforces the need for sex as a biological factor in pharmacogenomic studies. The co-occurrence of female-specific increased oxymorphone and state-dependent reward learning suggests that this minor yet potent and efficacious metabolite of oxycodone could increase opioid interoception and drug-cue associative learning of opioid reward which has implications for cue-induced relapse of drug-seeking behavior.

category: pharmacology and toxicology