title: Development of a fluorescent quantitative real-time polymerase chain reaction assay for the detection of novel-goose parvovirus in vivo

abstract:
ObjectivesTo develop a sensitive, highly specific fluorescent quantitative real-time PCR assay for accurate detection and quantification of novel-goose parvovirus (N-GPV) in vitro and in vivo.

MethodsSpecific primers was designed based on N-GPV inverted terminal repeats region; virus RNA (DFV, NDV, AIV, DHV-1, DHV-3) and virus DNA (MDPV, GPV, N-GPV) were extracted, cDNA (DFV, NDV, AIV, DHV-1, DHV-3) were prepared from viral RNAs using M-MLV Reverse Transcriptase, and prepared cDNA (DFV, NDV, AIV, DHV-1, DHV-3) and DNA (MDPV, GPV, N-GPV) amplified by real-time PCR; the sensitivity, specificity and reproducibility of established real-time PCR methods were evaluated, and finally we validated the reliability of real-time PCR methods in ducklings models in vivo.

ResultsThe standard curve of established real-time PCR had a good linearity (slope was -0.3098, Y-intercept was 37.865, efficiency of standard curve was 0.995); the detection limit of established real-time PCR for N-GPV was 10 copies/reaction. The sensitivity of real-time PCR was 10 copies/L, which was 1000 times higher than conventional gel-based PCR assay. The results of intra-assay CVs (0.04-0.74%) and inter-assay CVs (0.16-0.53%) showed that the real-time PCR assay had an excellent repeatability. This method also could efficiently detect viral load in heart, liver, spleen, lung, kidney, pancreas, bursa of Fabricius, brain, blood and excrement from ducklings models after N-GPV infection from 6h to 28 days, which could provided us a dynamic distribution observation of N-GPV viral load using this real-time PCR assay in vivo.

ConclusionIn the study, we developed a high sensitive, specific and reproducible real-time PCR assay for N-GPV detection. The established real-time PCR assay was suitable for parvovirus detection and quantification simultaneously, no matter sample obtained from blood, internal organs or ileac contents; the present work may provide insight into the pathogenesis of N-GPV and will contributes to better understanding of this newly emerged novel GPV related virus in cherry valley ducks.

category: zoology


title: Small RNA profiling in Mycobacterium insights into stress adapt ability

abstract: Mycobacteria would encounter a number of environment changes during infection, and respond to it using different mechanisms. sRNA is a posttranscriptionally regulatory system for the function of genes and has been investigated in many other bacteria. Here, we used Mycobacterium tuberculosis and Mycobacterium bovis BCG infection models and sequenced the whole bacterial RNAs before and after host cells infection. Comparison of differential expressed sRNAs, by using GO and KEGG, and target predication, was carried out. Six pathogenically relevant stresses, drug resistance test, growth rate and morphology were used for screening and identify sRNAs. From these data, we identified a subset of sRNAs that are differentially expressed in multiple infection groups and stress conditions. We found that many of them were associated with lipid metabolism. Among them, ncBCG427, was significantly down-regulated when BCG entered into macrophages, and was associated with increase of biofilm formation and changed in drug susceptibility. Then, reduction of virulence possibility depends on regulating lipid metabolism.


title: One-week escitalopram intake shifts excitation-inhibition balance in the healthy female brain

abstract: Neural health relies on cortical excitation-inhibition balance (EIB), with disrupted EIB underlying circuit dysfunction in several neuropsychiatric disorders. Previous research suggests links between increased cortical excitation and neuroplasticity induced by selective serotonin reuptake inhibitors (SSRIs). Whether there are modulations of EIB following SSRI-administration in the healthy human brain, however, remains unclear. To this end, we assessed changes in EIB following longitudinal escitalopram-intake. In a randomized, double-blind study protocol, a sample of 59 healthy female individuals on oral contraceptives underwent three resting-state electroencephalography recordings after daily administration of 20 mg escitalopram (n = 28) or placebo (n = 31) at baseline, after single dose, and after 1 week (steady state). We assessed 1/f slope of the power spectrum, a marker of EIB, compared individual trajectories of 1/f slope changes contrasting single dose and 1-week drug intake, and tested the relationship of escitalopram plasma levels and cortical excitatory and inhibitory balance shifts. Escitalopram-intake associated with decreased 1/f slope, indicating an EIB shift in favor of excitation. Furthermore, 1/f slope at baseline and after single dose of escitalopram predicted 1/f slope at steady state. Higher plasma escitalopram levels at single dose associated with better maintenance of these EIB changes throughout the drug administration week. Characterizing changes in 1/f slope during longitudinal SSRI-intake in healthy female individuals, we show that escitalopram shifted EIB in favor of excitation. These findings demonstrate the potential for 1/f slope to predict individual cortical responsivity to SSRIs and widen the neuroimaging lens by testing an interventional psychopharmacological design in a clearly-defined endocrinological state.

category: neuroscience


title: Neural excitation/inhibition imbalance and the treatment of severe depression

abstract: An influential hypothesis holds that depression is related to a neural excitation/inhibition imbalance, but its role in the treatment of depression remains unclear. Here, we show that unmedicated patients with severe depression demonstrated reduced inhibition of brain-wide resting-state networks relative to healthy controls. Patients using antidepressants showed inhibition that was higher than unmedicated patients and comparable to controls, but they still suffered from severe depression. Subsequent treatment with electroconvulsive therapy (ECT) reduced depressive symptoms, but its effectiveness did not depend on changes in network inhibition. Concomitant pharmacotherapy increased the effectiveness of ECT, but only when the strength of neural inhibition before ECT was within the normal range and not when inhibition was excessive. These findings suggest that reversing the excitation/inhibition imbalance may not be sufficient nor necessary for the effective treatment of severe depression, and that brain-state informed pharmacotherapy management may enhance the effectiveness of ECT.


title: Nucleolar stress controls mutant Huntingtin toxicity and monitors Huntington disease progression

abstract: Transcriptional and cellular stress surveillance deficits are hallmarks of Huntingtons disease (HD), a fatal autosomal dominant neurodegenerative disorder, caused by a pathological expansion of CAG repeats in the Huntingtin (HTT) gene. The nucleolus, a dynamic nuclear biomolecular condensate and the site of ribosomal RNA (rRNA) transcription, is implicated in the cellular stress response and in protein quality control. While the exact pathomechanisms of HD remain unclear, the impact of nucleolar dysfunction on HD pathophysiology in vivo is elusive. Here we identified aberrant maturation of rRNA and decreased translational rate in association with human mutant Huntingtin (mHTT) expression. Genetic disruption of nucleolar integrity in vulnerable striatal neurons of the R6/2 HD mouse model decreases mHTT disperse state in the nucleus, exacerbating the motor deficits. The protein nucleophosmin 1 (NPM1), important for nucleolar integrity and rRNA maturation, loses its nucleolar localization. NPM1 de-localization occurs in the striatum and in the skeletal muscle of the progressive zQ175 knock-in HD mouse model, mimicking the phenotype of HD patients in skeletal muscle biopsies. Taken together, we showed that nucleolar integrity regulates the formation of mHTT inclusions in vivo, and identified NPM1 as a novel, readily detectable peripheral histopathological marker of HD progression.


title: NSUN2-mediated m5C methylation of IRF3 mRNA negatively regulates type I interferon responses

abstract: 5-Methylcytosine (m5C) is a widespread post-transcriptional RNA modification and is reported to be involved in manifold cellular responses and biological processes through regulating RNA metabolism. However, its regulatory role in antiviral innate immunity has not yet been elucidated. Here, we report that NSUN2, a typical m5C methyltransferase, can negatively regulate type I interferon responses during viral infection. NSUN2 specifically mediates m5C methylation of IRF3 mRNA and accelerates its degradation, resulting in low levels of IRF3 and downstream IFN-{beta} production. Knockout or knockdown of NSUN2 could enhance type I interferon responses and downstream ISG expression after viral infection in vitro. And in vivo, the antiviral innate responses is more dramatically enhanced in Nsun2+/- mice than in Nsun2+/+ mice. Four highly m5C methylated cytosines in IRF3 mRNA were identified, and their mutation could enhance the cellular IRF3 mRNA levels. Moreover, infection with Sendai virus (SeV), vesicular stomatitis virus (VSV), herpes simplex virus 1 (HSV-1), Zika virus (ZIKV), or especially SARS-CoV-2 resulted in a reduction in endogenous levels of NSUN2. Together, our findings reveal that NSUN2 serves as a negative regulator of interferon response by accelerating the fast turnover of IRF3 mRNA, while endogenous NSUN2 levels decrease after viral infection to boost antiviral responses for the effective elimination of viruses. Our results suggest a paradigm of innate antiviral immune responses ingeniously involving NSUN2-mediated m5C modification.

category: immunology


title: A mouse-adapted SARS-CoV-2 strain replicating in standard laboratory mice.

abstract: SARS-CoV-2 has infected almost 200 million humans and caused over 4 million deaths worldwide. Evaluating countermeasures and improving our understanding of COVID-19 pathophysiology require access to animal models that replicate the hallmarks of human disease. Mouse infection with SARS-CoV-2 is limited by poor affinity between the virus spike protein and its cellular receptor ACE2. We have developed by serial passages the MACo3 virus strain which efficiently replicates in the lungs of standard mouse strains and induces age-dependent lung lesions. Compared to other mouse-adapted strains and severe mouse models, infection with MACo3 results in mild to moderate disease and will be useful to investigate the role of host genetics and other factors modulating COVID-19 severity.

category: microbiology


title: Borgs are giant extrachromosomal elements with the potential to augment methane oxidation

abstract: Anaerobic methane oxidation exerts a key control on greenhouse gas emissions 1, yet factors that modulate the activity of microorganisms performing this function remain little explored. In studying groundwater, sediments, and wetland soil where methane production and oxidation occur, we discovered extraordinarily large, diverse DNA sequences that primarily encode hypothetical proteins. Four curated, complete genomes are linear, up to ~1 Mbp in length and share genome organization, including replicore structure, long inverted terminal repeats, and genome-wide unique perfect tandem direct repeats that are intergenic or generate amino acid repeats. We infer that these are a new type of archaeal extrachromosomal element with a distinct evolutionary origin. Gene sequence similarity, phylogeny, and local divergence of sequence composition indicate that many of their genes were assimilated from methane-oxidizing Methanoperedens archaea. We refer to these elements as "Borgs". We identified at least 19 different Borg types coexisting with Methanoperedens in four distinct ecosystems. Borg genes expand redox and respiratory capacity (e.g., clusters of multiheme cytochromes), ability to respond to changing environmental conditions, and likely augment Methanoperedens capacity for methane oxidation (e.g., methyl coenzyme M reductase). By this process, Borgs could play a previously unrecognized role in controlling greenhouse gas emissions.


title: A whole transcriptomic approach reveals novel mechanisms of organophosphate and pyrethroid resistance in Anopheles arabiensis from Ethiopia

abstract:
The development of insecticide resistance in malaria vectors is of increasing concern in Ethiopia because of its potential implications for vector control failure. To better elucidate the specificity of resistance mechanisms and to facilitate the design of control strategies that minimize the likelihood of selecting for cross-resistance, a whole transcriptomic approach was used to explore gene expression patterns in a multi-insecticide resistant population of Anopheles arabiensis from Oromia Region, Ethiopia. This field population was resistant to the diagnostic doses of malathion (average mortality of 71.9%) and permethrin (77.4%), with pools of survivors and unexposed individuals analyzed using Illumina RNA-sequencing, alongside insecticide susceptible reference strains. This population also demonstrated deltamethrin resistance but complete susceptibility to alpha-cypermethrin, bendiocarb and propoxur, providing a phenotypic basis for detecting insecticide-specific resistance mechanisms. Transcriptomic data revealed overexpression of genes including cytochrome P450s, glutathione-s-transferases and carboxylesterases (including CYP4C36, CYP6AA1, CYP6M2, CYP6M3, CYP6P4, CYP9K1, CYP9L1, GSTD3, GSTE2, GSTE3, GSTE4, GSTE5, GSTE7 and two carboxylesterases) that were shared between malathion and permethrin survivors. We also identified nineteen highly overexpressed cuticular-associated proteins (including CYP4G16, CYP4G17 and chitinase) and eighteen salivary gland proteins (including D7r4 short form salivary protein), which may be contributing to a non-specific resistance phenotype by either enhancing the cuticular barrier or promoting binding and sequestration of insecticides, respectively. These findings provide novel insights into the molecular basis of insecticide resistance in this lesser well-characterized major malaria vector species.

ImportanceInsecticide-resistant mosquito populations remain a significant challenge to global malaria vector control. While substantial progress has been made unraveling resistance mechanisms in major vector species, such as Anopheles gambiae and An. funestus, comparatively less is known about An. arabiensis populations. Using a whole transcriptomic approach, we investigated genes associated with resistance to insecticides used to control An. arabiensis in Ethiopia. Study findings revealed shared detoxification genes between organophosphate- and pyrethroid-resistant vectors and highly overexpressed cuticular-associated proteins and salivary gland proteins, which may play a role in enhancing insecticide resistance. The whole transcriptomic analysis detected novel resistance-associated genes, which warrant functional validation to determine their specificity to particular insecticides and their potential to confer cross-resistance between different insecticides with the same mode of action. These genes may contribute to the development of diagnostic markers to monitor insecticide resistance dynamics in the field.

category: molecular biology


title: Predictors of the long-term conservation value of sites in a mechanistic metacommunity model

abstract: Conserving biodiversity often requires deciding which sites to prioritise for protection. Predicting the impact of habitat loss is a major challenge, however, since impacts can be distant from the perturbation in both space and time. Here we study the long-term impacts of habitat loss in a mechanistic metacommunity model in terms of both immediate extinctions and secondary species losses. We find that biomass-at-site, closely related to site area, is a poor predictor of long-term regional species losses following site removal. Knowledge of the compositional distinctness (average between-site Bray-Curtis dissimilarity) of the removed community can markedly improve the prediction of impacts at the regional scale, even when biotic responses play out at substantial spatial or temporal distance from the removed site. Fitting our model to empirical species-by-site tables describing Andean diatoms and Brazilian lichen-fungi, we show that compositional distinctness surpasses area as a predictor of long-term species losses in the empirically relevant parameter range. Our results robustly demonstrate that site area alone is not sufficient to gauge conservation priorities; analysis of compositional distinctness permits improved prioritisation at low cost.

category: ecology


title: Community composition exceeds area as a predictor of long-term conservation value


title: Mutations in Drosophila tRNA processing factors cause phenotypes similar to Pontocerebellar Hypoplasia

abstract: Mature tRNAs are generated by multiple RNA processing events, which can include the excision of intervening sequences. The tRNA splicing endonuclease (TSEN) complex is responsible for cleaving these intron-containing pre-tRNA transcripts. In humans, TSEN copurifies with CLP1, an RNA kinase. Despite extensive work on CLP1, its in vivo connection to tRNA splicing remains unclear. Interestingly, mutations in CLP1 or TSEN genes cause neurological diseases in humans that are collectively termed Pontocerebellar Hypoplasia (PCH). In mice, loss of Clp1 kinase activity results in premature death, microcephaly and progressive loss of motor function. To determine if similar phenotypes are observed in Drosophila, we characterized mutations in crowded-by-cid (cbc), the CLP1 ortholog, as well as in the fly ortholog of human TSEN54. Analyses of organismal viability, larval locomotion and brain size revealed that mutations in both cbc and Tsen54 phenocopy those in mammals in several details. In addition to an overall reduction in brain lobe size, we also found increased cell death in mutant larval brains. Ubiquitous or tissue-specific knockdown of cbc in neurons and muscles reduced viability and locomotor function. These findings indicate that we can successfully model PCH in a genetically-tractable invertebrate.

category: genetics


title: Selective whole genome amplification as a tool to enrich specimens with low Treponema pallidum genomic DNA copies for whole genome sequencing

abstract:
Downstream next generation sequencing (NGS) of the syphilis spirochete Treponema pallidum subspecies pallidum (T. pallidum) is hindered by low bacterial loads and the overwhelming presence of background metagenomic DNA in clinical specimens. In this study, we investigated selective whole genome amplification (SWGA) utilizing multiple displacement amplification (MDA) in conjunction with custom oligonucleotides with an increased specificity for the T. pallidum genome, and the capture and removal of CpG-methylated host DNA using the NEBNext(R) Microbiome DNA Enrichment Kit followed by MDA with the REPLI-g Single Cell Kit as enrichment methods to improve the yields of T. pallidum DNA in isolates and lesion specimens from syphilis patients. Sequencing was performed using the Illumina MiSeq v2 500 cycle or NovaSeq 6000 SP platform. These two enrichment methods led to 93-98% genome coverage at 5 reads/site in 5 clinical specimens from the United States and rabbit propagated isolates, containing >14 T. pallidum genomic copies/l of sample for SWGA and >129 genomic copies/l for CpG methylation capture with MDA. Variant analysis using sequencing data derived from SWGA-enriched specimens, showed that all 5 clinical strains had the A2058G mutation associated with azithromycin resistance. SWGA is a robust method that allows direct whole genome sequencing (WGS) of specimens containing very low numbers of T. pallidum, which have been challenging until now.

ImportanceSyphilis is a sexually transmitted, disseminated acute and chronic infection caused by the bacterial pathogen Treponema pallidum subspecies pallidum. Primary syphilis typically presents as single or multiple mucocutaneous lesions, and if left untreated, can progress through multiple stages with varied clinical manifestations. Molecular studies often rely on direct amplification of DNA sequences from clinical specimens; however, this can be impacted by inadequate samples due to disease progression or timing of patients seeking clinical care. While genotyping has provided important data on circulating strains over the past two decades, WGS data is needed to better understand strain diversity, perform evolutionary tracing, and monitor antimicrobial resistance markers. The significance of our research is the development of a SWGA DNA enrichment method that expands the range of clinical specimens that can be directly sequenced to include samples with low numbers of T. pallidum.

category: genomics


title: Single-cell bisulfite sequencing of spermatozoa from lean and obese humans reveals potential for the transmission of epimutations

abstract: Epigenetic marks in gametes modulate developmental programming after fertilization. Spermatozoa from obese men exhibit distinct epigenetic signatures compared to lean men, however, whether epigenetic differences are concentrated in a sub-population of spermatozoa or spread across the ejaculate population is unknown. Here, by using whole-genome single-cell bisulfite sequencing on 87 motile spermatozoa from 8 individuals (4 lean and 4 obese), we found that spermatozoa within single ejaculates are highly heterogeneous and contain subsets of spermatozoa with marked imprinting defects. Comparing lean and obese subjects, we discovered methylation differences across two large CpG dense regions located near PPM1D and LINC01237. These findings confirm that sperm DNA methylation is altered in human obesity and indicate that single ejaculates contain subpopulations of spermatozoa carrying distinct DNA methylation patterns. Distinct epigenetic patterns of spermatozoa within an ejaculate may result in different intergenerational effects and therefore influence strategies aiming to prevent epigenetic-related disorders in the offspring.


title: Tn5 transposase-based epigenomic profiling methods are prone to open chromatin bias

abstract: Epigenetic studies of rare biological samples like mammalian oocytes and preimplantation embryos require low input or even single cell epigenomic profiling methods. To reduce sample loss and avoid inefficient immunoprecipitation, several chromatin immuno-cleavage-based methods using Tn5 transposase fused with Protein A/G have been developed to profile histone modifications and transcription factor bindings using small number of cells. The Tn5 transposase-based epigenomic profiling methods are featured with simple library construction steps in the same tube, by taking advantage of Tn5 transposases capability of simultaneous DNA fragmentation and adaptor ligation. However, the Tn5 transposase prefers to cut open chromatin regions. Our comparative analysis shows that Tn5 transposase-based profiling methods are prone to open chromatin bias. The high false positive signals due to biased cleavage in open chromatin could cause misinterpretation of signal distributions and dynamics. Rigorous validation is needed when employing and interpreting results from Tn5 transposase-based epigenomic profiling methods.


title: Biomechanical effects of the addition of a precision constraint on a collective load carriage task

abstract:
1Team lifting is a complex and collective motor task that possesses both motor and cognitive components. The purpose of this study was to investigate to what extent the biomechanics of a collective load carriage is affected when a dyad of individuals is performing a carrying task with an additional accuracy constraint. Ten dyads performed a first condition in which they collectively transported a load (CC), and a second one in which they transported the same load while maintaining a ball in a target position on its top (PC).

The recovery-rate, amplitude, and period of the center-of-mass (COM) trajectory were computed for the whole system (dyad + table = PACS). We analyzed the forces and moments exerted at each joint of the upper limbs of the subjects. We observed a decrease in the overall performance of the dyads when the Precision task was added, i.e., i) the velocity and amplitude of CoMPACS decreased by 1,7% and 5,8%, respectively, ii) inter-subject variability of the Moment-Cost-Function decreased by 95% and recovery rate decreased by 19,2% during PC. A kinetic synergy analysis showed that the subjects reorganized their coordination in the PC.

Our results demonstrate that adding a precision task affects the economy of collective load carriage. Notwithstanding, the joint moments at the upper-limbs are better balanced and co-vary more across the paired subjects during the precision task. Our study results may find applications in domains such as Ergonomics, Robotics-developments, and Rehabilitation.

category: bioengineering


title: Successful microbial colonization of space using an anti-aggregation strategy

abstract: Many organisms live in habitats with limited nutrients or space, competition for these resources is ubiquitous. Although spatial factors related to populations manner of colonizing space influences its success in spatial competition, what these factors are and to what extent they influence the outcome remains under-explored. Here, we applied a simulated competitive model to explore the spatial factors affecting outcomes of competition for space. By quantifying spatial factors using  Space Accessibility, we show that colonizing space in an anti-aggregation manner contributes to microbial competitive success. We also find that the competitive edge derived from being anti-aggregation in colonizing space, which results in a higher  Space Accessibility, neutralizes the disadvantage arising from either lower growth rate or lower initial abundance. These findings shed light on the role of space colonization manners on maintaining biodiversity within ecosystems and provide novel insights critical for understanding how competition for space drives evolutionary innovation.


title: BioCCP.jl: Collecting Coupons in combinatorial biotechnology

abstract: In combinatorial biotechnology, it is crucial for screening experiments to sufficiently cover the design space. In the BioCCP.jl package, we provide functions for minimum sample size determination based on the mathematical framework coined the Coupon Collector Problem. BioCCP.jl, including source code, documentation and a Pluto notebook is available at https://github.com/kirstvh/BioCCP.


title: Effects of blue monochromatic light directed at one eye of pregnant horse mares on gestation, parturition and foal maturity

abstract: Blue light directed at one eye advances the equine ovulatory season but can also advance foaling. In this study, effects of blue LED light on pregnancy outcome were assessed. Twenty mares with singleton pregnancies were studied over two consecutive years in a cross-over design. In one year, mares received an extended photoperiod using 50 lux of blue LED light (468 nm) directed at a single eye from 08:00 until 23:00 daily via head-worn light masks starting mid-December and in the other year remained untreated as controls. Gestation was shorter in blue LED light-treated (333.2{+/-}1.0 days,{+/-}SEM) than in control pregnancies (337.9{+/-}1.8 days, p<0.05). Colostral IgG content was lower in treated than in control pregnancies (p<0.05) but was adequate to ensure sufficient IgG transfer to newborn foals with a single exception. Foals born to blue LED light-treated mares had lower wither heights (102.5{+/-}1.1 vs. 105.2{+/-}1.0 cm, p<0.01), similar weights (55.0{+/-}1.3 vs. 55.5{+/-}1.1 kg) and took less time to stand after birth than control foals (39{+/-}3 vs. 56{+/-}4 min, p<0.05). The neutrophil/lymphocyte ratio, was higher in foals born to blue LED light-treated mares than controls (3.2{+/-}0.2 vs. 2.7{+/-}0.2; p<0.05). Foals born to blue LED light-treated mares had reduced hair length compared to controls (13.1{+/-}0.8 vs. 20.9{+/-}0.1 mm; p<0.001) and hair regrowth in treated mares was reduced (p<0.05). Maternal plasma prolactin concentration was increased in treated mares (p=0.05) and peaked at foaling (p<0.001). In conclusion, blue LED light directed at one eye advanced foaling and influenced height, hair coat and maturity in foals.

category: physiology


title: Sphingolipids are involved in Pieris brassicae egg-induced cell death in Arabidopsis thaliana

abstract: In Brassicaceae, hypersensitive-like (HR-like) cell death is a central component of direct defenses triggered against eggs of the large white butterfly Pieris brassicae. The signaling pathway leading to HR-like in Arabidopsis is mainly dependent on salicylic acid (SA) accumulation, but downstream components are unclear. Here, we found that treatment with P. brassicae egg extract (EE) trigger changes in expression of sphingolipid metabolism genes in Arabidopsis and Brassica nigra. Disruption of ceramide synthase activity led to a significant decrease of EE-induced HR-like whereas SA signaling and reactive oxygen species levels were unchanged, suggesting that ceramides are downstream activators of HR-like. Sphingolipid quantifications showed that ceramides with C16:0 side-chains accumulated in both species, and this response was independent on SA accumulation. Finally, we provide genetic evidence that the modification of fatty acyl chains of sphingolipids modulates HR-like. Altogether, these results show that sphingolipids play a key and specific role during insect egg-triggered HR-like.

category: plant biology


title: A novel imaging ligand as a biomarker for mutant huntingtin-lowering in Huntington's disease

abstract: Huntingtons disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin (HTT) gene that encodes the pathologic mutant HTT (mHTT) protein with an expanded polyglutamine (PolyQ) tract. While several therapeutic programs targeting mHTT expression have advanced to clinical evaluation, no method is currently available to visualize mHTT levels in the living brain. Here we demonstrate the development of a positron emission tomography (PET) imaging radioligand with high affinity and selectivity for mHTT aggregates. This small molecule radiolabeled with 11C ([11C]CHDI-180R) enables non-invasive monitoring of mHTT pathology in the brain and can track region-and time-dependent suppression of mHTT in response to therapeutic interventions targeting mHTT expression. We further show that therapeutic agents that lower mHTT in the striatum have a functional restorative effect that can be measured by preservation of striatal imaging markers, enabling a translational path to assess the functional effect of mHTT lowering.


title: Zygotic contractility awakening during mouse preimplantation development

abstract: Actomyosin contractility is a major engine of preimplantation morphogenesis, which starts at the 8-cell stage during mouse embryonic development. Contractility becomes first visible with the appearance of periodic cortical waves of contraction (PeCoWaCo), which travel around blastomeres in an oscillatory fashion. How contractility of the mouse embryo becomes active remains unknown. We have taken advantage of PeCoWaCo to study the awakening of contractility during preimplantation development. We find that PeCoWaCo become detectable in most embryos only after the 2ndcleavage and gradually increase their oscillation frequency with each successive cleavage. To test the influence of cell size reduction during cleavage divisions, we use cell fusion and fragmentation to manipulate cell size across a 20-60 m range. We find that the stepwise reduction in cell size caused by cleavage divisions does not explain the presence of PeCoWaCo or their accelerating rhythm. Instead, we discover that blastomeres gradually decrease their surface tensions until the 8-cell stage and that artificially softening cells enhances PeCoWaCo prematurely. Therefore, during cleavage stages, cortical softening awakens zygotic contractility before preimplantation morphogenesis.

category: developmental biology


title: scSGL: Signed Graph Learning for Single-Cell Gene Regulatory Network Inference

abstract:
MotivationElucidating the topology of gene regulatory networks (GRNs) from large single-cell RNA sequencing (scRNAseq) datasets, while effectively capturing its inherent cell-cycle heterogeneity and dropouts, is currently one of the most pressing problems in computational systems biology. Recently, graph learning (GL) approaches based on graph signal processing (GSP) have been developed to infer graph topology from signals defined on graphs. However, existing GL methods are not suitable for learning signed graphs, which represent a characteristic feature of GRNs, as they account for both activating and inhibitory relationships between genes. They are also incapable of handling high proportion of zero values, which represent dropouts in single cell experiments. To this end, we propose a novel signed GL approach, scSGL, that learns GRNs based on the assumption of the smoothness and non-smoothness of gene expressions over activating and inhibitory edges, respectively. scSGL is then extended with kernels to take the nonlinearity of co-expressions into account and handle high proportion of dropouts. From GSP perspective, this extension corresponds to assuming smoothness/non-smoothness of graph signals in a higher dimensional space defined by the kernel. The proposed approach is formulated as a non-convex optimization problem and solved using an efficient ADMM framework.

ResultsIn our experiments on simulated and real single cell datasets, scSGL compares favorably with other single cell gene regulatory network reconstruction algorithms.

AvailabilityThe scSGL code and analysis scripts are available at (https://github.com/Single-Cell-Graph-Learning/scSGL).

category: bioinformatics


title: A Phosphorus-Limitation Induced, Functionally Conserved DUF506 Protein is a Repressor of Root Hair Elongation in Arabidopsis thaliana

abstract: Root hairs (RHs) function in nutrient and water acquisition, root metabolite exudation, soil anchorage and plant-microbe interactions. Longer or more abundant RHs are potential breeding traits for developing crops that are more resource-use efficient and can improve soil health. RH elongation is controlled by both environmental and endogenous factors. While many genes are known to promote RH elongation, relatively little is known about genes and mechanisms that constrain RH growth. Here we demonstrate that a DOMAIN OF UNKNOWN FUNCTION 506 (DUF506) protein, AT3G25240, negatively regulates Arabidopsis thaliana RH growth. The AT3G25240 gene is strongly and specifically induced during P-limitation. Mutants of this gene, which we call REPRESSOR OF EXCESSIVE ROOT HAIR ELONGATION 1 (RXR1), have much longer RHs, while over-expression of the gene results in much shorter RHs. RXR1 physically interacts with a Rab-GTPase (RXR2), and an rxr2 mutant phenocopies the rxr1 mutant. Overexpression of a Brachypodium distachyon RXR1 homolog resulted in repression of RH elongation in Brachypodium. Taken together, our results reveal a DUF506-GTPase module with a prominent role in repression of RH elongation that is conserved in monocots and dicots.


title: TGFβ signalling is required to maintain pluripotency of human naïve pluripotent stem cells

abstract: The signalling pathways that maintain primed human pluripotent stem cells (hPSCs) have been well characterised, revealing a critical role for TGF{beta}/Activin/Nodal signalling. In contrast, the signalling requirements of naive human pluripotency have not been fully established. Here, we demonstrate that TGF{beta} signalling is required to maintain naive hPSCs. The downstream effector proteins - SMAD2/3 - bind common sites in naive and primed hPSCs, including shared pluripotency genes. In naive hPSCs, SMAD2/3 additionally bind to active regulatory regions near to naive pluripotency genes. Inhibiting TGF{beta} signalling in naive hPSCs causes the downregulation of SMAD2/3-target genes and pluripotency exit. Single-cell analyses reveal that naive and primed hPSCs follow different transcriptional trajectories after inhibition of TGF{beta} signalling. Primed hPSCs differentiate into neuroectoderm cells, whereas naive hPSCs transition into trophectoderm. These results establish that there is a continuum for TGF{beta} pathway function in human pluripotency spanning a developmental window from naive to primed states.

category: cell biology


title: Condensate-mediated reactivation of mumps virus infection under stress

abstract:
Many viruses can establish long-term persistent infections in the human host and cause chronic diseases. Here we combine cell biology, whole-cell proteomics and cryo-electron tomography to uncover how cellular stress disrupts the host-virus equilibrium in persistent infection and induces viral replication, using a model of negative-stranded RNA viruses, the mumps virus. We show that phosphorylation of the largely disordered viral phosphoprotein coincides with increased partitioning of viral polymerase into pre-formed liquid-like condensates and the formation of a stable replication machinery. By obtaining the first atomic models for the authentic mumps virus nucleocapsid, we elucidate a concomitant conformational change that exposes the viral genome to its replication machinery. These events that occur within viral condensates upon stress, together with concerted down-regulation of the host antiviral defense, provide an environment that supports up-regulation of viral replication and constitute a stress-mediated switch that disrupts the host-virus equilibrium in persistent infection.

In BriefA multi-scale approach uncovers molecular and structural basis of how cellular stress provokes activation of persistent viral infection mediated by biomolecular condensates.


title: Molecular mechanisms of stress-induced reactivation in mumps virus condensates


title: Splicing Factor SRSF1 Deficiency in the Liver Triggers NASH-like Pathology via R-Loop Induced DNA Damage and Cell Death

abstract: Regulation of RNA processing contributes profoundly to tissue development and physiology. Here, we report that serine-arginine-rich splicing factor 1 (SRSF1) is essential for hepatocyte function and survival. Although SRSF1 is mainly known for its many roles in mRNA metabolism, it is also crucial for maintaining genome stability. We show that acute liver damage in the setting of targeted SRSF1 deletion in mice is primarily mediated by the excessive formation of deleterious RNA-DNA hybrids (R-loops), which induce DNA damage. Combining hepatocyte-specific transcriptome, proteome, and RNA binding analyses, we demonstrate that widespread genotoxic stress following SRSF1 depletion results in global inhibition of mRNA transcription and protein synthesis, leading to impaired metabolism and trafficking of lipids. Lipid accumulation in SRSF1-deficient hepatocytes is followed by necroptotic cell death, inflammation, and fibrosis, resulting in NASH-like liver pathology. Importantly, SRSF1-depleted human liver cancer cells recapitulate this pathogenesis illustrating a conserved and fundamental role for SRSF1 in preserving genome integrity and tissue homeostasis. Thus, our study uncovers how accumulation of detrimental R-loops impedes hepatocellular gene expression, triggering metabolic derangements and liver failure.


title: Functional analysis of Mmd2 and related PAQR genes during sex determination in mice

abstract: Sex determination in eutherian mammals is controlled by the Y-linked gene Sry, which drives the formation of testes in male embryos. Despite extensive study, the genetic steps linking Sry action and male sex determination remain largely unknown. Here, we focused on Mmd2, a gene that encodes a member of the progestin and adipoQ receptor (PAQR) family. We show that Mmd2 is expressed during the sex-determining period in XY but not XX gonads, specifically in the Sertoli cell lineage which orchestrates early testis development. Analysis of knockout mice deficient in Sox9 and Sf1 revealed that Mmd2 operates downstream of these known sex-determining genes. However, when we used CRISPR to ablate Mmd2 in the mouse, fetal testis development appeared to progress normally. To determine if other genes might have compensated for the loss of Mmd2, we identified the closely related PAQR family members Paqr8 and Mmd as also being expressed during testis development. We used CRISPR to generate mouse strains deficient in Paqr8 and Mmd, but both knockout lines appeared phenotypically normal and fertile. Finally, we generated Mmd2;Mmd and Mmd2;Paqr8 double-null embryos and again observed normal testis development. These results may reflect functional redundancy among these factors. Our findings highlight the difficulties involved in identifying genes with a functional role in sex determination and gonadal development through expression screening and loss-of-function analyses of individual candidate genes, and may help to explain the paucity of genes in which variations have been found to cause human disorders/differences of sex development.


title: De novo spatiotemporal modelling of cell-type signatures identifies novel cell populations in the developmental human heart

abstract: With the emergence of high throughput single cell techniques, the understanding of cellular diversity in biologically complex processes has rapidly increased. The next step towards comprehension of e.g. key organs in the mammal development is to obtain spatiotemporal atlases of the cellular diversity. However, targeted cell typing approaches relying on existing single cell data achieve incomplete and biased maps that could mask the molecular and cellular heterogeneity present in a tissue slide. Here we applied spage2vec, a de novo approach to spatially resolve and characterize cellular diversity during human heart development. We obtained well defined spatial maps of tissue samples from 4.5 to 9 post conception weeks, not biased by probabilistic cell typing approaches. We found previously unreported molecular diversity within cardiomyocytes and epicardial cells and identified their characteristic expression signatures by matching them with specific subpopulations found in single cell RNA sequencing datasets.


title: Minimal synthetic enhancers reveal control of the probability of transcriptional engagement and its timing by a morphogen gradient

abstract: How enhancers interpret morphogen gradients to generate spatial patterns of gene expression is a central question in developmental biology. Although recent studies have begun to elucidate that enhancers can dictate whether, when, and at what rate a promoter will engage in transcription, the complexity of endogenous enhancers calls for theoretical models with too many free parameters to quantitatively dissect these regulatory strategies. To overcome this limitation, we established a minimal synthetic enhancer system in embryos of the fruit 2y Drosophila melanogaster. Here, a gradient of the Dorsal activator is read by a single Dorsal binding site. By quantifying transcriptional activity using live imaging, our experiments revealed that this single Dorsal binding site is capable of regulating whether promoters engage in transcription in a Dorsal concentration-speci1c manner. By modulating binding-site aZnity, we determined that a genes decision to engage in transcription and its transcriptional onset time can be explained by a simple theoretical model where the promoter has to traverse multiple kinetic barriers before transcription can ensue. The experimental platform developed here pushes the boundaries of live-imaging in studying gene regulation in the early embryo by enabling the quanti1cation of the transcriptional activity driven by a single transcription factor binding site, and making it possible to build more complex enhancers from the ground up in the context of a dialogue between theory and experiment.


title: A Massively Parallel Trafficking Assay Accurately Predicts Loss of Channel Function in KCNH2 Variants

abstract: High throughput genomics has greatly facilitated identification of genetic variants. However, determining which variants contribute to disease causation is challenging with more than half of all missense variants now classified as variants of uncertain significance (VUS). A VUS leaves patients and their clinicians unable to utilize the variant information in clinical decision-making. In long QT syndrome type 2, KCNH2 channel function is directly associated with disease presentation. Therefore, functional phenotyping of KCNH2 variants can provide direct evidence to aid variant classification. Here, we investigated the expression of all codon variants in exon 2 of KCNH2 using a massively parallel trafficking assay and for a subset of 458 single nucleotide variants compared the results with peak tail current density and gating using automated patch clamp electrophysiology. Trafficking could correctly classify loss of peak tail current density variants with an AUC reaching 0.94 compared to AUCs of 0.75 to 0.8 for in silico variant classifiers. We suggest massively parallel trafficking assays can provide prospective and accurate functional assessment for all missense variants in KCNH2 and most likely many other ion channels and membrane proteins.

category: biophysics


title: Unconventional tonicity-regulated nuclear trafficking of NFAT5 mediated by KPNB1, XPOT and RUVBL2

abstract: NFAT5 is the only known mammalian tonicity-responsive transcription factor functionally implicated in diverse physiological and pathological processes. NFAT5 activity is tightly regulated by extracellular tonicity but the underlying mechanisms remain elusive. We demonstrated that NFAT5 enters the nucleus via the nuclear pore complex. We also found that NFAT5 utilizes a non-canonical nuclear localization signal (NFAT5-NLS) for nuclear imports. siRNA screening revealed that karyopherin {beta}1 (KPNB1) drives nuclear import of NFAT5 via directly interacting with NFAT5-NLS. Proteomics analysis and siRNA screening further revealed that nuclear export of NFAT5 under hypotonicity is mediated by Exportin-T, and that it requires RuvB-Like AAA type ATPase 2 (RUVBL2) as an indispensable chaperone. Our findings have identified KPNB1 and RUVBL2 as key molecules responsible for the unconventional tonicity-regulated nucleocytoplasmic shuttling of NFAT5. These findings offer an opportunity for developing novel NFAT5 targeting strategies that are potentially useful for the treatment of diseases associated with NFAT5 dysregulation.


title: Targeting the nervous system of the parasitic worm, Haemonchus contortus, with quercetin.

abstract:
High Prevalence of infection rate, limited choice of drugs and emerging resistance against these leads to an pressing need for the development of new antihelminthic drugs and drug targets. However, limited understanding of the physiology of worms has delayed the process substantially. Here, for the first time, we are reporting the tissue morphology of Haemonchus contortus, and target its nervous system with a quercetin, a naturally occurring flavonoid. Quercetin showed anthelmintic activity against all developmental stages of the nematode. Histological analysis demonstrated damage of various body parts including isthmus, brut, pseudocoele, and other organs. Mechanistic studies revealed the generation of oxidative stress and alteration of the stress response enzyme activities. Importantly, the time-dependent imaging of ROS-generation revealed the neuronal system as the primary target of quercetin in the adult worms, which eventually leads to the paralysis and death of the worms. This work demonstrates neuronal system as a novel drug target for quercetin.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/451932v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@9204caorg.highwire.dtl.DTLVardef@789005org.highwire.dtl.DTLVardef@1375539org.highwire.dtl.DTLVardef@1bc4c47_HPS_FORMAT_FIGEXP  M_FIG Graphical Abstract

Schematic representation showing generation of Reactive Oxygen Stress in the nervous system of H. contortus due to quercetin treatment.

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category: biochemistry


title: Structural basis for carbapenam C-alkylation by TokK, a B12-dependent radical SAM enzyme

abstract:
Cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes acting during carbapenem antibiotic biosynthesis carry out radical-mediated methyl transfers that underlie the therapeutic usefulness of these essential medicines. Here we present x-ray crystal structures of TokK, which are representative of this functional class, containing its two metallocofactors and determined in the presence and absence of carbapenam substrate. The structures give the first visualization of a cobalamin-dependent radical SAM methylase that employs the radical mechanism shared by a vast majority of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggests that substrate positioning governs the rate of each methylation event.

One Sentence SummaryStructural insight into a cobalamin-dependent radical SAM methylase that performs three sequential radical-mediated methylations to install the C6 side chain of a carbapenem antibiotic.


title: The SARS-CoV-2 spike reversibly samples an open-trimer conformation exposing novel epitopes

abstract:
Current COVID-19 vaccines and many clinical diagnostics are based on the structure and function of the SARS-CoV-2 spike ectodomain. Using hydrogen deuterium exchange mass spectrometry, we have uncovered that, in addition to the prefusion structure determined by cryo-EM, this protein adopts an alternative conformation that interconverts slowly with the canonical prefusion structure. This new conformation--an open trimer-- contains easily accessible RBDs. It exposes the conserved trimer interface buried in the prefusion conformation, thus exposing potential epitopes for pan-coronavirus antibody and ligand recognition. The population of this state and kinetics of interconversion are modulated by temperature, receptor binding, antibody binding, and sequence variants observed in the natural population. Knowledge of the structure and populations of this conformation will help improve existing diagnostics, therapeutics, and vaccines.

One Sentence SummaryAn alternative conformation of SARS-CoV-2 spike ectodomain modulated by temperature, binding, and sequence variants.


title: A patient-driven clinicogenomic partnership through the Metastatic Prostate Cancer Project

abstract:
Molecular profiling studies have enabled numerous discoveries for metastatic prostate cancer (MPC), but they have mostly occurred in academic medical institutions focused on select patient populations. We developed the Metastatic Prostate Cancer Project (MPCproject, mpcproject.org), a patient-partnered initiative to empower MPC patients living anywhere in the U.S. and Canada to participate in molecular research and contribute directly to translational discovery. Here we present clinicogenomic results from our partnership with the first 706 MPCproject participants. We found that a patient-centered and remote research strategy enhanced engagement with patients in rural and medically underserved areas. Furthermore, patient-reported data achieved 90% consistency with abstracted health records for therapies and provided a mechanism for patient-partners to share information about their cancer experience not documented in medical records. Among the molecular profiling data from 333 patient-partners (n = 573 samples), whole exome sequencing of 63 tumor samples obtained from hospitals across the U.S. and Canada and 19 plasma cell-free DNA (cfDNA) samples from blood donated remotely recapitulated known findings in MPC and enabled longitudinal study of prostate cancer evolution. Inexpensive ultra-low coverage whole genome sequencing of 318 cfDNA samples from donated blood revealed clinically relevant genomic changes like AR amplification, even in the context of low tumor burden. Collectively, this study illustrates the power of a longitudinal partnership with patients to generate a more representative clinical and molecular understanding of MPC.

NoteTo assist our patient-partners and the wider MPC community interpret the results of this study, we have included a glossary of terms in the Supplementary Materials.

category: cancer biology


title: Enhanced chemotaxis through spatially-regulated absolute concentration robustness

abstract: Chemotaxis, the directional motility of cells in response to spatial gradients of chemical cues, is a fundamental process behind a wide range of biological events, including the innate immune response and cancer metastasis. Recent advances in cell biology have shown that the protrusions that enable amoeboid cells to move are driven by the stochastic threshold crossings of an underlying excitable system. As a cell encounters a chemoattractant gradient, the size of this threshold is regulated spatially so that the crossings are biased towards the front of the cell. For efficient directional migration, cells must limit undesirable lateral and rear-directed protrusions. The inclusion of a control mechanism to suppress these unwanted firings would enhance chemotactic efficiency. It is known that absolute concentration robustness (ACR) exerts tight control over the mean and variance of species concentration. Here, we demonstrate how the coupling of the ACR mechanism to the cellular signaling machinery reduces the likelihood of threshold crossings in the excitable system. Moreover, we show that using the cells innate gradient sensing apparatus to direct the action of ACR to the rear, suppresses the lateral movement of the cells and that this results in improved chemotactic performance.

category: synthetic biology


title: Tracking neural markers of template formation and implementation in attentional inhibition under different distractor consistency

abstract: Performing visual search tasks requires optimal attention deployment to promote targets and inhibit distractors. Rejection templates based on the distractors feature can be built to constrain the search process. We measured electroencephalography (EEG) of human participants of both sexes when they performed a visual search task in conditions where the distractor cues were constant within a block (fixed-cueing) or changed on a trial-by-trial basis (varied-cueing). In the fixed-cueing condition, sustained decoding of the cued colors could be achieved during the retention interval and the participants with higher decoding accuracy showed larger suppression benefit of the distractor cueing in the search period. In the varied-cueing condition, the cued color could only be transiently decoded after its onset and the higher decoding accuracy was observed from the participants who demonstrated lower suppression benefit. The differential neural representations of the to-be-ignored color in the two cueing conditions as well as their reverse associations with behavioral performance implied that rejection templates were formed in the fixed-cueing condition but not in the varied-cueing condition. Additionally, we observed stronger posterior alpha lateralization and mid-frontal theta/beta power during the retention interval of the varied-cueing condition, indicating the cognitive costs in template formation caused by the trialwise change of distractor colors. Taken together, our findings revealed the neural markers associated with the critical roles of distractor consistency in linking template formation to successful inhibition.

category: neuroscience


title: The organization of the gravity-sensing system in zebrafish

abstract: Motor circuits develop in sequence from those governing fast movements to those governing slow. Here we examine whether upstream sensory circuits are organized by similar principles. Using serial-section electron microscopy in larval zebrafish, we generated a complete map of the gravity-sensing (utricular) system from the inner ear to the brainstem. We find that both sensory tuning and developmental sequence are organizing principles of vestibular topography. Patterned rostrocaudal innervation from hair cells to afferents creates a directional tuning map in the utricular ganglion, forming segregated pathways for rostral and caudal tilt. Furthermore, the mediolateral axis of the ganglion is linked to both developmental sequence and temporal kinetics. Early-born pathways carrying phasic information preferentially excite fast escape circuits, whereas later-born pathways carrying tonic signals excite slower postural and oculomotor circuits. These results demonstrate that vestibular circuits are organized by tuning direction and kinetics, aligning them with downstream motor circuits and behaviors.


title: Neurons in the Nucleus Tractus Solitarius are Selective to the Orientation of Gastric Electrical Stimulation

abstract: Gastric electrical stimulation (GES) is a bioelectric intervention for gastroparesis, obesity, and other functional gastrointestinal disorders. In a potential mechanism of action, GES activates the nerve endings of vagal afferent neurons and induces the vago-vagal reflex through the nucleus tractus solitarius (NTS) in the brainstem. However, it is unclear where and how to stimulate in order to optimize the vagal afferent responses. To address this question with electrophysiology in rats, we applied mild electrical currents to two serosal targets on the distal forestomach with dense distributions of vagal intramuscular arrays that innervated the circular and longitudinal smooth muscle layers. During stimulation, we recorded single and multi-unit responses from gastric neurons in NTS and evaluated how the recorded responses depended on the stimulus orientation and amplitude. We found that NTS responses were highly selective to the stimulus orientation for a range of stimulus amplitudes. The strongest responses were observed when the applied current flowed in the same direction as the intramuscular arrays in parallel with the underlying smooth muscle fibers. Our results suggest that gastric neurons in NTS may encode the orientation-specific activity of gastric smooth muscles relayed by vagal afferent neurons. This finding suggests that the orientation of GES is critical to effective engagement of vagal afferents and should be considered in light of the structural phenotypes of vagal terminals in the stomach.


title: Gastric Neurons in the Nucleus Tractus Solitarius are Selective to the Orientation of Gastric Electrical Stimulation


title: Pili Expression in Geobacter sulfurreducens Lacking the Putative Gene for the PilB Pilus Assembly Motor

abstract:
Multiple lines of evidence suggest that electrically conductive pili (e-pili) are an important conduit for long-range electron transport in Geobacter sulfurreducens, a common model microbe for the study of extracellular electron transport mechanisms. One strategy to study the function of e-pili has been to delete the gene for PilB, the pilus assembly motor protein, in order to prevent e-pili expression. However, we found that e-pili are still expressed after the gene for PilB is deleted. Conducting probe atomic force microscopy revealed filaments with the same diameter and similar current-voltage response as e-pili harvested from wild-type G. sulfurreducens or when e-pili are heterologously expressed from the G. sulfurreducens pilin gene in E. coli. Immunogold labeling demonstrated that a G. sulfurreducens strain expressing e-pili with a His-tag continued to express His-tag labelled e-pili when the PilB gene was deleted. Strains with the PilB gene deleted produced maximum current densities comparable to wild-type controls. These results demonstrate that deleting the gene for PilB is not an appropriate strategy for constructing strains of G. sulfurreducens without e-pili, necessitating a reinterpretation of the results of previous studies that have employed this approach.

ImportanceGeobacter sulfurreducens is a model microbe for the study of biogeochemically and technologically significant processes such as the reduction of Fe(III) oxides in soils and sediments; bioelectrochemical applications that produce electric current from waste organic matter or drive useful processes with the consumption of renewable electricity; direct interspecies electron transfer in anaerobic digestors and methanogenic soils and sediments; and metal corrosion. The phenotypes associated with gene deletions are an important strategy for determining the mechanisms for extracellular electron transfer in G. sulfurreducens. The results reported here demonstrate that a gene deletion previously thought to prevent the expression of electrically conductive pili in G. sulfurreducens does not have the intended result. Conductive pili continue to be expressed. This finding is important for interpreting the results of several previous studies on conductive pili function.


title: Glby, is a PBP with β-lactamase activity and is required for in vivo viability of M. abscessus

abstract: The prevalence of Mycobacteroides abscessus, Mab, (also known as Mycobacterium abscessus) has been increasing steadily globally. Patients with structural lung conditions such as bronchiectasis, cystic fibrosis and chronic obstructive pulmonary disease are at high risk of developing pulmonary Mab disease. The disease is often recurrent as the current treatment regimen is considered sub-efficacious. The cell wall peptidoglycan of bacteria is required for their viability and its biosynthetic pathway is enriched in proteins whose inhibition is the basis for two of the most widely used classes of antibiotics to treat bacterial infections. The peptidoglycan of Mab is distinct from that of most bacteria as its synthesis involves penicillin binding proteins (PBP) and L,D-transpeptidases. Here, we demonstrate that Mab gene locus MAB_3167c encodes a PBP (hereafter referred to as Glby) and is required for normal planktonic growth in liquid broth. Glby exhibits a strong {beta}-lactamase activity and is sensitive to {beta}-lactamase inhibitors. In a mouse model of pulmonary Mab disease, mutant lacking this gene was unable to proliferate, gradually cleared and undetectable after three weeks. In a collection of 1.046 Mab clinical isolates, there is evidence that changes in amino acid sequence that compromise Glby function are not favored. These evidences suggest that an agent that can inhibit Glby in vivo has the potential to be an efficacious treatment against Mab disease.


title: Bacterial type 1A topoisomerases maintain the stability of the genome by preventing and dealing with R-loop-and nucleotide excision repair-dependent topological stress.

abstract: E. coli type 1A topoisomerases (topos), topo I (topA) and topo III (topB) have both relaxation and decatenation activities. B. subtilis and E. coli topA topB null cells can survive owing to DNA amplifications allowing overproduction of topo IV, the main cellular decatenase that can also relax supercoiling. We show that overproducing human topo IB, a relaxase but not a decatenase, can substitute for topo IV in allowing E. coli topA null but not topA topB null cells to survive. Deleting topB exacerbates phenotypes of topA null mutants including hypernegative supercoiling, R-loop formation, and RNase HI-sensitive replication, phenotypes that are not corrected by topo IV overproduction. These phenotypes lead to Ter DNA amplification causing a chromosome segregation defect that is corrected by topo IV overproduction. Furthermore, topA topB null mutants not overproducing topo IV acquire uvrB or uvrC mutations, revealing a nucleotide excision repair (NER)-dependent problem with replication fork progression. Thus, type IA topos maintain the stability of the genome by providing essential relaxase and decatenase activities to prevent and solve topological stress related to R-loops and NER. Moreover, excess R-loop formation is well tolerated in cells that have enough topoisomerase activity to support the subsequent replication-related topological stress.


title: Dataset on the mass spectrometry-based proteomic profiling of mouse embryonic fibroblasts from a wild type and DYT-TOR1A mouse model of dystonia, basally and during stress

abstract: Here, we present quantitative subcellular compartment-specific proteomic data from wildtype and DYT-TOR1A heterozygous mouse embryonic fibroblasts (MEFs) basally and following thapsigargin treatment [1]. In this experiment, we generated MEFs from wild type and a heterozygous DYT-TOR1A mouse model of dystonia. Subsequently, these MEF cultures were treated with either 1 M thapsigargin or dimethylsulfoxide vehicle for six hours. Following treatment, the cells were fractionated into nuclear and cytosolic fractions. Liquid chromatography, tandem mass spectrometry (LC/MS/MS)-based proteomic profiling identified 65,056 unique peptides and 4801 unique proteins across all samples. The data presented here provide subcellular compartment-specific proteomic information within a dystonia model system both basally and under cellular stress. These data can inform future experiments focused on studying the function of TorsinA, the protein encoded by TOR1A, and its potential role in nucleocytoplasmic transport and proteostasis. In addition, the information in this article can also inform future mechanistic studies investigating the relationship between DYT-TOR1A dystonia and the cellular stress response to advance understanding of the pathogenesis of dystonia.


title: REPINs are facultative genomic symbionts of bacterial genomes

abstract: Mobile genetic elements (MGEs), such as transposons and insertion sequences, propagate within bacterial genomes, but persistence times in individual lineages are short. For long-term survival, MGEs must continuously invade new hosts by horizontal transfer. Theoretically, MGEs that persist for millions of years in single lineages, and are thus subject to vertical inheritance, should not exist. Here we make the case for an exception -- a class of MGE termed REPIN. REPINs are non-autonomous MGEs whose duplication depends on non-jumping RAYT transposases. Comparisons of REPINs and typical MGEs show that replication rates of REPINs are orders of magnitude lower, REPIN population size fluctuations correlate with changes in available genome space, REPIN conservation depends on RAYT function, and REPIN diversity accumulates within host lineages. From these data it follows that REPINs persist for millions of years within single host lineages. Such long-term persistence is expected to generate conflicts arising from the diverging effects of selection acting simultaneously on REPINs and host genomes. Evidence of conflict comes from analyses of REPIN abundance and diversity in two distantly related bacterial species. Taken together, our data lead to the conclusion that REPINs are ancient Darwinian replicators that have evolved enduring, beneficial relationships, with eubacterial genomes.

category: evolutionary biology


title: Ancient Darwinian replicators nested within eubacterial genomes


title: Allometry, sexual selection, and evolutionary lines of least resistance shaped the evolution of exaggerated sexual traits within the genus Tyrannus

abstract: Variational properties hold a fundamental role in shaping biological evolution, exerting control over the magnitude and direction of evolutionary change elicited by microevolutionary processes that sort variation, such as selection or drift. We studied the Tyrannus genus, as a model for examining the conditions and drivers that facilitate the repeated evolution of exaggerated, secondary sexual traits in the face of significant functional limitations. We study the role of allometry, sexual selection, and their interaction on the diversification of tail morphology in the genus, assessing whether and how they promoted or constrained phenotypic evolution. The exaggerated and functionally-constrained long feathers of deep-forked species, T. savana and T. forficatus, independently diverged from the rest of the genus following the same direction of main interspecific variation common to the entire cluster of species. However, at a macroevolutionary scale those axes summarising both sexual dimorphism and allometric variation of the deep-forked species were aligned with the between-species maximum variation axis of non deep-forked species. Thus, we are presenting evidence of amplified divergence via the co-option and reorientation of allometric shape variation involved in a sexual selection process that repeatedly drove morphology along a historically favoured direction of cladogenetic evolution.


title: Structure of ATP synthase from ESKAPE pathogen Acinetobacter baumannii

abstract:
Acinetobacter baumannii is a clinically relevant pathogen which causes multi-drug resistant, hospital-acquired infections and is a top priority target for antibiotic development. Cryo-EM structures of the A. baumannii F1Fo-ATP synthase in three conformational states reveal unique features, which represent attractive sites for the development of novel therapeutics.

One sentence summaryStructure of Acinetobacter baumannii ATP synthase


title: Benchmarking long-read genome sequence alignment tools for human genomics applications

abstract:
BackgroundThe utility of long-read genome sequencing platforms has been shown in many fields including whole genome assembly, metagenomics, and amplicon sequencing. Less clear is the applicability of long reads to reference-guided human genomics, the foundation of genomic medicine. Here, we benchmark available platform-agnostic alignment tools on datasets from nanopore and single-molecule real-time platforms to understand their suitability in producing a genome representation.

ResultsFor this study, we leveraged publicly-available data from sample NA12878 generated on Oxford Nanopore and Pacific Biosciences platforms. Each tool that was benchmarked, including GraphMap, minimap2, and NGMLR, produced the same alignment file each time. However, the different tools widely disagreed on which reads to leave unaligned, affecting the end genome coverage and the number and locations of discoverable breakpoints. Only minimap2 was computationally lightweight enough for use at scale. No alignment from one tool independently resolved all large structural variants (10,000-100,000 basepairs) present in the Database of Genome Variants (DGV) for sample NA12878. For variants larger than 1,000,000 basepairs, nanopore sequence aligned with minimap2 and NGMLR, and single-molecule real-time sequence aligned with NGMLR contained more breakpoints than are present in DGV.

ConclusionsWhen computational resources are not a limiting factor, it should be best practice to use an analysis pipeline that generates alignments with both minimap2 and NGMLR, as neither results in a comprehensive genome representation. When computational resources are limited, use of minimap2 for human genome alignment produces files sufficient to answer hypotheses and generate new questions.


title: Allosteric effect of nanobody binding on ligand-specific active states of the β2-Adrenergic Receptor

abstract:
Nanobody binding stabilizes the active state of G-protein-coupled receptor (GPCR) and modulates its affinity for bound ligands. However, the atomic level basis for this allosteric regulation remains elusive. Here, we investigate the conformational changes induced by the binding of a nanobody (Nb80) on the active-like {beta}2 adrenergic receptor ({beta}2AR) via enhanced sampling molecular dynamics simulations. Dimensionality reduction analysis shows that Nb80 stabilizes a highly active state of the {beta}2AR with a ~14 [A] outward movement of transmembrane helix 6 and close proximity of transmembrane (TM) helices 5 and 7. This is further supported by the residues located at hotspots located on TMs 5, 6 and 7, as shown by supervised machine learning methods. Besides, ligand-specific subtle differences in the conformations assumed by intercellular loop 2 and extracellular loop 2 are captured from the trajectories of various ligand-bound receptors in the presence of Nb80. Dynamic network analysis further reveals that Nb80 binding can enhance the communication between the binding sites of Nb80 and of the ligand. We identify unique allosteric signal transmission mechanisms between the Nb80-binding site and the extracellular domains in presence of full agonist and G-protein biased partial agonist, respectively. Altogether, our results provide insights into the effect of intracellular binding partners on the GPCR activation mechanism, which could be useful for structure-based drug discovery.

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title: Viscoelastic Parameterization of Human Skin Cells to Characterize Material Behavior at Multiple Timescales

abstract: Countless biophysical studies have sought distinct markers in the cellular mechanical response that could be linked to morphogenesis, homeostasis, and disease. Here, a novel iterative-fitting methodology is used to investigate the viscoelastic behavior at multiple relaxation times of human skin cells under physiologically relevant conditions. Past investigations often involved parameterizing linear elastic relationships and assuming purely Hertzian contact mechanics. However, linear elastic treatment fails to capture and properly account for the rich temporal information available in datasets. We demonstrate the performance superiority of the proposed iterative viscoelastic characterization method over standard open-search approaches. Our viscoelastic measurements revealed that 2D adherent metastatic melanoma cells exhibit reduced elasticity compared to normal counterparts--melanocytes and fibroblasts, whereas are significantly less viscous than only fibroblasts over timescales spanning three orders of magnitude. Interestingly, melanocytes are stiffer than melanoma cells, while being the less viscous cells measured. The measured loss angle indicates clear differential viscoelastic responses across multiple timescales between the measured cells. We propose the use of viscoelastic properties at multiple timescales as a mechanical biomarker of diseases. Altogether, this method provides new insight into the complex viscoelastic behavior of metastatic melanoma cells relevant to better understanding cancer metastasis aggression.


title: Adolescent Brain Cognitive Development (ABCD) Community MRI Collection and Utilities

abstract: The Adolescent Brain Cognitive Development Study (ABCD), a 10 year longitudinal neuroimaging study of the largest population based and demographically distributed cohort of 9-10 year olds (N=11,877), was designed to overcome reproducibility limitations of prior child mental health studies. Besides the fantastic wealth of research opportunities, the extremely large size of the ABCD data set also creates enormous data storage, processing, and analysis challenges for researchers. To ensure data privacy and safety, researchers are not currently able to share neuroimaging data derivatives through the central repository at the National Data Archive (NDA). However, sharing derived data amongst researchers laterally can powerfully accelerate scientific progress, to ensure the maximum public benefit is derived from the ABCD study. To simultaneously promote collaboration and data safety, we developed the ABCD-BIDS Community Collection (ABCC), which includes both curated processed data and software utilities for further analyses. The ABCC also enables researchers to upload their own custom-processed versions of ABCD data and derivatives for sharing with the research community. This NeuroResource is meant to serve as the companion guide for the ABCC. In section we describe the ABCC. Section II highlights ABCC utilities that help researchers access, share, and analyze ABCD data, while section III provides two exemplar reproducibility analyses using ABCC utilities. We hope that adoption of the ABCCs data-safe, open-science framework will boost access and reproducibility, thus facilitating progress in child and adolescent mental health research.


title: DYT-TOR1A Subcellular Proteomics Reveals Selective Vulnerability of the Nuclear Proteome to Cell Stress

abstract:
TorsinA is a AAA+ ATPase that shuttles between the ER lumen and outer nuclear envelope in an ATP-dependent manner and is functionally implicated in nucleocytoplasmic transport. We hypothesized that the DYT-TOR1A dystonia disease-causing variant, {Delta}E TorsinA, may therefore disrupt the normal subcellular distribution of proteins between the nuclear and cytosolic compartments. To test this hypothesis, we performed proteomic analysis on nuclear and cytosolic subcellular fractions from DYT-TOR1A and wildtype mouse embryonic fibroblasts (MEFs). We further examined the compartmental proteomes following exposure to thapsigargin (Tg), an endoplasmic reticulum (ER) stressor, because DYT-TOR1A dystonia models have previously shown abnormalities in cellular stress responses. Across both subcellular compartments, proteomes of DYT-TOR1A cells showed basal state disruptions consistent with an activated stress response, and in response to thapsigargin, a blunted stress response. However, the DYT-TOR1A nuclear proteome under Tg cell stress showed the most pronounced and disproportionate degree of protein disruptions - 3-fold greater than all other conditions. The affected proteins extended beyond those typically associated with stress responses, including enrichments for processes critical for neuronal synaptic function. These findings highlight the advantage of subcellular proteomics to reveal events that localize to discrete subcellular compartments and refine thinking about the mechanisms and significance of cell stress in DYT-TOR1A pathogenesis.

HighlightsO_LIThe DYT-TOR1A nuclear proteome under cell stress showed 3-fold greater protein disruptions.
C_LIO_LIDYT-TOR1A MEFs show basal proteome alterations consistent with cell stress.
C_LIO_LIThapsigargin modulation of WT stress-responsive proteins is blunted in DYT-TOR1A MEFs.
C_LIO_LITorsinB was identified as part of the cell-stress responsive proteome in WT MEFs.
C_LI

Graphical Abstract

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title: Longitudinal changes in brain activation underlying reading fluency

abstract: Reading fluency - the speed and accuracy of reading connected text - is foundational to educational success. The current longitudinal study investigates the neural correlates of fluency development using a connected-text paradigm with an individualized presentation rate. Twenty-six children completed a functional MRI task in 1st/2nd grade (time 1) and again 1-2 years later (time 2). There was a longitudinal increase in activation in the ventral occipito-temporal (vOT) cortex from time 1 to time 2. This increase was also associated with improvements in reading fluency skills and modulated by individual speed demands. These findings highlight the reciprocal relationship of the vOT region with reading proficiency and its importance for supporting the developmental transition to fluent reading. These results have implications for developing effective interventions to target increased automaticity in reading.


title: CARPOOL: A library-based platform to rapidly identify next generation chimeric antigen receptors

abstract: CD19-targeted CAR therapies have successfully treated B cell leukemias and lymphomas, but many responders later relapse or experience toxicities. CAR intracellular domains (ICDs) are key to converting antigen recognition into anti-tumor effector functions. Despite the many possible immune signaling domain combinations that could be included in CARs, almost all CARs currently rely upon CD3{zeta}, CD28, and/or 4-1BB signaling. To explore the signaling potential of CAR ICDs, we generated a library of 700,000 CD19 CAR molecules with diverse signaling domains and developed a high throughput screening platform to enable optimization of CAR signaling for anti-tumor functions. Our strategy identifies CARs with novel signaling domain combinations that elicit distinct T cell behaviors from a clinically available CAR, including enhanced proliferation and persistence, lower exhaustion, potent cytotoxicity in an in vitro tumor rechallenge condition, and comparable tumor control in vivo. This approach is readily adaptable to numerous disease models, cell types, and selection conditions, making it a promising tool for rapidly improving adoptive cell therapies and expanding their utility to new disease indications.

category: immunology


title: Genetic similarity enhances the strength of the relationship between gut bacteria and host DNA methylation

abstract: Factors such as host age, sex, diet, health status and genotype constitute the environmental envelope shaping microbial communities in the hosts gut. It has also been proposed that gut microbiota may be influenced by host epigenetics. Although the relationship between the hosts genotype/epigenotype and its associated microbiota has been the focus of a number of recent studies, the relative importance of these interactions and their biological relevance are still poorly understood. We used methylation-sensitive genotyping by sequencing to genotype and epigenotype invasive cane toads (Rhinella marina) from the species Australian range-core (three sites) and the invasion-front (three sites), and 16S rRNA gene sequencing to characterize their gut bacterial communities. We tested the effect of host genotype and epigenotype (i.e., methylome) on gut bacterial communities. Our results indicate that the genotypes, epigenotypes and gut communities of the range-core and invasion-front are significantly different from one another. We found a positive association between host pairwise genetic and epigenetic distances. More importantly, a positive relationship was found between the hosts epigenetic and gut bacterial pairwise distances. Interestingly, this association was stronger in individuals with low genetic differentiation. Our findings suggest that in range-expanding populations, where individuals are often genetically similar, the interaction between gut bacterial communities and host methylome may provide a mechanism through which invaders increase the plasticity of their response to novel environments, potentially increasing their invasion success.

category: molecular biology


title: Molecular spikes: a gold standard for single-cell RNA counting

abstract: Molecule counting is central to single-cell sequencing, yet no experimental strategy to evaluate counting performance exists. Here, we introduce molecular spikes, novel RNA spike-ins containing inbuilt unique molecular identifiers that we use to identify critical experimental and computational conditions for accurate RNA counting across single-cell RNA-sequencing methods. The molecular spikes are a new gold standard that can be widely used to validate RNA counting in single cells.


title: Scalable full-transcript coverage single cell RNA sequencing with Smart-seq3xpress

abstract: Plate-based single-cell RNA-sequencing methods with full-transcript coverage typically excel at sensitivity but are more costly and time-consuming. Here, we miniaturized and streamlined the Smart-seq3 protocol for drastically reduced cost and increased throughput. Applying Smart-seq3xpress to 16,349 human peripheral blood mononuclear cells revealed a highly granular atlas complete with both common and rare cell types whose identification previously relied on additional protein measurements or the integration with a reference atlas.


title: Directional Gaussian Mixture Models of the gut microbiome elucidate microbial spatial structure

abstract:
The gut microbiome is spatially heterogeneous, with environmental niches contributing to the distribution and composition of microbial populations. A recently developed mapping technology, MaPS-seq, aims to characterize the spatial organization of the gut microbiome by providing data about local microbial populations. However, information about the global arrangement of these populations is lost by MaPS-seq. To address this, we propose a class of Gaussian Mixture Models (GMM) with spatial dependencies between mixture components in order to computationally recover the relative spatial arrangement of microbial communities. We demonstrate on synthetic data that our spatial models can identify global spatial dynamics, accurately cluster data, and improve parameter inference over a naive GMM. We applied our model to three MaPS-Seq datasets taken from varying regions of the mouse intestine. On cecal and distal colon datasets, we find our model accurately recapitulates known spatial behaviors of the gut microbiome, including compositional differences between mucus and lumen-associated populations. Our model also seem to capture the role of a pH gradient on microbial populations in the mouse ileum and proposes new behaviors as well.

ImportanceThe spatial arrangement of the microbes in the gut microbiome is a defining characteristic of its behavior. Various experimental studies have attempted to provide glimpses into the mechanisms that contribute to microbial arrangements. However, many of these descriptions are qualitative. We developed a computational method that takes microbial spatial data and learns many of the experimentally validated spatial factors. We can then use our model to propose previously unknown spatial behaviors. Our results demonstrate that the gut microbiome, while exceptionally large, has predictable spatial patterns that can be used to help us understand its role in health and disease.

Code availabilitygithub.com/amepas/Spatial_Mbiome


title: Coupled protein synthesis and ribosome-guided piRNA processing on mRNAs

abstract: PIWI-interacting small RNAs (piRNAs) protect the germline genome and are essential for fertility. Previously, we showed that ribosomes guide the biogenesis of piRNAs from long non-coding RNAs (lncRNAs) after translating the short open reading frames (ORFs) near their 5 cap. It remained unclear, however, how ribosomes proceed downstream of ORFs and how piRNA precursors distinguish from other RNAs. It is thus important to test whether a short ORF length is required for substrate recognition for ribosome guided-piRNA biogenesis. Here, we characterized a poorly understood class of piRNAs that originate from the 3 untranslated regions (3UTRs) of protein coding genes in mice and chickens. We demonstrate that their precursors are full-length mRNAs and that post-termination 80S ribosomes guide piRNA production on 3UTRs after translation of upstream long ORFs. Similar to non-sense mediated decay (NMD), piRNA biogenesis degrades mRNA right after pioneer rounds of translation and fine-tunes protein production from mRNAs. Interestingly, however, we found that NMD, along with other surveillance pathways for ribosome recycling are temporally sequestered during the pachytene stage to allow for robust piRNA production. Although 3UTR piRNA precursor mRNAs code for distinct proteins in mice and chickens, they all harbor embedded transposable elements (TEs) and produce piRNAs that cleave TEs, suggesting that TE suppression, rather than the function of proteins, is the primary evolutionary force maintaining a subset of mRNAs as piRNA precursors. Altogether, we discover a function of the piRNA pathway in fine-tuning protein production and reveal a conserved, general piRNA biogenesis mechanism that recognizes translating RNAs regardless of their ORF length in amniotes.


title: Distributed and multifaceted effects of threat and safety

abstract: Sustained anticipation of unpredictable aversive events generates anticipatory processing that is central to anxiety. In the present functional Magnetic Resonance Study (fMRI) study, we examined how sustained threat is processed in the human brain. We used a relatively large sample (N = 109) and employed a Bayesian multilevel analysis approach to contrast threat and safe periods. Our analyses demonstrated that the effect of sustained threat is heterogeneous and distributed across the brain. Thus, the impact of threat is widespread, and not restricted to a small set of putatively emotion-related regions, such as the amygdala and the bed nucleus of the stria terminalis. Both transient and sustained, and increased and decreased responses during threat were observed. Our study reveals that transitioning between threat and safe states, and vice versa, leads to a widespread switch in brain responding that involves most of the brain.


title: Bacteria are everywhere, even in your COI marker gene data!

abstract:
The mitochondrial cytochrome C oxidase subunit I gene (COI) is commonly used in eDNA metabarcoding studies, especially for assessing metazoan diversity. Yet, a great number of COI operational taxonomic units or/and amplicon sequence variants are retrieved from such studies and referred to as "dark matter", and do not get a taxonomic assignment with a reference sequence. For a thorough investigation of this dark matter, we have developed the Dark mAtteR iNvestigator (DARN) software tool. A reference COI-oriented phylogenetic tree was built from 1,240 consensus sequences covering all the three domains of life, with more than 80% of those representing eukaryotic taxa. With respect to eukaryotes, consensus sequences at the family level were constructed from 183,330 retrieved from the Midori reference 2 database. Similarly, sequences from 559 bacterial genera and 41 archaeal were retrieved from the BOLD database. DARN makes use of the phylogenetic tree to investigate and quantify pre-processed sequences of amplicon samples to provide both a tabular and a graphical overview of phylogenetic assignments. To evaluate DARN, both environmental and bulk metabarcoding samples from different aquatic environments using various primer sets were analysed. We demonstrate that a large proportion of non-target prokaryotic organisms such as bacteria and archaea are also amplified in eDNA samples and we suggest bacterial COI sequences to be included in the reference databases used for the taxonomy assignment to allow for further analyses of dark matter. DARN source code is available on GitHub at https://github.com/hariszaf/darn and you may find it as a Docker at https://hub.docker.com/r/hariszaf/darn.

Author summaryDARN is a software approach aiming to provide further insight in the COI amplicon data coming from environmental samples. Building a COI-oriented reference phylogeny tree is a challenging task especially considering the small number of microbial curated COI sequences deposited in reference databases; e.g ~4,000 bacterial and ~150 archaeal in BOLD. Apparently, as more and more such sequences are collated, the DARN approach improves. To provide a more interactive way of communicating both our approach and our results, we strongly suggest the reader to visit this Google Collab notebook where all steps are described step by step and also this GitHub page where our results are demonstrated. Our approach corroborates the known presence of microbial sequences in COI environmental sequencing samples and highlights the need for curated bacterial and archaeal COI sequences and their integration into reference databases (i.e. Midori, BOLD, etc). We argue that DARN will benefit researchers as a quality control tool for their sequenced samples in terms of distinguishing eukaryotic from non-eukaryotic OTUs/ASVs, but also in terms of understanding the unknown unknowns.


title: Molecular architecture of the human tRNA ligase complex

abstract: RtcB enzymes are RNA ligases that play essential roles in tRNA splicing, unfolded protein response, and RNA repair. In metazoa, RtcB functions as part of a five-subunit tRNA ligase complex (tRNA-LC) along with Ddx1, Cgi-99, Fam98B and Ashwin. The human tRNA-LC or its individual subunits have been implicated in additional cellular processes including microRNA maturation, viral replication, DNA double-strand break repair and mRNA transport. Here we present a biochemical analysis of the inter-subunit interactions within the human tRNA-LC along with crystal structures of the catalytic subunit RTCB and the N-terminal domain of CGI-99. We show that the core of the human tRNA-LC is assembled from RTCB and the C-terminal alpha-helical regions of DDX1, CGI-99, and FAM98B, all of which are required for complex integrity. The N-terminal domain of CGI-99 displays structural homology to calponin-homology domains, and CGI-99 and FAM98B associate via their N-terminal domains to form a stable subcomplex. The crystal structure of GMP-bound RTCB reveals divalent metal coordination geometry in the active site, providing insights into its catalytic mechanism. Collectively, these findings shed light on the molecular architecture and mechanism of the human tRNA ligase complex, and provide a structural framework for understanding its functions in cellular RNA metabolism.


title: Serotonin GPCR-based biosensing modalities in yeast

abstract:
Serotonin is a key neurotransmitter involved in numerous physiological processes and serves as an important precursor for manufacturing bioactive indoleamines and alkaloids used in the treatment of human pathologies. In humans, serotonin sensing and signaling can occur by 12 G protein-coupled receptors (GPCRs) coupled to G proteins. To systematically assess serotonin GPCR signaling, we characterized reporter gene expression of a 144-sized library encoding all 12 human serotonin GPCRs in combination with 12 different G proteins in yeast exposed to serotonin. For the 5-HT4 receptor, we observe 25- and 64-fold changes in EC50 values and dynamic reporter gene outputs, respectively. Furthermore, we show that optimal biosensing designs enable high-resolution sensing of serotonin produced in yeast, as well as provide a platform for characterization of 19 serotonin GPCR polymorphisms found in human populations. Taken together, our study highlights serotonin biosensing modalities of relevance to both biotechnological and human health applications.

HighlightsO_LIHuman serotonin G protein-coupled receptors display promiscuous G coupling in yeast
C_LIO_LIG-coupled serotonin receptors display up to 64-fold changes in reporter expression output
C_LIO_LIDifferences in G protein evokes 25- and 2-fold difference in EC50 and sensitivity, respectively
C_LIO_LISerotonin receptor 5-HT4 and human SNP variants display physiologically relevant EC50 values in yeast
C_LIO_LI5-HT4 can be applied for high-resolution biosensing of serotonin produced from yeast
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title: Attention-based convolutional neural networks for protein-protein interaction site prediction

abstract: Proteins usually perform their cellular functions by interacting with other proteins. Accurate identification of protein-protein interaction sites (PPIs) from sequence is import for designing new drugs and developing novel therapeutics. A lot of computational models for PPIs prediction have been developed because experimental methods are slow and expensive. Most models employ a sliding window approach in which local neighbors are concatenated to present a target residue. However, those neighbors are not been distinguished by pairwise information between a neighbor and the target. In this study, we propose a novel PPIs prediction model AttCNNPPISP, which combines attention mechanism and convolutional neural networks (CNNs). The attention mechanism dynamically captures the pairwise correlation of each neighbor-target pair within a sliding window, and therefore makes a better understanding of the local environment of target residue. And then, CNNs take the local representation as input to make prediction. Experiments are employed on several public benchmark datasets. Compared with the state-of-the-art models, AttCNNPPISP significantly improves the prediction performance. Also, the experimental results demonstrate that the attention mechanism is effective in terms of constructing comprehensive context information of target residue.


title: Protein-Protein Interaction Site Prediction Based on Attention Mechanism and Convolutional Neural Networks


title: Assessing Reproducibility of High-throughput Experiments in the Case of Missing Data.

abstract:
High-throughput experiments are an essential part of modern biological and biomedical research. The outcomes of high-throughput biological experiments often have a lot of missing observations due to signals below detection levels. For example, most single-cell RNA-seq (scRNA-seq) protocols experience high levels of dropout due to the small amount of starting material, leading to a majority of reported expression levels being zero. Though missing data contain information about reproducibility, they are often excluded in the reproducibility assessment, potentially generating misleading assessments.

In this paper, we develop a regression model to assess how the reproducibility of high-throughput experiments is affected by the choices of operational factors (e.g., platform or sequencing depth) when a large number of measurements are missing. Using a latent variable approach, we extend correspondence curve regression (CCR), a recently proposed method for assessing the effects of operational factors to reproducibility, to incorporate missing values. Using simulations, we show that our method is more accurate in detecting differences in reproducibility than existing measures of reproducibility. We illustrate the usefulness of our method using a single-cell RNA-seq dataset collected on HCT116 cells. We compare the reproducibility of different library preparation platforms and study the effect of sequencing depth on reproducibility, thereby determining the cost-effective sequencing depth that is required to achieve sufficient reproducibility.