title: Genotype-phenotype correlation analysis and therapeutic development using a patient stem cell-derived disease model of Wolfram syndrome

abstract:
Wolfram syndrome is a rare genetic disorder largely caused by pathogenic variants in the WFS1 gene and manifested by diabetes mellitus, optic nerve atrophy, and progressive neurodegeneration. Recent genetic and clinical findings have revealed Wolfram syndrome as a spectrum disorder. Therefore, a genotype-phenotype correlation analysis is needed for diagnosis and therapeutic development. Here, we focus on the WFS1 c.1672C>T, p.R558C variant which is highly prevalent in the Ashkenazi-Jewish population. Clinical investigation indicates that subjects carrying the homozygous WFS1 c.1672C>T, p.R558C variant show mild forms of Wolfram syndrome phenotypes. Expression of WFS1 p.R558C is more stable compared to the other known recessive pathogenic variants associated with Wolfram syndrome. Stem cell-derived islets (SC-islets) homozygous for WFS1 c.1672C>T variant recapitulates genotype-related Wolfram phenotypes, which are milder than those of SC-islets with compound heterozygous WFS1 c.1672C>T (p.R558C), c.2654C>T (p.P885L). Enhancing residual WFS1 function by a combination treatment of chemical chaperones, sodium 4-phenylbutyrate (4-PBA) and tauroursodeoxycholic acid (TUDCA), mitigates detrimental effects caused by the WFS1 c.1672C>T, p.R558C variant and restored SC-islet function. Thus, the WFS1 c.1672C>T, p.R558C variant causes a mild form of Wolfram syndrome phenotypes, which can be remitted with a combination treatment of chemical chaperones. We demonstrate that our patient stem cell-derived disease model provides a valuable platform for further genotype-phenotype analysis and therapeutic development for Wolfram syndrome.

One sentence summaryDevelopment of personalized therapy for Wolfram syndrome using genetics and iPSC model.


title: Generation and network analysis of an RNA-seq transcriptional atlas for the rat.

abstract: The laboratory rat is an important model for biomedical research. To generate a comprehensive rat transcriptomic atlas, we curated and down-loaded 7700 rat RNA-seq datasets from public repositories, down-sampled them to a common depth and quantified expression. Data from 590 rat tissues and cells, averaged from each Bioproject, can be visualised and queried at http://biogps.org/ratatlas. Gene correlation network (GCN) analysis revealed clusters of transcripts that were tissue or cell-type restricted and contained transcription factors implicated in lineage determination. Other clusters were enriched for transcripts associated with biological processes. Many of these clusters overlap with previous data from analysis of other species whilst some (e.g. expressed specifically in immune cells, retina/pineal gland, pituitary and germ cells) are unique to these data. GCN on large subsets of the data related specifically to liver, nervous system, kidney, musculoskeletal system and cardiovascular system enabled deconvolution of cell-type specific signatures. The approach is extensible and the dataset can be used as a point of reference from which to analyse the transcriptomes of cell types and tissues that have not yet been sampled. Sets of strictly co-expressed transcripts provide a resource for critical interpretation of single cell RNA-seq data.

category: genomics


title: A combinatorial indexing strategy for epigenomic profiling of plant single cells

abstract: Understanding how cis-regulatory elements facilitate gene expression is a key question in biology. Recent advances in single-cell genomics have led to the discovery of cell-specific chromatin landscapes that underlie transcription programs. However, the high equipment and reagent costs of commercial systems limit their applications for many laboratories. In this study, we profiled the Arabidopsis root single-cell epigenome using a combinatorial index and dual PCR barcode strategy without the need of any specialized equipment. We generated chromatin accessibility profiles for 13,576 Arabidopsis thaliana root nuclei with an average of 12,784 unique Tn5 integrations per cell and 85% of the Tn5 insertions localizing to discrete accessible chromatin regions. Comparison with data generated from a commercial microfluidic platform revealed that our method is capable of unbiased identification of cell type-specific chromatin accessibility with improved throughput, quality, and efficiency. We anticipate that by removing cost, instrument, and other technical obstacles, this combinatorial indexing method will be a valuable tool for routine investigation of single-cell epigenomes and usher new insight into plant growth, development and their interactions with the environment.


title: Do You Want to Build a Genome? Benchmarking Hybrid Bacterial Genome Assembly Methods

abstract:
Long read sequencing technologies now allow routine highly contiguous assembly of bacterial genomes. However, because of the lower accuracy of some long read data, it is often combined with short read data (e.g. Illumina), to improve assembly quality. There are a number of methods available for producing such hybrid assemblies. Here we use Illumina and Oxford Nanopore (ONT) data from 49 natural isolates of Escherichia coli to characterise differences in assembly accuracy for five assembly methods (Canu, Unicycler, Raven, Flye, and Redbean). We evaluate assembly accuracy using five metrics designed to measure structural accuracy and sequence accuracy (indel and substitution frequency). We assess structural accuracy by quantifying (1) the contiguity of chromosomes and plasmids; (2) the fraction of concordantly mapped Illumina reads withheld from the assembly; and (3) whether rRNA operons are correctly oriented. We assess indel and substitution frequency by quantifying (1) the fraction of open reading frames that appear truncated and (2) the number of variants that are called using Illumina reads only. Applying these assembly metrics to a large number of E. coli strains, we find that different assembly methods offer different advantages. In particular, we find that Unicycler assemblies have the highest sequence accuracy in non-repetitive regions, while Flye and Raven tend to be the most structurally accurate. In addition, we find that there are unidentified strain-specific characteristics that affect ONT consensus accuracy, despite individual reads having similar levels of accuracy. The differences in consensus accuracy of the ONT reads can preclude accurate assembly regardless of assembly method. These results provide quantitative insight into the best approaches for hybrid assembly of bacterial genomes and the expected levels of structural and sequence accuracy. They also show that there are intrinsic idiosyncratic strain-level differences that inhibit accurate long read bacterial genome assembly. However, we also show it is possible to diagnose problematic assemblies, even in the absence of ground truth, by comparing long-read first and short-read first assemblies.

Author NotesAll supporting data, code and protocols have been provided within the article or through supplementary data files. The supporting code is available from the GitHub repository https://github.com/GeorgiaBreckell/assembly_pipeline. nine supplementary figures and three supplementary tables are available with the online version of this article.

Data summarySequence data and genome assemblies for the natural isolates are available at https://www.ebi.ac.uk/ena/browser/view/PRJEB36951. Genome assemblies for additional E. coli strains used here are available from NCBI: (MG1655, SE11, REL606, CFT073, W, IA136, O157:H7-EDL933)


title: Temporal variation in the behaviour of a cooperatively breeding bird, Jungle Babbler (Argya striata) at diel and seasonal scale

abstract: Time is an important and limited resource that can drive the trade-off between various essential activities in the lives of animals. Group-living animals need to perform different behaviour to meet their individual needs and also participate in group activities. They must, therefore, partition the available time between these activities which may vary considerably with environmental and ecological conditions. We examined time-activity budget of a cooperative passerine, Jungle Babbler (Argya striata) and how their behaviour vary across diel and seasonal scales. A repertoire of 13 behaviour was recorded of which 12 behaviour that occur throughout the year were examined further in detail. This included individual behaviour such as foraging, grooming, rest, shower and group behaviour such as allogrooming, movement, play, sentinel, mobbing and inter-group fight. Our results indicate that most of the time (about 70%) was spent performing individual behaviour and the remaining time (about 30%) was allocated to social behaviour. We also found almost all behaviour varied across diel and seasonal scale with respect to proportion of time spent performing them. This highlights the impact of environmental factors on how animals partition their time to perform various activities. Our study also lays the foundation for future studies examining the role of ecological factors such as habitat type and predation pressure in driving these patterns of behaviour in Jungle Babblers.

category: animal behavior and cognition


title: Proposed Mechanism for Monomethylarsonate Reductase Activity of Human Omega-class Glutathione Transferase GSTO1

abstract: Contamination of drinking water with toxic inorganic arsenic is a major public health issue. The mechanisms of enzymes and transporters in arsenic elimination are therefore of interest. The human omega-class glutathione transferases have been previously shown to possess monomethylarsonate (V) reductase activity. To further understanding of this activity, molecular dynamics of human GSTO1-1 bound to glutathione with a monomethylarsonate isostere were simulated to reveal putative monomethylarsonate binding sites on the enzyme. The major binding site is in the active site, adjacent to the glutathione binding site. Based on this and previously reported biochemical data, a reaction mechanism for this enzyme is proposed. Further insights were gained from comparison of the human omega-class GSTs to homologs from a range of animals.

category: biochemistry


title: OptoAssay - Light-controlled Dynamic Bioassay Using Optogenetic Switches

abstract: Circumventing the limitations of current bioassays, we introduce the first light-controlled assay, the OptoAssay, towards wash- and pump-free point-of-care diagnostics. Extending the capabilities of standard bioassays with light-dependent and reversible interaction of optogenetic switches, OptoAssays enable a bi-directional movement of assay components, only by changing the wavelength of light. Combined with smartphones, OptoAssays obviate the need for external flow control systems like pumps or valves and signal readout devices.

category: bioengineering


title: Sincast: a computational framework to predict cell identities in single cell transcriptomes using bulk atlases as references

abstract:
Characterizing the molecular identity of a cell is an essential step in single cell RNA-sequencing (scRNA-seq) data analysis. Numerous tools exist for predicting cell identity using single cell reference atlases. However, many challenges remain, including correcting for inherent batch effects between reference and query data and insufficient phenotype data from the reference. One solution is to project single cell data onto established bulk reference atlases to leverage their rich phenotype information.

Sincast is a computational framework to query scRNA-seq data based on bulk reference atlases. Prior to projection, single cell data are transformed to be directly comparable to bulk data, either with pseudo-bulk aggregation or graph-based imputation to address sparse single cell expression profiles. Sincast avoids batch effect correction, and cell identity is predicted along a continuum to highlight new cell states not found in the reference atlas.

In several case study scenarios, we show that Sincast projects single cells into the correct biological niches in the expression space of the bulk reference atlas. We demonstrate the effectiveness of our imputation approach that was specifically developed for querying scRNA-seq data based on bulk reference atlases. We show that Sincast is an efficient and powerful tool for single cell profiling that will facilitate downstream analysis of scRNA-seq data.

category: bioinformatics


title: Spacemake: processing and analysis of large-scale spatial transcriptomics data

abstract: Spatial sequencing methods increasingly gain popularity within RNA biology studies. State-of-the-art techniques can read mRNA expression levels from tissue sections and at the same time register information about the original locations of the molecules in the tissue. The resulting datasets are processed and analyzed by accompanying software which, however, is incompatible across inputs from different technologies. Here, we present spacemake, a modular, robust and scalable spatial transcriptomics pipeline built in snakemake and python. Spacemake is designed to handle all major spatial transcriptomics datasets and can be readily configured to run on other technologies. It can process and analyze several samples in parallel, even if they stem from different experimental methods. Spacemakes unified framework enables reproducible data processing from raw sequencing data to automatically generated downstream analysis reports. Moreover, spacemake is built with a modular design and offers additional functionality such as sample merging, saturation analysis and analysis of long-reads as separate modules. Moreover, spacemake employs novoSpaRc to integrate spatial and single-cell transcriptomics data, resulting in increased gene counts for the spatial dataset. Spacemake is open-source, extendable and can be readily integrated with existing computational workflows.


title: Differential richness inference for 16S rRNA marker gene surveys

abstract:
Individual and environmental health outcomes are frequently linked to changes in the diversity of associated microbial communities. This makes deriving health indicators based on microbiome diversity measures essential.

While microbiome data generated using high throughput 16S rRNA marker gene surveys are appealing for this purpose, 16S surveys also generate a plethora of spurious microbial taxa. When this artificial inflation in the observed number of taxa (i.e., richness, a diversity measure) is ignored, we find that changes in the abundance of detected taxa confound current methods for inferring differences in richness.

Here we argue that the evidence of our own experiments, theory guided exploratory data analyses and existing literature, support the conclusion that most sub-genus discoveries are spurious artifacts of clustering 16S sequencing reads. We proceed based on this finding to model a 16S surveys systematic patterns of sub-genus taxa generation as a function of genus abundance to derive a robust control for false taxa accumulation.

Such controls unlock classical regression approaches for highly flexible differential richness inference at various levels of the surveyed microbial assemblage: from sample groups to specific taxa collections. The proposed methodology for differential richness inference is available through an R package, Prokounter.

Package availability: https://github.com/mskb01/prokounter


title: The Recombination Landscape of the Khoe-San - the Upper Limits of Recombination Divergence in Humans

abstract: Recombination maps are important resources for epidemiological and evolutionary analyses, however, there are currently no recombination maps representing any African population outside of those with West African ancestry. We inferred the demographic history for the Nama, an indigenous Khoe-San population of southern Africa, and derived a novel, population-specific recombination map from the whole genome sequencing of 54 Nama individuals. We hypothesized that there are no publicly available recombination maps representative of the Nama, considering the deep population divergence and subsequent isolation of the Khoe-San from other African groups. We showed that the recombination landscape of the Nama does not cluster with any continental groups with publicly available representative recombination maps. Finally, we used selection scans as an example of how fine-scale differences between the Nama recombination map and the combined Phase II HapMap recombination map can impact the outcome of selection scans.


title: Torque force in the kinesin stroke drives coupled yaw axis and orbital rotations of kinesin coated gold nanorods

abstract: Kinesin motor domains generate impulses of force and movement that have both translational and rotational components, raising the question of how the rotational component contributes to motor functions. We used a new assay in which kinesin-coated gold nanorods (kinesin-GNRs) move on suspended microtubules, for two plus-end-directed and weakly-processive kinesins: single-headed KIF1A, dimeric ZEN-4. Polarization of the light scattered by two types of kinesin-GNRs periodically oscillated as they orbited the microtubule along a left-handed helical trajectory. Our analyses revealed that each kinesin-GNR unidirectionally rotates about its yaw axis as it translocates, and that the period of this yaw-axis rotation corresponds to two periods of its left-handed helical orbit around the microtubule axis. Theoretical analyses suggest that the yaw-axis rotation enhances biased lateral displacement of the kinesin team. Our study reveals biaxial rotation as a new mode of motility in kinesin teams that may help the team to sidestep roadblocks.

category: biophysics


title: Coupled yawing and orbital rotation in kinesin motility revealed by polarization measurement of gold nanorods


title: The effects of chronic consumption of lipid-rich and delipidated bovine dairy milk on brown adipose tissue volume in wild-type mice

abstract: Brown adipose tissue (BAT) activation is associated with increased energy expenditure by inducing non-shivering thermogenesis. Ingestion of a milk fat globule membrane (MFGM) supplement and a high calorie diet are reported gateways into BAT activation. However, little is known about the effect of MFGM and high calorie diets on BAT volume. To gain insight into this, mice were maintained on a high fat (HF) or low-fat (LF) diet in conjunction with either full-cream (FC) or skim bovine dairy milk (BDM). After being maintained on their respective diets for 13 weeks, body composition, including BAT volume, was measured using X-ray microtomography. A high calorie diet resulted in an increase in BAT volume and mice consuming a HF diet in conjunction with FC BDM had significantly greater BAT volume than all other groups. Conversely, mice consuming a HF diet in addition to skim milk had lower BAT volume compared to the HF control. The data presented suggests that consumption of a high calorie diet in conjunction with FC BDM increases BAT volume in wild-type mice. This study may provide valuable insight into future studies investigating BAT volume and BAT activity in relation to environmental factors including diet.

category: physiology


title: Bayesian Metamodeling of pancreatic islet architecture and functional dynamics

abstract: The pancreatic islet (islet of Langerhans) is a mini-organ comprising several thousand endocrine cells, functioning jointly to maintain normoglycemia. Cellular networks within an islet were shown to influence its function in health and disease, but there are major gaps in our quantitative understanding of such architecture-function relations. Comprehensive modeling of an islet architecture and function requires the integration of vast amounts of information obtained through different experimental and theoretical approaches. To address this challenge, our lab has recently developed Bayesian metamodeling, a general approach for modeling complex systems by integrating heterogeneous input models. Here, we further developed metamodeling and applied it to construct a metamodel of a pancreatic islet. The metamodel relates islet architecture and function by combining a Monte-Carlo model of architecture trained on islet imaging data; and an ordinary differential equations (ODEs) mathematical model of function trained on calcium imaging, hormone imaging, and electrophysiological data. These input models are converted to a standardized statistical representation relying on Probabilistic Graphical Models; coupled by modeling their mutual relations with the physical world; and finally, harmonized through backpropagation. We validate the metamodel using existing data and use it to derive a testable hypothesis regarding the functional effect of varying intercellular connections. Since metamodeling currently requires substantial expert intervention, we also develop an automation tool for converting models to PGMs (step I) using feedforward neural networks. This automation is a first step towards automating the entire metamodeling process, working towards collaborative science through sharing of expertise, resources, data, and models.

category: systems biology


title: Moth species richness in an upland tropical rainforest: A citizen scientist assisted study

abstract: Diversity studies on moths in Australia are rare, presenting various shortfalls in knowledge that impedes and understanding of their biodiversity values and their conservation. In particular, the Wet Tropics of Australia deserves attention, given the paucity of systematic moth surveys in the region and its World Heritage Area status. To fill this knowledge gap, we conducted a study to observe moths on 191 nights over a main one-year survey period at an upland rainforest locality, and uploaded all observations on iNaturalist. We also compiled other incidental observations in the general locality by other observers and observations outside the survey period. In total, we document 4,434 observations of moths represented by 1041 distinct moth morphospecies. Of these, 703 are formally named species of moths, 146 to genus and 255 to higher taxonomic designations above genus level. Despite the rather intensive main survey effort, our results suggest that we have yet to reach a plateau in documenting the moth species richness of the locality. Using this study as a model, we show that the iNaturalist platform serves as an effective means to document and digitally curate biodiversity values at a locality, whilst providing complete data transparency and enabling broader community engagement of citizen scientists. We recommend the use of iNaturalist for future moth inventories, and as a resource for follow up meta-analyses of regional moth diversity and distributions.

category: zoology


title: Morphology of immatures of the thelytokous ant, Monomorium triviale Wheeler (Formicidae: Myrmicinae: Solenopsidini) with descriptions of the larval caste dimorphism

abstract: The ant genus Monomorium is one of the most species-rich but taxonomically problematic groups in the hyperdiverse subfamily Myrmicinae. An East Asian species, M. triviale Wheeler, produces both reproductive queens and sterile workers via obligate thelytokous parthenogenesis. Here, we describe the immature forms of M. triviale based on light and scanning electron microscopy observations, with a note on the striking caste dimorphism in the last larval instar. The last-instar queen larvae were easily recognized by their large size, "aphaenogastroid" body shape, and rows of doorknob-like tubercles on the lateral and dorsal body surface. This type of queen-specific structure has not been found in ants in general, let alone congeneric species found in Japan. In stark contrast to the queen larvae, worker larvae showed a "pheidoloid" body shape and a body surface similar to other ants. The worker larvae were estimated to have three instars, consistent with previously described congeners. The pupae of both castes had no cocoon, a characteristic commonly described in other Myrmicinae species. In total, the developmental period from egg to adult worker averaged 59 days under 25{degrees}C. We discuss possible functions of the tubercles of queen larvae based on previous studies.


title: Morphology of immatures of the thelytokous ant, Monomorium triviale Wheeler (Formicidae: Myrmicinae: Solenopsidini) with descriptions of the extraordinary last-instar queen larvae


title: Interneuron Control of C. elegans Developmental Decision-making

abstract: Animals integrate external stimuli to shape their physiological responses throughout development. In adverse environments, Caenorhabditis elegans larvae can enter a stress-resistant diapause state with arrested metabolism and reproductive physiology. Amphid sensory neurons feed into both rapid chemotactic and short-term foraging mode decisions, mediated by amphid and premotor interneurons, as well as the long-term diapause decision. We identify amphid interneurons that integrate pheromone cues and propagate this information via a neuropeptidergic pathways to influence larval developmental fate, bypassing the pre-motor system. AIA interneuron-derived FLP-2 neuropeptide signaling promotes reproductive growth and AIA activity is suppressed by pheromone. FLP-2 acts antagonistically to the insulin-like INS-1. FLP-2s growth promoting effects are inhibited by upstream metabotropic glutamatergic signaling and mediated by the broadly-expressed neuropeptide receptor NPR-30. Conversely, the AIB interneurons and their neuropeptide receptor NPR-9/GALR2 promote diapause entry. These neuropeptidergic outputs allow reuse of parts of a sensory system for a decision with a distinct timescale.

category: neuroscience


title: Developmental dynamics of the neural crest-mesenchymal axis in creating the thymic microenvironment

abstract:
The thymic stroma is composed of epithelial and non-epithelial cells that collectively provide separate microenvironments controlling the homing of blood-born precursors to the tissue, and their subsequent differentiation to functionally mature and correctly selected T cells. While thymic epithelial cells are well characterized for their role in thymopoiesis, a comparably comprehensive analysis of the non-epithelial thymic stroma is lacking. Here we explore at single cell resolution the complex composition and dynamic changes that occur over time in the non-epithelial stromal compartment. We detail across different developmental stages in human and mouse thymus, and in an experimental model of Di George syndrome, the most common form of human thymic hypoplasia, the separate transcriptomes of mouse mesothelium, fibroblasts, neural crest cells, endothelial and vascular mural cells. The detected gene expression signatures identify novel stromal subtypes and relate their individual molecular profiles to separate differentiation trajectories and functions. Specifically, we demonstrate an abundance and unprecedented heterogeneity of diverse fibroblast subtypes that emerge at discrete developmental stages and vary in their expression of key regulatory signalling circuits and components of the extracellular matrix. Taken together, these findings highlight the dynamic complexity of the non-epithelial thymus stroma and link the cells specific gene expression profiles to separate instructive roles essential for normal thymus organogenesis and tissue maintenance.

TeaserSingle cell profiling of thymic stroma identifies a dynamic contribution from neural crest cells to the thymic mesenchyme.

category: immunology


title: Therapeutic potential of macrophage colony-stimulating factor (CSF1) in chronic liver disease

abstract:
Resident and recruited macrophages control the development and proliferation of the liver. We showed previously in multiple species that treatment with a macrophage colony stimulating factor (CSF1)-Fc fusion protein initiated hepatocyte proliferation and promoted repair in models of acute hepatic injury in mice. Here we investigated the impact of CSF1-Fc on resolution of advanced fibrosis and liver regeneration, utilizing a non-resolving toxin-induced model of chronic liver injury and fibrosis in C57BL/6J mice. Co-administration of CSF1-Fc with exposure to thioacetamide (TAA) exacerbated inflammation consistent with monocyte contributions to initiation of pathology. After removal of TAA, either acute or chronic CSF1-Fc treatment promoted liver growth, prevented progression and promoted resolution of fibrosis. Acute CSF1-Fc treatment was also anti-fibrotic and pro-regenerative in a model of partial hepatectomy in mice with established fibrosis. The beneficial impacts of CSF1-Fc treatment were associated with monocyte-macrophage recruitment and increased expression of remodeling enzymes and growth factors. These studies indicate that CSF1-dependent macrophages contribute to both initiation and resolution of fibrotic injury and that CSF1-Fc has therapeutic potential in human liver disease.

Summary statementMacrophages contribute to both progression and resolution of chronic tissue injury and fibrogenesis. Administration of a macrophage growth factor promoted liver regeneration and resolution of advanced liver fibrosis in mice.


title: A mixed blessing of viruses in wastewater treatment plants

abstract: Activated sludge of wastewater treatment plants harbors a very high diversity of both microorganisms and viruses, wherein the latter control microbial dynamics and metabolisms by infection and lysis of cells. However, it remains poorly understood how viruses impact the biochemical processes of activated sludge, for example in terms of treatment efficiency and pollutant removal. Using metagenomic and metatranscriptomic deep sequencing, the present study recovered thousands of viral sequences from activated sludge samples of three conventional wastewater treatment plants. Gene-sharing network indicated that most of viruses could not be assigned to known viral genera, implying activated sludge as an underexplored reservoir for new viruses and viral diversity. in silico Predictions of virus-host linkages demonstrated that infected microbial hosts, mostly belonging to bacteria, were transcriptionally active and able to hydrolyze polymers including starches, celluloses, and proteins. Some viruses encode auxiliary metabolic genes (AMGs) involved in carbon, nitrogen, and sulfur cycling, and antibiotic resistance genes (ARGs) for resistance to multiple drugs. The former group of virus-encoded genes (i.e. AMGs) may enhance the biodegradation of contaminants like starches and celluloses, suggesting a positive role for viruses in strengthening the performance of activated sludge. However, the latter group (i.e. ARGs) would be disseminated to different microorganisms using viruses as gene shuttles, demonstrating the possibility for viruses to facilitate the spread of antibiotic resistance in the environment. Collectively, this study highlights the mixed blessing of viruses in wastewater treatment plants, and deciphers how they manipulate the biochemical processes in the activated sludge, with implications for both environmental protection and ecosystem security.

category: microbiology


title: Heteromerization of alkaline-sensitive two-pore domain potassium channels

abstract: Two-pore domain (K2P) potassium channels are active as dimers. They produce inhibitory currents regulated by a variety of stimuli. Among them, TALK1, TALK2 and TASK2 form a subfamily of structurally related K2P channels stimulated by extracellular alkalosis. The human genes encoding them are clustered on chromosomal region 6p21. They are expressed in different tissues including the pancreas. By analyzing single cell transcriptomic data, we show that these channels are co-expressed in insulin-secreting pancreatic {beta} cells. By different approaches we show that they form functional heterodimers. Heteromerization of TALK2 with TALK1 or with TASK2 endorses TALK2 with sensitivity to extracellular alkalosis in the physiological range. The association of TASK2 with TALK1 and TALK2 increases their unitary conductance. These results provide a new example of heteromerization in the K2P channel family expanding the range of their potential physiological and pathophysiological roles.

category: molecular biology


title: OaAEP1-mediated PNA-protein conjugation enables erasable imaging of membrane protein

abstract:
Methods to efficiently and site-specifically conjugate proteins to nucleic acids could enable exciting application in bioanalytics and biotechnology. Here, we report the use of the strict protein ligase to covalently ligate a protein to a peptide nucleic acid (PNA). The rapid ligation requires only a short N-terminal GL dipeptide in target protein and a C-terminal NGL tripeptide in PNA. We demonstrate the versatility of this approach by conjugating three different types of proteins with a PNA strand. The biostable PNA strand then serves as a generic landing platform for nucleic acid hybridization. Lastly, we show the erasable imaging of EGFR on HEK293 cell membrane through toehold-mediated strand displacement. This work provides a controlled tool for precise conjugation of proteins with nucleic acids through an extremely small peptide linker and facilitates further study of membrane proteins.

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title: Multifaceted control of E-cadherin dynamics by the Adaptor Protein Complex 1 during epithelial morphogenesis

abstract:
Intracellular trafficking regulates the distribution of transmembrane proteins including the key determinants of epithelial polarity and adhesion. The Adaptor Protein 1 (AP-1) complex is the key regulator of vesicle sorting, which binds many specific cargos. We examined roles of the AP-1 complex in epithelial morphogenesis, using the Drosophila wing as a paradigm. We found that AP-1 knockdown leads to ectopic tissue folding, which is consistent with the observed defects in integrin targeting to the basal cell-extracellular matrix adhesion sites. This occurs concurrently with an integrin-independent induction of cell death, which counteracts elevated proliferation and prevents hyperplasia. We discovered a distinct pool of AP-1, which localizes at the subapical Adherens Junctions. Upon AP-1 knockdown, E-cadherin is hyperinternalized from these junctions and becomes enriched at the Golgi and recycling endosomes. We then provide evidence that E-cadherin hyperinternalization acts upstream of cell death in a potential tumour-suppressive mechanism. Simultaneously, cells compensate for elevated internalization of E-cadherin by increasing its expression to maintain cell-cell adhesion.

Author SummaryThe epithelium is one of the four types of tissues found in animals and is essential for the normal development and maintenance of multicellular organisms. In this tissue, the adhesion protein E-cadherin helps keep cells together and facilitates the coordination of their behaviours. E-cadherin is highly dynamic and undergoes constant turnover by intracellular trafficking machinery. Here, we describe the contributions of one of the core components of intracellular trafficking, the AP-1 complex to E-cadherin dynamics. Using epithelial cells from Drosophila melanogaster, we discover that the AP-1 complex limits E-cadherin internalization from the plasma membrane, which is consistent with the localization of a distinct pool of the AP-1 complex at the sites of E-cadherin cell-cell adhesion. We also show that increased E-cadherin internalization triggers programmed cell death, preventing the tissue from hyperplastic overgrowth in a potential tumour-suppressive mechanism. At the same time, cells compensate for the reduction in the membrane presentation of E-cadherin by increasing its expression, therefore protecting tissue integrity.

category: cell biology


title: ETV2 regulates PARP-1 binding protein to induce ER stress-mediated cell death in tuberin-deficient cells

abstract: Lymphangioleiomyomatosis (LAM) is a rare progressive disease, characterized by mutations in the tuberous sclerosis complex genes (Tsc1 or Tsc2), and hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1). The effectiveness of mTORC1 inhibitors is limited by their lack of cytotoxic effects. Here, we report that E26 transformation specific (ETS) Variant Transcription Factor 2 (ETV2) is a critical regulator of Tsc2-deficient cell survival. Nuclear localization of ETV2 in Tsc2-deficient cells is mTORC1-independent and is enhanced by spleen tyrosine kinase (Syk) inhibition. In the nucleus, ETV2 transcriptionally regulates poly(ADP-ribose) polymerase 1 binding protein (PARPBP), a coregulator of transcription, mRNA and protein expression. Silencing of ETV2 or PARPBP in Tsc2-deficient cells induced ER-stress and increased cell death in vitro and in vivo. We also found ETV2 expression in human cells with loss of heterozygosity for TSC2 lending support to the translational relevance of our findings. In conclusion, we report a novel signaling axis unique to Syk-inhibition is mTORC1-independent and promotes a cytocidal response in Tsc2-deficient cells, and therefore, maybe a potential alternative therapeutic target in LAM.


title: Structural Basis for Mis18 Complex Assembly: Implications for Centromere Maintenance

abstract: The centromere, defined by the enrichment of CENP-A (a Histone H3 variant)-containing nucleosomes, is a specialised chromosomal locus that acts as a microtubule attachment site. During each round of the cell cycle, CENP-A levels undergo DNA replication-mediated dilution. To maintain centromere identity, CENP-A levels must be restored. A central player mediating this process is the Mis18 complex (composed of Mis18, Mis18{beta} and Mis18BP1), which recruits the CENP-A specific chaperone HJURP to centromeres for CENP-A deposition. Here, using a multipronged approach we provide the structural basis for the assembly of the Mis18 complex. We show that the Mis18/{beta} hetero-trimer (2 Mis18:1 Mis18{beta}) is assembled by the formation of a triple helical bundle with a Mis18/{beta}Yippee hetero-dimer and Mis18Yippee monomer on opposite ends. Two such Mis18/{beta} hetero-trimers, each bound to a Mis18BP1, assemble as a hetero-octamer via Mis18Yippee homo-dimerisation. Evaluation of structure-guided separation of function mutants in cells reveal structural determinants essential for Mis18 complex assembly and function.


title: Visceral organ morphogenesis via calcium-patterned muscle contractions

abstract: How organs achieve their final shape is a problem at the interface between physics and developmental biology. Organs often involve multiple interacting tissue layers that must be coordinated to orchestrate the complex shape changes of development. Intense study uncovered genetic, and physical ingredients driving the form of mono layer tissue. Yet, tracing dynamics across tissue layers, and scales - from cell to tissue, to entire organs - remains an outstanding challenge. Here, we study the midgut of Drosophila embryos as a model visceral organ, to reconstruct in toto the dynamics of multi-layer organ formation in vivo. Using light-sheet microscopy, genetics, computer vision, and tissue cartography, we extract individual tissue layers to map the time course of shape across scales from cells to organ. We identify the kinematic mechanism driving the shape change due to tissue layer interactions by linking out-of-plane motion to active contraction patterns, revealing a convergent extension process in which cells deform as they flow into deepening folds. Acute perturbations of contractility in the muscle layer using non-neuronal optogenetics reveals that these contraction patterns are due to muscle activity, which induces cell shape changes in the adjacent endoderm layer. This induction cascade relies on high frequency calcium pulses in the muscle layer, under the control of hox genes. Inhibition of targets of calcium involved in myosin phosphorylation abolishes constrictions. Our study of multi-layer organogenesis reveals how genetic patterning in one layer triggers a dynamic molecular mechanism to control a physical process in the adjacent layer, to orchestrate whole-organ shape change.

category: developmental biology


title: Low-coverage reduced representation sequencing reveals subtle within-island genetic structure in Aldabra giant tortoises

abstract: Aldabrachelys gigantea (Aldabra giant tortoise) is one of only two giant tortoise species left in the world and survives as a single wild population of over 100,000 individuals on Aldabra Atoll, Seychelles. Despite this large current population size, the species faces an uncertain future because of its extremely restricted distribution range and high vulnerability to the projected consequences of climate change. Captive-bred A. gigantea are increasingly used in rewilding programs across the region, where they are introduced to replace extinct giant tortoises in an attempt to functionally resurrect degraded island ecosystems. However, there has been little consideration of the current levels of genetic variation and differentiation within and among the islands on Aldabra. As previous microsatellite studies were inconclusive, we combined low-coverage and double digest restriction associated DNA (ddRAD) sequencing to analyze samples from 33 tortoises (11 from each main island). Using 5,426 variant sites within the tortoise genome, we detected patterns of population structure within two of the three studied islands, but no differentiation between the islands. These unexpected results highlight the importance of using genome-wide genetic markers to capture higher-resolution genetic structure to inform future management plans, even in a seemingly panmictic population. We show that low-coverage ddRAD sequencing provides an affordable alternative approach to conservation genomic projects of non-model species with large genomes.

category: ecology


title: Climatic curves as predictors in MaxEnt niche modeling

abstract: Environmental niche modelling (ENM) uses different types of variables to predict species occurrence. In widespread use are variables derived from climatic curves, i.e., average annual changes in some climatic parameter. This study shows how to use the climatic curves themselves as ENM predictors. The key step is projection of the curves constituent variables on a suitable spline basis, which preserves time-ordering of the variables and supports smoothness of predictions. Complexity of the model is controlled by sensible choice of the spline basis, followed by lasso regularization in model fitting.


title: Whole-genomes from the extinct Xerces Blue butterfly reveal low diversity and long-term population decline

abstract: The Xerces Blue (Glaucopsyche xerces) is considered to be the first butterfly to become extinct at global scale in historical times. It was notable for its chalky lavender wings with conspicuous white spots on the ventral wings. The last individuals were collected in their restricted habitat, in the dunes near the Presidio military base in San Francisco, in 1941. To explore the demographic history of this iconic butterfly and to better understand why it went extinct, we sequenced at medium coverage the genomes of four 80 to 100-year-old Xerces Blue specimens and seven historic specimens of its closest relative, the Silvery Blue (G. lygdamus). We compared these to a novel annotated genome of the Green-Underside Blue (G. alexis). Phylogenetic relationships inferred from complete mitochondrial genomes indicate that Xerces Blue was a distinct species that diverged from the Silvery Blue lineage at least 850,000 years ago. Using nuclear genomes, we show that both species experienced population growth during the MIS 7 interglacial period, but the Xerces Blue decreased to a very low effective population size subsequently, a trend opposite to that observed in the Silvery Blue. Runs of homozygosity in the Xerces Blue were significantly greater than in the Silvery Blue, suggesting a higher incidence of inbreeding. In addition, the Xerces Blue carried a higher proportion of derived, putatively deleterious amino acid-changing alleles than the Silvery Blue. These results demonstrate that the Xerces Blue experienced more than 100 thousand years of population decline, prior to its human-induced final extinction.

category: evolutionary biology


title: Divergent evolutionary trajectories shape the postmating transcriptional profiles of conspecific and heterospecifically mated females

abstract: Postmating-prezygotic (PMPZ) reproductive isolation is hypothesized to result from divergent coevolutionary trajectories of sexual selection and/or sexual conflict in isolated populations (coevolutionary divergence model). However, the genetic basis of PMPZ incompatibilities between species is poorly understood. Here, we use a comparative framework to test predictions of the coevolutionary divergence model using a large transcriptomic dataset generated from con- and heterospecifically mated Drosophila mojavensis and D. arizonae female reproductive tracts. We found striking divergence between the species in the female postmating transcriptional response to conspecific mating, including differences in differential expression (DE), alternative splicing (AS), and intron retention (IR). As predicted, heterospecific matings produced disrupted transcriptional profiles, but the overall patterns of misregulation were different between the reciprocal crosses. Moreover, we found a positive correlation between postmating transcriptional divergence between species and levels of transcriptional disruption in heterospecific crosses, indicating that mating-responsive genes that have diverged more in expression also have more disrupted transcriptional profiles in heterospecifically mated females. Overall, our results are consistent with predictions of the coevolutionary divergence model and lay the foundation for future studies aimed at identifying specific genes involved in PMPZ incompatibilities and the evolutionary forces that have contributed to their divergence in closely related species.


title: Ancient mitogenomes unravel massive genetic diversity loss during near extinction of Alpine ibex

abstract: Population bottlenecks can have dramatic consequences for the health and long-term survival of a species. A recent bottleneck event can also largely obscure our understanding of standing variation prior to the contraction. Historic population sizes can be modeled based on extant genomics, however uncertainty increases with the severity of the bottleneck. Integrating ancient genomes provides a powerful complement to retrace the evolution of genetic diversity through population fluctuations. Here, we recover 15 high-quality mitogenomes of the once nearly extinct Alpine ibex spanning 8601 {+/-} 33 BP to 1919 CE and combine these with 60 published modern genomes. Coalescent demography simulations based on modern genomes indicate population fluctuations matching major climatic change over the past millennia. Using ancient genomes, we show that mitochondrial haplotype diversity has been reduced to a fifth of the pre-bottleneck diversity with several highly differentiated mitochondrial lineages having co-existed historically. The main collapse of mitochondrial diversity coincided with human settlement expansions in the Middle Ages. The near extinction severely reduced the mitochondrial diversity. After recovery, one lineage was spread and nearly fixed across the Alps due to recolonization efforts. Contrary to expectations, we show that a second ancestral mitochondrial lineage has survived in an isolated population further south. Our study highlights that a combined approach integrating genomic data of ancient, historic and extant populations unravels major long-term population fluctuations.


title: Cocaine-induced locomotor activation differs across six sets of inbred mouse substrains

abstract:
Cocaine use disorders (CUD) are devastating for affected individuals and impose a significant burden on society, but there are currently no FDA-approved therapies. The development of novel and effective treatments has been hindered by substantial gaps in our knowledge about the etiology of these disorders. The risk for developing a CUD is influenced by genetics, the environment and complex interactions between the two. Identifying specific genes and environmental risk factors that increase CUD risk would provide an avenue for the development of novel treatments.

Rodent models of addiction-relevant behaviors have been a valuable tool for studying the genetics of response to drugs of abuse. Traditional genetic mapping using genetically and phenotypically divergent inbred mice has been successful in identifying numerous chromosomal regions that influence addiction-relevant behaviors, but these strategies rarely result in identification of the causal gene or genetic variant. To overcome this challenge, reduced complexity crosses (RCC) between closely related inbred mouse substrains have been proposed as a method for rapidly identifying and validating functional variants. The RCC approach is dependent on identifying phenotypic differences between substrains. To date, however, the study of addiction-relevant behaviors has been limited to very few sets of substrains, mostly comprising the C57BL/6 lineage.

The present study expands upon the current literature to assess cocaine-induced locomotor activation in 20 inbred mouse substrains representing six inbred strain lineages (A/J, BALB/c, FVB/N, C3H/He, DBA/2 and NOD) that were either bred in-house or supplied directly by a commercial vendor. To our knowledge, we are the first to identify significant differences in cocaine-induced locomotor response in several of these inbred substrains. The identification of substrain differences allows for the initiation of RCC populations to more rapidly identify specific genetic variants associated with acute cocaine response. The observation of behavioral profiles that differ between mice generated in-house and those that are vendor-supplied also presents an opportunity to investigate the influence of environmental factors on cocaine-induced locomotor activity.

category: genetics


title: Phase of neural oscillations as a reference frame for attention-based routing in visual cortex

abstract: Selective attention allows the brain to efficiently process the image projected onto the retina, selectively focusing neural processing resources on behaviorally relevant visual information. While previous studies have documented the crucial role of the action potential rate of single neurons in relaying such information, little is known about how the activity of single neurons relative to their neighboring network contributes to the efficient representation of attended stimuli and transmission of this information to downstream areas. Here, we show in the dorsal visual pathway of monkeys (medial superior temporal (MST) area) that neurons fire spikes preferentially at a specific phase of the ongoing population beta (~20 Hz) oscillations of the surrounding local network. This preferred spiking phase shifts towards a later phase when monkeys selectively attend towards (rather than away from) the receptive field of the neuron. This shift of the locking phase is positively correlated with the speed at which animals report a visual change. Furthermore, our computational modelling suggests that neural networks can manipulate the preferred phase of coupling by imposing differential synaptic delays on postsynaptic potentials. This distinction between the locking phase of neurons activated by the spatially attended stimulus vs. that of neurons activated by the unattended stimulus, may enable the neural system to discriminate relevant from irrelevant sensory inputs and consequently filter out distracting stimuli information by aligning the spikes which convey relevant/irrelevant information to distinct phases linked to periods of better/worse perceptual sensitivity for higher cortices. This strategy may be used to reserve the narrow windows of highest perceptual efficacy to the processing of the most behaviorally relevant information, ensuring highly efficient responses to attended sensory events.

category: animal behavior and cognition


title: Structural insights into the activation of autoinhibited human lipid flippase ATP8B1 upon substrate binding

abstract: The human P4-type ATPase ATP8B1 in complex with the auxiliary noncatalytic protein CDC50A or CDC50B mediates the transport of cell membrane lipids from the outer to the inner membrane leaflet, which is crucial to maintain the asymmetry of membrane lipid. Its dysfunction usually leads to imbalance of bile acid circulation, and eventually causing intrahepatic cholestasis diseases. Here we found that both ATP8B1-CDC50A and ATP8B1-CDC50B possess a higher ATPase activity in the presence of the most favored substrate phosphatidylserine (PS); and moreover, the PS-stimulated activity could be augmented upon the addition of bile acids. The cryo-electron microscopy structures of ATP8B1-CDC50A at 3.36 [A] and ATP8B1-CDC50B at 3.39 [A] enabled us to capture an unprecedented phosphorylated and autoinhibited state, with the N- and C-terminal tails separately inserting into the cytoplasmic inter-domain clefts of ATP8B1. The PS-bound ATP8B1-CDC50A structure at 3.98 [A] indicated the autoinhibited state could be released upon PS binding. Structural analysis combined with mutagenesis revealed the residues that determine the substrate specificity, and a unique positively charged loop in the phosphorylated domain of ATP8B1 for the recruitment of bile acids. Altogether, we updated the Post-Albers transport cycle, with an extra autoinhibited state of ATP8B1, which could be activated upon substrate binding. These findings not only provide structural insights into the ATP8B1-mediated restoration of human membrane lipid asymmetry during bile acid circulation, but also advance our understanding on the molecular mechanism of P-type ATPases.

category: biochemistry


title: Light contamination in stable isotope-labelled internal peptide standards is frequent and a potential source of false discovery and quantitation error in proteomics

abstract: In mass spectrometry-based proteomics, heavy internal standards are used to validate target peptide detections and to calibrate peptide quantification. Here we report light contamination present in heavy labelled synthetic peptides of high isotopic enrichment. Application of such peptides as assay-internal standards potentially compromises the detection and quantification especially of low abundant cellular peptides. Therefore, it is important to adopt guidelines to prevent false-positive identifications of endogenous light peptides as well as errors in their quantification from biological samples.


title: Electrical signature of heterogeneous human mesenchymal stem cells

abstract: Human mesenchymal stem cells (hMSCs) have gained traction in transplantation therapy due to their immunomodulatory, paracrine, immune-evasive, and multipotent differentiation potential. Given the heterogeneous nature of hMSCs, therapeutic treatments and robust in vivo and in vitro experiments require additional biomarkers to ensure reproducibility when using these stem cells. In this work, we utilized dielectrophoresis (DEP), a label-free electrokinetic phenomenon, to investigate and quantify the heterogeneity of hMSCs derived from the bone marrow (BM) and adipose tissue (AD). Through computer simulation, we identified that the transient slope of the DEP force spectra can be used as a metric of heterogeneity. The electrical properties of BM-hMSCs were compared to homogeneous mouse fibroblasts (NIH-3T3), human fibroblasts (WS1), and human embryonic kidney cells (HEK-293). BM-hMSCs DEP profile was most different from HEK-293 cells. We compared the DEP profiles of BM-hMSCs and AD-hMSCs and found they have similar membrane capacitances, differing cytoplasm conductivity, and transient slopes. Inducing both populations to differentiate into adipocyte and osteocyte cells revealed they behave differently in response to differentiation-inducing cytokines. Histology and RT-qPCR analyses of the differentiation-related genes revealed differences in heterogeneity between BM-hMSCs and AD-hMSCs. The differentiation profiles correlate well with the DEP profiles developed and indicate that these BM-hMSCs have higher differentiation potential than AD-hMSCs. Our results demonstrate using DEP, membrane capacitance, cytoplasm conductivity, and transient slope can uniquely characterize the inherent heterogeneity of hMSCs to guide robust and reproducible stem cell transplantation therapies.

category: bioengineering


title: From the origin and molecular diversity of the amastins, to the origin and diversity of intracellular parasitism from human Trypanosomatids

abstract:
The large families of amastins from Leishmania donovani, L. infantum, L. major, L. braziliensis and Trypanosoma cruzi are strongly associated with the evolution of intracellular parasitism of rich cells in human MHC.1 molecules such as the macrophages, dendritic cells, and Langerhans cells by these parasites, recognize the MHC-1 molecules as host receptor. The internalization and transport of the paraste in the cytoplas of infected cell is facilitated by the MHC-1 recycle and endosome formation drag and transport the parasite in the cytoplasm of infected cell. The microbody amastins participate as coreceptor potency the infection, the tropism of L. major and L. braziliensis by the cells from the skin is facilitated by two molecular interactions, the first molecular interaction is faclitated by the amastins interact the human MHC-1 molecules, and the second molecular interaction is facilitated by the numerous microbody amastins; which also participate in the biogenesis of the small prasitophorous vcuole from L. major, and large parasitophorous vacuole from L. braziliensis.

All amastins from these parasites developed deactivation domains, in different grade L. donovani develop an amastin surface coat specialized in deactivation of infected macrophages heavily glycosylated developed 38 amastins with 38 glycosylation Asp. N-Glycosylation sites and 45 N-glucosamina glycosylation sites, whereas L. infantum, L. major and L. braziliensis developed one half of glycosylated amastins in asparagine N-glycosylation sites, and T. cruzi did not developed none glycosylated amastin.

The amastins surface coat from L. donovani is rich in phosphorylation sites, developed 45 amastins with 45 casein kinase II phosphorylations sites, and 48 amastins with 48 protein kinase phosphorylation sites. L. infantum, L. braziliensis, and T. cruzi developed 32, 42, and 8 amastins, with 94, 114, 21 casein kinase II phosphorylation sites; in similar way developed 35,38, 11 amastins with 89,78, and 22 protein kinase phosphorylation sites. The family of amastins from L. donovani develop 137 phosphoserines. and 128 phosphothreonine, L. major developed 14 phosphoserine and 4 phosphothreonine; L. infantum 1 phophoserine and 7 phosphothreonine; L. braziliensis did not developed phosphoserine and phosphothreonine and T. cruzi 4 phosphoserine and 4 phosphothreonine. The results show that amastin surface coat is equiped with numerous phosphorylations sites atractive for phosphohrylases from the infected host contribute with the dephosphorylation and deactivation of infectetd host cells.

The amastins from L. major develop a membrane amastin with laminin G domain, which can interact with the collagen and heparin sulfate proteoglycan sites from the extracellular matrix of the skin tissue. Furthermore develop 14 amastins with tyrosine sulfation site, evade the activation of receptor of chemokines and the activation of the immune response by chemokines.

There is an alternative mechanism of polarization of the immune response from protective TH1 to non protective TH2.

The parasite nutrition is mediated by amastins that dissimilate the MHC-1 molecules and other subsets of proteins, the dissimilation products can be translocated through of the parasite cell membrane and employed as nutrient source.

category: bioinformatics


title: Enhanced fluorescence from semiconductor quantum dot-labelled cells excited at 280 nm

abstract: Semiconductor quantum dots (QDs) have significant advantages over more traditional fluorophores used in fluorescence microscopy including reduced photobleaching, long-term photostability and high quantum yields, but due to limitations in light sources and optics, are often excited far from their optimum excitation wavelengths in the deep-UV. Here, we present a quantitative comparison of the excitation of semiconductor QDs at a wavelength of 280 nm, compared to the longer wavelength of 365 nm, within a cellular environment. We report increased fluorescence intensity and enhanced image quality when using 280 nm excitation compared to 365 nm excitation for cell imaging across multiple datasets, with a highest average fluorescence intensity increase of 3.59-fold. We also find no significant increase in photobleaching of QDs associated with 280 nm excitation.

category: biophysics


title: Disassembly of hemidesmosomes promotes tumorigenesis in PTEN-negative prostate cancer by targeting plectin into focal adhesions

abstract: Loss of 6{beta}4-dependent hemidesmosomes has been observed during prostate cancer progression. However, the significance and underlying mechanisms by which aberrant hemidesmosome assembly may modulate tumorigenesis remain elusive. Using an extensive CRISPR/Cas9-mediated genetic engineering approaches in different prostate cancer cell lines combined with in vivo tumorigenesis studies in mice, bone marrow-on-chip assays and bioinformatics, as well as histological analysis of prostate cancer patient cohorts, we demonstrated that simultaneous loss of PTEN and hemidesmosomes induced several tumorigenic properties including proliferation, migration, resistance to anoikis, apoptosis, and drug treatment in vitro, and increased metastatic capacity in vivo. Our studies showed that these effects were driven by activation of EGFR/PI3K/Akt and FAK/Src-pathways and were abolished by plectin downregulation. Therefore, dual loss of PTEN and hemidesmosomes may have diagnostic value helping to stratify prostate cancer patients with high risk for development of aggressive disease and highlight plectin as a potential therapeutic target in prostate cancer.

category: cancer biology


title: Dosage of duplicated and antifunctionalized homeobox proteins influences spikelet development in barley

abstract: Illuminating the mechanisms of inflorescence architecture of grain crops that feed our world may strengthen the goal towards sustainable agriculture. Lateral spikelet development of barley (Hordeum vulgare L.) is such an example of a floral architectural trait regulated by VRS1 (Vulgare Row-type Spike 1 or Six-rowed Spike 1, syn. HvHOX1). Its lateral spikelet-specific expression and the quantitative nature of suppressing spikelet development were previously shown in barley. However, the mechanistic function of this gene and its paralog HvHOX2 on spikelet development is still fragmentary.Here, we show that these duplicated transcription factors (TFs) have contrasting nucleotide diversity in various barley genotypes and several Hordeum species. Despite this difference, both proteins retain their basic properties of the homeodomain leucine zipper class I family of TFs. During spikelet development, these genes exhibit similar spatiotemporal expression patterns yet with anticyclic expression levels. A gene co-expression network analysis suggested that both have an ancestral relationship but their functions appear antagonistic to each other, i.e., HvHOX1 suppresses whereas HvHOX2 rather promotes spikelet development. Our transgenic promoter-swap analysis showed that HvHOX2 can restore suppressed lateral spikelets when expression levels are increased; however, at its low endogenous expression level, HvHOX2 appears dispensable for spikelet development. Collectively, this study proposes that the dosage of the two antagonistic TFs, HvHOX1 and HvHOX2, influence spikelet development in barley.

category: plant biology


title: BBX16 mediates the repression of seedling photomorphogenesis downstream of the GUN1-GLK1 module during retrograde signaling

abstract:
O_LIPlastid-to-nucleus retrograde signals (RS) initiated by dysfunctional chloroplasts impact photomorphogenic development. We previously showed that the transcription factor GLK1 acts downstream of the RS-regulator GUN1 in photodamaging conditions to regulate not only the well-established expression of photosynthesis-associated nuclear genes (PhANGs) but also to regulate seedling morphogenesis. Specifically, the GUN1/GLK1 module inhibits the light-induced PIF-repressed transcriptional network to suppress cotyledon development when chloroplast integrity is compromised, modulating the area exposed to potentially damaging high light. However, how the GUN1/GLK1 module inhibits photomorphogenesis upon chloroplast damage remained undefined.
C_LIO_LIHere, we report the identification of BBX16 as a novel direct target of GLK1. BBX16 is induced and promotes photomorphogenesis in moderate light and it is repressed via GUN1/GLK1 after chloroplast damage. Additionally, we show that BBX16 represents a regulatory branching point downstream of GUN1/GLK1 in the regulation of PhANG expression and seedling development upon RS activation.
C_LIO_LIThe gun1 phenotype in lincomycin and the gun1-like phenotype of GLK1OX are markedly suppressed in gun1bbx16 and GLK1OXbbx16.
C_LIO_LIThis study identifies BBX16 as the first member of the BBX family involved in RS, and defines a molecular bifurcation mechanism operated by GLK1/BBX16 to optimize seedling deetiolation, and to ensure photoprotection in unfavorable light conditions.
C_LI


title: Generation of a mitochondrial protein compendium in Dictyostelium discoideum

abstract: The social amoeba Dictyostelium discoideum is a well-established model to study numerous cellular processes including cell motility, chemotaxis, and differentiation. As energy metabolism is involved in these processes, mitochondrial genetics and bioenergetics are of interest, though many features of Dictyostelium mitochondria differ from metazoans. A comprehensive inventory of mitochondrial proteins is critical to understanding mitochondrial processes and their involvement in various cellular pathways. Here, we utilized high-throughput multiplexed protein quantitation and homology analyses to generate a high-confidence mitochondrial protein compendium. Our proteomic approach, which utilizes quantitative mass spectrometry in combination with mathematical modeling, was validated through mitochondrial targeting sequence prediction and live-cell imaging. Our final compendium consists of 1082 proteins. Within our D. discoideum mitochondrial proteome, we identify many proteins that are not present in humans, yeasts, or the ancestral alpha-proteobacteria, which can serve as a foundation for future investigations into the unique mitochondria of Dictyostelium. Additionally, we leverage our compendium to highlight the complexity of metabolic reprogramming during starvation-induced development. Our compendium lays a foundation to investigate mitochondrial processes that are unique in protists, as well as for future studies to understand the functions of conserved mitochondrial proteins in health and diseases using D. discoideum as the model.

category: systems biology


title: Modelling novelty detection in the thalamocortical loop

abstract:
In complex natural environments, sensory systems are constantly exposed to a large stream of inputs. Novel or rare stimuli, which are often associated with behaviorally important events, are typically processed differently than the steady sensory background, which has less relevance. Neural signatures of such differential processing, commonly referred to as novelty detection, have been identified on the level of EEG recordings as mismatch negativity and the level of single neurons as stimulus-specific adaptation. Here, we propose a multi-scale recurrent network with synaptic depression to explain how novelty detection can arise in the whisker-related part of the somatosensory thalamocortical loop. The architecture and dynamics of the model presume that neurons in cortical layer 6 adapt, via synaptic depression, specifically to a frequently presented stimulus, resulting in reduced population activity in the corresponding cortical column when compared with the population activity evoked by a rare stimulus. This difference in population activity is then projected from the cortex to the thalamus and amplified through the interaction between neurons of the primary and reticular nuclei of the thalamus, resulting in spindle-like, rhythmic oscillations. These differentially activated thalamic oscillations are forwarded to cortical layer 4 as a late secondary response that is specific to rare stimuli that violate a particular stimulus pattern. Model results show a strong analogy between this late single neuron activity and EEG-based mismatch negativity in terms of their common sensitivity to presentation context and timescales of response latency, as observed experimentally. Our results indicate that adaptation in L6 can establish the thalamocortical dynamics that produce signatures of SSA and MMN and suggest a mechanistic model of novelty detection that could generalize to other sensory modalities.

Author summaryCortical sensory neurons have been shown to be capable of novelty detection, that is they respond more vigorously when a novel, unexpected stimulus is presented, and less so when the stimulus is part of a predictable sequence. However, the neural mechanism underlying this capability is not yet fully understood. Here, we developed a thalamocortical network model that accounts for novelty detection and reproduces physiologically observed neural response patterns in the somatosensory cortex. Specifically, our results demonstrate that the novelty signal arises from the complex recurrent interplay between thalamic neurons and cortical neurons in layers 4 and 6. This work therefore provides a concrete mechanism that can serve as a starting point for further investigating the neural circuit mechanisms underlying novelty detection.

category: neuroscience


title: Stable Cell Clones Harboring Self-Replicating SARS-CoV-2 RNAs for Drug Screen

abstract:
The development of antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been hampered by the lack of efficient cell-based replication systems that are amenable to high-throughput screens in biosafety level 2 laboratories. Here we report that stable cell clones harboring autonomously replicating SARS-CoV-2 RNAs without S, M, E genes can be efficiently derived from the baby hamster kidney (BHK-21) cell line when a pair of mutations were introduced into the non-structural protein 1 (Nsp1) of SARS-CoV-2 to ameliorate cellular toxicity associated with virus replication. In a proof-of-concept experiment we screened a 273-compound library using replicon cells and identified three compounds as novel inhibitors of SARS-CoV-2 replication. Altogether, this work establishes a robust, cell-based system for genetic and functional analyses of SARS-CoV-2 replication and for the development of antiviral drugs.

IMPORTANCESARS-CoV-2 replicon systems that have been reported up to date were unsuccessful in deriving stable cell lines harboring non-cytopathic replicons. The transient expression of viral sgmRNA or a reporter gene makes it impractical for industry-scale screening of large compound libraries using these systems. Here, for the first time, we derived stable cell clones harboring the SARS-CoV-2 replicon. These clones may now be conveniently cultured in a standard BSL-2 laboratory for high throughput screen of compound libraries. This achievement represents a ground-breaking discovery that will greatly accelerate the pace of developing treatments for COVID-19.


title: ATM-dependent formation of a novel chromatin compartment regulates the Response to DNA Double Strand Breaks and the biogenesis of translocations

abstract: DNA Double-Strand Breaks (DSBs) repair is essential to safeguard genome integrity but the contribution of chromosome folding into this process remains elusive. Here we unveiled basic principles of chromosome dynamics upon DSBs in mammalian cells, controlled by key kinases from the DNA Damage Response. We report that ATM is responsible for the reinforcement of topologically associating domains (TAD) that experience a DSB. ATM further drives the formation of a new chromatin sub-compartment ("D" compartment) upon clustering of damaged TADs decorated with {gamma}H2AX and 53BP1. "D" compartment formation mostly occurs in G1, is independent of cohesin and is enhanced upon DNA-PK pharmacological inhibition. Importantly, a subset of DNA damage responsive genes that are upregulated following DSBs also physically localize in the D sub-compartment and this ensures their optimal activation, providing a function for DSB clustering in activating the DNA Damage Response. However, these DSB-induced changes in genome organization also come at the expense of an increased translocations rate, which we could also detect on cancer genomes. Overall, our work provides a function for DSB-induced compartmentalization in orchestrating the DNA Damage Response and highlights the critical impact of chromosome architecture in genomic instability.


title: Krüppel-like factor 4 is required for development and regeneration of germline and yolk cells from somatic stem cells in planarians

abstract: Sexually reproducing animals segregate their germline from their soma. In addition to gamete-producing gonads, planarian and parasitic flatworm reproduction relies on yolk-cell-generating accessory reproductive organs (vitellaria) supporting development of yolkless oocytes. Despite the importance of vitellaria for flatworm reproduction (and parasite transmission), little is known about this unique evolutionary innovation. Here we examine reproductive system development in the planarian Schmidtea mediterranea, in which pluripotent stem cells generate both somatic and germ cell lineages. We show that a homolog of the pluripotency factor Klf4 is expressed in primordial germ cells, presumptive germline stem cells, and yolk-cell progenitors. klf4 knockdown animals fail to specify or maintain germ cells; surprisingly, they also fail to maintain yolk cells. We find that yolk cells display germ-cell-like attributes and that vitellaria are structurally analogous to gonads. In addition to identifying a new proliferative cell population in planarians (yolk cell progenitors) and defining its niche, our work provides evidence supporting the hypothesis that flatworm germ cells and yolk cells share a common evolutionary origin.


title: From group to individual - Genotyping by pool sequencing eusocial colonies

abstract:
BackgroundEusocial insects play a central role in many ecosystems, and particularly the important pollinator honeybee (Apis mellifera). One approach to facilitate their study in molecular genetics, is to consider whole colonies as single individuals by combining DNA of multiple individuals in a single pool sequencing experiment. Such a technique comes with the drawback of producing data requiring dedicated analytical methods to be fully exploited. Despite this limitation, pool sequencing data has been shown to be informative and cost-effective when working on random mating populations. Here, we present new statistical methods for exploiting pool sequencing data of eusocial colonies in order to reconstruct the genotype of the colony founder, the queen. This leverages the possibility to monitor genetic diversity, perform genomic-based studies or implement selective breeding.

ResultsUsing simulations and honeybee real data, we show that the methods allow for a fast and accurate estimation of the genetic ancestry, with correlations of 0.9 with that obtained from individual genotyping, and for an accurate reconstruction of the queen genotype, with 2% genotyping error. We further validate the inference using experimental data on colonies with both pool sequencing and individual genotyping of drones.

ConclusionIn this study we present statistical models to accurately estimate the genetic ancestry and reconstruct the genotype of the queen from pool sequencing data from workers of an eusocial colony. Such information allows to exploit pool sequencing for traditional population genetics, association studies and selective breeding. While validated in Apis mellifera, these methods are applicable to other eusocial hymenoptera species.

category: genomics


title: Identification of known and novel long non-coding RNAs potentially responsible for the effects of BMD GWAS loci

abstract: Osteoporosis, characterized by low bone mineral density (BMD), is the most common complex disease affecting bone and constitutes a major societal health problem. Genome-wide association studies (GWASs) have identified over 1100 associations influencing BMD. It has been shown that perturbations to long non-coding RNAs (lncRNAs) influence BMD and the activities of bone cells; however, the extent to which lncRNAs are involved in the genetic regulation of BMD is unknown. Here, we combined the analysis of allelic imbalance (AI) in human acetabular bone fragments with a transcriptome-wide association study (TWAS) and expression quantitative trait loci (eQTL) colocalization analysis using data from the Genotype-Tissue Expression (GTEx) project to identify lncRNAs potentially responsible for GWAS associations. We identified 27 lncRNAs in bone that are located in proximity to a BMD GWAS association and harbor SNPs demonstrating AI. Using GTEx data we identified an additional 31 lncRNAs whose expression was associated (FDR correction<0.05) with BMD through TWAS and had a colocalizing eQTL (regional colocalization probability (RCP)>0.1). The 58 lncRNAs are located in 43 BMD associations. To further support a causal role for the identified lncRNAs, we show that 23 of the 58 lncRNAs are differentially expressed as a function of osteoblast differentiation. Our approach identifies lncRNAs that are potentially responsible for BMD GWAS associations and suggest that lncRNAs play a role in the genetics of osteoporosis.


title: DOCKGROUND Membrane Protein-Protein Set

abstract: Membrane proteins play essential role in cellular mechanisms. Despite that and the major progress in experimental structure determination, they are still significantly underrepresented in Protein Data Bank. Thus, computational approaches to protein structure determination, which are important in general, are especially valuable in the case of membrane proteins and protein-protein assemblies. Due to a number of reasons, not the least of which is much greater availability of structural data, the main focus of structure prediction techniques has been on soluble proteins. Structure prediction of protein-protein complexes is a well-developed field of study. However, because of the differences in physicochemical environment in the membranes and the spatial constraints of the membranes, the generic protein-protein docking approaches are not optimal for the membrane proteins. Thus, specialized computational methods for docking of the membrane proteins must be developed. Development and benchmarking of such methods requires high-quality datasets of membrane protein-protein complexes. In this study we present a new dataset of 456 non-redundant alpha helical binary complexes. The set is significantly larger and more representative than previously developed ones. In the future, this set will become the basis for the development of docking and scoring benchmarks, similar to the ones developed for soluble proteins in the DOCKGROUND resource http://dockground.compbio.ku.edu.


title: The anterior cruciate ligament in murine post-traumatic osteoarthritis: markers and mechanics

abstract: 1Osteoarthritis (OA) is a whole joint disease that affects all knee joint tissues. Ligaments, a matrix-rich connective tissue, play an important mechanical function that stabilises the knee joint and yet their role in OA is not well studied. Recent studies have shown that ligament extracellular matrix (ECM) structure is compromised in the early stages of OA, but it remains unclear how this affects ligament function and biomechanics. In this study, the aim was to investigate the structural, cellular and viscoelastic changes in the anterior cruciate ligament (ACL) in a murine non-invasive post-traumatic OA (PTOA) model. Non-invasive mechanical loading of the knee joint of C57BL/6J mice (10-week-old) was used as a PTOA model. Knee joints were analysed for joint space mineralisation and the ACLs were assessed with histology and mechanical testing. PTOA knee joints had a 33-46% increase in joint space mineralisation and PTOA knee joint ACLs exhibited ECM modifications, including collagen birefringence and COL2 and proteoglycan deposition. ECM changes were associated with cells expressing chondrogenic markers (SOX9 and RUNX2) expanding from the tibial enthesis to the ACL midbody. Viscoelastic and mechanical changes in the ACLs from PTOA knee joints included a 20-21% decrease in tangent modulus at 2MPa of stress, and a decrease strain rate sensitivity at higher strain rates and a significant increase in relaxation during stress-relaxation, but no changes to hysteresis and ultimate load to failure. These results demonstrate that ACL pathology and viscoelastic function is compromised in murine PTOA knee joints and provides further evidence of the important role of ligaments in the knee joint organ in health and disease.

category: pathology


title: Nuclear progesterone receptor regulates blood coagulation facilitating ovulation in zebrafish

abstract: Ovulation is a dramatic remodeling process that includes rupture of blood capillaries and clotting, but coagulation is not thought to directly regulate this process. Herein, we report remarkable increases of coagulation factors V (f5, [~]3145-fold) and tissue factor (f3a, [~]120-fold) in zebrafish ovarian follicle cells during ovulation. This increase was mediated through the nuclear progestin receptor (Pgr), which is essential for ovulation in zebrafish, and was totally abolished in ovarian follicular cells from pgr-/- mutants. In addition, promoter activities of f5 and f3a were significantly enhanced by progestin (DHP) via Pgr. Similar regulation of human F5 promoter activity was induced via human PGRB, suggesting a conserved mechanism. Site-directed mutagenesis of the zebrafish f5 promoter further demonstrated a direct regulation of coagulation factors via progestin response elements. Moreover, a stark increase of erythrocytes occurred in capillaries meshed in wildtype preovulatory follicles but was absent in pgr-/- mutants. Interestingly, anticoagulants significantly inhibited ovulation both in vitro and in vivo, respectively. Furthermore, reduced fecundity was observed in f5+/- female zebrafish. Taken together, our study provides plausible evidence for steroid regulation of coagulation factors, and a new hypothesis for blood clotting triggered ovulation in vertebrates.

category: physiology


title: Nuclear Progestin Receptor Mediated Linkage of Blood Coagulation and Ovulation


title: Sexual dimorphic responses of C57BL/6 mice to Fisetin or Dasatinib and Quercetin cocktail oral treatment

abstract: Senolytic treatment in aged mice clears senescent cell burden leading to functional improvements. We hypothesized that administering senotherapeutics in young adulthood of mice would slow physiological markers of aging through mid-life. C57BL/6 mice were treated monthly with either Fisetin or a Dasatinib (D) plus Quercetin (Q) cocktail from 4-13 months of age. Fisetin treated male mice had reduced senescence-associated secretory phenotype (SASP), enhanced glucose and energy metabolism, improved cognitive performance, and increased hippocampal expression of adiponectin 1 receptor and glucose transporter 4. D+Q treated females had increased SASP expression along with accumulation of white adipose tissue, reduced energy metabolism, and cognitive performance. Senotherapeutics in young adulthood, has beneficial, negligible, or detrimental effects in mice dependent upon sex and treatment.


title: Feasibility evaluation of transtympanic laser stimulation of the cochlea from the outer ear

abstract: Infrared laser stimulation has been studied as an alternative approach to auditory prostheses. This study evaluated the feasibility of infrared laser stimulation of the cochlea from the outer ear bypassing the middle ear function. An optic fiber was inserted into the ear canal and a laser was used to irradiate the cochlea through the tympanic membrane in Mongolian gerbil. A pulsed infrared laser (10.1 mJ/cm2) and clicking sound (70 peak-to-peak equivalent sound pressure level) were presented to the animals. The amplitude of the laser-evoked cochlear response was systematically decreased following insertion of a filter between the tympanic membrane and cochlea; however, the auditory-evoked cochlear response did not decrease. The filter was removed and the laser-evoked response returned to around the original level. The amplitude ratio and the relative change in response amplitude before and during filter insertion significantly decreased as the absorbance of the infrared filter increased. These results indicate that laser irradiation could bypass the function of the middle ear and directly activate the cochlea. Therefore, an auditory prosthesis based on laser stimulation represents a possible noninvasive alternative to conventional auditory prostheses requiring surgical implants.


title: ACPA-IgG variable domain glycosylation increases before the onset of rheumatoid arthritis and stabilizes thereafter; a cross-sectional study encompassing over 1500 samples

abstract:
ObjectiveThe autoimmune response in rheumatoid arthritis (RA) is marked by anti-citrullinated protein antibodies (ACPA). A remarkable feature of ACPA-IgG is the abundant expression of N-linked glycans in the variable domain. Nonetheless, the presence of ACPA variable domain glycans (VDG) across disease stages and its response to therapy is poorly described. To understand its dynamics, we investigated the abundance of ACPA-IgG VDG in 1574 samples from individuals in different clinical disease stages.

MethodsUsing liquid chromatography, we analyzed ACPA-IgG VDG profiles of 7 different cohorts from Japan, Canada, the Netherlands and Sweden. We assessed 184 healthy, 228 pre-symptomatic, 277 arthralgia, 305 patients at RA-onset and 117 RA-patients 4, 8 and 12 months after disease onset. Additionally, we measured VDG of 234 samples from RA-patients that did or did not achieve long-term drug-free remission (DFR) during up to 16 years follow-up.

ResultsOur data show that ACPA-IgG VDG significantly increases (p<0.0001) towards disease-onset and associates with ACPA-levels and epitope spreading pre-diagnosis. A slight increase in VDG was observed in established RA and a moderate influence of treatment. Individuals who later achieved DFR displayed reduced ACPA-IgG VDG already at RA-onset.

ConclusionThe abundance of ACPA-IgG VDG rises towards RA-onset and correlates with maturation of the ACPA-response. Although, ACPA-IgG VDG levels are rather stable in established disease, a lower degree at RA-onset correlates with DFR. Even though the underlying biological mechanisms are still elusive, our data support the concept that VDG relates to an expansion of the ACPA-response pre-disease and contributes to disease-development.

category: immunology


title: A comparative analysis of memory B cell and antibody responses against Plasmodium falciparum merozoite surface protein 1 in children and adults from Uganda

abstract: Memory B cells (MBCs) and plasma antibodies against Plasmodium falciparum merozoite antigens are important components of the protective immune response against malaria. To gain understanding of how responses against P. falciparum develop in these two arms of the humoral immune system, we evaluated MBC and antibody responses against the most abundant merozoite antigen, merozoite surface protein 1 (MSP1), in individuals from a region in Uganda with high P. falciparum transmission. Our results showed that MSP1-specific B cells in adults with immunological protection against malaria were predominantly IgG+ classical MBCs, while children with incomplete protection mainly harbored IgM+ MSP1-specific classical MBCs. In contrast, anti-MSP1 plasma IgM reactivity was minimal in both children and adults. Instead, both groups showed high plasma IgG reactivity against MSP1 and whole merozoites, with broadening of the response against non-3D7 strains in adults. The antibodies encoded by MSP1-specific IgG+ MBCs carried high levels of amino acid substitutions and recognized relatively conserved epitopes on the highly variable MSP1 protein. Proteomics analysis of MSP119-specific IgG in plasma of an adult revealed a limited repertoire of anti-MSP1 antibodies, most of which were IgG1 or IgG3. Similar to MSP1- specific MBCs, anti-MSP1 IgGs had relatively high levels of amino acid substitutions and their sequences were predominantly found in classical MBCs, not atypical MBCs. Collectively, these results showed evolution of the MSP1-specific humoral immune response with cumulative P. falciparum exposure, with a shift from IgM+ to IgG+ B cell memory, diversification of B cells from germline, and stronger recognition of MSP1 variants by the plasma IgG repertoire.


title: Recurrent urinary tract infection and estrogen shape the taxonomic ecology and functional potential of the postmenopausal urobiome

abstract: Community-acquired urinary tract infection (UTI) is among the most common bacterial infections observed in humans. Postmenopausal women are a rapidly growing and underserved demographic group who are severely affected by recurrent UTI (rUTI) with a >50% recurrence rate. In this population, rUTI can persist for years, reducing quality of life and imposing a significant healthcare burden. rUTI is most often treated by antibiotics, but development of antibiotic resistance and allergy limit therapeutic options. The female urinary microbiome (urobiome) has been identified as a key component of the urogenital environment. However, compositional and functional changes in the urobiome underlying rUTI susceptibility in postmenopausal women are not well understood. Here, we used a controlled, cross-sectional cohort of postmenopausal women, to interrogate changes in urobiome structure and function linked to rUTI susceptibility by whole genome metagenomic sequencing (WGMS), advanced urine culture, estrogen metabolite profiling, and antibiotic sensitivity testing. Overall, we detected 276 bacterial, archaeal, and fungal species representing 106 genera. We find a putative commensal population consisting of species known to protect against bacterial vaginosis, such as Lactobacillus crispatus, within the urobiome of postmenopausal women who do not experience UTI. Integration of clinical metadata detected an almost exclusive enrichment of lactobacilli, including L. crispatus and L. vaginalis, in women taking estrogen hormone therapy (EHT). Integrating quantitative metabolite profiling of urinary estrogens with WGMS, we observed robust correlations between urobiome taxa, such as Bifidobacterium breve and L. crispatus, and urinary estrogen conjugate concentrations in women with no history of UTI that were absent in women with rUTI history. We further used functional metagenomic profiling and patient-derived isolate phenotyping to identify microbial metabolic pathways, antimicrobial resistance genes (ARGs), and clinically relevant antimicrobial resistance phenotypes enriched between disease-states. Our data indicate that distinct urobiome metabolic and ARG signatures are associated with current rUTI status and history. Importantly, we observed that rUTI history leaves an imprint of enriched ARGs even in women not currently experiencing UTI. Taken together, our data suggests that rUTI history and estrogen strongly shape the functional and taxonomic composition of the urobiome in postmenopausal women.

category: microbiology


title: A full-length infectious cDNA clone of a dsRNA totivirus-like virus

abstract: Totivirus-like viruses are icosahedral non-enveloped double-stranded (ds)RNA viruses belonging to a group recently discovered and provisionally assigned in the Totiviridae family. Unlike fungal and protozoan Totiviridae viruses, these totivirus-like viruses infect a diverse spectrum of metazoan hosts and currently have enormous impacts on fisheries and agriculture. We developed the first totivirus-like virus Omono River virus (OmRV) infectious full-length DNA clone and produce the infectious particles using an RNA-transcript-based method. Unlike the parent wild-type particles from nature, the reverse-genetically-generated OmRV particles had an indistinguishable cytopathic effect, infectivity, and morphology. The established system is one of the few systems that have been reported for generating a non-segmented dsRNA virus DNA clone.


title: A simple and general approach to control the activity of DNA processing enzymes

abstract: DNA processing enzymes, such as DNA polymerases and endonucleases, have found many applications in biotechnology, molecular diagnostics, and synthetic biology, among others. The development of enzymes with controllable activity, such as hot-start or light-activatable versions, has boosted their applications and improved the sensitivity and specificity of the existing ones. However, current approaches to produce controllable enzymes are experimentally demanding to develop and case specific. Here, we introduce a simple and general method to design light-start DNA processing enzymes. In order to prove its versatility, we applied our method to three DNA polymerases commonly used in biotechnology, including the Phi29 (mesophilic), Taq and Pfu polymerases, and one restriction enzyme. Light-start enzymes showed suppressed polymerase, exonuclease and endonuclease activity until they were re-activated by an UV pulse. Finally, we applied our enzymes to common molecular biology assays, and showed comparable performance to commercial hot-start enzymes.

category: molecular biology


title: Lipid nanoparticles incorporating a GalNAc ligand enable in vivo liver ANGPTL3 editing in wild-type and somatic LDLR knockout non-human primates

abstract: Standard lipid nanoparticles (LNPs) deliver gene editing cargoes to hepatocytes through receptor-mediated uptake via the low-density lipoprotein receptor (LDLR). Homozygous familial hypercholesterolemia (HoFH) is a morbid genetic disease characterized by complete or near-complete LDLR deficiency, markedly elevated blood low-density lipoprotein cholesterol (LDL-C) levels, and premature atherosclerotic cardiovascular disease. In order to enable in vivo liver gene editing in HoFH patients, we developed a novel LNP delivery technology that incorporates a targeting ligand--N-acetylgalactosamine (GalNAc)--which binds to the asialoglycoprotein receptor (ASGPR). In a cynomolgus monkey (Macaca fascicularis) non-human primate (NHP) model of HoFH created by somatic knockout of the LDLR gene via CRISPR-Cas9, treatment with GalNAc-LNPs formulated with an adenine base editor mRNA and a guide RNA (gRNA) targeting the ANGPTL3 gene yielded ~60% whole-liver editing and ~94% reduction of blood ANGPTL3 protein levels, whereas standard LNPs yielded minimal editing. Moreover, in wild-type NHPs, the editing achieved by GalNAc-LNPs compared favorably to that achieved by standard LNPs, suggesting that GalNAc-LNP delivery technology may prove useful across a range of in vivo therapeutic applications targeting the liver.


title: Genetic continuity of Indo-Iranian speakers since the Iron Age in southern Central Asia.

abstract: Since prehistoric times, South Central Asia has been at the crossroads of the movement of people, culture, and goods. Today, the Central Asias populations are divided into two cultural and linguistic groups: the Indo-Iranian and the Turko-Mongolian groups. Previous genetic studies unveiled that migrations from East Asia contributed to the spread of Turko-Mongolian populations in Central Asia and the partial replacement of the Indo-Iranian population. However, little is known about the origin of the latter. To shed light on this, we compare the genetic data on two current-day populations- Yaghnobis and Tajiks - with genome-wide data from published ancient individuals. The present Indo-Iranian populations from Central Asia display a strong genetic continuity with Iron Age samples from Turkmenistan and Tajikistan. We model Yaghnobis as a mixture of 93% Iron Age individual from Turkmenistan and 7% from Baikal. For the Tajiks, we observe a higher Baikal ancestry and an additional admixture event with a South Asian population. Our results, therefore, suggest that in addition to a complex history, Central Asia shows a remarkable genetic continuity since the Iron Age, with only limited gene flow.


title: Block aligner: fast and flexible pairwise sequence alignment with SIMD-accelerated adaptive blocks

abstract:
BackgroundThe Smith-Waterman-Gotoh alignment algorithm is the most popular method for comparing biological sequences. Recently, Single Instruction Multiple Data methods have been used to speed up alignment. However, these algorithms have limitations like being optimized for specific scoring schemes, cannot handle large gaps, or require quadratic time computation.

ResultsWe propose a new algorithm called block aligner for aligning nucleotide and protein sequences. It greedily shifts and grows a block of computed scores to span large gaps within the aligned sequences. This greedy approach is able to only compute a fraction of the DP matrix. In exchange for these features, there is no guarantee that the computed scores are accurate compared to full DP. However, in our experiments, we show that block aligner performs accurately on various realistic datasets, and it is up to 9 times faster than the popular Farrars algorithm for protein global alignments.

ConclusionsOur algorithm has applications in computing global alignments and X-drop alignments on proteins and long reads. It is available as a Rust library at https://github.com/Daniel-Liu-c0deb0t/block-aligner.