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bioRxiv2023

Identifying and tracking mobile elements in evolving compost communities yields insights into the nanobiome

Microbial evolution is driven by rapid changes in gene content mediated by horizontal gene transfer (HGT). While mobile genetic elements (MGEs) are important drivers of gene flux, the nanobiome - the zoo of Darwinian replicators that depend on microbial hosts - remains poorly characterised. New experimental approaches and analyses are necessary to advance our understanding beyond simple pairwise MGE-host interactions. To detect horizontal transfer, a bioinformatic pipeline (xenoseq) was developed to cross-compare metagenomic samples, which was then applied to metagenomic data from evolving compost communities. These communities were routinely exposed to an "MGE cocktail" derived from allopatric communities. We show that this results in horizontal transfer of a multitude of previously undetected MGEs, including bacteriophages, phage-plasmids, megaplasmids, and even nanobacteria. Sequences that spread from one community to another are shown to disproportionally carry characteristics of phages and insertion-sequences, i.e., traits of canonically parasitic MGEs. We also show that one particularly prolific mobile element - a 313 kb plasmid - correlates substantially with rates of ammonia production, which under nitrogen limitation is likely beneficial. Taken together, our data show that new experimental strategies combined with bioinformatic analyses of metagenomic data stand to provide insight into the drivers of microbial community evolution.

microbiology↗

bioRxiv2023

Comprehensive single-cell genome analysis at nucleotide resolution using the PTA Analysis Toolbox

Detection of somatic mutations in single cells has been severely hampered by technical limitations of whole genome amplification. Novel technologies including primary template-directed amplification (PTA) significantly improved the accuracy of single-cell whole genome sequencing (WGS), but still generate hundreds of artefacts per amplification reaction. We developed a comprehensive bioinformatic workflow, called the PTA Analysis Toolkit (PTATO), to accurately detect single base substitutions, small insertions and deletions (indels) and structural variants in PTA-based WGS data. PTATO includes a machine learning approach to distinguish PTA-artefacts from true mutations with high sensitivity (up to 90% for base substitution and 95% for indels), outperforming existing bioinformatic approaches. Using PTATO, we demonstrate that many hematopoietic stem and progenitor cells of patients with Fanconi anemia, which cannot be analyzed using regular WGS technologies, have normal somatic single base substitution burdens, but increased numbers of deletions. Our results show that PTATO enables studying somatic mutagenesis in the genomes of single cells with unprecedented sensitivity and accuracy.

genomics↗

bioRxiv2023

A global Corynebacterium diphtheriae genomic framework sheds light on current diphtheria reemergence

BackgroundDiphtheria, caused by Corynebacterium diphtheriae, reemerges in Europe since 2022. Genomic sequencing can inform on transmission routes and genotypes of concern, but currently, no standard approach exists to detect clinically important genomic features and to interpret emergence in the global C. diphtheriae population framework. MethodsWe developed the bioinformatics pipeline DIPHTOSCAN (available at https://gitlab.pasteur.fr/BEBP/diphtoscan) to extract from genomes of Corynebacteria of the diphtheriae species complex, medically relevant features including tox gene presence and disruption. We analyzed 101 human C. diphtheriae isolates collected in 2022 in metropolitan and overseas France (France-2022). To define the population background of this emergence, we sequenced 379 additional isolates (mainly from France, 2018-2021) and collated 870 publicly-available genomes. ResultsThe France-2022 isolates comprised 45 tox-positive (44 toxigenic) isolates, mostly imported, belonging to 10 sublineages (<500 distinct core genes). The global dataset comprised 245 sublineages and 33.9% tox- positive genomes, with DIPHTOSCAN predicting non-toxigenicity in 16.0% of these. 12% of the global isolates, and 43.6% of France-2022 ones, were multidrug resistant. Convergence of toxigenicity with penicillin and erythromycin resistance was observed in 2 isolates from France-2022. Phylogenetic lineages Gravis and Mitis contrasted strikingly in their pathogenicity-associated genes. ConclusionsThis work provides a bioinformatics tool and global population framework to analyze C. diphtheriae genomes, revealing important heterogeneities in virulence and resistance features. Emerging genotypes combining toxigenicity and first-line antimicrobial resistance represent novel threats. Genomic epidemiology studies of C. diphtheriae should be intensified globally to improve understanding of reemergence and spatial spread.

microbiology↗

bioRxiv2023

Androgen Receptor driven gene score identifies tumors with Epithelial to Mesenchymal Transition features in triple negative breast cancer

BackgroundAndrogen receptor (AR) is considered marker associated with better prognosis within hormone receptor positive tumors. Its role in triple negative tumors however is controversial showing both better and worse prognosis and different methods are used for identification of AR driven tumors. Conflicting results could be due to intrinsic molecular differences or scoring method for AR positivity. We attempted to develop an AR driven gene score and examined its utility in subtypes of breast cancer (BC). MethodsA bioinformatic pipeline was constructed and applied on publicly available microarray data sets obtained from AR positive BC cell lines treated with dihydrotestosterone (DHT). Expression levels of genes identified through the pipeline along with set of epithelial mesenchymal transition (EMT) markers, proliferation associated genes and enzymes involved in intracrinal androgen metabolism was evaluated in a cohort of Indian Breast cancer patients. Tumors were divided into AR high and low based on the gene score and association with clinical parameters, circulating androgens, disease free survival, proliferation and EMT markers were examined, all results were further validated in external public datasets. ResultsAR driven gene score was calculated as average expression of the 6 genes selected through bioinformatic analysis. 53% (133/249) tumors were classified as AR high by the gene score and had significantly better clinical parameters such as higher age, smaller tumor size, lower grade and lower proliferation. Tumors with high AR driven gene score had significantly better disease-free survival (mean survival time of 86.13 vs 72.69 months, log rank p=0.032) when compared to the AR low tumors. A subset analysis within TNBC (N=66) showed 36% (24/66) were AR high and had significantly higher expression of EMT markers (p=0.024). Though circulating levels of total testosterone was not different between the groups, intratumoral levels of 5 alfa reductase (SRD5A1) was significantly high in tumors with high AR driven score. ConclusionRole of AR in breast cancer is debatable and difficult to decipher with protein detection alone. Our results support the context dependent function of AR in driving better prognosis, while identifying its role in driving EMT within TNBC tumors.

cancer biology↗

bioRxiv2023

ALOX15, a new potential functional target of lung adenocarcinoma

PurposeThe purpose of this study is to explore whether the down-regulation of ALOX15 is related to the stage, differentiation and prognosis of lung adenocarcinoma, whether the overexpression of ALOX15 can inhibit tumor proliferation and metastasis, and whether it is related to the functional target of lung adenocarcinoma(LUAD). MethodsFirstly, bioinformatics of lung cancer patients was analyzed using the TCGA database to study the differential expression of ALOX15 in LUAD and its effect on the survival of LUAD. Then, 50 clinical tissue samples of LUAD were collected to detect the expression of ALOX15 and its relationship with the differentiation degree and stage of lung cancer. Finally, the relationship between the expression of ALOX15 and the proliferation and differentiation of LUAD cell lines (NCI-H1944, A549, PC9) with different degrees of differentiation and the construction of ALOX15 overexpression stable lines was detected. ResultsALOX15 bioinformatics analysis showed that ALOX15 decreased significantly in the early stage of LUAD and had no correlation with the survival of lung cancer patients. ALOX15 was downregulated in LUAD with low differentiation and metastasis in LUAD. LUAD cell lines showed that the lower the degree of differentiation, the lower the expression of ALOX15, and the overexpression of ALOX15 in LUAD cells inhibited the proliferation and migration of cancer cells. ConclusionThese results suggest that the expression of ALOX15 is closely related to the differentiation, proliferation, and metastasis of LUAD, and that upregulation of ALOX15 may inhibit the development of LUAD, suggesting that ALOX15 is a potential biological therapeutic target. Author summaryO_ST_ABSWhy was this study done?C_ST_ABSO_LILung cancer is one of the most frequently diagnosed cancers in the world and the leading cause of cancer-related deaths worldwide. C_LIO_LILung cancer is a heterogeneous disease with a wide range of clinicopathologic features. Lung cancer is roughly divided into non-small cell lung cancer (85% of all diagnoses) and small cell lung cancer (15% of all diagnoses). Adenocarcinoma is a common subtype of non-small cell lung cancer, and its recurrence rate is high, and the prognosis is poor. Therefore, the pathogenesis and characteristics of adenocarcinoma are studied and explored. C_LI What did the researchers do and find?O_LIBiological information database was used to analyze the expression level of arachidonic acid-15-lipoxygenase (ALOX15) in lung adenocarcinoma, and then the expression differences were discussed through clinical samples and cell experiments. C_LIO_LILow ALOX15 expression was detected in lung adenocarcinoma (LAUD) patients compared with normal tissues, and ALOX15 levels influenced LUAD development. C_LIO_LIBy infecting A549 with lentivirus and overexpressing ALOX15 of A549 and PC-9 with PC9, it was found that ALOX15 inhibited the proliferation of tumor cells C_LI What do these findings mean?O_LIarachidonic acid-15-lipoxygenase may be one novel potential biomarker for LUAD and a potential therapeutic target C_LI

cancer biology↗

bioRxiv2023

Exploring the evolution and function of Canoes intrinsically disordered region in linking cell-cell junctions to the cytoskeleton during embryonic morphogenesis

One central question for cell and developmental biologists is defining how epithelial cells can change shape and move during embryonic development without tearing tissues apart. This requires robust yet dynamic connections of cells to one another, via the cell-cell adherens junction, and of junctions to the actin and myosin cytoskeleton, which generates force. The last decade revealed that these connections involve a multivalent network of proteins, rather than a simple linear pathway. We focus on Drosophila Canoe, homolog of mammalian Afadin, as a model for defining the underlying mechanisms. Canoe and Afadin are complex, multidomain proteins that share multiple domains with defined and undefined binding partners. Both also share a long carboxy-terminal intrinsically disordered region (IDR), whose function is less well defined. IDRs are found in many proteins assembled into large multiprotein complexes. We have combined bioinformatic analysis and the use of a series of canoe mutants with early stop codons to explore the evolution and function of the IDR. Our bioinformatic analysis reveals that the IDRs of Canoe and Afadin differ dramatically in sequence and sequence properties. When we looked over shorter evolutionary time scales, we identified multiple conserved motifs. Some of these are predicted by AlphaFold to be alpha-helical, and two correspond to known protein interaction sites for alpha-catenin and F-actin. We next identified the lesions in a series of eighteen canoe mutants, which have early stop codons across the entire protein coding sequence. Analysis of their phenotypes are consistent with the idea that the IDR, including its C-terminal conserved motifs, are important for protein function. These data provide the foundation for further analysis of IDR function.

cell biology↗

bioRxiv2023

Phased chromosome-scale genome assembly of an asexual, allopolyploid root-knot nematode reveals complex subgenomic structure

We present the chromosome-scale genome of the allopolyploid root-knot nematode Meloidogyne javanica. We show that the M. javanica genome is predominantly allotetraploid, comprising two subgenomes, A and B, that most likely originated from hybridisation of two ancestral parental species. The assembly is annotated using full-length non-chimeric transcripts, comparison to reference databases, and ab initio prediction techniques, and the subgenomes are phased using ancestral k-mer spectral analysis. Subgenome B appears to show greater fission of chromosomal contigs, and while there is substantial synteny between subgenomes, we also identify regions lacking synteny that may have diverged in the ancestral genomes prior to or following hybridisation. Indels are common both between alleles within a subgenome, and between the A and B subgenomes, suggesting the M. javanica genome exists in a dynamic hypo-tetraploidy where copy number can vary along the chromosome. This annotated and phased genome assembly forms a significant resource for understanding the origins and genetics of these globally important plant pathogens. Author SummaryRoot-knot nematodes represent one of the most significant crop parasites globally. Despite their agricultural importance, only rudimentary genomic resources have been published to date, leaving a gap in the understanding of genetic mechanisms driving genome evolution and crop virulence. Here, we have used modern genomic and bioinformatic approaches to create a chromosome-scale reference genome to investigate the origins and genomic constitution of the root-knot nematode species Meloidogyne javanica. This species reproduces by ameiotic parthenogenesis and has an allopolyploid genome and is among the most damaging plant parasitic nematodes with a large and evolving plant host range. Utilising modern long-range DNA sequencing and bioinformatics approaches, we successfully phased the genome into its constituent subgenomes, a first for this agriculturally important clade. While we find the genomic landscape is mostly syntenic between subgenomes, we identified regions of minimal similarity, and highlight structural divergence between subgenomes. We demonstrate that this species was originally tetraploid, but insertions and deletions have been the major force in generating diversity, resulting in a hypo-tetraploid genome with local variations in ploidy.

genomics↗

bioRxiv2023

Systems analysis of miR-199a/b-5p and multiple miR-199a/b-5p targets during chondrogenesis

Changes in chondrocyte gene expression can contribute to the development of osteoarthritis (OA), and so recognition of the regulative processes during chondrogenesis can lead to a better understanding of OA. microRNAs (miRNAs) are key regulators of gene expression in chondrocytes/OA and we have used a combined experimental, bioinformatic, and systems biology approach to explore the multiple miRNA-mRNA interactions that regulate chondrogenesis. A longitudinal chondrogenesis bioinformatic analysis identified paralogues miR-199a-5p and miR-199b-5p as pro-chondrogenic regulators. Experimental work demonstrated alteration of miR-199a-5p or miR-199b-5p expression led to significant inverse modulation of key chondrogenic genes and extracellular matrix production. miR-199a/b-5p targets FZD6, ITGA3 and CAV1 were identified by inhibition experiments and verified as direct targets by luciferase assay. The experimental work was used to generate and parameterize a multi-miRNA 14-day chondrogenesis kinetic model to be used as a repository for the experimental work and as a resource for further investigation of this system. This is the first multi-miRNA model of a chondrogenesis-based system, and highlights the complex relationships between regulatory miRNAs, and their target mRNAs.

systems biology↗

bioRxiv2023

Open-loop lab-on-a-chip technology enables remote computer science training in Latinx life sciences students

Despite many interventions, science education remains highly inequitable throughout the world. Among all life sciences fields, Bioinformatics and Computational Biology suffer from the strongest underrepresentation of racial and gender minorities. Internet-enabled project-based learning (PBL) has the potential to reach underserved communities and increase the diversity of the scientific workforce. Here, we demonstrate the use of lab-on-a-chip (LoC) technologies to train Latinx life science undergraduate students in concepts of computer programming by taking advantage of open-loop cloud-integrated LoCs. We developed a context-aware curriculum to train students at over 8,000 km from the experimental site. We showed that this approach was sufficient to develop programming skills and increase the interest of students in continuing careers in Bioinformatics. Altogether, we conclude that LoC-based Internet-enabled PBL can become a powerful tool to train Latinx students and increase the diversity in STEM.

scientific communication and education↗

bioRxiv2023

A multi-network approach to Alzheimers Disease gene prioritization complements cis-regulatory prioritization with molecular quantitative trait loci

Gene prioritization within mapped disease-risk loci from genome-wide association studies (GWAS) remains one of the central bioinformatic challenges of human genetics. This problem is abundantly clear in Alzheimers Disease (AD) which has several dozen risk loci, but no therapeutically effective drug target. Dominant strategies emphasize alignment between molecular quantitative trait loci (mQTLs) and disease risk loci, under the assumption that cis-regulatory drivers of gene expression or protein abundance mediate disease risk. However, mQTL data do not capture clinically relevant time points or they derive from bulk tissue. These limitations are particularly significant in complex diseases like AD where access to diseased tissue occurs only in end-stage disease, while genetically encoded risk events accumulate over a lifetime. Network-based functional predictions, where bioinformatic databases of gene interaction networks are used to learn disease-associated gene networks to prioritize genes, complement mQTL-based prioritization. The choice of input network, however, can have a profound impact on the output gene rankings, and the optimal tissue network may not be known a priori. Here, we develop a natural extension of the popular NetWAS approach to gene prioritization that allows us to combine information from multiple networks at once. We applied our multi-network (MNFP) approach to AD GWAS data to prioritize candidate genes and compared the results to baseline, single-network models. Finally, we applied the models to prioritize genes in recently mapped AD risk loci and compared our prioritizations to the state-of-the-art mQTL approach used to functionally prioritize genes within those loci. We observed a significant concordance between the top candidates prioritized by our MNFP method and those prioritized by the mQTL approach. Our results show that network-based functional predictions are a strong complement to mQTL-based approaches and are significant to the AD genetics community as they provide a strong functional rationale to mechanistically follow-up novel AD-risk candidates. Author SummaryRisk genes give us insight into the failing molecular mechanisms that drive disease phenotypes. However, these risk genes are several layers of complexity removed from the emergent phenotypes they are influencing, the p-value that denotes their risk status gives little insight into the functional implications of that risk, and it is not clear when that risk gene may be having its effect. Methods like network-based functional prediction start to address several of these limitations by contextualizing risk genes in their broader genetic neighborhood within disease-relevant tissues. For complex diseases like Alzheimers, there are many possible relevant tissues incorporating everything from individual brain cell types to whole lobes of the brain. The work in this paper expands upon the traditional network-based functional prediction approach by considering a genes connections in multiple relevant tissue networks to prioritize candidate genes. Unlike traditional genetic risk studies, this kind prioritization benefits the Alzheimers genetics community as it provides a strong functional rationale to mechanistically follow-up on novel gene candidates.

genetics↗

bioRxiv2023

Benchmarking of Hi-C tools for scaffolding de novo genome assemblies

The implementation of Hi-C reads in the de novo genome assembly allows to order large regions of the genome in scaffolds, obtaining chromosome-level assemblies. Several bioinformatics tools have been developed for genome scaffolding with Hi-C, and all have pros and cons which need to be carefully evaluated before adoption. We developed assemblyQC, a bash pipeline that combines QUAST, BUSCO, Merqury and, optionally, Liftoff, plus a gene positioning validation script to evaluate and benchmark the performance of three scaffolders, 3d-dna, SALSA2, and YaHS, on two de novo assembly of Arabidopsis thaliana obtained from the same raw PacBio HiFi and ONT data. In our analysis, YaHS proved to be the best-performing bioinformatic tool for scaffolding of de novo genome assembly.

genomics↗

bioRxiv2023

The PALB2 DNA binding domain is an intrinsically disordered recombinase.

The Partner and Localizer of BRCA2 (PALB2) is a scaffold protein that links BRCA1 with BRCA2 to initiate homologous recombination (HR). PALB2 interaction with DNA strongly enhances HR efficiency in cells. The PALB2 DNA-binding domain (PALB2-DBD) supports strand exchange, a complex multistep reaction conducted by only a few proteins such as RecA-like recombinases and Rad52. Using bioinformatics analysis, small-angle X-ray scattering, circular dichroism, and electron paramagnetic spectroscopy, we determined that PALB2-DBD is an intrinsically disordered region (IDR) forming compact molten globule-like dimer. IDRs contribute to oligomerization synergistically with the coiled-coil interaction. Using confocal single-molecule FRET we demonstrated that PALB2-DBD compacts single-stranded DNA even in the absence of DNA secondary structures. The compaction is bimodal, oligomerization-dependent, and is driven by IDRs, suggesting a novel strand exchange mechanism. Intrinsically disordered proteins (IDPs) are prevalent in the human proteome. Novel DNA binding properties of PALB2-DBD and the complexity of strand exchange mechanism significantly expands the functional repertoire of IDPs. Multivalent interactions and bioinformatics analysis suggest that PALB2 function is likely to depend on formation of protein-nucleic acids condensates. Similar intrinsically disordered DBDs may use chaperone-like mechanism to aid formation and resolution of DNA and RNA multichain intermediates during DNA replication, repair and recombination.

biophysics↗

bioRxiv2023

Rapid molecular species identification of mammalian scat samples using nanopore adaptive sampling

AO_SCPLOWBSTRACTC_SCPLOWAccurate species identification is essential to mammalogy. Despite this necessity, rapid and accurate identification of cryptic, understudied, and elusive mammals remains challenging. Traditional barcoding of mitochondrial genes is standard for molecular identification but requires time-consuming wet-lab methodologies. Recent bioinformatic advancements for nanopore sequencing data offer exciting opportunities for non-invasive and field-based identification of mammals. Nanopore adaptive sampling (NAS), a PCR-free method, selectively sequences regions of DNA according to user-specified reference databases. Here, we utilized NAS to enrich mammalian mitochondrial genome sequencing to identify species. Fecal DNA extractions were sequenced from nine mammals, several collected in collaboration with Minnesota Tribal Nations, to demonstrate utility for NAS-barcoding of non-invasive samples. By mapping to the entire National Center for Biotechnology Information (NCBI) mammalian mitochondrial reference genome database and bioinformatically analyzing highly similar matches, we successfully produced species identifications for all of our fecal samples. Eight of nine species identifications matched previous PCR or animal/fecal morphological identifications. For the ninth species, our genetic data indicate a misidentification stemming from the original study. Our approach has a range of applications, particularly field-based wildlife research, conservation, disease surveillance, and monitoring of wildlife trade. Of importance to Minnesota tribes is invasive species monitoring, detections, and confirmation as climate impacts causes changes in biodiversity and shifts in species distributions. The rapid assessment techniques described here will be useful as new introductions and range expansions of native and invasive species may first be detected by the presence of signs such as scat rather than direct observations and will be helpful for chronically understaffed tribal natural resources agencies.

genomics↗

bioRxiv2023

Transcriptome of left ventricle and sinoatrial node in young and old C57 mice

Advancing age is the most important risk factor for cardiovascular diseases (CVDs). Two types of cells within the heart pacemaker, sinoatrial node (SAN), and within the left ventricle (LV) control two crucial characteristics of heart function, heart beat rate and contraction strength. As age advances, the hearts structure becomes remodeled, and SAN and LV cell functions deteriorate, thus increasing the risk for CVDs. However, the different molecular features of age-associated changes in SAN and LV cells have never been compared in omics scale in the context of aging. We applied deep RNA sequencing to four groups of samples, young LV, old LV, young SAN and old SAN, followed by numerous bioinformatic analyses. In addition to profiling the differences in gene expression patterns between the two heart chambers (LV vs. SAN), we also identified the chamber-specific concordant or discordant age-associated changes in: (1) genes linked to energy production related to cardiomyocyte contraction, (2) genes related to post-transcriptional processing, (3) genes involved in KEGG longevity regulating pathway, (4) prolongevity and antilongevity genes recorded and curated in the GenAge database, and (5) CVD marker genes. Our bioinformatic analysis also predicted the regulation activities and mapped the expression of upstream regulators including transcription regulators and post-transcriptional regulator miRNAs. This comprehensive analysis promotes our understanding of regulation of heart functions and will enable discovery of gene-specific therapeutic targets of CVDs in advanced age.

systems biology↗

bioRxiv2023

The Effects of Nitric Oxide on Choroidal Gene Expression

PurposeNitric oxide (NO) is recognized as an important biological mediator that controls several physiological functions, and evidence is now emerging that this molecule may play a significant role in the postnatal control of ocular growth and myopia development. We therefore sought to understand the role that nitric oxide plays in visually-guided ocular growth in order to gain insight into the underlying mechanisms of this process. MethodsChoroids were incubated in organ culture in the presence of the NO donor, PAPA-NONOate (1.5 mM). Following RNA extraction, bulk RNA-seq was used to quantify and compare choroidal gene expression in the presence and absence of PAPA-NONOate. We used bioinformatics to identify enriched canonical pathways, predicted diseases and functions, and regulatory effects of NO in the choroid. ResultsUpon treatment of normal chick choroids with the NO donor, PAPA-NONOate, we identified a total of 837 differentially expressed genes (259 upregulated genes, 578 down-regulated genes) compared with untreated controls. Among these, the top five upregulated genes were LSMEM1, STEAP4, HSPB9, and CCL19, and the top five down-regulated genes were CDCA3, SMC2, a novel gene (ENSALGALG00000050836), an uncharacterized gene (LOC107054158), and SPAG5. Bioinformatics predicted that NO treatment will activate pathways involved in cell and organismal death, necrosis, and cardiovascular system development, and inhibit pathways involved in cell proliferation, cell movement, and gene expression. ConclusionsThe findings reported herein may provide insight into possible effects of NO in the choroid during visually regulated eye growth, and help to identify targeted therapies for the treatment of myopia and other ocular diseases.

cell biology↗

bioRxiv2023

Integrative analysis of CAKUT multi-omics data

Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) is the leading cause of childhood end-stage renal disease and a significant cause of chronic kidney disease in adults. Genetic and environmental factors are known to influence CAKUT development, but the currently known disease mechanism remains incomplete. Our goal is to identify affected pathways and networks in CAKUT, and thereby aid in getting a better understanding of its pathophysiology. Multi-omics experiments, including amniotic fluid miRNome, peptidome, and proteome analyses, can shed light on foetal kidney development in non-severe CAKUT patients compared to severe CAKUT cases. We performed FAIRification of these omics data sets to facilitate their integration with external data resources. Furthermore, we analysed and integrated the omics data sets using three different bioinformatics strategies. The three bioinformatics analyses provided complementary features, but all pointed towards an important role for collagen in CAKUT development. We published the three analysis strategies as containerized workflows. These workflows can be applied to other FAIR data sets and help gaining knowledge on other rare diseases.

systems biology↗

bioRxiv2023

Structure and Biosynthesis of Hectoramide B, a Linear Depsipeptide from the Marine Cyanobacterium Moorena producens JHB Discovered via Co-culture with Candida albicans

The tropical marine cyanobacterium Moorena producens JHB is a prolific source of secondary metabolites with potential biomedical utility. Previous studies of this strain led to the discovery of several novel compounds such as the hectochlorins and jamaicamides; however, bioinformatic analyses of its genome suggested that there were many more cryptic biosynthetic gene clusters yet to be characterized. To potentially stimulate the production of novel compounds from this strain, it was co-cultured with Candida albicans. From this experiment, we observed the increased production of a new compound that we characterize here as hectoramide B. Bioinformatic analysis of the M. producens JHB genome enabled the identification of a putative biosynthetic gene cluster responsible for hectoramide B biosynthesis. This work demonstrates that co-culture competition experiments can be a valuable method to facilitate the discovery of novel natural products from cyanobacteria. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/547815v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1b507d4org.highwire.dtl.DTLVardef@152376org.highwire.dtl.DTLVardef@1cb410dorg.highwire.dtl.DTLVardef@11bcabc_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Crk/Crkl regulates early angiogenesis in mouse embryos by accelerating endothelial cell maturation

RationaleUbiquitously expressed cytoplasmic adaptors CRK and CRKL mediate multiple signaling pathways in mammalian embryogenesis. They are also associated with cardiovascular defects occurring in Miller-Dieker syndrome and 22q11.2 deletion syndrome, respectively. The embryonic mesoderm contributes to the formation of the cardiovascular system, yet the roles that Crk and Crkl play there are not understood on a single cell level. ObjectivesTo determine functions of Crk and Crkl in the embryonic mesoderm during early mouse vascular development. Secondly, we will examine the molecular mechanisms responsible for early embryonic endothelial cell (EC) defects by performing single cell RNA-sequencing (scRNA-seq) and in vivo validation experiments. Methods and ResultsInactivation of both Crk and Crkl together using Mesp1Cre resulted embryonic lethality with severe vascular defects. Although vasculogenesis appeared normal, angiogenesis was disrupted both in the yolk sac and embryo proper, leading to disorganized vascular networks. We performed scRNA-seq of the Mesp1Cre mesodermal lineage and found that there was upregulation of a great number of angiogenesis and cell migration related genes in ECs in the mutants, including NOTCH signaling genes such as Dll4 and Hey1. Further bioinformatic analysis of EC subpopulations identified a relative increase in the number of more differentiated angiogenic ECs and decrease in EC progenitors. Consistent with this, we identified an expansion of Dll4 expressing cells within abnormal arteries, in vivo. Also, our bioinformatic data indicates that there is dysregulated expression of lineage genes that promote EC differentiation causing accelerated cell fate progression during EC differentiation. ConclusionsOur results show that Crk and Crkl are crucial for regulating early embryonic angiogenesis. Combined inactivation of Crk/Crkl caused precocious EC maturation with an increase of atypical differentiated angiogenic ECs and failed vascular remodeling. This is in part due to increased NOTCH signaling and altered expression of cell migration genes.

developmental biology↗