title: Differential growth rates and in vitro drug susceptibility to currently used drugs for multiple isolates of Naegleria fowleri

abstract: The free-living amoeba, Naegleria fowleri, which typically dwells within warm, freshwater environments, can opportunistically cause Primary Amoebic Meningoencephalitis (PAM), a disease with a mortality rate of >98%, even with the administration of the best available drug regimens. The lack of positive outcomes for PAM has prompted a push for the discovery and development of more effective therapeutics, but most studies only utilize one or two clinical isolates in their drug discovery assays. The inability to assess possible heterogenic responses to drugs among isolates from varying geographical regions hinders progress in the field due to a lack of proven universal efficacy for novel therapeutics. Herein we conducted drug efficacy and growth rate determinations for 11 different clinical isolates, including one obtained from a successful treatment outcome, by applying a previously developed CellTiter-Glo 2.0 screening technique and flow cytometry. We found some significant differences in the susceptibility of these isolates to 7 of 8 different drugs tested, all of which comprise the cocktail that is recommended to physicians by the Centers for Disease Control. We also discovered significant variances in growth rates among isolates which draws attention to the dissidence among the amoebae populations collected from different patients. The findings of this study reiterate the need for inclusion of additional clinical isolates of varying genotypes in drug assays and highlight the necessity for more targeted therapeutics with universal efficacy across N. fowleri isolates. Our data establishes a needed baseline for drug susceptibility among clinical isolates and provides a segue for future combination therapy studies as well as research related to phenotypic or genetic differences that could shed light on mechanisms of action or predispositions to specific drugs.


title: SARS-CoV-2 variants exhibit increased kinetic stability of open spike conformations as an evolutionary strategy

abstract: SARS-CoV-2 variants of concern harbor mutations in the Spike (S) glycoprotein that confer more efficient transmission and dampen the efficacy of COVID-19 vaccines and antibody therapies. S mediates virus entry and is the primary target for antibody responses. Structural studies of soluble S variants have revealed an increased propensity towards conformations accessible to receptor human Angiotensin-Converting Enzyme 2 (hACE2). However, real-time observations of conformational dynamics that govern the structural equilibriums of the S variants have been lacking. Here, we report single-molecule Forster Resonance Energy Transfer (smFRET) studies of S variants containing critical mutations, including D614G and E484K, in the context of virus particles. Investigated variants predominantly occupied more open hACE2-accessible conformations, agreeing with previous structures of soluble trimers. Additionally, these S variants exhibited decelerated transitions in hACE2-accessible/bound states. Our finding of increased S kinetic stability in the open conformation provides a new perspective on SARS-CoV-2 adaptation to the human population.

category: microbiology


title: Conserved α-helix-3 is crucial for structure and functions of Rad6 E2 ubiquitin-conjugating enzymes.

abstract: Rad6, an E2 ubiquitin-conjugating enzyme conserved from yeast to humans, functions in transcription, genome maintenance and proteostasis. The contributions of many conserved secondary structures of Rad6 and its human homologs UBE2A and UBE2B to their biological functions are not understood. A mutant RAD6 allele with a missense substitution at alanine-126 (A126) of helix-3 that causes defects in telomeric gene silencing, DNA repair and protein degradation was reported over two decades ago. Here, using a combination of genetics, biochemical, biophysical, and computational approaches, we discovered that helix-3 A126 mutations compromise the ability of Rad6 to ubiquitinate target proteins without disrupting interactions with partner E3 ubiquitin-ligases that are required for their various biological functions in vivo. Explaining the defective in vitro or in vivo ubiquitination activities, molecular dynamics simulations and NMR showed that helix-3 A126 mutations cause local disorder of the catalytic pocket of Rad6 in addition to disorganizing the global structure of the protein to decrease its stability in vivo. We also show that helix-3 A126 mutations deform the structures of UBE2A and UBE2B, the human Rad6 homologs, and compromise the in vitro ubiquitination activity and folding of UBE2B. In summary, our studies reveal that the conserved helix-3 is a crucial structural constituent that controls the organization of catalytic pockets, enzymatic activities, and biological functions of the Rad6-family E2 ubiquitin-conjugating enzymes.

category: molecular biology


title: Consequences of alanine-126 mutations in helix-3 on structure and functions of Rad6 E2 ubiquitin-conjugating enzymes


title: Histone H1 regulates non-coding RNA turnover on chromatin in a m6A-dependent manner

abstract: Linker histones are highly abundant chromatin-associated proteins with well-established structural roles in chromatin and as general transcriptional repressors. In addition, it has been long proposed that histone H1 exerts context-specific effects on gene expression. Here, we have identified a new function of histone H1 in chromatin structure and transcription using a range of genomic approaches. We show that histone H1-depleted cells accumulate nascent non-coding RNAs on chromatin, suggesting that histone H1 prevents non-coding RNA transcription and regulates non-coding transcript turnover on chromatin. Accumulated non-coding transcripts have reduced levels of m6A modification and cause replication-transcription conflicts. Accordingly, altering the m6A RNA methylation pathway rescues the replicative phenotype of H1 loss. This work unveils unexpected regulatory roles of histone H1 on non-coding RNA turnover and m6A deposition, highlighting the intimate relationship between chromatin conformation, RNA metabolism and DNA replication to maintain genome performance.


title: Highly resolved spatial transcriptomics for detection of rare events in cells

abstract: Single-cell spatial transcriptomics technologies leveraged the potential to transcriptionally landscape sophisticated reactions in cells. Current methods to delineate such complex interplay lack the flexibility in rapid target adaptation and are particularly restricted in detecting rare transcripts. We developed a multiplex single-cell RNA In-situ hybridization technique, called  Molecular Cartography (MC) that can be easily tailored to specific applications and, by providing unprecedented sensitivity, specificity and resolution, is particularly suitable in tracing rare events at a subcellular level. Using a SARS-CoV-2 infection model, MC allows the discernment of single events in host-pathogen interactions, dissects primary from secondary responses, and illustrates differences in antiviral signaling pathways affected by SARS-CoV-2, simultaneously in various cell types.


title: SLX4 dampens MutSα-dependent mismatch repair

abstract: The tumour suppressor SLX4 plays multiple roles in the maintenance of genome stability, acting as a scaffold for structure-specific endonucleases and other DNA repair proteins. It directly interacts with the mismatch repair (MMR) protein MSH2 but the significance of this interaction remained unknown until recent findings showing that MutS{beta} (MSH2-MSH3) stimulates in vitro the SLX4-dependent Holliday junction resolvase activity. Here, we characterize the mode of interaction between SLX4 and MSH2, which relies on an MSH2-interacting peptide (SHIP box) that drives interaction of SLX4 with both MutS{beta} and MutS (MSH2-MSH6). While we show that this MSH2 binding domain is dispensable for the well-established role of SLX4 in interstrand crosslink repair, we find that it mediates inhibition of MutS-dependent MMR by SLX4, unravelling an unanticipated function of SLX4.


title: Locus-Specific DNA Methylation Editing in Mammalian Cells using a CRISPR-Based System

abstract: DNA methylation is a key epigenetic modification implicated in the pathogenesis of numerous human diseases, including cancer development and metastasis. Gene promoter methylation changes are widely associated with transcriptional deregulation and disease progression. The advent of CRISPR-based technologies has provided a powerful toolkit for locus-specific manipulation of the epigenome. Here, we describe a comprehensive global workflow for the design and application of a dCas9-SunTag-based tool for editing a DNA methylation locus in human melanoma cells, alongside protocols for downstream techniques used to evaluate subsequent methylation and gene expression changes in methylation-edited cells. Using transient system delivery, we demonstrate both highly efficacious methylation and demethylation of the EBF3 promoter, a putative epigenetic driver of melanoma metastasis, achieving up to 304.00% gain of methylation and 99.99% relative demethylation, respectively. Further, we employ a novel, targeted screening approach to confirm minimal off-target activity and high on-target specificity of our editing sys-tem within our target locus.


title: The neurodevelopmental disorder-linked PHF14 complex that forms biomolecular condensates detects DNA damage and promotes repair

abstract: Numerous chromatin-associated proteins have been linked to neurodevelopmental disorders, yet their molecular functions often remain elusive. PHF14, HMG20A, TCF20 and RAI1 are components of a putative chromatin-associated complex and have been implicated in neurological disorders. Here, we found that Phf14 knockout embryonic stem cells and neural progenitor cells exhibit impaired cell cycle progression and proliferation, inadequate protection of stalled replication forks, and decreased DNA repair. The PHF14 complex rapidly assembles at DNA damage sites and binds to DNA through HMG20A. The PHF14 complex forms DNA-containing phase separated droplets in vitro, where TCF20 facilitates droplet formation. Furthermore, TCF20 maintenance at DNA damage sites is destabilized upon pathological mutation. Our results suggest that the PHF14 complex contributes to DNA damage repair by sensing damaged sites and forming biomolecular condensates, thus supporting cell cycle progression, especially in neural progenitor cells whose spatiotemporal pool is critical for proper brain development.


title: Ferroptosis Regulation by the NGLY1/NFE2L1 Pathway

abstract:
Ferroptosis is an oxidative form of non-apoptotic cell death whose transcriptional regulation is poorly understood. Capncollar (CNC) transcription factors including Nuclear Factor Erythroid-2 Related Factor 1 (NFE2L1/NRF1) and NFE2L2 (NRF2) are important regulators of oxidative stress responses. Here, we report that NFE2L1 expression inhibits ferroptosis, independent of NFE2L2. NFE2L1 inhibits ferroptosis by promoting expression of the key anti-ferroptotic lipid hydroperoxidase glutathione peroxidase 4 (GPX4). NFE2L1 abundance and function are regulated post-translationally by N-glycosylation. Functional maturation of NFE2L1 requires deglycosylation by cytosolic peptide:N-glycanase 1 (NGLY1). We find that loss of NGLY1 or NFE2L1 enhances ferroptosis sensitivity. Expression of wild-type NGLY1 but not a disease-associated NGLY1 mutant inhibits ferroptosis, and this effect is dependent on the presence of NFE2L1. Enhanced ferroptosis sensitivity in NFE2L1 and NFE2L2 knockout cells can be potently reverted by expression of an NFE2L1 mutant containing eight asparagine-to-aspartate protein sequence substitutions, which mimic NGLY1-catalyzed sequence editing. Enhanced ferroptosis sensitivity in NGLY1/NFE2L1 pathway mutants could also be reversed by overexpression of NFE2L2. These results suggest that ferroptosis sensitivity is regulated by NGLY1-catalyzed NFE2L1 deglycosylation, and highlight a broad role for CNC transcription factors in ferroptosis regulation.

Significance StatementFerroptosis is an oxidative form of cell death whose biochemical regulation remains incompletely understood. NFE2L1/NRF1 is a capncollar (CNC) transcription factor whose role in ferroptosis regulation is unclear. Unlike the CNC family member NFE2L2/NRF2, NFE2L1 is an N-glycoprotein whose abundance is regulated by post-translational deglycosylation catalyzed by the enzyme peptide:N-glycanase 1 (NGLY1). Our results indicate that NGLY1-mediated NFE2L1 deglycosylation, resulting in  editing of the NFE2L1 amino acid sequence, is necessary for NFE2L1 to inhibit ferroptosis. Mechanistically, NFE2L1 inhibits ferroptosis by via the anti-ferroptotic protein GPX4. This work demonstrates that CNC transcription factors beyond NFE2L2 can regulate ferroptosis. This work may suggest a role of misregulation of ferroptosis in NGLY1 deficiency, an ultrarare genetic disorder.

category: cell biology


title: Mutating novel interaction sites in NRP1 reduces SARS-CoV-2 spike protein internalization

abstract: The global pandemic of Coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 has become a severe global health problem because of its rapid spread(1). Both angiotensin-converting enzyme 2 and neuropilin 1 provide initial viral binding sites for SARS-CoV-2 (2, 3). Here, we show that three cysteine residues located in a1/a2 and b1 domains of neuropilin 1 are necessary for SARS-CoV-2 spike protein internalization in human cells. Mutating cysteines C82, C104 and C147 altered neuropilin 1 stability and binding ability as well as cellular internalization and lysosomal translocation of the spike protein. This resulted in up to 4 times reduction in spike protein load in cells for the original, alpha and delta SARS-CoV-2 variants even in the presence of the endogenous angiotensin-converting enzyme 2 receptor. Transcriptome analysis of cells transfected with mutated NRP1 revealed significantly reduced expression of genes involved in viral infection and replication, including eight members of the ribosomal protein L, ten members of ribosomal protein S and five members of the proteasome {beta} subunit family proteins. We also observed higher expression of genes involved in suppression of inflammation and endoplasmic reticulum associated degradation. These observations suggest that these cysteines offer viable targets for therapies against COVID-19.

category: genetics


title: Covid-19 genomic analysis reveals clusters of emerging sublineages within the delta variant

abstract: The emerging SARS-CoV-2 variants may potentially have enhanced transmissibility and virulence of the virus, and impacts on performance of diagnostic tools and efficacy of vaccines. Genomic surveillance provides an opportunity to detect and characterize new mutations early enough for effective deployment of control strategies. Here, genomic data from Germany and United Kingdom were examined for genetic diversity by assessing gene mutations and inferring phylogeny, and simplifying genomic data workflow for faster visualization and interpretation. Delta variant sublineages were grouped into seven distinct clusters of spike mutations located in N-terminal domain of S1 region (T95I, D138H, *D142G, Y145H and A222V) and S2 region (T719I and *N950D). The most predominant cluster was T95I mutation, with the highest frequencies (71.1% - 83.9%) in Wales, England and Scotland, and the least frequencies (8.9% - 12.1%) in Germany. Two mutations, *D142G and *N950D here described as *reverse mutations and T719I mutation, were largely unique to Germany. In a month, frequencies of D142G had increased from 55.6% to 67.8 % in Germany. Additionally, a cluster of D142G+T719I/T mutation went up from 27.7% to 34.1%, while a T95I+ D142G+N950D/N cluster rose from 19.2% to 26.2%. Although, two distinct clusters of T95I+D138H (2.6% - 3.8%) and T95I+Y145H+A222V (2.5% - 8.5%) mutations were present in all the countries, they were most predominant in Wales and Scotland respectively. Results suggest divergent evolutionary trajectories between the clusters of T95I mutation and those of D142G mutation. These findings provide insights into underlying dynamics of evolution of the delta variant. Future studies may evaluate the epidemiological and biological implications of these sublineages.

category: genomics


title: Altered Cardiolipin Metabolism is Associated with Cardiac Mitochondrial Dysfunction in Pulmonary Vascular Remodeled Perinatal Rat Pups

abstract:
BackgroundPulmonary vascular remodeling (PVR) in utero results in the development of heart failure (HF). The alterations that occur in cardiac lipid and mitochondrial bioenergetics during the development of in utero PVR was unknown.

MethodsPVR was induced in pups in utero by exposure of pregnant dams to indomethacin and hypoxia. Cardiac lipids, echocardiographic function and cardiomyocyte mitochondrial function were subsequently examined.

ResultsPerinatal rat pups with PVR exhibited elevated left and right cardiac ventricular internal dimensions and reduced ejection fraction and fractional shortening compared to controls. Cardiac myocytes from these pups exhibited increased glycolytic capacity and glycolytic reserve compared to controls. However, respiration with glucose as substrate was unaltered. Fatty acid oxidation and ATP-insensitive respiration were increased in isolated cardiac myocytes from these pups compared to controls indicating mitochondrial dysfunction. Although abundance of mitochondrial respiratory complexes were unaltered, increased trilinoleoyl-lysocardiolipin levels in these pups was observed. A compensatory increase in both cardiolipin (CL) and phosphatidylethanolamine (PE) content were observed due to increased synthesis of these phospholipids.

ConclusionAlterations in cardiac cardiolipin and phospholipid metabolism in PVR rat pups is associated with the mitochondrial bioenergetic and cardiac functional defects observed in their hearts.

Impact statement- Phospholipid metabolism was examined in pulmonary vascular remodeling in perinatal rat pups.
- Pulmonary vascular remodeling was induced in utero by treating pregnant dams with hypoxia and indomethacin at 19-21 days of gestation.
- The offspring exhibited altered pulmonary arterial remodeling with subsequent cardiac hypertrophy, ventricular dysfunction, cardiac myocyte mitochondrial dysfunction with altered fatty acid utilization.
- In addition, the offspring exhibited elevated cardiolipin, lysocardiolipin and phosphatidylethanolamine content which may potentially contribute to the cardiac mitochondrial dysfunction.

category: biochemistry


title: Localization of the putative recombinase Pf-int to the apicoplast of Plasmodium falciparum

abstract: Diseases caused by apicomplexan parasites, such as malaria and toxoplasmosis cause [~]200 million (worldwide) and 1 million (Europe) infections, respectively, every year. Apicomplexa possess a non-photosynthetic organelle homologous to the plant chloroplast, the so-called apicoplast, that is essential for their growth and survival. This study focused on the Int recombinase, the first protein discovered in Plasmodium spp. with the features of a site-specific recombinase, and which has an apicoplast targeting leader sequence at its amino-terminus. Int is conserved amongst several apicomplexan parasites. In the human toxoplasmosis parasite, Toxoplasma, Int localizes to the apicoplast and Pf-Int, the P. falciparum member, belongs to the group of non-mutable essential genes in P. falciparum. A conserved protein that has been shown to be essential at least in one species and that localizes to an essential organelle may become a novel drug target. Therefore, the aim of this study was to confirm the sub-cellular localization of Int in the human malaria parasite P. falciparum. Using western blot analysis and immunofluorescence microscopy of P. falciparum asexual blood stages, we observed that Int partially co-localized with the apicoplast (to discrete foci adjacent to the nucleus).


title: Topoisomerase II poisons inhibit vertebrate DNA replication through distinct mechanisms

abstract: Topoisomerase II (Top2) unlinks chromosomes during vertebrate DNA replication. Top2  poisons are widely-used chemotherapeutics that stabilize Top2 complexes on DNA, leading to cytotoxic DNA breaks. However, it is unclear how these drugs affect DNA replication, which is a major target of Top2 poisons. Using Xenopus egg extracts, we show that the Top2 poisons etoposide and doxorubicin both inhibit DNA replication through different mechanisms. Etoposide induces Top2-dependent DNA breaks and induces Top2-dependent fork stalling by trapping Top2 behind replication forks. In contrast, doxorubicin does not lead to appreciable break formation and instead intercalates into parental DNA to inhibit replication fork progression. In human cells, etoposide stalls replication forks in a Top2-dependent manner, while doxorubicin stalls forks independently of Top2. However, both drugs exhibit Top2-dependent cytotoxicity. Thus, despite shared genetic requirements for cytotoxicity etoposide and doxorubicin inhibit DNA replication through distinct mechanisms.


title: Single-particle cryo-EM structures from iDPC-STEM at near-atomic resolution

abstract: Electron cryo-microscopy (cryo-EM) is becoming one of the routine tools for structure determination of biological macromolecules. Commonly, molecular images are obtained by conventional transmission electron microcopy (CTEM) using underfocus and subsequently computationally combined into a high-resolution 3D structure. Here, we apply scanning transmission electron microscopy (STEM) using the integrated differential phase contrast mode also known as iDPC-STEM to the cryo-EM test specimen of tobacco mosaic virus (TMV). The micrographs show complete contrast transfer to high resolution and enable the cryo-EM structure determination at 3.5 [A] resolution using single-particle based helical reconstruction. A series of cryo-EM TMV maps was resolved at near-atomic resolution taken at different convergence semi-angle (CSA) beams and share identical features with maps obtained by CTEM of a previously acquired same-sized TMV data set. The associated map B-factors from iDPC-STEM match those obtained by CTEM recordings using 2nd generation direct electron detection devices. These data show that STEM imaging in general, and in particular the iDPC-STEM approach, can be applied to vitrified single-particle specimens to determine near-atomic resolution cryo-EM structures of biological macromolecules.

category: biophysics


title: Dietary Proanthocyanidins Exert Localized Immunomodulatory Effects in Porcine Pulmonary and Gastrointestinal Tissues during Ascaris suum-induced Type 2 inflammation

abstract: Bioactive dietary components may considerably influence intestinal health and resistance to enteric disease. Proanthocyanidins (PAC) are dietary polyphenols with putative health-promoting activity that have been increasingly studied for their anti-inflammatory and immunomodulatory effects. However, whether dietary PAC can regulate type-2 immune function and inflammation at mucosal surfaces remains unclear. Here, we investigated whether diets supplemented with purified PAC modulated pulmonary and intestinal mucosal immune responses during infection with the helminth parasite Ascaris suum in pigs. A. suum infection induced a type 2-biased immune response in lung and intestinal tissues, characterized by pulmonary granulocytosis, increased Th2/Th1 T cell ratios in tracheal-bronchial lymph nodes, intestinal eosinophilia, and modulation of genes involved in mucosal barrier function and immunity. We observed that PAC had only minor effects on pulmonary immune responses, regardless of concurrent A. suum infection. However, RNA-sequencing of intestinal tissues revealed that dietary PAC significantly enhanced transcriptional responses related to immune function, antioxidant responses, and cellular stress activity, both in uninfected and A. suum-infected animals. A. suum infection and dietary PAC both induced distinct changes in gut microbiota composition, primarily in the jejunum and colon, respectively. Notably, PAC substantially increased Limosilactobacillus reuteri abundance in the colon of both naive and A. suum-infected animals. Thus, dietary PAC may have distinct beneficial effects on intestinal health during infection with mucosal pathogens, whilst having limited activity to modulate naturally-induced type-2 pulmonary inflammation. Our results shed further light on the mechanisms underlying the health-promoting properties of PAC-rich foods, and may aid in the design of novel dietary supplements to regulate mucosal inflammatory responses in the gastrointestinal tract.

category: immunology


title: Macrophage Innate Training Induced by IL-4 Activation Enhances OXPHOS Driven Anti-Mycobacterial Responses

abstract: Macrophages are key innate immune cells for determining the outcome of Mycobacterium tuberculosis infection. Polarization with IFN{gamma} and LPS into the "classically activated" M1 macrophage enhances pro-inflammatory and microbicidal responses, important for eradicating the bacterium. By contrast, "alternatively activated" M2 macrophages, polarized with IL-4, oppose bactericidal mechanisms and allow mycobacterial growth. These activation states are accompanied by distinct metabolic profiles, where M1 macrophages favor near exclusive use of glycolysis, whereas M2 macrophages up-regulate oxidative phosphorylation (OXPHOS). Here we demonstrate that activation with IL-4 counterintuitively induces protective innate memory against mycobacterial challenge. This was associated with enhanced pro-inflammatory cytokine responses and killing capacity. Moreover, despite this switch towards a phenotype that is more akin to classical activation, IL-4 trained macrophages do not demonstrate M1-typical metabolism, instead retaining heightened use of OXPHOS. Moreover, inhibition of OXPHOS with oligomycin, 2-deoxy glucose or BPTES all impeded heightened pro-inflammatory cytokine responses from IL-4 trained macrophages. Lastly, this work identifies that IL-10 negatively regulates protective IL-4 training, impeding pro-inflammatory and bactericidal mechanisms. In summary, this work provides new and unexpected insight into alternative macrophage activation states in the context of mycobacterial infection.


title: CD20+ T cells are associated with inflammatory responses in experimental arthritis.

abstract: CD20+ T cells comprise a small but highly inflammatory subset that has been implicated in autoimmunity, including rheumatoid arthritis (RA). We sought to characterise the CD20+ T cell subset at the site of inflammation in murine collagen-induced arthritis (CIA) model of RA and investigate the phenotype and functional relevance of CD3+CD20+ T cells in the lymph nodes and arthritic joints using flow cytometry and immunohistochemistry. We demonstrate that CD3+CD4+CD20+ and CD3+CD8+CD20+ T cells are expanded in the draining lymph nodes of CIA mice. In addition, compared to naive mice and those that did not develop clinical symptoms, CD20 expressing T cells of arthritic mice produced increased levels of pro-inflammatory cytokines (GM-CSF, TNF-a, IL-17, and INF-g). Notably, CD3+CD4+CD20+ and CD3+CD8+CD20+ T cells of disease mice were enriched with CXCR5+PD-1+ T follicular helper cells and CXCR5-PD-1+ peripheral T helper cells, subsets of T cells that have been implicated in promoting B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues in RA. Importantly, CD3+CD20+ T cells were detected in the inflamed regions in the lymph nodes and paws of arthritic mice. Our findings suggest that CD20+ T cells are associated with inflammatory responses in the arthritic joint and may exacerbate pathology by promoting inflammatory B cell responses.


title: Glycosuria alters uropathogenic Escherichia coli global gene expression and virulence

abstract:
Uropathogenic Escherichia coli (UPEC) is the principal etiology of more than half of urinary tract infections (UTI) in humans with diabetes mellitus. Epidemiological data and studies in mouse model of ascending UTI have elucidated various host factors responsible for increasing the susceptibility of diabetic hosts to UPEC-UTI. In contrast, the nature of alterations in UPEC physiology mediated by diabetic urinary microenvironment and the contributions of altered UPEC physiology in shaping UPEC-UTI pathogenesis in diabetes have not been examined. Our central hypothesis is that glycosuria directly induces urinary virulence of UPEC. We compared virulence characteristics and gene expression in human UPEC strains UTI89 (cystitis) and CFT073 (pyelonephritis) exposed for 2h, in vitro to human urine either in the presence or absence of glycosuria (600mg/dl glucose). Compared to control UPEC exposed to nutrient-rich culture medium LB, glycosuria-exposed UPEC exhibited significant increase in biofilm formation and reduction in the hemagglutination of Guinea pig erythrocytes (a surrogate measure of type 1 piliation). In addition, analysis of UTI89 transcriptome by RNA sequencing revealed that 2h-long, in vitro exposure to glycosuria also significantly alters expression of virulence and metabolic genes central to urinary virulence of UPEC. In summary, our results provide novel insights into how glycosuria-mediated early changes in UPEC fitness may facilitate UTI pathogenesis in the diabetic urinary microenvironment.

IMPORTANCEUropathogenic Escherichia coli (UPEC) is an important causative agent of urinary tract infections in diabetic humans. We examined the effects of in vitro exposure to glycosuria (presence of glucose in urine) on the virulence and gene expression by UPEC. Our results show that glycosuria rapidly (in 2h) alters UPEC gene expression, induces biofilm formation, and suppresses hemagglutination. These results offer a novel insight into the pathogenesis of UPEC in the urinary tract.


title: Secondary structure of subgenomic RNA M of SARS-CoV-2

abstract: SARS-CoV-2 belongs the Coronavirinae family. As other coronaviruses, SARS-CoV-2 is enveloped and possesses positive-sense, single-stranded RNA genome of [~] 30 kb. Genome RNA is used as the template for replication and transcription. During these processes, positive-sense genomic RNA (gRNA) and subgenomic RNAs (sgRNAs) are created. Several studies showed importance of genomic RNA secondary structure in SARS-CoV-2 replication. However, the structure of sgRNAs have remained largely unsolved so far. In this study, we performed probing of sgRNA M of SARS-CoV-2 in vitro. This is the first experimentally informed secondary structure model of sgRNA M, which presents features likely to be important in sgRNA M function. The knowledge about sgRNA M provides insights to better understand virus biology and could be used for designing new therapeutics.


title: A bacterial Argonaute with efficient DNA and RNA cleavage activity guided by small DNA and RNA

abstract: Argonaute proteins are widespread in prokaryotes and eukaryotes. Most prokaryotic Argonaute proteins (pAgos) use 5P-gDNA to target complementary DNA. However, more and more studies on the properties of pAgos make their functions more diversified. Previously reported pAgos only possess several forms of high activity in all eight cleavage patterns, which limits their practical applications. Here, we described a unique pAgo from Marinitoga hydrogenitolerans (MhAgo) with eight cleavage activities. MhAgo can utilize all four types of guides (5OH-gDNA, 5P-gDNA, 5OH-gRNA, and 5P-gRNA) for ssDNA and RNA cleavage. Further studies demonstrated that MhAgo had high activities with 16-21 nt guides and no obvious preferences for the 5-end nucleotides of 5OH-guides. Unexpectedly, MhAgo had different preferences for the 5-end nucleotides of 5P-guides depending on the types of targets. Although the specificity of MhAgo was related to the types of guides, single mismatches in the central and 3-supplementary regions of guides greatly reduced the cleavage efficiency. Additionally, the electrophoretic mobility shift assay (EMSA) demonstrated MhAgo had the weakest affinity for 5P-gRNA:tRNA duplex, which was consistent with its cleavage efficiency. In conclusion, MhAgo is highly active under a wide range of conditions and can be used for programmable endonucleolytic cleavage of both ssDNA and RNA substrates. The abundant biochemical characteristics of MhAgo broaden our understanding of pAgos and expand the potential application in nucleic acids manipulations.


title: gp130/STAT3 signaling is required for homeostatic proliferation and anabolism in postnatal growth plate and articular chondrocytes

abstract: Growth of long bones and vertebrae is maintained postnatally by a long-lasting pool of progenitor cells. Little is known about the molecular mechanisms that regulate the output and maintenance of the cells that give rise to mature cartilage. Here we demonstrate that postnatal chondrocyte-specific deletion of a transcription factor Stat3 results in severely reduced proliferation coupled with increased hypertrophy, growth plate fusion, stunting and signs of progressive dysfunction of the articular cartilage. This effect is dimorphic, with females more strongly affected than males. Chondrocyte-specific deletion of the IL-6 family cytokine receptor gp130, which activates Stat3, phenocopied Stat3-deletion; deletion of Lifr, one of many co-receptors that signals through gp130, resulted in a milder phenotype. These data define a new molecular circuit that regulates chondrogenic cell maintenance and output and reveals a novel, hitherto unrecognized function of IL-6 cytokines in the skeletal system with direct implications for skeletal development and regeneration.


title: Aged human iPSC-RPE organoid cultures display hallmarks of drusen formation

abstract: Age-related macular degeneration (AMD) is among the most common causes of irreversible vision loss. Disease progression is strongly associated with age-related pathological changes of retinal pigment epithelial (RPE) cells, such as accumulation of intracellular lipid-containing cell debris, extracellular lipid-rich deposits (drusen) and collagen-rich basal laminar deposits. Current AMD models provide a limited understanding of the complex pathomechanisms, revealing the lack of adequate physiological human AMD models. In this study, we developed an in vitro model applicable for the exploration of AMD pathomechanisms and risk factors for AMD progression and drusen formation. Advanced 3D culturing technologies allow long-term cultivation of hiPSC-derived RPE organoids (RPEorg) for up to 360 days, which is the time frame necessary for the development of an AMD-like phenotype. Aged RPEorg exhibit hallmarks of AMD and age-related alterations such as increased autofluorescence, accumulation of lipid droplets, calcification, and the formation of extracellular clusters of the drusen-associated proteins such as apolipoprotein E (APOE) and tissue inhibitor of metalloproteinases 3 (TIMP3). Electron microscopy further reveals drusen-like extracellular deposits mimicking the signs of late drusen formation and AMD progression. In summary, our results demonstrate that hiPSC-derived 3D RPEorg provide a promising model to study age-associated RPE pathology and drusen formation. We show here that RPEorg are applicable for disease modelling studies and early stages of drug development and provide the opportunity to uncover inter-individual genetic and epigenetic factors that alter the course of the disease.


title: WNT/B-catenin dependant alteration of cortical neurogenesis in a human stem cell model of SETBP1 disorder

abstract:
Disruptions of SETBP1 (SET binding protein 1) on 18q12.3 by heterozygous gene deletion or loss-of-function variants cause SETBP1 disorder. Clinical features are frequently associated with moderate to severe intellectual disability, autistic traits and speech and motor delays. Despite SETBP1 association with neurodevelopmental disorders, little is known about its role in brain development. Using CRISPR/CAS9 genome editing technology, we generated a SETBP1 deletion model in human embryonic stem cells (hESCs), and examined the effects of SETBP1-deficiency in in vitro derived neural progenitors (NPCs) and neurons using a battery of cellular assays, genome wide transcriptomic profiling and drug-based phenotypic rescue.

SETBP1-deficient NPCs exhibit protracted proliferation and distorted layer-specific neuronal differentiation with overall decrease in neurogenesis. Genome wide transcriptome profiling and protein biochemical analysis showed that SETBP1 deletion led to enhanced activation of WNT/{beta}-catenin signaling. Crucially, treatment of the SETBP1-deficient NPCs with a small molecule WNT inhibitor XAV939 restored hyper canonical {beta}-catenin activity and rescued cortical neuronal differentiation.

Our study establishes a novel regulatory link between SETBP1 and WNT/{beta}-catenin signaling during human cortical neurogenesis and provides mechanistic insights into structural abnormalities and potential therapeutic avenues for SETBP1 disorder.

category: developmental biology


title: Reversing the miRNA -5p/-3p stoichiometry reveals physiological roles and targets of miR-140 miRNAs

abstract: The chondrocyte specific miR-140 miRNAs are necessary for normal endochondral bone growth in mice. miR-140 deficiency causes dwarfism and craniofacial deformity. However, the physiologically important targets of miR-140 miRNAs are still unclear. The miR-140 gene (Mir140) encodes three chondrocyte-specific microRNAs, miR-140-5p, derived from the 5 strand of primary miR-140, and miR140-3p.1 and -3p.2, derived from the 3 strand of primary miR-140. miR-140-3p miRNAs are ten times more abundant than miR-140-5p likely due to the non-preferential loading of miR-140-5p to Argonaute proteins. To differentiate the role of miR-140-5p and -3p miRNAs in endochondral bone development, two distinct mouse models, miR140-C>T, in which the first nucleotide of miR-140-5p was altered from cytosine to uridine, and miR140-CG, where the first two nucleotides of miR-140-3p were changed to cytosine and guanine, were created. These changes are expected to alter Argonaute protein loading preference of -5p and -3p to increase -5p loading and decrease -3p loading without changing the function of miR140-5p. These models presented a mild delay in epiphyseal development with delayed chondrocyte maturation. Using RNA-sequencing analysis of the two models, direct targets of miR140-5p, including Wnt11, were identified. Disruption of the predicted miR140-5p binding site in the 3 untranslated region of Wnt11 was shown to increase Wnt11 mRNA expression and caused a modest acceleration of epiphyseal development. These results show that the relative abundance of miRNA-5p and -3p can be altered by changing the first nucleotide of miRNAs in vivo, and this method can be useful to identify physiologically important miRNA targets.


title: Modulation of pre-mRNA structure by hnRNP proteins regulates alternative splicing of MALT1

abstract: Alternative splicing is controlled by differential binding of trans-acting RNA binding proteins (RBPs) to cis-regulatory elements in intronic and exonic pre-mRNA regions 1-3. How secondary structure in the pre-mRNA transcripts affects recognition by RBPs and determines alternative exon usage is poorly understood. The MALT1 paracaspase is a key component of signaling pathways that mediate innate and adaptive immune responses 4. Alternative splicing of MALT1 exon7 is critical for controlling optimal T cell activation 5,6. Here, we demonstrate that processing of the MALT1 pre-mRNA depends on RNA structural elements that shield the 5 and 3 splice sites of the alternatively spliced exon7. By combining biochemical analyses with chemical probing and NMR we show that the RBPs hnRNP U and hnRNP L bind competitively and with comparable affinities to identical stem-loop RNA structures flanking the 5 and 3 splice sites of MALT1 exon7. While hnRNP U stabilizes RNA stem-loop conformations that maintain exon7 skipping, hnRNP L unwinds these RNA elements to facilitate recruitment of the essential splicing factor U2AF2 to promote exon7 inclusion. Our data represent a paradigm for the control of splice site selection by differential RBP binding and modulation of pre-mRNA structure.


title: Intelligent Resolution: Integrating Cryo-EM with AI-driven Multi-resolution Simulations to Observe the SARS-CoV-2 Replication-Transcription Machinery in Action

abstract: The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) replication transcription complex (RTC) is a multi-domain protein responsible for replicating and transcribing the viral mRNA inside a human cell. Attacking RTC function with pharmaceutical compounds is a pathway to treating COVID-19. Conventional tools, e.g., cryo-electron microscopy and all-atom molecular dynamics (AAMD), do not provide sufficiently high resolution or timescale to capture important dynamics of this molecular machine. Consequently, we develop an innovative workflow that bridges the gap between these resolutions, using mesoscale fluctuating finite element analysis (FFEA) continuum simulations and a hierarchy of AI-methods that continually learn and infer features for maintaining consistency between AAMD and FFEA simulations. We leverage a multi-site distributed workflow manager to orchestrate AI, FFEA, and AAMD jobs, providing optimal resource utilization across HPC centers. Our study provides unprecedented access to study the SARS-CoV-2 RTC machinery, while providing general capability for AI-enabled multi-resolution simulations at scale.


title: APOBEC mutagenesis is low in most types of non-B DNA structures, unlike other types of cancer mutagenesis

abstract: While somatic mutations are known to be enriched in genome regions with non-canonical DNA secondary structure, the impact of particular mutagens still needs to be elucidated. Here, we demonstrate that in human cancers, the APOBEC mutagenesis is not enriched in direct repeats, mirror repeats, short tandem repeats, and G-quadruplexes, and even decreased below its level in B-DNA for cancer samples with very high APOBEC activity. In contrast, we observe that the APOBEC-induced mutational density is positively associated with APOBEC activity in inverted repeats (cruciform structures), where the impact of cytosine at the 3-end of the hairpin loop is substantial. Surprisingly, the APOBEC-signature mutation density per TC motif in the single-stranded DNA of a G-quadruplex (G4) is lower than in the four-stranded part of G4 and in B-DNA. The APOBEC mutagenesis, as well as the UV-mutagenesis in melanoma samples are absent in Z-DNA regions, due to depletion of their mutational signature motifs.

category: cancer biology


title: In vivo versus in silico assessment of potentially pathogenic missense variants in human reproductive genes

abstract: Infertility is a highly heterogeneous condition, with genetic causes estimated to be involved in approximately half of the cases. High-throughput sequencing (HTS) approaches are becoming an increasingly important tool for genetic diagnosis of diseases, including idiopathic infertility. Nevertheless, most rare or minor alleles revealed by HTS are classified as variants of uncertain significance (VUS). Interpreting the functional impacts of VUS is challenging but profoundly important for clinical management and genetic counseling. To determine the consequences of segregating polymorphisms in key fertility genes, we functionally evaluated 8 missense variants in the genes ANKRD31, BRDT, DMC1, EXOI, FKBP6, MSH4 and SEPT12 by generating genome-edited mouse models. Six variants were classified as deleterious by most functional prediction algorithms, and two disrupted a protein-protein interaction in the yeast 2 hybrid assay. Even though these genes are known to be essential for normal meiosis or spermiogenesis in mice, none of the tested human variants compromised fertility or gametogenesis in the mouse models. These results should be useful for genetic diagnoses of infertile patients, but they also underscore the need for more effective VUS categorization. To this end, we evaluated the performance of 10 widely used pathogenicity prediction algorithms in classifying missense variants within fertility-related genes from two sources: 1) the ClinVar database, and 2) those functionally tested in mice. We found that all the algorithms performed poorly in terms of predicting phenotypes of mouse-modeled variants. These studies highlight the importance of functional validation of potential infertility-causing genetic variants.


title: High levels of origin licensing during Xenopus cleavage divisions ensures complete and timely genome duplication

abstract: Cells face several challenges to completing genome duplication. One challenge is the irreversible stalling of converging replication forks ( double fork stalls). Cell types that cannot delay mitotic entry must also ensure that no replication origins are too far apart (the  random gap problem). We show how these challenges can be met in early Xenopus embryos by the very abundant licensing of replication origins: one MCM2-7 double hexamer every [~]250 bp. Licensing does not change nucleosome spacing, consistent with MCM2-7 being assembled onto inter-nucleosomal linker DNA. We show that later embryonic development can occur successfully with a per-cell cycle failure rate of <0.2% in early embryos. The high density of licensed origins in the early embryo reduces cell cycle failures from random gaps and from double fork stalls to levels compatible with subsequent development, suggesting that Xenopus early embryonic cells can ensure complete genome duplication without requiring unconventional replication mechanisms.


title: Rapid carbon accumulation at a saltmarsh restored by managed realignment far exceeds carbon emitted in site construction

abstract: Increasing attention is being paid to the carbon sequestration and storage services provided by coastal blue carbon ecosystems such as saltmarshes. Sites restored by managed realignment, where existing sea walls are breached to reinstate tidal inundation to the land behind, have considerable potential to accumulate carbon through deposition of sediment brought in by the tide and burial of vegetation in the site. While this potential has been recognised, it is not yet a common motivating factor for saltmarsh restoration, partly due to uncertainties about the rate of carbon accumulation and how this balances against the greenhouse gases emitted during site construction. We use a combination of field measurements over four years and remote sensing to quantify carbon accumulation at a large managed realignment site, Steart Marshes, UK. Sediment accumulated rapidly at Steart Marshes (mean of 75 mm yr-1) and had a high carbon content (4.4% total carbon, 2.2% total organic carbon), resulting in carbon accumulation of 36.6 t ha-1 yr-1 total carbon (19.4 t ha- 1 yr-1 total organic carbon). This rate of carbon accumulation is an order of magnitude higher than reported in many other restored saltmarshes, and is higher although more similar to values previously reported from another hypertidal system (Bay of Fundy, Canada). The estimated carbon emissions associated with the construction of the site were [~]2-4% of the observed carbon accumulation during the study period, supporting the view that managed realignment projects in such settings are likely to have significant carbon accumulation benefits. We outline further considerations that are needed to move towards a full carbon budget for saltmarsh restoration.

category: ecology


title: Applying And Promoting Open Science In Ecology - Surveyed Drivers And Challenges

abstract: Open Science (OS) comprises a variety of practices and principles that are broadly intended to improve the quality and transparency of research, and the concept is gaining traction. Since OS has multiple facets and still lacks a unifying definition, it may be interpreted quite differently among practitioners. Moreover, successfully implementing OS broadly throughout science requires a better understanding of the conditions that facilitate or hinder OS engagement, and in particular, how practitioners learn OS in the first place. We addressed these issues by surveying OS practitioners that attended a workshop hosted by the Living Norway Ecological Data Network in 2020. The survey contained scaled-response and open-ended questions, allowing for a mixed-methods approach. Out of 128 registered participants we obtained survey responses from 60 individuals. Responses indicated usage and sharing of data and code, as well as open access publications, as the OS aspects most frequently engaged with. Men and those affiliated with academic institutions reported more frequent engagement with OS than women and those with other affiliations. When it came to learning OS practices, only a minority of respondents reported having encountered OS in their own formal education. Consistent with this, a majority of respondents viewed OS as less important in their teaching than in their research and supervision. Even so, many of the respondents suggestions for what would help or hinder individual OS engagement included more knowledge, guidelines, resource availability and social and structural support; indicating that formal instruction can facilitate individual OS engagement. We suggest that the time is ripe to incorporate OS in teaching and learning, as this can yield substantial benefits to OS practitioners, student learning, and ultimately, the objectives advanced by the OS movement.

category: scientific communication and education


title: Time-based binding as a solution to and a limitation for flexible cognition

abstract: Why cant we keep as many items as we want in working memory? It has long been debated whether this resource limitation is a bug (a downside of our fallible biological system) or instead a feature (an optimal response to a computational problem). We propose that the resource limitation is a consequence of a useful feature. Specifically, we propose that flexible cognition requires time-based binding, and time-based binding necessarily limits the number of (bound) memoranda that can be stored simultaneously. Time-based binding is most naturally instantiated via neural oscillations, for which there exists ample experimental evidence. We report simulations that illustrate this theory and that relate it to empirical data. We also compare the theory to several other (feature and bug) resource theories.

category: neuroscience


title: Interactions between natural selection and recombination shape the genomic landscape of introgression

abstract: Hybridization between lineages that have not reached complete reproductive isolation appears more and more like a common phenomenon. Indeed, speciation genomics studies have now extensively shown that many species genomes have hybrid ancestry. However, genomic patterns of introgression are often heterogeneous across the genome. In many organisms, a positive correlation between introgression levels and recombination rate has been observed. It is usually explained by the purging of deleterious introgressed material due to incompatibilities. However, the opposite relationship was observed in a North American population of Drosophila melanogaster with admixed European and African ancestry. In order to explore how directional and epistatic selection can impact the relationship between introgression and recombination, we performed forward simulations of whole D. melanogaster genomes reflecting the North American populations history. Our results revealed that the simplest models of positive selection often yield negative correlations between introgression and recombination such as the one observed in D. melanogaster. We also confirmed that incompatibilities tend to produce positive introgression-recombination correlations. And yet, we identify parameter space under each model where the predicted correlation is reversed. These findings deepen our understanding of the evolutionary forces that may shape patterns of ancestry across genomes, and they strengthen the foundation for future studies aimed at estimating genome-wide parameters of selection in admixed populations.

category: evolutionary biology


title: Shorter constant work rate cycling tests as proxies for longer tests in highly trained cyclists

abstract:
Severe-intensity constant work rate (CWR) cycling tests are useful for monitoring training progression and adaptation as they impose significant physiological and psychological strain and thus simulate the high-intensity competition environment. However, fatiguing tests require substantial recovery and may disrupt athlete training or competition preparation. Therefore, the development of a brief, minimally fatiguing test providing comparable information is desirable.

PurposeTo determine whether physiological variables measured during, and functional decline in maximal power output immediately after, a 2-min CWR test can act as a proxy for 4-min test outcomes.

MethodsPhysiological stress was monitored and pre-to-post-CWR changes in 10-s sprint power computed (to estimate performance fatigability) during 2- and 4-min CWR tests in high-level cyclists.

ResultsThe 2-min CWR test evoked a smaller decline in sprint mechanical power (32% vs. 47%, p<0.001), however both the physiological variables and sprint mechanical power were independently and strongly correlated between 2- and 4-min tests. Differences in V{middle dot}O2peak and blood lactate concentration in both CWR tests were strongly associated with the decline in sprint mechanical power.

ConclusionPhysiological variables measured during, and the loss in sprint mechanical power measured after, a severe-intensity 2-min CWR test were less than in the 4-min test. Yet strong correlations between 2- and 4-min test outcomes indicated that the 2-min test can be used as a proxy for the longer test. Because shorter tests are less strenuous, they should have less impact on training and competition preparation and may therefore be more practically applicable within the elite performance environment.

category: physiology


title: Requirements for mammalian promoters to decode transcription factor dynamics

abstract: In response to different stimuli many transcription factors (TFs) display different activation dynamics that trigger the expression of specific sets of target genes, suggesting that promoters have a way to decode them. Combining optogenetics, deep learning-based image analysis and mathematical modeling, we find that decoding of TF dynamics occurs only when the coupling between TF binding and transcription pre-initiation complex formation is inefficient and that the ability of a promoter to decode TF dynamics gets amplified by inefficient translation initiation. Furthermore, we propose a theoretical mechanism based on phase separation that would allow a promoter to be activated better by pulsatile than sustained TF signals. These results provide an understanding on how TF dynamics are decoded in mammalian cells, which is important to develop optimal strategies to counteract disease conditions, and suggest ways to achieve multiplexing in synthetic pathways.

category: systems biology


title: Hippocampus-striatum wiring diagram revealed by directed stepwise polysynaptic tracing

abstract:
The hippocampus and the striatum represent two major systems in the brain for learning, memory and navigation. Although they were traditionally considered as two parallel systems responsible for distinct types of learning or navigation, increasing evidence indicates a close synergetic or competitive interaction between them. Both the hippocampus and the striatum consist of multiple anatomical and functional domains. Besides the limited direct projection from the hippocampus to the ventral striatum, most of the functional interaction between them may be mediated by polysynaptic projections. Polysynaptic connectivity has been difficult to examine due to a lack of methods to continuously track the pathways in a controlled manner. Here we developed a novel approach for directed stepwise polysynaptic tracing by reconstituting a replication-deficient retrograde transneuronal virus - pseudorabies virus lacking gene IE180 (PRV{Delta}IE). We minimized PRV neurotoxicity by temporally restricting viral replication; and enabled both anatomical tracing and functional analysis of the circuits. With these tools, we delineated a hippocampus-striatum wiring diagram, which consists of pathways from specific functional domains in the hippocampus to corresponding domains in the striatum via distinct intermediate regions. This polysynaptic wring diagram provides a structural foundation for further elucidation of the interaction between the hippocampus and the stratum in multiple brain functions.

Significance StatementWe created a new method for controlled stepwise tracing of polysynaptic neuronal circuits which also enables functional analysis of the circuits. With this method, we revealed the polysynaptic wiring diagram between the hippocampus and the striatum, two major brain structures centrally involved in learning, memory and spatial navigation. This wiring diagram demonstrates how specific anatomical domains in the hippocampus are connected to the specific domains in the striatum via distinct intermediate brain regions; thus it will help to elucidate the functional interaction between these two major memory and navigation systems. Our new method can be broadly applied to many other brain circuits for anatomical and functional analysis.


title: Representations of temporal community structure in hippocampus and precuneus predict inductive reasoning decisions

abstract: Our understanding of the world is shaped by inferences about underlying structure. For example, at the gym, you might notice that the same people tend to arrive around the same time and infer that they are friends that work out together. Consistent with this idea, after participants are presented with a temporal sequence of objects that follows an underlying community structure, they are biased to infer that objects from the same community share the same properties. Here, we used fMRI to measure neural representations of objects after temporal community structure learning and examine how these representations support inference about object relationships. We found that community structure learning affected inferred object similarity: when asked to spatially group items based on their experience, participants tended to group together objects from the same community. Neural representations in perirhinal cortex predicted individual differences in object grouping, suggesting that high-level object representations are affected by temporal community learning. Furthermore, participants were biased to infer that objects from the same community would share the same properties. Using computational modeling of temporal learning and inference decisions, we found that inductive reasoning is influenced by both detailed knowledge of temporal statistics and abstract knowledge of the temporal communities. The fidelity of temporal community representations in hippocampus and precuneus predicted the degree to which temporal community membership biased reasoning decisions. Our results suggest that temporal knowledge is represented at multiple levels of abstraction, and that perirhinal cortex, hippocampus, and precuneus may support inference based on this knowledge.


title: Benchmarking functional connectivity by the structure and geometry of the human brain

abstract: The brains structural connectivity supports the propagation of electrical impulses, manifesting as patterns of co-activation, termed functional connectivity. Functional connectivity emerges from the underlying sparse structural connections, particularly through poly-synaptic communication. As a result, functional connections between brain regions without direct structural links are numerous, but their organization is not completely understood. Here we investigate the organization of functional connections without direct structural links. We develop a simple, data-driven method to benchmark functional connections with respect to their underlying structural and geometric embedding. We then use this method to re-weigh and re-express functional connectivity. We find evidence of unexpectedly strong functional connectivity within the canonical intrinsic networks of the brain. We also find unexpectedly strong functional connectivity at the apex of the unimodal-transmodal hierarchy. Our results suggest that both phenomena - functional modules and functional hierarchies - emerge from functional interactions that transcend the underlying structure and geometry. These findings also potentially explain recent reports that structural and functional connectivity gradually diverge in transmodal cortex. Collectively, we show how structural connectivity and geometry can be used as a natural frame of reference with which to study functional connectivity patterns in the brain.


title: White matter microstructural and morphometric alterations in autism: Implications for intellectual and verbal capabilities

abstract:
BackgroundNeuroimage literature of autism spectrum disorder (ASD) has a moderate-to-high risk of bias, partially because those combined with intellectual impairment (II) and/or minimally verbal (MV) status are generally ignored. We aimed to provide more comprehensive insights into white matter alterations of ASD, inclusive of individuals with II (ASD-II-Only) or MV expression (ASD-MV).

MethodsSixty-five participants with ASD (ASD-Whole; 16.6{+/-}5.9 years; comprising 34 intellectually able youth, ASD-IA, and 31 intellectually impaired youth, ASD-II, including 24 ASD-II-Only plus 7 ASD-MV) and 38 demographic-matched typically developing controls (TDC; 17.3{+/-}5.6 years) were scanned in accelerated diffusion-weighted MRI. Fixel-based analysis was undertaken to investigate the categorical differences in fiber density (FD), fiber cross-section (FC), and a combined index (FDC), and brain-symptom/cognition associations.

ResultsASD-Whole had reduced FD in the anterior and posterior corpus callosum and left cerebellum Crus I, and smaller FDC in right cerebellum Crus II, compared to TDC. ASD-II, relative to TDC, showed almost identical alterations to those from ASD-Whole vs. TDC. ASD-II-Only had greater FD/FDC in the isthmus-splenium of callosum than ASD-MV. Autistic severity negatively correlated with FC in right Crus I. Non-verbal full-scale IQ positively correlated with FC/FDC in cerebellum VI. FD/FDC of the right dorsolateral prefrontal cortex showed a diagnosis-by-executive function interaction.

LimitationsWe could not preclude the potential effects of age and sex from the ASD cohort, although statistical tests suggested that these factors were not influential. Our results could be confounded by variable psychiatric comorbidities and psychotropic medication uses in our ASD participants recruited from outpatient clinics, which is nevertheless closer to a real-world presentation of ASD. The outcomes related to ASD-MV were considered preliminaries due to the small sample size within this subgroup.

Finally, our study design did not include intellectual impairment-only participants without ASD to disentangle the mixture of autistic and intellectual symptoms.

ConclusionsASD-associated white matter alterations appear driven by individuals with II and potentially further by MV. Results suggest that changes in the corpus callosum and cerebellum are key for psychopathology and cognition associated with ASD. Our work highlights an essential to include understudied sub-populations on the spectrum in research.


title: White matter microstructural and morphometric alterations in autism: Implications for intellectual capabilities


title: Transcriptome-wide Association Study and eQTL colocalization identify potentially causal genes responsible for bone mineral density GWAS associations

abstract: Genome-wide association studies (GWASs) for bone mineral density (BMD) have identified over 1,100 associations to date. However, identifying causal genes implicated by such studies has been challenging. Recent advances in the development of transcriptome reference datasets and computational approaches such as transcriptome-wide association studies (TWASs) and expression quantitative trait loci (eQTL) colocalization have proven to be informative in identifying putatively causal genes underlying GWAS associations. Here, we used TWAS/eQTL colocalization in conjunction with transcriptomic data from the Genotype-Tissue Expression (GTEx) project to identify potentially causal genes for the largest BMD GWAS performed to date. Using this approach, we identified 512 genes as significant (Bonferroni <= 0.05) using both TWAS and eQTL colocalization. This set of genes was enriched for regulators of BMD and members of bone relevant biological processes. To investigate the significance of our findings, we selected PPP6R3, the gene with the strongest support from our analysis which was not previously implicated in the regulation of BMD, for further investigation. We observed that Ppp6r3 deletion in mice decreased BMD. In this work, we provide an updated resource of putatively causal BMD genes and demonstrate that PPP6R3 is a putatively causal BMD GWAS gene. These data increase our understanding of the genetics of BMD and provide further evidence for the utility of combined TWAS/colocalization approaches in untangling the genetics of complex traits.


title: Improved Unsupervised Representation Learning of Spatial Transcriptomic Data with Sparse Filtering

abstract: We have developed representation learning methods, specifically to address the constraints and advantages of complex spatial data. Sparse filtering (SFt), uses principles of sparsity and mutual information to build representations from both global and local features from a minimal list of samples. Critically, the samples that comprise each representation are listed and ranked by informativeness. We used the Allen Mouse Brain Atlas gene expression data for prototyping and established performance metrics based on representation accuracy to labeled anatomy. SFt, implemented with the PyTorch machine learning libraries for Python, returned the most accurate reconstruction of anatomical ground truth of any method tested. SFt generated gene lists could be further compressed, retaining 95% of informativeness with only 580 genes. Finally, we build classifiers capable of parsing anatomy with >95% accuracy using only 10 derived genes. Sparse learning is a powerful, but underexplored means to derive biologically meaningful representations from complex datasets and a quantitative basis for compressed sensing of classifiable phenomena. SFt should be considered as an alternative to PCA or manifold learning for any high dimensional dataset and the basis for future spatial learning algorithms.

category: bioinformatics


title: Sparse Representation Learning Derives Biological Features with Explicit Gene Weights from the Allen Mouse Brain Atlas


title: A penalized linear mixed model with generalized method of moments for complex phenotype prediction

abstract: Linear mixed models have long been the method of choice for risk prediction analysis on high-dimensional genomic data. However, it remains computationally challenging to simultaneously model a large amount of genetic variants that can be noise or have predictive effects of complex forms. In this work, we have developed a penalized linear mixed model with generalized method of moments (pLMMGMM) estimators for prediction analysis. pLM-MGMM is built within the linear mixed model framework, where random effects are used to model the joint predictive effects from all genetic variants within a region. Fundamentally different from existing methods that usually focus on linear relationships and use empirical criteria for feature screening, pLMMGMM can jointly consider a large number of genetic regions and efficiently select those harboring variants with both linear and non-linear predictive effects. Through theoretical investigations, we have shown that our method has the selection consistency, estimation consistency and asymptotic normality. Through extensive simulations and the analysis of PET-imaging outcomes, we have demonstrated that pLMMGMM outperformed existing models and it can accurately detect regions that harbor risk factors with various forms of predictive effects.


title: phylogatR: Phylogeographic data aggregation and repurposing

abstract: Patterns of genetic diversity within species contain information about the history of that species, including how they have responded to historical climate change and how easily the organism is able to disperse across its habitat. More than 40,000 phylogeographic and population genetic investigations have been published to date, each collecting genetic data from hundreds of samples. Despite these millions of data points, meta-analyses are challenging because the synthesis of results across hundreds of studies, each using different methods and forms of analysis, is a daunting and time-consuming task. It is more efficient to proceed by repurposing existing data and using automated data analysis. To facilitate data repurposing, we created a database (phylogatR) that aggregates data from different sources and conducts automated multiple sequence alignments and data curation to provide users with nearly ready-to-analyze sets of data for thousands of species. Two types of scientific research will be made easier by phylogatR, large meta-analyses of thousands of species that can address classic questions in evolutionary biology and ecology and student- or citizen-science based investigations that will introduce a broad range of people to the analysis of genetic data. phylogatR enhances the value of existing data via the creation of software and web-based tools that enable these data to be recycled and reanalyzed and increase accessibility to big data for research labs and classroom instructors with limited computational expertise and resources.


title: Re-assessing the phylogenetic status and evolutionary relationship of Forest Owlet  using genomic data

abstract:
Phylogenetic relationships are often challenging to resolve in recent/younger lineage when only a few loci are used. Ultra Conserved Elements (UCE) are highly conserved regions across taxa that help resolve shallow and deep divergences. We utilized UCEs harvested from whole genomes to assess the phylogenetic position and taxonomic affiliation of an endangered endemic owlet in the family Strigidae - the Forest Owlet Athene blewitti. The taxonomic placement of this species has been revised multiple times. A multigene study attempted to address the question but showed a discrepancy across datasets in its placement of the species within genus Athene. We assembled a dataset of 5018 nuclear UCE loci with increased taxon sampling. Forest Owlet was found to be an early split from the Athene clade but sister to other Athene; and consistent across three approaches - maximum likelihood, bayesian, and the multispecies coalescence. Divergence dating using fossil calibrations suggest that the Athene lineage split from its ancestor about 7.6Mya, and the Forest Owlet diverged about 5.2Mya, consistent with previous multigene approaches. Despite osteological differences from other Athene, we suggest the placement of the Forest Owlet as a member of the Athene to emphasize its evolutionary relationship.

Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

HIGHLIGHTSO_LIPhylogenomics using genome-wide nuclear markers yielded a well-supported topology for Athene and Glaucidium lineages.
C_LIO_LIThree different methods of phylogenetic tree construction showed that Forest Owlet is an early split from all other Athene species.
C_LIO_LIDivergence dating in the bayesian framework puts the Forest Owlet age between 5.0my to 5.5my.
C_LI


title: Anatomy of the Heart with the Highest Heart Rate

abstract: Shrews occupy the lower extreme of the seven orders of magnitude mammals range in size. Their hearts are large relative to body weight and heart rate can exceed a thousand beats a minute. To investigate whether cardiac traits that are typical mammalian scale to these extremes, we assessed the heart of three species of shrew (genus Sorex) following the sequential segmental analysis developed for human hearts. Using micro-computed tomography we describe the overall structure and find, in agreement with previous studies, a large and elongate ventricle. The atrial and ventricular septums and the atrioventricular and arterial valves are typically mammalian. The ventricular walls comprise mostly compact myocardium and especially the right ventricle has few trabeculations on the luminal side. A developmental process of compaction is thought to reduce trabeculations in mammals, but in embryonic shrews the volume of trabeculations increase for every gestational stage, only slower than the compact volume. By expression of Hcn4, we identify a sinus node and an atrioventricular conduction axis which is continuous with the ventricular septal crest. Outstanding traits include pulmonary venous sleeve myocardium that reaches farther into the lungs than in any other mammals. Typical proportions of coronary arteries-to-aorta do not scale and the shrew coronary arteries are proportionally enormous, presumably to avoid the high resistance to blood flow of narrow vessels. In conclusion, most cardiac traits do scale to the miniscule shrews. The shrew heart, nevertheless, stands out by its relative size, elongation, proportionally large coronary vessels, and extent of pulmonary venous myocardium.

category: zoology


title: Specific N-glycans regulate an extracellular adhesion complex during somatosensory dendrite patterning

abstract: N-glycans are molecularly diverse sugars borne by over 70% of proteins transiting the secretory pathway and have been implicated in protein folding, stability, and localization. Mutations in genes important for N-glycosylation result in congenital disorders of glycosylation that are often associated with intellectual disability. Here, we show that structurally distinct N-glycans regulate the activity of an extracellular protein complex involved in patterning of somatosensory dendrites in Caenorhabditis elegans. Specifically, aman-2/Golgi alpha-mannosidase II, a conserved key enzyme in the biosynthesis of specific N-glycans regulates the activity of the Menorin adhesion complex without obviously affecting protein stability and localization of its components. AMAN-2 functions cell-autonomously to ensure decoration of the neuronal transmembrane receptor DMA-1/LRR-TM with high-mannose/hybrid N-glycans. Moreover, distinct types of N-glycans on specific N-glycosylation sites regulate the DMA-1/LRR-TM receptor, which together with three other extracellular proteins forms the Menorin adhesion complex. In summary, specific N-glycan structures regulate dendrite patterning by coordinating the activity of an extracellular adhesion complex suggesting that the molecular diversity of N-glycans can contribute to developmental specificity in the nervous system.


title: Realization of phosphorylation hypothesis of sleep by mammalian CaMKIIβ

abstract: The reduced sleep duration observed in Camk2a and Camk2b knockout mice revealed the role of Ca2+/calmodulin-dependent protein kinase II (CaMKII)/CAMKII{beta} as sleep-promoting kinases and lead to the phosphorylation hypothesis of sleep. However, the underlying mechanism of sleep regulation by kinases and protein phosphorylation is largely unknown. Here, we demonstrate that the phosphorylation states of CaMKII{beta} regulates sleep duration and sleep needs. Importantly, the activation or inhibition of CaMKII{beta} can increase or decrease sleep duration by almost two-fold, supporting the role of CaMKII{beta} as a core sleep regulator in mammals. This sleep regulation depends on the kinase activity of CaMKII{beta} in excitatory neurons. Furthermore, CaMKII{beta} mutants mimicking different phosphorylation states can regulate various sleep steps including sleep induction, sleep maintenance, and sleep cancelation. Key CaMKII{beta} residues responsible for the mode switch undergo ordered (auto-)phosphorylation. We thus propose that ordered multi-site phosphorylation of CaMKII{beta} underlies multi-step sleep regulation in mammals.


title: Distinct dendritic Ca2+ spike forms produce opposing input-output transformations in rat CA3 pyramidal cells

abstract: Proper integration of different inputs targeting the dendritic tree of CA3 pyramidal cells (CA3PCs) is critical for associative learning and recall. Dendritic Ca2+ spikes have been proposed to perform associative computations in other PC types, by detecting conjunctive activation of different afferent input pathways, initiating afterdepolarization (ADP) and triggering burst firing. Implementation of such operations fundamentally depends on the actual biophysical properties of dendritic Ca2+ spikes; yet little is known about these properties in dendrites of CA3PCs. Using dendritic patch-clamp recordings and two-photon Ca2+ imaging in acute slices from male rats we report that, unlike CA1PCs, distal apical trunk dendrites of CA3PCs exhibit distinct forms of dendritic Ca2+ spikes. Besides ADP-type global Ca2+ spikes, a majority of dendrites expresses a novel, fast Ca2+ spike type that is initiated locally without backpropagating action potentials, can recruit additional Na+ currents, and is compartmentalized to the activated dendritic subtree. Occurrence of the different Ca2+ spike types correlates with dendritic structure, indicating morpho-functional heterogeneity among CA3PCs. Importantly, ADPs and dendritically initiated spikes produce opposing somatic output: bursts versus strictly single action potentials, respectively. The uncovered variability of dendritic Ca2+ spikes may underlie heterogeneous input-output transformation and bursting properties of CA3PCs, and might specifically contribute to key associative and non-associative computations performed by the CA3 network.


title: Cross-feeding shapes both competition and cooperation in microbial ecosystems

abstract: Recent work suggests that cross-feeding - the secretion and consumption of metabolic biproducts by microbes - is essential for understanding microbial ecology. Yet how cross-feeding and competition combine to give rise to ecosystem-level properties remains poorly understood. To address this question, we analytically analyze the Microbial Consumer Resource Model (MiCRM), a prominent ecological model commonly used to study microbial communities. Our mean-field solution exploits the fact that unlike replicas, the cavity method does not require the existence of a Lyapunov function. We use our solution to derive new species-packing bounds for diverse ecosystems in the presence of cross-feeding, as well as simple expressions for species richness and the abundance of secreted resources as a function of cross-feeding (metabolic leakage) and competition. Our results show how a complex interplay between competition for resources and cooperation resulting from metabolic exchange combine to shape the properties of microbial ecosystems.


title: Highly similar sequence and structure yet different biophysical behaviour: A computational study of two triosephosphate isomerases

abstract: Homodimeric triosephosphate isomerases (TIM) from Trypanosoma cruzi (TcTIM) and Trypanosoma brucei (TbTIM) have a markedly similar amino acid sequences and three-dimensional structures. However, several of their biophysical parameters, such as their susceptibility to sulfhydryl agents and their reactivation speed after being denatured, have significant differences. The causes of these differences were explored with microsecond-scale molecular dynamics (MD) simulations of three different TIM proteins: TcTIM, TbTIM and a chimeric protein, Mut1. We examined their electrostatic interactions and explored the impact of simulation length on them. The same salt bridge between catalytic residues Lys 14 and Glu 98 was observed in all three proteins but key differences were found in other interactions that the catalytic amino acids form. In particular, a cation-{pi} interaction between catalytic amino acids Lys 14 and His 96, and both a salt bridge and a hydrogen bond between catalytic Glu168 and residue Arg100, were only observed in TcTIM. Furthermore, although TcTIM forms less hydrogen bonds than TbTIM and Mut1, its hydrogen bond network spans almost the entire protein, connecting the residues in both monomers. This work provides new insight on the mechanisms that give rise to the different behaviour of these proteins. The results also show the importance of long simulations.


title: STOICHIOMETRY OF THE SODIUM PUMP-PHOSPHOLEMMAN REGULATORY COMPLEX

abstract: The sodium-potassium ATPase (NKA) establishes ion gradients that facilitate many physiological processes. In the heart, NKA activity is regulated by its interaction with phospholemman (PLM, FXYD1). Here we used a novel fluorescence lifetime-based assay to investigate the structure, stoichiometry, and affinity of the NKA-PLM regulatory complex. We observed concentration dependent association of the subunits of NKA-PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit followed by lower affinity alpha-alpha and alpha-PLM interactions. The data provide the first evidence that the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits in intact cells. Docking and molecular dynamics simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha-alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with stoichiometry (alpha-beta-PLM)2.


title: Control of protein activity by photoinduced spin polarized charge reorganization

abstract: Biomolecules within the living cell are subject to extensive electrical fields, particularly next to membranes. Indeed, a role for bioelectricity has been well established at the organismal level. While the importance of electrostatics in protein functions such as protein-protein association and enzymatic activity has been well documented, very little is known on how biomolecules respond to external electric fields, or in other words, what may be the potential contribution of polarizability to protein function. Here we use phototriggered charge injection from a site-specifically attached ruthenium photosensitizer to directly demonstrate the effects of charge redistribution within a protein. We find that binding of an antibody to phosphoglycerate kinase (PGK) is increased two folds under illumination. Remarkably, illumination is found to suppress the enzymatic activity of PGK by a factor as large as three. These responses are sensitive to the photosensitizer position on the protein. Surprisingly, left (but not right) circularly polarized light elicits these responses, indicating that the electrons involved in the observed dynamics are spin polarized, due to spin filtration by protein chiral structures. Our results directly establish the contribution of electrical polarization and the importance of the spin-dependent charge reorganization in the function of proteins. Future experiments with phototriggered charge injection will allow delineation of charge rearrangement pathways within proteins and will further depict their effects on protein function.


title: Oral glucose feeding enhances adherence of quiescent lymphocytes to fibronectin via non-canonical insulin signalling.

abstract:
Impaired glucose metabolism is associated with chronic inflammation, aberrant immunity and anomalous leukocyte trafficking. Conversely, infusion of functional immune cells restores glucose metabolism. Despite being exposed to periodic alterations in blood insulin levels upon fasting and feeding, studies exploring the physiological effects of these hormonal changes on quiescent circulating lymphocytes are missing. Here we find that oral glucose load in healthy men and mice enhance adherence of circulating peripheral blood mononuclear cells (PBMCs) to fibronectin. This led to increased homing of post-load PBMCs to injured blood vessels. Cell culture based experiments on Jurkat-T cells and PBMCs demonstrated that insulin elicits these adhesive effects through a non-canonical signalling involving insulin growth factor-1 receptor (IGF-1R) and phospholipase C gamma-1 (PLC{gamma}-1) mediated activation of integrin {beta}1. Our findings point to the relevance of post-prandial insulin spikes in regulating homing of circulating T-cells to various organs for tissue repair and immunity.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/464163v1_ufig1.gif" ALT="Figure 1">
View larger version (30K):
org.highwire.dtl.DTLVardef@11edce6org.highwire.dtl.DTLVardef@1389835org.highwire.dtl.DTLVardef@90c96eorg.highwire.dtl.DTLVardef@1988a63_HPS_FORMAT_FIGEXP  M_FIG Insulin mediates fibronectin adherence of lymphocytes through non-canonical signalling.

Insulin mediates auto-phosphorylation of IGF-1 receptor at Tyr1135 leading to activation of PLC-{gamma}1 through Tyr783 phosphorylation, which in turn leads to the activation of integrin 1 through intracellular calcium to ultimately enhance adhesion of quiescent lymphocytes to fibronectin.

C_FIG


title: Architect: a tool for producing high-quality metabolic models through improved enzyme annotation

abstract:
MotivationConstraints-based modeling is a powerful framework for understanding growth of organisms. Results from such simulation experiments can be affected at least in part by the quality of the metabolic models used. Reconstructing a metabolic network manually can produce a high-quality metabolic model but is a time-consuming task. At the same time, current methods for automating the process typically transfer metabolic function based on sequence similarity, a process known to produce many false positives.

ResultsWe created Architect, a pipeline for automatic metabolic model reconstruction from protein sequences. First, it performs enzyme annotation through an ensemble approach, whereby a likelihood score is computed for an EC prediction based on predictions from existing tools; for this step, our method shows both increased precision and recall compared to individual tools. Next, Architect uses these annotations to construct a high-quality metabolic network which is then gap-filled based on likelihood scores from the ensemble approach. The resulting metabolic model is output in SBML format, suitable for constraints-based analyses. Through comparisons of enzyme annotations and curated metabolic models, we demonstrate improved performance of Architect over other state-of-the-art tools.

AvailabilityCode for Architect is available at https://github.com/ParkinsonLab/Architect.

Contactjohn.parkinson@utoronto.ca

Supplementary informationSupplementary data are available at Bioinformatics online.


title: Getting out of a mammalian egg: the egg tooth and caruncle of the echidna

abstract: In the short-beaked echidna, Tachyglossus aculeatus, after an initial period of in utero development, the egg is laid in the pouch and incubated for 10 days. During this time, the fetuses develop an egg tooth and caruncle to help them hatch. However, there are only a few historical references that describe the development of the monotreme egg tooth. Using unprecedented access to echidna pre- and post-hatching tissues, the egg tooth and caruncle were assessed by micro-CT, histology and immunofluorescence, to map the changes at the morphological and molecular level. Unlike mammalian tooth germs that develop by invagination of a placode, the echidna egg tooth developed by evagination, similar to that of the first teeth in some reptiles. The egg tooth ankylosed to the premaxilla, rather than forming a mammalian thecodont attachment, with loss of the egg tooth post-hatching associated with high levels of odontoclasts, and apoptosis. The caruncle formed as a separate mineralisation from the adjacent nasal capsule, and as observed in birds and turtles, the nasal region epithelium expressed markers of cornification. Together, this highlights that the monotreme egg tooth shares many similarities with reptilian teeth, suggesting that this tooth is conserved from a common ancestor of mammals and reptiles.


title: The extrachromosomal circular DNAs of the rice blast pathogen Magnaporthe oryzae contain a wide variety of LTR retrotransposons, genes, and effectors

abstract: One of the ways genomes respond to stress is by shedding extrachromosomal circular DNAs (eccDNAs). EccDNAs can contain genes and dramatically increase their copy number. They can also reinsert into the genome, generating structural variation. They have been shown to provide a source of phenotypic and genotypic plasticity in several species. However, whole-circularome studies have so far been limited to a few model organisms. Fungal plant pathogens are a serious threat to global food security in part because of their rapid adaptation to disease prevention strategies. Understanding the mechanisms fungal pathogens use to escape disease control is paramount to curbing their threat. We present a whole circularome sequencing study of the rice blast pathogen Magnaporthe oryzae. We find that M. oryzae has a highly diverse circularome containing many genes and showing evidence of large LTR retrotransposon activity. We find that genes enriched on eccDNAs in M. oryzae occur in genomic regions prone to presence-absence variation and that disease associated genes are frequently on eccDNAs. Finally, we find that a subset of genes is never present on eccDNAs, which indicates that the presence of these genes on eccDNAs is selected against.


title: Lineage Recording Reveals the Phylodynamics, Plasticity and Paths of Tumor Evolution

abstract: Tumor evolution is driven by the progressive acquisition of genetic and epigenetic alterations that enable uncontrolled growth, expansion to neighboring and distal tissues, and therapeutic resistance. The study of phylogenetic relationships between cancer cells provides key insights into these processes. Here, we introduced an evolving lineage-tracing system with a single-cell RNA-seq readout into a mouse model of Kras;Trp53(KP)-driven lung adenocarcinoma which enabled us to track tumor evolution from single transformed cells to metastatic tumors at unprecedented resolution. We found that loss of the initial, stable alveolar-type2-like state was accompanied by transient increase in plasticity. This was followed by adoption of distinct fitness-associated transcriptional programs which enable rapid expansion and ultimately clonal sweep of rare, stable subclones capable of metastasizing to distant sites. Finally, we showed that tumors develop through stereotypical evolutionary trajectories, and perturbing additional tumor suppressors accelerates tumor progression by creating novel evolutionary paths. Overall, our study elucidates the hierarchical nature of tumor evolution, and more broadly enables the in-depth study of tumor progression.


title: Loss of KRASG12D feedback regulation involving splicing factor SRSF1 accelerates pancreatic cancer

abstract:
The gene encoding KRAS GTPase is recurrently mutated in pancreatic ductal adenocarcinoma (PDAC), triggering the formation of precursor lesions, i.e., acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN). However, the majority of pancreatic cells from KC (LSL-KrasG12D/+; Pdx-1-Cre) mice expressing the KrasG12D mutation remain morphologically normal for a long time, suggesting the existence of compensatory feedback mechanisms that buffer aberrant KrasG12D signaling, and that additional steps are required for disrupting cell homeostasis and promoting transformation. Here we report a feedback mechanism in which the ubiquitously expressed splicing factor SRSF1--which is associated with cell transformation in multiple cell types--is downregulated in the majority of morphologically normal pancreas cells with the KrasG12D mutation. Conversely, increasing SRSF1 expression disrupts cell homeostasis by activating MAPK signaling, in part by regulating alternative splicing and mRNA stability of interleukin 1 receptor type 1 (Il1r1). This disruption in homeostasis in turn accelerates KrasG12D-mediated PDAC initiation and progression. Our results demonstrate the involvement of SRSF1 in the pancreatic-cell homeostatic response against the KrasG12D mutation, dysregulation of which facilitates PDAC initiation.

One-Sentence SummarySplicing factor SRSF1 is involved in KRASG12D feedback regulation and pancreatic-cancer tumorigenesis.


title: A molecular framework for proximal secondary vein branching in the Arabidopsis thaliana embryo

abstract:
O_LIThe establishment of a closed vascular network in foliar organs is achieved through the coordinated specification of newly recruited procambial cells, their proliferation and elongation. An important, yet poorly understood component of this process, is secondary vein branching; a mechanism employed in Arabidopsis thaliana cotyledons to extend vascular tissues throughout the organs surface by secondary vein formation.
C_LIO_LITo investigate the underlying molecular mechanism in vein branching, we analyzed at a single-cell level the discontinuous vein network of cotyledon vascular pattern 2 (cvp2) cvp2-like 1 (cvl1). Utilizing live-cell imaging and genetic approaches we uncovered two distinct branching mechanisms during embryogenesis.
C_LIO_LISimilar to wild type, distal veins in cvp2 cvl1 embryos emerged from the bifurcation of cell files contained in the midvein. However, the branching events giving rise to proximal veins are absent in this mutant. Restoration of proximal branching in cvp2 cvl1 cotyledons could be achieved by increasing OCTOPUS dosage as well as by silencing of RECEPTOR LIKE PROTEIN KINASE 2 (RPK2) expression. The RPK2-mediated restriction of proximal branching is auxin and CLE-independent.
C_LIO_LIOur work defines a genetic network conferring plasticity to Arabidopsis embryos to adapt the spatial configuration of vascular tissues to organ growth.
C_LI

category: plant biology


title: Wheat grain width: A clue for re-exploring visual indicators of grain weight

abstract: Mean grain weight (MGW) is among the most frequently measured parameters in wheat breeding and physiology. Although in the recent decades, various wheat grain analyses (e.g. counting, and determining the size, color, or shape features) have been facilitated thanks to the automated image processing systems, MGW estimations has been limited to using few number of image-derived indices; i.e. mainly the linear or power models developed based on the projected area (Area). Following a preliminary observation which indicated the potential of grain width in improving the predictions, the present study was conducted to explore potentially more efficient indices for increasing the precision of image-based MGW estimations. For this purpose, an image archive of the grains was processed, which was harvested from a two-year field experiment carried out with 3 replicates under two irrigation conditions and included 15 cultivar mixture treatments (so the archive was consisted of 180 images taken from an overall number of more than 72000 grains). It was observed that among the more than 30 evaluated indices of grain size and shape, indicators of grain width (i.e. Minor & MinFeret) along with 8 other empirical indices had a higher correlation with MGW, compared with Area. The most precise MGW predictions were obtained using the AreaxCircularity, PerimeterxCircularity, and Area/Perimeter indices. In general, two main common factors were detected in the structure of the major indices, i.e. either grain width or the Area/Perimeter ratio. Moreover, comparative efficiency of the superior indices almost remained stable across the 4 environmental conditions. Eventually, using the selected indices, ten simple linear models were developed and validated for MGW prediction, which indicated a relatively higher precision than the current Area-based models. The considerable effect of enhancing image resolution on the precision of the models has been also evidenced. It is expected that the findings of the present study improve the precision of the image-based MGW estimations, and consequently facilitate wheat breeding and physiological assessments.