bluuebunny/crossref_metadata_2025 huggingface dataset:
cazy_webscraper: local compilation and interrogation of comprehensive CAZyme datasets
Authors: Hobbs Emma E. M. , Gloster Tracey M. , Pritchard Leighton
Published: December 2022

Carbohydrate Active enZymes (CAZymes) are pivotal in biological processes including energy metabolism, cell structure maintenance, signalling and pathogen recognition. Bioinformatic prediction and mining of CAZymes improves our understanding of these activities, and enables discovery of candidates of interest for industrial biotechnology, particularly the processing of organic waste for biofuel production. CAZy (www.cazy.org) is a high-quality, manually-curated and authoritative database of CAZymes that is often the starting point for these analyses. Automated querying, and integration of CAZy data with other public datasets would constitute a powerful resource for mining and exploring CAZyme diversity. However, CAZy does not itself provide methods to automate queries, or integrate annotation data from other sources (except by following hyperlinks) to support further analysis.

To overcome these limitations we developedcazy_webscraper, a command-line tool that retrieves data from CAZy and other online resources to build a local, shareable, and reproducible database that augments and extends the authoritative CAZy database.cazy_webscraper’s integration of curated CAZyme annotations with their corresponding protein sequences, up to date taxonomy assignments, and protein structure data facilitates automated large-scale and targeted bioinformatic CAZyme family analysis and candidate screening. This tool has found widespread uptake in the community, with over 20,000 downloads.

We demonstrate the use and application ofcazy_webscraperto: (i) augment, update and correct CAZy database accessions; (ii) explore taxonomic distribution of CAZymes recorded in CAZy, identifying underrepresented taxa and unusual CAZy class distributions; and (iii) investigate three CAZymes having potential biotechnological application for degradation of biomass, but lacking a representative structure in the PDB database. We describe in general howcazy_webscraperfacilitates functional, structural and evolutionary studies to aid identification of candidate enzymes for further characterisation, and specifically note that CAZy provides supporting evidence for recent expansion of the Auxiliary Activities (AA) CAZy family in eukaryotes, consistent with functions potentially specific to eukaryotic lifestyles.

Supplementary information

cazy_webscrapersource code is available athttps://github.com/HobnobMancer/cazy_webscraper, and online documentation is provided athttps://cazywebscraper.readthedocs.io.
Selective volumetric excitation and imaging for single molecule localization microscopy in multicellular systems
Authors: Galgani Tommaso , Fedala Yasmina , Zapata Romeo , Caccianini Laura , Viasnoff Virgile , Sibarita Jean-Baptiste , Galland Rémi , Dahan Maxime , Hajj Bassam
Published: December 2022

Light sheet fluorescence microscopy (LSFM) has become a leading standard in high-resolution imaging of living samples in 2- and 3-dimensions. Biological samples are however not restricted to a single observation plane and several molecular processes evolve rapidly in 3D. The conventional mechanical scanning required in LSFM limits the range of observable dynamics and are usually restricted in resolution. Here we introduce a new strategy for instantaneous volumetric excitation and volumetric imaging of single-molecules in cell aggregates. The technique combines, for the first time, the use of light sheet microscopy and multifocus microscopy (MFM) and enables a volumetric 4D imaging of biological samples with single-molecule resolution. We engineered the excitation beam to yield a modular and uniform excitation matching the observable detection range of MFM. The strength of the method is highlighted with examples of single-molecule 3D tracking and 3D super-resolution imaging in multicellular samples.
Development of non-alcoholic steatohepatitis is associated with gut microbiota but not oxysterol synthesis
Authors: Wyss Jacqueline , Raselli Tina , Wyss Annika , Telzerov Anja , Rogler Gerhard , Krupka Niklas , Yilmaz Bahtiyar , Schmidt Thomas SB , Misselwitz Benjamin
Published: December 2022

Liver steatosis is the most frequent liver disorder and its advanced stage, non-alcoholic steatohepatitis (NASH), will soon become the main reason for liver fibrosis and cirrhosis. The “multiple hits hypothesis” suggests that progression from simple steatosis to NASH is triggered by multiple factors including the gut microbiota composition. The Epstein Barr virus induced gene 2 (EBI2) is a receptor for the oxysterol 7a, 25-dihydroxycholesterol synthesized by the enzymes CH25H and CYP7B1. EBI2 and its ligand control activation of immune cells in secondary lymphoid organs and the gut. Here we show a concurrent study of the microbial dysregulation and perturbation of the EBI2 axis in a mice model of NASH.

We used mice with wildtype, or littermates with CH25H-/-, EBI2-/-, or CYP7B1-/-genotypes fed with a high-fat diet (HFD) containing high amounts of fat, cholesterol, and fructose for 20 weeks to induce liver steatosis and NASH. Fecal and small intestinal microbiota samples were collected, and microbiota signatures were compared according to genotype and NASH disease state.

We found pronounced differences in microbiota composition of mice with HFD developing NASH compared to mice did not developing NASH. In mice with NASH, we identified significantly increased 33 taxa mainly belonging to the Clostridiales order and/ or the family, and significantly decreased 17 taxa. Using an Elastic Net algorithm, we suggest a microbiota signature that predicts NASH in animals with a HFD from the microbiota composition with moderate accuracy (area under the receiver operator characteristics curve=0.64). In contrast, no microbiota differences regarding the studied genotypes (wildtype vs knock-out CH25H-/-, EBI2-/-, or CYP7B1-/-) were observed.

In conclusion, our data confirm previous studies identifying the intestinal microbiota composition as a relevant marker for NASH pathogenesis. Further, no link of the EBI2 – oxysterol axis to the intestinal microbiota was detectable in the current study.
Specific interaction between Group BStreptococcusCC17 hypervirulent clone and phagocytes
Authors: Bourrel Anne-Sophie , Picart Amandine , Fernandez Jose-Carlos , Hays Constantin , Mignon Virginie , Saubaméa Bruno , Poyart Claire , Fouet Agnès , Tazi Asmaa , Guignot Julie
Published: December 2022

Streptococcus agalactiaealso named Group BStreptococcus(GBS) is the most significant pathogen causing invasive infections, such as bacteremia and meningitis, in neonates. Worldwide epidemiological studies have shown that a particular clonal complex (CC) of capsular serotype III, the CC17, is strongly associated with meningitis in neonates and is therefore designated as the hypervirulent clone. Macrophages are a permissive niche for intracellular bacteria of all GBS clones. In this study we deciphered the specific interaction of GBS CC17 strains with macrophages. Our study revealed that CC17 strains are phagocytosed at a higher rate than GBS non-CC17 strains by human monocytes and macrophages both in cellular models and primary cells. CC17 enhanced phagocytosis is due to an initial enhanced-attachment step to macrophages mediated by the CC17 specific surface protein HvgA and the PI-2b pilus (Spb1). We showed that two different inhibitors of scavenger receptors (fucoidan and poly(I)) specifically inhibited CC17 adhesion and phagocytosis while not affecting those of non-CC17 strains. Once phagocytosed, both CC17 and non-CC17 strains remained in a LAMP-1 positive vacuole that ultimately fuses with lysosomes where they can survive at similar rates. Finally, both strains displayed a basal egress which occurs independently from actin and microtubule networks. Our findings provide new insights into the interplay between the hypervirulent GBS CC17 and major players of the host’s innate immune response. This enhanced adhesion leading to higher phagocytosis could reflect a peculiar capacity of the CC17 lineage to subvert the host immune defenses, establish a niche for persistence or disseminate.
Leaf ontogeny steers ethylene and auxin crosstalk to regulate leaf epinasty during waterlogging of tomato
Authors: Geldhof B. , Pattyn J. , Mohorović P. , Van den Broeck K. , Everaerts V. , Novák O. , Van de Poel B.
Published: December 2022

Developing leaves undergo a vast array of age-related changes as they mature. These include physiological, hormonal and morphological changes that determine their adaptation plasticity towards adverse conditions. Waterlogging induces leaf epinasty in tomato, and the magnitude of leaf bending is intricately related to the age-dependent cellular and hormonal response. We now show that ethylene, the master regulator of epinasty, is differentially regulated throughout leaf development, giving rise to age-dependent epinastic responses. Young leaves have a higher basal ethylene production, but are less responsive to waterlogging-induced epinasty, as they have a higher capacity to convert the root-borne and mobilized ACC into the inactive conjugate MACC. Ethylene stimulates cell elongation relatively more at the adaxial petiole side, by activating auxin biosynthesis and locally inhibiting its transport through PIN4 and PIN9 in older and mature leaves. As a result, auxins accumulate in the petiole base of these leaves and enforce partially irreversible epinastic bending upon waterlogging. Young leaves maintain their potential to transport auxins, both locally and through the vascular tissue, leading to enhanced flexibility to dampen the epinastic response and a faster upwards repositioning during reoxygenation. This mechanism also explains the observed reduction of epinasty during and its recovery after waterlogging in theanthocyanin reduced(are) andNever ripe(Nr) mutants, both characterized by higher auxin flow. Our work has demonstrated that waterlogging activates intricate hormonal crosstalk between ethylene and auxin, controlled in an age-dependent way.
Mechanisms that promote the evolution of cross-reactive antibodies upon vaccination with designed influenza immunogens
Authors: Yang Leerang , Caradonna Timothy M. , Schmidt Aaron G. , Chakraborty Arup K.
Published: December 2022

Immunogens that elicit broadly neutralizing antibodies targeting the conserved receptor-binding site (RBS) on influenza hemagglutinin (HA) may serve as a universal influenza vaccine candidate. Here, we developed a computational model to interrogate antibody evolution by affinity maturation after immunization with two types of immunogens: a chimeric heterotrimeric ‘HAtCh’ antigen that is enriched for the RBS epitope relative to other B cell epitopes, and a cocktail composed of three non-epitope-enriched homotrimeric antigens that comprise the HAtCh. Experiments in mice (Caradonna et al.) find that the chimeric antigen outperforms the cocktail for eliciting RBS-directed antibodies. We show that this result follows from an interplay between how B cells engage these antigens and interact with diverse T helper cells, and requires T cell-mediated selection of germinal center B cells to be a stringent constraint. Our results shed new light on antibody evolution, and highlight how immunogen design and T cells modulate vaccination outcomes.
Dimerization thermodynamics govern biological phase separation
Authors: Dubach J. Matthew
Published: December 2022

Many intrinsically disordered proteins/regions (IDR) can phase separate into a liquid state (biomolecular condensates) and/or a solid state (aggregates and amyloids). De-mixing in liquid-like phase separation carries an entropic cost that must be balanced by an energetic advantage of condensing. Yet, the mechanism of this energetic advantage remains unclear. Here, it is shown that an increase in dimerization binding interactions through liquid-like condensation can create overall energetic minimums when a system phase separates. Yet, the dimer binding interactions must be rapid explaining why IDRs are so prominent in phase separations. Numerous experimental examples that can only be described through dimer interactions support the dimer model presented here. The transition from condensates to aggregates and amyloids under certain conditions also appears to be driven by dimer abundance. Overall, the dimer model provides a novel theory that solves the thermodynamic mechanism of protein phase separation into biomolecular condensates, aggregates and amyloids.
High-sensitivity calcium biosensor on the mitochondrial surface reveals that IP3R channels participate in the reticular Ca2+leak towards mitochondria
Authors: Gouriou Yves , Gonnot Fabrice , Gomez Ludovic , Bidaux Gabriel
Published: December 2022

Genetically encoded biosensors based on fluorescent proteins (FPs) are widely used to monitor dynamics and sub-cellular spatial distribution of calcium ion (Ca2+) fluxes and their role in intracellular signaling pathways. The development of different mutations in the Ca2+-sensitive elements of the cameleon probes has allowed sensitive range of Ca2+measurements in almost all cellular compartments. Region of the endoplasmic reticulum (ER) tethered to mitochondria, named as the mitochondrial-associated membranes (MAMs), has received an extended attention since the last 5 years. Indeed, as MAMs are essential for calcium homeostasis and mitochondrial function, molecular tools have been developed to assess quantitatively Ca2+levels in the MAMs. However, sensitivity of the first generation Ca2+biosensors on the surface of the outer-mitochondrial membrane (OMM)do not allow to measure μM or sub-μM changes in Ca2+concentration which prevents to measure the native activity (unstimulated exogenously) of endogenous channels. In this study, we assembled a new ratiometric highly sensitive Ca2+biosensor expressed on the surface of the outer-mitochondrial membrane (OMM). It allows the detection of smaller differences than the previous biosensor in or at proximity of the MAMs. Noteworthy, we demonstrated that IP3-receptors have an endogenous activity which participate to the Ca2+leak channel on the surface of the OMM during hypoxia or when SERCA activity is blocked.
The E-cadherin-ESR1-GRPR axis defines a sex-specific metastatic pathway in melanoma
Authors: Raymond Jérémy H. , Pouteaux Marie , Petit Valérie , Aktary Zackie , Luciani Flavie , Wehbe Maria , Gizzi Patrick , Bourban Claire , Martianov Igor , Davidson Irwin , Tomasetto Catherine-Laure , Betzer Florence-Mahuteau , Vergier Béatrice , Larue Lionel , Delmas Véronique
Published: December 2022

Although tremendous progress has been made in understanding the mechanisms leading to cancer, those governing metastases are still poorly understood. E-cadherin (Ecad) is a cell-cell adhesion molecule essential for tissue homeostasis, and its loss often correlates with the dissemination of human cancers. However, whether and how the loss of Ecad triggers the full metastatic program is largely unknown. Here, we show that the loss of Ecad promotes melanoma lung metastases in females. The loss of Ecad, after the induction of estrogen receptor α (ERα) expression, activates gastrin-releasing peptide receptor (GRPR) expression. GRPR promotes cellular processes essential for metastasis formation through G□qand YAP1 signaling and its pharmacological inhibition reduces metastasisin vivo. This study reveals an Ecad-ERα-GRPR metastatic sex dimorphism axis in melanoma that is conserved in human breast cancer and provides proof of concept that the G-coupled receptor GRPR is a therapeutic target for metastasis.
Phen2Disease: A Phenotype-driven Semantic Similarity-based Integrated Model for Disease and Gene Prioritization
Authors: Zhai Weiqi , Huang Xiaodi , Shen Nan , Zhu Shanfeng
Published: December 2022

By utilizing the Human Phenotype Ontology (HPO), recent approaches to prioritizing disease-causing genes for patients become popular. However, these approaches do not comprehensively use information about phenotypes of diseases and patients. We present a new method called Phen2Disease that calculates similarity scores between two phenotype sets of patients and diseases by which to prioritize diseases and genes. Specifically, we calculate three scores of information content-based similarities using the phenotypes, and their combination as the respective benchmarks, and integrate them as a final score. Comprehensive experiments were conducted on six real data cohorts with 2051 cases and two simulated data cohorts with 1000 cases. Compared with the three state-of-the-art methods, if we only use phenotype information and HPO knowledge base, Phen2Disease outperformed all of them, particularly in cohorts with the less average numbers of HPO terms. We have found that patients with higher information content scores had more specific information so their predictions would be more accurate. In addition, Phen2Disease has high interpretability with ranked diseases and patient HPO terms provided.
Viral burdens are associated with age and viral variant in a population-representative study of SARS-CoV-2 that accounts for time-since-infection related sampling bias
Authors: Fryer Helen R. , Golubchik Tanya , Hall Matthew , Fraser Christophe , Hinch Robert , Ferretti Luca , Thomson Laura , Nurtay Anel , Pellis Lorenzo , MackIntyre-Cockett George , Trebes Amy , Buck David , Piazza Paolo , Green Angela , Lonie Lorne J , Smith Darren , Bashton Matthew , Crown Matthew , Nelson Andrew , McCann Clare M. , Tariq Adnan Mohammed , Dos Santos Rui Nunes , Richards Zack ,   The COVID-19 Genomics UK (COG-UK) consortium, Bonsall David , Lythgoe Katrina A.
Published: December 2022

In this study, we evaluated the impact of viral variant, in addition to other variables, on within-host viral burdens, by analysing cycle threshold (Ct) values derived from nose and throat swabs, collected as part of the UK COVID-19 Infection Survey. Because viral burden distributions determined from community survey data can be biased due to the impact of variant epidemiology on the time-since-infection of samples, we developed a method to explicitly adjust observed Ct value distributions to account for the expected bias. Analysing the adjusted Ct values using partial least squares regression, we found that among unvaccinated individuals with no known prior infection, the average Ct value was 0.94 lower among Alpha variant infections, compared those with the predecessor strain, B.1.177. However, among vaccinated individuals, it was 0.34 lower among Delta variant infections, compared to those with the Alpha variant. In addition, the average Ct value decreased by 0.20 for every 10 year age increment of the infected individual. In summary, within-host viral burdens are associated with age, in addition to the interplay of vaccination status and viral variant.
Water shortage reducesPHYTOCHROME INTERACTING FACTOR 4, 5and3expression and shade avoidance in Arabidopsis
Authors: Semmoloni Mariana , Rojas Cecilia Costigliolo , Yan Yan , Cao Xiaofeng , Casal Jorge J.
Published: December 2022

In agricultural crops, forests and grasslands, water deficit often occurs in the presence of cues from neighbouring vegetation. However, most studies have addressed separately the mechanisms of plant growth responses to these two aspects of the environment. Here we show that transferringArabidopsis thalianaseedlings to agar containing polyethylene glycol (PEG) to restrict water availability reduces hypocotyl growth responses to shade without simultaneous affecting cotyledon expansion or its response to shade. Water restriction diminished the activity of thePHYTOCHROME INTERACTING FACTOR 4 (PIF4), PIF5, PIF3andPIF3-LIKE 1gene promoters, particularly in seedlings exposed to simulated shade. The response ofPIF4expression to PEG required the presence of its positive morning regulatorsCIRCADIAN CLOCK ASSOCIATED 1 (CCA1)andLATE ELONGATED HYPOCOTYL (LHY), which also reduced their expression in response to PEG. Water restriction diminished the nuclear abundance of PIF4 in hypocotyl cells only in the seedlings exposed to shade. In addition to the changes inPIF4levels, post-transcriptional processes also contributed to the response to PEG. Hypocotyl growth showed significant triple interaction among water availability, shade and the presence of PIF4, PIF5 and PIF3. Collectively, these results unveil PIFs as a hub that interlinks shade and drought information to control growth.
The dynamic linkage between intact provirus integration sites and the host functional genome property alongside HIV-1 infections associated with antiretroviral therapy
Authors: Chen Heng-Chang
Published: December 2022

The HIV-1 latent reservoir harboring replication-competent proviruses, is the major barrier in the quest for a HIV-1 infection cure. HIV-1 infection at all stages of disease progression is associated with immune activation and dysfunctional production of proinflammatory soluble factors (cytokines and chemokines) and it is expected that during HIV-1 infection different immune components and immune cells, in turn, participate in immune responses, subsequently activating downstream biological pathways. However, whether HIV-1 infections activate only specific pathways or result in the global activation of functional pathways is presently not fully understood. Therefore, in this work, I used genes targeted by intact proviruses from published datasets to seek enriched immunologic signatures and host biological pathways alongside HIV-1 infections. I observed that different compositions of immune cell types and proinflammatory soluble factors appeared alongside HIV-1 infections associated with antiretroviral therapy based on the over-representation analysis. Moreover, KEGG pathways relevant to “cancer specific type”, “immune system”, “infectious disease viral” and “signal transduction” were frequently enriched in HIV-1-infected individuals subjected to antiretroviral therapy.
Photoactivatable Large Stokes Shift Fluorophores for Multicolor Nanoscopy
Authors: Likhotkin Ilya , Lincoln Richard , Bossi Mariano L. , Butkevich Alexey N. , Hell Stefan W.
Published: December 2022

We designed caging-group-free photoactivatable live-cell permeant dyes with red fluorescence emission and ∼100 nm Stokes shifts based on a 1-vinyl-10-silaxanthone imine core structure. The proposed fluorophores undergo byproduct-free one- and two-photon activation, are suitable for multicolor fluorescence microscopy in fixed and living cells and are compatible with super-resolution techniques such as STED (stimulated emission depletion) and PALM (photoactivated localization microscopy). Use of photoactivatable labels for strain-promoted azide-alkyne cycloaddition and self-labeling protein tags (HaloTag, SNAP-tag), and duplexing of imaging channel with another large Stokes shift dye have been demonstrated.
Impacts of ocean acidification and warming on post-larval growth and metabolism in two populations of the great scallop (Pecten maximusL.)
Authors: Harney E. , Rastrick S.P.S. , Artigaud S. , Pisapia J. , Bernay B. , Miner P. , Pichereau V. , Strand Ø. , Boudry P. , Charrier G.
Published: December 2022

Ocean acidification and warming are key stressors for many marine organisms. Some organisms display physiological acclimatisation or plasticity, but this may vary across species ranges, especially if populations are adapted to local climatic conditions. Understanding how acclimatisation potential varies among populations is therefore important in predicting species responses to climate change. We carried out a common garden experiment to investigate how different populations of the economically important great scallop (Pecten maximus) from France and Norway responded to variation in temperature andpCO2concentration. After acclimation, post-larval scallops (spat) were reared for 31 days at one of two temperatures (13°C and 19°C) under either ambient or elevatedpCO2(pH 8.0 and pH 7.7). We combined measures of proteomic, metabolic, and phenotypic traits to produce an integrative picture of how physiological plasticity varies between the populations. The proteome of French spat showed significant sensitivity to environmental variation, with 12 metabolic, structural and stress-response proteins responding to temperature and/orpCO2. Principal component analysis revealed seven energy metabolism proteins in French spat that were consistent with countering ROS stress under elevated temperature. Oxygen uptake in French spat did not change under elevated temperature, but increased under elevatedpCO2. In contrast, Norwegian spat reduced oxygen uptake under both elevated temperature andpCO2. Metabolic plasticity seemingly allowed French scallops to maintain greater energy availability for growth than Norwegian spat. However, increased physiological plasticity and growth in French spat may come at a cost, as French (but not Norwegian) spat showed reduced survival under elevated temperature.

Summary Statement

Juvenile scallops from France and Norway differ in their response to warming and acidification. French scallops show more physiological plasticity, adjusting their proteome and metabolism in order to maintain growth.
PSIP1/LEDGF reduces R-loops at transcription sites to maintain genome integrity
Authors: Jayakumar Sundarraj , Patel Manthan , Boulet Fanny , Aziz Hadicha , Brooke Greg N. , Tummala Hemanth , Pradeepa Madapura M
Published: December 2022

R-loops that accumulate at transcription sites pose a persistent threat to genome integrity. PSIP1 is a chromatin protein associated with transcriptional elongation complex, possesses histone chaperone activity, and is implicated in recruiting RNA processing and DNA repair factors to transcription sites. Here, we show that PSIP1 interacts with R-loops and other proteins involved in R-loop homeostasis, including PARP1. Genome-wide mapping of PSIP1, R-loops and γ-H2AX in PSIP1-depleted human and mouse cell lines revealed an accumulation of R-loops and DNA damage at gene promoters in the absence of PSIP1. R-loop accumulation causes local transcriptional arrest and transcription-replication conflict, leading to DNA damage. PSIP1 depletion increases 53BP1 foci and reduces RAD51 foci, suggesting altered DNA repair choice. Furthermore, PSIP1 depletion increases the sensitivity of cancer cells to PARP1 inhibitors and DNA-damaging agents that induce R-loop-induced DNA damage. These findings provide fresh insights into the mechanism through which PSIP1 maintains genome integrity at the site of transcription.
Prioritized polycystic kidney disease drug targets and repurposing candidates from pre-cystic and cystic mousePkd2model gene expression reversion
Authors: Wilk Elizabeth J. , Howton Timothy C. , Fisher Jennifer L. , Oza Vishal H. , Brownlee Ryan T. , McPherson Kasi C. , Cleary Hannah L. , Yoder Bradley K. , George James F. , Mrug Michal , Lasseigne Brittany N.
Published: December 2022

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most prevalent monogenic human diseases. It is mostly caused by pathogenic variants inPKD1orPKD2genes that encode interacting transmembrane proteins polycystin-1 (PC1) and polycystin-2 (PC2). Among many pathogenic processes described in ADPKD, those associated with cAMP signaling, inflammation, and metabolic reprogramming appear to regulate the disease manifestations. Tolvaptan, a vasopressin receptor-2 antagonist that regulates cAMP pathway, is the only FDA-approved ADPKD therapeutic. Tolvaptan reduces renal cyst growth and kidney function loss, but it is not tolerated by many patients and is associated with idiosyncratic liver toxicity. Therefore, additional therapeutic options for ADPKD treatment are needed. As drug repurposing of FDA-approved drug candidates can significantly decrease the time and cost associated with traditional drug discovery, we used the computational approach signature reversion to detect inversely related drug response gene expression signatures from the Library of Integrated Network-Based Cellular Signatures (LINCS) database and identified compounds predicted to reverse disease-associated transcriptomic signatures in three publicly availablePkd2kidney transcriptomic data sets of mouse ADPKD models. We focused on a pre-cystic model for signature reversion, as it was less impacted by confounding secondary disease mechanisms in ADPKD, and then compared the resulting candidates’ target differential expression in the two cystic mouse models. We further prioritized these drug candidates based on their known mechanism of action, FDA status, targets, and by functional enrichment analysis. With this in-silico approach, we prioritized 29 unique drug targets differentially expressed inPkd2ADPKD cystic models and 16 prioritized drug repurposing candidates that target them, including bromocriptine and mirtazapine, which can be further tested in-vitro and in-vivo. Collectively, these indicate drug targets and repurposing candidates that may effectively treat pre-cystic as well as cystic ADPKD.

Abstract Figure

Graphical abstract of the study created with Biorender.com.
Native extracellular matrix probes to target patient- and tissue- specific cell-microenvironment interactions by force spectroscopy
Authors: Holuigue H. , Nacci L. , Di Chiaro P. , Chighizola M. , Locatelli I. , Schulte C. , Alfano M. , Diaferia G.R. , Podestà A.
Published: December 2022

Atomic Force Microscopy (AFM) is successfully used for the quantitative investigation of the cellular mechanosensing of the microenvironment. To this purpose, several force spectroscopy approaches aim at measuring the adhesive forces between two living cells and also between a cell and a suitable reproduction of the extracellular matrix (ECM), typically exploiting tips suitably functionalised with single components (e.g. collagen, fibronectin) of the ECM. However, these probes only poorly reproduce the complexity of the native cellular microenvironment and consequently of the biological interactions.

We developed a novel approach to produce AFM probes that faithfully retain the structural and biochemical complexity of the ECM; this was achieved by attaching to an AFM cantilever a micrometric slice of native decellularised ECM, which was cut by laser microdissection. We demonstrate that these probes preserve the morphological, mechanical, and chemical heterogeneity of the ECM.

Native ECM probes can be used in force spectroscopy experiments aimed at targeting cell-microenvironment interactions. Here, we demonstrate the feasibility of dissecting mechanotransductive cell-ECM interactions in the 10 pN range. As proof-of-principle, we tested a rat bladder ECM probe against the AY-27 rat bladder cancer cell line. On the one hand, we obtained reproducible results using different probes derived from the same ECM regions; on the other hand, we detected differences in the adhesion patterns of distinct bladder ECM regions, such as submucosa and detrusor, in line with the disparities in composition and biophysical properties of these ECM regions.

Our results demonstrate that native ECM probes, produced from patient-specific regions of organs and tissues, can be used to investigate cell-microenvironment interactions and early mechanotransductive processes by force spectroscopy. This opens new possibilities in the field of personalised medicine.
Growth/differentiation factor 15 controls number of ependymal and neural stem cells in the ventricular-subventricular zone
Authors: Baur Katja , Carrillo-García Carmen , Şan Şeydanur , von Hahn Manja , Strelau Jens , Hölzl-Wenig Gabriele , Mandl Claudia , Ciccolini Francesca
Published: December 2022

Late in neural development, the expression of growth/differentiation factor (GDF) 15 increases in the germinal epithelium of the murine ganglionic eminence (GE), especially in progenitors with characteristics of neural stem cells (NSCs). However, the function of GDF15 in this region is still unknown. We here show that apical progenitors in the E18 GE also express the GDF15 receptor and that ablation of GDF15 promotes proliferation and cell cycle progression of apically and subapically dividing progenitors. A similar phenotype was also observed in the adult ventricular subventricular zone (V-SVZ). At both ages, increased proliferation leads to the transient generation of more neuronal progenitors, which is compensated by cell death, and to a permanent increase in the number of ependymal cells and apical NSCs. We also found that GDF15 receptor-expressing cells display immunoreactivity for the epidermal growth factor receptor (EGFR), which is also involved in progenitor proliferation, and that manipulation of GDF15 affects the expression of EGFR in mutant progenitors. Moreover, our data indicate that EGFR signalling in WT and mutant progenitors relies on distinct transduction modes. However, only exposure to exogenous GDF15, but not to EGF, normalized proliferation and the number of apical progenitors, indicating that alteration in EGFR signalling is not the main mechanism by which GDF15 affects proliferation in the embryonic GE.

Taken together, GDF15 directly regulates proliferation of apical progenitors in the developing GE, thereby affecting the number of total ependymal cells and NSCs in this region.
An Autoimmune Transcriptional Circuit Driving Foxp3+Regulatory T cell Dysfunction
Authors: Sumida Tomokazu S. , Lincoln Matthew R. , He Liang , Park Yongjin , Ota Mineto , Stillwell Helen A. , Leissa Greta A. , Fujio Keishi , Kulminski Alexander M. , Epstein Charles B. , Bernstein Bradley E. , Kellis Manolis , Hafler David A.
Published: December 2022

Autoimmune diseases, among the most common disorders of young adults, are mediated by genetic and environmental factors. While CD4+Foxp3+regulatory T cells (Tregs) play a central role in preventing autoimmunity, the molecular mechanism underlying their dysfunction is unknown. Here, we performed comprehensive transcriptomic and epigenomic profiling of Tregs in the autoimmune disease multiple sclerosis (MS) to identify central transcriptional programs regulating human autoimmunity. We discovered that upregulation of a primate-specific shortPRDM1isoform (PRDM1-S) inducesSGK1independent from evolutionally conserved longPRDM1, leading to destabilization of Foxp3 and Treg dysfunction. This aberrantPRDM1-S/SGK1axis is shared among other autoimmune diseases. Furthermore, by chromatin landscape profiling in MS Tregs we identified aPRDM1-Sspecificcis-regulatory element associated with enriched binding of AP-1/IRF transcription factors. Our study identifies evolutionally emergedPRDM1-Sand epigenetic priming of AP-1/IRF as key drivers of pathogenic Treg programs leading to human autoimmune disease.
Structural insights into HIV-1 polyanion-dependent capsid lattice formation revealed by single particle cryo-EM
Authors: Highland Carolyn M. , Tan Aaron , Ricaña Clifton L. , Briggs John A.G. , Dick Robert A.
Published: December 2022

The HIV-1 capsid houses the viral genome and interacts extensively with host cell proteins throughout the viral life cycle. It is composed of capsid protein (CA), which assembles into a conical fullerene lattice composed of roughly 200 CA hexamers and 12 CA pentamers. Previous structural analyses of individual CA hexamers and pentamers have provided valuable insight into capsid structure and function, but high-resolution information about these assemblies in the broader context of the capsid lattice is lacking. In this study, we combined cryo-electron tomography and single particle analysis cryo-electron microscopy to determine high-resolution structures of continuous regions of the capsid lattice containing both hexamers and pentamers. We also developed a new method ofin vitrolattice assembly that enabled us to directly study the lattice under a wider range of conditions than has previously been possible. Using this approach, we identified a critical role for inositol hexakisphosphate (IP6) in pentamer formation and determined the structure of the CA lattice bound to the capsid-targeting antiretroviral drug GS-6207 (Lenacapvir). Our work reveals new structural details of the mature HIV-1 CA lattice and establishes the combination of lattice templating and single particle analysis as a robust strategy for studying retroviral capsid structure and capsid interactions with host proteins and antiviral compounds.

Significance statement

The mature HIV-1 capsid is composed of the capsid (CA) protein arranged in a conical lattice of hexamers and pentamers. Numerous structures of individual CA hexamers and pentamers alone have been published, but the molecular details of these assemblies in a more global, lattice-wide context are lacking. Here, we present high-resolution cryo-electron microscopy structures of continuous regions of the capsid lattice containing both hexamers and pentamers. We also describe key differences in the assembly and structures of these oligomers that have important implications for understanding retroviral maturation and for ongoing efforts to pharmacologically target the HIV-1 capsid.
Subcapsular sinus macrophage sensing of extracellular matrix rigidity alters membrane topography and immune complex mobility
Authors: Iliopoulou Maro , Bajur Anna T. , McArthur Hannah C. W. , Coyle Carl , Ajao Favour , Köchl Robert , Cope Andrew P. , Spillane Katelyn M.
Published: December 2022

Subcapsular sinus macrophages (SSMs) play a key role in immune defence by forming immunological barriers that control the transport of pathogens from lymph into lymph node follicles. SSMs participate in antibody responses by presenting antigens directly to naive B cells and by supplying antigens to follicular dendritic cells to propagate germinal centre reactions. Despite the prominent roles that SSMs play during immune responses, little is known about their cell biology because they are technically challenging to isolate and studyin vitro. Here, we used multi-colour fluorescence microscopy to identify lymph nodederived SSMs in culture. We focused on the role of SSMs as antigen-presenting cells and found that their actin cytoskeleton regulates the spatial organisation and mobility of immune complexes displayed on the cell surface. Moreover, we determined that SSMs are mechanosensitive cells that respond to changes in extracellular matrix (ECM) rigidity by altering the architecture of the actin cytoskeleton, leading to changes in cell morphology, membrane topography, and immune complex mobility. Our results reveal a new mechanism regulating physical aspects of antigen presentation by antigen-presenting cells, which may have implications for B cell activation and antibody responses.
Rhythmic temporal coordination of neural activity prevents representational conflict during working memory
Authors: Abdalaziz Miral , Redding Zach V. , Fiebelkorn Ian C.
Published: December 2022

Selective attention1is characterized by alternating states associated with either attentional sampling or attentional shifting, helping to avoid functional conflicts by isolating function-specific neural activity in time2–5. We hypothesized that such rhythmic temporal coordination might also help to avoid representational conflicts during working memory6. Multiple items can be simultaneously held in working memory, and these items can be represented by overlapping neural populations7–9. Traditional theories propose that short-term storage of to-be-remembered items occurs through persistent neural activity10–12, but when neurons are simultaneously representing multiple items, persistent activity creates a potential for representational conflicts. In comparison, more recent, ‘activity-silent’ theories of working memory propose that synaptic changes also contribute to the short-term storage of to-be-remembered items13–16. Transient bursts in neural activity17, rather than persistent activity, could serve to occasionally refresh these synaptic changes. Here, we used EEG and response times (RTs) to test whether rhythmic temporal coordination helps to isolate neural activity associated with different to-be-remembered items, which would help to avoid representational conflicts. Consistent with this hypothesis, we report that the relative strength of different item representations alternates over time as a function of frequency-specific phase. Although RTs were linked to theta (~6Hz) and beta (~25 Hz) phase during a memory delay, the relative strength of item representations only alternated as a function of beta phase. The present findings (i) are consistent with rhythmic temporal coordination being a general mechanism for avoiding either functional or representational conflicts during cognitive processes, and (ii) inform models describing the role of oscillatory dynamics in organizing working memory13,18–21.
Two structural switches in HIV-1 capsid regulate capsid curvature and host factor binding
Authors: Stacey James C.V. , Tan Aaron , Lu John M. , James Leo C. , Dick Robert A. , Briggs John A.G.
Published: December 2022

The mature HIV-1 capsid protects the viral genome and interacts with host proteins to travel from the cell periphery into the nucleus. To achieve this, the capsid protein, CA, constructs conical capsids from a lattice of hexamers and pentamers, and engages in and then relinquishes multiple interactions with cellular proteins in an orchestrated fashion. Cellular host factors including Nup153, CPSF6 and Sec24C engage the same pocket within CA hexamers. How CA assembles pentamers and hexamers of different curvatures, how CA oligomerization states or curvature might modulate host-protein interactions, and how binding of multiple co-factors to a single site is coordinated, all remain to be elucidated. Here, we have resolved the structure of the mature HIV-1 CA pentamer and hexamer from conical CA-IP6polyhedra to high resolution. We have determined structures of hexamers in the context of multiple lattice curvatures and number of pentamer contacts. Comparison of these structures, bound or not to host protein peptides, revealed two structural switches within HIV-1 CA that modulate peptide binding according to CA lattice curvature and whether CA is hexameric or pentameric. These observations suggest that the conical HIV-1 capsid has different host-protein binding properties at different positions on its surface, which may facilitate cell entry and represent an evolutionary advantage of conical morphology.

Significance statement

HIV-1 particles contain a characteristic, conical capsid that shields the genome from the cellular immune system and recruits cellular proteins to direct the capsid to the nucleus. The cone forms from hexamers of CA protein, and twelve pentamers that accommodate curvature. We obtained detailed 3D models of pentamers and hexamers at positions on capsid surfaces with different curvatures. We find two places in CA that switch conformation according to the local capsid curvature and whether CA is in a pentamer or hexamer. We also obtained models of CA bound to peptides from cellular proteins. The data show how switches in CA help it form a cone shape, and interact differently with cellular proteins at different positions on the cone surface.
Purifying and balancing selection on embryonic semi-lethal haplotypes in a wild mammal
Authors: Stoffel M.A. , Johnston S.E. , Pilkington J.G. , Pemberton J.M
Published: December 2022

Embryonic lethal mutations are arguably the earliest and most severe manifestation of inbreeding depression, but their impact on wild populations is not well understood. Here, we combined genomic, fitness and life-history data from 5,925 wild Soay sheep sampled over nearly three decades to explore the impact of embryonic lethal mutations and their evolutionary dynamics. We searched for haplotypes which in their homozygous state are unusually rare in the offspring of known carrier parents and found three putatively semi-lethal haplotypes with 27-46% fewer homozygous offspring than expected. Two of these haplotypes are decreasing in frequency, and gene-dropping simulations through the pedigree suggest that this is partially due to purifying selection. In contrast, the frequency of the third semi-lethal haplotype remains relatively stable over time. We show that the haplotype could be maintained by balancing selection because it is also associated with increased postnatal survival and body weight and because its cumulative frequency change is lower than in most drift-only simulations. Our study highlights embryonic mutations as a largely neglected contributor to inbreeding depression and provides a rare example of how harmful genetic variation can be maintained through balancing selection in a wild mammal population.
SLC26A3 (DRA) is stimulated in a synergistic, intracellular Ca2+dependent manner by cAMP AND ATP in intestinal epithelial cells
Authors: Sarker Rafiquel , Lin Ruxian , Singh Varsha , Donowitz Mark , Tse Chung Ming
Published: December 2022

In polarized intestinal epithelial cells, DRA is a brush border (BB) Cl-/HCO3-exchanger that is part of neutral NaCl absorption under baseline conditions but in cAMP driven diarrheas it is stimulated and contributes to increased anion secretion. To further understand regulation of DRA in conditions mimicking some diarrheal diseases, differentiated Caco-2/BBE cells were exposed to forskolin and ATP. Forskolin and ATP both acutely stimulated DRA in a concentration dependent manner. Forskolin at 1µM and ATP at 0.25 µM had minimal to no effect on DRA activity given individually; however, together, they stimulated DRA activity to levels seen with maximum concentrations of forskolin and ATP alone. In Caco-2/BBE cells expressing the Ca2+indicator GCaMP6s, ATP alone increased Ca2+in a concentration dependent manner, while forskolin (1 µM), that by itself did not significantly alter Ca2+, followed by 0.25 µM ATP produced a large increase in Ca2+that was ∼equal to the elevation caused by 1 µM ATP. BAPTA-AM pretreatment prevented the ATP and forskolin/ATP synergistic increased DRA activity and the increase in intracellular Ca2+caused by ATP/forskolin. Conclusion: In Caco-2/BBE cells subthreshold concentrations of forskolin (cAMP) and ATP (Ca2+) synergistically increased intracellular Ca2+and stimulated DRA activity with both being blocked by BAPTA-AM pretreatment. Diarrheal diseases such as bile acid diarrhea, in which both cAMP and Ca2+are elevated, are likely to be associated with stimulated DRA activity contributing to increased anion secretion, while separation of DRA from NHE3 contributes to reduced NaCl absorption.
Dynamic DNA methylation turnover in gene bodies is associated with enhanced gene expression plasticity in plants
Authors: Williams Clara J. , Dai Dawei , Tran Kevin A. , Monroe J. Grey , Williams Ben P
Published: December 2022

Background

In several eukaryotes, DNA methylation occurs within the coding regions of many genes, termed gene body methylation (GbM). Whereas the role of DNA methylation on the silencing of transposons and repetitive DNA is well understood, gene body methylation is not associated with transcriptional repression, and its biological importance has remained unclear.

Results

We report a newly discovered type of GbM in plants, which is under constitutive addition and removal by dynamic methylation modifiers in all cells, including the germline. Methylation at Dynamic GbM genes is removed by the DRDD demethylation pathway and added by an unknown source of de novo methylation, most likely the maintenance methyltransferase MET1. We show that the Dynamic GbM state is present at homologous genes across divergent lineages spanning over 100 million years, indicating evolutionary conservation. We demonstrate that Dynamic GbM is tightly associated with the presence of a promoter or regulatory chromatin state within the gene body, in contrast to other gene body methylated genes. We find Dynamic GbM is associated with enhanced gene expression plasticity across development and diverse physiological conditions, whereas stably methylated GbM genes exhibit reduced plasticity. Dynamic GbM genes exhibit reduced dynamic range indrddmutants, indicating a causal link between DNA demethylation and enhanced gene expression plasticity.

Conclusions

This study proposes a new model for GbM in regulating gene expression plasticity, including a newly discovered type of GbM in which increased gene expression plasticity is associated with the activity of DNA methylation writers and erasers and the enrichment of a regulatory chromatin state.