bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular confirmation ofAnopheles stephensiin the Al Hudaydah Governorate, Yemen, 2021-2022
Authors: Assada Methaq , Al-Hadi Mohammed , Esmail Mohammed A. , Al-Jurban Jamil , Alkawri Abdulsamad , Shamsan Arif , Gomein Payton , Samake Jeanne N. , Aljasari Adel , Awash Abdullah A. , Al Eryani Samira M. , Carter Tamar E.
Published: February 2024

Anopheles stephensiis an invasive malaria vector in Africa. To determine the status of the mosquito in Yemen,An. stephensivector surveillance and molecular confirmation was conducted in Al Hudaydah Governorate in 2021 and 2022. Mosquito larvae were collected in suspected man-made breeding habitats in Ah Dahi and Zabid city in 2021 and 2022, respectively. Mosquitoes morphologically identified asAn. stephensiunderwent molecular confirmation through PCR assays, sequencing, and phylogenetic analysis of the cytochrome oxidase subunit I (COI) gene and internal transcribed spacer 2 locus (ITS2). Analysis confirmedAn. stephensiidentification for the majority of samples (39/41), with two COI haplotypes detected: one newly reported haplotype and one haplotype common to Northeast Ethiopia and Somaliland. No clustering withAn. stephensifrom the Arabian Peninsula was observed. These findings provide preliminary insight into the diversity ofAn. stephensi in Yemen and the connection betweenAn. stephensiin Yemen and East Africa.
Arabidopsis Gluconolactonase, the First Enzyme Involved in Ascorbate Biosynthesis Localized in the Chloroplast Protects Plants from Light Stress
Authors: Yactayo-Chang Jessica P. , Nepal Nirman , Aboobucker Siddique I. , Medina-Jiménez Karina , Wilkie Austin , Teoh Thomas K. , Wilson Gwendolyn A. , Lorence Argelia
Published: February 2024

Vitamin C (L-ascorbic acid, AsA) is the most abundant water-soluble antioxidant in plants. Ascorbate scavenges free radicals, is an enzyme cofactor, and a donor and acceptor of electrons in the chloroplast. Ascorbate protects tissues against damage caused by reactive oxygen species (ROS) produced through normal metabolism or generated from stress. The inositol route to AsA involves four enzymes:myo-inositol oxygenase, glucuronate reductase, gluconolactonase (GNL), and L-gulono-1,4-lactone oxidase. The third enzyme, GNL, has been characterized in rat and bacteria but not in plants. Eighteen putative GNLs were identified in Arabidopsis, one of which,AtGNL,is interesting because it possesses a chloroplastic signal peptide. Plastids can accumulate up to 50 m M As A but until now no chloroplastic AsA biosynthetic genes have been described. This study includes the characterization of the first plant GNL enzymein vitroandin planta. A knockout on this gene had lower foliar As A and stunted growth compared to controls. The functional gene restored the phenotype of the knockout, and those restored plants had higher AsA content, enhanced photosynthetic capacity, and higher seed yield. These results highlight the importance ofAtGNL in As A formation and in maintaining a healthy redox balance in the leaves particularly under low light stress.
Career Navigator: An online platform to streamline professional development and career education for graduate bioscientists
Authors: Rudlaff Rachel M. , Adhikary Utsarga , Khairani Candrika D. , Emmans Daniel S. , Gutlerner Johanna L. , Heustis Ronald J.
Published: February 2024

Graduate professional development is a highly dynamic enterprise that prepares graduate students for personal and career success in a variety of fields, including the biosciences. National policies, funding awards, and institutional programs have generated myriad tools and services for graduate bioscience students, including new learning resources, events, connections to prospective employers, and opportunities to strengthen academic and professional portfolios. These interventions are welcome and have done much to enhance graduate bioscience training, but they may also be overwhelming for trainees. To streamline professional development and career education information for the bioscience graduate students at our institution, we tested a model where we built a centralized web portal of career development resources. Here we present our strategy and best practices for website design. We show data that students preferred a centralized online portal over other forms of resource communication; that programming, paired communication and environmental factors (e.g. remote learning and work as in the COVID-19 pandemic) combined to increase sustained engagement with the site; and that harnessing website analytics is an effective way to measure site utilization and generate insights on programming and resource development. This data, in turn, fits into broader priorities to evaluate interventions in graduate bioscience education.
Interpretable deep learning reveals the sequence rules of Hippo signaling
Authors: Dalal Khyati , McAnany Charles , Weilert Melanie , McKinney Mary Cathleen , Krueger Sabrina , Zeitlinger Julia
Published: February 2024

The response to signaling pathways is highly context-specific, and identifying the transcription factors and mechanisms that are responsible is very challenging. Using the Hippo pathway in mouse trophoblast stem cells as a model, we show here that this information is encoded incis-regulatory sequences and can be learned from high-resolution binding data of signaling transcription factors. Using interpretable deep learning, we show that the binding levels of TEAD4 and YAP1 are enhanced in a distance-dependent manner by cell type-specific transcription factors, including TFAP2C. We also discovered that strictly spacedTead doublemotifs are widespread highly active canonical response elements that mediate cooperativity by promoting labile TEAD4 protein-protein interactions on DNA. These syntax rules and mechanisms apply genome-wide and allow us to predict how small sequence changes alter the activity of enhancersin vivo. This illustrates the power of interpretable deep learning to decode canonical and cell type-specific sequence rules of signaling pathways.

Graphical abstract
Electron-Activated Dissociation and Collision-Induced Dissociation Glycopeptide Fragmentation for Improved Glycoproteomics
Authors: Macauslane Kyle L. , Pegg Cassandra L. , Nouwens Amanda S. , Kerr Edward D. , Seitanidou Joy , Schulz Benjamin L.
Published: February 2024

Tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS) has proven a versatile tool for the identification and quantification of proteins and their post-translational modifications (PTMs). Protein glycosylation is a critical PTM for the stability and biological function of many proteins, but full characterisation of site-specific glycosylation of proteins remains analytically challenging. Collision induced dissociation (CID) is the most common fragmentation method used in LC-MS/MS workflows, but loss of labile modifications render CID inappropriate for detailed characterisation of site-specific glycosylation. Electron-based dissociation (ExD) methods provide alternatives that retain intact glycopeptide fragments for unambiguous site localisation, but these methods often underperform CID due to increased reaction times and reduced efficiency. Electron activated dissociation (EAD) is another strategy for glycopeptide fragmentation. Here, we use a ZenoTOF 7600 SCIEX instrument to compare the performance of various fragmentation techniques for the analysis of a complex mixture of mammalianO- andN-glycopeptides. We found CID fragmentation identified the most glycopeptides and generally produced higher quality spectra, but EAD provided improved confidence in glycosylation site localisation. Supplementing EAD with CID fragmentation (EAciD) further increased the number and quality of glycopeptide identifications, while retaining localisation confidence. These methods will be useful for glycoproteomics workflows for either optimal glycopeptide identification or characterisation.
Fluid Shear Stress-Induced Changes in Megalin Trafficking Enhance Endocytic Capacity in Proximal Tubule Cells
Authors: Lackner Emily M , Cowan Isabella A , Long Kimberly R , Weisz Ora A , Shipman Katherine E
Published: February 2024

Proximal tubule (PT) cells maintain a high-capacity apical endocytic pathway to recover essentially all proteins that escape the glomerular filtration barrier. The multiligand receptors megalin and cubilin play pivotal roles in the endocytic uptake of normally filtered proteins in PT cells but also contribute to the uptake of nephrotoxic drugs, including aminoglycosides. We previously demonstrated that opossum kidney (OK) cells cultured under continuous fluid shear stress (FSS) are superior to cells cultured under static conditions in recapitulating essential functional properties of PT cells in vivo. To identify drivers of the high-capacity, efficient endocytic pathway in the PT, we compared FSS-cultured OK cells with less endocytically active static-cultured OK cells. Megalin and cubilin expression are increased, and endocytic uptake of albumin in FSS-cultured cells is >5-fold higher compared with cells cultured under static conditions. To understand how differences in receptor expression, distribution, and trafficking rates contribute to increased uptake, we used biochemical, morphological, and mathematical modeling approaches to compare megalin traffic in FSS- versus static-cultured OK cells. Our model predicts that culturing cells under FSS increases the rates of all steps in megalin trafficking. Importantly, the model explains why, despite seemingly counterintuitive observations (a reduced fraction of megalin at the cell surface, higher colocalization with lysosomes, and a shorter half-life of surface-tagged megalin in FSS-cultured cells), uptake of albumin is dramatically increased compared with static-grown cells. We also show that FSS-cultured OK cells more accurately exhibit the mechanisms that mediate uptake of nephrotoxic drugs in vivo compared with static-grown cells. This culture model thus provides a useful platform to understand drug uptake mechanisms, with implications for developing interventions in nephrotoxic injury prevention.
Loss of the scavenger receptor MARCO results in uncontrolled vomocytosis of fungi from macrophages
Authors: Onyishi Chinaemerem U. , Fejer Gyorgy , Mukhopadhyay Subhankar , Gordon Siamon , May Robin C.
Published: February 2024

Vomocytosis, also known as nonlytic exocytosis, is a process whereby fully phagocytosed microbes are expelled from phagocytes without discernible damage to either the phagocyte or microbe. Although this phenomenon was first described in the opportunistic fungal pathogenCryptococcus neoformansin 2006, to date, mechanistic studies have been hampered by an inability to reliably stimulate or inhibit vomocytosis. Here we present the fortuitous discovery that macrophages lacking the scavenger receptor MAcrophage Receptor with COllagenous domain (MARCO), exhibit near-total vomocytosis of internalised cryptococci within a few hours of infection. Our findings suggest that MARCO’s role in modulating vomocytosis is independent of its role as a phagocytic receptor and instead may be driven by variation in cytoskeletal arrangement between wildtype and MARCO-deficient macrophages.
RhizoBindingSites v2.0 is a bioinformatic database of DNA motifs potentially involved in transcriptional regulation deduced from sites of the itself genome
Authors: Taboada-Castro Hermenegildo , Hernández-Álvarez Alfredo José , Castro-Mondragón Jaime A. , Encarnación Guevara Sergio M.
Published: February 2024

RhizoBindingSites is ade novodepurified database of conserved DNA motifs potentially involved in the transcriptional regulation of theRhizobium,Sinorhizobium,Bradyrhizobium,Azorhizobium,andMesorhizobiumrepresentative symbiotic species, deduced from the upstream regulatory sequences of orthologous genes (O-matrices) from the Rhizobiales taxon. The sites collected with O-matrices per gene per genome from RhizoBindingSites were used to deduce matrices using the dyad-Regulatory Sequence Analysis Tool (RSAT) method, giving rise to novel S-matrices for the construction of the RizoBindingSites v2.0 database. A comparison of the S-matrix logos showed a greater frequency and/or re-definition of specific-position nucleotides found in the O-matrices. Moreover, S-matrices were better at detecting genes in the genome and there was a greater number of transcription factors (TFs) in the vicinity than O-matrices, corresponding to a more significant genomic coverage for S-matrices. The homology between the matrices of TFs from a genome showed inter-regulation between the clustered TFs. In addition, matrices of AraC, ArsR, GntR, and LysR ortholog TFs showed different motifs, suggesting distinct regulation. Benchmarking showed 72%, 68%, and 81% of common genes per regulon for O-matrices and approximately 14% less common genes with S-matrices ofRhizobium etliCFN42,R. leguminosarumbv.viciae3841, andSinorhizobium meliloti1021. These data were deposited in RhizoBindingSites and the RhizoBindingSites v2.0 database (http://rhizobindingsites.ccg.unam.mx/).
Senataxin modulates resistance to cisplatin through an R-loop mediated mechanism in HPV-associated Head and Neck Squamous Cell Carcinoma
Authors: Crane Hannah , Carr Ian , Hunter Keith D , El-Khamisy Sherif F.
Published: February 2024

Introduction

Oropharyngeal Squamous Cell Carcinoma (OPSCC) is a site defined subtype of head and neck cancer with two distinct clinical subtypes: HPV-associated (HPV+) and HPV-independent (HPV-); both of which are commonly treated with chemoradiotherapy involving cisplatin. Cisplatin creates DNA crosslinks, which lead to eventual cell death via apoptosis. Clinical outcomes in HPV-OPSCC are poor and although HPV+ has an improved response to therapy, a subset of patients suffer from distant metastases, with a poor prognosis. Therefore, there is a need to understand the molecular basis underlying treatment resistance. A common mechanism of chemotherapy resistance is upregulation of DNA repair, and a major source of endogenous DNA damage are DNA/RNA hybrids, known as R-loops. R-loops are three stranded DNA/RNA hybrids formed in the genome as a by- product of transcription and are normally transient; however, they can persist and become a source of genomic instability. The contribution of R-loops to the development of cisplatin resistance in OPSCC is unknown.

Methods

HPV+ and HPV- cisplatin resistant cell lines were developed, and RNA-sequencing was used to investigate changes in gene expression. Changes in R-loop dynamics were explored using slot blots and DRIP-qPCR. The effect of depleting known R-loop regulators on cisplatin sensitivity was assessed using siRNA. R-loop burden in a cohort of HPV+ and HPV- OPSCC tumours was explored using S9.6 immunohistochemistry.

Results

Development of cisplatin resistant clones led to changes in gene expression consistent with resistance, alongside alterations in the expression of known R-loop regulators. Both HPV+ and HPV- resistant cells had elevated global R-loop levels and in HPV+ resistant cells there was a corresponding upregulation of the R-loop resolving protein, senataxin, which was not observed in HPV- resistant cells. Depletion of senataxin led to increased sensitivity to cisplatin in both HPV+ and HPV- resistant cells, however, the effect was greater in HPV+ cells. Quantification of R-loop levels by S9.6 immunohistochemistry revealed that HPV+ tumours and tumours with bone metastases had a higher R-loop burden.

Conclusion

R-loops are involved in modulating sensitivity to cisplatin and may represent a potential therapeutic target.
Contractile to extensile transitions and mechanical adaptability enabled by activity in cytoskeletal structures
Authors: Lamtyugina Alexandra , Banerjee Deb Sankar , Qiu Yuqing , Vaikuntanathan Suriyanarayanan
Published: February 2024

In this study, we investigate the response of self-assembled cytoskeletal structures to external mechanical perturbations, focusing on filament and crosslinker mixtures in two dimensions. By applying external forces at the microscopic scale, our work, employing agent-based models and a coarse-grained thermodynamic theory, reveals that molecular motor action enables the cytoskeletal structures to robustly adapt to changes in external forcing conditions. Specifically, under the influence of external forces, self-assembled active asters transform into bundle-like structures, and active bundle assemblies elongate further in a reproducible and regular manner, demonstrating robust responses compared to passive assemblies where no regulated qualitative morphological change was observed. A minimal thermodynamic theory, using an effective temperature concept, elucidates the adaptive properties of active assemblies. Furthermore, we explore the distinct mechanical responses resulting from morphological differences, deriving a simple form to approximate active stress as a function of the mesoscopic architecture. The results highlight the association between morphological transitions from aster to bundle and changes in the nature of active stress from contractile to extensile, confirming predictions through agent-based simulations. These findings contribute to a deeper understanding of the intricate interplay between cytoskeletal morphologies and their mechanical responses under external forces.
Building predictive Markov State Models of ion channel permeation from Molecular Dynamics
Authors: Catacuzzeno Luigi , Leonardi Maria Vittoria , Franciolini Fabio , Domene Carmen , Michelucci Antonio , Furini Simone
Published: February 2024

Molecular dynamics (MD) simulation of biological processes has always been a very challenging task due to the long timescales of the processes involved and the challenges associated with handling the large amount of output data. Markov State Models (MSMs) have been recently introduced as a powerful tool in this area of research, as they provide a mechanistically comprehensible synthesis of the large amount of MD data and, at the same time, can be used to estimate experimental properties of biological processes. Of the many studies on protein simulation and the MSM-assisted approach, only a few have addressed ion channel permeation and, more importantly, none of these have tried to build a model capable to predict the currents passing through the channels, which are ultimately crucial for comparing simulations with experimental results. Herein, we propose a method for building an MSM of ion channel permeation that correctly evaluates the current flowing through the channel. This was done by including in the model the definition of a flux matrix carrying information on the charge moving across the channel, suitably built to be used in conjunction with the transition matrix to predict the ion current. The proposed method is also able to drastically reduce the number of states so to obtain an MSM simple enough to be easily understood. Finally, we applied the method to the KcsA channel, obtaining a four-state MSM capable of accurately reproducing the single channel ion current from microseconds MD trajectories.
NINJ1 regulates ferroptosis via xCT antiporter interaction and CoA modulation
Authors: Chen Ssu-Yu , Lin Chao-Chieh , Wu Jianli , Chen Yubin , Wang Ya-En , Setayeshpour Yasaman , Mestre Alexander , Chi Jen-Tsan
Published: February 2024

Ninjurin-1 (NINJ1), initially identified as a stress-induced protein in neurons, recently emerged as a key mediator of plasma membrane rupture during apoptosis, necrosis, and pyroptosis. However, its involvement in ferroptosis remains unknown. Here, we demonstrate that NINJ1 also plays a crucial role in ferroptosis, but through a distinct mechanism. NINJ1 knockdown significantly protected cancer cells against ferroptosis induced by xCT inhibitors but no other classes of ferroptosis-inducing compounds (FINs). Glycine, known to inhibit canonical NINJ1-mediated membrane rupture in other cell deaths, had no impact on ferroptosis. A compound screen revealed that NINJ1-mediated ferroptosis protection can be abolished by pantothenate kinase inhibitor (PANKi), buthionine sulfoximine (BSO), and diethylmaleate (DEM). These results suggest that this ferroptosis protection is mediated via Coenzyme A (CoA) and glutathione (GSH), both of which were found to be elevated upon NINJ1 knockdown. Furthermore, we discovered that NINJ1 interacts with the xCT antiporter, which is responsible for cystine uptake for the biosynthesis of CoA and GSH. The removal of NINJ1 increased xCT levels and stability, enhanced cystine uptake, and contributed to elevated CoA and GSH levels, collectively contributing to ferroptosis protection. These findings reveal that NINJ1 regulates ferroptosis via a non-canonical mechanism, distinct from other regulated cell deaths.

Graphical abstract
Ubiquitinated histone H2B as gatekeeper of the nucleosome acidic patch
Authors: Hicks Chad W. , Rahman Sanim , Gloor Susan L. , Fields James K. , Husby Natalia Ledo , Vaidya Anup , Maier Keith E. , Morgan Michael , Keogh Michael-Christopher , Wolberger Cynthia
Published: February 2024

Monoubiquitination of histones H2B-K120 (H2BK120ub) and H2A-K119 (H2AK119ub) play opposing roles in regulating transcription and chromatin compaction. H2BK120ub is a hallmark of actively transcribed euchromatin, while H2AK119ub is highly enriched in transcriptionally repressed heterochromatin. Whereas H2BK120ub is known to stimulate the binding or activity of various chromatin-modifying enzymes, this post-translational modification (PTM) also interferes with the binding of several proteins to the nucleosome H2A/H2B acidic patch via an unknown mechanism. Here we report cryoEM structures of an H2BK120ub nucleosome showing that ubiquitin adopts discrete positions that occlude the acidic patch. Molecular dynamics simulations show that ubiquitin remains stably positioned over this nucleosome region. By contrast, our cryoEM structures of H2AK119ub nucleosomes show ubiquitin adopting discrete positions that minimally occlude the acidic patch. Consistent with these observations, H2BK120ub, but not H2AK119ub, abrogates nucleosome interactions with acidic patch-binding proteins RCC1 and LANA, and single-domain antibodies specific to this region. Our results suggest a mechanism by which H2BK120ub serves as a gatekeeper to the acidic patch and point to distinct roles for histone H2AK119 and H2BK120 ubiquitination in regulating protein binding to nucleosomes.
Identification of Alpha-Gal conjugated Lipids in Saliva of Lone-Star Tick (Amblyomma americanum)
Authors: Sharma Surendra Raj , Hussain Sabir , Choudhary Shailesh K. , Commins Scott P. , Karim Shahid
Published: February 2024

Alpha-Gal Syndrome (AGS) is a delayed allergic reaction triggered by IgE antibodies targeting galactose-α-1,3-galactose (α-gal), prevalent in red meat. Its global significance has increased, with over 450,000 estimated cases in the United States alone. AGS is linked to tick bites, causing sensitization and elevated α-gal specific IgE levels. However, the precise mechanisms and tick intrinsic factors contributing to AGS development post-tick bites remain unclear. This study aims to characterize the alphagal conjugated lipid antigens inAmblyomma americanum(Am. americanum) salivary glands and saliva. Nanospray ionization mass spectrometry (NSI-MS) analysis revealed the identification of α-gal bound lipid antigens inAm. americanumsaliva. Additionally, activation of basophils by isolated alpha-gal bound lipids and proteins provides evidence of their antigenic capabilities.
Modern Insights into Muscle Glycogen Phosphorylase Activity
Authors: Kiriaev Leonit , Oakhill Jonathan S. , Tiong Chrystal F. , Seto Jane T. , Crossman Vanessa G. , Quinlan Kate G.R. , North Kathryn N. , Houweling Peter J. , Ling Naomi X.Y.
Published: February 2024

Recent identification of new human muscle glycogen phosphorylation sites has renewed interest in understanding human variations in the regulation of glycogen metabolism and glucose homeostasis. This paper presents a detailed method for the measurement of glycogen phosphorylase (GPh) activity in skeletal muscle. Our approach incorporates modifications to existing radiolabelling assays, optimizing specificity and sensitivity while enabling the assessment of both active and total enzyme activity levels. The utilization of radioisotope tracers and scintillation counting ensures accurate quantification of GPh activity, which we use to validate a previously published reduction in GPh activity in anActn3deficient mouse model. Moreover, we introduce a step-by-step guide for data acquisition, highlight the use of appropriate homogenization, discuss the need for allosteric activators/inhibitors and the importance of assay optimization to record a GPh activity assay for skeletal muscle. In conclusion, our refined method not only contributes to a deeper understanding of glycogen metabolism in muscle tissue but also provides a framework for future investigations, underscoring its role in advancing research on glycogen utilization and glucose homeostasis.

NEW & NOTEWORTHY

The study optimizes the glycogen phosphorylase radiolabelled activity assay, unveiling nuances in muscle homogenization, sample dilution, and caffeine inclusion. The research introduces standardized conditions, enhancing assay reliability and reproducibility across mouse strains to reveal sex specific variations in GPh activity and underscore novel distinctions in an Actn3 deficient mouse model. These findings advance our understanding of muscle glycogen metabolism, offering a crucial tool for researchers and facilitating meaningful inter-laboratory comparisons.
Another fly diuretic hormone: tachykinins increase fluid and ion transport by adultDrosophila melanogasterMalpighian ‘renal’ tubules
Authors: Agard Marishia A. , Zandawala Meet , Paluzzi Jean-Paul V.
Published: February 2024

Insects like the model organismDrosophila melanogastermust modulate their internal physiology to withstand changes in temperature and availability of water and food. Regulation of the excretory system by peptidergic hormones is one mechanism by which insects maintain their internal homeostasis. Tachykinins are a family of neuropeptides that have been shown to stimulate fluid secretion from the Malpighian ‘renal’ tubules (MTs) in some insect species, but it is unclear if that is the case in the fruit fly,D. melanogaster. A central objective of the current study was to examine the physiological role of tachykinin signaling in the MTs of adultD. melanogaster. Using the genetic toolbox available in this model organism along within vitroand whole animal bioassays, our results indicate thatDrosophilatachykinins (DTKs) function as diuretic hormones by binding to the DTK receptor (DTKR) expressed in stellate cells of the MTs. Specifically, DTK activates cation and anion transport across the stimulated MTs, which impairs their survival in response to desiccation due to their inability to conserve water. Thus, besides their previously described roles in neuromodulation of pathways controlling locomotion and food search, olfactory processing, aggression, lipid metabolism and metabolic stress, processing of noxious stimuli and hormone release, DTKs also appear to function asbona fideendocrine factors regulating the excretory system and appear essential for the maintenance of hydromineral balance.

Summary statement

Drosophilatachykinins are diuretic hormones in the fly that regulate activity of the MTs and, consequently, contribute towards the maintenance of ionic and osmotic homeostasis.
Generation of diversity in the blue cheese moldPenicillium roquefortiand identification of pleiotropic QTL for key cheese-making phenotypes
Authors: Caron Thibault , Crequer Ewen , Le Piver Mélanie , Le Prieur Stéphanie , Brunel Sammy , Snirc Alodie , Cueff Gwennina , Roueyre Daniel , Place Michel , Chassard Christophe , Simon Adeline , Rodriguez de la Vega Ricardo , Coton Monika , Coton Emmanuel , Foulongne-Oriol Marie , Branca Antoine , Giraud Tatiana
Published: February 2024

Elucidating the genomic architecture of quantitative traits is essential for our understanding of adaptation and for breeding in domesticated organisms.Penicillium roquefortiis the mold used worldwide for the blue cheese maturation, contributing to flavors through proteolytic and lipolytic activities. The two domesticated cheese populations display very little genetic diversity, but are differentiated and carry opposite mating types. We produced haploid F1 progenies from five crosses, using parents belonging to cheese and non-cheese populations. Analyses of high-quality genome assemblies of the parental strains revealed five large translocations, two having occurred via a circular intermediate. Offspring genotyping with genotype-by-sequencing (GBS) revealed several genomic regions with segregation distortion, possibly linked to degeneration in cheese lineages. We found transgressions for several traits relevant for cheese making, with offspring having more extreme trait values than parental strains. We identified quantitative trait loci (QTLs) for colony color, lipolysis, proteolysis, extrolite production, including mycotoxins, but not for growth rates. Some genomic regions appeared rich in QTLs for both lipid and protein metabolism, and other regions for the production of multiple extrolites, indicating that QTLs have pleiotropic impacts. Some QTLs corresponded to known biosynthetic gene clusters, e.g., for the production of melanin or extrolites. F1 hybrids constitute valuable strains for cheese producers, with new traits and genetic diversity, and allowed identifying target genomic regions for traits important in cheese making, paving the way for strain improvement. The findings further contribute to our understanding of the genetic mechanisms underlying rapid adaptation, revealing convergent adaptation targeting major regulators.
Enhancement of colorectal cancer therapy through interruption of the HSF1-HSP90 axis by p53 activation or cell cycle inhibition
Authors: Isermann Tamara , Schneider Kim Lucia , Wegwitz Florian , De Oliveira Tiago , Conradi Lena-Christin , Volk Valery , Feuerhake Friedrich , Papke Björn , Stintzing Sebastian , Mundt Bettina , Kühnel Florian , Moll Ute M. , Schulz-Heddergott Ramona
Published: February 2024

The stress-associated molecular chaperone system is an actionable target in cancer therapies. It is ubiquitously upregulated in cancer tissues and enables tumorigenicity by stabilizing hundreds of oncoproteins and disturbing the stoichiometry of protein complexes. Most inhibitors target the key component heat-shock protein 90 (HSP90). However, although classical HSP90 inhibitors are highly tumor-selective, they fail in phase 3 clinical oncology trials. These failures are at least partly due to an interference with a negative feedback loop by HSP90 inhibition, known as heat-shock response (HSR): in response to HSP90 inhibition there is compensatory synthesis of stress-inducible chaperones, mediated by the transcription factor heat-shock factor 1 (HSF1). We recently identified that wildtype p53 (p53) actively reduces the HSR by repressing HSF1 via a p21-CDK4/6-MAPK-HSF1 axis. Here we test the hypothesis that in HSP90-based therapies simultaneous p53 activation or direct cell cycle inhibition interrupts the deleterious HSF1-HSR axis and improves the efficiency of HSP90 inhibitors.

Indeed, we find that the clinically relevant p53 activator Idasanutlin suppresses the HSF1-HSR activity in HSP90 inhibitor-based therapies. This combination synergistically reduces cell viability and accelerates cell death in p53-proficient colorectal cancer (CRC) cells, murine tumor-derived organoids and patient-derived organoids (PDOs). Mechanistically, upon combination therapy human CRC cells strongly upregulate p53-associated pathways, apoptosis, and inflammatory immune pathways. Likewise, in the chemical AOM/DSS CRC model in mice, dual HSF1-HSP90 inhibition strongly represses tumor growth and remodels immune cell composition, yet displays only minor toxicities in mice and normal mucosa-derived organoids. Importantly, inhibition of the cyclin dependent kinases 4 and 6 (CDK4/6) under HSP90 inhibition phenocopies synergistic repression of the HSR in p53-proficient CRC cells. Even more important, in p53-deficient (mutp53-harboring) CRC cells, an HSP90 inhibition in combination with CDK4/6 inhibitors similarly suppresses the HSF1-HSR system and reduces cancer growth. Likewise, p53-mutated PDOs strongly respond to dual HSF1-HSP90 pathway inhibition and thus, providing a strategy to target CRC independent of the p53 status.

In sum, activating p53 (in p53-proficient cancer cells) or inhibiting CDK4/6 (independent of the p53 status) provide new options to improve the clinical outcome of HSP90-based therapies and to enhance colorectal cancer therapy.
PL-PatchSurfer3: Improved Structure-Based Virtual Screening for Structure Variation Using 3D Zernike Descriptors
Authors: Shin Woong-Hee , Kihara Daisuke
Published: February 2024

Structure-based virtual screening (SBVS) is a widely used method in silico drug discovery. It necessitates a receptor structure or binding site to predict the binding pose and fitness of a ligand. Therefore, the performance of the SBVS is affected by the protein conformation. The most frequently used method in SBVS is the protein-ligand docking program, which utilizes atomic distance-based scoring functions. Hence, they are highly prone to sensitivity towards variation in receptor structure, and it is reported that the conformational change significantly drops the performance of the docking program.

To address the problem, we have introduced a novel program of SBVS, named PL-PatchSurfer. This program makes use of molecular surface patches and the Zernike descriptor. The surfaces of the pocket and ligand are segmented into several patches by the program. These patches are then mapped with physico-chemical properties such as shape and electrostatic potential before being converted into the Zernike descriptor, which is rotationally invariant. A complementarity between the protein and the ligand is assessed by comparing the descriptors and geometric distribution of the patches in the molecules. A benchmarking study showed that PL-PatchSurfer2 was able to screen active molecules regardless of the receptor structure change with fast speed. However, the program could not achieve high performance for the targets that the hydrogen bonding feature is important such as nuclear hormone receptors.

In this paper, we present the newer version of PL-PatchSurfer, PL-PatchSurfer3, which incorporates two new features: a change in the definition of hydrogen bond complementarity and consideration of visibility that contains curvature information of a patch. Our evaluation demonstrates that the new program outperforms its predecessor and other SBVS methods while retaining its characteristic tolerance to receptor structure changes. Interested individuals can access the program atkiharalab.org/plps3.
Optimising the production of dsRNA biocontrols in microbial systems using multiple transcriptional terminators
Authors: Ross Sebastian J. , Owen Gareth R. , Hough James , Philips Annelies , Maddelein Wendy , Ray John , Kilby Peter M. , Dickman Mark J.
Published: February 2024

Crop pests and pathogens annually cause over $100 billion in global crop damage, with insects consuming 5-20% of major grain crops. Current crop pest and disease control strategies rely on insecticidal and fungicidal sprays, plant genetic resistance, transgenes and agricultural practices. dsRNA is emerging as a novel sustainable method of plant protection as an alternative to traditional chemical pesticides. Successful commercialisation of dsRNA based biocontrols requires the economical production of large quantities of dsRNA combined with suitable delivery methods to ensure RNAi efficacy against the target pest. In this study, we have optimised the design of plasmid DNA constructs to produce dsRNA biocontrols inE. coli, by employing a wide range of alternative synthetic transcriptional terminators prior to measurement of dsRNA yield. We demonstrate that a 7.8-fold increase of dsRNA was achieved using triple synthetic transcriptional terminators within a dual T7 dsRNA production system compared to the absence of transcriptional terminators. Moreover, our data demonstrates that batch fermentation production dsRNA using multiple transcriptional terminators is scalable and generates significantly higher yields of dsRNA generated in the absence of transcriptional terminators at both small-scale batch culture and large-scale fermentation. In addition, we show that application of these dsRNA biocontrols expressed inE. colicells results in increased insect mortality. Finally, novel mass spectrometry analysis was performed to determine the precise sites of transcriptional termination at the different transcriptional terminators providing important further mechanistic insight.
Exploring the Viral Landscape of Saffron through Metatranscriptomic Analysis
Authors: Martinez-Fajardo Cristian , Navarro-Simarro Pablo , Morote Lucia , Rubio-Moraga Angela , Mondejar-Lopez Maria , Niza Enrique , Argandona Javier , Ahrazem Oussama , Gomez-Gomez Lourdes , Lopez-Jimenez Alberto Jose
Published: February 2024

Saffron (Crocus sativus L.), a historically significant crop valued for its nutraceutical properties, has been poorly explored from a phytosanitary perspective. This study conducted a thorough examination of viruses affecting saffron samples from Spanish cultivars, using high-throughput sequencing alongside a systematic survey of transcriptomic datasets from Crocus sativus at the Sequence Read Archive. Our analysis unveiled a broad diversity and abundance, identifying 17 viruses across the 45 analyzed libraries, some of which were highly prevalent. This includes known saffron-infecting viruses and previously unreported ones. In addition, we discovered novel viruses from the Alphaflexiviridae, Betaflexiviridae, Potyviridae, Solemoviridae, and Geminiviridae families, with some present in libraries from various locations. These findings indicate that the saffron-associated virome is more complex than previously reported, emphasizing the potential of phytosanitary analysis to enhance saffron productivity.
An efficientin vivo-inducible CRISPR interference system for group AStreptococcusgenetic analysis and pathogenesis studies
Authors: Bjånes Elisabet , Stream Alexandra , Janssen Axel B. , Gibson Paddy S. , Bravo Afonso M. , Dahesh Samira , Baker Jonathon L. , Varble Andrew , Nizet Victor , Veening Jan-Willem
Published: February 2024

While genome-wide transposon mutagenesis screens have identified numerous essential genes in the significant human pathogenStreptococcus pyogenes(group AStreptococcusor GAS), many of their functions remain elusive. This knowledge gap is attributed in part to the limited molecular toolbox for controlling GAS gene expression and the bacterium’s poor genetic transformability. CRISPR interference (CRISPRi), using catalytically inactive GAS Cas9 (dCas9), is a powerful approach to specifically repress gene expression in both bacteria and eukaryotes, but ironically has never been harnessed for controlled gene expression in GAS. In this study, we present a highly transformable and fully virulent serotype M1T1 GAS strain and introduce a doxycycline-inducible CRISPRi system for efficient repression of bacterial gene expression. We demonstrate highly efficient, oligo-based sgRNA cloning directly to GAS, enabling the construction of a gene knockdown strain in just two days, in contrast to the several weeks typically required. The system is shown to be titratable and functional bothin vitroandin vivousing a murine model of GAS infection. Furthermore, we provide directin vivoevidence that the expression of the conserved cell division geneftsZis essential for GAS virulence, highlighting its promise as a target for emerging FtsZ-inhibitors. Finally, we introduce SpyBrowse (https://veeninglab.com/SpyBrowse), a comprehensive and user-friendly online resource for visually inspecting and exploring GAS genetic features. The tools and methodologies described in this work are poised to facilitate fundamental research in GAS, contribute to vaccine development, and aid in the discovery of antibiotic targets.

Significance statement

While GAS remains a predominant cause of bacterial infections worldwide, there are limited genetic tools available to study its basic cell biology. Here, we bridge this gap by creating a highly transformable, fully virulent M1T1 GAS strain. In addition, we established a tight and titratable doxycycline-inducible system and developed CRISPR interference for controlled gene expression in GAS. We show that CRISPRi is functionalin vivoin a mouse infection model. Additionally, we present SpyBrowse, an intuitive and accessible genome browser (https://veeninglab.com/SpyBrowse). Overall, this work overcomes significant technical challenges of working with GAS, and together with SpyBrowse, represents a valuable resource for researchers in the GAS field.
Chromosome-level genome assembly and annotation of the social amoebaDictyostelium firmibasis
Authors: Edelbroek Bart , Kjellin Jonas , Jerlström-Hultqvist Jon , Koskiniemi Sanna , Söderbom Fredrik
Published: February 2024

Dicytostelium firmibasisis a member of Dictyostelia, a group of social amoebae that upon starvation display aggregative multicellularity where the amoebae transition from uni-to multicellular life. TheD. firmibasisgenome assembly that is currently available is of limited use due to its low contiguity, large number of undetermined bases, and lack of annotations. Here we used Nanopore long read sequencing, complemented with Illumina sequencing, and developmental transcriptomics as well as small RNA-sequencing, to present a new, fully annotated, chromosome-levelD. firmibasisgenome assembly. The new assembly contains no undetermined bases, and consists mainly of six large contigs representing the chromosomes, as well as a complete mitochondrial genome. This new genome assembly will be a valuable tool, allowing comprehensive comparison toDictyostelium discoideum, the dictyostelid genetically tractable model. Further, the new genome will be important for studies of evolutionary processes governing the transition from unicellular to multicellular organisms and will aid in the sequencing and annotation of other dictyostelids genomes, many of which are currently of poor quality.
Habitat isolation diminishes potential of self-organised pattern formation to promote local diversity in metacommunities
Authors: Philipp Louica , Klauschies Toni , Guill Christian
Published: February 2024

Progressive destruction and isolation of natural habitat is a major threat to biodiversity worldwide. In this study we use a trophic metacommunity model with complex, spatially explicit structure to address how the interaction of local and regional processes affects the functional diversity of autotroph (producer) communities within and between individual habitat patches. One important driver of biodiversity in metacommunities is spatial heterogeneity of the environment, as it enables source-sink dynamics between patches. Besides a-priori differences in the environmental conditions, heterogeneous distributions of resources and species biomasses can also emerge through self-organised pattern formation caused by scale-dependent feedback between local trophic and regional dispersal dynamics. We show that this emergent heterogeneity can enhance the functional diversity of local autotroph communities by jointly strengthening source-sink dynamics and reducing stabilising selection pressure. Our results indicate that this effect is particularly strong in highly connected metacommunities, while metacommunity size (number of patches) alone plays a lesser role. We demonstrate that the positive effect on local diversity is driven by an eco-evo-spatial feedback loop that is fueled by the asynchronous biomass- and trait dynamics between the patches created by self-organised pattern formation. In highly connected metacommunities, oscillatory biomass patterns with particularly large amplitude strengthen this feedback loop. Our findings are highly relevant in the light of anthropogenic habitat changes that often destroy dispersal pathways, thereby increasing habitat isolation, lowering overall connectance of metacommunities and ultimately threatening the biodiversity in local habitats. Only a joint investigation of the contributing ecological, evolutionary, and spatial mechanisms in complex model systems can yield comprehensive understanding of these processes, allowing for the development of strategies to mitigate adverse anthropogenic influence.
Pre-bound State Discovered in the Unbinding Pathway of Fluorinated Variants of the Trypsin-BPTI Complex Using Random Acceleration Molecular Dynamics Simulations
Authors: Wehrhan Leon , Keller Bettina G.
Published: February 2024

The serine protease trypsin forms a tightly bound inhibitor complex with Bovine Pancreatic Trypsin Inhibitor (BPTI). The complex is stabilized by the P1 residue Lys15, which interacts with the negatively charged amino acids at the bottom of the S1 pocket. Truncating the P1 residue of wildtype BPTI to alpha-aminobutyric acid (Abu) leaves a complex with moderate inhibitor strength, which is held in place by additional hydrogen bonds at the protein-protein interface. Fluorination of the Abu residue partially restores inhibitor strength. The mechanism with which fluorination can restore the inhibitor strength is unknown and accurate computational investigation requires knowledge of the binding and unbinding pathways. The preferred unbinding pathway is likely to be complex, as encounter states have been described before and unrestrained Umbrella Sampling simulations of these complexes suggest additional energetic minima. Here, we use Random Acceleration Molecular Dynamics to find a new metastable state in the unbinding pathway of Abu-BPTI variants and wildtype BPTI from trypsin, which we call the pre-bound state. The pre-bound state and the fully bound state differ by a substantial shift in the position, a slight shift in the orientation of the the BPTI variants and change in the interaction pattern. Particularly important is the breaking of three hydrogen bonds around Arg17. Fluorination of the P1 residue lowers the energy barrier of the transition between fully bound state and pre-bound state and also lowers the energy minimum of the pre-bound state. While the effect of fluorination is in general difficult to quantify, here it is in part caused by a favorable stabilization of a hydrogen bond between Gln194 and Cys14. The interaction pattern of the pre-bound state offers insight into the inhibitory mechanism of BPTI and might add valuable information for the design serine protease inhibitors.
Non-cell-autonomous regulation of germline proteostasis by insulin/IGF-1 signaling via the intestinal peptide transporter PEPT-1
Authors: Muhammad Tahir , Edwards Stacey L. , Morphis Allison C. , Johnson Mary V. , Oliveira Vitor De , Chamera Tomasz , Liu Siyan , Nguyen Ngoc Gia Tuong , Li Jian
Published: February 2024

Gametogenesis involves active protein synthesis and heavily relies on proteostasis. How animals regulate germline proteostasis at the organismal level is poorly understood. TakingC. elegansas a model, we show that germline proteostasis requires coupled activities of HSF-1-dependent protein folding and insulin/IGF-1 signaling controlled protein synthesis. Depletion of HSF-1 from germ cells impairs chaperone gene expression, causing protein degradation and aggregation and, consequently, declines in fecundity and gamete quality. Reduced insulin/IGF-1 signaling confers germ cells’ resilience to limited protein folding capacity and proteotoxic stress by lowering ribosome biogenesis and the rate of translation. Interestingly, insulin/IGF-1 signaling promotes the expression of the evolutionarily conserved intestinal peptide transporter PEPT-1 via its downstream transcription factor FOXO/DAF-16, therefore allowing dietary proteins to be incorporated into an amino acid pool that fuels protein synthesis in the germline. We propose that this non-cell-autonomous pathway plays a critical role in regulating proteostasis in gametogenesis.

Teaser

Insulin/IGF-1 signaling regulates proteostasis in gametogenesis via the control of dietary protein absorption.