bluuebunny/crossref_metadata_2025 huggingface dataset:
Validation of the ACMG/AMP guidelines-based seven-category variant classification system
Authors: Chen Jian-Min , Masson Emmanuelle , Zou Wen-Bin , Liao Zhuan , Génin Emmanuelle , Cooper David N. , Férec Claude
Published: January 2023

Background

One shortcoming of employing the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP)-recommended five-category variant classification scheme (“pathogenic”, “likely pathogenic”, “uncertain significance”, “likely benign” and “benign”) in medical genetics lies in the scheme’s inherent inability to deal properly with variants that fall midway between “pathogenic” and “benign”. Employing chronic pancreatitis as a disease model, and focusing on the four most studied chronic pancreatitis-related genes, we recently expanded the five-category ACMG/AMP scheme into a seven-category variant classification system. With the addition of two new classificatory categories, “predisposing” and “likely predisposing”, our seven-category system promises to provide improved classification for the entire spectrum of variants in any disease-causing gene. The applicability and practical utility of our seven-category variant classification system however remains to be demonstrated in other disease/gene contexts, and this has been the aim of the current analysis.

Results

We have sought to demonstrate the potential universality of pathological variants that could be ascribed the new variant terminology (‘predisposing’) by trialing it across three Mendelian disease contexts (i.e., autosomal dominant, autosomal recessive and X-linked). To this end, we firstly employed illustrative genes/variants characteristic of these three contexts. On the basis of our own knowledge and expertise, we identified a series of variants that fitted well with our “predisposing” category, including “hypomorphic” variants in thePKD1gene and “variants of varying clinical consequence” in theCFTRgene. These examples, followed by reasonable extrapolations, enabled us to infer the widespread occurrence of “predisposing” variants in disease-causing genes. Such “predisposing” variants are likely to contribute significantly to the complexity of human genetic disease and may account not only for a considerable proportion of the unexplained cases of monogenic and oligogenic disease but also for much of the “missing heritability” characteristic of complex disease.

Conclusion

Employing an evidence-based approach together with reasonable extrapolations, we demonstrate both the applicability and utility of our seven-category variant classification system for disease-causing genes. The recognition of the new “predisposing” category not only has immediate implications for variant detection and interpretation but should also have important consequences for reproductive genetic counseling.
NGS-enriched activated sludge modelling of industrial wastewater treatment plant
Authors: Prawira Negara M A , Keesman K J , Euverink G J W , Jayawardhana B
Published: January 2023

Metagenomics advances with the Next Generation Sequencing (NGS) technology offer detailed insights into the microbial communities and their activities in a Wastewater Treatment Plant (WWTP). Since it has been shown recently that the microbial communities can be related to process data through machine learning, we investigate in this paper the enrichment of Activated Sludge Model 1 (ASM1) using time-series NGS data. We first present a modified ASM1 (mod-ASM1) to describe the industrial wastewater treatment at North Water’s WWTP facility in Delfzijl, the Netherlands. Subsequently, we identify the parameters for the ten weeks (weeks 40-50, 2014) of process data from North Water WWTP with prior parameters from the recommended ones from IWA. We further established a subset of parameters that are correlated to NGS data. Based on this relationship, a parameter-varying mod-ASM1 is obtained where the parameter variation is directly linked to the NGS data. We validate the NGS-enriched mod-ASM1 in the prediction of process data in the subsequent three weeks (weeks 50-53, 2014). While the enriched mod-ASM1 gives a good estimation of the COD effluent data, it cannot capture the production of nitrogen, which is often missed when the static model is deployed.
Robust and fast multicolor Single Molecule Localization Microscopy using spectral separation and demixing
Authors: Friedl Karoline , Mau Adrien , Caorsi Valentina , Bourg Nicolas , Lévêque-Fort Sandrine , Leterrier Christophe
Published: January 2023

Single Molecule Localization Microscopy (SMLM) is a straightforward approach to reach sub-50 nm resolution using techniques such as Stochastic Optical Reconstruction Microscopy (STORM) or DNA-Point Accumulation for Imaging in Nanoscale Topography (PAINT), and to resolve the arrangement of cellular components in their native environment. However, SMLM acquisitions are slow, particularly for multicolor experiments where channels are usually acquired in sequence. In this work, we evaluate two approaches to speed-up multicolor SMLM using a module splitting the fluorescence emission toward two cameras: simultaneous 2-color PAINT (S2C-PAINT) that images spectrally-separated red and far-red imager strands on each camera, and spectral demixing STORM (SD-STORM) that uses spectrally-close far-red fluorophores imaged on both cameras before assigning each localization to a channel by demixing. For each approach, we carefully evaluate the crosstalk between channels using three types of samples: DNA origami nanorulers of different sizes, single-target labeled cells, or cells labeled for multiple targets. We then devise experiments to assess how crosstalk can potentially affect the detection of biologically-relevant subdiffraction patterns. Finally, we show how these approaches can be combined with astigmatism to obtain three-dimensional data, and how SD-STORM can be extended three-color imaging, making spectral separation and demixing attractive options for robust and versatile multicolor SMLM investigations.
Exploring the genetic diversity of the Japanese Population: Insights from a Large-Scale Whole Genome Sequencing Analysis
Authors: Kawai Yosuke , Watanabe Yusuke , Omae Yosuke , Miyahara Reiko , Khor Seik-Soon , Noiri Eisei , Kitajima Koji , Shimanuki Hideyuki , Gatanaga Hiroyuki , Hata Kenichiro , Hattori Kotaro , Iida Aritoshi , Ishibashi-Ueda Hatsue , Kaname Tadashi , Kanto Tatsuya , Matsumura Ryo , Miyo Kengo , Noguchi Michio , Ozaki Kouichi , Sugiyama Masaya , Takahashi Ayako , Tokuda Haruhiko , Tomita Tsutomu , Umezawa Akihiro , Watanabe Hiroshi , Yoshida Sumiko , Goto Yu-ichi , Maruoka Yutaka , Matsubara Yoichi , Niida Shumpei , Mizokami Masashi , Tokunaga Katsushi
Published: January 2023

The Japanese archipelago is a terminal location for human migration, and the contemporary Japanese people represent a unique population whose genomic diversity has been shaped by multiple migrations from Eurasia. Through high-coverage whole-genome sequencing (WGS) analysis of 9,850 samples from the National Center Biobank Network, we analyzed the genomic characteristics that define the genetic makeup of the modern Japanese population from a population genetics perspective. The dataset comprised populations from the Ryukyu Islands and other parts of the Japanese archipelago (Hondo). Low frequency detrimental or pathogenic variants were found in these populations. The Hondo population underwent two episodes of population decline during the Jomon period, corresponding to the Late Neolithic, and the Edo period, corresponding to the Early Modern era, while the Ryukyu population experienced a population decline during the shell midden period of the Late Neolithic in this region. Genes related to alcohol and lipid metabolism were affected by positive natural selection. Two genes related to alcohol metabolism were found to be 12,500 years out of phase with the time when they began to be affected by positive natural selection; this finding indicates that the genomic diversity of Japanese people has been shaped by events closely related to agriculture and food production.

Author summary

The human population in the Japanese archipelago exhibits significant genetic diversity, with the Ryukyu Islands and other parts of the archipelago (Hondo) having undergone distinct evolutionary paths that have contributed to the genetic divergence of the populations in each region. In this study, whole genome sequencing of healthy individuals from national research hospital biobanks was utilized to investigate the genetic diversity of the Japanese population. Haplotypes were inferred from the genomic data, and a thorough population genetic analysis was conducted. The results indicated not only genetic differentiation between Hondo and the Ryukyu Islands, but also marked differences in past population size. In addition, gene genealogies were inferred from the haplotypes, and the patterns were scrutinized for evidence of natural selection. This analysis revealed unique traces of natural selection in East Asian populations, many of which were believed to be linked to dietary changes brought about by agriculture and food production.
The spectral slope as a marker of excitation/inhibition ratio and cognitive functioning in multiple sclerosis
Authors: Akbarian Fahimeh , Rossi Chiara , Costers Lars , D’hooghe Marie B , D’haeseleer Miguel , Nagels Guy , Schependom Jeroen Van
Published: January 2023

Background

Multiple sclerosis (MS) is a neurodegenerative disease characterized by neuronal and synaptic loss, resulting in an imbalance of excitatory and inhibitory synaptic transmission and potentially cognitive impairment. Current methods for measuring the excitation/inhibition (E/I) ratio are mostly invasive, but recent research combining neurocomputational modeling with measurements of local field potentials has indicated that the slope with which the power spectrum of neuronal activity captured by electro- and/or magnetoencephalography rolls off, is a non-invasive biomarker of the E/I ratio. A steeper roll- off is associated with a stronger inhibition. This novel method can be applied to assess the E/I ratio in people with multiple sclerosis (pwMS), detect the effect of medication such as benzodiazepines, and explore its utility as a biomarker for cognition.

Methods

We recruited 44 healthy control subjects and 95 people with multiple sclerosis (pwMS) who underwent resting-state magnetoencephalographic recordings. The 1/f spectral slope of the neural power spectra was calculated for each subject and for each brain region.

Results

As expected, the spectral slope was significantly steeper in pwMS treated with benzodiazepines (BZDs) compared to pwMS not receiving BZDs (p = 0.01). In the sub-cohort of pwMS not treated with BZDs, we observed a steeper slope in cognitively impaired pwMS compared to cognitively preserved pwMS (p = 0.01) and healthy subjects (p = 0.02). Furthermore, we observed a significant correlation between 1/f spectral slope and verbal and spatial working memory functioning in the brain regions located in the prefrontal and parietal cortex.

Conclusions

In this study, we highlighted the value of the spectral slope in MS by quantifying the effect of benzodiazepines and by putting it forward as a potential biomarker of cognitive deficits in pwMS.
Topological design principle for the robustness of necroptosis biphasic, emergent, and coexistent (BEC) dynamics
Authors: Xu Fei , Li Xiang , Wu Rui , Qi Hong , Jin Jun , Liu Zhilong , Wu Yuning , Lin Hai , Shen Chuansheng , Shuai Jianwei
Published: January 2023

Biphasic dynamics, the variable-dependent ability to enhance or restrain biological function, is prevalent in natural systems. Accompanied by biphasic dynamics, necroptosis signaling dominated by RIP1 also appears emergent and coexistent dynamics. Here, we identify the RIP1-RIP3-C8 incoherent feedforward loop embedded with positive feedback of RIP3 to RIP1 is the core topology, and the scale-free feature of RIP3 peak value dictates necroptosis BEC dynamics. Entropy production is introduced to quantify the uncertainty of coexistent dynamics. RIP3 auto-phosphorylation is further determined as a complementary process for robustly attaining necroptosis BEC dynamics. Through screening all possible two- and three-node circuit topologies, a complete atlas of three-node circuit BEC dynamics is generated and only three minimal circuits emerge as robust solutions, proving incoherent feedforward loop is the core topology. Overall, through highlighting a finite set of circuits, this study yields guiding principles for mapping, modulating, and designing circuits for BEC dynamics in biological systems.
All Quiet on the Western Front? The evolutionary history of monogeneans (Dactylogyridae:Cichlidogyrus, Onchobdella) infecting a West and Central African tribe of cichlid fishes (Chromidotilapiini)
Authors: Moons Tanisha , Kmentová Nikol , Pariselle Antoine , Artois Tom , Bert Wim , Vanhove Maarten P. M. , Cruz-Laufer Armando J.
Published: January 2023

Owing to the largely unexplored diversity of metazoan parasites, their speciation mechanisms and the circumstances under which such speciation occurs—in allopatry or sympatry—remain vastly understudied. Cichlids and their monogenean flatworm parasites have previously served as study system for macroevolutionary processes, e.g. for the role of East African host radiations on parasite communities. Here, we investigate the diversity and evolution of the poorly explored monogeneans infecting a West and Central African lineage of cichlid fishes: Chromidotilapiini Greenwood, 1987, which is the most species-rich tribe of cichlids in this region. We screened gills of 149 host specimens (27 species) from natural history collections and measured systematically informative characters of the sclerotised attachment and reproductive organs of the parasites. Ten monogenean species (Dactylogyridae:CichlidogyrusandOnchobdella) were found, eight of which are described and one redescribed herein. The phylogenetic positions of chromidotilapiines-infecting species ofCichlidogyruswere inferred through a parsimony analysis of the morphological characters. Furthermore, we employed machine learning algorithms to detect morphological features associated with the main lineages ofCichlidogyrus. Although the results of these experimental algorithms remain inconclusive, the parsimony analysis indicates that West and Central African lineages ofCichlidogyrusandOnchobdellaare monophyletic, unlike the paraphyletic host lineages. Several instances of host sharing suggest occurrences of intra-host speciation (sympatry) and host switching (allopatry). Some morphological variation was recorded that may also indicate the presence of species complexes. We conclude that collection material can provide important insights on parasite evolution despite the lack of well-preserved DNA material.

Data availability statement

Type material was deposited in the invertebrate collection of the Royal Museum for Central Africa (Tervuren, Belgium) (RMCA) (RMCA_VERMES_XXXXX–XXX), the collection of the Research Group Zoology: Biodiversity and Toxicology of Hasselt University (Diepenbeek, Belgium) (HU XXX–XXX), the Finnish Museum of Natural History (Helsinki, Finland) (MZH XXXXX–XXX), and the Iziko South African Museum (Cape Town, South Africa) (SAMC-XXXXXXX-XXX). The morphological data that support the findings of this study are openly available in MorphoBank atwww.morphobank.org, at https://dx.doi.org/XXXXXXXX. Phylogenetic trees and data matrices for the analysis in TNT are included as additional data in MorphoBank.
The Sin3B chromatin modifier restricts cell cycle progression to dictate hematopoietic stem cell differentiation
Authors: Calderon Alexander , Mestvirishvili Tamara , Boccalatte Francesco , Ruggles Kelly V. , David Gregory
Published: January 2023

Abstract/Summary

To maintain blood homeostasis, millions of terminally differentiated effector cells are produced every day. At the apex of this massive and constant blood production lie hematopoietic stem cells (HSCs), a rare cell type harboring unique self-renewal and multipotent properties. A key feature of HSCs is their ability to temporarily exit the cell cycle in a state termed quiescence. Defective control of cell cycle progression can eventually lead to bone marrow failure or malignant transformation. It is thought that HSCs must re-enter the cell cycle in order to commit to terminal differentiation. However, the molecular mechanisms tying cell cycle re-entry to cell fate commitment in HSCs remain elusive. Here, we identify the chromatin-associated Sin3B protein as a molecular link between cell cycle progression and differentiation in HSCs. We demonstrate that Sin3B is necessary for HSCs’ commitment to differentiation, but dispensable for their self-renewal or survival. Single cell transcriptional profiling of hematopoietic stem and progenitor cells (HSPCs) inactivated for Sin3B reveals aberrant cell cycle gene expression, consistent with the observed aberrant progression through the G1phase of the cell cycle. The defective cell cycle control elicited upon Sin3B inactivation correlates with the engagement of discrete signaling programs, including aberrant expression of cell adhesion molecules and essential components of the interferon signaling cascade in LT-HSCs. Additionally, chromatin accessibility profiling in LT-HSCs reveals the Sin3B-dependent accessibility of genomic elements controlling HSC differentiation, suggesting a functional link between cell cycle progression, and priming of hematopoietic stem cells for differentiation. Together, these results point to controlled progression through the G1phase of the cell cycle as a likely regulator of HSC lineage commitment through the modulation of chromatin features.
AGAMOUS mediates timing of guard cell formation during gynoecium development
Authors: Brazel Ailbhe J. , Fattorini Róisín , McCarthy Jesse , Franzen Rainer , Rümpler Florian , Coupland George , Ó’Maoiléidigh Diarmuid S.
Published: January 2023

InArabidopsis thaliana, stomata are composed of two guard cells that control the aperture of a central pore to facilitate gas exchange between the plant and its environment, which is particularly important during photosynthesis. Although leaves are the primary photosynthetic organs of higher plants, floral organs are also photosynthetically active. In the Brassicaceae, evidence suggests that silique photosynthesis is important for optimal seed oil content. A group of transcription factors containing MADS DNA binding domains is necessary and sufficient to confer floral organ identity. Elegant models, such as the ABCE model of flower development and the floral quartet model, have been instrumental in describing the molecular mechanisms by which these floral organ identity proteins govern flower development. However, we lack a complete understanding of how the floral organ identity genes interact with the underlying leaf development program. Here, we show that the MADS domain transcription factor AGAMOUS (AG) represses stomatal development on the gynoecial valves, so that maturation of stomatal complexes coincides with fertilization. We present evidence that this regulation by AG is mediated by direct transcriptional repression of the master regulator of the stomatal lineage,MUTE, and that this interaction is conserved among the Brassicaceae. This work extends on our understanding of the mechanisms underlying floral organ formation and provides a framework to decipher the mechanisms that control floral organ photosynthesis.
Utility of long-read sequencing for All of Us
Authors: Mahmoud M. , Huang Y. , Garimella K. , Audano P. A. , Wan W. , Prasad N. , Handsaker R. E. , Hall S. , Pionzio A. , Schatz M. C. , Talkowski M. E. , Eichler E. E. , Levy S. E. , Sedlazeck F. J.
Published: January 2023

TheAll of Us(AoU) initiative aims to sequence the genomes of over one million Americans from diverse ethnic backgrounds to improve personalized medical care. In a recent technical pilot, we compared the performance of traditional short-read sequencing with long-read sequencing in a small cohort of samples from the HapMap project and two AoU control samples representing eight datasets. Our analysis revealed substantial differences in the ability of these technologies to accurately sequence complex medically relevant genes, particularly in terms of gene coverage and pathogenic variant identification. We also considered the advantages and challenges of using low coverage sequencing to increase sample numbers in large cohort analysis. Our results show that HiFi reads produced the most accurate results for both small and large variants. Further, we present a cloud-based pipeline to optimize SNV, indel and SV calling at scale for long-reads analysis. These results will lead to widespread improvements across AoU.
Why do some fungi want to be sterile? The role of dysfunctional Pro1 in the rice blast fungus
Authors: Uchida Momotaka , Konishi Takahiro , Fujigasaki Ayaka , Kita Kohtetsu , Arie Tsutomu , Teraoka Tohru , Arazoe Takayuki , Kamakura Takashi
Published: January 2023

Although sexual reproduction is widespread in eukaryotes, some fungal species can only reproduce asexually. Therefore, loss of sexual reproduction may confer survival advantages under certain conditions in certain species. In the rice blast fungusPyricularia(Magnaporthe)oryzae, several isolates from the region of origin retain mating ability (female fertility), but most isolates are female sterile. Therefore, it is hypothesized that female fertility was lost during its spread from the origin to the rest of the world, andP. oryzaeis an ideal biological model for studying the cause of the evolutionary shift in the reproductive mode. Here, we show that functional mutations of Pro1, a global transcriptional regulator of mating-related genes in filamentous fungi, is one cause of loss of female fertility in this fungus. Employing backcrossing between female-fertile and female-sterile field isolates, we identified the putative genomic region involved in female sterility by comparative genomics between the genomes of F4female-fertile and -sterile progenies. Further genotyping, linkage, and functional analyses revealed that the functional mutation of Pro1 causes the loss of female fertility. RNA sequencing analysis showed that Pro1 regulates global gene expression, including that of several mating-related genes. The dysfunctional Pro1 did not affect the infection processes, such as conidial germination, appressorium formation, and penetration, but conidial release from conidiophores was increased. Furthermore, various types of mutations in Pro1 were detected in geographically distantP. oryzae, including pandemic isolates of wheat blast fungus. These results provide the first evidence that loss of female fertility may be advantageous to the life cycle of some plant pathogenic fungi.

Significance

Many pathogenic and industrial fungi are thought to have abdicated sexual reproduction, but the mechanisms and biological importance have been a long-standing mystery. Discovering why such fungi lost fertility is important to understand their survival strategies. Here, we revealed the genetic basis of how the rice blast fungus lost female fertility in nature and how this affects the life cycle. This has important implications for understanding evolution of blast pathogens and for developing an effective management strategy to control blast disease before a pandemic. Our findings also provide an additional perspective on advantages of asexual reproduction in some eukaryotes.
The Effect of Glycerol on Microbial Community in Industrial Wastewater Treatment Plant
Authors: Negara M A Prawira , Jayawardhana B , Euverink G J W
Published: January 2023

In this paper, we developed and analyzed lab-scale reactors that model the industrial saline wastewater treatment plant (SWWTP) used in North Water SWWTP in Delfzijl, the Netherlands. This industrial wastewater treatment plant is different from a typical municipal wastewater treatment plant, where the wastewater has some recalcitrant chemicals that are hard to degrade and contains a high COD-to-nitrogen ratio and a high concentration of NaCl. The process also differs from other standard industrial wastewater plants where the anaerobic process precedes the aerobic process. The proposed lab-scale reactors are shown to be stable and able to represent the studied industrial SWWTP where glycerol is present in abundance, and there is no similar lab-scale model that has investigated the effect of glycerol on the process. The removal of COD (glycerol) and nitrogen in the system and the changes in the microbial community in both reactors was followed over time. Based on the data, we were able to study the growth of the microbial population that is present in the sludge. The result of the experiment showed that glycerol and ammonia were completely removed, and some nitrate was left in the effluent. At the end of the experiment, we determined that the order Actinomycetales dominated the anaerobic reactor since it is known as the organisms that use glycerol as the carbon source and is quite tolerant with a high salt concentration in the influent. On the other hand, the order Flavobacteriales dominated the aerobic reactor as it is correlated with the ammonia concentration.
The effects of social experience on host gut microbiome in male zebrafish (Danio rerio)
Authors: Scott Emily , Brewer Michael S , Peralta Ariane L , Issa Fadi A
Published: January 2023

Although the gut and the brain vastly differ in physiological function, they have been interlinked in a variety of different neurological and behavioral disorders. The bacteria that comprise the gut microbiome communicate and influence the function of various physiological processes within the body including nervous system function. However, the effects of social experience in the context of dominance and chronic stress on gut microbiome remain poorly understood. Here, we examined whether social experience impacts the host zebrafish (Danio rerio) gut microbiome. We studied how social dominance during the first two weeks of social interactions changed the composition of zebrafish gut microbiome by comparing gut bacterial composition, diversity and relative abundance among socially dominant, submissive, social isolates, and control group-housed communal fish. Using amplicon sequencing of the 16S rRNA gene, we report that social dominance significantly affects host gut bacterial community composition but not bacterial diversity. At the genus-level,Aeromonasand unclassified Enterobacteriaceae relative abundance decreased in dominant individuals while commensal bacteria (e.g.,ExiguobacteriumandCetobacterium)increased in relative abundance. Conversely, the relative abundance ofPsychrobacterandAcinetobacterwas increased in subordinates, isolates, and communal fish compared to dominant fish. The shift in commensal and pathogenic bacteria highlights the impact of social experience and the accompanying stress on gut microbiome with potentially similar effects in other social organisms.

IMPORTANCE

Disruptions in the gut microbiome negatively impact various systems in the body. Recently, the gut microbiome has been associated with neurological deficits in both behavioral and neurodegenerative disorders. Given the increasing prevalence in diagnosis of both neurological disease and behavioral disorders, researching the link between social behaviors and the gut microbiome is critical to better understand how the gut and the brain communicate during healthy and diseased states. Our research findings demonstrate the effects of social dominance and chronic stress on host gut microbiome composition. By identifying variations in bacterial relative abundance based on social experience and associated stress, a broader understanding ofpathogenic(e.g., Enterobacteriaceae,Aeromonas) versus commensal communities (e.g.,Exiguobacterium, Cetobacterium) and related host physiology can be inferred.
The association between academic pressure and adolescent mental health problems: A systematic review
Authors: Steare Thomas , Muñoz Carolina Gutiérrez , Sullivan Alice , Lewis Gemma
Published: January 2023

Background

Academic pressure is a potential contributor to adolescent mental health problems, but international evidence on this association has never been synthesised.

Methods

We conducted the first systematic review on the association between academic pressure and adolescent depression, anxiety, self-harm, suicidality, suicide attempts and suicide. We searched MEDLINE, PsycINFO, ERIC and Web of Science (core collection) up to November 24, 2022, for studies of school-going children or adolescents, which measured academic pressure or timing within the school year as the exposure and depression, anxiety, self-harm, or suicidal ideation, attempts or suicide as outcomes. Risk of bias was assessed using the Mixed Methods Appraisal Tool. We used narrative synthesis to summarise the evidence. The review was prospectively registered with PROSPERO (CRD42021232702).

Results

We included 52 studies primarily from Asia (n=26) and Europe (n=20). Most studies assessed mixed anxiety and depressive symptoms (n=20) or depressive symptoms (n=19). Forty-eight studies found evidence of a positive association between academic pressure or timing within the school year and at least one mental health outcome.

Limitations

Most studies were cross-sectional (n=39), adjusted for a narrow range of confounders or had other limitations which limited the strength of causal inferences.

Conclusions

We found evidence that academic pressure is a potential candidate for public health interventions which could prevent adolescent mental health problems. Large population-based cohort studies are needed to investigate whether academic pressure is a causal risk factor that should be targeted in school- and policy-based interventions.

Funding

UCL Health of the Public; Wellcome Institutional Strategic Support Fund.
Phenotyping of lymphoproliferative tumours generated in xenografts of non-small cell lung cancer
Authors: Pearce David R. , Akarca Ayse U. , De Maeyer Roel P. H. , Kostina Emily , Huebner Ariana , Sivakumar Monica , Karasaki Takahiro , Shah Kavina , Janes Sam M. , McGranahan Nicholas , Reddy Venkat , Akbar Arne N. , Moore David A. , Marafioti Teresa , Swanton Charles , Hynds Robert E.
Published: January 2023

Patient-derived xenograft (PDX) models involve the engraftment of tumour tissue in immunocompromised mice and represent an important pre-clinixtcal oncology research. A limitation of non-small cell lung cancer (NSCLC) PDX model derivation in NOD-scidIL2Rgammanull(NSG) mice is that a subset of initial engraftments are of lymphocytic, rather than tumour origin. In the lung TRACERx PDX pipeline, lymphoproliferations occurred in 17.8% of lung adenocarcinoma and 10% of lung squamous cell carcinoma transplantations, despite none of these patients having a prior or subsequent clinical history of lymphoproliferative disease. Lymphoproliferations were predominantly human CD20+ B cells and had the immunophenotype expected for post-transplantation diffuse large B cell lymphoma. All lymphoproliferations expressed Epstein-Barr-encoded RNAs (EBER). Analysis of immunoglobulin light chain gene rearrangements in three tumours where multiple tumour regions had resulted in lymphoproliferations suggested that each had independent clonal origins. Overall, these data suggest the presence of B cell clones with lymphoproliferative potential within primary NSCLC tumours that are under continuous immune surveillance. Since these cells can be expanded following transplantation into NSG mice, our data highlight the value of quality control measures to identify lymphoproliferations within xenograft pipelines and support the incorporation of strategies to minimise lymphoproliferations during the early stages of xenograft establishment pipelines. To present the histology data herein, we developed a Python-based tool for generating patient-level pathology overview figures from whole-slide image files; PATHOverview is available on GitHub (https://github.com/EpiCENTR-Lab/PATHOverview).
Thermoneutral housing does not accelerate metabolic dysfunction-associated fatty liver disease in male or female mice fed a Western diet
Authors: Nunes Julia R.C. , Smith Tyler K.T. , Ghorbani Peyman , O’Dwyer Conor , Trzaskalski Natasha A. , Dergham Habiba , Pember Ciara , Kilgour Marisa K. , Mulvihill Erin E. , Fullerton Morgan D.
Published: January 2023

Objective

Metabolic dysfunction-associated fatty liver disease (MAFLD) represents a growing cause of mortality and morbidity and encompasses a spectrum of liver pathologies. Potential therapeutic targets have been identified and are currently being pre-clinically and clinically tested. However, while dozens of preclinical models have been developed to recapitulate various stages of MAFLD, few achieve fibrosis using an experimental design that mimics human pathogenesis. We sought to clarify whether the combination of thermoneutral (TN) housing and consumption of a classical Western diet (WD) would accelerate the onset of MAFLD and progression in male and female mice.

Methods

Male and female C57Bl/6J mice were fed a nutrient-matched low-fat control or Western diet (41% Kcal from fat, 43% carbohydrate and 0.2% cholesterol; WD) starting at ∼12 wk of age for a further 16 wk. Mice were divided and housed with littermates at either standard temperature (TS; 22°C) or thermoneutral conditions (TN; ∼29°C). Mice underwent tests for glucose tolerance, insulin sensitivity and body composition, as well as intestinal permeability. Following tissue harvest, circulating and liver markers of hepatic disease progression toward steatosis and fibrosis were determined.

Results

While male mice housed at TNand fed a WD were significantly heavier than TS-housed control animals, no other differences in body weight or composition were observed. WD-fed females housed under TNconditions had higher circulating LDL-cholesterol; however, there were no other significant differences between TNand TS-housing in circulating or hepatic lipid levels. While WD-fed TNmales had higher ALT levels, no other differences in markers of liver injury or disease progression were observed. Moreover, females housed at TNconditions and fed a WD remained significantly protected against the induction of fibrosis compared to male counterparts. Interestingly, sex-specific differences were observed in markers of glucose and insulin tolerance, where TNhousing and WD feeding resulted in hyperglycemia and impaired insulin responsiveness in both sexes, but glucose intolerance only in male mice.

Conclusions

While TNhousing has been demonstrated to exacerbate high fat-induced hepatic steatosis and inflammation in male and female mice, coupling TNhousing with a WD for 16 wk was not sufficient to augment fatty liver progression in male or female mice.

Highlights

Thermoneutral housing and Western diet feeding does not progress to NASH

Female mice are not more susceptible to obesity induced fatty liver under these conditions

Temperature and diet had sex-specific effects on glucose tolerance and insulin sensitivity
SynBa: Improved estimation of drug combination synergies with uncertainty quantification
Authors: Zhang Haoting , Ek Carl Henrik , Rattray Magnus , Milo Marta
Published: January 2023

Motivation

There exists a range of different quantification frameworks to estimate the synergistic effect of drug combinations. The diversity and disagreement in estimates make it challenging to determine which combinations from a large drug screening should be proceeded with. Furthermore, the lack of accurate uncertainty quantification for those estimates precludes the choice of optimal drug combinations based on the most favourable synergistic effect.

Results

In this work, we propose SynBa, a flexible Bayesian approach to estimate the uncertainty of the synergistic efficacy and potency of drug combinations, so that actionable decisions can be derived from the model outputs. The actionability is enabled by incorporating the Hill equation into SynBa, so that the parameters representing the potency and the efficacy can be preserved. Existing knowledge may be conveniently inserted due to the flexibility of the prior, as shown by the empirical Beta prior defined for the normalised maximal inhibition. Through experiments on large combination screenings and comparison against benchmark methods, we show that SynBa provides improved accuracy of dose-response predictions and better-calibrated uncertainty estimation for the parameters and the predictions.

Availability

The code for SynBa is available athttps://github.com/HaotingZhang1/SynBa. The datasets are publicly available (DOI of DREAM: 10.7303/syn4231880; DOI of the NCI-ALMANAC subset: 10.5281/zenodo.4135059).

Contact

hz381@cam.ac.uk
KDM6 demethylases mediate EWSR1-FLI1-driven oncogenic transformation in Ewing Sarcoma
Authors: Figuerola-Bou Elisabet , Rios-Astorch Carla , Blanco Enrique , Sánchez-Jiménez María , Táboas Pablo , Fernández Guerau , Gómez Soledad , Muñoz Oscar , Castellano-Escuder Pol , Pérez-Jaume Sara , Prada Estela , Mateo-Lozano Silvia , Riggi Nicolo , Avgustinova Alexandra , Lavarino Cinzia , Di Croce Luciano , Sánchez-Molina Sara , Mora Jaume
Published: January 2023

Ewing Sarcoma (EwS) is an aggressive bone and soft tissue tumor driven by the fusion oncoprotein EWSR1-FLI1. This aberrant transcription factor binds to GGAA microsatellites, causing epigenetic reprogramming through the formation of active neo-enhancers in a permissive cellular context. Inhibition of the oncogene remains challenging and current efforts instead seek to exploit emergent epigenetic treatments targeting EWSR1-FLI1 cofactors. Here, stemming from the genome-wide redistribution of H3K27me3 upon expression of EWSR1-FLI1 in pediatric hMSC, we unravel the contribution of the H3K27me3 demethylases KDM6A and KDM6B in transcriptional activation at EWSR1-FLI1 enhancers. We found that KDM6A has a demethylase-independent role in recruiting the SWI/SNF member BRG1 at EWSR1-FLI1-primed enhancers containing single GGAA motif, which is critical for EwS tumor growth. Conversely, KDM6B demethylates H3K27me3 at EWSR1-FLI1-active enhancers containing multimeric GGAA repeats and its deletion synergizes with EZH2 inhibitors. Our results highlight KDM6 demethylases as EWSR1-FLI1 cofactors with potential for future targeted therapies.
Amino acid transporter SLC7A5 regulates Paneth cell function to affect the intestinal inflammatory response
Authors: Bao Lingyu , Fu Liezhen , Su Yijun , Chen Zuojia , Peng Zhaoyi , Sun Lulu , Gonzalez Frank J. , Wu Chuan , Zhang Hongen , Shi Bingyin , Shi Yun-Bo
Published: January 2023

The intestine is critical for not only processing and resorbing nutrients but also protecting the organism from the environment. These functions are mainly carried out by the epithelium, which is constantly being self-renewed. Many genes and pathways can influence intestinal epithelial cell proliferation. Among them is mTORC1, whose activation increases cell proliferation. Here, we report the first intestinal epithelial cell-specific knockout (ΔIEC) of an amino acid transporter capable of activating mTORC1. We show that the transporter, SLC7A5, is highly expressed in mouse intestinal crypt andSlc7a5ΔIECreduces mTORC1 signaling. Surprisingly,Slc7a5ΔIECmice have increased cell proliferation but reduced secretory cells, particularly mature Paneth cells. scRNA-seq and electron microscopic analyses revealed dedifferentiation of Paneth cells inSlc7a5ΔIECmice, leading to markedly reduced secretory granules with little effect on Paneth cell number. We further show thatSlc7a5ΔIECmice are prone to experimental colitis. Thus, SLC7A5 regulates secretory cell differentiation to affect stem cell niche and/or inflammatory response to regulate cell proliferation.
Nuclease-dead S.aureusCas9 downregulates alpha-synuclein and reduces mtDNA damage and oxidative stress levels in patient-derived stem cell model of Parkinson’s disease
Authors: Sastre Danuta , Zafar Faria , Torres C. Alejandra Morato , Piper Desiree , Kirik Deniz , Sanders Laurie H. , Qi Stanley , Schüle Birgitt
Published: January 2023

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases, but no disease modifying therapies have been successful in clinical translation presenting a major unmet medical need. A promising target is alpha-synuclein or its aggregated form, which accumulates in the brain of PD patients as Lewy bodies. While it is not entirely clear which alpha-synuclein protein species is disease relevant, mere overexpression of alpha-synuclein in hereditary forms leads to neurodegeneration.

To specifically address gene regulation of alpha-synuclein, we developed a CRISPR interference (CRISPRi) system based on the nuclease deadS. aureusCas9 (SadCas9) fused with the transcriptional repressor domain Krueppel-associated box to controllably repress alpha-synuclein expression at the transcriptional level. We screened single guide (sg)RNAs across theSNCApromoter and identified several sgRNAs that mediate downregulation of alpha-synuclein at varying levels. CRISPRi downregulation of alpha-synuclein in iPSC-derived neuronal cultures from a patient with anSNCAgenomic triplication showed functional recovery by reduction of oxidative stress and mitochondrial DNA damage.

Our results are proof-of-conceptin vitrofor precision medicine by targeting theSNCAgene promoter. TheSNCACRISPRi approach presents a new model to understand safe levels of alpha-synuclein downregulation and a novel therapeutic strategy for PD and related alpha-synucleinopathies.
Spatially Resolved Transcriptomic Analysis of the Germinating Barley Grain
Authors: Peirats-Llobet Marta , Yi Changyu , Liew Lim Chee , Berkowitz Oliver , Narsai Reena , Lewsey Mathew G. , Whelan James
Published: January 2023

Seeds, which provide a major source of calories for humans, are a unique stage of a flowering plant ‘s lifecycle. During seed germination the embryo reactivates rapidly and goes through major developmental transitions to become a seedling. This requires extensive and complex spatiotemporal coordination of cell and tissue activity. Existing gene expression profiling methods, such as laser capture microdissection followed by RNA-seq and single-cell RNA-seq, suffer from either low throughput or the loss of spatial information about the cells analysed. Spatial transcriptomics methods couple high throughput analysis of gene expression simultaneously with the ability to record the spatial location of each individual region analysed. We developed a spatial transcriptomics workflow for germinating barley grain to better understand the spatiotemporal control of gene expression within individual seed cell types. More than 14,000 genes were differentially regulated across 0, 1, 3, 6 and 24 hours after imbibition. This approach enabled us to observe that many functional categories displayed specific spatial expression patterns that could be resolved at a sub-tissue level. Individual aquaporin gene family members, important for water and ion transport, had specific spatial expression patterns over time, as well as genes related to cell wall modification, membrane transport and transcription factors. Using spatial autocorrelation algorithms, we were able to identify auxin transport genes that had increasingly focused expression within subdomains of the embryo over germination time, suggestive of a role in establishment of the embryo axis. Together, our data provides an unprecedented spatially resolved cellular map for barley grain germination and specific genes to target for functional genomics to define cellular restricted processes in tissues during germination. The data can be viewed athttps://spatial.latrobe.edu.au/.
Deep Learning Assisted Smartphone-based Quantitative Microscopy for Label-free Hematological Analysis
Authors: Huang Bingxin , Kang Lei , Tsang Victor T. C. , Lo Claudia T. K. , Wong Terence T. W.
Published: January 2023

Hematologists evaluate alterations in blood cell enumeration and morphology to confirm the peripheral blood smear findings through manual microscopic examination. However, routine peripheral blood smear analysis is both time-consuming and labor-intensive. Here, we propose a smartphone-based autofluorescence microscopy (Smart-AM) system for imaging label-free blood smears at sub-cellular resolution and performing hematological analysis. Smart-AM enables rapid, high-quality, and label-free visualization of morphological features of different blood cells (leukocytes, erythrocytes, and thrombocytes) and abnormal variations in blood cells. Moreover, assisted with deep learning algorithms, this technique can automatically detect and classify different leukocytes with high accuracy, and transform the autofluorescence images into virtual Giemsa-stained images maintaining significant cellular features. The proposed technique is portable, cost-effective, and user-friendly, making it significant for broad point-of-care applications.
A time- and space-resolved nuclear receptor atlas in mouse liver
Authors: Zummo Francesco Paolo , Berthier Alexandre , Gheeraert Céline , Vinod Manjula , Bobowski-Gérard Marie , Molendi-Coste Olivier , Pineau Laurent , Jung Matthieu , Guille Loic , Chevalier-Dubois Julie , Dombrowicz David , Staels Bart , Eeckhoute Jérôme , Lefebvre Philippe
Published: January 2023

The unique functional versatility of the liver is paramount for organismal homeostasis. Both liver development and adult functions are controlled by tightly regulated transcription factor networks, within which nuclear receptors regulate essential functions of parenchymal and non-parenchymal cells. Acting as transcription factors sensitive to extracellular cues such as steroidal hormones, lipid metabolites, xenobiotics… and modulated by intracellular signaling pathways, nuclear receptors orchestrate many aspects of hepatic physiology. While liver functional zonation and adaptability to fluctuating conditions are known to rely on a sophisticated cellular architecture, a comprehensive knowledge of nuclear receptor functions in the different liver cell types is still lacking. As a first step toward the accurate mapping of nuclear receptor functions in mouse liver, we characterized their levels of expression in whole liver as a function of time and diet, and explored nuclear receptor isoform expression in hepatocytes, cholangiocytes, Kupffer cells, hepatic stellate cells and liver sinusoidal cells. In addition, we leveraged liver single cell RNAseq studies to provide here an up-to-date compendium of nuclear receptor expression in mouse liver in space and time.
Multimodal characterization of antigen-specific CD8+T cells across SARS-CoV-2 vaccination and infection
Authors: Zhang Bingjie , Upadhyay Rabi , Hao Yuhan , Samanovic Marie I. , Herati Ramin S. , Blair John , Axelrad Jordan , Mulligan Mark J. , Littman Dan R. , Satija Rahul
Published: January 2023

The human immune response to SARS-CoV-2 antigen after infection or vaccination is defined by the durable production of antibodies and T cells. Population-based monitoring typically focuses on antibody titer, but there is a need for improved characterization and quantification of T cell responses. Here, we utilize multimodal sequencing technologies to perform a longitudinal analysis of circulating human leukocytes collected before and after BNT162b2 immunization. Our data reveal distinct subpopulations of CD8+T cells which reliably appear 28 days after prime vaccination (7 days post boost). Using a suite of cross-modality integration tools, we define their transcriptome, accessible chromatin landscape, and immunophenotype, and identify unique biomarkers within each modality. By leveraging DNA-oligo-tagged peptide-MHC multimers and T cell receptor sequencing, we demonstrate that this vaccine-induced population is SARS-CoV-2 antigen-specific and capable of rapid clonal expansion. Moreover, we also identify these CD8+populations in scRNA-seq datasets from COVID-19 patients and find that their relative frequency and differentiation outcomes are predictive of subsequent clinical outcomes. Our work contributes to our understanding of T cell immunity, and highlights the potential for integrative and multimodal analysis to characterize rare cell populations.