bluuebunny/crossref_metadata_2025 huggingface dataset:
The nutrient-sensing Rag-GTPase complex in B cells controls humoral immunity via TFEB/TFE3-dependent mitochondrial fitness
Authors: Zhu Xingxing , Wu Yue , Li Yanfeng , Zhou Xian , Watzlawik Jens O. , Chen Yin Maggie , Raybuck Ariel L. , Billadeau Daniel , Shapiro Virginia , Springer Wolfdieter , Sun Jie , Boothby Mark R. , Zeng Hu
Published: March 2024

During the humoral immune response, B cells undergo rapid metabolic reprogramming with a high demand for nutrients, which are vital to sustain the formation of the germinal centers (GCs). Rag-GTPases sense amino acid availability to modulate the mechanistic target of rapamycin complex 1 (mTORC1) pathway and suppress transcription factor EB (TFEB) and transcription factor enhancer 3 (TFE3), members of the microphthalmia (MiT/TFE) family of HLH-leucine zipper transcription factors. However, how Rag-GTPases coordinate amino acid sensing, mTORC1 activation, and TFEB/TFE3 activity in humoral immunity remains undefined. Here, we show that B cell-intrinsic Rag-GTPases are critical for the development and activation of B cells. RagA/RagB deficient B cells fail to form GCs, produce antibodies, and generate plasmablasts in both T-dependent (TD) and T-independent (TI) humoral immune responses. Deletion of RagA/RagB in GC B cells leads to abnormal dark zone (DZ) to light zone (LZ) ratio and reduced affinity maturation. Mechanistically, the Rag-GTPase complex constrains TFEB/TFE3 activity to prevent mitophagy dysregulation and maintain mitochondrial fitness in B cells, which are independent of canonical mTORC1 activation. TFEB/TFE3 deletion restores B cell development, GC formation in Peyer’s patches and TI humoral immunity, but not TD humoral immunity in the absence of Rag-GTPases. Collectively, our data establish Rag-GTPase-TFEB/TFE3 pathway as an mTORC1 independent mechanism to coordinating nutrient sensing and mitochondrial metabolism in B cells.

One sentence summary

Rag-GTPases restrain TFEB/TFE3 to prevent abnormal mitophagy, maintain mitochondrial fitness, and support B cell development, Peyer’s patch germinal center formation, and T-independent humoral immunity.
Biogenesis of circular RNAsin vitroandin vivofrom theDrosophila Nk2.1/scarecrowgene
Authors: Son Suhyeon , Jeong Hyunjin , Lee Gyunghee , Park Jae H. , Yoo Siuk
Published: March 2024

scarecrow(scro) encodes a fly homolog of mammalianNkx2.1that is vital for early fly development as well as for optic lobe development. Interestingly,scrowas reported to produce a circular RNA (circRNA). In this study, we identified 12 differentscrocircRNAs, which are either mono- or multi-exonic forms. The most abundant forms are circE2 carrying the second exon only and bi-exonic circE3-E4. Levels of circE2 show an age-dependent increase in adult heads, supporting a general trend of high accumulation of circRNAs in aged fly brains. Aligning sequences of introns flanking exons uncovered two pairs of intronic complementary sequences (ICSs); one pair residing in introns 1 and 2 and the other in introns 2 and 4. The first pair was demonstrated to be essential for the circE2 production in cell-based assays; furthermore, deletion of the region including potential ICS components in the intron-2 reducedin vivoproduction of circE2 and circE3-E4 by 80%, indicating them to be essential for the biogenesis of these isoforms. Besides the ICS, the intron regions immediately abutting exons seemed to be responsible for a basal level of circRNA formation. Moreover, the replacement ofscro-ICS with those derived fromlaccase2was comparably effective inscro-circRNA production, buttressing the importance of the hairpin-loop structure formed by ICS for the biogenesis of circRNA. Lastly, overexpressedscroaffected outcomes of both linear and circular RNAs from the endogenousscrolocus, suggesting that Scro plays a direct or indirect role in regulating expression levels of either or both forms.
An injury-induced tissue niche shaped by mesenchymal plasticity coordinates the regenerative and disease response in the lung
Authors: Jones Dakota L. , Morley Michael P. , Li Xinyuan , Ying Yun , Cardenas-Diaz Fabian L. , Li Shanru , Zhou Su , Schaefer Sarah E. , Chembazhi Ullas V. , Nottingham Ana , Lin Susan , Cantu Edward , Diamond Joshua M. , Basil Maria C. , Vaughan Andrew E. , Morrisey Edward E.
Published: March 2024

Severe lung injury causes basal stem cells to migrate and outcompete alveolar stem cells resulting in dysplastic repair and a loss of gas exchange function. This “stem cell collision” is part of a multistep process that is now revealed to generate aninjury-inducedtissue niche (iTCH) containing Keratin 5+ epithelial cells and plastic Pdgfra+ mesenchymal cells. Temporal and spatial single cell analysis reveals that iTCHs are governed by mesenchymal proliferation and Notch signaling, which suppresses Wnt and Fgf signaling in iTCHs. Conversely, loss of Notch in iTCHs rewires alveolar signaling patterns to promote euplastic regeneration and gas exchange. The signaling patterns of iTCHs can differentially phenotype fibrotic from degenerative human lung diseases, through apposing flows of FGF and WNT signaling. These data reveal the emergence of an injury and disease associated iTCH in the lung and the ability of using iTCH specific signaling patterns to discriminate human lung disease phenotypes.
Genome assembly variation and its implications for gene discovery in nematode species
Authors: Mariene Grace M. , Wasmuth James D.
Published: March 2024

Genome assemblers are a critical component of genome science, but the choice of assembly software and protocols can be daunting. Here, we investigate genome assembly variation and its implications for gene discovery across three nematode species—Caenorhabditis bovis,Haemonchus contortus, andHeligmosomoides bakeri—highlighting the critical interplay between assembly choice and downstream genomic analysis. Selecting popular genome assemblers, we generated multiple assemblies for each species, analyzing their structure, completeness, and effect on gene family analysis. Our findings demonstrate that assembly variations can significantly affect gene family composition, with notable differences in critical gene families likecyp,gst,ugt, andnhr. Despite broadly similar performance using various assembly metrics, comparisons of assemblies with a single species revealed underlying structural rearrangements and inconsistencies in gene content. This emphasizes the imperative for continuous refinement of genomic resources. Our findings advocate for a cautious and informed approach to genome assembly and annotation to ensure reliable and insightful genomic interpretations.
A Micro-scale Humanized Ventilator-on-a-Chip to Examine the Injurious Effects of Mechanical Ventilation
Authors: Gabela-Zuniga Basia , Shukla Vasudha C. , Bobba Christopher , Higuita-Castro Natalia , Powell Heather M. , Englert Joshua A. , Ghadiali Samir N.
Published: March 2024

Patients with compromised respiratory function frequently require mechanical ventilation to survive. Unfortunately, non-uniform ventilation of injured lungs generates complex mechanical forces that lead to ventilator induced lung injury (VILI). Although investigators have developed lung-on-a-chip systems to simulate normal respiration, modeling the complex mechanics of VILI as well as the subsequent recovery phase is a challenge. Here we present a novel humanized in vitro ventilator-on-a-chip (VOC) model of the lung microenvironment that simulates the different types of injurious forces generated in the lung during mechanical ventilation. We used transepithelial/endothelial electrical resistance (TEER) measurements to investigate how individual and simultaneous application of the different mechanical forces alters real-time changes in barrier integrity during and after injury. We find that compressive stress (i.e. barotrauma) does not significantly alter barrier integrity while over-distention (20% cyclic radial strain, volutrauma) results in decreased barrier integrity that quickly recovers upon removal of mechanical stress. Conversely, surface tension forces generated during airway reopening (atelectrauma), result in a rapid loss of barrier integrity with a delayed recovery relative to volutrauma. Simultaneous application of cyclic stretching (volutrauma) and airway reopening (atelectrauma), indicate that the surface tension forces associated with reopening fluid-occluded lung regions is the primary driver of barrier disruption. Thus, our novel VOC system can monitor the effects of different types of injurious forces on barrier disruption and recovery in real-time and can be used to identify the biomechanical mechanisms of VILI.
Bio-guided isolation of a new sesquiterpene fromArtemisia cinawith anthelmintic activity againstHaemonchus contortusL3infective larvae
Authors: Arango-De-la Pava Luis David , González-Cortázar Manasés , Zamilpa Alejandro , Cuéllar-Ordaz Jorge Alfredo , de la Cruz-Cruz Héctor Alejandro , Higuera-Piedrahita Rosa Isabel , López-Arellano Raquel
Published: March 2024

Haemonchus contortusis a blood-feeding gastrointestinal parasite that impacts grazing sheep, causing economic losses in animal production and presenting anthelmintic resistance, requiring alternative antiparasitic treatments, including the exploration of plant-based anthelmintics.Artemisia cina(Asteraceae) is a plant whosen-hexane (n-HE) and ethyl acetate extract (EAE) exhibits anthelmintic activity againstH. contortus, being then-HE more active. With the aim of discovering additional bioactive metabolites, a chemical analysis was performed on EAE, which presented a LC90of 3.30 mg/mL and allowed the isolation of 11-[(1R,5S,7R,8R,10S,)-1,8-dihydroxy-5,10-dimethyl-4-oxodecahydroazulen-7-yl] acrylic acid, a new sesquiterpene that was identified through one and two-dimensional NMR. The compound was named cinic acid and displayed a CL50of 0.13 (0.11 -0.14) mg/mL and CL90of 0.40 (0.37 - 0.44) mg/mL, which, compared with EAE larvicidal activity, was 256-fold more active at LC50and 15.71-fold at LC90. In this study, a new sesquiterpene with anthelmintic effects againstH. contortusL3infective larvae was isolated from the EAE ofArtemisia cina.

Author summary

Haemonchus contortusis a hematophagous gastrointestinal parasite that affects grazing sheep. Due to its feeding habits, it induces anemia, poor digestion, diarrhea, and weight loss in animals, potentially leading to death in young individuals and causing economic losses in animal production. Moreover, it demonstrates resistance to drugs, making it imperative to explore alternative antiparasitic treatments againstH. contortus, such as the discovery and development of plant-based anthelmintics. In this work, we explore the ethyl acetate extract (EAE) ofArtemisia cinain the search of bioactive compounds. A new sesquiterpene was separated through a bio-guided isolation monitoring the larvicidal effect againstH. ContortusL3infective larvae and was named cinic acid. These findings suggest that the EAE could be promising candidate for the development of a plant-based pharmaceutical preparation with notable anthelmintic activity againstH. contortus.
Investigation of the Association of Acute Pancreatitis Outcomes with Social Vulnerability Indicators
Authors: Chhoda Ankit , Liyen-Cartelle Anabel , Noriega Marco , Anderson Kelsey , Zuberi Shaharyar A. , Sur Alana , Olivares Miriam , Kelly Jill , Freedman Steven D. , Rabinowitz Loren G. , Sheth Sunil G.
Published: February 2024

BACKGROUND AND AIM

Geospatial analyses integrate location-based sociodemographic data, offering a promising approach to investigate the impact of social determinants on acute pancreatitis (AP) outcomes. This study aimed to examine the association of social vulnerability index (SVI) and its constituent 16 attributes in 4 domains (socioeconomic status, household composition and disability, minority status and language, and housing type and transportation), with outcomes of patients with AP.

METHODS

This study included AP patients hospitalized between 1/1/2008 and 12/31/2018 and recorded their demographics and clinical outcomes. Physical addresses were geocoded to determine SVI, a composite variable which was ranked and divided into quartiles (I to IV: IV representing the highest vulnerability).

RESULT

In 824 patients [age of 53.0±10 years and 48.2% females], with 993 AP-related hospitalizations, we noted significantly higher prevalence of no/federal/state insurance(P<0.001)and racial minorities(P<0.001)in patients residing in communities with higher SVI. A non-significant trend of higher 30-day admission rate amongst patients from higher SVI regions (III/IV: 27(18.0%)/31(18.9%)vs.I/II: 40(13.8%)/36(16.3%);p=0.49) was noted. We observed a significant association of alcohol withdrawal with residence in areas with higher SVI despite adjustment for age, body mass index, and comorbidities (OR:1.62[95%CI:1.19-2.22];p=0.003). However, we observed no association of SVI with severity of AP, inpatient opioid use, length of stay, and mortality.

CONCLUSION

We noted significantly higher alcohol withdrawal in patients residing in areas with higher SVI ranks although AP severity and other outcomes lacked significant association with SVI. Given our findings further investigation of various social determinants of health in AP is warranted in large sized prospective studies.
A SNP-based honey bee paternity assignment test for evaluating the effectiveness of mating stations and its application to the Ataun valley, Basque Country, Spain
Authors: Parejo Melanie , Galartza Egoitz , Momeni Jamal , Gorrochategui-Ortega June , Farajzadeh Leila , Wegener Jakob , Bienefeld Kaspar , Zarraonaindia Iratxe , Estonba Andone
Published: March 2024

Geographically isolated mating stations are deployed across Europe to facilitate controlled mating with selected drone-producing colonies. To assess the reliability of these stations, we developed a paternity assignment test using a custom Illumina genotyping chip with 6457 SNPs based on two metrics (number of mismatch alleles and kinship). The method demonstrated remarkable accuracy during validation with an independent dataset of known parent-offspring pairs, with an accuracy rate of 97.7%. We then applied the developed paternity assignment test to theApis mellifera iberiensismating station in the Ataun valley, Basque Country, Spain, in 2021. Drone-producing colonies in the valley were sampled and genotyped, as well as 156 worker offspring of queens mated at the station, and 56 drones collected in the drone congregation area. Out of the 156 worker samples, we could assign paternity of 120 (76.9%) to one of the drone-producing colonies in the valley, while 23.1% were of unknown patriline. Out of the 56 drones collected in the air, 52 (92.9%) were assigned to drone-producing colonies. We were also able to determine the colonies and apiaries that made the most significant contributions to the matings. This information aids in effective apiary management, including the selection of suitable mating station locations and the positioning of drone-producing colonies therein. Overall, our SNP-based paternity assignment test offers a valuable tool for evaluating mating station effectiveness across Europe, crucial for advancing breeding objectives in honey bee populations.
Endometriotic lesions exhibit distinct metabolic signature compared to paired eutopic endometrium at the single-cell level
Authors: Sarsenova Meruert , Lawarde Ankita , Pathare Amruta D. S. , Saare Merli , Modhukur Vijayachitra , Soplepmann Pille , Terasmaa Anton , Käämbre Tuuli , Gemzell-Danielsson Kristina , Lalitkumar Parameswaran Grace Luther , Salumets Andres , Peters Maire
Published: March 2024

Current therapeutics of endometriosis are limited to hormonal action on endometriotic lesions to disrupt their growth. Based on the recent findings of the high utilization of glycolysis over oxidative metabolism (Warburg-like effect) in endometriotic lesions, a new strategy of nonhormonal management by addressing cellular metabolism has been proposed. However, it remains unclear which cell types are metabolically altered and contribute to endometriotic lesion growth for targeting them with metabolic drugs. Using single-cell RNA-sequencing, we investigated the activity of twelve metabolic pathways and genes involved in steroidogenesis in paired samples of eutopic endometrium (EuE) and peritoneal lesions (ectopic endometrium, EcE) from women with confirmed endometriosis. We detected nine major cell clusters in both EuE and EcE. The metabolic pathways were differentially regulated in perivascular, stromal and to a lesser extent in endothelial cell clusters, with the highest changes in AMP-activated protein kinase signaling, Hypoxia-Inducible Factor-1 signaling, glutathione metabolism, oxidative phosphorylation, and glycolysis/gluconeogenesis. We identified a transcriptomic co-activation of glycolysis and oxidative metabolism in perivascular and stromal cells of EcE compared with EuE, suggesting that metabolic reprogramming may play a critical role in maintaining cell growth and survival of endometriotic lesions. Additionally, progesterone receptor was significantly downregulated in perivascular and endothelial cells of EcE. The expression of estrogen receptor 1 was significantly reduced in perivascular, stromal and endothelial cells of EcE. In parallel, perivascular cells exhibited a high expression of estrogen receptor 2 andHSD17B8gene that encodes for protein converting estrone (E1) to estradiol (E2), while in endothelial cellsHSD17B2gene coding for enzyme converting E2 to E1 was downregulated. Overall, our results identified different expression patterns of energy metabolic pathways and steroidogenesis-related genes in perivascular, stromal, and endothelial cells in EcE compared with EuE. Perivascular cells, known to contribute to the restoration of endometrial stroma and angiogenesis, can be a potential target for non-hormonal treatment of endometriosis.
Exploring a large cancer cell line RNA-sequencing dataset with k-mers
Authors: Bessière Chloé , Xue Haoliang , Guibert Benoit , Boureux Anthony , Rufflé Florence , Viot Julien , Chikhi Rayan , Salson Mikaël , Marchet Camille , Commes Thérèse , Gautheret Daniel
Published: March 2024

Analyzing the immense diversity of RNA isoforms in large RNA-seq repositories requires laborious data processing using specialized tools. Indexing techniques based on k-mers have previously been effective at searching for RNA sequences across thousands of RNA-seq libraries but falling short of enabling direct RNA quantification. We show here that RNAs queried in the form of k-mer sets can be quantified in seconds, with a precision akin to that of conventional RNA quantification methods. We showcase several applications by exploring an index of the Cancer Cell Line Encyclopedia (CCLE) collection consisting of 1019 RNA-seq samples. Non-reference RNA sequences such as RNAs harboring driver mutations and fusions, splicing isoforms or RNAs derived from repetitive elements, can be retrieved with high accuracy. Moreover, we show that k-mer indexing offers a powerful means to reveal variant RNAs induced by specific gene alterations, for instance in splicing factors. A web server allows public queries in CCLE and other indexes:https://transipedia.fr. Code is provided to allow users to set up their own server from any RNA-seq dataset.
Detecting Genetic Interactions with Visible Neural Networks
Authors: van Hilten Arno , Melograna Federico , Fan Bowen , Niessen Wiro , van Steen Kristel , Roshchupkin Gennady
Published: March 2024

Non-linear interactions among single nucleotide polymorphisms (SNPs), genes, and pathways play an important role in human diseases, but identifying these interactions is a challenging task. Neural networks are state-of-the-art predictors in many domains due to their ability to analyze big data and model complex patterns, including non-linear interactions. In genetics, visible neural networks are gaining popularity as they provide insight into the most important SNPs, genes and pathways for prediction. Visible neural networks use prior knowledge (e.g. gene and pathway annotations) to define the connections between nodes in the network, making them sparse and interpretable. Currently, most of these networks provide measures for the importance of SNPs, genes, and pathways but lack details on the nature of the interactions. In this paper, we explore different methods to detect non-linear interactions with visible neural networks. We adapted and sped up existing methods, created a comprehensive benchmark with simulated data from GAMETES and EpiGEN, and demonstrated that these methods can extract multiple types of interactions from trained visible neural networks. Finally, we applied these methods to a genome-wide case-control study of inflammatory bowel disease and found high consistency of the epistasis pairs candidates between the interpretation methods. The follow-up association test on these candidate pairs identified seven significant epistasis pairs.
Cognate Antigen Engagement Induces HIV-1 Expression In CD4+T Cells From People On Long-Term ART
Authors: Moskovljevic Milica , Dragoni Filippo , Board Nathan L. , Wu Fengting , Lai Jun , Zhang Hao , White James R. , Hoh Rebecca , Lynn Kenneth , Tebas Pablo , Mounzer Karam , Deeks Steven G. , Montaner Luis J. , Siliciano Janet D. , Simonetti Francesco R. , Siliciano Robert F.
Published: March 2024

Despite antiretroviral therapy (ART), HIV-1 persists in latently-infected CD4+T cells, preventing cure. Antigens drive the proliferation of infected cells, precluding latent reservoir decay. However, the relationship between antigen recognition and HIV-1 gene expression is poorly understood since most studies of latency reversal use agents that induce non-specific global T cell activation. Here, we isolated rare CD4+T cells responding to cytomegalovirus (CMV) or HIV-1 Gag antigens from participants on long-term ART and assessed T cell activation and HIV-1 RNA expression upon co-culture with autologous dendritic cells (DCs) presenting cognate antigens. Physiological presentation of cognate antigens induced broad T cell activation (median 42-fold increase in CD154+CD69+cells) and significantly increased HIV-1 transcription (median 4-fold), mostly through the induction of rare cells with higher viral expression. Thus, despite low proviral inducibility, physiologic antigen recognition can promote HIV-1 expression, potentially contributing to spontaneous reservoir activity on ART and viral rebound upon ART interruption.
Intracellular biomacromolecule delivery by stimuli responsive protein vesicles loaded by hydrophobic ion pairing
Authors: Gray Mikaela A. , de Janon Alejandro , Seeler Michelle , Heller William T. , Panoskaltsis Nicki , Mantalaris Athanasios , Champion Julie A.
Published: March 2024

Therapeutic biomacromolecules are highly specific, which results in controlled therapeutic effect and less toxicity than small molecules. However, proteins and nucleic acids are large and have significant surface hydrophilicity and charge, thus cannot diffuse into cells. These chemical features render them poorly encapsulated by nanoparticles. Protein vesicles are self-assembling nanoparticles made by warming elastin-like polypeptide (ELP) fused to an arginine-rich leucine zipper and a globular protein fused to a glutamate-rich leucine zipper. To impart stimuli-responsive disassembly and small size, ELP was modified to include histidine and tyrosine residues. Additionally, hydrophobic ion pairing (HIP) was used to load and release protein and siRNA cargos requiring endosomal escape. HIP vesicles enabled delivery of cytochrome c, a cytosolically active protein, and significant reduction in viability in traditional two-dimensional (2D) human cancer cell line culture and a biomimetic three-dimensional (3D) organoid model of acute myeloid leukemia. They also delivered siRNA to knockdown protein expression in a murine fibroblast cell line. By examining uptake of positive and negatively charged fluorescent protein cargos loaded by HIP, this work revealed the necessity of HIP for cargo release and how HIP influences protein vesicle self-assembly using microscopy, small angle x-ray scattering, and nanoparticle tracking analysis. HIP protein vesicles have the potential to broaden the use of intracellular proteins for various diseases and extend protein vesicles to deliver other biomacromolecules.
Spatial control of sensory adaptation modulates mechanosensing inPseudomonas aeruginosa
Authors: Patino Ramiro , Kühn Marco J. , Macmillan Henriette , Inclan Yuki F. , Chavez Ivan , Von Dollen John , Johnson Jeffrey R. , Swaney Danielle L. , Krogan Nevan J. , Persat Alexandre , Engel Joanne N.
Published: February 2024

Sensory signaling pathways use adaptation to dynamically respond to changes in their environment. Here, we report the mechanism of sensory adaptation in the Pil-Chp mechanosensory system, which the important human pathogenPseudomonas aeruginosauses to sense mechanical stimuli during surface exploration. Using biochemistry, genetics, and cell biology, we discovered that the enzymes responsible for adaptation, a methyltransferase and a methylesterase, are segregated to opposing cell poles asP. aeruginosaexplore surfaces. By coordinating the localization of both enzymes, we found that the Pil-Chp response regulators influence local receptor methylation, the molecular basis of bacterial sensory adaptation. We propose a model in which adaptation during mechanosensing spatially resets local receptor methylation, and thus Pil-Chp signaling, to modulate the pathway outputs, which are involved inP. aeruginosavirulence. Despite decades of bacterial sensory adaptation studies, our work has uncovered an unrecognized mechanism that bacteria use to achieve adaptation to sensory stimuli.
Spatial transcriptomics profiling of gallbladder adenocarcinoma: a detailed two-case study of progression from precursor lesions to cancer
Authors: Pirenne Sophie , Manzano-Núñez Fátima , Loriot Axelle , Cordi Sabine , Desmet Lieven , Aydin Selda , Hubert Catherine , Toffoli Sébastien , Limaye Nisha , Sempoux Christine , Komuta Mina , Gatto Laurent , Lemaigre Frédéric P.
Published: March 2024

Background

Most studies on tumour progression from precursor lesion toward gallbladder adenocarcinoma investigate lesions sampled from distinct patients, providing an overarching view of pathogenic cascades. Whether this reflects the tumourigenic process in individual patients remains insufficiently explored. Genomic and epigenomic studies suggest that a subset of gallbladder cancers originate from biliary intraepithelial neoplasia (BilIN) precursor lesions, whereas others form independently from BilINs. Spatial transcriptomic data supporting these conclusions are missing. Moreover, multiple areas with precursor or adenocarcinoma lesions can be detected within the same pathological sample. Yet, knowledge about intra-patient variability of such lesions is lacking.

Methods

To characterise the spatial transcriptomics of gallbladder cancer tumourigenesis in individual patients, we selected two patients with distinct cancer aetiology and whose samples simultaneously displayed multiple areas of normal epithelium, BilINs and adenocarcinoma. Using GeoMx digital spatial profiling, we characterised the whole transcriptome of a high number of regions of interest (ROIs) per sample in the two patients (24 and 32 ROIs respectively), with each ROI covering approximately 200 cells of normal epithelium, low-grade BilIN, high-grade BilIN or adenocarcinoma. Human gallbladder organoids and cell-ine derived tumours were used to investigate the tumour-promoting role of genes.

Results

Spatial transcriptomics revealed that each type of lesion displayed limited intra-patient transcriptomic variability. Our data further suggest that adenocarcinoma derived from high-grade BilIN in one patient and from low-grade BilIN in the other patient, with co-existing high-grade BilIN evolving via a distinct process in the latter case. The two patients displayed distinct sequences of signalling pathway activation during tumour progression, but Semaphorin 4A (SEMA4A) expression was repressed in both patients. Using human gallbladder-derived organoids and cell line-derived tumours, we provide evidence that repression ofSEMA4Apromotes pseudostratification of the epithelium and enhances cell migration and survival.

Conclusion

Gallbladder adenocarcinoma can develop according to patient-specific processes, and limited intra-patient variability of precursor and cancer lesions was noticed. Our data suggest that repression ofSEMA4Acan promote tumour progression. They also highlight the need to gain gene expression data in addition to histological information to avoid understimating the risk of low-grade preneoplastic lesions.
N-formylation modifies membrane damage associated to PSMα3 interfacial fibrillation
Authors: Bonnecaze Laura , Jumel Katlyn , Vial Anthony , Khemtemourian Lucie , Feuillie Cecile , Molinari Michael , Lecomte Sophie , Mathelie-Guinlet Marion
Published: March 2024

The virulence of Staphylococcus aureus, a multi-drug resistant pathogen, notably depends on the expression of the phenol soluble modulins α3 (PSMα3) peptides, able to self-assemble into amyloid-like cross-α fibrils. Despite remarkable advances evidencing the crucial, yet insufficient, role of fibrils in PSMα3 cytotoxic activities towards host cells, the relationship between its molecular structures, assembly propensities, and modes of action remains an open intriguing problem. In this study, combining Atomic Force Microscopy (AFM) imaging and infrared spectroscopy, we first demonstrated in vitro that the charge provided by the N-terminal capping of PSMα3 alters its interactions with model membranes of controlled lipid composition, without compromising its fibrillation kinetics or morphology. N-formylation eventually dictates PSMα3 - membrane binding via electrostatic interactions with the lipid head groups. Furthermore, PSMα3 insertion within the lipid bilayer is favoured by hydrophobic interactions with the lipid acyl chains, only in the fluid-phase of membranes, and not in the gel-like ordered domains. Strikingly, our real-time AFM imaging emphasizes how intermediate protofibrillar entities, formed along PSMα3 self-assembly and promoted at the membrane interface, likely disrupt membrane integrity via peptide accumulation, and subsequent membrane thinning in a peptide concentration and lipid-dependent manner. Overall, our multiscale and multimodal approach sheds new light on the key roles of N-formylation and intermediate self-assembling entities, rather than mature fibrils, in dictating deleterious interactions of PSMα3 with specific membrane lipids, likely underscoring its ultimate cellular toxicity in vivo, and in turn S. aureus pathogenesis.
Methods for imaging intracellular calcium signals in the mouse mammary epithelium in 2- and 3-dimensions
Authors: Folacci Mathilde , Chalmers Silke B. , Davis Felicity M.
Published: March 2024

The mammary gland has a central role in optimal mammalian development and survival. Contractions of smooth muscle-like basal (or myoepithelial) cells in the functionally mature mammary gland in response to oxytocin are essential for milk ejection and are tightly regulated by intracellular calcium (Ca2+). Using mice expressing a genetically encoded Ca2+indicator (GCaMP6f), we present in this chapter a method to visualise at high spatiotemporal resolution changes in intracellular Ca2+in mammary epithelial cells, bothin vitro(2D) andex vivo(3D). The procedure to optimally prepare mammary tissue and primary cells is presented in detail.
Benchmarking transcriptional deconvolution methods for estimating tissue- and cell type-specific extracellular vesicle abundances
Authors: Larsen Jannik Hjortshøj , Jensen Iben Skov , Svenningsen Per
Published: March 2024

Extracellular vesicles (EVs) contain cell-derived lipids, proteins, and RNAs; however, the challenge to determine the tissue- and cell type-specific EV abundances in body fluids remains a significant hurdle for our understanding of EV biology. While tissue- and cell type-specific EV abundances can be estimated by matching the EV’s transcriptome to a tissue’s/cell type’s expression signature using deconvolutional methods, a comparative assessment of deconvolution methods’ performance on EV transcriptome data is currently lacking. We benchmarked 11 deconvolution methods using data from 4 cell lines and their EVs,in silicomixtures, 118 human plasma, and 88 urine EVs. We identified deconvolution methods that estimated cell type-specific abundances of pure andin silicomixed cell line-derived EV samples with high accuracy. Using data from two urine EV cohorts with different EV isolation procedures, four deconvolution methods produced highly similar results. The four methods were also highly concordant in their tissue-specific plasma EV abundance estimates. We identified driving factors for deconvolution accuracy and highlight the importance of implementing biological knowledge in creating the tissue/cell type signature. Overall, our analyses demonstrate that the deconvolution algorithms DWLS and CIBERSORTx produce highly similar and accurate estimates of tissue- and cell type-specific EV abundances in biological fluids.
The NLRP3 inflammasome is essential for IL-18 production in a murine model of macrophage activation syndrome
Authors: Gleeson Tara A. , Kaiser Christina , Lawrence Catherine B. , Brough David , Allan Stuart M. , Green Jack P.
Published: March 2024

Hyperinflammatory disease is associated with an aberrant immune response resulting in cytokine storm. One such instance of hyperinflammatory disease is known as macrophage activation syndrome (MAS). The pathology of MAS can be characterised by significantly elevated serum levels of interleukin (IL)-18 and interferon (IFN)-γ. Given the role for IL-18 in MAS, we sought to establish the role of inflammasomes in the disease process. Using a murine model of CpG-DNA induced MAS, we discovered that the expression of the NLRP3 inflammasome was increased and correlated with IL-18 production. Inhibition of the NLRP3 inflammasome, or downstream caspase-1, prevented MAS-mediated upregulation of plasma IL-18 but interestingly did not alleviate key features of hyperinflammatory disease including hyperferritinaemia and splenomegaly. Furthermore IL-1 receptor blockade with IL-1Ra did not prevent the development of CpG-induced MAS, despite being clinically effective in the treatment of MAS. These data demonstrate that in the development of MAS, the NLRP3 inflammasome was essential for the elevation in plasma IL-18, a key cytokine in clinical cases of MAS, but was not a driving factor in the pathogenesis of CpG-induced MAS.
Measuring the orientation of segmented Deep Brain Stimulation leads using electroencephalography
Authors: Jacob Alan Bince , Krueger Marie T. , Akram Harith , Aristovich Kirill , Litvak Vladimir
Published: March 2024

Segmented Deep Brain Stimulation (DBS) leads enable current steering in specific directions, but this comes with an increased level of programming complexity. Precise measurement of lead orientation is crucial for facilitating stimulation programming. Presently employed methods involve radiation technology posing inherent risks and limitations. Additionally, the potential rotation of leads post-implantation may require repeated measurements. To address these challenges, we propose an outpatient-friendly, radiation-free method using post-operative imaging-informed electroencephalography (EEG). The method was tested in an EEG phantom yielding maximal errors of under 10 ° with under 30s of data. It works with as few as 4 EEG electrodes with only a small error increase. Measurement variance was of the order of a few degrees, indicating that the method could reach this precision if all the sources of bias are removed. Thus, with optimised hardware, software and measurement protocol, the method is feasible for routine use in a clinical setting.
SIRT2-Mediated ACSS2 K271 Deacetylation Suppresses Lipogenesis Under Nutrient Stress
Authors: Karim Rezwana , Teng Wendi , Behram Cameron , Lin Hening
Published: March 2024

De novo lipogenesis is associated with the development of human diseases such as cancer, diabetes, and obesity. At the core of lipogenesis lies acetyl coenzyme A (CoA), a metabolite that plays a crucial role in fatty acid synthesis. One of the pathways contributing to the production of cytosolic acetyl-CoA is mediated by acetyl-CoA synthetase 2 (ACSS2). Here, we reveal that when cells encounter nutrient stress, particularly a deficiency in amino acids, Sirtuin 2 (SIRT2) catalyzes the deacetylation of ACSS2 at the lysine residue K271. This results in K271 ubiquitination and subsequently proteasomal degradation of ACSS2. Substitution of K271 leads to decreased ubiquitination of ACSS2, increased ACSS2 protein level, and thus increased lipogenesis. Our study uncovers a mechanism that cells employ to efficiently manage lipogenesis during periods of nutrient stress.
Comprehensive analysis of insertion sequences within rRNA genes of CPR bacteria and biochemical characterization of a homing endonuclease encoded by these sequences
Authors: Tsurumaki Megumi , Sato Asako , Saito Motofumi , Kanai Akio
Published: March 2024

The Candidate Phyla Radiation (CPR) represents an extensive bacterial clade comprising primarily uncultured lineages and is distinguished from other bacteria by a significant prevalence of insertion sequences (ISs) within their rRNA genes. However, our understanding of the taxonomic distribution and characteristics of these ISs remains limited. In this study, we used a comprehensive approach to systematically determine the nature of the rRNA ISs in CPR bacteria. The analysis of hundreds of rRNA gene sequences across 65 CPR phyla revealed that ISs are present in 48% of 16S rRNA genes and 82% of 23S rRNA genes, indicating a broad distribution across the CPR clade, with exceptions in the 16S and 23S rRNA genes of Saccharibacteria and the 16S rRNA genes of Peregrinibacteria. Over half the ISs display a group-I-intron-like structure, whereas specific 16S rRNA gene ISs display features reminiscent of group II introns. The ISs frequently encode proteins with homing endonuclease (HE) domains, centered around the LAGLIDADG motif. The LAGLIDADG HE (LHE) proteins encoded by the rRNA ISs of CPR bacteria predominantly have a single-domain structure, deviating from the usual single- or double-domain configuration observed in typical prokaryotic LHEs. Experimental analysis of one LHE protein, I-ShaI fromCa. Shapirobacteria, confirmed that its endonuclease activity targets the DNA sequence of its insertion site, and chemical cross-linking experiments demonstrated its capacity to form homodimers. These results provide robust evidence supporting the hypothesis that the explosive proliferation of rRNA ISs in CPR bacteria was facilitated by mechanisms involving LHEs.

IMPORTANCE

Insertion sequences (ISs) in rRNA genes are relatively limited and infrequent in standard bacteria. With a comprehensive bioinformatic analysis, we show that in CPR bacteria, which are characterized by a high frequency of ISs, these ISs occur in 48% of 16S rRNA genes and 82% of 23S rRNA genes. We also report the systematic and biochemical characterization of the LAGLIDADG homing endonucleases (LHEs) encoded by these ISs in the first such analysis of the CPR bacteria. This study significantly extends our understanding of the phylogenetic positions of rRNA ISs within CPR bacteria and the biochemical features of their LHEs.
An overview of GabRat edge disruption and its new extensions for unbiased quantification of disruptive camouflaging patterns using randomization technique
Authors: Tanahashi Masahiko , Lin Min-Chen , Lin Chung-Ping
Published: March 2024

Disruptive colorations are camouflaging patterns that use contrasting colorations to interrupt the continuously of object’s edge and disturb the observer’s visual recognition. The GabRat method has been introduced and widely used to quantify the strength of edge disruption. The original GabRat method requires a composite image where a target object is placed on a particular background. It computes the intensities of ’frequency components’ parallel and perpendicular to the edge direction at each edge point using Gabor filters, and summarize the ratios of these two intensities around the perimeter of the shape. However, we found that the original GabRat method has an issue which produces false signals and biases to overestimating the GabRat value depending on the edge angle. Here, we introduce GabRat-R, which can diminish that angle dependency using Gabor filters with randomized base angles. Additionally, we developed GabRat-RR, which iteratively places a target object on a background with random positions and rotation angles to average the effects of the heterogeneity and anisotropy of background. Compared with the original GabRat, our GabRat-R and GabRat-RR programs run more efficiently using multithreading techniques. GabRat-R and GabRat-RR were freely available inNatsumushi2.0 software of the authors’ website.
Generative Haplotype Prediction Outperforms Statistical Methods for Small Variant Detection in NGS Data
Authors: O’Fallon Brendan , Bolia Ashini , Durtschi Jacob , Yang Luobin , Fredrickson Eric , Best Hunter
Published: March 2024

Detection of germline variants in next-generation sequencing data is an essential component of modern genomics analysis. Variant detection tools typically rely on statistical algorithms such as de Bruijn graphs or Hidden Markov Models, and are often coupled with heuristic techniques and thresholds to maximize accuracy. Here, we introduce a new approach that replaces these handcrafted statistical techniques with a single deep generative model. The model’s input is the set of reads aligning to a single genomic region, and the model produces two sets of output tokens, each representing the nucleotide sequence of a germline haplotype. Using a standard transformer-based encoder and double-decoder architecture, our model learns to construct germline haplotypes in a generative fashion identical to modern Large Language Models (LLMs). We train our model on 37 Whole Genome Sequences (WGS) from Genome-in-a-Bottle samples, and demonstrate that our method learns to produce accurate haplotypes with correct phase and genotype for all classes of small variants. We compare our method, called Jenever, to FreeBayes, GATK HaplotypeCaller, Clair3 and DeepVariant, and demonstrate that our method has superior overall accuracy compared to other methods. AtF1-maximizing quality thresholds, our model delivers the highest sensitivity, precision, and the fewest genotyping errors for insertion and deletion variants. For single nucleotide variants our model demonstrates the highest sensitivity but at somewhat lower precision, and achieves the highest overallF1 score among all callers we tested.
Enterohemorrhagic Escherichia coli (EHEC) Disrupts Intestinal Barrier Integrity in Translational Canine Stem Cell-Derived Monolayers
Authors: Nagao Itsuma , Kawasaki Minae , Goyama Takashi , Kim Hyun Jung , Call Douglas R. , Ambrosini Yoko M.
Published: March 2024

This study addresses the gap in translatablein vitromodels for investigating EnterohemorrhagicE. coli(EHEC) infections, particularly relevant to both canine and human health. EHEC is known to induce acute colitis in dogs, leading to symptoms like hemorrhagic diarrhea and hemolytic uremic syndrome, similar to those observed in humans. However, understanding the pathophysiology and developing treatment strategies have been challenging due to the lack of effective models that replicate the clinical disease caused by EHEC in both species. Our approach involved the development of colonoid-derived monolayers using intestinal tissues from healthy, client-owned dogs. These monolayers were exposed to EHEC and the impact of EHEC was assessed through several techniques, including trans-epithelial electrical resistance (TEER) measurement, immunofluorescence staining for junction proteins and mucus, and scanning electron microscopy for morphological analysis. Modified culture with saline, which was intended to prevent bacterial overgrowth, maintained barrier integrity and cell differentiation. EHEC infection led to significant decreases in TEER and ZO-1 expression, but not in E- cadherin levels or mucus production. Additionally, EHEC elicited a notable increase in TNF-α production, highlighting its distinct impact on canine intestinal epithelial cells compared to non-pathogenicE. coli. These findings closely replicatein vivoobservations in dogs and humans with EHEC enteropathy, validating the canine colonoid-derived monolayer system as a translational model to study host- pathogen interactions in EHEC and potentially other clinically significant enteric pathogens.
Distinct roles of the major binding residues in the cation-binding pocket of MelB
Authors: Hariharan Parameswaran , Bakhtiiari Amirhossein , Liang Ruibin , Guan Lan
Published: March 2024

Salmonella entericaserovar Typhimurium melibiose permease (MelBSt) is a prototype of the major facilitator superfamily (MFS) transporters, which play important roles in human health and diseases. MelBStcatalyzed the symport of galactosides with either H+, Li+, or Na+, but prefers the coupling with Na+. Previously, we determined the structures of the inward- and outward-facing conformation of MelBSt, as well as the molecular recognition for galactoside and Na+. However, the molecular mechanisms for H+- and Na+-coupled symport still remain poorly understood. We have solved two x-ray crystal structures of MelBStcation-binding site mutants D59C at an unliganded apo-state and D55C at a ligand-bound state, and both structures display the outward-facing conformations virtually identical as published previously. We determined the energetic contributions of three major Na+-binding residues in cation selectivity for Na+and H+by the free energy simulations. The D55C mutant converted MelBStto a solely H+-coupled symporter, and together with the free-energy perturbation calculation, Asp59 is affirmed to be the sole protonation site of MelBSt. Unexpectedly, the H+-coupled melibiose transport with poor activities at higher ΔpH and better activities at reversal ΔpH was observed, supporting that the membrane potential is the primary driving force for the H+-coupled symport mediated by MelBSt. This integrated study of crystal structure, bioenergetics, and free energy simulations, demonstrated the distinct roles of the major binding residues in the cation-binding pocket.
tRNA modifications tune decoding of codon pairs to prevent cellular quality control responses
Authors: Wu Jie , Eggers Cristian , Sin Olga , Koziej Łukasz , Mancilla Hector , Mollet Fabienne , Schöler Hans R. , Drexler Hannes C.A. , Ranff Tristan , Fufezan Christian , Kraft Claudine , Glatt Sebastian , Bruder Jan M. , Leidel Sebastian A.
Published: March 2024

tRNA modifications tune translation rates and codon optimality, thereby optimizing co-translational protein folding, but how codon optimality defects trigger cellular phenotypes remains unclear. Here, we show that ribosomes stall at specific modification-dependent codon pairs, triggering ribosome collisions and inducing a coordinated and hierarchical response of cellular quality control pathways. Ribosome profiling reveals an unexpected functional diversity for wobble-uridine (U34) modifications during decoding. The same modification can have different effects at the A and P sites. Furthermore, modification-dependent stalling codon pairs induce ribosome collisions, triggering ribosome-associated quality control (RQC) to prevent protein aggregation by degrading aberrant nascent peptides and mRNAs. RQC inactivation stimulates the expression of molecular chaperones to remove protein aggregates. Our results show that loss of tRNA modifications primarily disrupts translation rates of suboptimal codon pairs and reveal the coordinated regulation and adaptability of cellular surveillance systems to ensure efficient and accurate protein synthesis and maintain protein homeostasis.