bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative pathogenesis of three Mayaro virus genotypes in the cynomolgus macaque
Authors: Hamilton Melissa M. , Webb Emily M. , Peterson Max C. , Patel Grishma , Porto Maciel , Orekov Tatyana , Erasmus Jesse H. , Finneyfrock Brad , Cook Anthony , Auguste Albert J. , Kar Swagata
Published: July 2024

Mayaro virus (MAYV), a mosquito-borne alphavirus, is considered an emerging threat to public health with epidemic potential. Phylogenetic studies show the existence of three MAYV genotypes. In this study, we provide a preliminary analysis of the pathogenesis of all three MAYV genotypes in cynomolgus macaques (Macaca facicularis, Mauritian origin). Significant MAYV-specific RNAemia and viremia were detected during acute infection in animals challenged intravenously with the three MAYV genotypes, and strong neutralizing antibody responses were observed. MAYV RNA was detected at high levels in lymphoid tissues, joint muscle and synovia over 1 month after infection, suggesting that this model could serve as a promising tool in studying MAYV-induced chronic arthralgia, which can persist for years. Significant leucopenia was observed across all MAYV genotypes, peaking with RNAemia. Notable differences in the severity of acute RNAemia and composition of cytokine responses were observed among the three MAYV genotypes. Our model showed no outward signs of clinical disease, but several major endpoints for future MAYV pathology and intervention studies are described. Disruptions to normal blood cell counts and cytokine responses were markedly distinct from those observed in macaque models of CHIKV infection, underlining the importance of developing non-human primate models specific to MAYV infection.
SARS-CoV-2 infects neurons, astrocytes, choroid plexus epithelial cells and pericytes of the human central nervous system in vitro
Authors: Haverty Ruth , McCormack Janet , Evans Christopher , Purves Kevin , O'Reilly Sophie , Gautier Virginie , Rochfort Keith , Fabre Aurelie , Fletcher Nicola F.
Published: July 2024

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is associated with neurological sequelae including haemorrhage, thrombosis and ischaemic necrosis and encephalitis. However, the mechanism by which this occurs is unclear. Neurological disease associated with COVID-19 has been proposed to occur following direct infection of the central nervous system and/or indirectly by local or systemic immune activation. We evaluated the expression of angiotensin-converting enzyme-2 and transmembrane protease, serine 2 (TMPRSS2) in brain tissue from five healthy human donors and observed low-level expression of these proteins in cells morphologically consistent with astrocytes, neurons and choroidal ependymal cells within the frontal cortex and medulla oblongata. Primary human astrocytes, neurons, choroid plexus epithelial cells and pericytes supported productive SARS-CoV-2 infection with ancestral, Alpha, Delta and Omicron variants. Infected cells supported the full viral life cycle, releasing infectious virus particles. In contrast, primary brain microvascular endothelial cells and microglia were refractory to SARS-CoV-2 infection. These data support a model whereby SARS-CoV-2 can infect human brain cells, and the mechanism of viral entry warrants further investigation.
Transient reemergence of Bordetella parapertussis in France in 2022
Authors: Bouchez Valérie , Toubiana Julie , Guillot Sophie , El Belghiti Fatima Aït , Landier Annie , Armatys Nathalie , Trombert-Paolantoni Sabine , Soares Anaïs , Rodrigues Carla , Brisse Sylvain ,   Parapertussis 2022 Study Group†
Published: July 2024

Between March and October 2022, a peak of detection of Bordetella parapertussis by qPCR, real-time PCR was observed in France.

Hypothesis/Gap Statement. Whether this peak was due to resurgence from previous circulating lineages or reintroduction into the country was unknown.

Objective. The objective of this study is to understand B. parapertussis-transient increase observed in France in 2022 whereas it had virtually stopped being reported since the start of the COVID-19 pandemic in 2020.

Methods. We analysed real-time PCR (qPCR) data from the two largest French outpatient laboratories performing whooping cough diagnosis and characterized all B. parapertussis isolates collected in the 2016–2022 period by the French National Reference Centre for Whooping Cough.

Results. Microbiological analyses reveal that 13 of 18 bacterial isolates collected in 2022 produce the vaccine antigen pertactin, whereas none of the 22 isolates collected in the 2016–2021 period did.

Conclusion. We hypothesize a re-introduction of B. parapertussis from regions of the world where whole-cell vaccines are still in use.
Structural Mapping of Polyclonal IgG Responses to HA After Influenza Virus Vaccination or Infection
Authors: León André Nicolás , Rodriguez Alesandra J. , Richey Sara T. , Torrents de la Peña Alba , Wolters Rachael M. , Jackson Abigail M. , Webb Katherine , Creech C. Buddy , Yoder Sandra , Mudd Philip A. , Crowe James E. , Han Julianna , Ward Andrew B.
Published: July 2024

Cellular and molecular characterization of immune responses elicited by influenza virus infection and seasonal vaccination have informed efforts to improve vaccine efficacy, breadth, and longevity. Here, we use negative stain electron microscopy polyclonal epitope mapping (nsEMPEM) to structurally characterize the humoral IgG antibody responses to hemagglutinin (HA) from human patients vaccinated with a seasonal quadrivalent flu vaccine or infected with influenza A viruses. Our data show that both vaccinated and infected patients had humoral IgGs targeting highly conserved regions on both H1 and H3 subtype HAs, including the stem and anchor, which are targets for universal influenza vaccine design. Responses against H1 predominantly targeted the central stem epitope in infected patients and vaccinated donors, whereas head epitopes were more prominently targeted on H3. Responses against H3 were less abundant, but a greater diversity of H3 epitopes were targeted relative to H1. While our analysis is limited by sample size, on average, vaccinated donors responded to a greater diversity of epitopes on both H1 and H3 than infected patients. These data establish a baseline for assessing polyclonal antibody responses in vaccination and infection, providing context for future vaccine trials and emphasizing the importance of carefully designing vaccines to boost protective responses towards conserved epitopes.
Selective Activation of GPCRs: Molecular Dynamics Shows Siponimod Binds but Fails to Activate S1PR2 Unlike S1PR1
Authors: Soniya Kumari , Nanduru Chanukya , Halder Antarip
Published: July 2024

Understanding drug selectivity is crucial for designing effective medications that target specific proteins while minimizing off-target interactions, thus optimizing therapeutic outcomes. Biochemical and cellular assays provide functional insights but often lack detailed molecular understanding of selectivity in large families of similar proteins. In such cases, computational studies can complement experimental approaches to fill gaps in understanding drug selectivity. In this study, Molecular Dynamics (MD) simulation methods have been used to examine the selective modulation of sphingosine-1-phosphate receptor 1 (S1PR1) over S1PR2 by Siponimod, an FDA-approved drug for Multiple Sclerosis. Both S1PR1 and S1PR2 belong to the same sub-family of G Protein-Coupled Receptors (GPCRs). Contrary to the previous hypothesis based on molecular docking, MD simulations showed that Siponimod can bind to S1PR2 with anin silico-determined affinity comparable to that of S1PR1. Comparing the dynamics of Siponimod-bound S1PR2 with S1P-bound S1PR2 revealed that the transmission switches necessary for downstream biological activity are activated by S1P but not by Siponimod, whereas both S1P and Siponimod activate those transmission switches in S1PR1. A few crucial residues in S1PR2 were also identified that can be leveraged to optimize molecules to bind selectively to S1PR1 over S1PR2. Through our study, we showed thatin silicoapproaches can help in understanding the ligand-induced structural changes and aid in developing and optimizing drugs for GPCRs, the most abundant class of membrane proteins in the human genome and the largest family of membrane receptors targeted by approved drugs.
Functional properties of a disease mutation for migraine in Kv2.1/6.4 channels
Authors: Tewari Debanjan , Sattler Christian , Benndorf Klaus
Published: July 2024

Voltage-gated potassium (Kv) channels are integral to cellular excitability, impacting the resting membrane potential, repolarization, and shaping action potentials in neurons and cardiac myocytes. Structurally, Kv channels are homo or heterotetramers comprising four α-subunits, each with six transmembrane segments (S1-S6). Silent Kv (KvS), includes Kv5.1, Kv6.1-6.4, Kv8.1-8.2, and Kv9.1-9.3, they do not form functional channels on their own but modulate the properties of heteromeric channels. Recent studies have identified the Kv6.4 subunit as a significant modulator within heteromeric channels, such as Kv2.16.4. The Kv2.16.4 heteromer exhibits altered biophysical properties, including a shift in voltage-dependent inactivation and a complex activation. Current genetic studies in migraine patients have revealed a single missense mutation in the Kv6.4 gene. The single missense mutation, L360P is in the highly conserved S4-S5 linker region. This study aims to demonstrate the biophysical effects of the L360P mutation in Kv2.1 6.4 channels with a fixed 2:2 stoichiometry, using monomeric (Kv2.1/6.4) and tandem dimer (Kv2.1_6.4) configurations. Our findings suggest that the L360P mutation significantly impacts the function and regulation of Kv2.1/6.4 channels, providing insights into the molecular mechanisms underlying channel dysfunction in migraine pathology.

Statement of significance

This study elucidates the biophysical properties of the Kv6.4 L360P mutation, providing insights into its role in Kv2.1 6.4 channel function. Given the high conservation of the leucine residue in the Kv channel family and its association with migraine, our findings have significant implications for understanding the molecular basis of migraine pathophysiology. By analyzing channels with a fixed 2:2 stoichiometry, we highlight the impact of the L360P mutation on channel gating and inactivation. This research advances the knowledge of the silent Kv6.4 channel mechanism and its role in pathophysiology.
Vcam1+ Fibro-adipogenic Progenitors Mark Fatty Infiltration in Chronic Limb Threatening Ischemia
Authors: Dai Qunsheng , Wan Changxin , Xu Yueyuan , Fei Kaileen , Olivere Lindsey A. , Garrett Brianna , Akers Leo , Peters Derek , Otto James , Kontos Christopher D. , Ji Zhiceng , Diao Yarui , Southerland Kevin W.
Published: July 2024

Skeletal muscle health and function is a critical determinant of clinical outcomes in patients with peripheral arterial disease (PAD). Herein, we identify fatty infiltration, the ectopic deposition of adipocytes in skeletal muscle, as a histological hallmark of end-stage PAD, also known as chronic limb threatening ischemia (CLTI). Leveraging single cell transcriptome mapping in mouse models of PAD, we identify a pro-adipogenic mesenchymal stromal cell population marked by expression of Vcam1 (termed Vcam1+ FAPs) that expands in the ischemic limb. Mechanistically, we identify Sfrp1 and Nr3c1 as regulators of Vcam1+ FAP adipogenic differentiation. Loss of Sfrp1 and Nr3c1 impair Vcam1+ FAP differentiation into adipocytesin vitro. Finally, we show that Vcam1+ FAPs are enriched in human CLTI patients. Collectively, our results identify a pro-adipogenic FAP subpopulation in CLTI patients and provide a potential therapeutic target for muscle regeneration in PAD.
A look beyond topography: transient phenomena ofEscherichia colicell division captured with high-speed in-line force mapping
Authors: Ganser Christian , Nishiguchi Shigetaka , Chan Feng-Yueh , Uchihashi Takayuki
Published: July 2024

Life on the nanoscale has been made accessible in recent decades by the development of techniques that are fast and non-invasive. High-speed atomic force microscopy (HS-AFM) is one such technique that has proven to shed light on elusive mechanisms involving single proteins. Extending HS-AFM to effortlessly incorporate mechanical property mapping while maintaining fast imaging speed allows us to look deeper than topography and reveal more details of the nanoscale mechanisms that govern life. Here, we present high-speed in-line force mapping (HS-iFM), which enables the recording of mechanical properties and topography maps with high spatiotemporal resolution. Employing this method, a detailed study of the dynamic nanoscale mechanical properties of livingEscherichia colibacteria reveals localized stiffening during division, intricate details of the division process, formation and diffusion of pores in the membrane, and the impact of depressurization of a cell. All of these phenomena were recorded with a frame time as low as 15 s and a spatial resolution of 5.5 nm/pixel in topography and 22 nm/pixel in force maps, allowing the capture of transient phenomena on bacterial surfaces in striking detail.
Faster model-based estimation of ancestry proportions
Authors: Santander Cindy G. , Refoyo Martinez Alba , Meisner Jonas
Published: July 2024

Ancestry estimation from genotype data in unrelated individuals has become an essential tool in population and medical genetics to understand demographic population histories and to model or correct for population structure. TheADMIXTUREsoftware is a widely used model-based approach to account for population stratification, however, it struggles with convergence issues and does not scale to modern human datasets or the large number of variants in whole-genome sequencing data. Likelihood-free approaches optimize a least square objective and have gained popularity in recent years due to their scalability. However, this comes at the cost of accuracy in the ancestry estimates in more complex admixture scenarios. We present a new model-based approach,fastmixture, which adopts aspects from likelihood-free approaches for parameter initialization, followed by a mini-batch expectation-maximization procedure to model the standard likelihood. In a simulation study, we demonstrate that the model-based approaches offastmixtureandADMIXTUREare significantly more accurate than recent and likelihood-free approaches. We further show thatfastmixtureruns approximately 30×faster thanADMIXTUREon both simulated and empirical data from the 1000 Genomes Project such that our model-based approach scales to much larger sample sizes than previously possible. Our software is freely available athttps://github.com/Rosemeis/fastmixture.
Mineral Phase Changes During Intervertebral Disc Degeneration
Authors: Yenen Theresa Banu , Jugdaohsingh Ravin , Thom William D. , Khan Sam , Solano Viviana Rojas , Lampronti Giulio , Brown Andy , Saez Janire , Corbetta Davide , Eminağa Salih , Cherubini Giunio Bruto , Powell Jonathan , Hughes Kate , Freeman Paul
Published: July 2024

Intervertebral disc disease is a common cause of pain and neurological deficits and is known to be associated with degeneration and calcification. Here we analysed samples of herniated disc material and compared it to material taken from non-herniated discs following surgical treatment in dogs. Our clinical approach to these cases allows collection of samples providing a unique opportunity for a case-controlled study such as this, an opportunity which is not available to the human neurosurgeon. We analysed all samples using Fourier transform infrared (FTIR) spectroscopy, as well as a proportion with X-ray diffraction (XRD) and transmission electron microscopy (TEM). FTIR spectra of the majority of herniated samples were consistent with the presence of crystalline hydroxyapatite, whereas most of the non-herniated discs showed spectra consistent with amorphous phosphate material. XRD analysis and TEM confirmed these findings and identified the amorphous material as amorphous calcium phosphate nanoparticle clusters of ∼ 20 nm diameter and the crystalline hydroxyapatite material as needles up to 100 nm in length.

The differences between the herniated and non-herniated discs indicate that the degenerative process involves a conversion of amorphous calcium phosphate into crystalline hydroxyapatite which precedes and may predispose the disc to herniate.
Slow diffusion and signal amplification on membranes regulated by phospholipase D
Authors: Honda Gen , Tanaka Chihuku , Sawai Satoshi , Yanagisawa Miho
Published: July 2024

Control of molecular diffusion is pivotal for highly fluidic membranes to serve as substrates for biochemical reactions and the self-assembly of molecular machinery driving membrane protrusions. Lateral diffusion in membranes depends on lipid composition, which is highly diverse and homeostatically controlled in living cells. Due to the complexity of the underlying processes, its impact on molecular diffusion remain largely unclear. In this study, we show that lipid diffusion in model membranes is markedly decreased in cytosolic extracts. The reduction in lipid diffusivity could be pharmacologically inhibited by targeting phospholipase D (PLD), and addition of PLD to membranes mimicked the reduction in diffusion. Phosphatidic acid, a direct product of PLD, diffused slowly in model membranes and reduced the diffusivity of surrounding lipids. Furthermore, we demonstrated that PLD specifically controls the lateral diffusion of a myristoylated protein in cells, possibly due to auxiliary electrostatic interactions between cationic residues located near the lipidated tail and anionic phospholipids. PLD controlled the size and lifetime of localized patches of phosphatidylinositol (3,4,5) triphosphates that specify regions of membrane protrusions. Overall, the results of this study suggest that PLD controls the lateral diffusion of certain membrane proteins, which play key roles in phosphoinositide signaling.

Significance Statement

In living cells, many biochemical reactions occur in confined regions on the membranes, facilitating the local occurrence of specific events, such as membrane protrusion. This is puzzling from a physical perspective because the membrane is a two-dimensional fluidic structure that should allow molecules to spread freely. Herein, we found that the fluidity of artificial membranes was markedly reduced by adding extracts from the cell cytoplasm. A lipid-modifying protein phospholipase D (PLD) was found to be responsible for this and it regulates the diffusion of membrane proteins in cells. This study suggests the novel role of PLD as a regulator of molecular diffusion and its impact on phosphoinositide production that serves as an important signal for cell deformation.
Live-cell single-molecule dynamics of eukaryotic RNA polymerase machineries
Authors: Ling Yick Hin , Liang Chloe , Wang Sixiang , Wu Carl
Published: July 2024

Eukaryotic gene expression in the nucleus is orchestrated by three RNA polymerases (RNAP-I, -II, and -III) and associated factors1,2. Despite extensive biochemical, genomic, structural, and imaging studies, the real-time dynamics of these transcription complexes remain obscure. Here, we employ single-molecule tracking in living yeast to assess the physiological kinetics of over 50 representative proteins encompassing all three RNAP machineries. Components of RNAPI and RNAPIII pre-initiation complexes (PICs) engage in long-lived interactions on chromatin, reflecting their roles for constitutive rRNA and tRNA synthesis, in contrast to the transient RNAPII PIC. We further report the dynamics of key components across the RNAPII transcription cycle2–5—factors for upstream regulation, elongation, histone modification, RNAPII C-terminal domain (CTD) modification, RNA processing, and termination—revealing unprecedented insights into the temporal landscape of RNAPII transcription. Strikingly, many elongation factors, previously thought to travel processively with RNAPII, display transient residence times, suggesting highly dynamic interactions rather than constant association. Systematic screening of RNAPII-associated factors shows that truncation of RNAPII-CTD substantially reduces U1 snRNP residence time and decreases intron retention in ribosomal protein genes, providing insights into how CTD length influences co-transcriptional splicing. Our findings establish a framework for dynamic chromatin interactions of RNA polymerase machineries in living cells.
Human Gastric Multi-Regional Assembloids Favour Functional Parietal Maturation and Allow Modelling of Antral Foveolar Hyperplasia
Authors: Jones Brendan C , Benedetti Giada , Calà Giuseppe , Tullie Lucinda , Simcock Ian C , Lutman Roberto , Balys Monika , Amiri Ramin , Sufi Jahangir , Arthurs Owen , Eaton Simon , Anderson Glenn , Elvassore Nicola , Li Vivian SW , Jones Kelsey DJ , Tape Christopher J. , Luni Camilla , Giobbe Giovanni Giuseppe , De Coppi Paolo
Published: July 2024

Patient-derived human organoids have the remarkable capacity to self-organise into more complex structures. However, to what extent gastric organoids can recapitulate human stomach physiological functions remain unexplored. Here, we report how region-specific gastric organoids can self-assemble into complex multi-regional assembloids showing functional response to drugs targeting the ATPase H+/K+ pump. The assembloids show preserved fundus, body, and antrum regional identity, and gastric-specific crosstalk pathways arise. The increased complexity and cross-communication between the different gastric regions, allow for the emergence of the elusive parietal cell type, responsible for the production of gastric acid, with functional response to drugs targeting the ATPase H+/K+ pump. Remarkably, we generated assembloids from PMM2-HIPKD-IBD paediatric patients (Phosphomannomutase 2 – Hyperinsulinemic hypoglycaemia and autosomal recessive polycystic kidney disease - Inflammatory bowel disease), a genetic condition found to be associated with unusual antral foveolar hyperplasia and hyperplastic polyposis. The cellular mechanisms behind such phenomena are poorly understood, and an exhaustive experimental model is needed. The ΔPMM2 multi-regional assembloid we have generated efficiently recapitulates hyperplastic-like antral regions, with decreased mucin secretion and glycosylated ATP4b, which results in impaired gastric acid secretion. Multi-regional gastric assembloids, generated using adult-stem cell-derived organoids, successfully recapitulate the structural and functional characteristics of the human stomach, offering a promising tool for studying gastric epithelial interactions and disease mechanisms previously challenging to investigate in primary models.
Priming lymphocyte responsiveness and differential T cell signaling in pediatric IBD patients withCannabisuse
Authors: Sanctuary Megan R. , Hudacheck Cinthia L. , Jones Ashleigh J. , Murphy Brittany V. , Welsh Nichole , Klawitter Jost , Hoffenberg Edward J. , Collins Colm B.
Published: July 2024

The prevalence of inflammatory bowel disease (IBD) has increased dramatically in recent years, particularly in pediatric populations. Successful remission with current therapies is limited and often transient, leading patients to seek alternative therapies for symptom relief, including the use of medical marijuana (Cannabis sativa). However, chronic cannabis use among IBD patients is associated with increased risk for surgical interventions. Therefore, determining the direct impact of cannabis use on immune modulation in IBD patients is of critical importance. Peripheral blood mononuclear cells of cannabis using and non-using pediatric IBD patients were phenotyped by flow cytometry and functionally assessed for their cytokine production profile. A phospho-kinase array was also performed to better understand changes in immune responses. Results were then compared with serum phytocannabinoid profiles of each patient to identify cannabinoid-correlated changes in immune responses.Results demonstrated elevated levels of a myriad of pro-inflammatory cytokines in users versus non-users. Differences in signaling cascades of activated T cells between users and non-users were also observed. A number of anti-inflammatory cytokines were inversely correlated with serum phytocannabinoids. These results suggest that cannabis exposure, which can desensitize cannabinoid receptors, may prime pro-inflammatory pathways in pediatric IBD patients.

Article Summary

This observational study examines the impact of chronic cannabis use on peripheral immune cell function in adolescent IBD patients from Children’s Hospital Colorado. Cannabis users displayed altered T cell phenotype, increased pro-inflammatory cytokine release and dephosphorylation of protective protein kinases.
Single cell profiling to determine influence of wheeze and early-life viral infection on developmental programming of airway epithelium
Authors: Berdnikovs Sergejs , Newcomb Dawn C , McKernan Kaitlin E , Kuehnle Shelby N , Haruna Nana-Fatima , Gebretsadik Tebeb , McKennan Christopher , Ma Siyuan , Cephus Jacqueline-Yvonne , Rosas-Salazar Christian , Anderson Larry J , Gern James E , Hartert Tina
Published: July 2024

Abstract

Although childhood asthma is in part an airway epithelial disorder, the development of the airway epithelium in asthma is not understood. We sought to characterize airway epithelial developmental phenotypes in those with and without recurrent wheeze and the impact of infant infection with respiratory syncytial virus (RSV). Nasal airway epithelial cells (NAECs) were collected at age 2-3 years from ana prioridesigned nested birth cohort of children from four mutually exclusive groups of wheezers/non-wheezers and RSV-infected/uninfected in the first year of life. NAECs were cultured in air-liquid interface differentiation conditions followed by a combined analysis of single cell RNA sequencing (scRNA-seq) andin vitroinfection with respiratory syncytial virus (RSV). NAECs from children with a wheeze phenotype were characterized by abnormal differentiation and basal cell activation of developmental pathways, plasticity in precursor differentiation and a delayed onset of maturation. NAECs from children with wheeze also had increased diversity of currently known RSV receptors and blunted anti-viral immune responses toin vitroinfection. The most dramatic changes in differentiation of cultured epithelium were observed in NAECs derived from children that had both wheeze and RSV in the first year of life. Together this suggests that airway epithelium in children with wheeze is developmentally reprogrammed and characterized by increased barrier permeability, decreased antiviral response, and increased RSV receptors, which may predispose to and amplify the effects of RSV infection in infancy and susceptibility to other asthma risk factors that interact with the airway mucosa.

Nasal airway epithelial cells from children with wheeze are characterized by altered development and increased susceptibility to RSV infection.
VTA μ–opioidergic neurons facilitate low sociability in protracted opioid withdrawal
Authors: Jo Adrienne Y. , Xie Yihan , Rodrigues Amrith , Sandoval Ortega Raquel Adaia , Creasy Kate Townsend , Beier Kevin T. , Blendy Julie A. , Corder Gregory
Published: July 2024

Opioids initiate dynamic maladaptation in brain reward and affect circuits that occur throughout chronic exposure and withdrawal that persist beyond cessation. Protracted withdrawal is characterized by negative affective behaviors such as heightened anxiety, irritability, dysphoria, and anhedonia, which pose a significant risk factor for relapse. While the ventral tegmental area (VTA) and mu-opioid receptors (MORs) are critical for opioid reinforcement, the specific contributions of VTAMORneurons in mediating protracted withdrawal-induced negative affect is not fully understood. In our study, we elucidate the role of VTAMORneurons in mediating negative affect and altered brain-wide neuronal activities following opioid exposure and withdrawal in male and female mice. Utilizing a chronic oral morphine administration model, we observe increased social deficit, anxiety-related, and despair-like behaviors during protracted withdrawal. VTAMORneurons show heightened neuronal FOS activation at the onset of withdrawal and connect to an array of brain regions that mediate reward and affective processes. Viral re-expression of MORs selectively within the VTA of MOR knockout mice demonstrates that the disrupted social interaction observed during protracted withdrawal is facilitated by this neural population, without affecting other protracted withdrawal behaviors. Lastly, VTAMORscontribute to heightened neuronal FOS activation in the anterior cingulate cortex (ACC) in response to an acute morphine challenge, suggesting their unique role in modulating ACC-specific neuronal activity. These findings identify VTAMORneurons as critical modulators of low sociability during protracted withdrawal and highlight their potential as a mechanistic target to alleviate negative affective behaviors associated with opioid withdrawal.

SIGNFICANCE

The compelling urge for relief from negative affective states during long-term opioid withdrawal presents a crucial challenge for maintaining abstinence. The ventral tegmental area (VTA) and its mu-opioid receptor-expressing (VTAMOR) neurons represent a critical target of opioidergic action that underlie dependence and withdrawal. Chronic activation of VTAMORneurons during opioid exposure induces maladaptations within these neurons and their structurally connected circuitries, which alter reward processing and contribute to negative affect. Using an oral morphine drinking paradigm to induce dependence, we demonstrate that withdrawal engages VTAMORneurons and identify this neuronal population as key mediators of opioid withdrawal-induced social deficits. These findings hold promise to inform development of targeted therapies aimed at alleviating negative affective states associated with protracted opioid withdrawal.
Evolutionary dynamics of genome structure and content among closely related bacteria
Authors: Molari Marco , Shaw Liam P. , Neher Richard A.
Published: July 2024

Bacterial genomes primarily diversify via gain, loss, and rearrangement of genetic material in their flexible accessory genome. Yet the dynamics of accessory genome evolution are very poorly understood, in contrast to the core genome where diversification is readily described by mutations and homologous recombination. Here, we tackle this problem for the case of very closely related genomes. We comprehensively describe genome evolution within n=222 genomes ofE. coliST131, which likely shared a common ancestor around one hundred years ago. After removing putative recombinant diversity, the total length of the phylogeny is 6000 core genome mutations. Within this diversity, we find 22 modifications to core genome synteny and estimate around 2000 structural changes within the accessory genome, i.e. one structural change for every 3 core genome mutations. 63% of loci with structural diversity could be resolved into individual gain and loss events with ten-fold more gains than losses, demonstrating a dominance of gains due to insertion sequences and prophage integration. Our results suggest the majority of synteny changes and insertions in bacterial genomes are likely deleterious and only persist for a short time before being removed by purifying selection.
The nuclear periphery confers repression on H3K9me2-marked genes and transposons to shape cell fate
Authors: Marin Harold , Simental Eric , Allen Charlie , Martin Eric , Panning Barbara , Al-Sady Bassem , Buchwalter Abigail
Published: July 2024

Heterochromatic loci marked by histone H3 lysine 9 dimethylation (H3K9me2) are enriched at the nuclear periphery in metazoans, but the effect of spatial position on heterochromatin function has not been defined. Here, we remove three nuclear lamins and lamin B receptor (LBR) in mouse embryonic stem cells (mESCs) and show that heterochromatin detaches from the nuclear periphery. Mutant mESCs sustain naïve pluripotency and maintain H3K9me2 across the genome but cannot repress H3K9me2-marked genes or transposons. Further, mutant cells fail to differentiate into epiblast-like cells (EpiLCs), a transition that requires the expansion of H3K9me2 across the genome. Mutant EpiLCs can silence naïve pluripotency genes and activate epiblast-stage genes. However, H3K9me2 cannot repress markers of alternative fates, including primitive endoderm. We conclude that the nuclear periphery controls the spatial position, dynamic remodeling, and repressive capacity of H3K9me2-marked heterochromatin to shape cell fate decisions.
Mitochondrial Fatty Acid Synthesis and Mecr Regulate CD4+ T Cell Function and Oxidative Metabolism
Authors: Steiner KayLee K , Young Arissa C , Patterson Andrew R. , Jennings Erin Q , Chi Channing , Hatem Zaid , Heintzman Darren , Sugiura Ayaka , Arner Emily N , Sewell Allison E , Madden Matthew Z , Okparaugo Richmond , Fallman Emilia V , Gibson-Corley Katherine , Voss Kelsey , Mogilenko Denis , Rathmell Jeffrey
Published: July 2024

Lipid metabolism is fundamental to CD4+ T cell metabolism yet remains poorly understood across subsets. Therefore, we performed targeted in vivo CRISPR/Cas9 screens to identify lipid-associated genes essential for T cell subset functions. These screens established mitochondrial fatty acid synthesis (mtFAS) genes Mecr, Mcat and Oxsm as highly impactful. Of these, the inborn error of metabolism gene Mecr was most dynamically regulated. Effector and memory T cells were reduced in Mecrfl/fl; Cd4cre mice, and MECR was required for activated CD4+ T cells to efficiently proliferate, differentiate, and survive. Mecr-deficient T cells also had decreased mitochondrial respiration, reduced TCA intermediates, and accumulated intracellular iron, which contributed to cell death and sensitivity to ferroptosis. Importantly, Mecr-deficient T cells exhibited fitness disadvantages in inflammatory, tumor, and infection models. mtFAS and MECR thus play important roles in activated T cells and may provide targets to modulate immune functions in inflammatory diseases. The immunological state of MECR- and mtFAS-deficient patients may also be compromised.
Optically Mapped Black Genomes: Distinct Structures and 22q11.2 Deletion Syndrome Mechanisms
Authors: Pastor Steven , Tran Oanh , Lapointe Ryan , Olali Arnold Z. , Wallace Douglas C. , Morrow Bernice , Zackai Elaine H. , McDonald-McGinn Donna M. , Emanuel Beverly S.
Published: July 2024

The genomic architecture of 22q11.2 Deletion Syndrome (22q11.2DS) has focused on analysis of white genomes. However, Black individuals appear to have a lower prevalence of 22q11.2DS compared to whites. To improve the understanding of different populations in relation to 22q11.2DS, optical mapping data from 106 genomes across various Black and white genomes were used to determine the organization of 22q11.2 genomic structures. This revealed extensive variability between the groups regarding copy number and orientation changes of the elements comprising the 22q11.2 low copy repeats (LCR22s). Several novel CNVs and whole haplotype configurations, private and of different prevalence to each group were detected. The diversity of CNVs within Black genomes compared to white genomes was especially striking. To determine the impact of this variability, Black families withde novo22q11.2DS probands were compared to white families. The highly variable configurations of Black and white haplotypes led to several unique non-allelic homologous recombination (NAHR) scenarios with recombinations at different loci. In particular, Black families had unique recombinations yet to be observed. Thus, the unique and highly variable haplotype configurations of LCR22s in Black individuals may play a role in their decreased incidence of 22q11.2DS.
Behavioral and anatomical effects of complex rearing after day 7 neonatal frontal lesions vary with sex
Authors: Gibb Robbin , Kolb Bryan
Published: July 2024

Rats received medial frontal lesions, or sham surgery, on postnatal day 7 (P7), and were placed in complex environments or standard lab housing at weaning. Three months later the animals were tested in a spatial learning task and in a skilled reaching task. Rats with P7 lesions had smaller deficits in spatial learning than similar adult operates, and males performed better than females. Complex housing further improved the performance of male, but not female, lesion rats. The opposite was seen in motor performance as female lesion rats performed better than males and benefited from experience whereas males did not. There was a correlation between behavior and dendritic change as complex housing reversed lesion-induced dendritic atrophy, an effect that was greater in males. P7 frontal males, but not females, showed an increase in spine density that was reversed by complex housing. Experience thus can affect functional and anatomical outcome after early brain injury, but the effects vary with sex.
Acute rapamycin treatment reveals novel mechanisms of behavioral, physiological, and functional dysfunction in a maternal inflammation mouse model of autism and sensory over-responsivity
Authors: Le Belle JE , Condro M , Cepeda C , Oikonomou KD , Tessema K , Dudley L , Schoenfield J , Kawaguchi R , Geschwind D , Silva AJ , Zhang Z , Shokat K , Harris NG , Kornblum HI
Published: July 2024

Maternal inflammatory response (MIR) during early gestation in mice induces a cascade of physiological and behavioral changes that have been associated with autism spectrum disorder (ASD). In a prior study and the current one, we find that mild MIR results in chronic systemic and neuro-inflammation, mTOR pathway activation, mild brain overgrowth followed by regionally specific volumetric changes, sensory processing dysregulation, and social and repetitive behavior abnormalities. Prior studies of rapamycin treatment in autism models have focused on chronic treatments that might be expected to alter or prevent physical brain changes. Here, we have focused on the acute effects of rapamycin to uncover novel mechanisms of dysfunction and related to mTOR pathway signaling. We find that within 2 hours, rapamycin treatment could rapidly rescue neuronal hyper-excitability, seizure susceptibility, functional network connectivity and brain community structure, and repetitive behaviors and sensory over-responsivity in adult offspring with persistent brain overgrowth. These CNS-mediated effects are also associated with alteration of the expression of several ASD-,ion channel-, and epilepsy-associated genes, in the same time frame. Our findings suggest that mTOR dysregulation in MIR offspring is a key contributor to various levels of brain dysfunction, including neuronal excitability, altered gene expression in multiple cell types, sensory functional network connectivity, and modulation of information flow. However, we demonstrate that the adult MIR brain is also amenable to rapid normalization of these functional changes which results in the rescue of both core and comorbid ASD behaviors in adult animals without requiring long-term physical alterations to the brain. Thus, restoring excitatory/inhibitory imbalance and sensory functional network modularity may be important targets for therapeutically addressing both primary sensory and social behavior phenotypes, and compensatory repetitive behavior phenotypes.
Endogenous FGFs drive ERK-dependent cell fate patterning in 2D human gastruloids
Authors: Jo Kyoung , Liu Zong-Yuan , Patel Gauri , Yu Zhiyuan , Yao LiAng , Teague Seth , Johnson Craig , Spence Jason , Heemskerk Idse
Published: July 2024

The role of FGF is the least understood of the morphogens driving mammalian gastrulation. Here we investigated the function of FGF in a stem cell model for human gastrulation known as a 2D gastruloid. We found a ring of FGF-dependent ERK activity that closely follows the emergence of primitive streak (PS)-like cells but expands further inward. We showed that this ERK activity pattern is required for PS-like differentiation and that loss of PS-like cells upon FGF receptor inhibition can be rescued by directly activating ERK. We further demonstrated that the ERK-ring depends on localized activation of basally localized FGF receptors (FGFR) by endogenous FGF gradients. We confirm and extend previous studies in analyzing expression of FGF pathway components, showing the main receptor to be FGFR1 and the key ligands FGF2/4/17, similar to the human and monkey embryo but different from the mouse. In situ hybridization and scRNA-seq revealed thatFGF4andFGF17expression colocalize with the PS marker TBXT but onlyFGF17is maintained in nascent mesoderm and endoderm. FGF4 and FGF17 reduction both reduced ERK activity and differentiation to PS-like cells and their derivatives, indicating overlapping function. Thus, we have identified a previously unknown role for FGF-dependent ERK signaling in 2D gastruloids and possibly the human embryo, driven by a mechanism where FGF4 and FGF17 signal through basally localized FGFR1 to induce PS-like cells.
KLF6 in Pulmonary Hypertension: The Dual Role of Friend and Foe
Authors: Alharbi Rehab , Keles Merve , Fernandes Nadia , Maude Hannah , Fellows Adam , Williams Richard D , Chen Chien-Nien , Lambie Natalie , Matthews Nicholas , Al-Sahaf May , Guo Minzhe , Zhao Lan , Lawrie Allan , Whitsett Jeffrey A. , Cebola Ines , Wojciak-Stothard Beata
Published: July 2024

Background: Pulmonary arterial hypertension (PAH) is a severe lung condition with unmet clinical needs, marked by endothelial damage, excessive repair, and arterial narrowing, though mechanisms remain unclear. Methods: This study investigates Kruppel-like transcription factor 6 (KLF6), known for its role in tissue injury response and cancer onset, in PAH through functional and expression analyses in human pulmonary artery endothelial cells (HPAECs) and human and rodent PAH lung tissues. Findings: KLF6 expression increased in early experimental PAH in response to hypoxia and inflammation, while the expression of endothelial homeostasis regulators KLF2 and KLF4, previously linked to PAH, decreased. KLF6 overexpression enhanced pulmonary endothelial survival and angiogenesis through broad transcriptomic remodelling, including changes in genes governing endothelial homeostasis and arterial identity (e.g., SOX17, ERG, BMPR2) and promoted human pulmonary artery smooth muscle cells (HPASMCs) proliferation, which was inhibited by bosentan and imatinib. KLF6 functional and transcriptomic responses differed from those of KLF2 and KLF4. Comparative analysis of RNA-seq PAH databases and spatial transcriptomic analysis of human idiopathic PAH (IPAH) tissues highlighted strong association of KLF6 with vascular remodelling, especially with the formation of angioproliferative (plexiform) lesions. High KLF6 expression was observed in IPAH vascular endothelium and IPAH blood-derived endothelial progenitor cells. Single nucleus RNA-seq in PAH associated with Alveolar Capillary Dysplasia confirmed disease-related elevated KLF6 expression in arterial endothelial cells. Interpretation: Accumulation and reorganization of KLF6+ endothelial cells characterize human PAH. KLF6 drives endothelial repair and an apoptosis-resistant, angioproliferative endothelial phenotype. Targeting KLF6 could be a novel therapeutic approach for PAH.
Quadrupia: Derivation of G-quadruplexes for organismal genomes across the tree of life
Authors: Chantzi Nikol , Nayak Akshatha , Baltoumas Fotis A. , Aplakidou Eleni , Liew Shiau Wei , Galuh Jesslyn Elvaretta , Patsakis Michail , Moeckel Camille , Mouratidis Ioannis , Sazed Saiful Arefeen , Guiblet Wilfried , Montgomery Austin , Karmiris-Obratanski Panagiotis , Guliang Wang , Zaravinos Apostolos , Vasquez Karen M , Kwok Chun Kit , Pavlopoulos Georgios , Georgakopoulos-Soares Ilias
Published: July 2024

G-quadruplex DNA structures exhibit a profound influence on essential biological processes, including transcription, replication, telomere maintenance, and genomic stability. These structures have demonstrably shaped organismal evolution. However, a comprehensive, organism-wide G-quadruplex map encompassing the diversity of life has remained elusive. Here, we introduce Quadrupia, the most extensive and well-characterized G-quadruplex database to date, facilitating the exploration of G-quadruplex structures across the evolutionary spectrum. Quadrupia has identified G-quadruplex sequences in 108,449 reference genomes, with a total of 140,181,277 G-quadruplexes. The database also hosts a collection of 319,784 G-quadruplex clusters of 20 or more members, annotated by taxonomic distributions, multiple sequence alignments, profile Hidden Markov Models and cross-references to G-quadruplex 3D structures. Examination of G-quadruplexes across functional genomic elements in different taxa indicates preferential orientation and positioning, with significant differences between individual taxonomic groups. For example, we find that G-quadruplexes in bacteria with a single replication origin display profound preference for the leading orientation. Finally, we experimentally validate the most frequently observed G-quadruplexes using CD-spectroscopy, UV melting, and fluorescent-based approaches. Quadrupia is publicly available through https://www.pavlopoulos-lab.org/quadrupia.
Ketogenesis supports hepatic polyunsaturated fatty acid homeostasis via fatty acid elongation
Authors: Queathem Eric D. , Moazzami Zahra , Stagg David B , Nelson Alisa B. , Fulghum Kyle , Hayir Abdirahman , Seay Alisha , Gillingham Jacob R. , d’Avignon D. Andre , Han Xianlin , Ruan Hai-Bin , Crawford Peter A. , Puchalska Patrycja
Published: July 2024

Therapeutic interventions targeting hepatic lipid metabolism in metabolic dysfunction-associated steatotic liver disease (MASLD) and steatohepatitis (MASH) remain elusive. Using mass spectrometry-based stable isotope tracing and shotgun lipidomics, we established a novel link between ketogenesis and MASLD pathophysiology. Our findings show that mouse liver and primary hepatocytes consume ketone bodies to support fatty acid (FA) biosynthesis via both de novo lipogenesis (DNL) and FA elongation. Analysis of13C-labeled FAs in hepatocytes lacking mitochondrial D-β-hydroxybutyrate dehydrogenase (BDH1) revealed a partial reliance on mitochondrial conversion of D-βOHB to acetoacetate (AcAc) for cytoplasmic DNL contribution, whereas FA elongation from ketone bodies was fully dependent on cytosolic acetoacetyl-CoA synthetase (AACS). Ketone bodies were essential for polyunsaturated FA (PUFA) homeostasis in hepatocytes, as loss of AACS diminished both free and esterified PUFAs. Ketogenic insufficiency depleted liver PUFAs and increased triacylglycerols, mimicking human MASLD, suggesting that ketogenesis supports PUFA homeostasis, and may mitigate MASLD-MASH progression in humans.
Repeated extrinsic and anisotropic mechanical inputs promote polarized adherens junction elongation
Authors: Yang Xinyi , Ferraro Teresa , Molnar Kelly , Pontabry Julien , Malanda Sam-Rayden , Maghelli Nicola , Royer Loïc , Grill Stephan W. , Myers Gene , Grigolon Silvia , Labouesse Michel
Published: July 2024

A key challenge in development is understanding how complex organisms physically coordinate the morphogenesis of multiple tissues. Here, using biophysical approaches, we investigate how muscles under the epidermis specifically stimulate the extension of anterior-posterior (AP-oriented) epidermal adherens junctions during lateC. elegansembryonic elongation. First, light-sheet imaging shows that asynchronous patterns of muscle contractions drive embryo rotations. In turn, junctions between the lateral and dorso-ventral epidermis repeatedly oscillate between a folded, hypotensed state, and an extended, hypertensed state. Second, FRAP (Fluorescence Recovery After Photobleaching) analysis of an E-cadherin::GFP construct shows that muscle contractions stimulate E-cadherin turnover. Moreover, a mechano-chemical model backed by genetic tests suggests that E-cadherin trafficking modulates junction elongation due to lower line tension. Altogether, our results illustrate how muscle contractions fluidize epidermal adherens junctions, which, combined with anisotropic tension in the epidermis, drives their polarized extension.