bluuebunny/crossref_metadata_2025 huggingface dataset:
Anti-mitochondrial antibodies in systemic sclerosis target enteric neurons and are associated with GI dysmotility
Authors: McMahan Zsuzsanna H. , Casciola-Rosen Livia , Kaniecki Timothy , Gutierrez-Alamillo Laura , Ming Su Hong , Seika Philippa , Kulkarni Subhash
Published: November 2024

Background

Most patients with systemic sclerosis (SSc) experience gastrointestinal (GI) dysmotility. The enteric nervous system (ENS) regulates GI motility, and its dysfunction causes dysmotility. A subset of SSc patients harbor autoantibodies against the M2 mitochondrial antigen (AM2A). Here, we investigate whether M2 is expressed by specific ENS cells, and if AM2A associate with GI dysmotility in SSc patients.

Methods

Sera from 154 well-characterized patients with SSc were screened for AM2A by ELISA. Clinical features and GI transit data were compared between AM2A-positive and negative patients. HepG2 cells were cultured with these sera and co-stained with AM2A.

Results

Nineteen of 147 patients (12.9%) were AM2A positive. AM2A positivity was significantly associated with slower transit in the esophagus (β -14.4, 95%CI -26.2, -2.6) and stomach (β -7.9, 95% CI -14.1, -1.6). Immunostaining demonstrated pan-mitochondrial antigens TOM-20 and M2 enrichment in human ENS neurons, specifically in mesoderm- derived enteric neurons (MENS). HepG2 cells cultured with SSc sera showed that SSc autoantibodies penetrate live cells and that AM2A and other SSc autoantibodies are localized to subcellular compartments containing target antigens.

Conclusion

AM2A in SSc patients associate with slower GI transit. MENs are enriched in mitochondria, suggesting enhanced susceptibility to mitochondrial dysfunction and associated GI dysmotility in SSc. Our finding that SSc autoantibodies penetrate live cellsin vitrosuggests that SSc-AM2A may penetrate MENsin vivo,driving ENS and GI dysfunction. Further studies are warranted to understand whether AM2As contribute to mitochondrial dysfunction, and whether mitochondrial dysfunction contributes to GI dysmotility in SSc.

Key messages

What is already known on this topic

subset of SSc patients have autoantibodies against the M2 mitochondrial antigen (AM2A). Whether AM2A antibodies inform the presence or severity of GI dysfunction in SSc is unknown.

What this study adds:

AM2A antibodies in SSc patients associate with slower upper GI transit.

Mitochondria are enriched in a recently identified mesoderm-derived lineage of enteric neurons (MENs), which play a major role in GI motility. This suggests that MENS may be more mitochondrial-dependent than other cell types, and thus more susceptible to mitochondrial dysfunction. This may contribute to dysmotility in AM2A-positive SSc patients.

SSc autoantibodies penetrate live cellsin vitroand bind to their target antigens intracellularly.

How this study might affect research, practice or policy

AM2A antibodies in SSc patients may penetrate MENs in vivo, driving ENS dysfunction and subsequent GI dysmotility

This potentially novel mechanism in SSc GI disease could inform our current approach to diagnosing and managing these patients.
Detection of Anti-Non-α-Gal Xenoreactive Antibodies in Human Blood Products
Authors: Ladowski Joseph M. , Hu Meghan , Yoon Janghoon , Chen Zheng , Knechtle Stuart , Jackson Annette , Kwun Jean
Published: November 2024

Background

Surgical bleeding is a risk in any solid organ transplant, and commonly addressed with the transfusion of human blood product to replace or supplement coagulation factors. It is unknown if these blood products would harm xenotransplanted pig organs in human recipients demonstrating coagulopathy. The aim of this study was to investigatein vitroif blood products such as fresh frozen plasma (FFP) or cryoprecipitate (cryo) contain xenoantibodies capable of cytotoxicity to GTKO pig cells.

Methods

We obtained 12 individual single donor (7 FFP and 5 cryo) blood products from our institution’s blood bank for testing. PBMCs were obtained from a GTKO/hCD55 pig for use as target cells. We performed a series of flow cytometry crossmatch and complement-dependent cytotoxicity assays.

Results

We found that all the tested blood products contained some degree of IgM and IgG xenoantibody. Tests using a 1:50 dilution revealed a significant decrease in IgM xenoantibody binding, but an increase the detection of IgG binding. Multiple preparations were capable of GTKO PBMC cytotoxicity but the level of antibody binding and cell death varied by preparation.

Conclusions

Both FFP and cryo contain IgM and IgG non-αGal xenoantibodies capable of killing GTKO PBMCs, though the level varies by preparation. While some centers utilize a genetic background with mutations in the three enzymes responsible for the known xenoantigens, others are investigating the GTKO pig as a potential option. These results suggest that a center pursuing a human xenotransplantation study with a GTKO genetic background should pre-screen blood products prior to administration.
Hematocrit and Progressive Stroke in Single Small Subcortical Infarction: Insights from the China Stroke Registry III
Authors: Lu Jiancong , Hankey Graeme J. , Zhang Xiaoli , Meng Xia , Zeng Weiqi , Jiang Jun , Wang Anxin , Tan Zefeng
Published: November 2024

Background and Purpose

Single Small Subcortical Infarction (SSSI) is a subtype of ischemic stroke with unique pathophysiology, often leading to poor outcomes. This study investigates the association between admission hematocrit (HCT) levels and the risk of progressive stroke in SSSI patients.

Methods

We conducted a retrospective cohort study using data from the China National Stroke Registry III, including 2,457 SSSI patients. Progressive stroke was defined as an increase in the NIHSS score by ≥2 points during hospitalization. Logistic regression models assessed the association between HCT levels and progressive stroke risk.

Results

Among 2,457 SSSI patients, 135 (5.50%) experienced progressive stroke. Higher HCT levels were independently associated with a lower risk of progressive stroke (adjusted OR 0.983, 95% CI 0.973-0.994, P = 0.002), with a potential dose-response relationship. The protective effect was more pronounced in patients with severe sensory deficits (P for interaction = 0.013) and younger age (<65 years) (P for interaction = 0.037). Lower HCT levels were also independently associated with increased mortality risk within 3 months after stroke (adjusted OR 0.937, 95% CI 0.892-0.984, P < 0.001).

Conclusions

Higher admission HCT levels are associated with a lower risk of progressive stroke in SSSI patients, particularly those with severe sensory deficits and younger age. Lower HCT levels may also increase early mortality risk. These findings highlight the importance of monitoring and optimizing HCT levels in SSSI management. Further research should explore the underlying mechanisms and therapeutic implications.
Affinity maturation of antibody responses is mediated by differential plasma cell proliferation
Authors: MacLean Andrew J. , Deimel Lachlan P. , Zhou Pengcheng , ElTanbouly Mohamed A. , Merkenschlager Julia , Ramos Victor , Santos Gabriela S. , Hägglöf Thomas , Mayer Christian T. , Hernandez Brianna , Gazumyan Anna , Nussenzweig Michel C.
Published: November 2024

Increased antibody affinity over time after vaccination, known as affinity maturation, is a prototypical feature of immune responses. Recent studies have shown that a diverse collection of B cells, producing antibodies with a wide spectrum of different affinities, are selected into the plasma cell (PC) pathway. How affinity-permissive selection enables PC affinity maturation remains unknown. Here we report that PC precursors (prePC) expressing high affinity antibodies receive higher levels of T follicular helper (Tfh)-derived help and divide at higher rates than their lower affinity counterparts once they leave the GC. Thus, differential cell division by selected prePCs accounts for how diverse precursors develop into a PC compartment that mediates serological affinity maturation.
Measuring Microbial Community-Wide Antibiotic Resistance Propagation via Natural Transformation in the Human Gut Microbiome
Authors: Chowdhury Nadratun N. , Forry Samuel P. , Servetas Stephanie L. , Hunter Monique E. , Dootz Jennifer N. , Dunkers Joy P. , Jackson Scott A.
Published: November 2024

1)

This work explored the role of natural transformation – a mechanism by which bacteria uptake and express extracellular genes - in driving antibiotic resistance propagation in the human gut microbiome. The model extracellular antibiotic resistance gene (eARG) – a plasmid containing a kanamycin resistance (kanR) gene and a green fluorescence protein (GFP) gene – was dosed into pooled and homogenized human stool and incubated anaerobically. Cellular uptake of the eARG was assessed via droplet digital PCR, the expression of newly acquired genes was assessed by culturing on selective media and fluorescent microscopy, newly resistant isolates were identified by long-read Nanopore sequencing and the impacts on the taxonomy of the gut microbiome was assessed using shotgun Illumina sequencing. Significant gene uptake of bothkanRand GFP was quantified in gut microbes, and extent of gene accumulation correlated with background kanamycin levels. Gut microbes dosed with background kanamycin expressed kanamycin resistance acquired by the eARG (as quantified by CFU on kanamycin-containing media). Newly resistant isolates, identified asEnterococcus faeciumby long-read sequencing, also expressed green fluorescence acquired from the eARG. Though compositional changes of the kanamycin-resistant subpopulation were observed in the gut microbiome in response to eARG and antibiotic exposure, these changes were not reproducible among replicates and trends in taxonomy due to transformation could not be identified. This comprehensive analysis therefore establishes the significant propagation of antibiotic resistance within the human gut microbiome due to eARG exposure, while evaluating the utility of various measurements in characterizing transformation in a complex microbial community.

2)

Importance

Infections from antibiotic resistant bacteria in the human gut microbiome are a growing public health concern. Antibiotic resistance may develop in gut microbiota from exposure to environmentally prevalent extracellular antibiotic resistance genes (eARGs). This work explores the impact of eARG exposure on a complex human gut microbial community. It quantifies significant accumulation and expression of eARG-borne genes by endogenous gut microorganisms, thereby demonstrating that natural transformation may play a role in resistance propagation in the human gut. It also demonstrates the highly variable changes in gut taxonomy in response to eARG exposure, implying that eARGs may impact gut composition and therefore downstream human health effects. These data may be useful in characterizing and mitigating resistance propagation in the human gut, and in general, the suite of genotypic and phenotypic measurements used constitute a quantitative framework to characterize the effects of perturbations on complex microbiomes.
Mapping essential somatic hypermutations in a CD4-binding site bNAb informs HIV-1 vaccine design
Authors: Dam Kim-Marie A. , Gristick Harry B. , Li Yancheng E. , Yang Zhi , Gnanapragasam Priyanthi N.P. , West Anthony P. , Seaman Michael S. , Bjorkman Pamela J.
Published: November 2024

HIV-1 broadly-neutralizing antibodies (bNAbs) targeting the CD4-binding site (CD4bs) contain rare features that pose challenges to elicit these bNAbs through vaccination. The IOMA-class of CD4bs bNAbs includes fewer rare features and somatic hypermutations (SHMs) to achieve broad neutralization, thus presenting a potentially accessible pathway for vaccine-induced bNAb development. Here, we created a library of IOMA variants in which each SHM was individually reverted to the inferred germline (iGL) counterpart to investigate the roles of SHMs in conferring IOMA’s neutralization potency and breadth. Impacts on neutralization for each variant were evaluated, and this information was used to design minimally-mutated IOMA-class variants (IOMAmin) that incorporated the fewest SHMs required for achieving IOMA’s neutralization breadth. A cryo-EM structure of an IOMAmin variant bound to Env was used to further interpret characteristics of IOMA variants to elucidate how IOMA’s structural features correlate with its neutralization mechanism, informing the design of IOMA-targeting immunogens.

Highlights

IOMAmin variants with reduced SHMs retain neutralization potency and breadth.

Cryo-EM structure reveals IOMAmin preserves key Env interactions despite fewer SHMs.

IOMA variants with mutations in CDRH3 and CDRL1 fail to improve neutralization.
Multiple pathways to red carotenoid coloration: House finches (Haemorhous mexicanus) do not use CYP2J19 to produce red plumage
Authors: Koch Rebecca E. , Truong Christy N. , Reeb Hannah R. , Joski Brooke H. , Hill Geoffrey E. , Zhang Yufeng , Toomey Matthew B.
Published: November 2024

The carotenoid-based colors of birds are a celebrated example of biological diversity and an important system for the study of evolution. Recently, a two-step mechanism, with the enzymes cytochrome P450 2J19 (CYP2J19) and 3-hydroxybutyrate dehydrogenase 1-like (BDH1L), was described for the biosynthesis of red ketocarotenoids from yellow dietary carotenoids in the retina and plumage of birds. A common assumption has been that all birds with ketocarotenoid-based plumage coloration used this CYP2J19/BDH1L mechanism to produce red feathers. We tested this assumption in house finches (Haemorhous mexicanus) by examining the catalytic function of the house finch homologs of these enzymes and tracking their expression in molting birds. We found that CYP2J19 and BDH1L did not catalyze the production of 3-hydroxy-echinenone (3-OH-echinenone), the primary red plumage pigment of house finches, when provided with common dietary carotenoid substrates. Moreover, gene expression analyses revealed little to no expression ofCYP2J19in liver tissue or growing feather follicles, the putative sites of pigment metabolism in molting house finches. Finally, although the hepatic mitochondria of house finches have high concentrations of 3-OH-echinenone, observations using fluorescent markers suggest that both CYP2J19 and BDH1L localize to the endomembrane system rather than the mitochondria. We propose that house finches and other birds that deposit 3-OH-echinenone as their primary red plumage pigment use an alternative enzymatic pathway to produce their characteristic red ketocarotenoid-based coloration.
Divergent reference genomes compromise the reconstruction of demographic histories, selection scans, and population genetic summary statistics
Authors: Akopyan Maria , Genchev Matthew , Armstrong Ellie E. , Mooney Jazlyn A.
Published: November 2024

Characterizing genetic variation in natural populations is central to evolutionary biology. However, most non-model organisms lack integral genomic resources such as reference genomes and recombination maps, limiting accurate evolutionary inference. Here, we explore the consequences of reference genome bias on the inference of genetic diversity, demographic histories, and recombination rates using gray foxes (Urocyon cinereoargenteus), which, like most members ofCanidae,are traditionally mapped to the dog (Canis lupus familiaris) reference genome. Whole genome sequence data from gray foxes were mapped to the gray fox reference genome and two heterospecific canid references (dog and Arctic fox;Vulpes lagopus). Our results reveal that reference bias significantly affects population genomic analyses. Mapping to the conspecific gray fox genome improved read pairing, increased detection of SNPs, especially rare variants, and reduced spurious variants. Estimates of nucleotide diversity (π) and genetic differentiation (FST) were higher using the gray fox genome. We observed that mapping to heterospecific references leads to underestimates of population sizes, distorted demographic trajectories, and more variable recombination rates. These effects are further complicated by population-specific biases, which vary in their magnitude and direction across populations, highlighting the need for tailored approaches to mitigate reference bias. Importantly,FSToutlier detection also differed among references, affecting functional interpretations. Collectively, this work addresses a critical gap in the rapidly expanding field of non-model species genomics by demonstrating the importance of using conspecific genomic resources in evolutionary research and illustrating how reliance on distantly related reference genomes can distort population genetic analyses.
The Potential of CCA-associated Bacteria to Fight Antimicrobial-Resistant Pathogens: a Genomic Survey
Authors: Lera-Lozano Diego , Ruiz-Toquica Jordan S. , Holt Matthew W. , Jones Elizabeth K. , Ritchie Kim B. , Medina Mónica , González-Pech Raúl A.
Published: November 2024

The global rise of antimicrobial resistance has intensified efforts in bioprospecting, with researchers increasingly exploring unique marine environments for novel antimicrobials. In line with this trend, our study focused on bacteria isolated from the unique microbiome of crustose coralline algae (CCA), which has yet to be investigated for antimicrobial discovery. In the present work, bacteria were isolated from the CCAHydrolithon boergesenii. After running antimicrobial assays against antibiotic-resistant human and marine pathogens, three isolates were selected for genome sequencing using the Oxford Nanopore technology. Genome mining of the high-quality assemblies revealed 100 putative Biosynthetic Gene Clusters (BGCs) across the three genomes. Further analysis uncovered BGCs potentially involved in the biosynthesis of novel antimicrobial compounds. Our study provides valuable resources for future research aimed at the discovery of novel antimicrobials, particularly in the face of the antibiotic-resistance global crisis and highlights the potential of specialized marine environments like CCA biofilms.
CLINICAL AND MOLECULAR CHARACTERISATION OF SLC31A1-RELATED NEURODEVELOPMENTAL DISORDER.
Authors: Julia-Palacios Natalia , Munoz-Pujol Gerard , Maroofian Reza , Bertoli-Avella Aida M. , Gomez-Chiari Marta , Muchart Jordi , Paredes-Fuentes Abraham J. , O'Callaghan Maria del Mar , Machado-Casas Irene S. , Cristian Ingrid , Morrison Jennifer , Garcia-Cazorla Angels , Codina Anna , Miryounesi Mohammad , Zonic Emir , Bauer Peter , Cheema Huma , Anjum Muhammad Nadeem , Al-Sannaa Nouriya , Elmaksoud Marwa , Ababneh Faroug , Alijanpour Sahar , Tonekaboni Seyed Hassan , Fayazi Afshin , Urbaniak Maria , Barba Uxia , Hoenicka Janet , Palau Francesc , Houlden Henry , Ortigoza-Escobar Juan Dario , Ribes Antonia , Santos-Ocana Carlos , Tyler Millie , Gaffney Patrick , Carroll Christopher , Tort Frederic , Wierenga Klaas J. , Webb Bryn , Artuch Rafael , Baide-Mairena Heidy , Urreizti Roser
Published: November 2024

Copper is indispensable for various metabolic processes, notably mitochondrial respiration. In humans, copper homeostasis hinges on transporters such as copper transporter 1 (CTR1), encoded by the SLC31A1 gene. Recently, bi-allelic mutations in SLC31A1 have been associated with a new neurodevelopmental disorder. This study presents clinical, genetic, and biochemical findings from 13 new cases across 10 families worldwide. RNA sequencing evaluated gene expression, and Western blotting assessed CTR1 protein levels. Additionally, mitochondrial respiratory capacity was measured via high-resolution respirometry. Affected individuals exhibited a distinct clinical phenotype characterized by early-onset epileptic encephalopathy, severe neurodevelopmental delay and hypotonia, with high mortality. Neuroimaging revealed significant brain atrophy and white matter abnormalities. Genetic analysis identified bi-allelic SLC31A1 variants, predominantly p.His120Gln in six cases and p.(Arg102Cys/His) in three cases. Functional studies in patient fibroblasts demonstrated impaired mitochondrial respiration. This study significantly broadens the clinical spectrum of this recently described syndrome, presenting as a severe developmental encephalopathy with high mortality risk, and suggests mitochondrial dysfunction as a potential pathomechanism. These findings contribute to the mounting evidence linking CTR1 dysfunction to neurodegeneration, underscoring the urgency for further therapeutic investigations.
Patient-Reported Treatment Outcomes in ME/CFS and Long COVID
Authors: Eckey Martha , Li Peng , Morrison Braxton , Davis Ronald W , Xiao Wenzhong
Published: November 2024

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and Long COVID are persistent multi-system illnesses affecting many patients. With no known effective FDA-approved treatments for either condition, patient-reported outcomes of treatments are invaluable for guiding management strategies in patient care and generating new avenues for research. Here, we present the results of an ME/CFS and Long COVID treatment survey with responses from 3,925 patients. We assessed the experiences of these patients with more than 150 treatments, as well as their demographics, symptoms, and comorbidities. Patients with each condition who participated in the study shared similar symptom profiles, including all the core symptoms of ME/CFS, e.g., 89.7% of ME/CFS and 79.4% of Long COVID reported post-exertional malaise (PEM). Treatments with the greatest perceived benefits were identified, which had varied effects on different core symptoms. In addition, treatment responses were significantly correlated (R² = 0.68) between the two patient groups. Patient subgroups with distinct profiles of symptoms and comorbidities showed varied responses to treatments, e.g., a POTS-dominant cluster benefiting from autonomic modulators and a cognitive-dysfunction cluster from CNS stimulants. This study underscores the symptomatic and therapeutic similarities between ME/CFS and Long COVID and highlights the commonalities and nuanced complexities of infection-associated chronic diseases and related conditions. Insights from patient-reported experiences, in the absence of approved treatments, provide urgently needed real-world evidence for targeted therapies in patient care and for developing future clinical trials.

(Disclaimer: The findings presented in this paper are based on patient-reported information and are intended for research purposes only. They should not be interpreted as medical advice. Patients are advised to consult their healthcare provider before initiating or altering any treatment.)
Incident benzodiazepine and Z-drug use and subsequent risk of alcohol- and drug-related problems: a nationwide matched cohort study with co-twin comparison
Authors: Wang Xinchen , Chang Zheng , Molero Yasmina , Isomura Kayoko , de la Cruz Lorena Fernández , Lichtenstein Paul , Kuja-Halkola Ralf , D’Onofrio Brian M , Quinn Patrick D , Larsson Henrik , Brikell Isabell , Hellner Clara , Hasselström Jan , Jayaram-Lindström Nitya , Mataix-Cols David , Sidorchuk Anna
Published: November 2024

Background

Despite considerable interest in the consequences of benzodiazepine and benzodiazepine-related Z-drug (BZDR) use, little is known about whether and how initiation of BZDR treatment contributes to the development of alcohol- and drug-related problems.

Aim

To examine the association of incident BZDR dispensing with subsequent development of broadly defined alcohol- and drug-related problems.

Methods

This nationwide register-based study included demographically matched and co-twin control cohorts. Among all Swedish residents aged ≥10 years and BZDR-naïve by 2007, 960,430 BZDR-recipients with incident dispensation in 2007-2019 and without any recorded pre-existing substance-related conditions were identified and matched (1:1) to nonrecipients from the general population. Twin BZDR-recipients (n=12,048) were linked to 12,579 unexposed co-twins. Outcomes included alcohol and drug use disorders, poisoning, deaths, and related suspected criminal offences. Flexible parametric survival models estimated outcome risks across up to 14 years of follow-up.

Results

In the demographically matched cohort (60% women, median age at BZDR initiation 51 years), incidence rates in BZDR-recipients and nonrecipients (per 1000 person-years) were 5.60 vs 2.79 for alcohol-related and 4.15 vs 1.23 for drug-related problems, respectively. In fully-adjusted models, relative risks were increased for alcohol- and drug-related problems (adjusted hazard ratio [95% confidence interval]: 1.56 [1.53-1.59] and 2.11 [2.05-2.17], respectively). The risks persisted within the co-twin comparison, different follow-ups, and all additional and sensitivity analyses.

Conclusions

BZDR initiation was associated with a small but robust increase in absolute and relative risks of developing alcohol- and drug-related problems. The findings contribute to evidence base for making decisions on BZDR treatment initiation.
De novo functional discovery of peptide-MHC restricted CARs from recombinase-constructed large-diversity monoclonal T cell libraries
Authors: Wang Zening , Sarkar Abhijit , Ge Xin
Published: November 2024

Chimeric antigen receptors (CAR) that mimic T cell receptors (TCR) on eliciting peptide-major histocompatibility complex (pMHC) specific T cell responses hold great promise in the development of immunotherapies against solid tumors, infections, and autoimmune diseases. However, broad applications of TCR-mimic (TCRm) CARs are hindered to date largely due to lack of a facile approach for the effective isolation of TCRm CARs. Here, we establish a highly efficient process forde novodiscovery of TCRm CARs from human naïve antibody repertories by combining recombinase-mediated large-diversity monoclonal library construction with T cell activation-based positive and negative screenings. Panels of highly functional TCRm CARs with peptide-specific recognition, minimal cross-reactivity, and low tonic signaling were rapidly identified towards MHC-restricted intracellular tumor-associated antigens MAGE-A3, NY-ESO-1, and MART-1. Transduced TCRm CAR-T cells exhibited pMHC-specific functional avidity, potent cytokine release, and efficacious and persistent cytotoxicity. The developed approach could be used to generate safe and potent immunotherapies targeting MHC-restricted antigens.
Development of antigen-dextramers for detection and evaluation of CAR T cells
Authors: Friis Rasmus U. W. , Ormhøj Maria , Krüger-Jensen Cecilie S. , Barden Markus , Ramanathan Keerthana , Hansen Mikkel R. , Abken Hinrich , Hadrup Sine R.
Published: November 2024

Background

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment landscape of hematologic cancers by engineering T cells to specifically target and destroy cancer cells. Monitoring CAR T cell activity and function is essential for optimizing therapeutic outcomes, but existing tools for CAR detection are often limited in specificity and functional assessment capability.

Methods

We developed antigen-dextramers by conjugating multiple CAR-specific antigens to a dextran backbone. The dextramers were compared to previously reported antigen-tetramers for their ability to stain and detect CAR T cells. Because these multimers incorporate the CAR target antigen, they uniquely enable assessment of CAR T cell functionality by facilitating binding and activation analyses. We tested the staining and functional properties of the multimers across a range of CAR constructs with different affinities, using flow cytometry, microscopy, and NFAT-luciferase reporter assays.

Results

The antigen-dextramers demonstrated high specificity and sensitivity in staining CAR T cells, with adjustable antigen density to optimize binding. Antigen-dextramers also enabled effective clustering and subsequent activation of CARs, showing their utility as both a staining and functional assessment tool. The dextramers revealed that CARs with different affinities and clustering tendencies displayed varied binding and activation in response to different antigen densities.

Conclusion

Antigen-dextramers offer a dual advantage as versatile reagents for both staining and functional analysis of CAR T cells. Their capacity to engage CARs with the specific antigen provides a valuable platform for evaluating CAR functionality, informing CAR design improvements, and enhancing therapeutic precision.
Raver1 linksRipk1RNA splicing to caspase-8-mediated pyroptotic cell death, inflammation, and pathogen resistance
Authors: Zhang Boyao , Orning Pontus , Lehman Jesse W. , Dinis Alexandre , Torres-Ulloa Leslie , Elling Roland , Kelliher Michelle A. , Bertin John , Proulx Megan K. , Ryan Liv , Kandasamy Richard , Espevik Terje , Pai Athma A. , Fitzgerald Katherine A. , Lien Egil
Published: November 2024

Multiple cell death and inflammatory signaling pathways converge on two critical factors: receptor interacting serine/threonine kinase 1 (RIPK1) and caspase-8. Careful regulation of these molecules is critical to control apoptosis, pyroptosis and inflammation. Here we discovered a pivotal role of Raver1 as an essential regulator ofRipk1pre-mRNA splicing, expression, and functionality, and the subsequent caspase-8-dependent inflammatory cell death. Macrophages fromRaver1-deficient mice exhibit altered splicing ofRipk1, accompanied by diminished cell death and reduced activation of caspase-8, Gasdermin D and E, caspase-1, as well as decreased interleukin-18 (IL-18) and IL-1ß production. These effects were triggered byYersiniabacteria, or by restraining TAK1 or IKKβ in the presence of LPS, TNF family members, or IFNγ. Consequently, animals lackingRaver1showed heightened susceptibility toYersiniainfection. Raver1 and RIPK1 also controlled the expression and function of the C-type lectin receptor Mincle. Our study underscores the critical regulatory role of Raver1 in modulating innate immune responses and highlights its significance in directingin vivoandin vitroinflammatory processes.

Significance

Caspase-8 and the kinase RIPK1 are at focal points of several inflammation and cell death pathways. Thus, a careful regulation of their actions is needed. Our work identifies the RNA splicing factor Raver1 as a critical factor directing the splicing ofRipk1in order to modulate RIPK1/caspase-8-driven pyroptosis, apoptosis and inflammation. Raver1 is central for macrophage responses toYersiniabacteria, initiated after blockade of kinases TAK1 and IKK, measured as activation of RIPK1, caspase-8, Gasdermin D, caspase-3, IL-1ß and IL-18. Importantly, Raver1 is necessary for host resistance toYersiniainfectionin vivo. We propose that Raver1 is key for correct tuning of RIPK1-caspase-8 dependent processes.
Reliable amplification of highly repetitive or low complexity sequence DNA enabled by superhelicase-mediated isothermal amplification
Authors: Kang Jimin , Rashid Fahad , Murray Peter J. , Merino-Urteaga Raquel , Gavrilov Momčilo , Shang Tiantian , Jo Wonyoung , Ahmed Arman , Aksel Tural , Barrick Doug , Berger James M. , Ha Taekjip
Published: November 2024

Polymerase Chain Reaction (PCR) requires thermal cycling to melt DNA and proceed through the subsequent cycles of DNA synthesis needed for exponential amplification. Previously, we engineered a superhelicase, with enhanced processivity and speed, to replace this traditional PCR melting step with enzymatic DNA unwinding while retaining desired PCR characteristics, such as multi-kb amplicon size and applicability to cloning and gene editing outcome assessment. This isothermal amplification method is named SHARP (SSB-Helicase Assisted Rapid PCR) because single-stranded DNA binding protein (SSB) and superhelicases are added to standard PCR reagents. Here, we show that SHARP can be effective for DNA sequences that PCR is unable to amplify or that produce side products of. SHARP is demonstrated to be capable of amplifying up to six identical repeats of the Widom 601 nucleosome positioning sequence and up to 35 identical repeats of ankyrin sequence. We also show that a sequence with 91% AT-content can be amplified using SHARP and that the amplification product can be validated using single-molecule optical tweezers experiments.
Design of high specificity binders for peptide-MHC-I complexes
Authors: Liu Bingxu , Greenwood Nathan F. , Bonzanini Julia E. , Motmaen Amir , Sharp Jazmin , Wang Chunyu , Visani Gian Marco , Vafeados Dionne K. , Roullier Nicole , Nourmohammad Armita , Garcia K. Christopher , Baker David
Published: November 2024

Class I MHC molecules present peptides derived from intracellular antigens on the cell surface for immune surveillance, and specific targeting of these peptide-MHC (pMHC) complexes could have considerable utility for treating diseases. Such targeting is challenging as it requires readout of the few outward facing peptide antigen residues and the avoidance of extensive contacts with the MHC carrier which is present on almost all cells. Here we describe the use of deep learning-based protein design tools tode novodesign small proteins that arc above the peptide binding groove of pMHC complexes and make extensive contacts with the peptide. We identify specific binders for ten target pMHCs which when displayed on yeast bind the on-target pMHC tetramer but not closely related peptides. For five targets, incorporation of designs into chimeric antigen receptors leads to T-cell activation by the cognate pMHC complexes well above the background from complexes with peptides derived from proteome. Our approach can generate high specificity binders starting from either experimental or predicted structures of the target pMHC complexes, and should be widely useful for both protein and cell based pMHC targeting.
Enhanced Brain-Heart Connectivity as a Precursor of Reduced State Anxiety After Therapeutic Virtual Reality Immersion
Authors: Sezer Idil , Moreau Paul , El Sayed Hussein Jomaa Mohamad , Godefroy Valérie , Batrancourt Bénédicte , Lévy Richard , Filipchuk Anton
Published: November 2024

State anxiety involves transient feelings of tension and nervousness in response to threats, which can escalate into anxiety disorders if persistent. Despite treatments, 30%-50% of individuals show limited improvement, and neurophysiological mechanisms of treatment responsiveness remain unclear, requiring the development of objective biomarkers.

In this study, we monitored multimodal electrophysiological parameters: heart rate variability (high-frequency, low-frequency, LF/HF ratio), EEG beta and alpha relative power, and brain-to-heart connectivity in participants with real-life state anxiety. Participants underwent a therapeutic intervention combining virtual-reality immersion, hypnotic script, and a breath control exercise. Real-life state anxiety was captured using the STAI-Y1 scale before and after the intervention.

We observed reduced anxiety immediately after the intervention in 16 out of 27 participants. While all participants, independently of their STAI-Y1 score, showed increased HRV low frequency power, only treatment-responders displayed increased overall autonomic tone (high and low frequency HRV), increased midline beta power and brain-to-heart connectivity. Notably, the LF/HF ratio showed a significant linear relationship with anxiety reduction, with higher ratios linked to greater therapeutic response.

These findings suggest that increased cognitive regulation of brain-to-heart connectivity could serve as a biomarker for therapeutic efficacy, with elevated midline beta power facilitating improved cardiac tone in responders.

Significance Statement

Elevated state anxiety can escalate into debilitating disorders, such as generalized anxiety disorder, yet treatment efficacy remains inconsistent, and reliable biomarkers predicting therapeutic outcomes are lacking. This study identifies key neural and physiological markers linked to effective anxiety reduction following a virtual reality-based non-pharmacological intervention in healthy participants with increased state anxiety. Anxiety reduction is associated with increased midline beta power, heightened heart rate variability (LF/HF ratio), and enhanced brain-to-heart connectivity. These findings highlight the role of brain-to-heart modulation and autonomic nervous system functioning in therapeutic response. By highlighting these biomarkers, this research aims at advancing our understanding of anxiety treatment mechanisms and offering insights into the development of biomarker-driven, scalable interventions.
A pharmacological chaperone stabilizer rescues the expression of the vast majority of pathogenic variants in a G protein-coupled receptor
Authors: Mighell Taylor L. , Lehner Ben
Published: November 2024

Reduced protein stability is the most frequent mechanism by which rare missense variants cause disease. A promising therapeutic avenue for treating destabilizing variants is pharmacological chaperones (PCs, also known as correctors or stabilizers), small molecules that bind to and stabilize target proteins. PCs have been approved as clinical treatments for specific variants, but protein energetics suggest their effects might be much more general. Here, we test this hypothesis for the first time by comprehensively quantifying PC efficacy for all missense variants in a human disease gene, the vasopressin 2 receptor (V2R), a G-protein coupled receptor in which loss-of-function variants cause nephrogenic diabetes insipidus (NDI). Strikingly, treatment with a PC rescues the expression of nearly all destabilized variants, with non-rescued variants identifying the drug’s binding site. Our results provide proof-of-principle that a single small molecule can rescue destabilizing variants throughout a protein’s structure. The application of this principle to other proteins should allow the development of effective therapies for many genetic diseases.
Cardiac Reprogramming with Drug Resistance Alleviates Doxorubicin-induced Cardiotoxicity in Mice and Pigs
Authors: Zhang Yixin , Li Weirun , Xiao Yingxian , Luo Li , Ai Jiacong , Mo Shushan , Li Lanya , Deng Junyao , Wang Xueyi , Li Qishan , Zeng Yan , Liu Huifang , Wang Fei , Li Zhenhua
Published: November 2024

Doxorubicin (Dox)-induced cardiotoxicity represents a significant clinical adverse effect associated with cancer chemotherapy treatment. The systematic toxicity associated with the currently available drug dexrazoxane has limited its clinical utility. Inspired by cancer drug resistance mechanisms, we propose a novel strategy termedTransientoverexpression ofp-glycoprotein (P-gp) forCardiacreprogramming (TopCare) to induce cardiac drug resistance as a treatment for cardiotoxicity. This approach involves reprogramming cardiomyocytes by delivering lipid nanoparticles (LNPs)-based mRNA therapeutics to induce temporary P-gp overexpression, which in turn reduces intracellular Dox levels and suppresses cytotoxic effects. Our results demonstrated that LNPs with P-gp mRNAs (P-gp LNPs) enhance P-gp expression in H9c2 cells, significantly lowering the cytotoxicity of both Dox and paclitaxel (PTX). In a mouse model with Dox-induced cardiotoxicity, it was observed that P-gp LNPs effectively promoted P-gp overexpression in cardiomyocytes, with a time-dependent decline in P-gp protein levels. This TopCare strategy resulted in improved survival rates, restored cardiac function, and reduced myocardial fibrosis and structural cardiac alterations. Furthermore, studies in large animals have shown that intrapericardial (iPC) injection of P-gp LNPs effectively mitigates adverse effects and restores cardiac function in pig models of Dox-induced cardiotoxicity. The significant cardioprotective effects achieved through cardiac drug resistance highlight the safety, efficacy, and clinical potential of the TopCare strategy for alleviating doxorubicin-induced cardiotoxicity.
Ecological Scaling of Temporal Fluctuations with Bacterial Abundance in Gut Microbiota Depends on Functional Properties of Individual Microbial Species and Bacterial Communities
Authors: Su Peng , Tchourine Konstantine , Vitkup Dennis
Published: November 2024

Macroecological relationships that describe various statistical associations between species’ abundances, their spatial, and temporal variability are among the most general laws in ecology and biology. One of the most commonly observed relationships is a power-law scaling between means and variances of temporal species abundances, known in ecology as Taylor’s law. Taylor’s law has been observed across many ecosystems, from diverse plant and animal ecosystems to complex microbial communities. While many mathematical models have been proposed to explain the potential origins of Taylor’s law, what determines its scaling exponents across species and ecosystems is not understood. Here, we use temporal trajectories of human and baboon gut microbiota to analyze the relationship between functional properties of individual bacterial species and microbial communities with the scaling of species-specific and community-level Taylor’s law. The species Taylor law characterizes – for each individual species – the relationship between the species’ temporal abundance means and temporal abundance variances across host organisms. On the other hand, community-level Taylor’s law characterizes – in each host organism – the scaling across multiple species between their temporal abundance means and temporal abundance variances. For community Taylor’s law, we find that the power law scaling is strongly associated with the microbiota abundance of certain nutrient-degrading enzymes in the gut. Notably, our results demonstrate that the availability of enzymes metabolizing starch glycogen significantly increases Taylor’s law scaling. We also find that species Taylor’s law depends on the individual species’ functional properties. Specifically, we observe lower Taylor’s law scaling for species with larger metabolic networks, for species that are able to grow on a larger number of carbon sources, and for species with particular metabolic functions, such as glutamine and folate metabolism. Overall, our study reveals that Taylor’s law scaling is strongly associated with the functional capabilities of bacterial communities and individual microbial species’ biosynthetic properties, which are likely related to their ecological roles in the gut microbiota.
Dynamic regulation of RNA Polymerase III transcription in mouse embryonic stem cells during heat shock stress
Authors: Nguyen Thomas F. , Kwan James Z.J. , Mitchell Jennifer E. , Cui Jieying H. , Teves Sheila S.
Published: November 2024

Cells respond to many different types of stresses by overhauling gene expression patterns, both at the transcriptional and translational level. Under heat stress, global transcription and translation are inhibited, while the expression of chaperone proteins are preferentially favored. As the direct link between mRNA transcription and protein translation, tRNA expression is intricately regulated during the stress response. Despite extensive research into the heat shock response (HSR), the regulation of tRNA expression by RNA Polymerase III (Pol III) transcription has yet to be fully elucidated in mammalian cells. Here, we examine the regulation of Pol III transcription during different stages of heat shock stress in mouse embryonic stem cells (mESCs). We observe that Pol III transcription is downregulated after 30 minutes of heat shock, followed by an overall increase in transcription after 60 minutes of heat shock. This effect is more evident in tRNAs, though other Pol III gene targets are also similarly affected. Notably, we show that the downregulation at 30 minutes of heat shock is independent of HSF1, the master transcription factor of the HSR, but that the subsequent increase in expression at 60 minutes requires HSF1. Taken together, these results demonstrate an adaptive RNA Pol III response to heat stress, and an intricate relationship between the canonical HSR and tRNA expression.

Article Summary

This study explores the regulation of RNA Polymerase III (Pol III) transcription during heat shock in mouse embryonic stem cells (mESCs). Results show that tRNA transcription is downregulated after 30 minutes of heat shock, but increases after 60 minutes, while other Pol III targets remain unaffected. Importantly, the initial downregulation is independent of heat shock factor 1 (HSF1), the key regulator of the heat shock response, but the subsequent increase in tRNA expression depends on HSF1. These findings reveal an adaptive mechanism of Pol III activity under heat stress, highlighting a complex interplay between heat shock response and tRNA expression.
Bridging immunotypes and enterotypes using a systems immunology approach
Authors: Affaticati Fabio , Ha My K. , Gehrmann Thies , Boeck Ilke De , Kuznetsova Maria , Vandoren Romi , Deuren Vincent Van , Jansens Hilde , Reu Hans De , Schippers Jolien , Peeters Karin , Bartholomeus Esther , Ierssel Sabrina Van , Coenen Samuel , Naesens Reinout , Ariën Kevin K. , Vercauteren Koen , Vlieghe Erika , Beutels Philippe , Damme Pierre Van , Goossens Herman , Lion Eva , Suls Arvid , Lebeer Sarah , Laukens Kris , Meysman Pieter , Ogunjimi Benson
Published: November 2024

Unveiling the systemic effects of disease and health requires an holistic approach that has mainly revolved around well established, directly determinable molecular relationships such as the protein synthesis cascade and epigenetic mechanisms. In this study, involving 394 individuals, we found direct linkage of branches spanning human biological functions often not studied in conjunction, using clinical data, gut microbial abundances, blood immune cell repertoires, blood transcriptomic and blood T cell receptor data. Contrary to current paradigms, we demonstrate that immunotypes and enterotypes are orthogonal, likely fulfilling distinct roles in maintaining homeostasis, only bridged via the blood transcriptome. We also identified two distinct inflammatory profiles: the first driven by interferon signalling and the other characterised by non-viral, NF-kB and IL-6 markers. Lastly, we present compelling data showing strong associations of the Ruminococcaceae and Christensenellaceae bacteria with a healthy immunotype and transcriptomic pattern, highlighting their potential role in immune health.

Graphical Abstract