bluuebunny/crossref_metadata_2025 huggingface dataset:
Accuracy of Ultrasound-Guided Attenuation Parameter for Diagnosing Hepatic Steatosis
Authors: Yagobian Shiva D. , Dasyam Navya , Minervini Marta , Tublin Mitchell , Behari Jaideep , Furlan Alessandro
Published: December 2024

The purpose of this study is to investigate the diagnostic accuracy of a new noninvasive imaging technique, ultrasound-guided attenuation parameter (UGAP), in diagnosing hepatic steatosis. This single-center retrospective study included 81 UGAP studies performed to guide therapy between July 2022 and June 2023 at a large academic medical center. Patients with either liver biopsy or Magnetic resonance–based proton-density fat fraction (MRI-PDFF) within 12 months of US-UGAP imaging, irrespective of order, were included. Patient demographics, body mass index, liver function tests, UGAP values, MRI-PDFF values, and liver biopsy results were obtained from a review of the medical records. Presence of steatosis was defined as PDFF >5.2% or >5% of hepatocytes with steatosis at pathology. Area under the ROC curve (AUROC) was used to measure UGAP accuracy for the detection of hepatic steatosis with statistical significance P < 0.05. There was a significant positive correlation between UGAP and MRI-PDFF (r = 0.463; P < 0.001; confidence interval [CI]: 0.220;0.651). The AUROC to differentiate absence of steatosis (n = 21) from presence of steatosis (n = 32) for UGAP with MRI as the gold standard was 0.760 (P < 0.001; CI: 0.623;0.867). A UGAP value >0.66 had 78% sensitivity and 67% specificity to identify steatosis presence on MRI-PDFF. The AUROC to differentiate absence of steatosis (n = 11) from presence of steatosis (n = 21) for UGAP with pathology as the gold standard was 0.894 (P < 0.001; CI: 0.734;0.974). A UGAP value >0.57 had 100% sensitivity and 64% specificity to identify steatosis presence at pathology. UGAP is an accurate measure for detecting the presence of hepatic steatosis and may be a noninvasive method for metabolic dysfunction-associated steatotic liver disease diagnosis and follow-up.
Computational modeling reveals biological mechanisms underlying the whisker-flick EEG
Authors: Tharayil Joseph , Isbister James B. , Neufeld Esra , Reimann Michael
Published: December 2024

Whisker flick stimulation is a commonly used protocol to investigate somatosensory processing in rodents. Neural activity in the brain evoked by whisker flicks produces a characteristic EEG waveform recorded at the skull, known as a somatosensory evoked potential. In this paper, we usein silicomodeling to identify the neural populations that serve as sources and targets of the synaptic currents contributing to this signal (presynaptic and postsynaptic populations, respectively). The initial positive deflection of the EEG waveform is driven largely by direct thalamic inputs to Layer 2/3 and Layer 5 pyramidal cells, though interestingly, L5-L5 inhibition plays a modulatory role, reducing the amplitude and width of the deflection. This suggests that increasing thalamocortical connectivity and decreasing L5-L5 inhibition may be responsible for some of the changes observed in the EEG waveform over the course of development. The negative deflection is driven by a more complex mix of sources, including both thalamic and recurrent cortical connectivity. We demonstrate that small changes to the local connectivity of the circuit, particularly to perisomatic inhibitory targeting, can have an important impact on the recorded EEG, without substantially affecting firing rates, suggesting that EEG may be useful in constrainingin siliconeural models.
Growth-coupled continuous directed evolution by MutaT7 enables efficient and automated enzyme engineering
Authors: Deng Yijie , Etheridge Kai , Ran Xinping , Maurais Hannah E. , Sarpeshkar Rahul
Published: December 2024

Traditional directed evolution is limited by labor-intensive iterative steps and low-throughput selection and screening. To address these challenges, we developed a growth-coupled continuous directed evolution (GCCDE) approach, enabling automated and efficient enzyme engineering. By linking enzyme activity to bacterial growth and utilizing the MutaT7 system, GCCDE combinesin vivomutagenesis and high-throughput selection of superior enzyme variants in a single process. To validate this approach, we evolved the thermostable enzyme CelB to enhance β-galactosidase activity at lower temperatures while maintaining thermal stability. CelB activity was coupled to the growth ofE. coli, allowing variants with improved activity to metabolize lactose more efficiently and promote faster bacterial growth in a minimal medium. Using a continuous culture system, we achieved automated mutagenesis and real-time selection of over 109variants per culture. Integratingin vitroandin vivomutagenesis further increased genetic diversity, yielding CelB variants with significantly enhanced low-temperature activity compared to the wild type while preserving thermostability. DNA sequencing identified key mutations likely responsible for improved substrate binding and catalytic turnover. This GCCDE approach is broadly applicable for optimizing diverse enzymes, demonstrating the potential of automated continuous evolution for industrial and research applications.

IMPORTANCE

Enzyme engineering aims to develop enzymes with improved or novel traits, but traditional methods are slow and require repetitive manual steps. This study presents a faster, automated protein engineering approach. We utilized anin vivomutagenesis technique, MutaT7 tools, to induce mutations in living bacteria and established a direct link between enzyme activity and bacterial growth. A continuous culture setup was used to enable growth-coupled high-throughput selection of better-performing variants. Bacteria with improved enzymes grew faster, selecting superior variants without manual intervention. Using this method, we engineered CelB with better performance at lower temperatures while maintaining high-temperature stability. The approach is adaptable to many enzymes. It offers a faster and more efficient solution for enzyme engineering. This system enables high-throughput mutagenesis and selection simultaneously, showing the power of automated continuous evolution to advance enzyme engineering.
Innovative Carnitine-Fed Rats Model Reveals Resveratrol Butyrate Ester’s Multimechanistic Role in Reducing TMAO and Cardiovascular Risk
Authors: Hou Chih-Yao , Liu Cai-Sian , Shih Ming-Kuei , Bhat Asif Ali , Tain You-Lin , Hsieh Chang-Wei , Chen Yu-Wei , Chen Shin-Yu
Published: December 2024

BACKGROUND

Trimethylamine-N-oxide (TMAO), a metabolite produced from dietary carnitine through gut microbiota, is a recognized risk factor for cardiovascular disease (CVD). High-fat diets and carnitine supplementation exacerbate TMAO levels and related risks, making them valuable in developing experimental models for studying CVD. Resveratrol butyrate ester (RBE) and its monomer ED4 have shown potential in reducing TMAO levels and improving cardiovascular outcomes through metabolic and microbial modulation, but their precise mechanisms remain unclear.

METHODS AND RESULTS

A novel animal model was established using 36 male Sprague-Dawley rats fed a high-fat diet supplemented with carnitine to elevate serum TMAO levels, simulating dietary-induced CVD risks. Rats were divided into six groups: control diet (CN), high-fat diet (HFD), high-fat diet with carnitine (HFDC), HFDC with dimethylbutanol (HFDCB), CN with ED4 (CNM), and HFDC with ED4 (HFDCM). Physiological parameters, serum lipid profiles, SCFA levels, microbiota composition, and gene expression (OCTN2 and FMO3) were analyzed. ED4 supplementation reduced serum TMAO levels by upregulating OCTN2 expression, promoting urinary TMAO excretion, and restoring SCFA levels. ED4 also modulated gut microbiota, reducing TMA-producing bacteria (e.g.,Bacteroides), and improved cardiovascular markers, including reduced blood lipid levels and fat accumulation. While dimethylbutanol inhibited FMO3 expression to reduce TMAO, ED4 acted primarily through carnitine utilization and microbiota modulation. Both treatments enhanced urinary TMAO excretion and altered gut microbiota composition.

CONCLUSIONS

This study introduced an innovative animal model combining high-fat and carnitine-enriched diets to study TMAO-related cardiovascular risks. ED4 demonstrated multimechanistic effects in reducing TMAO levels and CVD risk factors by modulating gut microbiota, restoring SCFA levels, and enhancing carnitine metabolism. These findings highlight ED4’s therapeutic potential in cardiovascular protection and metabolic regulation. However, further research is needed to elucidate the molecular pathways underlying ED4’s effects on TMAO metabolism and its broader translational applications.
Molecular flexibility of high molecular weight hyaluronic acid measured by NMR has a profound effect on invasion of cancer cells
Authors: Bashtanova Uliana , Kuraite Agne , Rajan Rakesh , Duer Melinda J
Published: December 2024

Extracellular hyaluronic acid (HA) has been shown to be important in cancer; low molecular weight HA typically correlates with cancer progression, high molecular weight HA with homeostasis. Here we show that even high molecular weight HA can induce cancer cell migration when it is highly diluted. HA-induced cell signalling is primarily through HA binding to the cell surface receptor, CD44. We show by NMR spectroscopy that at high dilution, high molecular weight HA molecules access the conformations needed for strong binding to CD44 on the tens of nanosecond timescale, the relevant timescale for induction of CD44 signalling. We further show that, in contrast, at higher concentrations, HA molecules have insufficient flexibility for strong CD44 binding. The high dilution HA condition correlates with profound changes in brain cancer cell morphology and proteome which supports cancer cell invasion. We hypothesize that the flexibility of HA molecules is central to HA-mediated cell signalling and that this concept can explain previous observations that the outcome of HA-mediated signalling depends on the HA molecular weight. HA dilution leading to stronger HA signalling may be important in understanding the role that oedema plays in cancer recurrence after primary surgery.
A universal single-cell transcriptomics atlas of human lung decodes multiple pulmonary diseases
Authors: Wu Fanjie , Cai Wenhao , Tang Hai , Zheng Shikang , Zhang Haiyue , Chen Yixin , Han Yutong , Zhou Dingli , Wang Ruihan , Ye Mingli , You Renke , Chen Amin , Li Jiaqi , Zhang Xuegong , Li Weizhong
Published: December 2024

Human lung is a complex organ susceptible to various diseases. Single-cell transcriptomic studies provide rich data to targeting specific research questions. Here, we present uniLUNG, the largest lung transcriptomic cell atlas, comprising over 10 million cells across 20 disease states and healthy controls. We ensembled a universal hierarchical annotation framework and conducted a full benchmarking of data integration to define a standardized nomenclature and marker genes for lung cell types. Using uniLUNG, we identified Lym-monocyte and T-like B cells, new cell types in specific lung diseases, confirming their existence by comparing with external single-cell atlases. Additionally, we discovered the NSCLC-like SCLC subpopulation, a transitional malignant cell population associated with the transition from NSCLC to SCLC, which was validated and further characterized in spatial dimensions, revealing its complex role in tumour progression. Overall, uniLUNG represents a comprehensive range of human lung cell diversity, providing valuable data resources and a reliable foundation for lung single-cell research.

HIGHLIGHTS

The largest scRNA atlas for human lung covers 10 million cells from 20 lung states.

A four-level universal cell annotation framework encompasses 120 lung cell types.

Comprehensive benchmarking on 18 strategies guides data integration.

Specific distribution of Lym-monocytes and T-like B cells in specific lung diseases.

The NSCLC-like SCLC subpopulation in transitional events of malignant cells from NSCLC to SCLC.
Exposure to novel females increases fecundity in adult male prairie voles
Authors: Hurd Jessica A. , Watanabe Yurika L. , Toben Gracie J. , Ford Alexandra K. , Miller Craig A. , Sergott Casey E. , Kelley Dale , Donaldson Zoe R. , McCullagh Elizabeth A.
Published: December 2024

Social circuitry of the mammalian brain can influence male reproductive physiology. This often manifests as plasticity in sperm production or allocation, particularly in response to male-male competition. However, socially mediated testicular plasticity has not been investigated with respect to mating and parental strategy. Testis mass and sperm production of sexually naïve and female-exposed adult male individuals of three rodent species were compared: the socially monogamous and paternal prairie vole (Microtus ochrogaster), the promiscuous and non-paternal meadow vole (Microtus pennsylvanicus), and the promiscuous and non-paternal house mouse (Mus musculus). Monogamously paired prairie vole males exhibited significantly larger testes and greater sperm production than naïve prairie vole males. Comparatively, there were no significant differences between naïve and monogamously paired male meadow voles or mice. To investigate the role of olfactory cues for regulating this phenomenon in prairie voles, a group of naïve males exposed to soiled bedding from novel females was used. These males were more similar to paired males than to naïve males not exposed to novel female odors, demonstrating a strong role of the social olfactory system. Further, while the predictions of sperm competition theory (species with greater female promiscuity have larger testes than closely related species with less female promiscuity) are consistent between naïve meadow voles and prairie voles, the prediction does not hold for monogamously paired prairie voles and meadow voles. This demonstrates the complexity of internal social dynamics and reproductive pressures which socially monogamous paternal males face and the evolutionary adaptions that may develop in response.
Deciphering single-cell heterogeneity and cellular ecosystem dynamics during prostate cancer progression
Authors: Zhao Faming , Zeng Jianming , Chen Canping , Zhao Xiaofan , Zhang Tingting , Thomas George V. , Sears Rosalie C. , Alumkal Joshi J. , Moran Amy E. , Mills Gordon B. , Nelson Peter S. , Xia Zheng
Published: December 2024

Prostate cancer (PC) progresses from benign epithelium through pre-malignant lesions, localized tumors, metastatic dissemination, and castration-resistant stages, with some cases exhibiting phenotype plasticity under therapeutic pressure. However, high-resolution insights into how cell phenotypes evolve across successive stages of PC remain limited. Here, we present the Prostate Cancer Cell Atlas (PCCAT) by integrating ∼710,000 single cells from 197 human samples covering a spectrum of tumor stages. This comprehensive analysis dissects the cellular landscape and characterizes key cell types and molecular features that associate with PC progression and prognosis. In malignant cells, we highlight a distinctive profile denoted by high Major Histocompatibility Complex (MHC) expression, low Androgen Receptor (AR) activity, and enhanced stemness programs associated with enzalutamide resistance. Moreover, we reveal several cell states strongly correlated with PC progression and adverse prognosis, including lineage plasticity-like malignant cells (LPCs), neuroendocrine tumor cells, pericytes, and matrix cancer-associated fibroblasts (mCAFs). Furthermore, we uncover shared cell states that underpin the immune suppressive tumor microenvironment in advanced PC, including activated regulatory T cells, exhausted CD8+ T cells, and SPP1-expressing macrophages. Lastly, we pinpoint a spatial niche composed of mCAFs and SPP1-expressing macrophages localized near the tumor boundary in aggressive PC, which correlates with poor prognosis. Overall, our work provides a valuable resource and offers deeper insights into the diverse cell states, dynamics, and functional characteristics involved in PC progression at single-cell resolution.
Electric field-induced pore constriction in the human Kv2.1 channel
Authors: Mandala Venkata Shiva , MacKinnon Roderick
Published: December 2024

Gating in voltage-dependent ion channels is regulated by the transmembrane voltage. This form of regulation is enabled by voltage sensing domains (VSDs) that respond to transmembrane voltage differences by changing their conformation and exerting force on the pore to open or close it. Here we use cryogenic electron microscopy to study the neuronal Kv2.1 channel in lipid vesicles with and without a voltage difference across the membrane. Hyperpolarizing voltage differences displace the positively charged S4 helix in the voltage sensor by one helical turn (∼5 Å). When this displacement occurs, the S4 helix changes its contact with the pore at two different interfaces. When these changes are observed in fewer than four voltage sensors the pore remains open, but when they are observed in all four voltage sensors the pore constricts. The constriction occurs because the S4 helix, as it displaces inward, squeezes the right-handed helical bundle of pore lining S6 helices. A similar conformational change occurs upon hyperpolarization of the EAG1 channel. Therefore, while Kv2.1 and EAG1 are from distinct architectural classes of voltage-dependent ion channels, called domain-swapped and nondomain-swapped, the manner in which the voltage sensors gate their pores is very similar.

Significance Statement

Our ability to transmit signals across long distances rapidly – for example an instruction from the brain to the muscles in our fingers – depends on electrical impulses that travel along nerve cells. These electrical signals are mediated by membrane proteins called voltage-dependent ion channels. These channels have voltage sensors, which are domains that sense the voltage difference across the cell membrane and switch the channel on or off accordingly. Scientists discovered two architectural classes of voltage-dependent ion channels distinguished by the different ways the voltage sensors attach to the pore. This study shows that the two architectures are not very different after all because they both solve the problem of regulation of the pore by voltage sensors in the same way.
Chromatin regulator HELLS mediates SSB repair and responses to DNA alkylation damage
Authors: Joseph Joyous T , Wright Christine M , Peixoto Estanislao , Khan Asad , Li Yong , Neidigk Jason S Romero , Decker Olivia , Imtiaz Azait , Jones Brianna A , Zhang Yangfeng , Shiabata Etsuko , Gradilone Sergio A , Lewis Zachary A , Contreras-Galindo Rafael , Sen Arko , Dutta Anindya , Czaja Wioletta
Published: December 2024

The SNF2 family chromatin remodeler HELLS has emerged as an important regulator of cell proliferation, genome stability, and several cancer pathways. Significant upregulation of HELLS has been reported in 33 human cancer types. While HELLS has been implicated in DNA damage response, its function in DNA repair is poorly understood. Here we report a new regulatory link between HELLS and single-strand break (SSB) repair in cellular responses to DNA alkylation damage. We found that loss of HELLS impairs SSB repair, and selectively sensitizes cells to DNA alkylating agents and PARP inhibitors (PARPi). Furthermore, we found that HELLS is co-expressed with PARP1 in cancer cells, and its loss is synthetic lethal with homologous recombination deficiency (HRD). This work unveils new functions of HELLS in modulating SSB repair and responses to clinically relevant DNA alkylation damage, thus offering new insights into the potential therapeutic value of targeting HELLS in cancer.

Graphical Abstract
Solution biophysics identifies lipid nanoparticle non-sphericity, polydispersity, and dependence on internal ordering for efficacious mRNA delivery
Authors: Padilla Marshall S. , Shepherd Sarah J. , Hanna Andrew R. , Kurnik Martin , Zhang Xujun , Chen Michelle , Byrnes James , Joseph Ryann A. , Yamagata Hannah M. , Ricciardi Adele S. , Mrksich Kaitlin , Issadore David , Gupta Kushol , Mitchell Michael J.
Published: December 2024

Lipid nanoparticles (LNPs) are the most advanced delivery system currently available for RNA therapeutics. Their development has accelerated since the success of Patisiran, the first siRNA-LNP therapeutic, and the mRNA vaccines that emerged during the COVID-19 pandemic. Designing LNPs with specific targeting, high potency, and minimal side effects is crucial for their successful clinical use. However, our understanding of how the composition and mixing method influences the structural, biophysical, and biological properties of the resulting LNPs remains limited, hindering the development of LNPs. Our lack of structural understanding extends from the physical and compositional polydispersity of LNPs, which render traditional characterization methods, such as dynamic light scattering (DLS), unable to accurately quantitate the physicochemical characteristics of LNPs. In this study, we address the challenge of structurally characterizing polydisperse LNP formulations using emerging solution-based biophysical methods that have higher resolution and provide biophysical data beyond size and polydispersity. These techniques include sedimentation velocity analytical ultracentrifugation (SV-AUC), field-flow fractionation followed by multi-angle light scattering (FFF-MALS), and size-exclusion chromatography in-line with synchrotron small-angle X-ray scattering (SEC-SAXS). Here, we show that the LNPs have intrinsic polydispersity in size, RNA loading, and shape, and that these parameters are dependent on both the formulation technique and lipid composition. Lastly, we demonstrate that these biophysical methods can be employed to predict transfection in human primary T cells, intravenous administration, and intramuscular administration by examining the relationship between mRNA translation and physicochemical characteristics. We envision that employing solution-based biophysical methods will be essential for determining LNP structure-function relationships, facilitating the creation of new design rules for LNPs.
Neurons and molecules involved in noxious light sensation inCaenorhabditis elegans
Authors: Dunkel Eva , Aoki Ichiro , Gottschalk Alexander
Published: December 2024

Ultraviolet (UV) light is a danger to unpigmented organisms, inducing photodamage of cells and DNA. The transparent nematodeCaenorhabditis elegans, despite having no eyes, detects light and exhibits negative phototaxis in order to evade sunlight. UV absorption is detected by the photosensor protein LITE-1, that also responds to reactive oxygen species. We investigated which neurons express LITE-1 and act as noxious photosensors and how they transmit this sensation to the nervous system to evoke escape behavior. We identified the interneuron AVG as a main focus of LITE-1 function in mediating the noxious light evoked escape behavior, with minor roles of the interneuron PVT, the sensory ASK neurons and touch receptor neurons. AVG is activated by blue light, and also its optogenetic stimulation causes escape behavior. Signaling from AVG involves chemical neurotransmission, likely directly to premotor interneurons, and to other cells, by extrasynaptic signaling through the neuropeptide NLP-10. NLP-10 signaling is not required for the acute response, but for maintaining responsiveness to repeated noxious stimuli. The source of NLP-10 in this context is largely AVG, however, also other cells contribute, possibly PVT. This work uncovers entry points of sensory information to the neuronal circuits mediating behavioral responses to noxious UV/blue light.

Article Summary

C. eleganssenses noxious light and induces escape behavior to avoid damage. The photosensor protein LITE-1 mediates this sensation but understanding responsible neural circuits is incomplete. We identified neurons expressing LITE-1 and identify AVG as the main site of action. The neurotransmitter GABA plays a role in acuteness of the response. AVG, and other cells, need to release the neuropeptide NLP-10 to maintain responsiveness to repeated noxious stimuli. These findings help understanding theC. elegansphotophobic response and will guide future work delineating the precise circuit pathways, as an example of how similar photosensation can evoke protective behavior in invertebrates.
Evaluating Genome Sequencing Strategies: Trio, Singleton, and Standard Testing in Rare Disease Diagnosis
Authors: Kaschta Daniel , Post Christina , Gaass Franziska , Greiten Bianca , Liegmann Anna-Sophie , Gembicki Rebecca , Pozojevic Jelena , Meyenborg Michelle , Wehnert Janne , Schau-Römer Katharina , Rust Franka , Salewski Maj-Britt , Schulz Kristin , Sreenivasan Varun , Balachandran Saranya , Händler Kristian , Yumiceba Veronica , Rösler Laelia , Dalski Andreas , Hoff Kirstin , Hornig Nadine , Köhler Juliane , Arriens Vincent , Utermann-Thüsing Caroline , Roberts Kimberly , Murga Penas Eva Maria , Zühlke Christine , Kautza-Lucht Monika , Dittmar Maike , Hüning Irina , Hellenbroich Yorck , Hanker Britta , Berge Valerie , Birgel Friederike , Rosenstiel Philip , Franke Andre , Fuß Janina , Löscher Britt-Sabina , Franzenburg Sören , Papingi Dzhoy , van der Ven Amelie , Wilson Sandra , Woitschach Rixa , Lisfeld Jasmin , Volk Alexander E. , Herget Theresia , Schlein Christian , Möllring Anna , Hoffmann Birga , Poggenburg Imke , Nommels Henning , Al-Tawil Milad , Herrmann Gloria , Recke Andreas , Toutouna Louiza , Hiort Olaf , Margraf Nils , Gehring Bettina , Muhle Hiltrud , Bäumer Tobias , Harder Lana , Münchau Alexander , Brüggemann Norbert , Vater Inga , Caliebe Almuth , Nagel Inga , Spielmann Malte
Published: December 2024

Purpose

Short-read genome sequencing (GS) is a comprehensive genetic testing method capable of detecting multiple variant types. Despite its technical advantages, systemic comparisons of singleton GS (sGS), trio GS (tGS), and exome sequencing-based standard-of-care (SoC) in real-world diagnostics remain limited.

Methods

We systematically compared sGS, tGS, and SoC genetic testing in 448 patients with rare diseases in a blinded, prospective study. Three independent teams evaluated the diagnostic yield, variant detection capabilities, and clinical feasibility of GS as a first-tier test. Diagnostic yield was assessed through both prospective and retrospective analyses.

Results

In prospective analyses, tGS achieved the highest diagnostic yield for likely pathogenic/pathogenic variants (36.8%) in a newly trained team, outperforming the experienced SoC team (36.0%) and the sGS team (30.4%). Retrospective analyses, accounting for technical variant detection and team experience differences, reported diagnostic yields of 38.6% for SoC, 41.3% for sGS, and 42.2% for tGS. GS excelled in identifying deep intronic, non-coding, and small copy-number variants missed by SoC. Notably, tGS additionally identified three de novo variants classified as likely pathogenic based on recent GeneMatcher collaborations and newly published gene-disease association studies.

Conclusion

GS, particularly tGS, demonstrated superior diagnostic performance, supporting its use as a first-tier genetic test. sGS offers a cost-effective alternative, enabling faster, more efficient diagnoses for rare disease patients.
LEF1-AS1 deregulation in the peripheral blood of patients with persistent post-COVID symptoms
Authors: Madè Alisia , Piella Santiago Nicolas , Ranucci Marco , Gaetano Carlo , Renna Laura Valentina , Cardani Rosanna , Spinetti Gaia , Martelli Fabio
Published: December 2024

Long COVID denotes the persistence of symptoms after acute SARS-CoV-2 infection lasting for at least two months without another identifiable cause. Affecting an estimated 15% of COVID-19 patients, long COVID manifests in a wide range of symptoms. Despite extensive research on its one-year effects, limited data exist beyond 12 months. Due to the different manifestations of long COVID, its diagnosis can be challenging. Identifying potential mechanistic contributors and biomarkers would be highly valuable. Recent studies have highlighted the potential of noncoding RNAs (ncRNAs) as biomarkers for disease stratification in COVID-19. Specifically, we have recently identified miR-144-3p and a subset of lncRNAs as candidates for assessing disease severity and outcomes in COVID-19. This study extends such investigations to 98 long COVID patients recruited 18 months after hospitalization, exploring the relationship between circulating ncRNA expression and persistent symptoms. While miR-144-3p, HCG18, and lncCEACAM21 expression did not differ between symptomatic and asymptomatic patients, LEF1-AS1 was downregulated in peripheral blood mononuclear cells (PBMCs) of symptomatic patients. Of note, multiple LEF1-AS1 isoforms and LEF1 sense transcript levels were reduced and negatively correlated with relevant clinical markers. While further studies are needed, our discoveries offer new perspectives for the diagnosis and management of persistent long COVID.
PrimalScheme: open-source community resources for low-cost viral genome sequencing
Authors: Kent Chris , Smith Andrew D , Tyson John , Stepniak Dominika , Kinganda-Lusamaki Eddy , Lee Tracy , Weaver Mia , Sparks Natalie , Landsdowne Lauren , Wilkinson Sam AJ , Brier Thomas , Colquhoun Rachel , O'Toole Aine N , Kingebeni Placide Mbala , Goodfellow Ian G , Rambaut Andrew , Loman Nicholas James , Quick Joshua
Published: December 2024

Viral genome sequencing using the ARTIC protocol has been a vital tool for understanding the spread of epidemics including Ebola, Zika, COVID-19 and Mpox and has seen widespread adoption due to its low cost and high sensitivity. Here, we describe PrimalScheme, an open-source toolkit and website that allows users to easily design primer schemes for amplicon sequencing of viruses and has generated over 67,000 primer schemes for a global community since 2017. In January 2020, PrimalScheme was used to rapidly generate a primer scheme for SARS-CoV-2, with primer pools distributed to researchers from 44 countries to help scale-up genomic surveillance efforts. Overall, these primers were used to generate an estimated 18M genome sequences and the protocols were viewed online ~250K times. To complement PrimalScheme, we have built PrimalScheme Labs, a scheme repository which allows users to find and share primers schemes as well as establishing a set of data standards. Through improvements to the primer design process, including the use of discrete primer clouds, we have expanded the use of amplicon sequencing to include diverse virus species. We demonstrate the utility of this approach through a high diversity pan-genotype Measles virus (MeV) scheme. We also demonstrate its use on a high sensitivity, short amplicon Monkeypox virus (MPXV) scheme with over 1000 primers, showing high genome recovery on low-titre clinical samples. These developments have implications for sequencing from samples such as wastewater, for genomic surveillance of endemic pathogens and in preparing for future pandemics.
Same-Slide Spatial Multi-Omics Integration Reveals Tumor Virus-Linked Spatial Reorganization of the Tumor Microenvironment
Authors: Yeo Yao Yu , Chang Yuzhou , Qiu Huaying , Yiu Stephanie Pei Tung , Michel Hendrik A , Wu Wenrui , Jin Xiaojie , Kure Shoko , Parmelee Lindsay , Luo Shuli , Cramer Precious , Lee Jia Le , Wang Yang , Yeung Jason , Ahmar Nourhan El , Simsek Berkay , Mohanna Razan , Van Orden McKayla , Lu Wesley , Livak Kenneth J , Li Shuqiang , Shahryari Jahanbanoo , Kingsley Leandra , Al-Humadi Reem N , Nasr Sahar , Nkosi Dingani , Sadigh Sam , Rock Philip , Frauenfeld Leonie , Kaufmann Louisa , Zhu Bokai , Basak Ankit , Dhanikonda Nagendra , Chan Chi Ngai , Krull Jordan , Cho Ye Won , Chen Chia-Yu , Lee Jia Ying Joey , Wang Hongbo , Zhao Bo , Loo Lit-Hsin , Kim David M , Boussiotis Vassiliki , Zhang Baochun , Shalek Alex K , Howitt Brooke , Signoretti Sabina , Schürch Christian M , Hodi F Stephan , Burack W Richard , Rodig Scott J , Ma Qin , Jiang Sizun
Published: December 2024

The advent of spatial transcriptomics and spatial proteomics have enabled profound insights into tissue organization to provide systems-level understanding of diseases. Both technologies currently remain largely independent, and emerging same slide spatial multi-omics approaches are generally limited in plex, spatial resolution, and analytical approaches. We introduce IN-situ DEtailed Phenotyping To High-resolution transcriptomics (IN-DEPTH), a streamlined and resource-effective approach compatible with various spatial platforms. This iterative approach first entails single-cell spatial proteomics and rapid analysis to guide subsequent spatial transcriptomics capture on the same slide without loss in RNA signal. To enable multi-modal insights not possible with current approaches, we introduce k-bandlimited Spectral Graph Cross-Correlation (SGCC) for integrative spatial multi-omics analysis. Application of IN-DEPTH and SGCC on lymphoid tissues demonstrated precise single-cell phenotyping and cell-type specific transcriptome capture, and accurately resolved the local and global transcriptome changes associated with the cellular organization of germinal centers. We then implemented IN-DEPTH and SGCC to dissect the tumor microenvironment (TME) of Epstein-Barr Virus (EBV)-positive and EBV-negative diffuse large B-cell lymphoma (DLBCL). Our results identified a key tumor-macrophage-CD4 T-cell immunomodulatory axis differently regulated between EBV-positive and EBV-negative DLBCL, and its central role in coordinating immune dysfunction and suppression. IN-DEPTH enables scalable, resource-efficient, and comprehensive spatial multi-omics dissection of tissues to advance clinically relevant discoveries.
Unexpected Activities of CYP152 Peroxygenases towards Non-carboxylic Substrates Reveal Novel Substrate Recognition Mechanism and Catalytic Versatility
Authors: Jiang Yuanyuan , Gong Piqian , Li Zhong , Li Yuxuan , Wang Binju , Peng Wei , Gao Xiang , Li Shengying
Published: December 2024

Exploring and exploiting the catalytic promiscuity of enzymes is a central topic and captivating challenge in enzymology. CYP152 peroxygenases are attractive biocatalysts for diverse reactions under mild conditions using H2O2as cofactor. However, their substrate scope is limited by a carboxyl group required for acid-base catalysis, following the well-accepted principle that heme-dependent H2O2-utilizing enzymes employ a carboxyl group within their active sites to facilitate H2O2activation. Herein, we reveal for the first time that several CYP152 family members can directly degrade various aromatic pollutants without any carboxyl group, exhibiting novel aromatic hydroxylation and dehalogenation activities. Through crystal structure analysis, isotope tracing experiments, and QM/MM calculations, we elucidate that the phenolic hydroxyl group activated by electron-withdrawing substituent(s) functionally replaces the carboxyl group, forming hydrogen bonds with the conserved arginine leading to Compound I formation. The oxygen atom of the newly formed hydroxyl group originates from water, bypassing the conventional oxygen rebound step. These findings provide first insights into the mechanisms of P450 peroxygenases towards non-carboxylic substrates, expanding our knowledge of biological C-H activation and C-halogen bond cleavage beyond canonical P450 reactions. This discovery holds immense potential for harnessing these enzymes in innovative strategies for industrial biocatalysis and environmental remediation.
MCL-1 regulates cellular transitions during oligodendrocyte development
Authors: Gil Melanie , Hanna Marina R , Gama Vivian
Published: December 2024

Oligodendrocytes are the myelinating cells of the central nervous system. Regulation of the early stages of oligodendrocyte development is critical to the function of the cell. Specifically, myelin sheath formation is an energetically demanding event that requires precision, as alterations may lead to dysmyelination. Recent work has established that fatty acid β-oxidation is required for the function of oligodendrocytes. We have shown that MCL-1, a well-characterized anti-apoptotic protein, is required for the development of oligodendrocytesin vivo. Further, it was recently uncovered that MCL-1 regulates long- chain fatty acid β-oxidation through its interaction with acyl-CoA synthetase long-chain family member 1 (ACSL1), an enzyme responsible for the conversion of long-chain fatty acids into acyl-CoA. Here, we introduce anin vitrosystem to isolate human stem cell- derived oligodendrocyte progenitor cells and investigate the involvement of MCL-1 during human oligodendrocyte development. Using this system, we pharmacologically inhibited MCL-1 in oligodendrocyte progenitor cells (OPCs) to elucidate the non-apoptotic function of the protein at this developmental stage. Additionally, we used a motor neuron co-culture system to investigate the downstream effects that MCL-1 inhibition has at later developmental stages when oligodendrocytes begin to contact axons and generate myelin basic protein. We demonstrate that the mitochondrial network changes in human oligodendrocyte development resemble those reportedin vivo. Our findings point to MCL-1 as a critical factor essential at the OPC stage for proper oligodendrocyte morphogenesis.
Heterologous Expression of the carbon monoxide dehydrogenase Gene fromClostridiumsp. to Enhance Acetic Acid and Alcohol Production from CO₂
Authors: Tharak Athmakuri , Suresh G , Kaveti Sreeram , Jain Nishant , Mohan S Venkata
Published: December 2024

This study evaluates the performance of carbon monoxide dehydrogenase (codh) embedded strains in bench-scale microbial electrochemical systems (MES) for CO₂ reduction to biofuels and biochemicals. CO2fermentation efficiency was evaluated by comparing the wild-typeClostridium acetobutylicum(Wild), a negative controlE. colistrain lacking thecodhgene (NC-BL21), and engineeredE. colistrain (Eng) alone and with IPTG induction (Eng+IPTG). Four electrochemical systems were used viz. Wild+E, NC-BL21+E, Eng+E, and Eng+IPTG+E, with a poised potential of 0.6 V applied to the working electrode. CO₂ and bicarbonate were supplemented to a total inorganic carbon (IC) concentration of 40 g/L, with a retention time of 60 h. The engineered strains demonstrated enhanced metabolic performance compared to the wild-type and negative control strains, yielding a maximum formic acid concentration of 2.1 g/L and acetic acid concentration of 7.8 g/L under the Eng+IPTG condition. Ethanol yield was highest at 3.9 g/L under the Eng+IPTG+E condition, substantially exceeding the 2.4 g/L acetic acid yield observed in the wild-type strain. The engineered strains showed superior cumulative yields (0.4075 g/g), improvedcodhcharge flux stability (60 vs. 5 for Wild), and upregulated expression of genes in the Wood-Ljungdahl pathway. Bioelectrochemical performance analysis demonstrated elevated reductive catalytic currents, enhanced CO₂ reduction, and optimal charge transfer kinetics. This study highlights the effectiveness of genetic and process engineering, particularlycodhoverexpression and IPTG induction, in optimizing microbial electrosynthesis for biofuel and biochemical production from C1 gases.
Restriction on Ku’s Inward Translocation Caps Telomere Ends
Authors: Mattarocci Stefano , Baconnais Sonia , Roisné-Hamelin Florian , Pobiega Sabrina , Alibert Olivier , Morin Vincent , Deshayes Alice , Veaute Xavier , Ropars Virginie , Chevreuil Maelenn , Mehringer Johannes , Busso Didier , Mazon Gerard , Fernandez Varela Paloma , Le Cam Éric , Charbonnier Jean-Baptiste , Cuniasse Philippe , Marcand Stéphane
Published: December 2024

Safeguarding chromosome ends against fusions via nonhomologous end joining (NHEJ) is essential for preserving genome integrity. Paradoxically, the conserved NHEJ core factor Ku binds telomere ends. How it is prevented from promoting NHEJ remains unclear, as does the mechanism that allows Ku to coexist with telomere-protective DNA binding proteins, e.g., Rap1 inSaccharomyces cerevisiae. Here, we reveal a direct role for Rap1 in the inhibition of Ku’s NHEJ function at telomeres. A single Rap1 molecule bound near a DNA end inhibits NHEJin vivowithout disrupting Ku presence. Consistent with this, Rap1 and Ku form a complex on short DNA duplexesin vitro. Cryo-EM and molecular modelling analysis of this complex shows that Rap1 obstructs Ku’s inward translocation on DNA – an essential step for NHEJ at broken ends. Nanopore sequencing of telomere fusions confirms the importance of this pathway in protecting native telomere ends. Collectively, our findings uncover a mechanism of telomere end protection mediated by restricting Ku’s inward translocation, a functional switch that prevents promiscuous NHEJ repair at telomeres.
Spatially ordered zygotic genome activation fulfills embryo quality control
Authors: Qian Wenchao , Chen Hui , Lee Hongju , Good Matthew C.
Published: December 2024

Early embryo development features autonomous, maternally-driven cell divisions that self- organize the multicellular blastula or blastocyst tissue. Maternal control cedes to the zygote starting with the onset of widespread zygotic genome activation (ZGA), which is essential for subsequent cell fate determination and morphogenesis. Intriguingly, although the onset of ZGA is highly regulated at the level of an embryo, it can be non-homogenous and precisely patterned at the single-cell level. We previously demonstrated a stereotyped spatial and temporal ordering of ZGA in a model vertebrate embryo. Unknown, however, was whether this precise ZGA patterning was required for development. To address this fundamental question, we devised a strategy to spatially control cell divisions in the embryo that perturb blastula embryo organization. We demonstrate the feasibility of spatially inverting the cell size pattern of embryos and find that these inverted embryos undergo a flipped pattern of ZGA. Mispatterned ZGA along the animal-vegetal axis causes embryo apoptosis, revealing that gastrula embryos have a built-in quality control system to sense inappropriate ZGA patterning, including regional defects in transcriptional onset. The quality control response is non-autonomous which may depend on anti-apoptotic signals that repress cell death outside of the animal hemisphere. These results reveal the requirement of properly patterned ZGA for normal development and the existence of an embryo quality control response exquisitely tuned to the spatial and temporal ordering of genome activation and zygotic gene expression.
Structural maturation of the matrix lattice is not required for HIV-1 particle infectivity
Authors: Chen Long , Hikichi Yuta , Rey Juan S. , Akil Caner , Zhu Yanan , Veler Hana , Shen Yao , Perilla Juan R. , Freed Eric O. , Zhang Peijun
Published: December 2024

HIV-1 assembly is initiated by the binding of Gag polyproteins to the inner leaflet of the plasma membrane, mediated by the myristylated matrix (MA) domain of Gag. Subsequent to membrane binding, Gag oligomerizes and buds as an immature, non-infectious virus particle, which, upon cleavage of the Gag precursor by the viral protease, transforms into a mature, infectious virion. During maturation, the MA lattice underlying the viral membrane undergoes a structural rearrangement and the newly released capsid (CA) protein forms a mature capsid that encloses the viral genome. While it is well established that formation of the mature capsid is essential to particle infectivity, the functional role of MA structural maturation remains unclear. Here, we examine MA maturation of an MA triple mutant, L20K/E73K/A82T, which exhibits distinct biochemical behaviours. The L20K/E73K/A82T mutant is a revertant derived by propagating the L20K mutant, which exhibits reduced infectivity and increased association of the Gag polyprotein with membranes. L20K/E73K/A82T replicates similarly to wild type but retains the increased Gag membrane binding properties of L20K. L20K/E73K/A82T MA also sediments to high-density fractions in sucrose gradients after detergent treatment under conditions that fully solubilize WT MA, suggesting enhanced MA-MA interactions. Cryo-electron tomography with subtomogram averaging reveals that the immature MA lattice of L20K/E73K/A82T closely resembles the wild type. However, mature virions of the triple mutant lack a detectable MA lattice, in stark contrast to both the wild type and L20K mutant. All-atom molecular dynamics simulations suggest that this absence results from destabilized inter-trimer interactions in the mature L20K/E73K/A82T MA. Furthermore, introducing additional mutations designed to disrupt the mature MA lattice does not impair particle infectivity. These findings suggest that an ordered, membrane-associated mature MA lattice is not essential for HIV-1 infectivity, providing new insights into the structural plasticity of the matrix during maturation and its functional role in the viral lifecycle.
Cell-associated viral community composition and its functional potential in a dimictic lake on the Canadian Shield
Authors: Collis Cody C. , Tromas Nicolas , Nissimov Jozef I.
Published: December 2024

The Turkey Lake Watershed (TLW), located in Northern Ontario, forms a cascading lake system which flows and eventually connects into Lake Superior. Historically, the TLW has been used extensively for studying the effects of acid rain and other aspects related to climate change. While microbes are integral components of all aquatic habitats, investigations into TLW’s microbial community are still relatively limited, and there are no studies that have specifically investigated the viral fraction of that community. One reason for this oversight is the difficulty in obtaining enough virus biomass from a remote location such as the TLW, which could then be used effectively in downstream sequencing and analysis. To overcome this, we investigated viral signatures associated with microbial cells, which requires less biomass at the time of sample collection. We performed a seasonal metagenomics study of Big Turkey Lake (BTL), one of the main lakes in the TLW, to examine cell-associated viral communities in the summer of 2018, spring-autumn of 2019, and winter of 2020. We evaluated viral diversity, predicted potential host identity, and performed an analysis of auxiliary metabolic genes (AMGs) to gain additional functional insights into the detected viruses and their potential impacts. Through our analysis, we reveal a dynamic viral community across seasons. The majority of viral contigs (56-70%) belonged to unclassified viruses. Of those that were classified, ∼2-25% were assigned to the recently abolishedCaudoviralesorder (nowCaudoviricetesclass), and∼1.5-19% were in the viral familyPhycodnaviridae;i.e., viruses which normally infect eukaryotic algae. Six draft viral metagenome-assembled genomes (vMAGs) with a genome size range of 11-45 kbp were also reconstructed and identified asCaudovirales. Interestingly, AMGs for lipid metabolism were only detected in the winter, suggestive of a potential niche specific role for these AMGs in Big Turkey Lake. Collectively, these insights provide an essential footing for further work on the role of viruses in this important freshwater habitat.