bluuebunny/crossref_metadata_2025 huggingface dataset:
E41K Mutation Activates Bruton’s Tyrosine Kinase by Stabilizing an Inositol Hexakisphosphate dependent Invisible Dimer
Authors: Chowdhury Subhankar , Chakraborty Manas Pratim , Roy Swarnendu , Gangopadhyay Kaustav , Das Rahul
Published: January 2024

Bruton’s tyrosine kinase (BTK) regulates diverse cellular signaling of the innate and adaptive immune system in response to microbial pathogens. Downregulation or constitutive activation of BTK is reported in patients with autoimmune diseases or various B-cell leukemias. BTK is a multidomain protein tyrosine kinase that adopts an Src-like autoinhibited conformation maintained by the interaction between the kinase and PH-TH domains. The PH-TH domain plays a central role in regulating BTK function. The BTK is activated by binding to PIP3at the plasma membrane upon stimulation by the B-cell receptor (BCR). The PIP3binding allows dimerization of the PH-TH domain and subsequent transphosphorylation of the activation loop. Alternatively, a recent study shows that the multivalent T-cell-independent (TI) antigen induces BCR response by activating BTK independently of PIP3binding. It was proposed that a transiently stable IP6-dependent PH-TH dimer may activate BTK during BCR activation by the TI antigens. However, no IP6-dependent PH-TH dimer has been identified yet. Here, we investigated a constitutively active PH-TH mutant (E41K) to determine if the elusive IP6-dependent PH-TH dimer exists. We showed that the constitutively active E41K mutation activates BTK by stabilizing the IP6-dependent PH-TH dimer. We observed that a downregulating mutation in the PH-TH domain (R28H) linked to X-linked agammaglobulinemia impairs BTK activation at the membrane and in the cytosol by preventing PH-TH dimerization. We conclude that the IP6dynamically remodels the BTK active fraction between the membrane and cytoplasm. Stimulating with IP6increases the cytosolic fraction of the activated BTK.
The interferon-ɣ inducible factor 16 (IFI16) restricts adeno-associated virus serotype 2 (AAV2) transduction in an immune-modulatory independent way
Authors: Sutter Sereina O. , Tobler Kurt , Seyffert Michael , Lkharrazi Anouk , Zöllig Joël , Schraner Elisabeth M. , Vogt Bernd , Büning Hildegard , Fraefel Cornel
Published: January 2024

We determined the transcription profile of AAV2-infected primary human fibroblasts. Subsequent analysis revealed that cells respond to AAV infection through changes in several significantly affected pathways including cell cycle regulation, chromatin modulation, and innate immune responses. Various assays were performed to validate selected differentially expressed genes and confirmed not only the quality but also the robustness of the raw data. One of the genes upregulated in AAV2 infected cells was the interferon-ɣ inducible factor 16 (IFI16). IFI16 is known as a multifunctional cytosolic and nuclear innate immune sensor for double-stranded, as well as single-stranded DNA, exerting its effects through various mechanisms, such as interferon response, epigenetic modifications, or transcriptional regulation. IFI16 thereby constitutes a restriction factor for many different viruses amongst them, as shown here, AAV2 and thereof derived vectors. Indeed, the post-transcriptional silencing ofIFI16significantly increased AAV2 transduction efficiency, independent of the structure of the virus/vector genome. We also show that IFI16 exerts its inhibitory effect on AAV2 transduction in an immune-modulatory independent way, by interfering with Sp1-dependent transactivation of wild-type AAV2 and AAV2 vector promoters.

IMPORTANCE

Adeno-associated virus (AAV) vectors are among the most frequently used viral vectors for gene therapy. The lack of pathogenicity of the parental virus, the long-term persistence as episomes in non-proliferating cells, and the availability of a variety of AAV serotypes differing in their cellular tropism are advantageous features of this biological nanoparticle. To deepen our understanding of virus-host interactions, especially in terms of innate immune responses, we present here the first transcriptome analysis of AAV serotype 2 (AAV2) infected human primary fibroblasts. Our findings indicate that the interferon-ɣ inducible factor 16 (IFI16) acts as an antiviral factor in AAV2 infection and AAV2 vector-mediated cell transduction in an immune-modulatory independent way by interrupting the Sp1-dependent gene expression from viral or vector genomes.
Whole-genome phylogenomics of the tinamous (Aves: Tinamidae): comparing gene tree estimation error between BUSCOs and UCEs illuminates rapid divergence with introgression
Authors: Musher Lukas J. , Catanach Therese A. , Valqui Thomas , Brumfield Robb T. , Aleixo Alexandre , Johnson Kevin P. , Weckstein Jason D.
Published: January 2024

Incomplete lineage sorting (ILS) and introgression increase genealogical discordance across the genome, which complicates phylogenetic inference. In such cases, identifying orthologs that result in gene trees with low estimation error is crucial because phylogenomic methods rely on accurate gene histories. We sequenced whole genomes of tinamous (Aves: Tinamidae) to reconstruct their interrelationships and dissect the sources of gene tree and species-tree discordance. We compared results based on five ortholog sets: (1) coding genes (BUSCOs), (2) ultraconserved elements (UCEs) with short flanking regions, (3) UCEs with intermediate flanks, (4) UCEs with long flanks, and (5) UCEs mapped to the Z-chromosome. We hypothesized that orthologs with more phylogenetically informative sites would result in more accurate species trees because the resulting gene trees contain lower stochastic error. Consistent with our hypothesis (and a large body of theory), we found that long UCEs had the most informative sites and lowest rates of error. Surprisingly, BUSCO gene trees contained high error compared to long UCEs, despite having many informative sites. Unlike UCEs, BUSCO gene sequences showed a positive association between the proportion of informative sites and gene tree error. Thus, the underlying properties of molecular evolution differ between BUSCO and UCE datasets, and these differences should be considered when selecting loci for phylogenomic analysis. Importantly, these results indicate stochastic error is not driving inaccurate gene tree estimation for BUSCO loci, instead suggesting a more problematic impact of systematic error in this data-type. Still, species trees from different datasets were mostly congruent. Only one clade, which has a history of ILS and introgression, exhibited substantial species-tree discordance across the different data sets. We suggest that agreement between the Z-chromosome dataset and that of long UCEs lends support to this topology because the Z-chromosome is expected to contain low rates of ILS and faster coalescent times due its relatively smaller effective population size. Overall, we present the most complete phylogeny for tinamous to date, identify an unrecognized species, and provide a case study for species-level phylogenomic analysis using whole-genomes.
Hybrid immunity to SARS-CoV-2 arises from serological recall of IgG antibodies distinctly imprinted by infection or vaccination
Authors: Voss William N. , Mallory Michael A. , Byrne Patrick O. , Marchioni Jeffrey M. , Knudson Sean A. , Powers John M. , Leist Sarah R. , Dadonaite Bernadeta , Townsend Douglas R. , Kain Jessica , Huang Yimin , Satterwhite Ed , Castillo Izabella N. , Mattocks Melissa , Paresi Chelsea , Munt Jennifer E. , Scobey Trevor , Seeger Allison , Premkumar Lakshmanane , Bloom Jesse D. , Georgiou George , McLellan Jason S. , Baric Ralph S. , Lavinder Jason J. , Ippolito Gregory C.
Published: January 2024

We used plasma IgG proteomics to study the molecular composition and temporal durability of polyclonal IgG antibodies triggered by ancestral SARS-CoV-2 infection, vaccination, or their combination ("hybrid immunity"). Infection, whether primary or post-vaccination, mainly triggered an anti-spike antibody response to the S2 domain, while vaccination predominantly induced anti-RBD antibodies. Immunological imprinting persisted after a secondary (hybrid) exposure, with >60% of the ensuing serological response originating from the initial antibodies generated during the first exposure. We highlight one instance where hybrid immunity arising from breakthrough infection resulted in a marked increase in the breadth and affinity of a highly abundant vaccination-elicited plasma IgG antibody, SC27. With an intrinsic binding affinity surpassing a theoretical maximum (KD< 5 pM), SC27 demonstrated potent neutralization of various SARS-CoV-2 variants and SARS-like zoonotic viruses (IC50∼0.1–1.75 nM) and provided robust protectionin vivo. Cryo-EM structural analysis unveiled that SC27 binds to the RBD class 1/4 epitope, with both VH and VL significantly contributing to the binding interface. These findings suggest that exceptionally broad and potent antibodies can be prevalent in plasma and can largely dictate the nature of serological neutralization.

HIGHLIGHTS

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Infection and vaccination elicit unique IgG antibody profiles at the molecular level

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Immunological imprinting varies between infection (S2/NTD) and vaccination (RBD)

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Hybrid immunity maintains the imprint of first infection or first vaccination

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Hybrid immune IgG plasma mAbs have superior neutralization potency and breadth
Minimizing variability in the filament middle cerebral artery occlusion model in C57BL/6 mice by surgical optimization - the PURE-MCAo Model
Authors: Yoshimura Sodai , Dorok Maximilian , Mamrak Uta , Wehn Antonia , Krestel Eva , Khalin Igor , Plesnila Nikolaus
Published: January 2024

BACKGROUND

In the intraluminal filament middle cerebral artery occlusion (fMCAo) model, there is considerable variability in infarct volumes, especially in C57BL/6 mice, which often lack the P1 segment of the posterior cerebral artery (PCA) and therefore develop not only MCA but also PCA area infarcts after fMCAo. Another factor contributing to infarct volume variability is collateral flow to the MCA area. The aim of this study was to establish an optimal surgical method to reduce the infarct volume variability in C57BL/6 mice.

METHODS

C57BL/6 mice were subjected to 60 min of fMCAo with cerebral blood flow monitored by laser Doppler fluxmetry. The influence of the common carotid artery (CCA) ligation, filament morphology, and the pterygopalatine artery (PPA) ligation on lesion volume and neurological severity score 24 hours after reperfusion were assessed.

RESULT

The use of filaments with appropriate length of coating and ligation of the PPA while maintaining perfusion of the CCA prevented the development of infarcts in the PCA area, resulted in pure MCA infarcts (68.3±14.5mm3, 26.1±3.6% of the hemisphere with Swanson’s correction) and reduced the variability of infarct volumes by more than half to 13.9% of the standard deviation divided by mean.

CONCLUSIONS

Using improved surgical methods with suitable filaments to induce MCA occlusion in mice, we were able to producePCA area-unaffectedreproducible infarcts exclusively in the MCA area with reduced variability (PURE-MCAo). Our results may thus help to increase the reproducibility of the fMCAo model and reduce the number of animals required in preclinical stroke research.
Towards a Buruli Ulcer Rapid Diagnostic Test that Targets Mycolactone
Authors: Siirin Marina , Pedram Bijan , Gonzalez-Moa Maria J. , Warryn Louisa , Yotsu Rie , Saunders Jean M. , Saunders Aaron E. , Baldwin Richard K. , Porter Jessica L. , Stinear Timothy P. , Cruz-Mata Israel , de Souza Dziedzom Komi , Pluschke Gerd , Biamonte Marco A.
Published: January 2024

We report the development of a prototype rapid diagnostic test for Buruli ulcer, an ulcerative necrotizing skin disease caused byM. ulcerans. The test was designed to detect mycolactone, a metabolite unique toM. ulcerans. The chief technical challenge was to develop a simple workflow to extract trace amounts of mycolactone from wound exudates collected with a swab and, after a concentration step, to visualize the mycolactone by means of a lateral flow assay. This was achieved by utilizing a mouse monoclonal antibody specific for mycolactone and magnetic gold nanoshells. The latter are a novel class of reporter particles consisting of a ferrite core, a silica gel middle layer that serves to decrease the overall density of the nanoparticle and facilitate its resuspension in aqueous media, and an outer layer of gold, which provides a dark coloration through plasmon resonance effects. These nanoparticles, once conjugated to the anti-mycolactone antibody, enable the immunomagnetic concentration of the targeted analyte and its detection by lateral flow assay. The test procedure can be conducted within 2 hours with a magnetic rack and no powered instrumentation is required. The test can detect as little as 3.5–7 ng of mycolactone collected on a swab.

Author summary

Buruli ulcer is a neglected tropical disease that affects the poorest of the poorest in Africa. Even young people can harbor large ulcers, with raw flesh directly exposed. While antibiotic treatments exist, there are no simple methods to diagnose the disease. Here, we attempted to detect mycolactone, a small molecule produced by the bacteria that causes Buruli ulcer. One difficulty was the sample type to be used, as the open wounds are sampled with a swab. The question was how to extract mycolactone from the swab and then detect it by means of a rapid diagnostic test, without using sophisticated equipment. We tried to answer this question by combining monoclonal antibodies specific for mycolactone and novel magnetic nanoparticles to concentrate and visualize mycolactone with a rapid test.
Hemoglobin-Albumin-Lymphocyte-Platelet (HALP) score as a predictive value of Incidental prostate cancer for patients going for Transurethral resection of the prostate (TURP): A Single Center Study
Authors: Bendari Ahmed
Published: January 2024

Aims

Prostate cancer is a significant health concern worldwide, and early detection is crucial for effective treatment. This study aimed to investigate the role of the Hemoglobin-Albumin-Lymphocyte-Platelet (HALP) score in detecting prostate cancer in patients undergoing Transurethral Resection of the Prostate (TURP). Additionally, comprehensive analysis was performed to explore clinical parameters associated with incidentally diagnosed prostate cancer post-TURP.

Methods

A total of 131 patients with symptomatic bladder outlet obstruction who underwent TURP were included in the study. The patients were divided into two groups: those with benign prostatic hyperplasia (BPH) and those with invasive prostatic carcinoma. The IPC group consisted of patients with both low-grade and high-grade IPC determined by Gleason score. Demographic data, including age, race, medical history, body mass index, smoking and alcohol status, and family history of prostate cancer, were evaluated. Postoperative measurement of specimen weight and prostate-specific antigen (PSA) levels were also analyzed.

Result

Results revealed that approximately 50% of patients had BPH, while the remaining 50% had IPC. Patients with IPC, particularly high-grade IPC, had significantly higher PSA levels and lower resected specimen weight compared to those with BPH. The HALP score, which incorporates hemoglobin, albumin, lymphocyte, and platelet levels, showed promise as a discriminatory tool for distinguishing between BPH and IPC, as well as between high-grade IPC and BPH/low-grade IPC. Logistic regression analysis identified increased PSA levels, decreased HALP score, and smaller specimen weight as independent predictive factors for IPC after TURP. Notably, HALP score was the only significant independent predictive factor associated with high-grade IPC.

Conclusion

These findings contribute to the understanding of risk factors and diagnostic tools for incidentally detected prostate cancer in patients with bladder outlet obstruction undergoing TURP. The HALP score, along with PSA levels and specimen weight, can aid in the early detection and management of prostate cancer. Further research is warranted to validate these findings and explore the clinical utility of the HALP score in predicting prostate cancer outcomes.
Comparison of the performance of multiple whole-genome sequence-based tools for the identification ofBacillus cereus sensu strictobiovar Thuringiensis
Authors: Chung Taejung , Salazar Abimel , Harm Grant , Johler Sophia , Carroll Laura M. , Kovac Jasna
Published: January 2024

TheBacillus cereus sensu stricto(s.s.) species comprises strains of biovarThuringiensis(Bt) known for their bioinsecticidal activity, as well as strains with foodborne pathogenic potential.Btstrains are identified (i) based on the production of insecticidal crystal proteins also known as Bt toxins or (ii) based on the presence ofcry,cyt, andvipgenes, which encode Bt toxins. Multiple bioinformatics tools have been developed for the detection of crystal protein-encoding genes based on whole-genome sequencing (WGS) data. However, the performance of these tools is yet to be evaluated using phenotypic data. Thus, the goal of this study was to assess the performance of four bioinformatics tools for the detection of crystal protein-encoding genes. The accuracy of sequence-based identification ofBtwas determined in reference to phenotypic microscope-based screening for production of crystal proteins. A total of 58 diverseB. cereus s.l.strains isolated from clinical, food, environmental, and commercial biopesticide products were underwent WGS. Isolates were examined for crystal protein production using phase contrast microscopy. Crystal protein-encoding genes were detected using BtToxin_Digger, BTyper3, IDOPS, and Cry_processor. Out of 58 isolates, the phenotypic production of crystal proteins was confirmed for 18 isolates. Specificity and sensitivity ofBtidentification based on sequences were 0.85 and 0.94 for BtToxin_Digger, 0.97 and 0.89 for BTyper3, 0.95 and 0.94 for IDOPS, and 0.88 and 1.00 for Cry_processor, respectively. Cry_processor predicted crystal protein production with highest specificity, and BtToxin_Digger and IDOPS predicted crystal protein production with the highest sensitivity. Three out of four tested bioinformatic tools performed well overall, with IDOPS achieving both high sensitivity and specificity (>0.90).

IMPORTANCE

Bacillus cereus s.s.biovarThuringiensis(Bt) is used as an organic biopesticide. It is differentiated from the foodborne pathogenBacillus cereus s.s.by the production of insecticidal crystal proteins. Thus, reliable genomic identification of biovarThuringiensisis necessary to ensure food safety and facilitate risk assessment. This study assessed the accuracy of WGS-based identification ofBtcompared to phenotypic microscopy-based screening for crystal protein production. Multiple bioinformatics tools were compared to assess their performance in predicting crystal protein production. Among them, IDOPS performed best overall at WGS- basedBtidentification.
Stepwise Neofunctionalization of the NF-κB Family Member c-Rel during Vertebrate Evolution
Authors: Daly Allison E. , Chang Abraham B. , Purbey Prabhat , Williams Kevin J. , Yeh George , Li Shuxing , Pope Scott D. , Venkatesh Byrappa , Redelings Benjamin D. , Li Sibon , Nguyen Kaylin , Rodrigues Joseph , Jorgensen Kelsey , Siggers Trevor , Chen Lin , Smale Stephen T.
Published: January 2024

Adaptive immunity and the five vertebrate NF-κB/Rel family members first appeared in cartilaginous fish, suggesting that divergence and specialization within the NF-κB family helped facilitate the evolution of adaptive immunity. One specialized function of the NF-κB c-Rel protein in macrophages is the activation ofIl12b, which encodes a key regulator of T-cell development. We found that c-Rel is a far more potent regulator ofIl12bthan of any other inducible genes in macrophages, with c-Rel regulation ofIl12bdependent on its heightened intrinsic DNA-binding affinity. c-Rel homodimers regulateIl12btranscription in part via motifs with little resemblance to canonical NF-κB motifs. ChIP-seq experiments further defined distinct c-Rel DNA-binding preferences genome-wide, and X-ray crystallography of a c-Rel/RelA chimeric protein identified key amino acid changes that support the unique c-Rel properties. Unexpectedly, these changes, along with the c-Rel/RelA binding affinity differences, were largely restricted to mammalian species. Together, our findings reveal how a transcription factor family member can undergo a structural transition at a late stage of vertebrate evolution, resulting in an increased intrinsic DNA binding affinity and with clear functional consequences, presumably to support the increasing complexity of immune regulation.
Artemisinin-resistantPlasmodium falciparumKelch13 mutant proteins display reduced heme-binding affinity and decreased artemisinin activation
Authors: Rahman Abdur , Tamseel Sabahat , Coppée Romain , Dutta Smritikana , Khan Nawaal , Faaiz Mohammad , Rastogi Harshita , Nath Jyoti Rani , Chowdhury Pramit , Ashish  , Clain Jérôme , Bhattacharjee Souvik
Published: January 2024

The rapid emergence of artemisinin resistance (ART-R) poses a challenge to global malaria control efforts. ART potency is triggered by ferrous iron- and/or heme-mediated cleavage of the endoperoxide bond to generate reactive heme-ART alkoxy radicals and covalent heme-ART adducts that alkylate parasite targets or inhibit the detoxification of heme into β-hematin crystals; both of which lead to parasite death. Mutations in theP. falciparumKelch-containing protein Kelch13 (PfKekch13) confer clinical ART-R, in which the resistant parasites exhibit impaired hemoglobin uptake, reduced heme yield, and thus decreased ART activation. However, a more direct involvement of PfKelch13 in heme-mediated ART activation has not been reported. Here, we show that recombinant, purified PfKelch13 wild-type (WT) protein displays measurable binding affinity for both iron and heme, the main effectors for ART activation. Comparative biochemical analyses further indicate weaker heme-binding affinities in the two Southeast Asian ART-R PfKelch13 mutants C580Y and R539T compared to the ART-sensitive WT and A578S mutant proteins, which ultimately translates into reduced yield of heme-ART derivatives. In conclusion, this study provides the first evidence for regulated ART activationviathe heme-binding propensity of PfKelch13, which may contribute towards modulating the level of ART-R in malaria parasites with PfKelch13 mutations.
DecodingKlebsiella pneumoniaein Poultry Chain: Unveiling Genetic Landscape, Antibiotic Resistance, and Biocide Tolerance in Non-Clinical Reservoirs
Authors: Mourão Joana , Magalhães Mafalda , Ribeiro-Almeida Marisa , Rebelo Andreia , Novais Carla , Peixe Luísa , Novais Ângela , Antunes Patrícia
Published: January 2024

The rise of antibiotic resistance in the food chain is influenced by the use of antimicrobial agents, such as antibiotics, metals, and biocides, throughout the entire farm-to-fork continuum. Besides, non-clinical reservoirs potentially contribute to the transmission of critical pathogens such as multidrug-resistant (MDR)Klebsiella pneumoniae. However, limited knowledge exists about the population structure and genomic diversity ofK. pneumoniaecirculating in conventional poultry production. We conducted a comprehensive characterization ofK. pneumoniaeacross the whole chicken production chain (flocks/environment/meat, 2019-2022), exploring factors beyond antibiotics, like copper and quaternary ammonium compounds (QACs). Clonal diversity and adaptive features ofK. pneumoniaewere characterized through cultural, molecular (FT-IR), and whole-genome-sequencing (WGS) approaches. All except one flock were positive forK. pneumoniaewith a significant increase (p < 0.05) from early to pre-slaughter stages, most persisting in chicken meat batches. Colistin-resistantK. pneumoniaerates were low (4%), while most samples carried MDR strains (67%) and copper-tolerant isolates (63%;sil+pcoclusters; MICCuSO4≥16mM), particularly at pre-slaughter. Benzalkonium chloride consistently exhibited activity inK. pneumoniae(MIC/MBC range=4-64mg/L) from diverse and representative strains independently of the presence/absence of genes linked to QACs tolerance. A polyclonalK. pneumoniaepopulation, discriminated by FT-IR and WGS, included various lineages dispersed throughout the chicken’s lifecycle at the farm (ST29-KL124, ST11-KL106, ST15-KL19, ST1228-KL38), until the meat (ST1-KL19, ST11-KL111, ST6405-KL109, and ST6406-CG147-KL111), or over years (ST631-49 KL109, ST6651-KL107, ST6406-CG147-KL111). Notably, some lineages were identical to those from human clinical isolates. WGS also revealed F-type multireplicon plasmids carryingsil+pco(copper) co-located withqacEΔ1±qacF(QACs) and antibiotic resistance genes like those disseminated in humans. In conclusion, chicken farms and their derived meat are significant reservoirs for diverseK. pneumoniaeclones enriched in antibiotic resistance and metal tolerance genes, some exhibiting genetic similarities with human clinical strains. Further research is imperative to unravel the factors influencingK. pneumoniaepersistence and dissemination within poultry production, contributing to improved food safety risk management. This study underscores the significance of understanding the interplay between antimicrobial control strategies and non-clinical sources to effectively address the spread of antimicrobial resistance.
Retinoic acid generates a beneficial microenvironment for liver progenitor cell activation in acute liver failure
Authors: Wang Sai , Link Frederik , Munker Stefan , Wang Wenjing , Feng Rilu , Liebe Roman , Li Yujia , Yao Ye , Liu Hui , Shao Chen , Ebert Matthias P. A. , Ding Huiguo , Dooley Steven , Weng Hong-Lei , Wang Shan-Shan
Published: January 2024

Objective

When massive necrosis occurs in acute liver failure (ALF), rapid expansion of hepatic stem cells called liver progenitor cells (LPC) in a process called ductular reaction (DR) is required for survival. The exact underlying mechanisms of this process are not known to date. In ALF, high levels of retinoic acid (RA), a molecule known for its pleiotropic roles in embryonic development, are secreted by activated hepatic stellate cells (HSCs). We hypothesized that RA plays a key role during DR in ALF.

Methods

RNA-Seq was performed to identify molecular signaling pathways affected by all-trans retinoid acid (atRA) treatment in HepaRG LPC cells. Functional assays for RA were performed in HepaRG cells with atRA treatment as well as co-culture with LX-2 cellsin vitro, and liver tissue of patients suffering from ALFin vivo.

Results

Under ALF conditions, activated HSCs secreted RA, inducing RARα nuclear translocation in LPCs. RNA-seq data and investigations in HepaRG cells revealed that atRA treatment activated the WNT-β-Catenin pathway, enhanced stemness genes (SOX9, AFP, et.al), promoted energy storage, and elevated the expression of ATP-binding cassette (ABC) transporters depending on RARα nuclear translocation. Further, atRA treatment-induced pathways were confirmed in a co-culture system of HepaRG with LX-2 cells. Patients with ALF who displayed RARα nuclear translocation in LPC had significantly better MELD scores than those without.

Conclusion

In ALF, RA secreted by activated hepatic stellate cells promotes LPC activation, a prerequisite for subsequent LPC-mediated liver regeneration.
CryoCycle your grids: Plunge vitrifying and reusing clipped grids to advance cryoEM democratization
Authors: Serbynovskyi Viacheslav , Wang Jing , Chua Eugene YD , Ishemgulova Aygul , Alink Lambertus M. , Budell William C. , Johnston Jake D. , Dubbeldam Charlie , Gonzalez Fabio A. , Rozovsky Sharon , Eng Edward T. , de Marco Alex , Noble Alex J.
Published: January 2024

CryoEM democratization is hampered by access to costly plunge-freezing supplies. We introduce methods, called CryoCycle, for reliably blotting, vitrifying, and reusing clipped cryoEM grids. We demonstrate that vitreous ice may be produced by plunging clipped grids with purified proteins into liquid ethane and that clipped grids may be reused several times for different protein samples. Furthermore, we demonstrate the vitrification of thin areas of cells prepared on gold-coated, pre-clipped grids.
ThirdPeak: A flexible tool designed for the robust analysis of two- and three-dimensional (single-molecule) tracking data
Authors: Müller Thomas , Meiser Elisabeth , Engstler Markus
Published: January 2024

ThirdPeak is an open-source tool tailored for the comprehensive analysis of two– and three-dimensional track data across various scales. Its versatile import options facilitate seamless integration into established workflows, and the user interface allows for swift visualization and analysis of the data. When applied to live-cell diffusion data, this software unveils the advantages of combining both 2D and 3D analysis, providing valuable insights into the understanding of biological processes.
Neurometabolic predictors of mental effort in the frontal cortex
Authors: Barakat Arthur , Clairis Nicolas , Brochard Jules , Pessiglione Mathias , Godin Jean-Philippe , Cuenoud Bernard , Xin Lijing , Sandi Carmen
Published: January 2024

Motivation drives individuals to overcome costs to achieve desired outcomes, such as rewards or avoidance of punishment, with significant variability across individuals. The dorsomedial prefrontal cortex/dorsal anterior cingulate cortex (dmPFC/dACC) and anterior insula are key brain regions implicated in effort-based decision-making. Here, we utilized proton magnetic resonance spectroscopy (1H-MRS) at 7 Tesla on 69 healthy participants in these brain regions to uncover the neurometabolic factors that influence these differences. We designed and applied an effort-based decision-making task requiring mental and physical effort to probe motivated behavior, complemented by computational modeling to extract key behavioral parameters. Gradient boosting machine learning was applied to explore the predictive role of specific metabolites in motivated behavior. Our results reveal that a model established on dmPFC/dACC metabolites explains decisions to exert high mental effort and sensitivity to mental effort. In particular, glutamate, aspartate, and lactate in dmPFC/dACC, three metabolites linked through the tricarboxylic acid cycle and glycolysis, were identified as key discriminative metabolites in the dmPFC/dACC, predictive of mental effort choices, underpinning energy supply and cognitive processes. Anterior insula metabolites did not significantly relate to effort-related decisions. Notably, glutamine and lactate levels between the periphery (plasma) and the dmPFC/dACC were correlated, suggesting a metabolic link between peripheral and central biomarkers of effort. Our findings provide novel insights into the neurometabolic underpinnings of motivated behavior and propose novel biomarkers for mental effort-based decision-making. Importantly, our study highlights the relevance of multivariate approaches in elucidating complex cognitive functions.
Single-domain antibodies reveal unique borreliacidal epitopes on the Lyme disease vaccine antigen, Outer surface protein A (OspA)
Authors: Vance David J , Basir Saiful , Piazza Carol Lyn , Willsey Graham , Haque H M Emranul , Tremblay Jacque M , Rudolph Michael J , Muriuki Beatrice , Cavacini Lisa A , Weis David D , Shoemaker Charles B , Mantis Nicholas J
Published: January 2024

Camelid-derived, single-domain antibodies (VHHs) have proven to be extremely powerful tools in defining the antigenic landscape of immunologically heterogeneous surface proteins. In this report, we generated a phage-displayed VHH library directed against the candidate Lyme disease vaccine antigen, Outer surface protein A (OspA). Two alpacas were immunized with recombinant OspA serotype 1 (ST1) fromBorrelia burgdorferisensu stricto strain B31, in combination with the canine vaccine RECOMBITEK®Lyme containing lipidated OspA. The phage library was subjected to two rounds of affinity enrichment (“panning”) against recombinant OspA, yielding 21 unique VHHs within two epitope bins, as determined through competition ELISAs with a panel of OspA-specific human monoclonal antibodies. Epitope refinement was conducted by hydrogen exchange-mass spectrometry (HX-MS). Six of the monovalent VHHs were expressed as human IgG1-Fc fusion proteins and shown to have functional properties associated with protective human monoclonal antibodies, includingB. burgdorferiagglutination, outer membrane damage, and complement-dependent borreliacidal activity. The VHHs displayed unique reactivity profiles with the seven OspA serotypes associated withB. burgdorferigenospecies in the United States and Europe consistent with there being conserved epitopes across OspA serotypes that should be considered when designing and evaluating multivalent Lyme disease vaccines.
Dynamic Nature ofStaphylococcus aureusType I Signal Peptidases
Authors: Madsen Jesper J. , Yu Wenqi
Published: January 2024

Molecular dynamics simulations are used to interrogate the dynamic nature ofStaphylococcus aureusType I signal peptidases, SpsA and SpsB, including the impact of the P29S mutation of SpsB. Fluctuations and plasticity-rigidity characteristics vary among the proteins, particularly in the extracellular domain. Intriguingly, the P29S mutation, which influences susceptibility to arylomycin antibiotics, affect the mechanically coupled motions in SpsB. The integrity of the active site is crucial for catalytic competency, and variations in sampled structural conformations among the proteins are consistent with diverse peptidase capabilities. We also explored the intricate interactions between the proteins and the modelS. aureusmembrane. It was observed that certain membrane-inserted residues in the loop around residue 50 (50s) and C-terminal loops, beyond the transmembrane domain, give rise to direct interactions with lipids in the bilayer membrane. Our findings are discussed in the context of functional knowledge about these signal peptidases, offering additional understanding of dynamic aspects relevant to some cellular processes with potential implications for drug targeting strategies.

Highlights

Dynamics ofS. aureusSpsA and SpsB (wild-type and P29S) is analyzed.

Differences in flexibility and plasticity are relevant for function.

P29S mutation decouples mechanical motions in SpsB.

Extracellular domain loops insert differently into the membrane in SpsA and SpsB.

S. aureusmodel membrane properties are elucidated.
A marine sponge-associated mycobacterium closely related toMycobacterium tuberculosis
Authors: Pidot Sacha J. , Klatt Stephan , Ates Louis S. , Frigui Wafa , Sayes Fadel , Majlessi Laleh , Izumi Hiroshi , Monk Ian R. , Porter Jessica L. , Bennett-Wood Vicki , Seemann Torsten , Otter Ashley , Taiaroa George , Cook Gregory M. , West Nicholas , Tobias Nicholas J. , Fuerst John A. , Stutz Michael D. , Pellegrini Marc , McConville Malcolm , Brosch Roland , Stinear Timothy P.
Published: January 2024

Reconstructing the evolutionary origins ofMycobacterium tuberculosis, the causative agent of human tuberculosis, has helped identify bacterial factors that have led to the tubercle bacillus becoming such a formidable human pathogen. Here we report the discovery and detailed characterization of an exceedingly slow growing mycobacterium that is closely related toM. tuberculosisfor which we have proposed the species nameMycobacterium spongiaesp. nov., (strain ID: FSD4b-SM). The bacterium was isolated from a marine sponge, taken from the waters of the Great Barrier Reef in Queensland, Australia. Comparative genomics revealed that, after the opportunistic human pathogenMycobacterium decipiens,M. spongiaeis the most closely related species to theM. tuberculosiscomplex reported to date, with 80% shared average nucleotide identity and extensive conservation of keyM. tuberculosisvirulence factors, including intact ESX secretion systems and associated effectors. Proteomic and lipidomic analyses showed that these conserved systems are functional in FSD4b-SM, but that it also produces cell wall lipids not previously reported in mycobacteria. We investigated the virulence potential of FSD4b-SM in mice and found that, while the bacteria persist in lungs for 56 days after intranasal infection, no overt pathology was detected. The similarities withM. tuberculosis, together with its lack of virulence, motivated us to investigate the potential of FSD4b-SM as a vaccine strain and as a genetic donor of the ESX-1 genetic locus to improve BCG immunogenicity. However, neither of these approaches resulted in superior protection againstM. tuberculosischallenge compared to BCG vaccination alone. The discovery ofM. spongiaeadds to our understanding of the emergence of theM. tuberculosiscomplex and it will be another useful resource to refine our understanding of the factors that shaped the evolution and pathogenesis ofM. tuberculosis.
Phylogenomics reveals coincident divergence between giant host sea anemones and the clownfish adaptive radiation
Authors: De Jode Aurelien , Quattrini Andrea M. , Chiodo Tommaso , Daly Marymegan , McFadden Catherine S. , Berumen Michael L. , Meyer Christopher P. , Mills Suzanne , Beldade Ricardo , Bartholomew Aaron , Scott Anna , Reimer James D , Yanagi Kensuke , Fuji Takuma , Rodríguez Estefanía , Titus Benjamin M.
Published: January 2024

The mutualism between clownfishes (or anemonefishes) and their giant host sea anemones are among the most immediately recognizable animal interactions on the planet and have attracted a great deal of popular and scientific attention [1-5]. However, our evolutionary understanding of this iconic symbiosis comes almost entirely from studies on clownfishes— a charismatic group of 28 described species in the genusAmphiprion[2]. Adaptation to venomous sea anemones (Anthozoa: Actiniaria) provided clownfishes with novel habitat space, ultimately triggering the adaptive radiation of the group [2]. Clownfishes diverged from their free-living ancestors 25-30 MYA with their adaptive radiation to sea anemones dating to 13.2 MYA [2, 3]. Far from being mere habitat space, the host sea anemones also receive substantial benefits from hosting clownfishes, making the mutualistic and co-dependent nature of the symbiosis well established [4, 5]. Yet the evolutionary consequences of mutualism with clownfishes have remained a mystery from the host perspective. Here we use bait-capture sequencing to fully resolve the evolutionary relationships among the 10 nominal species of clownfish-hosting sea anemones for the first time (Figure 1). Using time-calibrated divergence dating analyses we calculate divergence times of less than 25 MYA for each host species, with 9 of 10 host species having divergence times within the last 13 MYA (Figure 1). The clownfish-hosting sea anemones thus diversified coincidently with clownfishes, potentially facilitating the clownfish adaptive radiation, and providing the first strong evidence for co-evolutionary patterns in this iconic partnership.
PP2B-dependent cerebellar plasticity sets the amplitude of an innate reflex during juvenile development
Authors: Wu Bin , Post Laura , Lin Zhanmin , Schonewille Martijn
Published: January 2024

Throughout life, the cerebellum plays a central role in the coordination and optimization of movements, using cellular plasticity to adapt a range of behaviors. If these plasticity processes establish a fixed setpoint during development, or continuously adjust behaviors throughout life, is currently unclear. Here, by spatiotemporally manipulating the activity of protein phosphatase 2B (PP2B), an enzyme critical for cerebellar plasticity, we examined the consequences of disrupted plasticity on the performance and adaptation of the vestibulo-ocular reflex (VOR). We find that, in contrast to Purkinje cell specific deletion starting early postnatally, acute pharmacological as well as adult-onset genetic deletion of PP2B affects all forms of VOR adaptation, but not the level of VOR itself. Next, we show that Purkinje cell-specific genetic deletion of PP2B in juvenile mice leads to a progressive loss of the protein PP2B and a concurrent change in the VOR, in addition to the loss of adaptive abilities. Finally, re-expressing PP2B in adult mice that lack PP2B expression from early in development, rescues VOR adaptation, but does not affect the performance of the reflex. Together, our results indicate that chronic or acute, genetic or pharmacological block of PP2B disrupts the adaptation of the VOR. In contrast, only the absence of plasticity during cerebellar development affects the setpoint of VOR, an effect that cannot be corrected after maturation of the cerebellum. These findings suggest that cerebellar plasticity influences behavior in two ways, through direct control of behavioral adaptation and via long-term effects initiated in the juvenile period.

Significance Statement

Early damage to motor adaptation structures, such as the cerebellum, has been linked to neurodevelopmental disorders persisting into adulthood. Understanding these long-term effects requires disentangling the persistent, long-term effects of disrupted development from the acute, ongoing effects directly caused by the continuous presence of the disruption. Here, we demonstrate that disruptions during early development affect both basal level and adaptation, whereas late, adult-onset disruption of cerebellar plasticity only affects the ability to adapt, not the setpoint of an innate reflex. Our findings that specifically the absence of plasticity during cerebellar development affects the setpoint of VOR, which cannot be corrected by re-instating plasticity after maturation of the cerebellum, supports the concept of a sensitive developmental period for setting innate reflexes.
A potential role of inefficient and non-specific piRNA production from the whole transcriptome
Authors: Shoji Keisuke , Tomari Yukihide
Published: January 2024

PIWI-interacting RNAs (piRNAs) are small RNAs that play a critical role in repressing transposons in the germline by guiding PIWI proteins to complementary target sequences. It is well known that the 5’ ends of piRNAs are typically defined through cleavage by PIWI proteins or by the endonuclease Zucchini, and piRNAs are amplified via reciprocal cleavage of sense and antisense transcripts by PIWI proteins. Here, we show that low levels of sense-stranded piRNAs are produced from the whole transcriptome in silkworms, flies, and mice, in a manner independent from the known biogenesis pathways. Notably, this mechanism is utilized in silkworm cultured cells for the production of piRNAs derived from the extremely abundant sub-genomic RNAs ofBombyx morilatent virus, which have been suggested to play a role in suppressing viral replication. We propose that this inefficient and non-specific piRNA-production pathway acts as a primitive sensor for aberrantly abundant transcripts in the germline, potentially seeding piRNA sequence repertoire for more efficient and established biogenesis pathways.
A Deep Learning Pipeline for Mapping in situ Network-level Neurovascular Coupling in Multi-photon Fluorescence Microscopy
Authors: Rozak Matthew , Mester James , Attarpour Ahmadreza , Dorr Adrienne , Patel Shruti , Koletar Margaret , Hill Mary , McLaurin JoAnne , Goubran Maged , Stefanovic Bojana
Published: January 2024

1

Functional hyperaemia is a well-established hallmark of healthy brain function, whereby local brain blood flow adjusts in response to a change in the activity of the surrounding neurons. Although functional hyperemia has been extensively studied at the level of both tissue and individual vessels, vascular network-level coordination remains largely unknown. To bridge this gap, we developed a deep learning-based computational pipeline that uses two-photon fluorescence microscopy images of cerebral microcirculation to enable automated reconstruction and quantification of the geometric changes across the microvascular network, comprising hundreds of interconnected blood vessels, pre and post-activation of the neighbouring neurons. The pipeline’s utility was demonstrated in the Thy1-ChR2 optogenetic mouse model, where we observed network-wide vessel radius changes to depend on the photostimulation intensity, with both dilations and constrictions occurring across the cortical depth, at an average of 16.1±14.3 μm (mean±stddev) away from the most proximal neuron for dilations; and at 21.9±14.6 μm away for constrictions. We observed a significant heterogeneity of the vascular radius changes within vessels, with radius adjustment varying by an average of 24 ± 28% of the resting diameter, likely reflecting the heterogeneity of the distribution of contractile cells on the vessel walls. A graph theory-based network analysis revealed that the assortativity of adjacent blood vessel responses rose by 152 ± 65% at 4.3 mW/mm2of blue photostimulationvs. the control, with a 4% median increase in the efficiency of the capillary networks during this level of blue photostimulation in relation to the baseline. Interrogating individual vessels is thus not sufficient to predict how the blood flow is modulated in the network. Our computational pipeline, to be made openly available, enables tracking of the microvascular network geometry over time, relating caliber adjustments to vessel wall-associated cells’ state, and mapping network-level flow distribution impairments in experimental models of disease.
Optical conductivity and damping of plasmons due to electron-electron interaction
Authors: Sharma Prachi , Principi Alessandro , Vignale Giovanni , Maslov Dmitrii L.
Published: January 2024

We revisit the issue of plasmon damping due to electron-electron interaction. The plasmon linewidth can be related to the imaginary part of the charge susceptibility or, equivalently, to the real part of the optical conductivity, Reσ(q,ω). Approaching the problem first via a standard semiclassical Boltzmann equation, we show that Reσ(q,ω) of a two-dimensional (2D) electron gas scales as q2T2/ω4 for ω≪T, which agrees with the results of Principi [] and Sharma [] but disagrees with that of Mishchenko [], according to which Reσ(q,ω)∝q2T2/ω2. To resolve this disagreement, we rederive Reσ(q,ω) using the original method of Mishchenko for an arbitrary ratio ω/T and show that while the last term is, indeed, present, it is subleading to the q2T2/ω4 term. We give a physical interpretation of both leading and subleading contributions in terms of the shear and bulk viscosities of an electron liquid, respectively. We also calculate Reσ(q,ω) for a three-dimensional electron gas and doped monolayer graphene. We find that, all other parameters being equal, finite temperature has the strongest effect on the plasmon linewidth in graphene, where it scales as T4lnT for ω≪T.

Published by the American Physical Society
2024