bluuebunny/crossref_metadata_2025 huggingface dataset:
Progress in Combined Liver–lung Transplantation at a Single Center
Authors: Connor Ashton A. , Huang Howard J. , Mobley Constance M. , Graviss Edward A. , Nguyen Duc T. , Goodarzi Ahmad , Saharia Ashish , Yau Simon , Hobeika Mark J. , Suarez Erik E. , Moaddab Mozhgon , Brombosz Elizabeth W. , Moore Linda W. , Yi Stephanie G. , Gaber A. Osama , Ghobrial Rafik Mark
Published: April 2023

Background.

Combined liver–lung transplantation is an uncommon, although vital, procedure for patients with simultaneous end-stage lung and liver disease. The utility of lung–liver transplant has been questioned because of initial poor survival outcomes, particularly when compared with liver-alone transplant recipients.

Methods.

A single-center, retrospective review of the medical records of 19 adult lung–liver transplant recipients was conducted, comparing early recipients (2009–2014) with a recent cohort (2015–2021). Patients were also compared with the center’s single lung or liver transplant recipients.

Results.

Recent lung–liver recipients were older (P = 0.004), had a higher body mass index (P = 0.03), and were less likely to have ascites (P = 0.02), reflecting changes in the etiologies of lung and liver disease. Liver cold ischemia time was longer in the modern cohort (P = 0.004), and patients had a longer posttransplant length of hospitalization (P = 0.048). Overall survival was not statistically different between the 2 eras studied (P = 0.61), although 1-y survival was higher in the more recent group (90.9% versus 62.5%). Overall survival after lung–liver transplant was equivalent to lung-alone recipients and was significantly lower than liver-alone recipients (5-y survival: 52%, 51%, and 75%, respectively). Lung–liver recipient mortality was primarily driven by deaths within 6 mo of transplant due to infection and sepsis. Graft failure was not significantly different (liver: P = 0.06; lung: P = 0.74).

Conclusions.

The severity of illness in lung–liver recipients combined with the infrequency of the procedure supports its continued use. However, particular attention should be paid to patient selection, immunosuppression, and prophylaxis against infection to ensure proper utilization of scarce donor organs.
Rhizopus homothallicus, an emerging pathogen causing cavitary lung lesions
Authors: Taneja Juhi , Chatterjee Kuhu , Sachdeva Ruchi Arora , Abbas S. Zafar , Sen M.K.
Published: April 2023

Introduction.
Rhizopus homothallicus is an emerging pathogen that causes pulmonary mucormycosis.

Case Presentation. We report a case of pneumonia caused by R. homothallicus in a 54-year-old type 2 diabetic patient. The organism was isolated from bronchoalveolar lavage fluid and preliminarily identified by fungal morphology and finally by sequencing of the internal transcribed spacer region.

Conclusion. Mucormycosis may be associated with cavitary lung lesions against a backdrop of poorly controlled diabetes or other immunosuppressed states. Pulmonary mucormycosis may have variable clinical and radiological presentations. Therefore, strong clinical suspicion and prompt management can address the high fatality associated with the disease.
Analysis of the ARTIC V4 and V4.1 SARS-CoV-2 primers and their impact on the detection of Omicron BA.1 and BA.2 lineage-defining mutations
Authors: Ulhuq Fatima R. , Barge Madhuri , Falconer Kerry , Wild Jonathan , Fernandes Goncalo , Gallagher Abbie , McGinley Suzie , Sugadol Ahmad , Tariq Muhammad , Maloney Daniel , Kenicer Juliet , Dewar Rebecca , Templeton Kate , McHugh Martin P.
Published: April 2023

The ARTIC protocol uses a multiplexed PCR approach with two primer pools tiling the entire SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) genome. Primer pool updates are necessary for accurate amplicon sequencing of evolving SARS-CoV-2 variants with novel mutations. The suitability of the ARTIC V4 and updated V4.1 primer scheme was assessed using whole genome sequencing of Omicron from clinical samples using Oxford Nanopore Technology. Analysis of Omicron BA.1 genomes revealed that 93.22 % of clinical samples generated improved genome coverage at 50× read depth with V4.1 primers when compared to V4 primers. Additionally, the V4.1 primers improved coverage of BA.1 across amplicons 76 and 88, which resulted in the detection of the variant-defining mutations G22898A, A26530G and C26577G. The Omicron BA.2 sub-variant (VUI-22JAN-01) replaced BA.1 as the dominant variant by March 2022, and analysis of 168 clinical samples showed reduced coverage across amplicons 15 and 75. Upon further interrogation of primer binding sites, a mutation at C4321T [present in 163/168 (97 %) of samples] was identified as a possible cause of complete dropout of amplicon 15. Furthermore, two mutations were identified within the primer binding regions for amplicon 75: A22786C (present in 90 % of samples) and C22792T (present in 12.5 % of samples). Together, these mutations may result in reduced coverage of amplicon 75, and further primer updates would allow the identification of the two BA.2-defining mutations present in amplicon 75: A22688G and T22679C. This work highlights the need for ongoing surveillance of primer matches as circulating variants evolve and change.
A global genomic perspective on the multidrug-resistant Streptococcus pneumoniae 15A-CC63 sub-lineage following pneumococcal conjugate vaccine introduction
Authors: Hawkins Paulina A. , Chochua Sopio , Lo Stephanie W. , Belman Sophie , Antonio Martin , Kwambana-Adams Brenda , von Gottberg Anne , du Plessis Mignon , Cornick Jen , Beall Bernard , Breiman Robert F. , Bentley Stephen D. , McGee Lesley ,   The Global Pneumococcal Sequencing Consortium
Published: April 2023

The introduction of pneumococcal conjugate vaccines (PCV7, PCV10, PCV13) around the world has proved successful in preventing invasive pneumococcal disease. However, immunization against

Streptococcus pneumoniae

has led to serotype replacement by non-vaccine serotypes, including serotype 15A. Clonal complex 63 (CC63) is associated with many serotypes and has been reported in association with 15A after introduction of PCVs. A total of 865 CC63 isolates were included in this study, from the USA (n=391) and a global collection (n=474) from 1998–2019 and 1995–2018, respectively. We analysed the genomic sequences to identify serotypes and penicillin-binding protein (PBP) genes 1A, 2B and 2X, and other resistance determinants, to predict minimum inhibitory concentrations (MICs) against penicillin, erythromycin, clindamycin, co-trimoxazole and tetracycline. We conducted phylogenetic and spatiotemporal analyses to understand the evolutionary history of the 15A-CC63 sub-lineage. Overall, most (89.5 %, n=247) pre-PCV isolates in the CC63 cluster belonged to serotype 14, with 15A representing 6.5 % of isolates. Conversely, serotype 14 isolates represented 28.2 % of post-PCV CC63 isolates (n=618), whilst serotype 15A isolates represented 65.4 %. Dating of the CC63 lineage determined the most recent common ancestor emerged in the 1980s, suggesting the 15A-CC63 sub-lineage emerged from its closest serotype 14 ancestor prior to the development of pneumococcal vaccines. This sub-lineage was predominant in the USA, Israel and China. Multidrug resistance (to three or more drug classes) was widespread among isolates in this sub-lineage. We show that the CC63 lineage is globally distributed and most of the isolates are penicillin non-susceptible, and thus should be monitored.
Assessing GPT-4 for cell type annotation in single-cell RNA-seq analysis
Authors: Hou Wenpin , Ji Zhicheng
Published: April 2023

Cell type annotation is an essential step in single-cell RNA-seq analysis. However, it is a time-consuming process that often requires expertise in collecting canonical marker genes and manually annotating cell types. Automated cell type annotation methods typically require the acquisition of high-quality reference datasets and the development of additional pipelines. We assessed the performance of GPT-4, a highly potent large language model, for cell type annotation, and demonstrated that it can automatically and accurately annotate cell types by utilizing marker gene information generated from standard single-cell RNA-seq analysis pipelines. Evaluated across hundreds of tissue types and cell types, GPT-4 generates cell type annotations exhibiting strong concordance with manual annotations and has the potential to considerably reduce the effort and expertise needed in cell type annotation. We also developed GPTCelltype, an open-source R software package to facilitate cell type annotation by GPT-4.
Altered neutrophil extracellular traps in response toMycobacterium tuberculosisin persons living with HIV with no previous TB and negative TST and IGRA
Authors: Kroon EE , Correa-Macedo W , Evans R , Seeger A , Engelbrecht L , Kriel JA , Loos B , Okugbeni N , Orlova M , Cassart P , Kinnear CJ , Tromp GC , Möller M , Wilkinson RJ , Coussens AK , Schurr E , Hoal EG
Published: April 2023

Persons living with HIV (PLWH) have an increased risk for tuberculosis (TB). After prolonged and repeated exposure, some PLWH never develop TB and test persistently negative in tests of immune sensitization tuberculin skin test (TST) and interferon gamma release assays (IGRA) forMycobacterium tuberculosis(Mtb). This group has been identified and defined as HIV+ persistently TB, tuberculin and IGRA negative (HITTIN). To investigate potential innate mechanisms unique to individuals with the HITTIN phenotype we compared their neutrophilMtbinfection response to that of PLWH, with no TB history, but who test persistently IGRA positive, and tuberculin positive (HIT). Neutrophil samples from 17 HITTIN (PMNHITTIN) and 11 HIT (PMNHIT) were isolated and infected withMtbH37Rv for 1h and 6h. RNA was extracted and used for RNAseq analysis. At 1h ofMtbinfection, PMNHITTINdisplayed 151 significantly upregulated and 40 significantly downregulated differentially expressed genes (DEGs) and PMNHIT98 significantly upregulated and 11 significantly downregulated DEGs. At the 6h timepoint, PMNHITTINdisplayed 3106 significantly upregulated and 3548 significantly downregulated DEGs while PMNHIThad 3816 significantly up- and 3794 significantly downregulated DEGs. There was no significant differential transcriptional response at 1h between infected PMNHITTINand PMNHIT.However, when contrasting the log2FC 6h infection response toMtbfrom PMNHITTINagainst PMNHIT, 2285 genes showed significant differential response between the two groups. Apoptosis and NETosis were key pathways linked to the enrichment of genes in PMNHITTINwhen contrasted to PMNHITafter 6h infection withMtb. Fluorescence microscopy revealed relatively lower neutrophil extracellular trap formation and cell loss in PMNHITTINcompared to PMNHIT, showing that PMNHITTINhave a distinct response toMtb.
The X chromosome of insects predates the origin of Class Insecta
Authors: Toups Melissa A. , Vicoso Beatriz
Published: April 2023

Sex chromosomes have evolved independently multiple times, but why some are conserved for more than 100 million years whereas others turnover rapidly remains an open question. Here, we examine the homology of sex chromosomes across nine orders of insects, plus the outgroup springtails. We find that the X chromosome is shared among all insect orders and springtails; the only exception is in the Lepidoptera, which has lost the X and now has a ZZ/ZW sex chromosome system. Therefore, the ancestral insect X chromosome has persisted for more than 450 million years – the oldest known sex chromosome to date. Further, we suggest that the shrinking of gene content the Dipteran X chromosome has allowed for a burst of sex-chromosome turnover that is absent from other speciose insect orders.
Nuclear bodies protect phase separated proteins from degradation in stressed proteome
Authors: Jung Kwan Ho , Sun Jiarui , Hsiung Chia-Heng , Lian Xiaojun Lance , Liu Yu , Zhang Xin
Published: April 2023

RNA-binding proteins (RBPs) containing intrinsically disordered domains undergo liquid-liquid phase separation to form nuclear bodies under stress conditions. This process is also connected to the misfolding and aggregation of RBPs, which are associated with a series of neurodegenerative diseases. However, it remains elusive how folding states of RBPs changes upon the formation and maturation of nuclear bodies. Here, we describe SNAP-tag based imaging methods to visualize the folding states of RBPs in live cells via time-resolved quantitative microscopic analyses of their micropolarity and microviscosity. Using these imaging methods in conjunction with immunofluorescence imaging, we demonstrate that RBPs, represented by TDP-43, initially enters the PML nuclear bodies in its native state upon transient proteostasis stress, albeit it begins to misfolded during prolonged stress. Furthermore, we show that heat shock protein 70 co-enters the PML nuclear bodies to prevent the degradation of TDP-43 from the proteotoxic stress, thus revealing a previously unappreciated protective role of the PML nuclear bodies in the prevention of stress-induced degradation of TDP-43. In summary, our imaging methods described in the manuscript, for the first time, reveal the folding states of RBPs, which were previously challenging to study with conventional methods in nuclear bodies of live cells. This study uncovers the mechanistic correlations between the folding states of a protein and functions of nuclear bodies, in particular PML bodies. We envision that the imaging methods can be generally applied to elucidating the structural aspects of other proteins that exhibit granular structures under biological stimulus.
Acis-regulatory point mutation at a R2R3-Myb transcription factor contributes to speciation by reinforcement inPhlox drummondii
Authors: Garner Austin G. , Cameron Andrew , Berardi Andrea E. , Hopkins Robin
Published: April 2023

The process of reinforcement, whereby selection favors the evolution of increased reproductive trait divergence to reduce costly hybridization between species, has been well documented in nature, yet we know very little about how this process evolves at the molecular level. In this study, we combine functional characterization and genetic association tests to identify the mutational basis of reinforcement in the Texas wildflowerPhlox drummondii. P. drummondiievolved from light to dark flower color intensity by selection to stop hybridization with the closely related speciesP. cuspidata, and previous research suggests differential expression of a R2R3-Myb transcription factor underlies this phenotypic transition. Using gene-silencing experiments, we demonstrate expression of this transcription factor does control variation in flower color intensity. We then apply association mapping across a large genomic region flanking the R2R3-Myb gene and identified a point mutation within the gene’s promoter that is highly associated with flower color intensity in nature. Alleles at this mutation site match the expected patterns of dominance, create variation in predicted cis-regulatory motifs within the R2R3-Myb proximal promoter, and occur in the direction of evolution predicted for flower color variation in this system. By identifying the mutational basis of reinforcement in this system we demonstrate that, as predicted by theory, reproductive isolation can evolve despite gene flow through a very simple genetic basis.
Galectin-3 Mediates Vascular Dysfunction in Obesity by Regulating NADPH Oxidase 1
Authors: Padgett Caleb A. , Bátori Róbert K. , Speese Andrew C. , Rosewater Cody L. , Bush Weston B. , Derella Cassandra C. , Haigh Stephen B. , Sellers Hunter G. , Corley Zachary L. , West Madison A. , Mintz James D. , Ange Brittany B. , Harris Ryan A. , Brands Michael W. , Fulton David J. R. , Stepp David W.
Published: April 2023

Rationale

Obesity increases the risk of cardiovascular disease (CVD) through mechanisms that remain incompletely defined. Metabolic dysfunction, especially hyperglycemia, is thought to be a major contributor but how glucose impacts vascular function is unclear. Galectin-3 (GAL3) is a sugar binding lectin upregulated by hyperglycemia but its role as a causative mechanism of CVD remains poorly understood.

Objective

To determine the role of GAL3 in regulating microvascular endothelial vasodilation in obesity.

Methods and Results

GAL3 was markedly increased in the plasma of overweight and obese patients, as well as in the microvascular endothelium of diabetic patients. To investigate a role for GAL3 in CVD, mice deficient in GAL3 were bred with obesedb/dbmice to generate lean, lean GAL3 knockout (KO), obese, and obese GAL3 KO genotypes. GAL3 KO did not alter body mass, adiposity, glycemia or lipidemia, but normalized elevated markers of reactive oxygen species (TBARS) in plasma. Obese mice exhibited profound endothelial dysfunction and hypertension, both of which were rescued by GAL3 deletion. Isolated microvascular endothelial cells (EC) from obese mice had increased NOX1 expression, which we have previously shown to contribute to increased oxidative stress and endothelial dysfunction, and NOX1 levels were normalized in EC from obese mice lacking GAL3. EC-specific GAL3 knockout mice made obese using a novel AAV-approach recapitulated whole-body knockout studies, confirming that endothelial GAL3 drives obesity-induced NOX1 overexpression and endothelial dysfunction. Improved metabolism through increased muscle mass, enhanced insulin signaling, or metformin treatment, decreased microvascular GAL3 and NOX1. GAL3 increased NOX1 promoter activity and this was dependent on GAL3 oligomerization.

Conclusions

Deletion of GAL3 normalizes microvascular endothelial function in obesedb/dbmice, likely through a NOX1-mediated mechanism. Pathological levels of GAL3 and in turn, NOX1, are amenable to improvements in metabolic status, presenting a potential therapeutic target to ameliorate pathological cardiovascular consequences of obesity.

Figure 8.

Visual abstract
SIV-specific neutralizing antibody induction following selection of a PI3K drive-attenuatednefvariant
Authors: Yamamoto Hiroyuki , Matano Tetsuro
Published: April 2023

HIV and simian immunodeficiency virus (SIV) infections are known for impaired neutralizing antibody (NAb) responses. While sequential virus-host B cell interaction appears to be basally required for NAb induction, driver molecular signatures predisposing to NAb induction still remain largely unknown. Here we describe SIV-specific NAb induction following a virus-host interplay decreasing aberrant viral drive of phosphoinositide 3-kinase (PI3K). Screening of seventy difficult-to-neutralize SIVmac239-infected macaques found nine NAb-inducing animals, with seven selecting for a specific CD8+T-cell escape mutation in viralnefbefore NAb induction. This Nef-G63E mutation reduced excess Nef interaction-mediated drive of B-cell maturation-limiting PI3K/mammalian target of rapamycin complex 2 (mTORC2).In vivoimaging cytometry depicted preferential Nef perturbation of cognate Envelope-specific B cells, suggestive of polarized contact-dependent Nef transfer and corroborating cognate B-cell maturation post-mutant selection up to NAb induction. Results collectively exemplify a NAb induction pattern extrinsically reciprocal to human PI3K gain-of-function antibody-dysregulating disease, and indicate that harnessing the PI3K/mTORC2 axis may facilitate NAb induction against difficult-to-neutralize viruses including HIV/SIV.
Regulation of apico-basolateral trafficking polarity of homologous Copper-ATPases ATP7A and ATP7B
Authors: Ruturaj  , Mishra Monalisa , Saha Soumyendu , Maji Saptarshi , Rodriguez-Boulan Enrique , Schreiner Ryan , Gupta Arnab
Published: April 2023

We suggest a model of apico-basolateral sorting in polarized epithelia using homologous Cu-ATPases as membrane cargoes. In polarized epithelia, upon copper treatment, homologous copper-ATPases ATP7A and ATP7B traffic from trans-Golgi network (TGN) to basolateral and apical membranes respectively. We characterized sorting pathways of Cu-ATPases between TGN and plasma-membrane and identified the machinery involved. ATP7A and ATP7B reside on distinct domains of TGN and in high copper, ATP7A traffics directly to basolateral membrane, whereas ATP7B traverses common-recycling, apical-sorting and apical-recycling endosomesen-routeto apical membrane. Mass-spectrometry identified regulatory partners of ATP7A and ATP7B that include Adaptor Protein-1 complex. Upon knocking-out pan-AP-1, sorting of both copper-ATPases are disrupted. ATP7A loses polarity and localizes on both apical and basolateral surfaces in high copper. Contrastingly, ATP7B loses TGN-retention but retains apical polarity that becomes copper-independent. Using isoform-specific knockouts, we found that AP-1A provides directionality and TGN-retention for both Cu-ATPases, whereas, AP-1B governs polarized trafficking of ATP7B solely. Trafficking phenotypes of Wilson disease-causing ATP7B mutants that disrupts putative ATP7B-AP1 interaction further substantiates the role of AP-1 in apical sorting of ATP7B.

Summary statement

The Adapter Protein-1 (AP-1) isoforms AP-1A and AP-1B governs the apico-basolateral trafficking polarities of the homologous Copper-ATPases, ATP7A and ATP7B at the trans-Golgi network.
Scabrous is distributed via signaling filopodia to modulate Notch response during bristle patterning in Drosophila
Authors: Presser Adam , Freund Olivia , Hassapelis Theodora , Hunter Ginger L
Published: April 2023

During development, tissues must be patterned correctly in order to support tissue function and shape. The sensory bristles of the peripheral nervous system on the thorax of Drosophila melanogaster self-organize from an unpatterned epithelial tissue to a regular spot pattern during pupal stages. Wild type patterning requires Notch-mediated lateral inhibition. Scabrous is a protein that can bind to and modify Notch receptor activity. Scabrous can be secreted, but it is also known to be localized to basal signaling filopodia, or cytonemes, that play a role in long-range Notch signaling. Consistent with previous work, we show that Scabrous is primarily distributed basally, within the range of signaling filopodia extension. We show that signaling filopodia are required for the distribution of Scabrous protein during sensory bristle patterning stages. We show that the Notch response of epithelial cells is sensitive to the level of Scabrous protein being expressed by the sensory bristle precursor cell. Our findings at the cell-level suggest a model for how epithelial cells engaged in lateral inhibition at a distance are sensitive local levels of Scabrous protein.
Dissecting the molecular basis for the modulation of neurotransmitter GPCR signaling by GINIP
Authors: Luebbers Alex , Zhou Myles , Eyles Stephen J , Garcia-Marcos Mikel
Published: April 2023

It is well-established that activation of heterotrimeric G-proteins (Gαβγ) by G-protein-coupled receptors (GPCRs) stimulated by neurotransmitters is a key mechanism underlying neuromodulation. Much less is known about how G-protein regulation after receptor-mediated activation contributes to neuromodulation. Recent evidence indicates that the neuronal protein GINIP shapes GPCR inhibitory neuromodulation via a unique mechanism of G-protein regulation that controls neurological processes like pain and seizure susceptibility. However, the molecular basis of this mechanism remains ill-defined because the structural determinants of GINIP responsible for binding Gαi subunits and regulating G-protein signaling are not known. Here, we combined hydrogen-deuterium exchange mass-spectrometry, protein folding predictions, bioluminescence resonance energy transfer assays, and biochemical experiments to identify the first loop of the PHD domain of GINIP as an obligatory requirement for Gαi binding. Surprisingly, our results support a model in which GINIP undergoes a long-range conformational change to accommodate Gαi binding to this loop. Using cell-based assays, we demonstrate that specific amino acids in the first loop of the PHD domain are essential for the regulation of Gαi-GTP and free Gβγ signaling upon neurotransmitter GPCR stimulation. In summary, these findings shed light onto the molecular basis for a post-receptor mechanism of G-protein regulation that fine-tunes inhibitory neuromodulation.
Interplay betweenATRXandIDH1mutations governs innate immune responses in diffuse gliomas
Authors: Hariharan Seethalakshmi , Whitfield Benjamin T. , Pirozzi Christopher J. , Waitkus Matthew S. , Brown Michael C. , Bowie Michelle L. , Irvin David M. , Roso Kristen , Fuller Rebecca , Hostettler Janell , Dharmaiah Sharvari , Gibson Emiley A. , Briley Aaron , Mangoli Avani , Fraley Casey , Shobande Mariah , Stevenson Kevin , Zhang Gao , Malgulwar Prit Benny , Roberts Hannah , Roskoski Martin , Spasojevic Ivan , Keir Stephen T. , He Yiping , Castro Maria G. , Huse Jason T. , Ashley David M.
Published: April 2023

Stimulating the innate immune system has been explored as a therapeutic option for the treatment of gliomas. Inactivating mutations inATRX, defining molecular alterations inIDH-mutant astrocytomas, have been implicated in dysfunctional immune signaling. However, little is known about the interplay between ATRX loss andIDHmutation on innate immunity. To explore this, we generatedATRXknockout glioma models in the presence and absence of theIDH1R132Hmutation. ATRX-deficient glioma cells were sensitive to dsRNA-based innate immune agonism and exhibited impaired lethality and increased T-cell infiltrationin vivo. However, the presence ofIDH1R132Hdampened baseline expression of key innate immune genes and cytokines in a manner restored by genetic and pharmacological IDH1R132Hinhibition. IDH1R132Hco-expression did not interfere with theATRXKO-mediated sensitivity to dsRNA. Thus, ATRX loss primes cells for recognition of dsRNA, while IDH1R132Hreversibly masks this priming. This work reveals innate immunity as a therapeutic vulnerability of astrocytoma.
The Effect of Selected Coffee Constituents on Mitochon-Drial Dysfunction In An Early Alzheimer Disease Cell Model
Authors: Babylon Lukas , Limbeck Micha T. , Eckert Gunter P.
Published: April 2023

Alzheimer disease (AD) is an emerging medical problem worldwide without any cure yet. By 2050, more than 152 million people will be affected. AD is characterized by mitochondrial dys-function (MD) and increased amyloid beta (Aβ) levels. Coffee is one of the most commonly consumed beverages. It has many bioactive and neuroprotective ingredients of which caffeine (Cof), kahweohl (KW) and cafestol (CF) shows a variety of pharmacological properties such as anti-inflammatory and neuroprotective effects. Effects of Cof, KW, and CF were tested in a cel-lular model of AD on MD and Aβ. SH-SY5Y-APP695 cells were incubated with 50µM Cof, 1µM CF and 1µM KW for 24h. The energetic metabolite ATP was determined using a luciferase-catalyzed bioluminescence assay. The activity of mitochondrial respiration chain complexes was assessed by high-resolution respirometry using a Clarke electrode. Expression levels genes were deter-mined using quantitative real-time polymerase chain reaction (qRT-PCR). The levels of amyloid β-protein (Aβ1-40) were measured using homogeneous time-resolved fluorescence (HTRF). ROS levels, cAMP levels, and peroxidase activity were determined using a fluorescence assay. The combination of Cof, KW and CF significantly increased ATP levels. The combination had neither a significant effect on MMP, on activity of respiration chain complexes, nor on Aβ1-40levels. cAMP levels were slightly increased after incubation with the combination, but not the peroxi-dase activity. Pyruvate levels and the lactate-pyruvate-ration but not lactate levels were signifi- cantly enhanced. No effect was seen on the expression level of lactate dehydrogenase and py-ruvate dehydrogenase kinase. In some experiments we have tested the single substances. They showed significant results especially in ATP, lactate and pyruvate values compared to the con-trol. The combinations have a lesser effect on mitochondrial dysfunction in cells and none on Aβ production. Whereas ATP levels and pyruvate levels were significantly increased. This suggests a change in glycolysis in neuronal cells harbouring human genes relevant for AD.
The Cell Subtypes Selection by Genes (CSSG) algorithm for discovering cell populations in high resolution
Authors: Kubiś Jakub , Figiel Maciej
Published: April 2023

The recent massive improvements in transcriptomics and single-cell technologies have led to a rising volume of data and demand for advances in bioinformatics processing. Existing methods are not fully capable of discovering genetic markers responsible for high-resolution cellular tissue heterogeneity, cell lineages during organism development, and cell differentiation with rare intermediate populations. In response to demand, we have generated a new Cell Subtypes Selection by Genes (CSSG) algorithm which is supported by a dedicated and fully automatic JSEQ®pipeline. The new CSSG algorithm is iterative, parallel, and able to make decisions for discovering cell populations in tissues based on transcript occurrence in cells. The CSSG/JSEQ is complemented by a new strategy and specialized algorithm for the naming of cell populations. Our approach allows for high-resolution tracing of cell populations, finding relations and hierarchy between them, particularly important for complex tissues such as the brain. The pipeline allows the establishment of developmental, differentiation, and pathogenic trajectory and takes a “snapshot” of a current physiological or pathological cellular stage of the investigated organ at the transcriptional level.
Immune cells employ intermittent integrin-mediated traction forces for 3D migration
Authors: Czerwinski Tina , Bischof Lars , Böhringer David , Kara Sibel , Strissel Pamela L. , Strick Reiner , Winterl Alexander , Gerum Richard , Wittmann Ernst , Schneider Michael , Beckmann Matthias W. , Nusser Gina , Wiesinger Manuel , Budday Silvia , Lux Anja , Voskens Caroline , Fabry Ben , Mark Christoph
Published: April 2023

To reach targets outside the bloodstream, immune cells can extravasate and migrate through connective tissue. During tissue infiltration, immune cells migrate in an amoeboid fashion, characterized by weak matrix adhesions and low traction forces, that allows them to achieve high migration speeds of up to 10µm/min. How immune cells reconcile amoeboid migration with the need to overcome steric hindrance in dense matrices is currently not understood. Here we show that immune cells can switch from their default amoeboid migration mode to a contractile, mesenchymal-like migration mode when moving through fibrous human amniotic membrane (HAM) tissue. We subsequently study immune cell migration in reconstituted 3D collagen networks with known mechanical properties and pore sizes and apply time-lapse confocal reflection microscopy to obtain simultaneous measurements of migration speed, directional persistence, and cell contractility. We find that NK92 (natural killer) cells are highly mechanoresponsive and exert substantial acto-myosin driven, integrin-mediated contractile forces of up to 100 nN on the extracellular matrix during short contractile phases. This burst-like contractile behavior is also found in primary B, T, NK cells, neutrophils, and monocytes, and is tightly related to the fraction of cells that appear to become stuck in narrow pores of the surrounding matrix. Our results demonstrate that steric hindrance guides the rapid regulation of integrin-mediated adhesion to the ECM in a large number of immune cell subtypes.
Simulations suggest a scaffolding mechanism of membrane deformation by the caveolin 8S complex
Authors: Vasquez Rodriguez Sayyid Yobhel , Lazaridis Themis
Published: April 2023

Caveolins form complexes of various sizes that deform membranes into polyhedral shapes. However, the recent structure of the 8S complex was disk-like with a flat membrane-binding surface. How can a flat complex deform membranes into nonplanar structures? Molecular dynamics simulations revealed that the 8S complex rapidly takes the form of a suction cup. Simulations on implicit membrane vesicles determined that binding is stronger when E140 gets protonated. In that case, the complex binds much more strongly to 5 and 10-nm radius vesicles. A concave membrane binding surface readily explains the membrane-deforming ability of caveolins by direct scaffolding. We propose that the 8S complex sits at the vertices of the caveolar polyhedra, rather than at the center of the polyhedral faces.

Significance

Caveolae are membrane invaginations that play important roles in eukaryotic cells. Crucial for their formation is the caveolin protein family, but the mechanism by which these proteins deform membranes is unclear. Molecular dynamics simulations revealed that a recently determined structure changes shape from flat to concave. With one titratable residue protonated, this structure binds more strongly to spherical membranes, thus providing a possible mechanism for the membrane-deforming ability of caveolins.
Collagen and actin network mediate antiviral immunity against Orsay in C. elegans intestinal cells
Authors: Zhou Ying , Zhong Weiwei , Tao Yizhi Jane
Published: April 2023

C. elegans is a free-living nematode that is widely used as a small animal model for studying fundamental biological processes and disease mechanisms. Since the discovery of the Orsay virus in 2011, C. elegans also holds the promise of dissecting virus-host interaction networks and innate antiviral immunity pathways in an intact animal. Orsay primarily targets the worm intestine, causing enlarged intestinal lumen as well as visible changes to infected cells such as liquefaction of cytoplasm and rearrangement of the terminal web. Previous studies of Orsay identified that C. elegans is able to mount antiviral responses by DRH-1/RIG-I mediated RNA interference and Intracellular Pathogen Response, a uridylyltransferase that destabilizes viral RNAs by 3′ end uridylation, and ubiquitin protein modifications and turnover. To comprehensively search for novel antiviral pathways inC. elegans, we performed genome-wide RNAi screens by bacterial feeding using existing bacterial RNAi libraries covering 94% of the entire genome. Out of the 106 antiviral genes identified, we investigated those in three new pathways: collagens, actin remodelers, and epigenetic regulators. By characterizing Orsay infection in RNAi and mutant worms, our results indicate that collagens likely form a physical barrier in intestine cells to inhibit viral infection by preventing Orsay entry. Furthermore, evidence suggests that the intestinal actin (act-5), which is regulated by actin remodeling proteins (unc-34,wve-1andwsp-1), a Rho GTPase (cdc-42) and chromatin remodelers (nurf-1andisw-1), also provides antiviral immunity against Orsay possibly through another physical barrier presented as the terminal web.
Glycolytic reprogramming fuels myeloid cell-driven hypercoagulability
Authors: Rehill Aisling M. , Leon Gemma , McCluskey Sean , Schoen Ingmar , Hernandez-Santana Yasmina , Annett Stephanie , Klavina Paula , Robson Tracy , Curtis Annie M. , Renné Thomas , Hussey Seamus , O’Donnell James S. , Walsh Patrick T. , Preston Roger J.S.
Published: April 2023

Background

Myeloid cell metabolic reprogramming is a hallmark of inflammatory disease, however, its role in inflammation-induced hypercoagulability is poorly understood.

Objective/Methods

Using novel myeloid cell-based global haemostasis assays and murine models of immunometabolic disease, we evaluated the role of inflammation-associated metabolic reprogramming in regulating blood coagulation.

Results

Glycolysis was essential for enhanced activated myeloid cell tissue factor expression and decryption, driving increased cell-dependent thrombin generation in response to inflammatory challenge. Similarly, inhibition of glycolysis enhanced activated macrophage fibrinolytic activity via reduced plasminogen activator inhibitor 1 (PAI-1)-activity. Macrophage polarisation or activation markedly increased endothelial protein C receptor (EPCR) expression on monocytes and macrophages, leading to increased myeloid cell-dependent protein C activation. Importantly, inflammation-dependent EPCR expression on tissue-resident macrophages was also observedin vivo. Adipose tissue macrophages from obese mice fed a high-fat diet exhibited significantly enhanced EPCR expression and APC generation compared to macrophages isolated from the adipose tissue of healthy mice. Similarly, the induction of colitis in mice prompted infiltration of EPCR+innate myeloid cells within inflamed colonic tissue that were absent from the intestinal tissue of healthy mice.

Conclusion

Collectively, this study identifies immunometabolic regulation of myeloid cell hypercoagulability, opening new therapeutic possibilities for targeted mitigation of thrombo-inflammatory disease.

ESSENTIALS

Inflammation-mediated glycolytic reprogramming enables myeloid cell-induced hypercoagulability and antifibrinolytic activity.

2-Deoxy-D-glucose (2-DG) inhibits the expression of transcription factors necessary for inflammation-induced procoagulant gene expression.

Myeloid cell membrane regulation of tissue factor procoagulant activity is glycolysis-dependent.

Activation of myeloid innate immunity dysregulates activated protein C anticoagulant pathway activity.
Structure and assembly of a bacterial gasdermin pore
Authors: Johnson Alex G. , Mayer Megan L. , Schaefer Stefan L. , McNamara-Bordewick Nora K. , Hummer Gerhard , Kranzusch Philip J.
Published: April 2023

In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis1–3. Studies of human and mouse GSDM pores reveal the functions and architectures of 24–33 protomers assemblies4–9, but the mechanism and evolutionary origin of membrane targeting and GSDM pore formation remain unknown. Here we determine a structure of a bacterial GSDM (bGSDM) pore and define a conserved mechanism of pore assembly. Engineering a panel of bGSDMs for site-specific proteolytic activation, we demonstrate that diverse bGSDMs form distinct pore sizes that range from smaller mammalian-like assemblies to exceptionally large pores containing >50 protomers. We determine a 3.3 Å cryo-EM structure of aVitiosangiumbGSDM in an active slinky-like oligomeric conformation and analyze bGSDM pores in a native lipid environment to create an atomic-level model of a full 52-mer bGSDM pore. Combining our structural analysis with molecular dynamics simulations and cellular assays, our results support a stepwise model of GSDM pore assembly and suggest that a covalently bound palmitoyl can leave a hydrophobic sheath and insert into the membrane before formation of the membrane-spanning β-strand regions. These results reveal the diversity of GSDM pores found in nature and explain the function of an ancient post-translational modification in enabling programmed host cell death.
Enhanced Peripheral Nerve Regeneration by Mechano-electrical Stimulation
Authors: Tai Youyi , Tonmoy Thamidul Islam , Win Shwe , Brinkley Natasha T. , Hyle Park B. , Nam Jin
Published: April 2023

To address limitations in current approaches for treating large peripheral nerve defects, this study evaluated the efficacy of functional material-mediated physical stimuli on peripheral nerve regeneration. Electrospun piezoelectric poly(vinylidene fluoride-trifluoroethylene) nanofibers were utilized to deliver mechanical actuation-activated electrical stimulation to nerve cells/tissues in a non-invasive manner. Using morphologically and piezoelectrically optimized nanofibers for neurite extension and Schwann cell maturation based on in vitro experiments, piezoelectric nerve conduits were implanted in a rat sciatic nerve transection model to bridge a critical-sized sciatic nerve defect (15 mm). A therapeutic shockwave system was utilized to activate the piezoelectric effect of the implanted nerve conduit on demand. The piezoelectric nerve conduit-mediated mechano-electrical stimulation induced enhanced peripheral nerve regeneration, resulting in full axon reconnection with myelin regeneration from the proximal to the distal ends over the critical-sized nerve gap. Furthermore, superior functional recovery was observed by walking track analysis and polarization-sensitive optical coherence tomography, demonstrating the excellent efficacy of the mechano-electrical stimulation strategy for treating peripheral nerve injuries.
Virally induced lipid droplets are a platform for innate immune signalling complexes
Authors: Monson EA , Laws JL , Telikani Z , Milligan AJ , Rozario AM , Amarasinghe I , Smith ML , Tran V , Dinh Q , Williamson N , Mechler A , Johnson C , Hofer MJ , Nie S , Whelan DR , Helbig KJ
Published: April 2023

Lipid droplets (LDs) are upregulated by host cells in the face of pathogen infection, however, the reason for this phenomenon remains largely unknown. Here, we demonstrate that virally induced LDs house a distinct and dynamic proteome containing key antiviral signalling pathway members, including the essential pattern recognition receptor; RIG-I, key adaptor proteins; STAT1 and STAT2 and prominent interferon inducible proteins; viperin and MX1. Changes in the LD proteome were underpinned by specific key changes in the lipidome of virally driven LDs, particularity in the phospholipid membrane. Following virus infection, key antiviral proteins formed complex protein-protein interactions on the LD surface, positioning this organelle as a key antiviral signalling platform for the first time. It is clear that dynamic regulation of both the proteome and the lipidome of LDs occurs rapidly following viral infection towards the initiation of a successful innate immune response.
Multiplexed Sequential Imaging in Living Cells with Orthogonal Fluorogenic RNA Aptamer/Dye Pairs
Authors: Zheng Ru , Wu Rigumula , Liu Yuanchang , Sun Zhining , Bagheri Yousef , Xue Zhaolin , Mi Lan , Tian Qian , Pho Raymond , Siddiqui Sidrat , Ren Kewei , You Mingxu
Published: April 2023

Single-cell detection of multiple target analytes is an important goal in cell biology. However, due to the spectral overlap of common fluorophores, multiplexed fluorescence imaging beyond two-to-three targets inside living cells remains a technical challenge. Herein, we introduce a multiplexed imaging strategy that enables live-cell target detection via sequential rounds of imaging-and-stripping process, which is named as “sequential Fluorogenic RNA Imaging-Enabled Sensor” (seqFRIES). In seqFRIES, multiple orthogonal fluorogenic RNA aptamers are genetically encoded inside cells, and then the corresponding cell membrane permeable dye molecules are added, imaged, and rapidly removed in consecutive detection cycles. As a proof-of-concept, we have identified in this study fivein vitroorthogonal fluorogenic RNA aptamer/dye pairs (>10-fold higher fluorescence signals), four of which can be used for highly orthogonal and multiplexed imaging in living bacterial and mammalian cells. After further optimizing the cellular fluorescence activation and deactivation kinetics of these RNA/dye pairs, the whole four-color semi-quantitative seqFRIES process can now be completed in ∼20 min. Meanwhile, seqFRIES-mediated simultaneous detection of two critical signaling molecules, guanosine tetraphosphate and cyclic diguanylate, was also achieved within individual living cells. We expect our validation of this new seqFRIES concept here will facilitate the further development and potential broad usage of these orthogonal fluorogenic RNA/dye pairs for highly multiplexed and dynamic cellular imaging and cell biology studies.
A Logic-incorporated Gene Regulatory Network Deciphers Principles in Cell Fate Decisions
Authors: Xue Gang , Zhang Xiaoyi , Li Wanqi , Zhang Lu , Zhang Zongxu , Zhou Xiaolin , Zhang Di , Zhang Lei , Li Zhiyuan
Published: April 2023

Organisms utilize gene regulatory networks (GRNs) to make fate decisions, but the regulatory mechanisms of transcription factors (TFs) in GRNs are exceedingly intricate. A longstanding question in this field is how these tangled interactions synergistically contribute to decision- making procedures. To comprehensively understand the role of regulatory logic in cell fate decisions, we constructed a logic-incorporated GRN model and examined its behavior under two distinct driving forces (noise-driven and signal-driven). Under the noise-driven mode, we distilled the relationship among fate bias, regulatory logic, and noise profile. Under the signal-driven mode, we bridged regulatory logic and progression-accuracy trade-off, and uncovered distinctive trajectories of reprogramming influenced by logic motifs. In differentiation, we characterized a special logic-dependent priming stage by the solution landscape. Finally, we applied our findings to decipher three biological instances: hematopoiesis, embryogenesis, and trans-differentiation. Orthogonal to the classical analysis of expression profile, we harnessed noise patterns to construct the GRN corresponding to fate transition. Our work presents a generalizable framework for top- down fate-decision studies and a practical approach to the taxonomy of cell fate decisions.
COMPLETE GENOME SEQUENCE OFVIBRIO SYNGNATHI SP. NOV., A FISH PATHOGEN, ISOLATED FROM THE KIEL FJORD
Authors: Chibani Cynthia Maria , Hertel Robert , Neumann-Schaal Meina , Goehlich Henry , Wagner Kim , Bunk Boyke , Spröer Cathrin , Overmann Jörg , Hoppert Michael , Marten Mareike , Roth Olivia , Liesegang Heiko , Wendling Carolin C.
Published: April 2023

A newVibriostrain K08M4Twas isolated from the broad-nosed pipefishSyngnathus typhlein the Kiel Fjord. Infection experiments revealed that K08M4Tis highly virulent for juvenile pipefish. Cells of strain K08M4Tare Gram-stain-negative, curved rod-shaped and motile by means of a single polar flagellum. The strain can grow aerobically at 9 to 40°C, at pH 4 to 10.5 and tolerates up to 12% (w/v) NaCl. The most prevalent (> 10%) cellular fatty acids of K08M4Twere C16:1ω7cand C16:0.Whole-genome comparisons revealed that K08M4Trepresents a separate evolutionary lineage which is distinct from otherVibriospecies and falls within theVibrio Splendidusclade. The genome is 4,886,292 bp in size, consists of two circular chromosomes (3,298,328 bp, 1,587,964 bp), and comprises 4,178 protein-coding genes and 175 RNA genes. In this study, we describe the phenotypic features of the new isolate and present the annotation and analysis of its complete genome sequence. Based on these data, the new isolate represents a new species for which we propose the nameVibrio syngnathi. The type strain is K08M4T(=DSM 109818T).

Supplementary material

One supplementary figure and six supplementary table are available with the online version of the Manuscript.
Analysis of the worldwide diversity ofXanthomonas hortorumpv.carotae, the agent of bacterial blight of carrot, reveals two distinct populations
Authors: Dupas Enora , Durand Karine , Jacques Marie-Agnès
Published: April 2023

Xanthomonas hortorumpv.carotaeis a pathogen responsible for the bacterial blight of carrot. While it infects carrot fields worldwide, its diversity remains unknown. Here, we validated by PCR the identification asX. hortorumpv.carotaeof most of the strains isolated from carrot symptoms. We study their diversity by sequencing seven housekeeping genes. The analysis confirmed the identity of most of the strains previously identified asX. hortorumpv.carotaeand highlighted the presence of two clusters inside the pathovarcarotae. The 18 non-X. hortorumpv.carotaehave been identified asXanthomonasfrom other species. As theX. hortorumpv.carotaestrains clustered only in two groups by this analysis, a new MLVA scheme was developed. It allowed to validate the subclusteration in two groups and observe diversity among them. At the same times based on this study and data from the literature, we proposed the strain CFBP 7900 as the neopathotype strain of the pathovar. The strain should replacement the holopathotype strain CFBP 4997, as it is no longer available in international collection.