Intranasal corticosteroids are effective medications against allergic rhinitis (AR). However, mucociliary clearance promptly eliminates these drugs from the nasal cavity and delays their onset of action. Therefore, a faster, longer-lasting therapeutic effect on the nasal mucosa is required to enhance the efficacy of AR management. Our previous study showed that polyarginine, a cell-penetrating peptide, can deliver cargo to nasal cells; moreover, polyarginine-mediated cell-nonspecific protein transduction into the nasal epithelium exhibited high transfection efficiency with minimal cytotoxicity. In this study, poly-arginine-fused forkhead box P3 (FOXP3) protein, the “master transcriptional regulator” of regulatory T cells (Tregs), was administered into the bilateral nasal cavities of the ovalbumin (OVA)-immunoglobulin E mouse model of AR. The effects of these proteins on AR following OVA administration were investigated using histopathological, nasal symptom, flow cytometry, and cytokine dot blot analyses. Polyarginine-mediated FOXP3 protein transduction induced Treg-like cell generation in the nasal epithelium and allergen tolerance. Overall, this study proposes FOXP3 activation-mediated Treg induction as a novel and potential therapeutic strategy for AR, providing a potential alternative to conventional intranasal drug application for nasal drug delivery. Allergic rhinitis (AR) is an inflammatory disease triggered by an immunoglobulin E (IgE)-mediated atopic response. It is characterized by nasal mucosal inflammation and manifests as rhinorrhea, sneezing, itching, and nasal congestion ,2. The rA possible solution for this issue is the use of cell-penetrating peptides (CPPs), a diverse family of short peptides that can enter several mammalian cell types ,10. Vari+ regulatory T cells (Tregs). Because the transfer of SLIT-induced Tregs provides tolerance against antigens, production of Tregs is necessary for SLIT efficacy [FOXP3) is widely recognized as a Treg marker and is designated as the “master transcriptional regulator” of Tregs [FOXP3 expression may stimulate the differentiation of naïve and memory CD4+ T cells into Tregs [FOXP3-mediated Treg induction is a potential therapeutic strategy for AR. To date, cell-based therapy such as adoptive cell transfer of engineered Tregs has provided an effective therapeutic alternative to combat autoimmune diseases. However, no studies have reported directly on the application of the FOXP3 gene or protein for treating AR.Sublingual immunotherapy (SLIT) is another AR-therapeutic strategy that is clinically used to treat type 1 allergies . SLIT inefficacy ,16. Forkof Tregs ,18. FOXPto Tregs . TherefoThe purpose of the current investigation was to explore the feasibility of 9R-mediated FOXP3 protein transduction into the nasal epithelium for therapeutic application in AR.FOXP3 and 9R-conjugated FOXP3 (FOXP3-9R) were constructed for attachment of 9R to the C-terminus of FOXP3 protein  [Plasmids encoding  protein . The plaMA, USA) ,20. ProtSix-week-old male ovalbumin (OVA)-specific IgE transgenic mice (“BALB/cA-Tg(IgE-H01-4)Rin Tg(IgE-kL01-4)Rin/Jcl”) were obtained from CLEA Japan Inc. . OVA-IgE mice are genetically modified BALB/c mice that exhibit both acute and chronic allergic reactions with persistent production of IgE upon intraperitoneal administration of egg albumin ,22. The FOXP3-9R and FOXP3 (control) proteins  were administered into the bilateral nasal cavities of the animals (5 mice/group) using a micropipette . Before nasal administration, the micropipette tip was positioned 1–2 mm from the entrance of the nasal cavity while mice were awake in the supine position. After nasal treatment, mice were placed in a prone posture for 30 min. We divided the OVA-IgE mice into 6 groups: FOXP3 96 h before OVA (FOXP3 injected 96 h before OVA administration), FOXP3-9R 96 h before OVA (FOXP3-9R injected 96 h before OVA administration), FOXP3 24 h before OVA (FOXP3 injected 24 h before OVA administration), FOXP3-9R 24 h before OVA (FOXP3-9R injected 24 h before OVA administration), FOXP3 immediately after OVA (FOXP3 injected immediately after OVA administration), and FOXP3-9R immediately after OVA groups (FOXP3-9R injected immediately after OVA administration). An overview of the experimental procedure is illustrated in Nasal itching in OVA-IgE mice was investigated to assess the allergic reaction induced by intraperitoneal administration of OVA. Following FOXP3-9R, FOXP3, or saline administration, mice were observed for itching for 10 min, after a 10 min adaptation period at each time point , on daysn = 5 per time point) were euthanized 3 h after OVA administration; none of the animals died before this point .+ T cells and APC-conjugated FOXP3+ T cells were positively selected using the CytoFLEX® flow cytometer  to isolate Tregs. The purity of sorted populations was estimated to be >99%. Fluorescence-activated cell sorting data were analyzed using the Kaluza 2.1 software package (Beckman Coulter) [To examine Treg expression, nasal and spleen cells were extracted from treated mice by gentle processing between the ends of two sterile frosted slides. Cell surface staining and flow cytometric analysis of FITC-conjugated mouse anti-CD4 (RM4-5), PE-conjugated mouse anti-CD25 (PC61.5), and APC-conjugated mouse or rat anti-FOXP3 (FJK-16s) expression were performed using the eBioscience FOXP3 “Mouse Regulatory T cell Staining Kit #2” (Thermo Fisher Scientific) as previously described . FITC-coTo examine the expression of cytokines, dot blot analysis was performed using a mouse cytokine antibody array kit  following the manufacturer’s instructions. Briefly, nasal epithelial tissues were homogenized and sonicated for 10 min to produce a uniform protein suspension and diluted in concentrations of 250 µg/mL using a BCA protein assay kit (Thermo Fisher Scientific) as previously described . After mp-values < 0.05 were considered statistically significant.GraphPad Prism version 9.50 for Windows  was utilized for all statistical analyses. Values are expressed as the mean ± standard error. Repeated measurements by two-way ANOVA with Tukey’s post hoc correction were used to determine variations among groups. After OVA administration, nasal itching in OVA-IgE mice was monitored at each time point. Nasal symptoms of AR began 1 h after administration, peaked at 3 h, and subsided by 48 h -4, IL-5, and IL-17 than in those that received FOXP3 24 h before OVA . Regarding the time course, compared to mice receiving FOXP3 immediately after OVA administration, mice receiving FOXP3-9R 24 h before OVA administration had lower levels of IL-4, IL-5, and IL-17 and higher levels of IL-9 and IL-13 in their CD4+ Path T cells . Moreover, mice receiving FOXP3-9R 96 h before OVA administration had lower levels of IL-2, IL-9, IL-10, IL-12p70, and IL-13 and higher levels of IL-4 and IL-5 than those receiving FOXP3 24 h before OVA administration .Nasal epithelium was isolated from the six experimental groups for performing bioassays; different ratios of Treg and CD4This study demonstrated that transduction of polyarginine-fused recombinant FOXP3 into the nasal epithelium of OVA-IgE transgenic mice may induce immune tolerance via homing of Tregs and activation of Th2, thereby preventing AR-related nasal symptoms. Administration of FOXP3 without polyarginine resulted in partial transduction; however, it did not alleviate the nasal symptoms. Similar to findings in previous studies, mice that underwent 9R-mediated protein transduction did not exhibit deterioration in terms of nasal symptoms or histopathological findings. In our study, AR-related histopathological findings, such as mucosal edema, increased inflammation, cilia loss, vascular dilatation, glandular hyperplasia, eosinophil infiltration in lamina propria, and dilatation and vacuolization in the pseudostratified columnar epithelium on the nasal mucosa surface, were observed in the FOXP3 and saline groups but not in the FOXP3-9R groups. Further, FOXP3-9R mice that maintained nasal mucosal integrity did not exhibit local side effects such as epithelial necrosis, irritation, or hemorrhage. Notably, the histopathological findings were substantially different between mice receiving FOXP3-9R and FOXP3. CPPs help cargo molecules penetrate cells and preserve their biological activity ,11. TherFOXP3 gene or FOXP3 protein.FOXP3 transduction can trigger immune tolerance against AR by inducing Treg-like cell production. FOXP3 also contributes to the maintenance of autoimmune system homeostasis. For example, extracellular adenosine is transported into the cytoplasm more easily by FOXP3 . In our The use of Tregs in therapeutic applications may involve risks, as Tregs transferred to patients can be altered into Th17 cells that lack regulatory functions ; Th17 ceThis study had several limitations. First, we did not monitor any long-term effects. As previously described for systemic administration, long-term observation may reveal tumorigenesis, off-target effects, and genesis of chronic inflammation through changing the T cell phenotype by immune responses . In addiOur study demonstrated that effective intranasal FOXP3-9R delivery into the nasal mucosa of mice induced Treg-like cells and generated anti-AR tolerance. Furthermore, cytokine assays indicated that FOXP3-9R transduction before OVA administration induced a SLIT-like response in the mouse nasal epithelium. These results suggest that CPP-mediated local protein transduction and immune tolerance therapy are potential alternatives to conventional protein transduction methods for the delivery of therapeutically relevant molecules for AR therapy. These findings shed light on new AR therapeutic approaches. However, further research is warranted for the definitive establishment of AR therapy using this strategy.


Individuals with COVID-19 who are immunocompromised have been reported to experience prolonged shedding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) compared to those who are immunocompetent. However, it remains unclear how long viable SARS-CoV-2 shedding persists in immunocompromised individuals during the era of widespread vaccination against COVID-19. This study aimed to evaluate the association between the duration of viable SARS-CoV-2 detection among immunocompromised individuals and their COVID-19 vaccination status.We retrospectively reviewed patients who admitted to our institute for COVID-19 treatment between November 2022 and March 2023. We defined immunocompromised individuals as those who had received corticosteroids, immunosuppressants, or cytotoxic chemotherapy. We performed daily COVID-19 rapid antigen detection tests (RAD) on immunocompromised patients from 10 days after the onset of COVID-19 until two consecutive negative results were obtained.Among 181 patients admitted for COVID-19 treatment, 22 immunocompromised patients participated in this study. Of these, 14 (64%), 6 (27%), and 10 (46%) patients received corticosteroids, immunosuppressants, and cytotoxic chemotherapy, respectively. Twelve (55%) patients were fully vaccinated. Nine (41%) patients required oxygen therapy for respiratory failure. All patients received antiviral therapy, with 5 (23%), 12 (55%), 5 (23%) patients receiving remdesivir, ritonavir-boosted nirmatrelvir, and molnupiravir, respectively. Three (14%) patients died from COVID-19. The median duration from COVID-19 onset to confirmation of negative RAD or death was 15.5 (interquartile range: 12 to 17) days. Until 20 days after the onset of COVID-19, 17 (77%) patients had achieved two consecutive negative RAD results. There was no association between vaccination status and the duration of positive RAD results (p = 0.59) or the proportion of patients achieving negative RAD results within 20 days of COVID-19 onset (P > 0.99).Most immunocompromised COVID-19 patients in our hospital achieved two consecutive negative RAD results within 20 days of COVID-19 onset. Vaccination status was not associated with the duration of detectable SARS-CoV-2 shedding.All Authors: No reported disclosures


MicrotubuleAssociatedProteins (MAPs) assist the assembly and function of mitotic spindles and interphase arrays. Nine MAP65 genes exist in the genome of the acentrosomal model plant,Arabidopsis thaliana, and the function of majority of these proteins is unclear. To address this knowledge gap, we demonstrate the localization ofA. thaliana MAP65-6andMAP65-7fusion proteins expressed from native promoters in interphase cells of developingA. thalianaseedlings. Analyses of these fusion proteins co-expressed with alpha-tubulin 6 reporters indicate thatMAP65-6andMAP65-7bind a subset of interphase microtubules. Co-expression of GFP:MAP65-6with mCherry:MAP65-2from native promoters inA. thaliana showed overlapping localization patterns on interphase microtubule bundles. Collectively, these data suggested thatMAP65-2, -6, and -7 bind cortical microtubule bundles in plant interphase microtubule arrays.Microtubules are essential components of eukaryotic cells. Myriad proteins associate with microtubules to facilitate the organization and operation of microtubule arrays. Various MicrotubuleAssociatedProteins (MAPs) assist microtubule array organization and function in numerous ways throughout the eukaryotic cell cycle. In addition to the mitotic spindle, plant cells organize the preprophase band and phragmoplast microtubule arrays that predict and execute cytokinesis, respectively . The plant interphase microtubule cytoskeleton transitions between multiple organizations in response to developmental cues, physiological states, and environmental stimuli . Multiple MAPs facilitate array organization and MAPs mediate the role of interphase microtubules in growth and morphogenesis through cell wall formation, differential growth, and coordination with the actin cytoskeletonArabidopsis thalianaMAP65-6andMAP65-7proteins are unknown. Specific MAP65 family members are known to facilitate mitosis by bundling antiparallel microtubules in the central mitotic spindle . Nine MAP65 genes (designatedMAP65-1throughMAP65-9) exist in the model plantA. thalianaand are split into five sub-paralogous groups . The role ofA. thalianaMAP65-3and 65-4 appear similar to canonical animal MAP65 proteins in antiparallel microtubule bundling during cell division .MAP65-1andMAP65-2act during axial cell elongation and cell proliferation  and may provide microtubule polymers protection from severing, depolymerization from salt and/or low temperatures .The cellular roles ofA. thalianaMAP65 family members exhibited different biochemical properties, subcellular localizations, and expression patterns within plants . Published reports of MAP65 subcellular localization are inconsistent which may be due to differences between immunolocalization and fluorescent reporter studies using heterologous and/or constitutive overexpression of fluorescent protein fusions in plants and cultured cells . For example,in vitrostudies suggestedA. thalianaMAP65-6crosslinks microtubules into a perpendicular meshwork whileMAP65-1bundles microtubules into linear arrays . Immunolocalization ofMAP65-6, however, indicated binding to linear microtubule arrays in the plant-specific preprophase band and phragmoplast . A separate antibody study indicated thatMAP65-6labeled mitochondria but not microtubules . The localization ofMAP65-7has not yet been published.Previous studies indicated thatMAP65-6andMAP65-7fluorescent reporter proteins expressed from native genomic promoters on interphase cortical microtubules inA. thalianaseedlings.MAP65-6(At2g019010) andMAP65-7(At1g14690) proteins are 79.8% identical at the amino acid level. We built N-terminal eGFP and mCherry fusions to bothA. thaliana MAP65-6andMAP65-7genomic sequences , as a C-terminal fusion may interfere with protein function . Native promoters were used to examine where expression occurred in the plant and because overexpression of microtubule associated proteins may disrupt protein localization patterns .Here we report the localization ofMAP65-6andMAP65-7were both expressed in the majority of light-grownA. thaliana epidermal hypocotyl cells and both labeled a subset of interphase cortical microtubules . While bothMAP65-6andMAP65-7fusion proteins bound apparent microtubule bundles within the cortical arrays, not all GFP:TUA6 or mCherry:TUA6 microtubule structures were MAP65 labeled . Apparently single cortical microtubules were not labeled withMAP65-6. mCherry:MAP65-6, mCherry:MAP65-7, and GFP:MAP65-7 fusion proteins localized to non-microtubule cytoplasmic structures , and co-localized with GFP:MAP65-6in seedling stems  shares 45.6% and 46.1% amino acid identity withMAP65-6andMAP65-7respectively.To better define the cortical microtubule subpopulation bound byg stems . WithinA. thalianaMAP65-6andMAP65-7fluorescent reporter protein fusions driven from native promoters in living cells decorated a subset of interphase microtubules , possibly due to cell type and/or cell cycle specific regulation. Further studies will determine the identity of the intracellular compartments labeled with theseMAP65-6andMAP65-7fusion proteins. Co-expression of these reporters with tubulin orMAP65-2reporters indicated thatMAP65-6decorated antiparallel bundles. Furthermore, parallel microtubule bundles in stomatal guard cells were not labeled with mCherry:MAP65-7. AsMAP65-6and MAP65-7 are nearly 80% similar to one another and show similar subcellular localizations, their functional roles may overlap. Further genetic analysis will be needed to determine protein function and assess potential redundancy with other MAP65 proteins in plant cells.In conclusion,tubules  and repConstruction of Arabidopsis thaliana plants expressing fusion proteins.MAP65-6(At2g01910) andMAP65-7(At1g14690) full genome sequences were cloned from wild type Columbia-0A. thalianagenomic DNA into p211 plasmid so the GFP coding sequence was N-terminal to the MAP65 coding sequence. The native promoter (2.5kB of upstream genomic DNA sequence upstream) of bothMAP65-6and 65-7 were cloned to drive the expression of the respective N-terminal GFP fusion. Each construct was transformed byAgrobacteriumfloral dip transformation into homozygous T-DNA SALK lines for the respective gene . Detailed protocols on plasmid and transgene construction, transformation, and selection, see Lucaset al2011.A. thalianaplants into liquid cultures ofAgrobacterium tumenfaciens harboring Ti plasmids with engineered transgenesA. tumenfacienscultures containing both transgenes. Mature seeds from dipped plants dried for two weeks and were then sown on 1/2x Murishage and Skoog nutrient agar plates agar plates supplemented with kanamycin and/or hygromycin antibiotics. Seeds were germinated in a Percival growth chamber for 12-14 days to select for genetic transformation. T1 seedlings resistant to antibiotics were transplanted to soil, grown to maturity, and produced progeny T2 seeds through self-fertilization. These T2 seeds were then germinated in 24 hours of light on nutrient agar plates, and 6-day old hypocotyls were imaged on a Leica SP5 confocal microscope. At least six T2 seedlings from five different T1 parent plants were viewed for each of four transformations . All seedlings showed similar microtubule localizations of MAP65-6 and MAP65-7 on interphase cortical microtubules.Transgenic lines were produced by dipping developing floral stems ofLive-cell Confocal Imaging of Reporter Gene FusionsSeedlings were imaged on a Leica SP5 microscope with 488-nm and 561-nm laser excitation lines to excite eGFP and mCherry reporter fusions, respectively. Cells were viewed with 63x water immersion lens (1.2 N.A.). Seedlings were mounted on glass slides in liquid nutrient media under 1.5 coverslips. Vacuum grease adhered the coverslip to the slide and protected the seedling from compression. Plants acclimated to slides for 20 minutes before imaging.


Results indicated that, in addition to hypercholesterolemia and hyperlipidemia, LPC levels were two to four times higher in apoE−/− mice compared to wild-type mice in C57BL/6 background, regardless of whether they were 8 or 16 weeks old. Sphingomyelin (SM) and CER were elevated three- to five-fold in apoE−/− mice both at the basal level and after 16 weeks when compared to wild-type mice. After HFD treatment, the difference in CER levels elevated more than ten-fold. Considering the atherogenic properties of LPC and CER, they may also contribute to the early onset of atherosclerosis in apoE−/− mice. In summary, the HFD-fed apoE−/− mouse shows elevated LPC and CER contents and is a suitable model for developing LPC- and CER-lowering therapies.Despite the availability and use of numerous cholesterol-lowering drugs, atherosclerotic cardiovascular disease (ASCVD) remains the leading cause of mortality globally. Many researchers have focused their effort on identifying modified lipoproteins. However, lipid moieties such as lysophosphatidylcholine (LPC) and ceramide (CER) contribute to atherogenic events. LPC and CER both cause endothelial mitochondrial dysfunction, leading to fatty acid and triglyceride (TG) accumulation. In addition, they cause immune cells to differentiate into proinflammatory phenotypes. To uncover alternative therapeutic approaches other than cholesterol- and TG-lowering medications, we conducted untargeted lipidomic investigations to assess the alteration of lipid profiles in  Herba patriniae, a phytomedicine, has been identified as a potential anti-atherosclerosis candidate by modulating the glycerophospholipid metabolic pathway  PAF more slowly than their wild-type control, due to competition by truncated oxidized phospholipids [apoE−/− mice in comparison to the NCD group. The explanation for this point needs further investigation.Both at baseline and after diet therapy, holipids . HoweverapoE−/− mice , 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (C18:0 LPC), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (d18:1/16:0 SM), N-stearoyl-D-erythro-sphingosylphosphorylcholine (d18:1/18:0 SM), N-palmitoyl-D-erythro-sphingosine (d18:1/16:0 ceramide), N-lignoceroyl-D-erythro-sphingosine (d18:1/24:0 ceramide) and ceramide/sphingolipid internal standard mixture I  C12 ceramide, 12:0 sphingomyelin, glucosyl(ß) C12 ceramide, 12:0 ceramide, 12:0 ceramide-1-P, 25:0 ceramide) were purchased from Avanti Polar Lipids . Standard stock solutions were dissolved in chloroform/methanol 1:1 (v/v) at a concentration of 1 mM and stored at −20 °C. Lipid standard mixtures were prepared freshly every day in lipid buffer  at a concentration of 25 μM and used immediately.1-palmitoyl-2-hydroxy-apoE−/− mice (C57BL/6-Apoeem1Narl/Narl) were purchased from National Applied Research Laboratories (NARL) Taiwan. Animals were kept in pathogen-free environments. Animals were acclimated until they were 8 weeks old. Following this, animals were divided into three groups Group I—C57BL/6 wild-type (n = 7) fed on the normal-chow diet (NCD), Group II— apoE−/− (n = 7) fed on NCD  and Group III— apoE−/− (n = 7) fed with HFD. The HFD was procured from Research Diets Inc.  and comprised 60 kcal% fat, 20 kcal% protein, and 20 kcal% carbohydrates per 100 g of pellet. Blood was drawn into anti-coagulated (K2-EDTA) collection tubes from the tail vein at baseline (8 weeks old) and after 8 weeks (16 weeks old).The Institutional Animal Care and Use Committee (IACUC) of Kaohsiung Medical University (KMU) approved these experiments (IACUC-109273). All tests were conducted in compliance with the institution’s regulations, using humane endpoints and adhering to ethical principles. Six-week-old male C57BL/6 (wild type) and g at 4 °C with soft braking. Plasma was collected after centrifugation for biochemical analysis including aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine (Cr), glucose, total cholesterol (TC), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C). The NARL Taiwan performed these tests using a Hitachi 7080 biochemical analyzer.Whole blood was gently centrifuged for 15 min at 800× g, 4 °C for 10 min. The upper aqueous solvent was discarded, and the lower organic phase was collected into a new glass vial, which was then evaporated with a nitrogen gas evaporator . It was then solubilized using sample buffer  and stored at −80 °C until LC/MSE analysis.Plasma lipids were extracted using the Folch technique ,59. In sE) system equipped with a charged surface hybrid (CSH™) C18 column [® UPLC  equipped with CSH™ 1.7 um, 2.1 mm × 10 cm C18 column under gradient conditions at a flow rate of 0.1 mL/min for 20 min at 55 °C. The mobile phase A was composed of 10 mM ammonium formate  and 0.1% (v/v) formic acid  in acetonitrile (ACN)  /H2O in the ratio 60:40. Mobile phase B contained 10 mM ammonium formate and 0.1% (v/v) formic acid in isopropanol (IPA) /ACN in the ratio 90:10 for molecule protonation. Mass spectrometry was carried out using a Xevo G2 QTOF  equipped with an electrospray ionization interface (ESI) and operated in the data-independent collection mode (MSE). Parallel ion fragmentation had been programmed to switch between low (4 eV) and high (35–55 eV) energies in the collision cell, and signals were collected from 250 to 1600 m/z utilizing leucine enkephalin (556.2771 Da) as the separate data channel lock mass calibrant. Data were processed by Progenesis QI® . Analysis modules were enhanced by updating chemical identifiers from LipidBlast [Total lipids were extracted and quantified using a liquid chromatography data-independent parallel-fragmentation mass spectrometry . GraphPad Prism Software version 9.2.0 was used for statistical analysis and statistical graphics. The differences between groups were evaluated using the two-tailed student apoE−/− mice, while the latter was substantially higher following HFD feeding. We conclude that HFD-fed apoE−/− mice are a suitable model for developing LPC- and CER-lowering ASCVD therapeutics. These bioactive lipids could play crucial roles in the etiology of ASCVD. The noteworthy findings of this study provide new insights into the treatment of ASCVD.Both bioactive lipids LPC and CER were significantly elevated in


LGALS3BP, displays broad antiviral activity. It reduces HIV-1 infectivity by interfering with Env maturation and virion incorporation, and increases survival of Influenza A virus-infected mice via antiviral innate immune signaling. Its antiviral potential in SARS-CoV-2 infection remains largely unknown. Here, we analyzed the expression of 90K/LGALS3BP in 44 hospitalized COVID-19 patients at multiple levels. We quantified 90K protein concentrations in serum and PBMCs as well as LGALS3BP mRNA levels. Complementary, we analyzed two single cell RNA-sequencing datasets for expression of LGALS3BP in respiratory specimens and PBMCs from COVID-19 patients. Finally, we analyzed the potential of 90K to interfere with SARS-CoV-2 infection of HEK293T/ACE2, Calu-3 and Caco-2 cells using authentic virus. 90K protein serum concentrations were significantly elevated in COVID-19 patients compared to uninfected sex- and age-matched controls. Furthermore, PBMC-associated concentrations of 90K protein were overall reduced by SARS-CoV-2 infection in vivo, suggesting enhanced secretion into the extracellular space. Mining of published PBMC scRNA-seq datasets uncovered monocyte-specific induction of LGALS3BP mRNA expression in COVID-19 patients. In functional assays, neither 90K overexpression in susceptible cell lines nor exogenous addition of purified 90K consistently inhibited SARS-CoV-2 infection. Our data suggests that 90K/LGALS3BP contributes to the global type I IFN response during SARS-CoV-2 infection in vivo without displaying detectable antiviral properties in vitro.Glycoprotein 90K, encoded by the interferon-stimulated gene The online version contains supplementary material available at 10.1007/s10238-023-01077-2. LGALS3BP) is a ubiquitously expressed cellular secreted glycoprotein with multiple antiviral activities. Expression of LGALS3BP is stimulated by IFNs, resulting in upregulated 90K serum concentrations in individuals with viral infections, including HIV-1 [+ T-cell-mediated cytotoxicity, and cytokine production [LGALS3BP expression and its reported association with viral infections in vivo, including its potential suitability as a prognostic marker for disease progression in HIV-1/AIDS [LGALS3BP in specimens of COVID-19 patients and uninfected individuals and probed for a potential direct antiviral role of 90K against SARS-CoV-2 infection.SARS-CoV-2 infects host cells via interaction of its spike protein with the ACE2 receptor , 2. The ng HIV-1 , HCV [6,ng HIV-1 , hantaving HIV-1 , and denng HIV-1 . 90K hasng HIV-1 . In adding HIV-1 . Furtheroduction . Defectioduction  and presoduction , 16 haveV-1/AIDS , we invePa-COVID-19 Study [Pa-COVID-19 is a prospective observational cohort study collecting data longitudinally from patients with confirmed COVID-19 weekly during their hospitalization. Data include, inter alia, epidemiological and demographic parameters, medical history, clinical course and morbidity [Hospitalized COVID-19 patients’ blood samples and clinical data were collected at Charité - Universitätsmedizin Berlin in the context of the 19 Study . Pa-COVIorbidity . Patientorbidity , Hepatitorbidity , or HIV-orbidity . Furtherorbidity .WHO 1: Asymptomatic infection.WHO 2: Symptomatic infection, ambulatory care.WHO 3: Hospitalization, no supplemental oxygen required.WHO 4: Supplemental oxygen required.WHO 5: High Flow or Continuous Positive Airway Pressure (CPAP) required.WHO 6: Mechanical ventilation required.WHO 7: Extracorporeal Membrane Oxygenation (ECMO) and/or Continuous RenalReplacement Therapy (CRRT) required.WHO 8: Death.We assessed COVID-19 disease severity using the World Health Organization (WHO) ordinal scale for clinical improvement on sampling day .WHO 1: AFor practical reasons, we designated patients admitted to general hospital wards as “mild COVID-19” (WHO 3-4) and patients admitted to intensive care units as “severe COVID-19” (WHO 5-7). Within the latter, patients receiving mechanical ventilation, ECMO or CRRT were designated as “critical COVID-19” (WHO 6-7). Patients with multiple WHO grades within the sampling period (6 out of 42 patients) were assigned the highest disease severity grade.—Germany’s largest population-based longitudinal cohort study [A control population was provided by the study center Berlin-Mitte of the German National Cohort .2. HEK293T/ACE2 cells were additionally supplemented with 13 µg/ml Blasticidin. Caco-2/90K cells were additionally supplemented with 10 µg/ml Puromycin.Calu-3 (ATCC HTB-55), Caco-2 (ATCC HTB-37), Vero E6 (ATCC CRL-1586), and HEK293T (ATCC CRL-3216) cells were cultured in DMEM (Sigma Aldrich) with 4.5 g/L glucose, supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% L-glutamine at 37 °C and 5% COg. PBMCs were then washed with PBS. Remaining erythrocytes were lysed with ACK-Lysis Buffer , followed by PBS washing. PBMC pellets were frozen at − 20 °C.PBMCs from COVID-19 patients and healthy controls from anonymized blood donors were isolated from 2–3 ml EDTA whole blood. Samples were mixed 1:1 with PBS and centrifuged on Pancoll (Pan Biotech) for 30 min at 200 × ® 20 concentrators (Sartorius Stedim Biotech). Virus stocks were diluted in OptiPro serum-free medium supplemented with 0.5% gelatine and PBS and stored at − 80 °C. Infectious titer was defined by plaque titration assay.SARS-CoV-2 strain Munich 984  was propagated on Vero E6 cells and concentrated using VivaspinVSV-G-pseudotyped lentiviral vector particles encoding human 90K were generated by calcium phosphate-based transfection of HEK293T cells with the packaging plasmid pCMV ΔR8.91 , the len5, 3 × 105, and 4.5 × 105 cells/ml, respectively. Cells were infected with SARS-CoV-2 (MOI 0.01). Virus inoculum was removed after one hour, cells were washed with PBS and resupplied with fresh medium. Where indicated, cells were pretreated with Remdesivir (20 µM), and treatment was continued until the end of the experiment.Calu-3, Caco-2, and HEK293T/ACE2 cells were seeded in 6-well plates at densities of 6 × 10Bar graphs indicate mean values and error bars indicate standard deviation. Whiskers in box plots indicate minimum to maximum values. Trends over time are displayed using locally weighted scatterplot smoothing (LOESS) estimates.To determine statistical significance between two independent groups, we used unpaired t-tests and paired t-tests when comparing cases and matched controls, assuming normal distribution. To analyze data with both independent and dependent observations due to longitudinal measurements of an individual, we used mixed effects models.Based on scRNA-seq data we used these models to compare expression between cases and controls, just as between mild cases, severe cases, and controls regarding ten cell types from PBMCs and 20 cell types from respiratory samples. As we consider all analyses to be exploratory, we did not adjust for multiple testing.Graph Pad Prism 9.1.2. Further statistical analyses were performed using R. The code utilized for the analysis will be available at https://github.com/GoffinetLab/SARS-CoV-2_90K_patient_study.When analyzing independent observations, graphs and statistical analyses were generated using p < 0.0001). Highest 90K concentrations were detected in patients classified WHO 5 . Analyzing all samples with respect to the sampling time point, we saw gradually decreasing 90K levels over time with highest levels at early infection stages  and similar rate of decline in patients with mild and severe disease .We quantified 90K concentrations in 119 serum samples from 42 hospitalized COVID-19 patients and 42 age- and sex-matched healthy controls (NAKO). Both mean . This difference was not observed in COVID-19 patients, which displayed enhanced 90K serum concentrations irrespective of age.Furthermore, we analyzed possible interrelations between 90K serum concentrations, viral RNA concentrations from nasopharyngeal swabs and anti-SARS-CoV-2 IgG/IgA titers and found no association . For demographic aspects, no sex-dependent differences in 90K serum levels were noted , but we recorded higher concentrations in older healthy individuals > 55 y compared to younger individuals  and found reduced concentrations in COVID-19 compared to healthy controls, regardless of disease severity  showed no upregulated LGALS3BP mRNA expression in PBMCs from COVID-19 patients remained unaltered as compared to healthy controls.In summary, in our cohort of SARS-CoV-2-infected individuals, 90K protein concentrations were enhanced in sera and reduced in PBMCs, while LGALS3BP mRNA expression patterns in individual cell types of PBMCs of COVID-19 patients that may be undetectable in our whole-PBMC analysis . Overall, expression was highest at early infection stages and decreased after symptom onset .We analyzed the expression of eq study . In contLGALS3BP expression, we infected Calu-3 cells with authentic SARS-CoV-2. We reached infection rates of 19% 48 h post infection as measured by flow cytometry . LGALS3BP expression was slightly induced after IFN-α2 treatment  but not after SARS-CoV-2 infection . In contrast to our findings in HEK293T/ACE2 cells, overexpression of 90K in Caco-2 cells neither reduced the concentration of viral RNA, nor diminished the expression of nucleocapsid and spike in SARS-CoV-2 producing cells, nor influenced spike processing Fig. B. ImportTo analyze a potential role of exogenously added 90K protein on SARS-CoV-2 infection, we pretreated Calu-3 and Caco-2 cells with 90K, for which signaling via cell surface proteins has been reported , 37–39 oLGALS3BP mRNA in monocytes, plasmablasts, and dendritic cells hint towards a complex, compartment- and cell type-specific regulation of 90K expression in SARS-CoV-2 infection in vivo. However, data for PBMC-associated 90K protein levels were gathered from a rather small cohort and should ideally be confirmed in a larger sample size study. It is conceivable that elevated serum 90K concentrations in COVID-19 originate from monocytes, even though our data do not exclude other 90K-producing cell types as alternative sources. Strikingly, scRNA-seq data from respiratory samples of COVID-19 patients showed no upregulated LGALS3BP mRNA expression, illustrating the multifaceted ability of SARS-CoV-2 to antagonize or evade cellular sensing machineries in productively infected cells [Our findings of upregulated 90K serum concentrations in COVID-19 with highest levels in severe disease courses are in line with proteomic and serological findings from hospitalized COVID-19 patients , 42. Oveed cells , 44.LGALS3BP is an ISG, enhanced 90K serum levels in patients with severe COVID-19 could possibly reflect exaggerated type I IFN responses. On the other hand, mounting of an efficient IFN-mediated antiviral state is associated with effective clearance of SARS-CoV-2 infection and milder course of the disease [LGALS3BP upregulation in our scRNA-seq analysis (cohort A), whereas mildly affected patients displayed distinct upregulation. However, this pattern could not be reproduced in cohort B, possibly due to technical differences in the scRNA-seq pipelines and/or unknown clinical differences such as medication or comorbidities. Nevertheless, since LGALS3BP is a prototypic ISG [LGALS3BP is part of, and does not necessarily imply that 90K is an active antiviral component of SARS-CoV-2 infection.The role of type I IFN in the context of COVID-19 has been discussed controversially and may be time point-dependent. On the one hand, overdriven IFN responses are thought to cause severe COVID-19 , 46. Sin disease . Accordiypic ISG , 48, itsIn line with this idea, we did not generate consistent evidence for an anti-SARS-CoV-2 activity of 90K that is exerted directly on SARS-CoV-2 infection. Mild inhibitory effects were detected in HEK293T/ACE2 cells heterologously expressing 90K, complementing former results suggesting a reduced susceptibility to transduction with lentiviral particles decorated with SARS-CoV-2 spike in the same cell line overexpressing 90K . The inaLGALS3BP in COVID-19 at multiple levels. We propose that 90K/LGALS3BP contributes to the global type I IFN response during SARS-CoV-2 infection in vivo without exerting biologically relevant, direct antiviral effects detectable in cell culture. Therefore, further investigations on 90K as a potential biomarker and/or potential immunomodulator of SARS-CoV-2 pathogenesis are warranted.In summary, our study describes the expression pattern of Supplementary file1 (DOCX 1883 KB)Below is the link to the electronic supplementary material.


TNNT2 messenger RNA (mRNA) expression in the iPSCs-induced cardiomyocytes (iPS-CMs) than in the iPSCs. Cardiac troponin Ts became progressively organized but cytoplasmic RYR2 and SCN10A aggregations occurred in the iPS-CMs. Proband-specific iPS-CMs showed decreased RYR2 and SCN10A mRNA expression. The RYR2-A1855D variant resulted in premature spontaneous sarcoplasmic reticular Ca2+ transients, Ca2+ oscillations and increased action potential durations. SCN10A-Q1362H did not confer any specific phenotype. However, the combined heterozygous RYR2-A1855D and SCN10A-Q1362H variants in the proband iPS-CMs resulted in accentuated Ca2+ homeostasis disorders, action potential prolongation and susceptibility to early afterdepolarizations at high stimulus frequencies. These findings attribute the clinical phenotype in the proband to effects of the heterozygous RYR2 variant exacerbated by heterozygous SCN10A modification.We illustrate use of induced pluripotent stem cells (iPSCs) as platforms for investigating cardiomyocyte phenotypes in a human family pedigree exemplified by novel heterozygous RYR2-A1855D and SCN10A-Q1362H variants occurring alone and in combination. The proband, a four-month-old boy, presented with polymorphic ventricular tachycardia. Genetic tests revealed double novel heterozygous RYR2-A1855D and SCN10A-Q1362H variants inherited from his father (F) and mother (M), respectively. His father showed ventricular premature beats; his mother was asymptomatic. Molecular biological characterizations demonstrated greater This article is part of the theme issue ‘The heartbeat: its molecular basis and physiological mechanisms’. These have involved channel proteins including those encoded by RYR2, CALM1, CALM2 and KCNJ2 and SCN5A  or result in a tendency to store overload-induced Ca2+ release. The enhanced SR diastolic Ca2+ release associated with RYR2 gain of function causes an intracellular Ca2+ overload during exercise or emotional stress i is a ubiquitous intracellular second messenger. Pathological unregulated SR Ca2+ release is a potential cause of life-threatening arrhythmias i termed Ca2+ oscillations (COs) i and systolic, and abnormal diastolic [Ca2+]i increases accompanying AP activity reflecting the consequent influx of extracellular Ca2+ or Ca2+ release from intracellular stores i-oscillations i abnormalities, and that this was exacerbated by the presence of the SCN10A-Q1362H variant. The latter was reflected in the increased severity of the resulting phenotype in the proband compared to the proband-F iPS-CMs. By contrast, the SCN10A-Q1362H variant alone in the proband-M iPS-CMs was without effect.Secondly, established reports associate aberrant SR Caractions . The preSCN10A loci with altered cardiac conduction reflected in PR and/or QRS intervals, AF i through the resulting increased net [Ca2+] influx, and decreases efflux owing to the shorter diastolic intervals [Fourthly, cardiac AP waveforms vary with, thereby adapting to, altered heart rates following increased adrenergic drive during exercise ,45. At t courses . Increasntervals . This mantervals . In the . 5INa-L increases the probability of EADs, which may trigger arrhythmias. In addition, enhanced INa-L may result in NCX-mediated Ca2+ release events. These could promote depolarizing transient inward currents (Iti), resulting in similarly pro-arrhythmic DADs [+ current, pro-arrhythmic diastolic Ca2+ release, as well as spontaneous APs. This led to suggestions that Nav1.8-dependent selective INa-L reduction could offer a novel antiarrhythmic therapeutic approach [in vitro remains an important issue when using these as a platform for such studies. Future studies could use the Nav1.8-blocker A-803467 and selective late sodium current inhibitors  in different experiment platforms for PVT.The present study using iPS-CMs has several limitations that are worth mentioning. By prolonging the duration of the AP, mic DADs . In addiapproach . However. 62+ handling caused by the double novel heterozygous RYR2-A1855D and SCN10A-Q1362H variants associated with a clinical pro-arrhythmic phenotype. It associated the RYR2-A1855D but not the SCN10A-Q1362H variant when either was expressed alone, with abnormal Ca2+ homeostasis and APD prolongation. However, the compound heterozygous RYR2-A1855D and SCN10A-Q1362H variants showed an accentuated Ca2+ homeostasis and APD phenotype, suggesting a possible basis for the clinical features of the originating patient family.In summary, the study investigates acute abnormalities of myocardial Ca


Intravenous drug use predisposes users to life-threatening bacterial infections primarily caused by gram-positive organisms. Studies have seen an uptrend in gram-negative injection-related infections in persons who inject drugs (PWID). Therefore, this study aimed to assess the microbiology of injection-related infections in PWID and evaluate risk factors that may predispose these patients to infections caused by gram-negative organisms.>18 years with bacterial growth on specimens collected within 72 hours of admission from September 1, 2021, to March 31, 2022. Data analysis utilized descriptive statistics, chi-square tests, and Mann-Whitney U tests where appropriate.This retrospective chart review of adult PWID hospitalized with an injection-related infection  included patients aged S. pyogenes (43%), and MSSA (9%). The gram-negative organisms isolated are shown in Figure 1. Approximately 84% of patients received overtreatment with an anti-pseudomonal agent; however, only 2% required its use. SSTIs with lower extremity involvement were found to be associated with gram-negative infections within this cohort, as shown in Table 1.A total of 259 patients were included in the study. 243 (93.8%) patients grew gram-positive organisms, while only 16 (6.2%) grew gram-negative organisms. The majority of patients were male (60%), the median age was 38 (IQR [33-44]), and 10% had a prior infection with MRSA. The distribution of injection-related infections included SSTIs (79.9%), bacteremia (34.7%), septic arthritis (12%), infective endocarditis (10.4%), osteomyelitis (8.5%), and epidural abscess (3.5%). The most commonly observed organisms were MRSA (36%), In this study, despite less than 10% of patients growing gram-negative organisms on culture, approximately 80% received gram-negative treatment. Knowledge of the microbiology of infections in PWID can aid prescribers in optimizing empiric therapy for injection-related infections and preserving the core principles of antimicrobial stewardship.Sara K. Schultz, MD FACP FIDSA, AbbVie: Advisor/Consultant


A proposed mechanism of the reaction of guanidinium chlorides with dimethyl acetylenedicarboxylate in a tandem aza-Michael addition reaction/intramolecular cyclization was investigated by DFT M06-2X and B3LYP computational approaches. The energies of the products were compared against the G3, M08-HX, M11, and wB97xD data or experimentally obtained product ratios. The structural diversity of the products was interpreted by the concurrent formation of different tautomers formed in situ upon deprotonation with a 2-chlorofumarate anion. A comparison of relative energies of the characteristic stationary points along the examined reaction paths indicated that the initial nucleophilic addition is energetically the most demanding process. The overall reaction is strongly exergonic, as predicted by both methods, which is primarily due to methanol elimination during the intramolecular cyclization step producing cyclic amide structures. Formation of a five-membered ring upon intramolecular cyclization is highly favored for the acyclic guanidine, while optimal product structure for the cyclic guanidines is based on a 1,5,7-triaza [4.3.0]-bicyclononane skeleton. Relative stabilities of the possible products calculated by the employed DFT methods were compared against the experimental product ratio. The best agreement was obtained for the M08-HX approach while the B3LYP approach provided somewhat better results than the M06-2X and M11 methods. Similarly to amines, guanidines can be alkylated with different nucleophiles, including alkyl halides oct-4-ene (TBO) imply higher nucleophilicity of the larger ring system -dec-1-ene  = 35.688) [Computational details: All the structures were fully optimized at the B3LYP/6-31G level of theory ,29,30,31 35.688) . The Gib 35.688) . These a 35.688) , and M08 35.688) ) encompaM11: M11(SMD)//aug-cc-pVTZ//M11(SMD)/6-31+GwB97xD: wB97xD(SMD)/aug-cc-pVTZ//wB97xD(SMD)/6-31+GM08-HX: M08-HX(SMD)/pcseg-3//M08-HX(SMD)/pcseg-2In the case of M06-HX, Jensen’s polarization consistent segmented contracted basis sets of double zeta (pcseg-2) and triple zeta (pcseg-3) quality  taken fr


An estimated 1-3% of children with SARS-CoV-2 infection will develop Post-COVID Conditions (PCC). This study evaluates mRNA COVID-19 vaccine impact on likelihood of PCC in children.>14 days before infection) and unvaccinated (no vaccine doses before infection). Vaccination status was verified through vaccine registry and/or medical records.A multi-site cohort of children enrolled 7/21/2021-9/1/2022 underwent weekly SARS-CoV-2 screening tests and were surveyed via self- or parental report 12/1/2022-5/31/2023 regarding PCC (defined as ≥1 new or on-going symptoms lasting ≥ 1 month after infection). Multivariable logistic regression was performed to estimate the occurrence of PCC by vaccination status among children aged 5–17 years whose first PCR-confirmed SARS-CoV-2 infection occurred in-study with Omicron variant, who completed the survey >60 days from infection, and who were vaccine age-eligible at time of infection per ACIP recommendations. Vaccination status was categorized as vaccinated , >2 PCC symptoms , and respiratory PCC symptoms  (Table 3).Of 622 participants surveyed, 5% (n=28) had PCC (Table 1) and 67% (n=474) were vaccinated (Table 2). Surveys were completed a median (IQR) of 203.7 days (119.0–293.0) after infection. Children with non-Hispanic Black race/ethnicity and good/fair/poor self-rated baseline health were more likely to report PCC. Children aged 12-18 years, Non-Hispanic Asian and White children, those reporting symptomatic SARS-CoV-2 infection, and those with excellent/very good self-rated baseline health were more likely to report vaccination When comparing children with and without PCC symptoms, COVID-19 mRNA vaccination was associated with a decreased likelihood of >1, >2, and respiratory PCC symptoms among vaccinated children, respectively. These findings support COVID-19 vaccination for children and may encourage increased pediatric vaccine uptake.In this study, mRNA COVID-19 vaccination appeared to be protective against PCC in children following Omicron SARS-CoV-2 infection. The adjusted ORs correspond to an estimated 34%, 48%, and 47% reduced likelihood of Lisa Gwynn, MBA, MSPH, Merck: Honoraria


Furthermore, LAIR-1 expression in the tumor compartment was significantly higher in patients with lung adenocarcinoma (LUAD) than those with lung squamous cell carcinoma subtype . Our results indicated that high tumor LAIR-1 expression in patients with LUAD is negatively associated with OS  highlighting its prognostic value in LUAD but not in other subtypes. The Pearson correlation between LAIR-1 and PD-L1 is 0.31; however, mutual exclusive staining pattern  was observed. Altogether, our data suggest that the combination therapy of anti-PD-1/PD-L1 with anti-LAIR-1 or the anti-LAIR-1 monotherapy alone may be promising cancer immunotherapeutic strategies.Targeting the interaction of leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) and its ligands has been shown to reinstate antitumor immunity. In addition, the introduction of the LAIR-1 decoy protein, LAIR-2, sensitizes previously resistant lung tumors to programmed death-1 (PD-1) blockade, indicating the potential of LAIR-1 as an alternative marker for anti-PD-1 resistance in lung cancer. Here, we assessed LAIR-1 as compared with programmed death-ligand 1 (PD-L1) expression in various tumors, with a focus on non–small cell lung cancer (NSCLC) and its histologic subtypes using multiplexed quantitative immunofluorescence (mQIF) in 287 (discovery cohort) and 144  patients with NSCLC. In addition, using multispectral imaging technology on mQIF images, we evaluated the localization of LAIR-1 on various cell types. We observed that CD14+/CD68+ cell densities and negative association of OS with high LAIR-1 expression in LUAD tumor subtype.The spatial, quantitative assessment of LAIR-1 in NSCLC shows positive association of OS with high LAIR-1 LAIR-1 gene which maps to a region of 19q13.4 (leukocyte receptor cluster), a member of both the immunoglobulin superfamily and the leukocyte-associated inhibitory receptor family. Its expression has been reported in many the immune cell subsets and thought to be restricted to immune cells  may likely preclude the use of a single predictive biomarker of response to immunotherapy. The main objective of this study was to investigate LAIR-1 protein expression using quantitative immunofluorescence (QIF) and to assess its prognostic value alone or in combination with PD-L1 in patients with early-stage non–small cell lung cancer (NSCLC). Furthermore, using multiplexed QIF (mQIF), we investigated LAIR-1 protein expression across different subtypes of NSCLC and identified its localization in various tumor and stromal cell types comprising the NSCLC TME. Understanding the relative abundance of tumor and stromal cells where LAIR-1 is expressed can be a predictive feature to guide therapeutic decision-making through composite assessment of biomarkers and/or treatment regimens.Formalin-fixed, paraffin-embedded (FFPE) tumor specimens represented in a tissue microarray (TMA) with NSCLC from two independent cohorts were analyzed in this study. First, multitumor TMA, YTMA-395, containing 295 cores from 12 different types of solid tumors and hematologic malignancies was tested to identify the predominant expression of LAIR-1 in different tumor types. A small lung test array, YTMA-295 containing 35 lung tumor cores was used to test four different anti-LAIR-1 antibody clones targeting nonoverlapping epitopes. The discovery cohort, Yale TMA (YTMA-423), contains the tissue specimens from 287 patients with early-stage NSCLC with tumors resected between 2011 and 2016. Quantitative titration of four monoclonal LAIR-1 antibody clones including E7X6I , NKTA25 (Santa Cruz Biotechnology), 1A10 , and 1E4 (NC) were performed. The optimized final concentration for each antibody, using the standard approach in our lab , was 0.22) were analyzed using phenoptrReports (Akoya Biosciences) in R.First, the AQUA  method was used to measure the protein expression on fluorescence images acquired using a PM-2000 system (Navigate BioPharma) as described previously . A totalhttps://portal.gdc.cancer.gov/). The clinical information was filtered to exclude the data from solid tissue normal, and only the samples from the primary tumor and metastatic samples were analyzed. LAIR-1 mean RNA expression across all tumor types was compared to select LAIR-1 highly expressing tumors for further analyses. For validation, single-cell RNA-seq data from three lung carcinomas were downloaded from https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE127465 (LAIR-1 on specific cell types.RNA sequencing (RNA-seq) data and the clinical metadata in read counts of nine human tumors were obtained from The Cancer Genome Atlas (TCGA) data portal  P < 0.05 and (**) P < 0.01.Statistical analyses were performed using GraphPad Prism 9.2.0 (GraphPad Software Inc.) and RStudio 2022.12.0+353 . Comparison between two continuous variables was assessed for linear association using Pearson correlation coefficient. The unpaired https://portal.gdc.cancer.gov/ and https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE127465  showed that LAIR-1 is highly expressed in both LUAD and lung squamous cell carcinoma  in the discovery cohort was noted, confirming its expression in both compartments  colocalized with LAIR-1 in serial TMA sections  and quantify the composition of tumor and the TME cells (cells densities/mmsections . LAIR-1 R = 0.84 .+ T cells (70%–80%), CD8+ T cells (80%–90%), CD56+ NK cells (95%–100%), CD19+ B cells (80%–90%), and CD14+ monocytes  but not the mean stromal expression (P = 0.59) in the discovery cohort (P = 0.06) and stroma (P = 0.09) was observed in the validation cohort (P = 0.56) nor stromal (P = 0.93) LAIR-1 expression in the whole discovery cohort significantly predicted OS. Similarly, neither stromal nor tumor LAIR-1 expression of patients with LUSC histologic subtype significantly predicted OS after surgery. In contrast, high LAIR-1 tumor expression in LUAD subtype was significantly associated with reduced OS  in the whole NSCLC discovery cohort (LUAD + LUSC + other). Consistent with the finding of tumor compartmentalized AQUA scores, patients with high LAIR-1+/CK+ tumor cell densities showed worse outcomes (HR = 1.5) in both discovery and validation cohorts  LAIR-1 mean RNA expression across nine tumor types. Representative QIF images of antibody validation using four different anti-LAIR-1 antibody clones (B) E7X61, (C) NKT25, (D) 1A10 and (E) 1E4 targeting non-overlapping epitopes in a lung test array (YTMA–295) containing 35 lung tumor cores. CK: green, LAIR-1: red, DAPI: blue. Three clones NKT25, 1A10 and 1E4 were tested earlier in the experiment hence the serial TMA sections and E7X61 was tested in a TMA a few sections away from the earlier tested TMAs later at the stage of the experiment. The fluorescent channels where each marker was acquired were shown in brackets. (F) Correlation of LAIR-1 protein expression scores measured by AQUA between four antibody clones. (G) Correlation plot showing the good reproducibility of staining by 1E4 clone between two independent experiments. (H) Correlation plot showing high expression LAIR-1 in both tumor and stroma.Click here for additional data file.Supplementary Table TS1Clinicopathological characteristics of NSCLC validation cohort (YTMA-250).Click here for additional data file.Supplementary Figure FS2Representative figures of staining by different antibody clones. Representative QIF images of different LAIR-1 clones staining targeting non-overlapping epitopes in a lung test array (YTMA–295) containing 35 lung tumor cores stained by (A) E7X61 clone, (B) NKT25 clone, (C) 1A10 clone and (D) 1E4 clone. (E) The staining quantification of the four LAIR-1 clones. CK: green, LAIR-1: red, DAPI: blue. The fluorescent channels where each marker was acquired were shown in brackets.Click here for additional data file.Supplementary Figure FS3LAIR-1 expression in different molecular subtypes of within Storma and Tumor. (A) Umap (Uniform Manifold Approximation and Projection) plot showing the expression of LAIR-1 RNA in patient lung tumor biopsies (n=7) derived single cell RNA-seq data. (B) Quantification of LAIR-1 expression in LM22 immune cells showing highest expression of LAIR-1 in macrophages.Click here for additional data file.Supplementary Figure FS4LAIR-1 expression in different compartments and its association with survival in Validation Cohort 250 (YTMA-250). (A) LAIR-1 protein expression in tumor and stroma compartments. (B) LAIR-1 tumor and stromal protein expression in both LUAD and LUSC-NSCLC subtypes. (C) Association of LAIR-1 expression in tumor and CD68 compartments with the outcome.Click here for additional data file.


Author Correction: Neurocritical Care (2023) 38:564–583 10.1007/s12028-023-01707-3The original article has been updated to insert the following sentence, before the line “The librarian search string..” in the Systematic Review Methodology paragraph.The in-depth literature search first conducted on February 20, 2019, included literature from 1946 onwards and was last updated on 3/16/2022.


Exhausted CD8+ T cells upregulate the expression of a wide diversity of coinhibitory immunoreceptors , such as PD-1, Tim-3, LAG-3, and TIGIT. Concomitantly, exhausted CD8+ T cells lose their potential to differentiate into functional memory cells and are characterized by hierarchical loss of effector function, leading to compromised tumor control and viral eradication [The functional exhaustion of CD8dication .+ T cells in the tumor microenvironment (TME) are highly heterogeneous, mainly consisting of subsets of TCF-1-expressing precursors of exhausted T (TPEX) cells and Tim-3-expressing terminally differentiated exhausted CD8+ T (TEX) cells [+ T-cell exhaustion in the TME [+ T-cell responses in the control of tumor progression upon PD-1/PD-L1 ICB treatment, especially in tumor-draining lymph nodes (TdLNs) [+ T cells in TdLNs (TdLN-TTSM) of both mice tumor models and human hepatocellular carcinoma patients and showed that TdLN-TTSM cells serve as primary responders to PD-1/PD-L1 ICB and exhibit superior tumor repression to that of TCF-1+ TPEX cells [Exhausted CD8X) cells . Immune  the TME –7. Howev (TdLNs) . RecentlEX cells .TSM cells were comparable to conventional memory T cells (TMEM) generated during acute viral infection in several aspects, including antigen-independent self-renewal and proliferation burst upon antigen reencounter. TTSM cells express the lymphoid homing molecule L-selectin (CD62L) and C-C chemokine receptor 7 (CCR7). Moreover, we noticed that genes associated with T-cell extravasation and chemotaxis were less enriched in TdLN-derived P14 cells than in TMEM P14 cells, indicating the potentially distinct circulating features between these two subsets [MEM cells can patrol between lymphoid organs, blood and peripheral tissues, while during chronic viral infection, TPEX cells are reported to largely reside in lymphoid tissues, with a very limited population in infected peripheral tissues [TSM cells derived from TdLNs during tumorigenesis has not yet been elucidated. Thus, herein, we sought to dissect the migratory pattern of TTSM cells.TdLN-T subsets . TMEM ce tissues . ImportaTSM cells during tumorigenesis, C57BL/6 mice (hereafter referred to as B6 mice) were first adoptively transferred with naive Tcf7 (encoding TCF-1 protein)-GFP P14 cells  harboring transgenic TCRs specific to the H-2Db Gp33-41 epitope from Tcf7-GFP knock-in reporter P14 mice, and then these B6 mice were subcutaneously inoculated with B16.F10 melanoma cells expressing the LCMV glycoprotein as a surrogate neoantigen (hereafter referred to as B16.Gp) as we previously reported [TSM P14 cells were sorted and retransferred into tumor-bearing recipients at Day 8 post B16.Gp inoculation  expression; notably, S1PR1 mediates lymphocyte egress from LNs [TSM cells also expressed CD69 and CD103  were first sorted as previously reported [−CD45.2+). After resting for 3 days, the vasculature of B16.Gp tumor-bearing mice (adoptively transferred with TTSM P14 cells on Day 6 post tumor implantation) were conjoined to those of tumor-matched mice (without P14 cell adoptive transfer) via parabiosis surgery. As a control, we performed the same surgery using LCMV-Arm+-acutely infected mice (P14 cells adoptively transferred) in which acute infection (>60 days) had cleared. Then, we tested whether donor P14 cells equilibrated between the parabionts 20 days later in the peripheral blood (PBMC), spleen (Arm+ infection), TME, and inguinal draining lymph node  , the frequency of tetramer-positive CD8+ T cells in the blood is very low compared to that in the spleen, and the majority of these circulating virus-specific CD8+ T cells in chronically infected mice were CD101−Tim-3+ CX3CR1+ transitory cells [+P14 cells from the host parabiont with those from the TdLNs of donor mice. Consistent with published data [+ T cells in TdLNs consisted of TCF1+TOX− TTSM cells and a small proportion of TCF1+TOX+ TPEX cells. In contrast, in PBMCs, a substantial proportion (~70%) of TTSM-derived P14 cells differentiated into TCF1-negative cells and upregulated the expression of exhaustion-associated markers, including PD-1, TOX, and CD39 -derived P14 cells, including the T(R) Fig. . In contnts Fig. . This smry cells , 19–22. hed data , antigenD39 Fig. . Furtherdes Fig. , suggestTSM and TMEM cells. TdLN-TTSM cells were predominantly found in TdLNs and seemed to be resident at these sites. Importantly, this study indicates that TdLNs might provide a unique niche in facilitating the differentiation and residency of TdLN-TTSM cells. However, more efforts are needed to further explore this possibility and examine how such lymphoid niches are generated and operated during tumorigenesis.Collectively, our study delineated the unique tissue distribution and different migratory patterns of TMaterials and MethodsSupplementary TableSupplementary FigureSupplementary Figure Legend


Epstein-Barr virus (EBV) and human herpesvirus 6 (HHV-6) infections are widespread in human populations. Here, I describe single-cell RNA sequencing of two lymphoblastoid cell lines harboring both episomal EBV and inherited chromosomally integrated HHV-6. Rare instances of HHV-6 expression appear enriched with EBV reactivation. Herpesviruses have ubiquitously spread among humans. Epstein-Barr virus (EBV), a gammaherpesvirus, infects ~90% of the population . The cirin vitro generates lymphoblastoid cell lines (LCLs) (Transformation of primary B cells by EBV s (LCLs)  that havs (LCLs) . These rThe EBV-immortalized lines HG00362 and HG01277  contain iciHHV-6B and iciHHV-6A, respectively . LCLs weNC_007605.1), and either HHV-6A U1102 (NC_001664.4) or HHV-6B Z29 (NC_000898.1). Viral genomes were appended as extra chromosomes.A pipeline optimized for simultaneously measuring viral and human transcripts , 13 was HHV-6 RNA is enriched with high levels of EBV gene expression . The majGSE230239, which links to NCBI Sequence Read Archive entries SRX20032490 and SRX20032491.These data have been deposited in the NCBI Gene Expression Omnibus , 18 unde


SARS-CoV-2 Omicron variants have increased vaccine-induced immune evasion and their emergence coincided with waning COVID-19 vaccine effectiveness (VE). The duration of immunity from the primary series and VE of booster doses against pediatric COVID-19 (Omicron)-associated hospitalizations has not been characterized in the United States.Using a case-control design, we examined VE against laboratory-confirmed COVID-19-associated hospitalizations, enrolling case-patients hospitalized for COVID-19 and SARS-CoV-2 test-negative controls with COVID-like illness from 31 hospitals in 23 states. VE was estimated through multivariable logistic regression by comparing the odds of antecedent primary series or booster COVID-19 mRNA vaccination within and beyond 60 days prior to hospitalization. Results were analyzed by age group (5–11 and 12–18 years) among patients admitted December 19, 2021–March 30, 2023.We enrolled 1,242 case-patients  and 1,309 controls. Among children aged 5-18 years, VE of 2 mRNA doses (complete primary series) against pediatric COVID-19 hospitalization was 64%  at 14-60 days after dose 2 ; VE waned to 38% (95% CI: 23–50%) at ≥60 days . Within 60 days of a monovalent booster dose, VE was 52% (95% CI: -3–77%); among children aged 5-11 years, VE of a bivalent booster dose was 79% (95% CI: 25-94%). Among case-patients who required life support or died, 122/164 (74%) were unvaccinated and only 3 (2%) had received a bivalent booster dose.VE estimates were only plotted if confidence interval width was <200. Adjusted models included ≥1 underlying medical condition (yes/no), age in years, census region, biweekly date of hospital admission, and social vulnerability index (SVI) score. Sex and race/ethnicity were tested as confounders in the full model but were dropped for absence of evidence of confounding.Abbreviations: ICU, intensive care unit; ECMO, extracorporeal membrane oxygenationCOVID-19 mRNA vaccination reduced the likelihood of pediatric COVID-19 hospitalization by >60%; VE waned but remained protective, even at a median of 213 days after dose 2. Although not reaching statistical significance due to limited power, monovalent boosters appeared to temporarily restore protection, and bivalent booster doses among 5–11-year-olds were nearly 80% protective against hospitalization. Nearly three quarters of children requiring life support or who died were unvaccinated.Regina Simeone, PhD, Pfizer: Stocks/Bonds Natasha B. Halasa, MD, MPH, Merck: Grant/Research Support|Quidell: Grant/Research Support|Quidell: donation of kits|Sanofi: Grant/Research Support|Sanofi: vaccine support


The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a zoonotic infectious virus that has caused significant outbreaks in the Middle East and beyond. Due to a highly mortality rate, easy transmission, and rapid spread of the MERS-CoV, it remains as a significant public health treat. There is currently no licensed vaccine available to protect against MERS-CoV.In this study, we investigated whether the proteolytic cleavage sites and fusion peptide domain of the MERS-CoV spike (S) protein could be a vaccine target to elicit the MERS-CoV S protein-specific antibody responses and confer immune protection against MERS-CoV infection. Our results demonstrate that immunization of the proteolytic cleavage sites and the fusion peptide domain using virus-like particle (VLP) induced the MERS-CoV S protein-specific IgG antibodies with capacity to neutralize pseudotyped MERS-CoV infection in vitro. Moreover, proteolytic cleavage sites and the fusion peptide VLP immunization showed a synergistic effect on the immune protection against MERS-CoV infection elicited by immunization with VLP expressing the receptor binding domain (RBD) of the S protein. Additionally, immune evasion of MERS-CoV RBD variants from anti-RBD sera was significantly controlled by anti-proteolytic cleavage sites and the fusion peptide sera.Our study demonstrates the potential of VLP immunization targeting the proteolytic cleavage sites and the fusion peptide and RBD domains of the MERS-CoV S protein for the development of effective treatments and vaccines against MERS-CoV and related variants. MERS-CoV is a coronavirus that was first identified in Saudi Arabia in 2012 . Geneticvia three key biochemical processes; (i) the protein-protein interaction between the S protein and host cell’s dipeptidyl peptidase IV (DPP4) receptor, (ii) the proteolytic cleavage of the S protein at the S1/S2 and S2’ sites, and (iii) the fusion of the viral envelope with the plasma membrane of the host cells  covering the proteolytic cleavage sites and the fusion peptide could be targeted to elicit the MERS-CoV S protein-specific antibodies and neutralizing antibodies in mice and could be elaborated with RBD targeting vaccine for synergistic enhancement in immune protection against MERS-CoV and related variants.C57BL/6J mice and OTII transgenic mice were purchased from DBL (South Korea) and obtained from in-house breeders, respectively. hDPP4 knock-in mice  were genThe coding sequences of the headless HA (D18-C59 and C291-Q528) of A/PR/8/1934 (Accession P03452) linked with 4XG, and the receptor binding domain  and proteolytic cleavage sites and the fusion peptide (E589-F972) regions of the MERS-CoV S protein (Accession YP_009047204) were synthesized and cloned into the CDS position for hen egg lysozyme (HEL) of HEL-GTMCD plasmid  were produced from HEK293T cells by transfecting GTMCD plasmids expressing HEL, headless HA of A/PR/8/1934, or RBD or proteolytic cleavage sites and the fusion peptide region of MERS-CoV with either OTII-Hgsyn or empty Hgsyn plasmid using plain DMEM (Welgene) and polyethylenimine (PEI) (PolyScience). After transfection, HEK293 T cells were cultured in complete DMEM , and supernatants were collected daily for 3 days and filtered. Pooled supernatants were ultra-centrifuged at 15,000 rpm for 1.5 hours. The resulting pellets were resuspended, aliquoted with plain DMEM, and stored at -80°C until use.2CO3 coating buffer were coated onto a 96-well plate (Nunc). After overnight incubation, the plate was washed and blocked with 5% PBS-T-F buffer . The plate was then incubated with anti-p24 antibody (Antibodies-online) followed by anti-mouse IgG-HRP (GeneTex) diluted in 2% PBS-T-F buffer . The p24 concentration was calculated using the standard curve generated from the recombinant p24 protein.To quantify the amount of VLPs, recombinant HIV1 p24 protein (Abcam) was used as standard . VLPs seTo produce pseudotyped MERS-CoV (pMERS-CoV), HEK293T were transfected with pLP1, pLP2, Luc2P pLVX IRES ZsGreen1, and the MERS-CoV S protein pcDNA3 plasmids. Site-directed mutagenesis using QuickChange lightning site-directed mutagenesis (Agilent) was performed to generate pMERS-CoV harboring D510G and I529T mutant S protein. After transfection, cells were cultured with complete DMEM media. Supernatants were collected daily for three consecutive days and filtered. The supernatants were then ultra-centrifuged at 15,000 rpm for 1.5 hours, and the precipitated pMERS-CoV were resuspended and aliquoted with plain DMEM. The aliquots were stored at -80°C until use.+ HEK293T cells were sorted and subsequently cultured in complete DMEM media with 0.2 μg/mL puromycin.HEK293T cells were transfected with DPP4-pEF-IRES-Puro plasmid and cultured using pre-warmed complete DMEM media with puromycin . Expression of hDPP4 was checked using anti-human CD26 APC antibody , and hDPP4VLPs that express the headless HA of A/PR/8/1934, HEL, or the RBD or proteolytic cleavage sites and the fusion peptide region of the MERS-CoV were mixed with Addavax (Invivogen) and injected subcutaneously into footpad under anesthesia. To examine CD4 T cell immune responses in an antigen-specific manner, 2.5e5 OTII CD4 T cells, isolated from the spleens of OTII TCRtg mice using Easysep mouse CD4 T cell isolation kit (Stem cell technologies), were adoptively transferred into C57BL/6J mice one day before immunization. For experiments involving booster immunizations, the mice were given VLPs fourteen days after the primary immunization.Popliteal lymph nodes (popLNs) of VLP-immunized or unimmunized mice were collected in complete DMEM media. Single cell suspensions were obtained after mincing the popLNs on cell strainers  using syringe plungers.The cells were stained with purified or fluorescent-conjugated antibodies against CD4 (RM 4-5), CD19 (6D5), APC-Streptavidin, CD8a (53-6.7), and CD19 (11-26C) from eBioscience; CD4 (RM 4-5), Fas (Jo2), PSGL-1 (2PH1), CD44 (IM7), CD45.1 (A20), CD8a (53-6.7), purified LE/AF rat-anti-mouse CXCR5 (2G8), biotin rat anti-mouse CD138 (281-2), and CD45R (B220) (RA3-6B2) from BD Biosciences; PNA (FL-1071) from Vector Laboratories; PD-1 (J43) from Invitrogen; biotin-SP(long spacer)-conjugated Affipure Goat anti-Rat IgG(H+L) from Jackson Immunoresearch). Three step CXCR5 staining for phenotypic identification of the Tfh cells was performed using purified anti-CXCR5 (2G8) antibody .via retro-orbital sinus under anesthesia before immunization or after VLP immunization. The blood was spun at 10,000 rpm for 40 minutes to collect serum. To measure A/PR/8/1934 HA- and MERS-CoV S protein-specific IgGs, a 96-well plate (Nunc) was coated with 0.5 μg/mL of A/PR/8/1934 HA protein (BEI resources) or with 1 μg/mL of the MERS-CoV S protein  in PBS overnight at 4°C. After wash and blocking with 0.05% PBS-T-B buffer , the serum samples, pre-diluted at 1:10 in 0.05% PBS-T-B buffer, were added to top wells, 3-fold serially diluted to the bottom, and incubated for 1.5 hours. After washing, anti-mouse IgG-HRP (Abcam) antibody diluted in 0.1% PBS-T buffer was added and incubated. TMB chromogen solution (Invitrogen) was added for colorimetric development, which was subsequently stopped with stop solution (Invitrogen). O.D. was measured by Sunrise Microplate Reader (Tecan) at a measurement wavelength of 450 nm and a reference wavelength of 570 nm.The mice were bled via retro-orbital sinus bleed were stored at -20°C. Prior to performing neutralization assays, the sera were thawed and heat-inactivated at 56°C for 30 minutes. The inactivated serum pre-diluted in 2% DMEM  underwent 4-fold serial dilution. Pseudotyped MERS-CoV was added to the mixture and incubated in 4°C cold room for 1 hour on a rotator. The mixture was then added to hDPP4+ HEK293T cells on a 96-well plate. After centrifugation of the plate at 1,500 rpm for 1 hour at 25°C, the media was replaced with 10% DMEM media . After 2 days of culture, the cells were washed with PBS and lysed with cell culture lysis buffer (Promega) and centrifuged at 2,000 rpm for 5 minutes. The supernatant was transferred to a 96-well white plate (SPL) with luciferase assay reagent (Promega). The luminescence was measured using a microplate reader (Tecan) with following conditions: wait time of 2 seconds and integration time of 1000 milliseconds.Serum samples obtained from mice 4 PFU of mouse adapted MERS-CoV  of A/PR/8/1934. hDPP4-Tg mice were subcutaneously immunized with VLPs expressing the proteolytic cleavage sites and the fusion peptide region of MERS-CoV or HEL on day 0 and day 14. On day 21, VLPs expressing the RBD region of MERS-CoV were administered to the immunized mice. Seven days later, both the immunized and unimmunized groups of mice were intranasally infected with 10MERS-CoV . A/PR/8/Either paired or unpaired student’s t-test and one-way ANOVA with Tukey’s multiple comparison test were used for statistical analyses using GraphPad Prism 7. P-values less than 0.05 were considered statistically significan .Virus-like particle (VLP) is an effective platform to deliver target antigens in vaccines due to its similar morphologic features to infectious viruses while lacking viral genome required for replication . As a prvia binding of the hemagglutinin (HA) protein to sialic acids present on the glycoproteins in the respiratory tract  as a negative control   that recognize highly conserved epitopes of viruses can neutralize various strains and have been identified in a small group of patients infected with HIV-1 and IAV , 33. TheThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by Seoul National University College of Medicine (SNUCM) and Seoul National University Hospital (SNUH).Conceptualization: YSC. Methodology: JO, JK, CK, N-HC, and YSC. Investigation: JO, JK, UP, KJ, N-HC, and YSC. Funding acquisition: NC and YSC. Project administration: YSC. Supervision: YSC. Writing: YSC. All authors contributed to the article and approved the submitted version.


Nature Communications 10.1038/s41467-023-38397-6, published online 9 May 2023Correction to: The original version of this article contained a duplication of Supplementary Fig. Updated Supplementary Information


MARCH1 mRNA. By recruiting β-TRCP, a component of the SCFβ-TRCP E3 ligase complex that targets phosphorylated β-catenin for degradation, Vpu increases β-catenin levels, which, in concert with TCF4/LEF, drives transcription of the MARCH1-targeting microRNAs. This potentiates HIV-1 infectivity as a result of increased Env incorporation into nascent virions. Pharmacological targeting of the β-catenin pathway inhibits Vpu-mediated upregulation of microRNAs-25 and -93 and restores MARCH1 restriction on HIV-1 infectivity. Overall, our findings highlight a novel mechanism by which HIV-1 counteracts MARCH1 by downregulating its expression via Vpu-mediated induction of microRNAs-25 and -93.The type 1 interferon-regulated E3 ubiquitin ligase MARCH1 reduces surface expression of HIV-1 envelope glycoproteins (Env) and their packaging into nascent virions, a condition that restricts viral infectivity. However, how HIV-1 counters this restriction, notably during infection of macrophages, remains unclear. Here, we show that the HIV-1 accessory protein Vpu increases the levels of microRNAs-25 and -93 to target MARCH1 mRNA. We also show that Vpu induces these cellular microRNAs in macrophages by hijacking the cellular β-catenin pathway. The notion that HIV-1 has evolved a mechanism to counteract MARCH1 restriction on viral infectivity underlines the importance of MARCH1 in the host antiviral response.In order to efficiently produce infectious viral particles, HIV must counter several restrictions exerted by host cell antiviral proteins. MARCH1 is a member of the MARCH protein family that restricts HIV infection by limiting the incorporation of viral envelope glycoproteins into nascent virions. Here, we identified two regulatory RNAs, microRNAs-25 and -93, induced by the HIV-1 accessory protein Vpu, that downregulate  Host organisms have evolved elaborate cell-autonomous defense mechanisms that rely on restriction factors to impede virus replication and spread. These antiviral factors, which frequently target specific components of viruses during their replication cycle, are often induced by interferons (IFNs) as part of the host innate immune response . Recentlβ-TrCP  > 2) as shown in Fig. S1. Among these 11 miRNAs, miRNA-25-3p  and miRNA-93-5p  stood out as by far as the most abundantly expressed  . We thus focused on these two miRNAs that were upregulated in HSA+ macrophages. The RNA transcriptomics data of 1,818 genes (−log10[Padj] > 2) obtained from total RNAseq analyses (P = 0.0286) by qRT-PCR of RNA samples derived from sorted cells of additional blood donors and using a different reporter HIV-1 construct expressing the green fluorescent protein (GFP).miRNAs are single-stranded non-coding regulatory RNA molecules of approximately 22 nucleotides that modulate gene expression in plants, mammals, and even viruses . They fuanalyses  of the sanalyses . Importahttp://ophid.utoronto.ca/mirDIP/) for relevant mRNA targets of miRNA-25 and/or miRNA-93 using the RNA transcriptomics data. Several downregulated mRNAs in HSA+ macrophages, as compared to mock-infected cells, were identified as potential targets of either or both miRNAs: AKT3 , FOXP1 , and MARCH1   by qRT-PCR. In contrast, we did not detect a significative reduction of AKT3 or FOXP1 mRNAs in GFP+ MDMs by qRT-PCR  . The red qRT-PCR . Accordiwww.targetscan.org) suggested that the 3′UTR of MARCH1 mRNA is a target for both miRNA-25 and miRNA-93. To verify this, we transiently expressed a construct encoding the Firefly Luciferase (F-Luc) gene fused to the 3′UTR of MARCH1 (P < 0.0001) or miRNA-93 (P = 0.0004) mimics, or combination of both (MARCH1 3′UTR sequences predicted to be targeted by miRNA-25 or miRNA-93 (MARCH1 mRNA were determined by qRT-PCR. The levels of MARCH1 mRNA were decreased by ~50% in miRNA-25 (P < 0.0001) or miRNA-93 (P < 0.0001) mimic-transfected macrophages, compared to controls; a combination of both mimics (P < 0.0001) achieved ~75% reduction. In contrast, levels of MARCH2 and MARCH8 mRNAs were not affected by these miRNA mimics  reduction in MARCH1 mRNA levels compared to control-treated cells. It is important to note that all our attempts to detect endogenous human MARCH1 proteins in macrophages using a validated anti-MARCH1 antibody  reduction in infectivity of viruses produced in antagomir-treated MDMs. Specifically, HIV-1 produced in MDMs treated with both miRNA-25 and miRNA-93 antagomirs was ~75% less infectious than virus from control cells  in Vpu that is necessary for β-TrCP binding and results in upregulation of the Wnt/β-catenin signaling pathway which drives expression of miRNA-93 (P = 0.0286) more miRNA-25 than uninfected cells (P = 0.0286) (MARCH1 mRNA (P = 0.0286) was dependent on the presence of Vpu that had the ability to recruit β-TrCP   .We have previously shown that miRNA-93 is among a group of miRNAs upregulated by the HIV-1 accessory protein Vpu via a mechanism that involves the recruitment of β-TrCP. The interaction is dependent upon a highly conserved motif (DSmiRNA-93 . We thermiRNA-93 . MDMs wemiRNA-93 . Infecteed cells . As expe 0.0286) . However 0.0286) . This in 0.0286) . Consistt β-TrCP . Importa 0.0286) . The enhMARCH1 mRNA and upregulate miRNA-25 and miRNA-93, even though these viruses do not encode for a vpu gene. These findings highlight the importance of counteracting MARCH1 during primate lentivirus infection of myeloid cells and underline the potential role of miRNA-25/miRNA-93 in this counteraction, although the mechanism driving miRNA-25/miRNA-93 upregulation during HIV-2 or SIVmac infection is likely to be different than in the case of HIV-1.Unexpectedly, we also found that HIV-2 and simian immunodeficiency virus from macaques (SIVmac)  infections also downregulate P < 0.0001, MARCH1 mRNA, as measured by qRT-PCR in miRNA-34c mimic-transfected MDMs , nor their infectivity .Our results suggested that miRNA-25 and miRNA-93 exert their enhancing effect on HIV-1 infectivity by downregulating MARCH1, prior to infection with WT or Vpu-defective HIV-1 and determined the infectivity of progeny viruses  had little effect on the infectivity of WT viruses  in Vpu-defective virus-infected MDMs and determined the infectivity of progeny viruses. As shown in MARCH1 on Vpu-defective virus infectivity was found to be basically BST2-independent. Finally, since HIV-1 cell-to-cell transmission is an important mode of viral dissemination .To further examine the impact of MARCH1 downregulation by miRNA-25 and miRNA-93 on HIV-1 infectivity, we treated MDMs with control or small interfering RNAs (siRNAs) that target  viruses , but resmination , we also compare , while iMARCH1 restricts cell-free HIV-1 infectivity as well as cell-to-cell transmission. They also further suggest that targeting MARCH1 by these Vpu-induced miRNAs in macrophages is an important factor in controlling the spreading of infection.Given that MDM infection by Vpu-defective HIV-1 does not lead to an upregulation of miRNA-25 and miRNA-93  and miRNA-93  levels, resulting in a concomitant increase in MARCH1 mRNA  as compared to vehicle-treated cells  gene  gene . These mM7) gene  and are M7) gene .MARCH1 was identified among the potential miRNA-25 and miRNA-93 targets that were modulated negatively in productively HIV-1-infected macrophages , the mechanisms of which remain undetermined and warrant further investigation. It is also noteworthy that MARCH1 is more inducible by IFN-I in macrophages than MARCH2 or MARCH8  did not involve BST2/tetherin -seronegative adults (of either gender).Renilla luciferase under the control of the actin promoter, was used instead of pMIR-REPORT-β-gal. In order to generate pMIR-REPORT-MARCH1-Luc, Pfu PCR was performed on cDNA of IL-10-activated pooled primary peripheral blood monocytes using primers M1utrSacI-F and M1utrMluI-R  and Qiagen , respectively.NL4-3-ADA--GFP-IRES-NEF, NL4-3-ADA-GFP-IRES-NEF-ΔVpu , NL4-3-AuI-R see , and theuI-R see  using thThe following antibodies were used in Western immunoblotting or confocal microscopy imaging analyses: mouse anti-p24 ; mouse anti-hsp90 ; Chessie 8 mouse anti-gp41 #13049 ; rabbit MDMs were obtained from peripheral blood mononuclear cells and characterized as previously described . TransfeCells were collected following a 15 min, 37°C incubation in phosphate-buffered saline (PBS)-EDTA and gentle scraping. Harvested cells were then fixed with 4% paraformaldehyde in PBS for GFP expression analyses or processed for flow immunocytometry. In this case, macrophages were washed in PBS containing 2% fetal bovine serum (FBS) (FACS buffer) and blocked on ice for 1 h in FACS buffer containing 2% goat serum, 2% rabbit serum, and a mix of human decomplemented plasma. Mouse primary antibodies were added directly into the cells in the blocking solution, incubated 1 h on ice and cells were washed two times in FACS buffer. Secondary fluorescent antibodies were then added for 30 min on ice, and cells were washed and fixed with 4% paraformaldehyde in PBS. Finally, cells were resuspended in PBS-EDTA and analyzed on a BD Fortessa cytometer (BD Biosciences) equipped with appropriate lasers. Detailed analyses were obtained using the FlowJo software package. Geometric mean fluorescence intensities were used.MDMs grown on coverslips were processed for confocal microscopy following transfection or infection. Cells were washed in PBS and fixed in 3% paraformaldehyde in PBS at room temperature, permeabilized with 0.2% Triton X-100 for 10 min and Fc receptors blocked either with blocking buffer (a mix of human decomplemented plasma or 1 mg/mL human IgGs in PBS) on ice. Samples were then incubated on ice for 1 h with primary antibodies diluted in blocking buffer, washed three times in wash buffer (PBS containing 2% FBS) and incubated with fluorochrome-labeled secondary antibodies for 1 h on ice, washed three times, and mounted on slides. In some cases, Cell Mask Deep Red stain (ThermoFisher) or DAPI was used to visualize cells. For HIV-1 gp41 staining, samples were examined using a Zeiss 710 confocal microscope using a 40×/1.3 oil PlanApo objective and images were acquired using the Zeiss Zen software package. For peroxisome staining, samples were examined using an Olympus 1 × 81 spinning-disc confocal microscope with a 60×/1.42 oil PlanApo objective. PEX3 immunofluorescence intensity was determined as previously described using Volocity 6.2.1 software .Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of macrophage or virus lysates and immunoblotting were performed as previously described . ProteinTC variations were calculated.Total cellular RNAs were extracted using RNeasy RNA extraction columns (Qiagen) according to the manufacturer’s instructions and stored at −80°C. For the isolation of productively infected (GFP-positive) and bystander (GFP-negative) macrophage populations, MDMs infected with NL4-3-ADA-GFP-IRES-NEF-based viruses (MOI of 1) were sorted using an Influx cell sorter (BD Biosciences), and GFP-positive and GFP-negative cells were directly recovered in RLT lysis buffer as previously described . In the P values computed using DESeq2. Volcano plots were generated using GraphPad Prism9 . The online search for miRNA and mRNA pairing was performed using mirDIP (http://ophid.utoronto.ca/mirDIP/).Details on how samples were obtained and processed for comparative mRNA expression of productively infected or bystander versus uninfected (mock infected) macrophage populations and the bioinformatics processing of sequences can be found in Deshiere et al. . These st test, or one-way ANOVA. Statistical significance is indicated in the figures .Statistical analyses were performed in GraphPad Prism9 using the nonparametric Mann-Whitney rank test, Student’s


Citrobacter freundii CF20-4P-1 and Escherichia coli EC20-4B-2 were isolated from edible Mastacembelidae in Vietnam. We present the draft genome sequences, and the complete plasmid genome sequencing was also performed by hybrid assembly sequencing of Oxford Nanopore and Illumina. The 137-kbp plasmid encoding the assembled blaNDM-1 was detected in both strains.Carbapenem-resistant  Citrobacter freundii and Escherichia coli harboring a common plasmid isolated from fresh edible river fish, Mastacembelidae, purchased in Ho Chi Minh City, Vietnam.As antibiotic-resistant bacteria (ARB) have reportedly been found in imported foods, the spread of plasmid-mediated ARB through food products is a concern . Here, wMastacembelidae fish were purchased from a retail market in Ho Chi Minh City, Vietnam; their gut contents (5 g) were mixed with buffered peptone water (BPW) (45 mL). After incubation at 37°C for 24 h, BPW (100 μL) was spread on CHROMagar ECC (CE)  containing meropenem (0.25 g/mL) and incubated at 37°C for 24 h. Several colonies were selected, and isolated Enterobacteriaceae were investigated for antibiotic susceptibility using the disk diffusion method  for next-generation sequencing using short-read sequencing and Genomic-Tip 100/G (Qiagen). The extracted DNA was analyzed using a Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit . For short- and long-read sequencing library preparation, the QIAseq FX DNA library UDI kit (Qiagen) and rapid barcoding kit  were used, and sequencing was performed using Illumina HiSeq  with a 2 × 150-bp paired-end protocol and MinION (Oxford) with flow cells . After we obtained short-read sequences, trimming and quality checks were conducted using fastp v0.20.0  and MinION  sequencing data was performed using Unicycler v0.5.0 -VI, and qacE were found in both bacterial plasmids  5 mL. After (BPW) 5 mL. Aftplasmids .26 and ISEc52 are located upstream and downstream of blaNDM-1, qacE, sul1, and aph(3′)-VI (Enterobacteriaceae.A comparison showed that the plasmids carried by both bacteria were the same. The mobile genetic factors insertion sequences ISh(3′)-VI . TherefoC. freundii CF20-4P-1 and E. coli EC20-4B-2 WGS, as well as pCF20-4P-1-2 and pEC20-4B-2-2 partial genome sequencing (PGS), were deposited in DDBJ and GenBank . The raw read SRA accession numbers are DRX382161, DRX382162, DRX382159, and DRX382160, with BioProject accession number PRJDB11927.


Aggregation of the RNA-binding protein, TDP-43, is the unifying hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. TDP-43-related neurodegeneration involves multiple changes to normal physiological TDP-43, which undergoes nuclear depletion, cytoplasmic mislocalisation, post-translational modification, and aberrant liquid–liquid phase separation, preceding inclusion formation. Along with toxic cytoplasmic aggregation, concurrent depletion and dysfunction of normal nuclear TDP-43 in cells with TDP-43 pathology is likely a key potentiator of neurodegeneration, but is not well understood. To define processes driving TDP-43 dysfunction, we used CRISPR/Cas9-mediated fluorescent tagging to investigate how disease-associated stressors and pathological TDP-43 alter abundance, localisation, self-assembly, aggregation, solubility, and mobility dynamics of normal nuclear TDP-43 over time in live cells. Oxidative stress stimulated liquid–liquid phase separation of endogenous TDP-43 into droplet-like puncta, or spherical shell-like anisosomes. Further, nuclear RNA-binding-ablated or acetylation-mimicking TDP-43 readily sequestered and depleted free normal nuclear TDP-43 into dynamic anisosomes, in which recruited endogenous TDP-43 proteins remained soluble and highly mobile. Large, phosphorylated inclusions formed by nuclear or cytoplasmic aggregation-prone TDP-43 mutants also caused sequestration, but rendered endogenous TDP-43 immobile and insoluble, indicating pathological transition. These findings suggest that RNA-binding deficiency and post-translational modifications including acetylation exacerbate TDP-43 aggregation and dysfunction by driving sequestration, mislocalisation, and depletion of normal nuclear TDP-43 in neurodegenerative diseases.The online version contains supplementary material available at 10.1007/s00018-023-04739-2. Pathological aggregation of TAR DNA-binding protein 43 (TDP-43) in neurons is a key hallmark of almost all amyotrophic lateral sclerosis (ALS) and approximately half of frontotemporal dementia (FTD) cases , 2. TDP-Post-translational modifications (PTMs) are a major feature of TDP-43 pathology in ALS and FTD, and likely play diverse roles in TDP-43 dysfunction, aggregation propensity, and neurotoxicity. For example, phosphorylation is a widely recognised hallmark of pathology progression –20. LessRNA deficiency promotes TDP-43 instability , 27, andAmong additional disease-associated cellular stressors, oxidative stress is observed in brain and spinal cord of ALS and FTD patients , 44–47, TARDBP expression is mediated by TDP-43 binding its own mRNA , mouse anti-FLAG MAb , and applied overnight at room temperature. Secondary antibodies were similarly prepared, including goat anti-Rabbit H+L AlexaFluor647 , and applied for 1.5 h at room temperature. Cells were counterstained with DAPI  for 10 min at room temperature, mounted onto microscope slides with ProLong Gold Antifade Mountant , and allowed to cure overnight at room temperature before microscopy.Confocal images were captured using either the Zeiss Laser Scanning Microscope (LSM) 510 META or Zeiss LSM 710. Both are equipped with a 63 × oil objective , blue laser diode for DAPI (405 nm), argon laser for AlexaFluor488 (488 nm), 561 nm laser for AlexaFluor555, and HeNe 633 nm laser for AlexaFluor647 excitation. Images were acquired using Zen software and all acquisition parameters were applied consistently for all samples in an experimental set. Image processing and intensity profile analyses were completed with FIJI. Automated and unbiased single-cell segmentation analyses were conducted using CellProfiler version 4.2.1  using cuP values < 0.05 deemed statistically significant. Live-cell imaging of nuclear TDP-43-mCherry expression over time included n = 3 independent repeats, recording 12 ROIs at 20 × magnification for each experimental condition. FRAP experiments also included n = 3 independent repeats, with at least 5 cells per replicate for each of the distinct TDP-43 assemblies studied. All immunoblot experiments included n = 3 independent repeats. Individual data points for each independent replicate are shown overlaid with a line at the mean. Statistically significant differences between the means were determined using a two-tailed paired t-test (where indicated in figure legends) for comparisons between only two groups, or a one-way ANOVA with a Tukey’s post hoc test for multiple comparisons in experiments with several groups.Data were represented as mean ± standard error about the mean (SEM). GraphPad Prism 9 was used for statistical analysis and preparation of graphs, with Supplementary file1 (XLSX 503 KB)Supplementary file2 (XLSX 79 KB)Supplementary file3 (XLSX 250 KB)Supplementary file4 (XLSX 76734 KB)Supplementary file5 (MP4 16010 KB)Supplementary file6 (MP4 9739 KB)Supplementary file7 (MP4 23411 KB)Supplementary file8 (PDF 9613 KB)Below is the link to the electronic supplementary material.


Correction: J Exp Clin Cancer Res42, 143 (2023)10.1186/s13046-023-02715-zFollowing publication of the original article , an erro(c) Well confluency of U87MG-shCtrl and -shGYS1 cells treated with different concentrations of GTPP, cultured in 5.6 mM glucose complete DMEM, was measured by Incucyte every 3 h.The correction does not affect the overall Conclusion of the article. The original article has been corrected.