Biomedicines

Background and AimsSevere alcoholic hepatitis (SAH) has a high mortality and corticosteroid therapy is effective in 60% patients. Reliable indicators of response to therapy and mortality in SAH are needed. A total of 223 SAH patients, 70 in derivative [50 responders (R) and 20 non-responders (NR)] and 153 in validation cohort [136R, 17NR] were subjected to plasma metabolic/meta-proteomic analysis using UHPLC-HRMS and validated using Machine-Learning (ML). Temporal metabolic changes were assessed using Weighted Metabolome Correlation Network Analysis (WMCNA). Functionality (inflammatory-nature, effect on membrane integrity and glucocorticoid receptor) of non-response indicator was assessed in-vitro on primary healthy neutrophils or mice enterocytes. Baseline plasma metabolomics and meta-proteomics clearly discriminated NR and showed significant increase in urobilinogen (3.6-fold), cholesterol sulfate (6.9-fold), Adenosine monophosphate (4.7-fold) and others (p<0.05, FC>1.5, FDR<0.01). Increase in alpha/beta diversity, biosynthesis of secondary metabolites was a characteristic feature of NR (p<0.05). NR were metabolically inactive however R showed temporal change in the metabolite expression post-corticosteroid therapy (p<0.05). Plasma urobilinogen predicted non-response [AUC=0.94] with a hazard-ratio of 1.5(1.2-1.6) and cut-off >0.07mg/ml segregated non-survivors (p<0.01) and showed >98% accuracy using ML. Plasma urobilinogen directly correlated with circulating bacterial peptides linked to bilirubin to urobilinogen metabolising bacteria (r2>0.7;p<0.05). Urobilinogen induced neutrophil activation, oxidative-stress and pro-inflammatory cytokines (CXCR1, NGAL, NOXO1, NOX4, IL15, TNF and others, p<0.05), promoted corticosteroid resistance by increasing the expression of GR-Beta and trans-repression genes under GR-alpha (inflammatory-NFkB, MAPK-MAP) and reducing GR-alpha, and transactivation (anti-inflammatory) gene levels. Urobilinogen also promoted leaky gut by deregulating intestinal membrane junction proteins. ConclusionPlasma metabolome/meta-proteome can stratify pre-therapy steroid response. Increase in plasma Urobilinogen pedals a vicious cycle of bacterial translocation and increase in inflammation and corticosteroid non-response in SAH patients.

NF2-related Schwannomatosis (NF2-SWN) is an inherited autosomal dominant disorder resulting from loss-of-function mutations in the NF2 gene, for which no effective treatment is currently available. Furthermore, truncating variants in NF2 are associated with the severest phenotype compared to in-frame or missense variants. Previously, a shorter NF2 isoform with exon 11 skipped (merlin_e-11), induced through antisense morpholino oligomers (PMOs), was able to partially rescue the deleterious effect of nonsense variants located at that exon in patients primary fibroblast. To test the potential of this approach into Schwann cells, those responsible of NF2-SWN tumours, we developed an iPSC-based model carrying heterozygous and homozygous truncating variants on exon 11 of the NF2 gene and differentiated them to Schwann cells forming spheroids. After 3 days of treatment, merlin_e-11 expression was induced in NF2-deficient cell lines. Furthermore, key pathways associated with NF2-deficiency in schwannomas, such as PI3K/Akt/mTORC and YAP levels, were recovered without signs of toxicity. These results confirm that the PMO treatment induces effective skipping of exon 11 in Schwann cell spheroids, generating an hypomorphic merlin_e-11 that has the capacity to partially rescue merlin-deficiency in NF2-SWN spheroid cell model and it being a potential treatment of patients harbouring truncating variants located in exon 11.

This study aimed to evaluate the prognostic value of quantitative analysis of {superscript 1}F-FDG positron emission tomography (PET)/computed tomography (CT) metabolic parameters in patients with pancreatic ductal adenocarcinoma (PDAC) after neoadjuvant chemotherapy (NACT). A retrospective analysis was conducted on the clinical and imaging data of 44 patients with pathologically confirmed PDAC who received NACT. All patients completed standard chemotherapy regimens and underwent {superscript 1}F-FDG PET/CT examinations within 2 weeks before and after chemotherapy. Multiple metabolic parameters of lesions were extracted, their percentage changes were calculated, and the optimal cut-off values for each parameter were determined. Kaplan-Meier survival analysis and Cox proportional hazards regression analysis were applied to explore the prognostic value of the metabolic parameters, and the prognostic stratification performance of PET Response Criteria in Solid Tumors (PERCIST) 1.0 was compared with that of Response Evaluation Criteria in Solid Tumors (RECIST) 1.1. PERCIST 1.0 demonstrated significantly superior prognostic stratification compared with RECIST 1.1. A peak standardized uptake value corrected for lean body mass (SULpeak2) > 3.07 and a percentage change in SULpeak between pre- and post-treatment scans ({Delta}SULpeak%) [&le;] 37.66% were identified as independent risk factors for poor prognosis. Furthermore, SUL-related parameters exhibited markedly better predictive efficacy than traditional metabolic parameters such as the standardized uptake value and metabolic tumor volume. Quantitative analysis of {superscript 1}F-FDG PET/CT metabolic parameters can effectively predict prognosis in PDAC after NACT, and PERCIST 1.0 is a more optimal criterion for efficacy and prognostic assessment. A post-NACT SULpeak > 3.07 and {Delta}SULpeak% [&le;] 37.66% were core independent indicators for predicting poor prognosis in these patients.

Circulating stem and progenitor cells (SPCs), including mesenchymal stromal cells (MSCs) and hematopoietic stem/progenitor cells (HSPCs), are mobilised after tissue injury but their temporal behaviour after hemorrhagic shock (HS) and relationship to cytokine milieus and outcome remain unclear. In a prospective observational cohort at JPN Apex Trauma Centre, AIIMS, New Delhi we studied 100 participants: 50 trauma patients with hemorrhagic shock and traumatic brain injury (HS index group), 25 trauma patients without HS, and 25 minor-injury controls. Peripheral blood was collected at admission (day 0) for all groups and additionally at days 3, 7 and 14 for the HS group. PBMCs were phenotyped by flow cytometry (HSPC markers: CD45, CD123, CD38, CD34; MSC markers: CD105, CD73, CD90) and serum SDF-1, VEGF-A, EGF, GRO- and GRO-{beta}, GM-CSF and G-CSF were measured by ELISA; group and time effects were evaluated with mixed-effects models and correlations by Spearman tests (two-tailed p<0.05). At admission, trauma patients without HS had significantly higher MSC and HSPC-like populations versus controls (p<0.0001). In the HS cohort SPC percentages rose modestly at day 0-3 then declined sharply by days 7-14 (time effect p<0.0001); non-survivors exhibited significantly higher early SPC and cytokine levels that persisted until death while survivors showed an early rise followed by decline (outcome and time interaction p<0.0001). All cytokines were up-regulated in trauma groups, peaked at day 0-3 in HS patients, and correlated positively with SPC counts (notably SDF-1, VEGF-A, G-CSF, Gro- and GM-CSF; Spearman p<0.05); higher early SPC and cytokine signatures associated with greater organ dysfunction (higher SOFA) and with timing of sepsis. These findings indicate that trauma provokes an early SPC and cytokine response that in HS is followed by later decline, and that persistent early elevation predicts worse outcomes, suggesting serial SPC and cytokine profiling may have prognostic value and identify an early therapeutic window for regenerative or immunomodulatory interventions.

ObjectiveCircular RNA(circRNA) is a kind of endogenous non-coding RNA, which may be related to the occurrence and development of cancer. Based on the GEO database, this paper constructs a circRNA as a competitive endogenous RNAs(ceRNAs) that binds with microRNAs (miRNAs) to affect and regulate the expression of target genes. The ceRNA regulatory network based on circRNA-miRNA-mRNA model plays an important role in tumor prognosis and treatment. This paper explores the mechanism of circRNA-related ceRNA regulatory network in gastric cancer. MethodCircRNA, miRNA and mRNA data sets related to gastric cancer were downloaded from The Gene Expression Omnibus (GEO), and the limma package of R software (R 4.2.1 version) was used to identify the differences between gastric cancer tissues and adjacent normal tissues of gastric cancer. DEcircRNA, DEmiRNAs, and DEmRNAs. Based on circBase database, we explored the interactions among circRNA, miRNA and mRNA, and constructed the circRNA-miRNA-mRNA ceRNA network by using Cytoscape_v3.8.0. Then KEGG, GO and survival analysis of ceRNA-related genes were performed. Then, the prognostic data of gastric cancer were extracted from the TCGA database to construct the prognostic subnetwork of gastric cancer. ResultsKEGG analysis of ceRNAs mRNA showed that the pathway was mainly enriched in IL-17 signaling pathway, TNF signaling pathway and so on, which affected the prognosis of gastric cancer. hsa_circ_0055521/hsa-miR-204-5p/FAP, (hsa_circ_0005051, hsa_circ_0007613, hsa_circ_0045602, hsa_circ_0034398, hsa_circ_0006089) /hsa-miR-32-3p/FNDC1 were the ceRNA networks related to the prognosis of gastric cancer collaterals. ConclusionThis study found that IL-17 signaling pathway and TNF signaling pathway may affect the occurrence, development and prognosis of gastric cancer. And hsa_circ_0055521/hsa-miR-204-5p/FAP, (hsa_circ_0005051, hsa_circ_0007613, hsa_circ_0045602, hsa_circ_0034398, hsa_circ_0006089) /hsa-miR-32-3p/FNDC1 two circrNa-based stomachs The cancer ceRNA prognostic network is a new prognostic related ceRNA network for gastric cancer. FNDC1 and FAP may be potential therapeutic targets for gastric cancer.

LIGHT, encoded by the TNFSF14 gene, is a cytokine belonging to the tumor necrosis factor superfamily. Upon binding to its receptors, HVEM and LTBR, it activates inflammatory responses. We used a single-molecule immunoassay to determine the circulating levels of free LIGHT in plasma from pediatric patients with Crohns disease (N = 183) and a panel of healthy pediatric reference samples (N = 9). LIGHT levels were greatly elevated in Crohns disease (average of 305 pg/ml versus 57 pg/ml in controls, P < 0.0001). We performed correlational analyses between LIGHT levels and the clinical characteristics of the Crohns cohort, including age, Montreal classification, family history, medical/surgical therapy, and routine blood test parameters. We found statistically significant correlation between white blood cell count and free LIGHT (P < 0.046). Our results support the hypothesis that elevated levels of the cytokine contribute to the pathology of Crohns disease and that therapies to neutralize free LIGHT with antibodies may be beneficial.

IntroductionLiver fibrosis can progress to cirrhosis, liver failure, or hepatocellular carcinoma, which often requires transplantation and burdens healthcare systems around the world. Advances in single-cell RNA sequencing and machine learning have enhanced the understanding of immune responses in many liver diseases particularly alcohol liver cirrhosis (ALC) and non-alcoholic steatohepatitis (NASH). This study aims to identify key biomarkers involved in these conditions and assess their potential as non-invasive diagnostic tools. MethodsTwo gene expression profiles GSE136103 and GSE115469 were used to conduct differential gene expression (DEG) analysis. Using the results from DEG analysis, we then applied two machine learning network-based algorithms, master regulator analysis (MRA) and weighted key driver analysis (wKDA), to identify potential biomarker genes for NASH and ALC. ResultsA total of 1,435 and 5,074 DEGs were identified for ALC and NASH compared to healthy controls, including 1,077 shared DEGs between the two diseases. The MRA showed HLA-DPA1, HLA-DRB1, IFI44L, ISG15, and CD74 as the potential master regulators of ALC and HLA-DPB1, HLA-DQB1, HLA-DRB5, PFN1, and TMSB4X as the potential master regulators of NASH. In addition, wKDA analysis indicated CD300A, FCGR2A, RGS1, HLA-DMB, and C1QA as the key drivers of ALC and INPP5D, NCKAP1L, RAC2, PTPRC, and TYROBP as key drivers of NASH. ConclusionThis study presented a comprehensive framework for analyzing single-cell RNA-seq data, demonstrating the potential of combining advanced network-based machine-learning techniques with conventional DEG analysis to uncover actionable prognostic markers for ALC and NASH with potential use as target biomarkers in drug development.

BackgroundBioactive adrenomedullin (bio-ADM) is a vasoactive peptide hormone that predicts clinical outcomes in heart failure - the main driver of adverse outcomes in cardiac amyloidosis (CA). This prospective observational study sought to assess the prognostic role of bio-ADM in CA. MethodsCA patients were enrolled from amyloid centres in Germany (observation cohort), Japan and the U.S. (combined validation cohort). Bio-ADM was quantified using the sphingotest(R) bio-ADM(R) assay. Associations of bio-ADM with all-cause death and major adverse cardiovascular events (MACE) over two years were assessed using Kaplan-Meier and Cox regression analyses. Likelihood ratio chi-squared tests for nested models evaluated whether adding bio-ADM improves validated prognostic staging systems. ResultsIn both the German observation cohort (n=86) and the combined validation cohort from Japan and the U.S. (n=124), elevated bio-ADM (>29 pg/mL) was associated with more frequent all-cause death and MACE. Bio-ADM remained independently associated with impaired overall (p<0.001) and MACE-free survival (p<0.001) after adjustment for age, sex, and established prognostic biomarkers in the entire cohort. Adding categorised bio-ADM (>29 pg/mL) significantly improved the prognostic accuracy of the National Amyloidosis Centre (C-index 0.674 to 0.787; p=0.002) and MayoATTR (C-index 0.662 to 0.757; p<0.001) staging systems for cardiac transthyretin amyloidosis (ATTR-CA). Adding bio-ADM to staging systems for cardiac immunoglobulin light chain amyloidosis (AL-CA) yielded no significant changes. ConclusionsBio-ADM is a promising prognostic biomarker, especially in ATTR-CA, where it improved risk stratification when added to established staging systems. Further research is needed to clarify its role as part of staging systems for AL-CA. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIBioactive Adrenomedullin (bio-ADM) is a novel prognostic biomarker for all-cause mortality and major adverse cardiovascular events (MACE) in patients with cardiac amyloidosis. C_LIO_LIIncorporating bio-ADM into established staging systems for cardiac transthyretin amyloidosis (ATTR-CA) improves their prognostic performance. Further research is required to determine the role of bio-ADM as part of staging systems for cardiac immunoglobulin light chain amyloidosis (AL-CA). C_LI What Are the Clinical Implications?O_LIUsing bio-ADM as an additional prognostic biomarker allows for more accurate risk-stratification and may thereby enhance individualized clinical management in patients with ATTR-CA. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/25341962v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@5a4b27org.highwire.dtl.DTLVardef@1fc0d6corg.highwire.dtl.DTLVardef@d53ac7org.highwire.dtl.DTLVardef@10619b7_HPS_FORMAT_FIGEXP M_FIG C_FIG This cross-continental multi-centre study, comprising a German observation cohort (n=86) and a combined validation cohort from Japan and the United States (n=124), sought to assess the prognostic value of bioactive Adrenomedullin (bio-ADM) in patients with cardiac amyloidosis (CA) and evaluate whether its incorporation into established staging systems improves risk-stratification. Therefore, patients were stratified into groups with low (i.e., [&le;]29pg/mL; n=43, 20.5%) and high (i.e., >29pg/mL; n=167, 79.5%) bio-ADM plasma levels at baseline and were then followed for the occurrence of major adverse cardiovascular events (MACE) or all-cause death over a period of up to two years. High bio-ADM plasma levels were associated with an elevated risk for MACE and all-cause death in both the observation and the validation cohort. Integrating bio-ADM into validated prognostic staging systems for cardiac transthyretin amyloidosis (ATTR-CA; NAC and MayoATTR staging systems) led to significant improvements in their prognostic accuracy. No such improvements were observed when adding bio-ADM to staging systems for cardiac immunoglobulin light chain amyloidosis (AL-CA; Mayo2004 and Mayo2012 staging systems). In conclusion, bio-ADM is a promising novel prognostic biomarker, especially in ATTR-CA, where it allows for improved risk-stratification and may thereby enhance individualized clinical management. Further research is needed to clarify the role of bio-ADM as part of staging systems for AL-CA.

CRE recombinase is a protein that recognizes and mediates site-specific recombination between loxP site sequences. The Cre/loxP recombination system has become a useful tool for genetic manipulation. Spatial regulation of recombination can be achieved by using cell type-specific promoters that drive expression of CRE in the tissue of interest. The temporal regulation can be obtained with CreER recombinase, which consists of Cre fused to mutated hormone-binding domain of the estrogen receptor (ER). In the more improved versions of the construct, the CRE-mediated gene regulation can be controlled both spatially and temporally, by combining tissue-specific expression of a CreER recombinase with its tamoxifen-dependent activity. We recently generated and characterized an astrocyte specific mutant of the lipolysis-stimulated lipoprotein receptor lsr gene by crossing Glast ERT2 mice with floxed lsr mice (El Hajj et al., 2022). During the behavioral analysis of generated mice, we identified specific hyperactive traits in the Glast ERT2 mice (CRE mice) that prevented them from being used as a control group. Here we further assessed the hyperactive trait of those CRE mice using a battery of behavioral tests. We showed that CRE mice exhibited hyperactive behavior combined with attention-deficit, sleep disturbance and impulsivity that affect their learning and memorization performances. These mice may therefore serve as a model to study attention deficit / hyperactivity disorder. Our work also pointed out the need for proper behavioral analysis of control groups in transgenic animal generation to avoid misinterpretation and misattribution of behavioral traits.

Base editing technique and PRIME editing techniques derived from the CRISPR/Cas9 discovery permit to modify selected nucleotides. We initially used the base editing technique to introduce in the APP gene the A673T mutation, which prevents the development of Alzheimers disease. Although the desired cytidine to thymidine mutation was inserted in up to 17% of the APP gene in HEK393T, there were also modifications of up to 20% of other nearby cytidines. More specific mutations of the APP gene were obtained with the PRIME editing technique. However, the best percentage of mutations was only 5.8%. The efficiency of the PRIME editing treatment was initially tested on the EMX1 gene. A single treatment produced the desired modification in 36% of the EMX1 gene. Three consecutive treatments increased the percentage of mutations to 50%. The PRIME editing technique was also used to insert specific point mutations in exons 9 and 35 of the DMD gene coding for the dystrophin gene and which is mutated in Duchenne Muscular Dystrophy (DMD). Up to 10% desired mutations of the DMD gene were obtained. Three repeated treatments increased the percentage of specific mutations to 16%. Given that there are thousands of nuclei inside a human muscle fiber and that the dystrophin nuclear domain is about 500 m, this level of modifications would be sufficient to produce a phenotype improvement in DMD patients.

Sickle cell disease (SCD) is due to a mutation in the {beta}-globin (HBB) gene causing the production of the toxic sickle hemoglobin (HbS, a2{beta}S2). Transplantation of autologous hematopoietic stem/progenitor cells (HSPCs) transduced with lentiviral vectors (LVs) expressing an anti-sickling {beta}-globin ({beta}AS) is a promising treatment; however, it is only partially effective and patients still present elevated HbS levels. Here, we developed a bifunctional LV expressing {beta}AS3-globin and an artificial microRNA (amiR) specifically downregulating {beta}S-globin expression with the aim of reducing HbS levels and favoring {beta}AS3 incorporation into Hb tetramers. Efficient transduction of SCD HSPC by the bifunctional LV led to a substantial decrease of {beta}S-globin transcripts in HSPC-derived erythroid cells, a significant reduction of HbS+ red cells and effective correction of the sickling phenotype, outperforming {beta}AS gene addition and BCL11A gene silencing strategies. The bifunctional LV showed a standard integration profile and neither the HSPC viability, engraftment and multi-lineage differentiation nor the erythroid transcriptome and miRNAome were affected by the treatment, confirming the safety of this therapeutic strategy. In conclusion, the combination of gene addition and gene silencing strategies can improve the efficacy of current LV-based therapeutic approaches without increasing the mutagenic vector load, thus representing a novel treatment for SCD.

Microbial metabolites affect the neuron system and muscle cell functions. Amyotrophic Lateral Sclerosis (ALS) is a multifactorial neuromuscular disease. Our previous study has demonstrated elevated intestinal inflammation and dysfunctional microbiome in ALS patients and an ALS mouse model (human-SOD1G93A transgenic mice). However, the metabolites in ALS progression are unknown. Using an unbiased global metabolomic measurement and targeted measurement, we investigated the longitudinal changes of fecal metabolites in the SOD1G93A mice over the course of 13 weeks. We compared the changes of metabolites and inflammatory response in age-matched WT and SOD1G93A mice treated with bacterial product butyrate. We found changes in carbohydrate levels, amino acid metabolism, and formation of gamma-glutamyl amino acids. Shifts in several microbially-contributed catabolites of aromatic amino acids agree with butyrate-induced changes in composition of gut microbiome. Declines in gamma-glutamyl amino acids in feces may stem from differential expression of GGT in response to butyrate administration. Due to signaling nature of amino acid-derived metabolites, these changes indicate changes in inflammation (e.g. histamine) and contribute to differences in systemic levels of neurotransmitters (e.g. GABA, glutamate). Butyrate treatment was able to restore some of the healthy metabolites in ALS mice. Moreover, microglia in the spinal cord were measured by the IBA1 staining. Butyrate treatment significantly suppressed the IBA1 level in the SOD1G93A mice. The serum IL-17 and LPS were significantly reduced in the butyrate treated SOD1G93A mice. We have demonstrated an inter-organ communications link among metabolites, inflammation, and ALS progression, suggesting the potential to use metabolites as ALS hallmarks and for treatment. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/476456v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@16d3e4dorg.highwire.dtl.DTLVardef@1155c05org.highwire.dtl.DTLVardef@14c6155org.highwire.dtl.DTLVardef@18a30d6_HPS_FORMAT_FIGEXP M_FIG We compared the changes of metabolites and inflammatory response in age-matched WT and SOD1G93A mice treated with bacterial product butyrate. Butyrate treatment was able to restore some of the healthy metabolites in ALS mice. Due to signaling nature of amino acid-derived metabolites, these changes indicate changes in inflammation and contribute to differences in systemic levels of neurotransmitters (e.g. GABA, glutamate). Moreover, butyrate treatment significantly suppressed the microglia IBA1 level and aggregated SOD1G93A in the SOD1G93A mice. The inflammatory cytokine, e.g serum IL-17, was significantly reduced in the butyrate treated SOD1G93A mice. We have demonstrated an inter-organ communications link among metabolites, inflammation, and ALS progression, suggesting the potential to use metabolites as ALS hallmarks and for treatment. C_FIG

BackgroundDelirium following cardiac surgery is common, morbid, and costly, but may be prevented with risk stratification and targeted intervention. Preoperative protein signatures may identify patients at increased risk for worse postoperative outcomes, including delirium. In this study, we aimed to identify plasma protein biomarkers and develop a predictive model for postoperative delirium in older patients undergoing cardiac surgery, while also uncovering possible pathophysiological mechanisms. MethodsSOMAscan analysis of 1,305 proteins in the plasma from 57 older adults undergoing cardiac surgery requiring cardiopulmonary bypass was conducted to define delirium-specific protein signatures at baseline (PREOP) and postoperative day 2 (POD2). Selected proteins were validated in 115 patients using the ELLA multiplex immunoassay platform. Proteins were combined with clinical and demographic variables to build multivariable models that estimate the risk of postoperative delirium and bring light to the underlying pathophysiology. ResultsA total of 115 and 85 proteins from SOMAscan analyses were found altered in delirious patients at PREOP and POD2, respectively (p<0.05). Using four criteria including associations with surgery, delirium, and biological plausibility, 12 biomarker candidates (Tukeys fold change (|tFC|)>1.4, Benjamini-Hochberg (BH)-p<0.01) were selected for ELLA multiplex validation. Eight proteins were significantly altered at PREOP, and seven proteins at POD2 (p<0.05), in patients who developed postoperative delirium compared to non-delirious patients. Statistical analyses of model fit resulted in the selection of a combination of age, sex, and three proteins (angiopoietin-2 (ANGPT2); C-C motif chemokine 5 (CCL5); and metalloproteinase inhibitor 1 (TIMP1); AUC=0.829) as the best performing predictive model for delirium at PREOP. The delirium-associated proteins identified as biomarker candidates are involved with inflammation, glial dysfunction, vascularization, and hemostasis, highlighting the multifactorial pathophysiology of delirium. ConclusionOur study proposes a model of postoperative delirium that includes a combination of older age, female sex, and altered levels of three proteins. Our results support the identification of patients at higher risk of developing postoperative delirium after cardiac surgery and provide insights on the underlying pathophysiology. ClinicalTrials.gov (NCT02546765).

Neurofibromatosis type 2 (NF2) is an autosomal dominant condition caused by loss of function variants in the NF2 gene, which codes for the protein Merlin, and characterized by the development of multiple tumours of the nervous system. The clinical presentation of the disease is variable and related to the type of the inherited germline variant. Here, we tested if PMOs could be used to correct the splice signalling caused by variants at +/-13 within the intron-exon boundary region. Here we show that the PMOs designed for these variants do not constitute a therapeutic approach. Furthermore, we evaluated the use of phosphorodiamidate morpholino oligomers (PMOs) to reduce the severity of the effects of NF2 truncating variants with the aim of generating milder hypomorphic isoforms in vitro through the induction of the in-frame deletion of the exon-carrying variant. We were able to specifically induce the skipping of exons 4, 8 and 11 maintaining the NF2 gene reading frame at cDNA level. Only the skipping of exon 11 produced a hypomorphic Merlin (Merlin-e11), able to partially rescue the observed phenotype in primary fibroblast cultures from NF2 patients, being encouraging for the treatment of patients harbouring truncating variants located in exon 11.

Organ fibrosis is one of the major causes of morbidity and mortality globally. Though fibrosis in genetic diseases such as Duchenne muscular dystrophy (DMD) may be attributed to the genetic defect, chronic microinflammation remains a key mechanism underlying such fibrosis, which also precedes both other organ fibrosis and post-organ transplant fibrosis. Having proven the anti-inflammatory, anti-fibrotic effects of Beta-1,3-1,6-glucan (Neu-REFIX) produced by N-163 strain of Aureobasidium Pullulans in earlier clinical and pre-clinical studies, we performed the current study to evaluate its effects on myocardial fibrosis. N-163 beta-glucan was administered to 45 mice in three groups, each fifteen animals, Gr. 1, normal mice, Gr.2, mdx mice as vehicle, Gr.3, mdx mice which were administered Neu REFIX beta-glucan orally. Evaluation of Collagen Type I (Col-I) in myocardium was performed by immunohistochemistry. Percentage of myocardium Col-I positive area of 6.42 {+/-} 2.67 significantly decreased in the Neu-REFIX group (4.32 {+/-} 1.78) (p-value < 0.01). As myocardial fibrosis has been shown to be reduced following treatment with N-163 beta glucan in a genetic, muscle structure anomaly disease such as DMD, in addition to adding value to DMD patients, in whom myocardial failure occurs in the advanced stages leading to pre-mature death, Neu-REFIX beta-glucan adjuvant treatment in the setting of solid organ transplantation may be of value to reduce the incidence of fibrosis which is a known feature of chronic allograft rejection leading to graft loss.

Background and aimsAlcohol-associated hepatitis (AH) is the most life-threatening form of alcohol-associated liver disease (ALD). AH is characterized by severe inflammation attributed to increased levels of ethanol, microbes or microbial components, and damage-associated molecular pattern (DAMP) molecules in the liver. HSPB1 (Heat Shock Protein Family B (Small) Member 1; also known as Hsp25/27) is a DAMP that is rapidly increased in and released from cells experiencing stress, including hepatocytes. The goal of this study was to define the role of HSPB1 in AH pathophysiology. MethodsSerum HSPB1 was measured in a retrospective study of 184 heathy controls (HC), heavy alcohol consumers (HA), patients with alcohol-associated cirrhosis (AC), and patients with AH recruited from major hospital centers. HSPB1 was also retrospectively evaluated in liver tissue from 10 HC and AH patients and an existing liver RNA-seq dataset. Finally, HSPB1 was investigated in a murine Lieber-DeCarli diet model of early ALD as well as cellular models of ethanol stress in hepatocytes and hepatocyte-macrophage communication during ethanol stress. ResultsCirculating HSPB1 was significantly increased in AH patients and levels positively correlated with disease-severity scores. Likewise, HSPB1 was increased in the liver of patients with severe AH and in the liver of ethanol-fed mice. In vitro, ethanol-stressed hepatocytes released HSPB1, which then triggered TNF-mediated inflammation in macrophages. Anti-HSPB1 antibody prevented TNF release from macrophages exposed to media conditioned by ethanol-stressed hepatocytes. ConclusionsOur findings support investigation of HSPB1 as both a biomarker and therapeutic target in ALD. Furthermore, this work demonstrates that anti-HSPB1 antibody is a rational approach to targeting HSPB1 with the potential to block inflammation and protect hepatocytes, without inactivating host defense. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/24313193v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@141816eorg.highwire.dtl.DTLVardef@1985d66org.highwire.dtl.DTLVardef@1c0ed4org.highwire.dtl.DTLVardef@118ca9_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIHSPB1 is significantly increased in serum and liver of patients with alcohol-associated hepatitis. C_LIO_LIEthanol consumption leads to early increases in HSPB1 in the mouse liver. C_LIO_LIHepatocytes subjected to ethanol stress release HSPB1 into the extracellular environment where it activates TNF-mediated inflammation in macrophages. C_LIO_LIAnti-HSPB1 antibody blocks hepatocyte-triggered TNF in a model of hepatocyte-macrophage communication during ethanol stress. C_LI

Purpose of the study: to describe the composition of the microbiota of the initial sections of the small intestine in patients with severe necrotizing acute pancreatitis. Objectives of the studyO_LIDetermine the composition of the microbiota of the initial sections of the small intestine upon admission to the ICU; C_LIO_LIDetermine the differences in the composition of the microbiota of the initial sections of the small intestine, depending on the timing of the onset of the disease. C_LI IntroductionDisturbance of intestinal homeostasis is a leading factor in the pathogenesis and progression of systemic inflammation in patients with severe acute pancreatitis. The development of systemic complications occurs due to both mesenteric hypoperfusion and dysregulation of intestinal motility, and the destruction of the intestinal barrier, with the translocation of bacterial bodies and their substrates. Which increases the risk of developing POI and increasing mortality. With the advent of methods for high-throughput sequencing of microbiome samples - for example, in the 16S rRNA format - the possibilities for studying the structure of microbial communities have significantly expanded. In this regard, there is more and more evidence of the relationship between the state of human health and microflora inhabiting various parts of the body. Materials and methodsThe study included 7 patients with a diagnosis of severe necrotizing acute pancreatitis (6 men, 1 woman), the mean age was 54.1 {+/-} 14.4 years. The patients were divided into two groups. Group 1 (n = 4) included patients admitted 2-4 days after the onset of a pain attack. Group 2 (n = 3) - patients admitted no later than 24 hours from the onset of the disease. The bacterial composition of jejunal wash samples was studied using 16S RNA sequencing. The severity of the condition was assessed using the integral scales APACHE II, SOFA, SAPS II. In patients of the main group, APACHE II was 22 {+/-} 2.83 points (18; 24), SOFA - 6.8 {+/-} 0.5 points (6; 7), SAPSII - 32.9 {+/-} 6.4 points (24.7; 40), in patients of the comparison group, APACHE II is 18.0 {+/-} 3.7 points (12; 22), SOFA - 4.0 {+/-} 2.6 points (2; 7), SAPSII - 24.4 {+/-} 5.0 points (20.9; 30.1). Material was collected at the time of installation of a sterile multifunctional intestinal catheter for Treitzs ligament, no later than 12 hours from the moment of admission to the ICU. At the time of sampling, patients were not receiving antibiotic therapy. ResultsA more severe course was associated with a reduced representation in the microbiome of the species Nesseria mucosa and Parvimonas micra inhabiting the mucosal layer, as well as Megasphaera micronuciformis. The share of Streptococcus genera (S. rubneri / parasanguinis / australis species) and Actinomyces and a number of genera from the Enterobacteriaceae family in such patients, on the contrary, was higher. Interest disclosureSample preparation, sequencing and analysis of these microbiome samples was carried out by Knomics LLC during a commercial project for VneshPromFarm LLC, the manufacturer of saline electrolyte solution (SES).

Autoantibodies targeting the endothelial protein C receptor (EPCR) have been associated with ulcerative colitis (UC). We aimed to assess a potential role of EPCR lipidation on the detection of anti-EPCR antibodies in inflammatory bowel disease (IBD). To this end, serum samples from patients with UC, Crohns disease, and healthy controls were analyzed using an in-house ELISA employing either native or delipidated EPCR. Overall, male UC patients showed significantly higher absorbance (mean=1.09) than CD patients (mean=0.50) and controls (mean=0.37). Remarkably, in males, the difference between UC patients and controls was highly significant (p=1.2e-07), as was the difference between UC and CD patients (p=5.7e-05). In women, the difference was less pronounced and only significant when comparing UC to controls (p=0.023). Replacement of native EPCR with delipidated EPCR in the ELISA procedure dropped detection and eliminated the UC-CD discrimination power (p=0.784). These findings indicate a role for the bound lipid as key determinant of male-biased, anti-EPCR reactivity, and support the diagnostic potential of this biomarker when assay conditions preserve the physiological lipid-bound state of EPCR.

Lung adenocarcinoma (LUAD), the primary subtype of Non-Small Cell Lung Cancer (NSCLC), accounts for 80% to 85% of cases. Due to suboptimal screening method, LUAD is often detected in late stage, leading to aggressive progression and poor outcomes. Therefore, early disease prognosis for the LUAD is high priority. In order to identify early detection biomarkers, we conducted a meta-analysis of mRNA expression TCGA and GTEx datasets from LUAD patients. A total of 795 differentially expressed genes (DEGs) were identified by exploring the Network-Analyst tool and utilizing combined effect size methods. DEGs refer to genes whose expression levels are significantly different (either higher or lower) compared to their normal baseline expression levels. KEGG pathway enrichment analysis highlighted the TNF signaling pathway as being prominently associated with these DEGs. Subsequently, using the MCODE and CytoHubba plugins in Cytoscape software, we filtered out the top 10 genes. Among these, SOX2 was the only gene exhibiting higher expression, while the others were downregulated. Consequently, our subsequent research focused on SOX2. Further transcription factor-gene network analysis revealed that enhancer of zeste homolog 2 (EZH2) is a significant partner of SOX2, potentially playing a crucial role in euchromatin-heterochromatin dynamics. Structure of SOX2 protein suggest that it is a non-druggable transcription factor, literature survey suggests the same; hence, we drove our focus to investigate on potential drug(s) targeting EZH2. Molecular docking analyses predicted most probable inhibitors of EZH2. We employed several predictive analysis tools and identified GSK343, as a promising inhibitor of EZH2.

The Hypothalmic-pituitary axis also known as the HPA axis is central to stress response. It also acts as the relay centre between the body and the brain. We analysed hypothalamic proteome from mice subjected to chronic social defeat paradigm using iTRAQ based quantitative proteomics in identify changes associated with stress response. We identified greater than 2000 proteins processing our samples analysed through Q-Exactive (Thermo) and Orbitrap Velos (Thermo) at 5% FDR. Analysis of data procured from the runs showed that the proteins whose levels were affected belonged primarily to mitochondrial and metabolic processes, translation, complement pathway among others. We also found increased levels of fibrinogen, myelin basic protein (MBP) and neurofilaments (NEFL, NEFM, NEFH) in the hypothalamus from socially defeated mice. Interestingly these proteins are found upregulated in blood and CSF of subjects exposed to trauma and stress. Since hypothalamus is in direct contact of blood and CSF, their utility as biomarkers in depression holds an impressive probability and should be validated in clinical samples.