Factor Activity Research Articles

Farnesol inhibits growth and development of Ustilaginoidea virens

AbstractRice false smut disease, caused by the pathogen Ustilaginoidea virens, specifically infects rice panicles. This disease leads to significant quantitative and qualitative losses in the rice industry. Farnesol, a naturally occurring terpene produced by plants, exhibits diverse toxic potentials against various fungi. Here, we investigate the in vitro inhibitory effects of farnesol on U. virens isolates. The results indicate that farnesol could retard conidial germination at a concentration of 20 μM and inhibit germination at concentrations exceeding 40 μM. Additionally, farnesol demonstrates inhibitory activity on the mycelial growth and adhesion of U. virens in a dose‐dependent manner. RNA‐seq data show that farnesol treatment results in the misregulation of a subset of genes involved in oxidoreductase activity, membrane components, molecular function, and transcription factor activity. Furthermore, farnesol induces a significant accumulation of reactive oxygen species, including superoxide anions, which may subsequently trigger cell death. Notably, farnesol also induces the expression of genes encoding GTPase activity of UvRas1 and UvRas2, leading to the expression of genes encoding adenylate cyclase, which promotes the synthesis of intracellular cAMP, and activates genes involved in MAPK pathway. Collectively, this study proposes a mechanism by which farnesol affects the growth and development of U. virens.

Ustilago maydis Trf2 ensures genome stability by antagonizing Blm-mediated telomere recombination: Fine-tuning DNA repair factor activity at telomeres through opposing regulations.

TRF2 is an essential and conserved double-strand telomere binding protein that stabilizes chromosome ends by suppressing DNA damage response and aberrant DNA repair. Herein we investigated the mechanisms and functions of the Trf2 ortholog in the basidiomycete fungus Ustilago maydis, which manifests strong resemblances to metazoans with regards to the telomere and DNA repair machinery. We showed that UmTrf2 binds to Blm in vitro and inhibits Blm-mediated unwinding of telomeric DNA substrates. Consistent with a similar inhibitory activity in vivo, over-expression of Trf2 induces telomere shortening, just like deletion of blm, which is required for efficient telomere replication. While the loss of Trf2 engenders growth arrest and multiple telomere aberrations, these defects are fully suppressed by the concurrent deletion of blm or mre11 (but not other DNA repair factors). Over-expression of Blm alone triggers aberrant telomere recombination and the accumulation of aberrant telomere structures, which are blocked by concurrent Trf2 over-expression. Together, these findings highlight the suppression of Blm as a key protective mechanism of Trf2. Notably, U. maydis harbors another double-strand telomere-binding protein (Tay1), which promotes Blm activity to ensure efficient replication. We found that deletion of tay1 partially suppresses the telomere aberration of Trf2-depleted cells. Our results thus point to opposing regulation of Blm helicase by telomere proteins as a strategy for optimizing both telomere maintenance and protection. We also show that aberrant transcription of both telomere G- and C-strand is a recurrent phenotype of telomere mutants, underscoring another potential similarity between double strand breaks and de-protected telomeres.

Muramyl dipeptide potentiates Staphylococcus aureus lipoteichoic acid-induced nitric oxide production via TLR2/NOD2/PAFR signaling pathways

Lipoteichoic acid (LTA) and peptidoglycan (PGN) are considered as key virulence factors of Staphylococcus aureus, which is a representative sepsis-causing Gram-positive pathogen. However, cooperative effect of S. aureus LTA and PGN on nitric oxide (NO) production is still unclear despite the pivotal roles of NO in initiation and progression of sepsis. We here evaluated the cooperative effects of S. aureus LTA (SaLTA) and muramyl dipeptide (MDP), the minimal structure of PGN, on NO production in both a mouse macrophage-like cell line, RAW 264.7 and mouse bone marrow-derived macrophages (BMMs). Although MDP alone did not affect NO production, MDP potently enhanced SaLTA-induced NO production via the expression of inducible NO synthases. The enhanced NO production was ameliorated in BMMs from TLR2-, CD14-, MyD88-, and NOD2-deficient mice. Moreover, the augmented SaLTA-induced NO production by MDP was attenuated by inhibitors specific for PAFR and MAP kinases. Furthermore, MDP also potently increased SaLTA-induced activities of STAT1, NF-κB, and AP-1 transcription factors, and specific inhibitors for these transcription factors suppressed the elevated NO production. Collectively, these results demonstrated that MDP potentiates SaLTA-induced NO production via TLR2/NOD2/PAFR, MAP kinases signaling axis, resulting in the activation of NF-κB, AP-1 and STAT1 transcription factors.

Hypoxia-Inducible Factor-2α Promotes Liver Fibrosis by Inducing Hepatocellular Death

The activation of hypoxia-inducible factors (HIF)-1α and 2α in the liver is closely linked to the progression of fatty liver diseases. Prior studies indicated that disrupting hepatocyte HIF-2α attenuates diet-induced hepatic steatosis, subsequently decreasing fibrosis. However, the direct role of hepatocyte HIF-2α in liver fibrosis has not been addressed. Hepatic HIF-2α expression was examined in mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis. Conditional hepatocyte Hif-2α knockout mice were employed to investigate the role of hepatocyte HIF-2α in fibrosis. Markers of apoptosis, proliferation, inflammation, and fibrosis were assessed through biochemical, molecular, and histological analyses. We found an induction of HIF-2α in CCL4-injected liver injury and fibrosis mouse models. Hepatocyte-specific deletion of HIF-2α attenuated stellate cell activation and fibrosis, with no significant difference in inflammation. Disrupting hepatocyte HIF-2α led to reduced injury-mediated hepatocellular apoptosis. Surviving hepatocytes exhibited hypertrophy, which was strongly associated with the activation of c-JUN signaling. Our study demonstrates a direct role of hepatocyte HIF-2α in liver fibrosis by promoting hepatocyte apoptosis. The reduction in apoptosis and induction of hepatocyte hypertrophy following HIF-2α disruption is closely linked to enhanced c-JUN signaling, a survival mechanism in response to liver injury. These findings highlight HIF-2α as a potential therapeutic target for liver fibrosis.

Perioperative Management of Hemophilia A: An Uncommon Inherited Blood Disorder

Hemophilia A is a rare hematological disorder caused by an inherited X-linked factor VIII deficiency. It can be responsible for significant perioperative bleeding with fatal consequences if not diagnosed and managed correctly in the preoperative phase. We report the case of a 6-month-old infant scheduled for a cure for bilateral inguinal hernia. During the pre-anesthetic consultation, given that the child was uncircumcised and had not yet reached walking age, a blood workout was made, which revealed a prolonged aPTT with a patient/control aPTT ratio of 3.27, without hemorrhagic signs. An activity work-up of clotting factors showed less than 1% of normal levels of active clotting factor VIII. Thus, the diagnosis of a severe form of hemophilia A was made. Recombinant factor VIII was administrated in the preoperative period, with an activity factor VIII control over 60%. The surgical procedure was uneventful, with minimal intraoperative bleeding. Our case emphasizes the importance of prophylactic treatment and a proper pre-anesthetic assessment, which are essential to establish an appropriate management strategy and avoid complications, most of which are fatal.

Integrative multi-omics analysis reveals a novel subtype of hepatocellular carcinoma with biological and clinical relevance

BackgroundHepatocellular carcinoma (HCC) is a highly heterogeneous tumor, and the development of accurate predictive models for prognosis and drug sensitivity remains challenging.MethodsWe integrated laboratory data and public cohorts to conduct a multi-omics analysis of HCC, which included bulk RNA sequencing, proteomic analysis, single-cell RNA sequencing (scRNA-seq), spatial transcriptomics sequencing (ST-seq), and genome sequencing. We constructed a tumor purity (TP) and tumor microenvironment (TME) prognostic risk model. Proteomic analysis validated the TP-TME-related signatures. Joint analysis of scRNA-seq and ST-seq revealed characteristic clusters associated with TP high-risk subtypes, and immunohistochemistry confirmed the expression of key genes. We conducted functional enrichment analysis, transcription factor activity inference, cell-cell interaction, drug efficacy analysis, and mutation information analysis to identify a novel subtype of HCC.ResultsOur analyses constructed a robust HCC prognostic risk prediction model. The patients with TP-TME high-risk subtypes predominantly exhibit hypoxia and activation of the Wnt/beta-catenin, Notch, and TGF-beta signaling pathways. Furthermore, we identified a novel subtype, XPO1+Epithelial. This subtype expresses signatures of the TP risk subtype and aligns with the biological behavior of high-risk patients. Additional analyses revealed that XPO1+Epithelial is influenced primarily by fibroblasts via ligand-receptor interactions, such as FN1-(ITGAV+ITGB1), and constitute a significant component of the TP-TME subtype. Moreover, XPO1+Epithelial interact with monocytes/macrophages, T/NK cells, and endothelial cells through ligand-receptor pairs, including MIF-(CD74+CXCR4), MIF-(CD74+CD44), and VEGFA-VEGFR1R2, respectively, thereby promoting the recruitment of immune-suppressive cells and angiogenesis. The ST-seq cohort treated with Tyrosine Kinase Inhibitors (TKIs) and Programmed Cell Death Protein 1 (PD-1) presented elevated levels of TP and TME risk subtype signature genes, as well as XPO1+Epithelial, T-cell, and endothelial cell infiltration in the treatment response group. Drug sensitivity analyses indicated that TP-TME high-risk subtypes, including sorafenib and pembrolizumab, were associated with sensitivity to multiple drugs. Further exploratory analyses revealed that CTLA4, PDCD1, and the cancer antigens MSLN, MUC1, EPCAM, and PROM1 presented significantly increase expression levels in the high-risk subtype group.ConclusionsThis study constructed a robust prognostic model for HCC and identified novel subgroups at the single-cell level, potentially assisting in the assessment of prognostic risk for HCC patients and facilitating personalized drug therapy.

Correlation between Phenotype and Coagulation Factor Activity Level in Rare Bleeding Disorders: A Systematic Review.

Rare bleeding disorders (RBDs) represent 3 to 5% of congenital bleeding disorders and are primarily inherited in an autosomal recessive manner, with increased prevalence in consanguineous populations. Clinically, RBDs can be accompanied by mild to severe bleeding episodes, often assessed using bleeding assessment tools (BATs) such as the International Society on Thrombosis and Hemostasis (ISTH)-BAT. However, the correlation between bleeding severity and coagulation factor activity levels remains inconsistent. This systematic review investigates this relationship to enhance understanding and improve management strategies for patients with RBD. This review adhered to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and was registered with the International Prospective Register for Systematic Reviews (PROSPERO) (CRD42024504537). Using the PICO (Population, Intervention, Comparator, and Outcomes) framework, the study focused on RBD patients to explore the correlation between coagulation factor activity levels and bleeding severity. A comprehensive search was conducted across PubMed, Scopus, and Web of Science until April 1, 2024, with data extracted on bleeding severity, phenotype, and coagulation factor activity levels. The analysis highlights complex and often inconsistent relationships between coagulation factor levels and the severity of bleeding. In cases of fibrinogen deficiency, three out of four studies (n = 73 of 111 cases, 66%) demonstrated a moderate to strong correlation between fibrinogen levels and bleeding severity. In prothrombin deficiency, one of two studies (n = 16 of 29 cases, 55%) found a strong correlation between FII levels and bleeding severity. Four of six studies (n = 106 of 139 cases, 76%) in FV deficiency found a weak or no correlation between factor activity and bleeding severity. In combined FV and FVIII deficiency, two of three studies (n = 26 of 60 cases, 43%) found a significant correlation between factor activity and bleeding severity. In FVII deficiency, four (of nine) studies with a study population of 325 patients (65%) found a weak correlation between factor activity and severity of bleeding. Almost all studies (five of six studies, n = 114 of 118 patients, 97%) in FX deficiency revealed a strong correlation between FX levels and bleeding severity. In FXI deficiency, most studies (five of seven studies, n = 254 patients, 93%) found a weak or no correlation between factor activity and bleeding severity or symptoms. For FXIII deficiency, there was a moderate to strong correlation between FXIII activity and bleeding severity in all three studies (n = 61 patients). In conclusion, despite current controversies, this review highlights a moderate or strong correlation between factor activity and bleeding severity in fibrinogen, FX, and FXIII deficiencies, but no correlation or weak correlation for FV, FVII, and FXI deficiencies. Further prospective studies with standardized BATs on a large number of patients are needed to better understand these relationships and optimize patient management.

Repurposing raltegravir for reducing inflammation and treating cancer: a bioinformatics analysis

Inflammation is a defensive mechanism that safeguards the human body against detrimental stimuli. Within this intricate process, ADAM17, a zinc-dependent metalloprotease, emerges as an indispensable element, fostering the activation of diverse inflammatory and growth factors within the organism. Nonetheless, ADAM17 malfunctions can augment the rate of growth, inflammatory factors, and subsequent damage. Thus, in this study, we examined and repurposed drugs to suppress the activity of ADAM17. To this end, we employed bioinformatics techniques such as molecular docking, molecular dynamics, and pharmacokinetic studies. Five FDA-approved drugs including Raltegravir, Conivaptan, Paclitaxel, Saquinavir, and Venetoclax with the ability to impede the activity of the ADAM17 metalloenzyme were identified. Moreover, these drugs did not include strong zinc-binding functional groups when verified by the ACE functional group finder. However, further in silico analysis has indicated that Raltegravir demonstrates a commendable interaction with the active site amino acids and exhibits the most favorable pharmacokinetic properties compared to others. Considering the results of bioinformatics tools, it can be concluded that Raltegravir as an antiviral drug could be repurposed to prevent severe inflammatory response and tumorigenesis resulting from ADAM17 malfunction.

Effective commercial activity of a company factors in digital economy

In modern world, digital economy affects all aspects of commercial activity, and successful application of digital innovations is becoming a key factor in companies’ competitiveness. Commercial organizations in their activities use software tools and the Internet, which significantly expands their opportunities for successful development. The key factors of effective commercial activity in the context of the rapid development of digital economy have been highlighted. Referring to the statistical data of the Federal State Statistics Service, the authors analyzed the trends in information technologies implementation in Russian economy. Special attention has been paid to reviewing the main difficulties that Russian companies face when trying to integrate modern digital solutions into their operations. Recommendations have been given on digitalization of key components of commercial activities, including customer services automation, e-commerce, digital supply chain management, big data analytics to optimize business strategies, and online marketing. The study is supported by the examples of specific tools and techniques that have already shown their effectiveness in practice for Russian companies adapting to digital economy. The research is based on the system and process approaches.

Cellular translational enhancer elements that recruit eukaryotic initiation factor.

Translation initiation is a highly regulated process which broadly affects eukaryotic gene expression. Eukaryotic initiation factor 3 (eIF3) is a central player in canonical and alternative pathways for ribosome recruitment. Here we have investigated how direct binding of eIF3 contributes to the large and regulated differences in protein output conferred by different 5'-untranslated regions (5'-UTRs) of cellular mRNAs. Using an unbiased high-throughput approach to determine the affinity of budding yeast eIF3 for native 5'-UTRs from 4,252 genes, we demonstrate that eIF3 binds specifically to a subset of 5'-UTRs that contain a short unstructured binding motif, AMAYAA. eIF3 binding mRNAs have higher ribosome density in growing cells and are preferentially translated under certain stress conditions, supporting the functional relevance of this interaction. Our results reveal a new class of translational enhancer and suggest a mechanism by which changes in core initiation factor activity enact mRNA-specific translation.

Network Pharmacology and In Vivo Experimental Verification of the Mechanism of the Qing'e Pill for Treating Intervertebral Disc Degeneration.

The Qing'e Pill (QEP) is widely used to alleviate low back pain and sciatica caused by Intervertebral Disc Degeneration (IDD). However, its active components, key targets, and molecular mechanisms are not fully understood. The aim of this study is to elucidate the molecular mechanisms through which the QEP improves IDD using database mining techniques. Active components and candidate targets of the QEP were identified using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and the Bioinformatics Analysis Tool for Molecular Mechanisms of Traditional Chinese Medicine. IDD-related targets were obtained from the GeneCards database, and liver- and kidney-specific genes were retrieved from the BioGPS database. The intersection of these candidate targets was analyzed to identify potential targets for the QEP in IDD. A protein-protein interaction network analysis was performed using STRING and Cytoscape 3.7.2 software. Core targets were further analyzed through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Molecular docking was used to assess the binding affinity of active components to candidate targets, and animal experiments were conducted for validation. We identified 65 potentially active components of the QEP that corresponded to 1,093 candidate targets, 2,108 IDD-related targets, and 1,113 liver- and kidney-specific genes. Key components included quercetin, berberine, isorhamnetin, and emodin. The primary candidate targets were Wnt5A, CTNNB1, IL-1β, MAPK14, MMP9, and MMP3. The GO and KEGG analyses revealed the involvement of these targets in Wnt signaling, TNF signaling, Wnt receptor activation, Frizzled binding, and Wnt-protein interactions. Molecular docking showed strong binding between these components and their targets. Animal experiments demonstrated that the QEP treatment significantly reduced the expression of Wnt5A, CTNNB1, IL-1β, MAPK14, MMP9, and MMP3 at high, medium, and low doses compared with the model group. The QEP alleviated IDD by modulating the Wnt/MAPK/MMP signaling pathways and reducing the release and activation of key factors.

Oxygen-controllable injectable hydrogel alleviates intervertebral disc degeneration by balancing extracellular matrix metabolism

Nucleus pulposus (NP) cells, situated at the core of intervertebral discs, have acclimated to a hypoxic environment, orchestrating the equilibrium of extracellular matrix metabolism (ECM) under the regulatory influence of hypoxia inducible factor-1α (HIF-1α). Neovascularization and increased oxygen content pose a threat, triggering ECM degradation and intervertebral disc degeneration (IVDD). To address this, our study devised an oxygen-controllable strategy, introducing laccase into an injectable and ultrasound-responsive gelatin/agarose hydrogel. Laccase-mediated reactions were employed to deplete oxygen, establishing a hypoxic microenvironment that upregulated HIF-1α expression. The activation of hypoxia-inducible factors significantly enhanced the expression of aggrecan and collagen II, concurrently suppressing Matrix metalloproteinases (MMP13) and A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS5) levels, thereby restoring the equilibrium of ECM metabolism. Simultaneously, the hydrogel facilitated the recruitment of stem cells into the NP through the controlled release of ATI2341, activating C-X-C chemokine receptor type 4 (CXCR4). Moreover, ultrasound amplification enhanced ATI2341 release, promoting the migration of NP stem cells. The hydrogel's efficacy in mitigating metabolic imbalances and inhibiting IVDD progression was substantiated in a rat puncture IVDD model through hydrogel injection into the discs. In conclusion, this hypoxia-inducible hydrogel, responsive to thermal stimuli from ultrasound, presents a promising avenue for IVDD treatment.

Optimized reporters for multiplexed detection of transcription factor activity.

In any given cell type, dozens of transcription factors (TFs) act in concert to control the activity of the genome by binding to specific DNA sequences in regulatory elements. Despite their considerable importance, we currently lack simple tools to directly measure the activity of many TFs in parallel. Massively parallel reporter assays (MPRAs) allow the detection of TF activities in a multiplexed fashion; however, we lack basic understanding to rationally design sensitive reporters for many TFs. Here, we use an MPRA to systematically optimize transcriptional reporters for 86 TFs and evaluate the specificity of all reporters across a wide array of TF perturbation conditions. We thus identified critical TF reporter design features and obtained highly sensitive and specific reporters for 62 TFs, many of which outperform available reporters. The resulting collection of "prime" TF reporters can be used to uncover TF regulatory networks and to illuminate signaling pathways. A record of this paper's transparent peer review process is included in the supplemental information.

An Activity-Based Sensing Approach to Multiplex Mapping of Labile Copper Pools by Stimulated Raman Scattering.

Molecular imaging with analyte-responsive probes offers a powerful chemical approach to studying biological processes. Many reagents for bioimaging employ a fluorescence readout, but the relatively broad emission bands of this modality and the need to alter the chemical structure of the fluorophore for different signal colors can potentially limit multiplex imaging. Here, we report a generalizable approach to multiplex analyte imaging by leveraging the comparably narrow spectral signatures of stimulated Raman scattering (SRS) in activity-based sensing (ABS) mode. We illustrate this concept with two copper Raman probes (CRPs), CRP2181 and CRP2153.2, that react selectively with loosely bound Cu(I/II) and Cu(II) ions, respectively, termed the labile copper pool, through copper-directed acyl imidazole (CDAI) chemistry. These reagents label proximal proteins in a copper-dependent manner using a dye scaffold bearing a 13C≡N or 13C≡15N isotopic SRS tag with nearly identical physiochemical properties in terms of shape and size. SRS imaging with the CRP reagents enables duplex monitoring of changes in intracellular labile Cu(I) and Cu(II) pools upon exogenous copper supplementation or copper depletion or genetic perturbations to copper transport proteins. Moreover, CRP imaging reveals reciprocal increases in labile Cu(II) pools upon decreases in activity of the antioxidant response nuclear factor-erythroid 2-related factor 2 (NRF2) in cellular models of lung adenocarcinoma. By showcasing the use of narrow-bandwidth ABS probes for multiplex imaging of copper pools in different oxidation states and identifying alterations in labile metal nutrient pools in cancer, this work establishes a foundation for broader SRS applications in analyte-responsive imaging in biological systems.

Validation of current commercially-available devices to thaw pooled, solvent/detergent-treated human plasma.

Proper thawing procedures are required to preserve the quality of human fresh frozen plasma (FFP). octaplasLG (Octapharma AG, Switzerland) is a frozen solution of solvent/detergent (S/D)-treated human plasma, produced to improve pathogen safety. This study aimed to validate the S/D plasma thawing process using the latest-generation of commercially-available thawing devices and to determine S/D plasma quality after thawing. Thawing of S/D plasma units was investigated using a microwave oven (Transfusio-therm 3000) and dry tempering systems (SAHARA 4 and Plasmatherm V). Times to defrost plasma and reach 30°C product temperature, using the 37°C thawing programmes, were defined for all devices. Thawed S/D plasma units were tested against product release parameters, as well as for hemostatic capacity tested indirectly via global coagulation parameters, coagulation factors, protease inhibitors and markers of activated coagulation. The fastest thawing was observed using the microwave oven; S/D plasma units were defrosted after 2-3 min. All S/D plasma units thawed by the different devices using optimised thawing conditions were clear and free of solid and gelatinous particles, indicating no local overheating or protein denaturation. Coagulation factor and inhibitor activities and hemostatic capacity of S/D plasma were comparable when thawed by the three different devices. With each device, all product parameters were within product release specification levels after thawing. S/D plasma can be thawed using the Transfusio-therm 3000, SAHARA 4 or Plasmatherm V thawing devices using the optimised settings defined for this plasma product, with no negative influence on plasma quality.

Andean Enriched NFKB1 Haplotype Reduces Inflammation and Improves Response to Ropeginterferon Alfa-2b in Polycythemia Vera (PV) and Essential Thrombocythemia (ET)

Chronic inflammation is a feature of polycythemia vera (PV), essential thrombocythemia (ET), and other myeloproliferative neoplasms (MPNs). NF-κB plays a key role in regulating pro-inflammatory cytokines. In PV and ET, activation of the NF-κB pathway is a primary driver of inflammation and augments hypoxia-inducible factors (HIFs) activity, increasing transcription of prothrombotic genes. Suppression of the NF-κB pathway has been shown to reduce disease burden in MPNs (Laranjeira, Blood, 2024).We investigated the genetic basis underlying elevated hemoglobin in Andean natives, Aymara, by using granulocyte transcriptome data from both Aymara and Europeans from La Paz, Bolivia. We found their differentially expressed and spliced genes are associated with inflammatory and HIFs pathways. We identified three novel, alternatively spliced NFKB1 transcripts (AS-NFKB1) in Aymara, with skipped exon 4, exon 5, or both. The AS-NFKB1 transcript with exon 5 skipped was translated into protein but showed a defect in nuclear translocation, while the other two AS-NFKB1 transcripts were not translated, indicating a partial or complete loss of NFKB1's function as an NF-κB suppressor. AS-NFKB1 also augmented activity of HIFs, explaining high Aymara hemoglobin. Using integrative analysis of whole-genome sequencing and granulocyte transcriptome, we identified splicing quantitative trait locus associated with AS-NFKB1; NFKB1 rs230511 is linked to high AS-NFKB1 expression. Its T allele (C/T or T/T genotype) is present in approximately 90% of Aymara but is also present in ~30% of Europeans, Hispanics, and Asians. Under TNF-induced inflammatory stress, unlike wild-type NFKB1, AS-NFKB1 suppressed inflammatory gene expression, suggesting a protective mechanism against inflammation (under review, Nature Communication).Thus, we examined NFKB1 rs230511 association with inflammatory markers and response to ropeginterferon alfa-2b (Ropeg) therapy in PV and ET. We genotyped 30 PV and 15 ET patients, assessing inflammatory gene expression (IL1B, CXCL8, IL6, IL15, and TNF) in neutrophils and platelets. In platelets, individuals with the C/T genotype showed lower levels of these inflammatory transcripts compared to those with the C/C genotype and IL1B, CXCL8, and TNF levels were also reduced in the T/T genotype. In neutrophils, IL1B and CXCL8 were decreased in the T/T genotype, and IL15 and TNF levels were lower in the C/T genotype.Given that AS-NFKB1 and NFKB1 rs230511 are linked to elevated transcriptional activity of HIFs, we analyzed the expression of HIF-target genes. LDHA, SLC2A1, and VEGFA transcripts were significantly lower in the C/T genotype in both PV and ET neutrophils and platelets. Since increased inflammation and HIF transcriptional activity are associated with thrombosis in PV and ET (Gangaraju, Blood Advances, 2020), we also measured expression levels of prothrombotic genes (F3, THBD, SELP, SERPINE1). In both platelets and granulocytes, F3 and THBD transcript levels were significantly lower in individuals with the C/T genotype compared to those with the C/C genotype.Ropeg modulates inflammation via interferon signaling and attacks PV and ET clones (Verger, Blood Cancer J., 2018). We found that the C/T genotype (58.6%) was more prevalent among patients who achieved complete hematological response (CHR) compared to the non-CHR cohort, which included patients who did not reach CHR or had cytopenia (33.3%) (p<0.0001). This finding also correlated with lower transcript levels of inflammatory, HIF-targeted, and thrombotic genes in the CHR group compared to non-CHR group.Our study indicates that presence of Aymara enriched NFKB1 rs230511 haplotype decreases PV and ET inflammatory, prothrombotic, and HIF's activity compared to NFKB1 wild allele. The NFKB1 rs230511 haplotype also modulates response to Ropeg in PV and ET. The presence of NFKB1 rs230511 T allele is associated with reduced expression of inflammatory and HIF-targeted genes, potentially decreasing inflammation and thrombosis risk in PV and ET. Patients with the C/T genotype were more likely to achieve CHR, suggesting this NFKB1 haplotype may impact Ropeg therapy efficacy and serve as a potential biomarker for response assessment in PV and ET patients. These novel insights offer valuable implications for both of our understanding of inflammatory and prothrombotic processes and may also lead to novel PV and ET therapies.

Inhibiting NLRP3 Inflammasome Activation to Alleviate Retinal Inflammation and Protect the Optic Nerve of OPTN(E50K)Mice.

OPTN (E50K) mutation is one of the significant pathogenic mutations in normal tension glaucoma (NTG). The molecular mechanism of NTG optic nerve injury is complex and diverse; its key mechanism is still unclear. The NLR family pyrin domain containing (NLRP3) inflammasome plays an essential role in the occurrence and development of inflammation. There is no report on whether NLRP3 inflammasome activation plays a crucial role in NTG optic nerve injury. Here, we explored the role of retinal inflammatory cascade reaction triggered by NLRP3 inflammasome activation in OPTN (E50K) mutated NTG optic nerve injury. This research may provide innovative strategies for effectively treating NTG optic nerve injury caused by OPTN (E50K) mutation. The R28 cell was constructed by AAV2 transfection, named GFP-R28, WT-R28, and E50K-R28 groups. Western blot, qPCR, and immunofluorescence were performed to measure the expression levels of the neurotrophic factors, the senescence indicators, the NLRP3-related indicators, the expression of the glial cell markers, and the inflammatory cytokines. Further, observe the changes in the above indicators in the WT-R28 and E50K-R28 groups after treatment with MCC950. Next, we compared the expression of neurotrophic factors and senescence indicators, NLRP3-related indicators, glial cell markers, and inflammatory factors between young and old WT and OPTN (E50K) mice. We examined the visual function of mice on days 1, 4 and 7. Furthermore, we observed the retinal morphology and the expression of neurotrophic factors and senescence indicators, NLRP3-related indicators, glial cell markers, and inflammatory factors between all groups were measured. We found that OPTN (E50K) mutations lead to NLRP3 inflammasome activation. The OPTN (E50K) mutant groups showed an inflammatory cascade, including glial cell activation and release of proinflammatory factors, leading to retinal structural and functional impairment in mice.MCC950 effectively inhibited the activation of the NLRP3 inflammasome and alleviated the retinal inflammatory cascade caused by the OPTN (E50K) mutation, ultimately improving visual function and retinal damage in mice. OPTN (E50K) mutation induces the activation of the NLRP3 inflammasome, which leads to a retinal inflammatory cascade. MCC950 can inhibit the activation of the NLRP3 inflammasome and retinal inflammatory cascade, improving visual function in OPTN (E50K) mutation mice.

Hepatitis B small surface protein hijacking Bip is initial and essential to promote lipid synthesis

To date, the molecular pathogenic mechanisms between HBsAg and liver metabolic disorders have not been fully understood. To explore the overall effects of HBsAg on liver tissues from HBV transgenic mice, proteome, interactome, and signal pathway analysis were employed to uncover the underlying mechanisms. Bioinformatics analysis of 191 differentially expressed proteins suggested that HBV upregulated the expression of multiple enzymes involved in lipid synthesis, and small HBs (SHBs) caused lipid accumulation in cells. Further studies showed that SHBs bound to binding immunoglobulin protein (Bip), which normally functions in cell homeostasis against the unfolded protein response (UPR) signaling via occupying inositol-requiring enzyme 1 (IRE1). Hijacking Bip by SHBs alleviated the inhibition of post-endoplasmic reticulum (ER) signaling and sequential activation of the IRE1 downstream transcription factors involved in lipid synthesis, such as spliced X-box binding protein 1 (sXBP1) and sterol regulatory element-binding protein 1 (SREBP1), leading to lipid metabolism disorder. The restoration of Bip can alleviate ER stress, and block the sequential post-ER signaling caused by SHBs. This study revealed a new pathway through which SHBs promote lipid disorder, and suggests that Bip may serve as a novel target for intervention in HBV related liver diseases. SignificanceIn this study, we found a new pathway promoting the lipid disorder by SHBs through quantitative proteomics studies, and Bip may serve as a novel target for intervention in HBV related liver diseases. These findings highlight a novel role of SHBs in regulating cell lipid metabolism and provide an insight into the relationship between HBV infection and liver fatty disorders, which may serve as a potential therapeutic target for intervention of HBV related liver diseases.

Pseudokinase STK40 promotes TH1 and TH17 cell differentiation by targeting FOXO transcription factors.

Inappropriate CD4+ T helper (TH) cell differentiation leads to progression of inflammatory and autoimmune diseases, yet the regulatory mechanisms governing stability and activity of transcription factors controlling TH cell differentiation remain elusive. Here, we describe how pseudokinase serine threonine kinase 40 (STK40) facilitates TH1/TH17 differentiation under pathological conditions. STK40 in T cells is dispensable for immune homeostasis in resting mice. However, mice with T cell-specific deletion of STK40 exhibit attenuated symptoms of experimental autoimmune encephalomyelitis and colitis, accompanied by diminished TH1 and TH17 cell differentiation. Mechanistically, STK40 facilitates K48-linked polyubiquitination and proteasomal degradation of FOXO1/4 through promoting their interaction with E3 ligase COP1. Inhibition of FOXO4 or FOXO1, respectively, restores differentiation potential of STK40-deficient TH1/TH17 cells. Together, our data suggest a crucial role of STK40 in TH1 and TH17 cell differentiation, thereby enabling better understanding of the molecular regulatory network of CD4+ T cell differentiation and providing effective targets for the treatment of autoimmune diseases.

Phospho-regulation of ASCL1-mediated chromatin opening during cellular reprogramming.

The proneural transcription factor ASCL1 regulates neurogenesis and drives somatic cell reprogramming into neurons. However, not all cell types can be reprogrammed by ASCL1, raising the questions of what provides competence and how we can overcome barriers to enable directed differentiation. Here, we investigate how levels of ASCL1 and its phosphorylation modulate its activity over progressive lineage restriction of embryonic stem cells. We find that inhibition of ASCL1 phosphorylation enhances reprogramming of both mesodermal and neuroectodermal cells, while pluripotent cells remain refractory to ASCL1-directed neuronal differentiation. By performing RNA-seq and ATAC-seq in neuroectoderm, we find that un(der)phosphorylated ASCL1 causes increased chromatin accessibility at sites proximal to neuronal genes, accompanied by their increased expression. Combined analysis of protein stability and proneural function of phosphomutant and phosphomimetic ASCL1 reveals that protein stability plays only a marginal role in regulating activity, while changes in amino acid charge cannot fully explain enhanced activity of the serine-proline mutant variants of ASCL1. Our work provides new insights into proneural factor activity and regulation and suggests ways to optimize reprogramming protocols in cancer and regenerative medicine.