Principal Target Research Articles

Yi-Qi-Jian-Pi-Xiao-Yu formula inhibits cisplatin-induced acute kidney injury through suppressing ferroptosis via STING-NCOA4-mediated ferritinophagy

BackgroundThe kidneys are the primary excretory organs for platinum drugs, making them susceptible to damage from these drugs. Cisplatin-induced acute kidney injury (CIAKI) is the most common side effect observed in patients undergoing clinical cisplatin treatment. A traditional Chinese medicinal preparation, the Yi-Qi-Jian-Pi-Xiao-Yu formula (YQJPXY), which is a modified formulation of the classical Chinese medicine formula Buyang Huanwu Decoction, has long been used in the treatment of clinical kidney diseases. It is expected to be used to ameliorate cisplatin-induced acute kidney injury. However, the mechanism of this YQJPXY for the treatment of cisplatin-induced acute kidney injury remains unclear. PurposeThe objective of this study is to examine the impact of the YQJPXY on the inhibition of ferroptosis in cisplatin-induced acute kidney injury and to elucidate the underlying mechanisms. MethodsThe active components of YQJPXY were analysed using UPLC-MS/MS. A comprehensive investigation was conducted to elucidate the effects and regulatory mechanisms of YQJPXY on CIAKI and ferroptosis in mice subjected to acute cisplatin treatment and in mice receiving cisplatin treatment after STING expression was inhibited using the STING inhibitor C176. The renoprotective effect of YQJPXY on cisplatin-treated mice was evaluated by measuring tissue damage, inflammation and pro-fibrosis. In addition, we employed network pharmacology and molecular docking methodologies to analyse the principal regulatory targets of YQJPXY. Furthermore, the expression of key proteins and markers of ferroptosis and iron metabolism, as well as the levels of key indicators related to STING-associated ferritinophagy, were examined by immunoblotting, immunohistochemistry, immunoprecipitation, quantitative real-time PCR (qPCR) and specific probes. ResultsThe results demonstrated that YQJPXY reduced the levels of indicators of injury, inflammation and pro-fibrosis in CIAKI mice, with renoprotective effects. Network pharmacological analyses revealed that ferroptosis might be the main biological process regulated by YQJPXY. Furthermore, molecular docking results indicated that STING might be a potential regulatory target of YQJPXY. Furthermore, YQJPXY treatment resulted in a significant reduction in MDA and 4-HNE levels, as well as the inhibition of ferroptosis and improvement in iron metabolic processes. Concomitantly, YQJPXY exhibited a robust protective effect on ferroptosis and iron metabolism homeostasis, as evidenced by its inhibitory action on ferritinophagy. Validation experiments utilising the cisplatin inhibitor C176 demonstrated that YQJPXY inhibits cisplatin-induced ferroptosis in kidney via STING-mediated ferritinophagy. ConclusionThese suggest that YQJPXY alleviates cisplatin-induced acute kidney injury through suppressing ferroptosis via STING-NCOA4-mediated Ferritinophagy.

Quantitative chemical proteomics reveals that phenethyl isothiocyanate covalently targets BID to promote apoptosis

Naturally occurring isothiocyanates (ITCs) found in cruciferous vegetables, such as benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), and sulforaphane (SFN), have attracted significant research interest for their promising anti-cancer activity in vitro and in vivo. While the induction of apoptosis is recognized to play a key role in the anti-cancer effects of ITCs, the specific protein targets and associated upstream events underlying ITC-induced apoptosis remain unknown. In this study, we present a set of chemical probes that are derived from BITC, PEITC, and SFN and equipped with bioorthogonal alkynyl handles to systematically profile the target proteins of ITCs in live cancer cells. Using a competition-based quantitative chemical proteomics approach, we identify a range of candidate target proteins of ITCs enriched in biological processes such as apoptosis. We show that BID, an apoptosis regulator of the Bcl-2 family, is covalently modified by ITCs on its N-terminal cysteines. Functional characterization demonstrates that covalent binding to N-terminal cysteines of BID by PEITC results in conformational changes of the protein and disruption of the self-inhibitory interaction between N- and C-terminal regions of BID, thus unleashing the highly active C-terminal segment to exert downstream pro-apoptotic effects. Consistently, PEITC promotes the cleavage and mitochondrial translocation of BID, leading to a strong induction of apoptosis. We further show that mutation of N-terminal cysteines impairs the N- and C-terminal interaction of BID, relieving the self-inhibition and enhancing its apoptotic activity. Overall, our chemical proteomics profiling and functional studies not only reveal BID as the principal target of PEITC in mediating upstream events for the induction of apoptosis, but also uncover a novel molecular mechanism involving N-terminal cysteines within the first helix of BID in regulating its pro-apoptotic potential.

The N-Terminal Region of the Transcription Factor E2F1 Contains a Novel Transactivation Domain and Recruits General Transcription Factor GTF2H2.

The transcription factor E2F1 is the principal target of the tumor suppressor pRB. E2F1 promotes cell proliferation by activating growth-promoting genes upon growth stimulation. In contrast, E2F1 contributes to tumor suppression by activating tumor suppressor genes, such as ARF, upon loss of pRB function, a major oncogenic change. The transactivation domain of E2F1 has previously been mapped to the C-terminal region. We show here that the N-terminal region of E2F1 is critical for the activation of tumor suppressor genes. Deletion of the N-terminal region dramatically compromised E2F1 activation of tumor suppressor genes. The N-terminal region showed transactivation ability when fused to the DNA-binding domain of GAL4. A search for novel interacting factors with the N-terminal region, using a yeast two-hybrid system, identified the general transcription factor GTF2H2. Overexpression of GTF2H2 enhanced E2F1 activation of tumor suppressor genes and induction of cell death. Conversely, the knockdown of GTF2H2 compromised both. E2F1 binding enhanced the binding of GTF2H2 to target promoters depending on the integrity of the N-terminal region. Taken together, these results suggest that the N-terminal region of E2F1 contains a novel transactivation domain that mediates the activation of tumor suppressor genes, at least in part, by recruiting GTF2H2.

Identifying AWaRe indicators for appropriate antibiotic use: a narrative review.

Quality indicators (QIs) are widely used tools for antibiotic stewardship programmes. The Access, Watch, Reserve (AWaRe) system has been developed by the WHO to classify antibiotics based on their spectrum of activity and potential selection of antibiotic resistance. This review aimed to identify existing indicators for optimal antibiotic use to inform the development of future AWaRe QIs. A literature search was performed in PubMed. We included articles describing QIs for hospital and primary healthcare antibiotic use. We extracted information about (i) the type of infection; (ii) setting; (iii) target for quality assessment; and (iv) methodology used for the development. We then identified the indicators that reflected the guidance provided in the AWaRe system. A total of 773 indicators for antibiotic use were identified. The management of health services and/or workers, the consumption of antibiotics, and antibiotic prescribing/dispensing were the principal targets for quality assessment. There was a similar distribution of indicators across primary and secondary care. For infection-specific indicators, about 50% focused on respiratory tract infections. Only a few QIs included information on review treatment or microbiological investigations. Although only 8 (1%) indicators directly cited the AWaRe system in the wording of the indicators, 445 (57.6%) indicators reflected the guidance provided in the AWaRe book. A high number of indicators for appropriate antibiotic use have been developed. However, few are currently based directly on the WHO AWaRe system. There is a clear need to develop globally applicable AWaRe based indicators that can be integrated into antibiotic stewardship programmes.

Deciphering the Role of SVP-Like Genes and Their Key Regulation Networks During Reproductive Cone Development in Pinus tabuliformis.

Reproductive development plays an essential role in the perpetuation of genetic material and environmental adaptation. In angiosperms, the Short Vegetative Phase (SVP) serves as a flowering repressor, influencing the development of floral organs. In this study, heterologous transformation of Arabidopsis thaliana with SVP-like genes (PtSVL1 and PtSVL2) derived from Pinus tabuliformis significantly impacted stamen formation and pollen fertility, without altering flowering time. Gene co-expression networks revealed that SVP-like and SOC1-like genes function as key coregulatory transcription factors during the initial stages of cone development in P. tabuliformis. Interestingly, the regulatory module of SOC1 regulated by SVP in angiosperms is absent in conifers and conifer SVP-like exercises its function in a form that is physically bound to SOC1-like. Furthermore, combining the yeast one-hybrid scanning with co-expression network analysis, revealed that SPLs and TPSs were the principal downstream target genes of PtSVL1. Notably, the PtSPL16 promoter is positively regulated by PtSVL1, and overexpression of PtSPL16 results in delayed flowering in Arabidopsis, suggesting that the PtSVL1-PtSPL16 module plays a crucial role in regulating reproductive development in conifers. Collectively, these findings enhance our understanding of the roles of SVP-like genes in conifers and the key regulatory networks centred on PtSVL1 during reproductive cone development.

Combining systems pharmacology, metabolomics, and transcriptomics to reveal the mechanism of Salvia miltiorrhiza-Cortex moutan herb pair for the treatment of ischemic stroke.

Ischemic stroke (IS), predominantly triggered by blockages in cerebral blood flow, is increasingly recognized as a critical public health issue. The combination of Salvia miltiorrhiza (SM) and Cortex moutan (CM), traditional herbs in Eastern medicine, are frequently used for managing heart and brain vascular conditions. However, the exact mechanisms by which this herb pair (SC) combats IS remain largely unexplored. This investigation focuses on pinpointing the active constituents in SC that contribute to its protective role and deciphering the mechanisms countering cerebral ischemia, particularly in a middle cerebral artery occlusion (MCAO) rat model. We employed UPLC-Q-TOF-MS/MS alongside network pharmacology for predicting SC's target actions against IS. Key ingredients were examined for their interaction with principal targets using molecular docking. The therapeutic impact was gauged through H&E, TUNEL, and Nissl staining, complemented by transcriptomic and metabolomic integration for mechanistic insights, with vital genes confirmed via western blot. UPLC-Q-TOF-MS/MS analysis revealed that the main components of SC included benzoylpaeoniflorin, salvianolic acid B, oxypaeoniflora, salvianolic acid A, and others. Network pharmacology analysis indicated that SC's mechanism in treating IS primarily involves inflammation, angiogenesis, and cell apoptosis-related pathways, potentially through targets such as AKT1, TNF, PTGS2, MMP9, PIK3CA, and VEGFA. Molecular docking underscored strong affinities between these constituents and their targets. Our empirical studies indicated SC's significant role in enhancing neuroprotection in IS, with transcriptomics suggesting the involvement of the VEGFA/PI3K/AKT pathway and metabolomics revealing improvements in various metabolic processes, including amino acids, glycerophospholipids, sphingomyelin, and fatty acids metabolisms.

Role of nucleobase-specific interactions in the binding and bending of DNA by human male sex determination factor SRY

Y-chromosome-encoded master transcription factor SRY functions in the embryogenesis of therian mammals to initiate male development. Through interactions of its conserved high mobility-group (HMG) box within a widened DNA minor groove, SRY and related Sox factors induce sharp bends at specific DNA target sites. Here, we present the crystal structure of the SRY HMG domain bound to a DNA site containing consensus element 5’-ATTGTT. The structure contains three complexes in the asymmetric unit; in each complex, SRY forms 10 hydrogen bonds with minor-groove base atoms in 5’-CATTGT/ACAATG-3’, shifting the recognition sequence by one base pair (italics). These nucleobase interactions involve conserved residues Arg7, Asn10, and Tyr74 on one side of intercalated Ile13 (the cantilever side chain), and Arg20, Asn32 and Ser36 on the other. Unlike the less-bent NMR structure, DNA bend angles of the distinct box-DNA complexes range from 69-84°, similar to those observed in homologous Sox domain-DNA structures. Electrophoretic studies indicate that respective substitutions of Asn32, Ser36 or Tyr74 by Ala exhibit slightly attenuated specific DNA-binding affinity and bend angles (70-73°) relative to WT (79°). By contrast, respective substitutions of Arg7, Asn10 or Arg20 by Ala markedly impaired DNA-binding affinity in association with much smaller DNA bend angles (53-65°). In a rodent cell-based model of the embryonic gonadal ridge, full-length SRY variants bearing these respective, Ala substitutions exhibited significantly decreased transcriptional activation of SRY’s principal target gene (Sox9). Together, our findings suggest that nucleobase-specific hydrogen bonds by SRY are critical for specific DNA binding, bending, and transcriptional activation.

Unraveling the molecular pathogenesis of Type 2 Diabetes and its impact on female infertility: A bioinformatics and systems biology approach

Type 2 diabetes mellitus (T2D) has been linked with female infertility (FI). Nevertheless, our understanding of the molecular hallmarks and underlying mechanisms remains elusive. This research article aimed to find the hub genes, pathways, transcription factors, and miRNA involved. For this study, softwares like cytoscape, string, Enrichr, FFL loop, etc., were utilized. This research article employed differentially expressed genes (DEGs) to identify multiple biological targets to understand the association between T2D and female infertility (FI). Between T2D and FI, we found 3869 differentially expressed genes. We have also analyzed different pathways like thyroid hormone signaling pathways, AGE-RAGE signaling pathways in diabetic complications and ubiquitin-mediated proteolysis through pathway analysis. Moreover, hub genes MED17, PRKCG, THRA, FOXO1, NCOA2, PLCG2, COL1A1, CXCL8, PRPF19, ANAPC5, UBE2I, XIAP and KEAP1 have been identified. Additionally, these hub genes were subjected to identify the miRNA-mRNA regulation network specific to T2D-associated female infertility. In the FFL study (Feed Forward Loop), transcription factor (SP1, NFKB1, RELA and FOX01), miRNA (has-mir-7-5p, has-let-7a-5p, hsa-mir-16-5p, hsa-mir-155-5p, has-mir-122-5p, has-let-7b-5p, has-mir-124-3p, has-mir-34a-5p, has-mir-130a-3p, has-let-7i-5p, and hsa-mir-27a-3p) and six genes (XIAP, THRA, NCOA2, MED17, FOXO1, and COL1A1) among the thirteen key genes were recognized as regulator and inhibitor. Our analysis reveals that these genes can serve as a significant biomarker for female infertility linked with Type 2 Diabetes, through the prioritization of candidate genes. This study gives us insight into the molecular and cellular mechanism of T2D-associated FI. This finding helps in developing novel therapeutic approaches and will improve efficacy and reduce side effects of the treatment. This research requires further experimental investigation of the principal targets.

Optical Detection of Underwater Propeller Wake Based on a Position-Sensitive Detector

The study of underwater vehicle wake detection is of significant importance within the field of target detection, localisation, and tracking of underwater vehicles. Given that propellers are the propellers of modern ships and underwater vehicles, the propeller wake field represents the principal target source for wake detection in underwater vehicles. The objective of this paper is to propose a method for measuring the wake of an underwater propeller based on a position-sensitive detector. A theoretical model of the relationship between the laser spot displacement and the change in the refractive index of the wake field is established on the basis of the principle of laser beam deflection. A prototype experimental setup for underwater propeller wake measurement was constructed based on the aforementioned optical measurement method. Furthermore, the simulation of the propeller wake flow field with strong density stratification and linear density stratification was conducted based on the experimental setup. Furthermore, experiments were conducted to detect the flow field of a propeller wake. The experimental results indicate that the wake dissipation times of the propeller in a strong density-stratified water environment are approximately 800 s and 750 s. Following the stabilisation of the wake field density, the laser spot position is observed to be stable at 0.341 mm and 0.441 mm, respectively, with a corresponding refractive index change of 2.99 × 10−6 RIU (refractive index unit) and 3.87 × 10−6 RIU, respectively. These experimental results are found to be in general agreement with the simulation results of the propeller wake field. A comparison of the experimental wake measurements based on the device with the wake measurements based on a CTD (conductivity–temperature–depth) device reveals a consistent trend. The realisation of this detection technique is of great significance for the advancement of research in the field of optical detection of underwater vehicle wake streams.

Exploration of Berberine Against Ulcerative Colitis via TLR4/NF-κB/HIF-1α Pathway by Bioinformatics and Experimental Validation.

This study aimed to delineate the molecular processes underlying the therapeutic effects of berberine on UC by employing network pharmacology tactics, molecular docking, and dynamic simulations supported by empirical validations both in vivo and in vitro. We systematically screened potential targets and relevant pathways affected by berberine for UC treatment from comprehensive databases, including GeneCards, DisGeNET, and GEO. Molecular docking and simulation protocols were used to assess the interaction stability between berberine and its principal targets. The predictions were validated using both a DSS-induced UC mouse model and a lipopolysaccharide (LPS)-stimulated NCM460 cellular inflammation model. Network pharmacology analysis revealed the regulatory effect of the TLR4/NF-κB/HIF-1α pathway in the ameliorative action of berberine in UC. Docking and simulation studies predicted the high-affinity interactions of berberine with pivotal targets: TLR4, NF-κB, HIF-1α, and the HIF inhibitor KC7F2. Moreover, in vivo analyses demonstrated that berberine attenuates clinical severity, as reflected by decreased disease activity index (DAI) scores, reduced weight loss, and mitigated intestinal inflammation in DSS-challenged mice. These outcomes include suppression of the proinflammatory cytokines IL-6 and TNF-α and downregulation of TLR4/NF-κB/HIF-1α mRNA and protein levels. Correspondingly, in vitro findings indicate that berberine decreases cellular inflammatory injury and suppresses TLR4/NF-κB/HIF-1α signaling, with notable effectiveness similar to that of the HIF-1α inhibitor KC7F2. Through network pharmacology analysis and experimental substantiation, this study confirmed that berberine enhances UC treatment outcomes by inhibiting the TLR4/NF-κB/HIF-1α axis, thereby mitigating inflammatory reactions and improving colonic pathology.

An efficient plasmid-based system for the recovery of recombinant vesicular stomatitis virus encoding foreign glycoproteins

Viral glycoproteins mediate entry into host cells, thereby dictating host range and pathogenesis. In addition, they constitute the principal target of neutralizing antibody responses, making them important antigens in vaccine development. Recombinant vesicular stomatitis virus (VSV) encoding foreign glycoproteins can provide a convenient and safe surrogate system to interrogate the function, evolution, and antigenicity of viral glycoproteins from viruses that are difficult to manipulate or those requiring high biosafety level containment. However, the production of recombinant VSV can be technically challenging. In this work, we present an efficient and robust plasmid-based system for the production of recombinant VSV encoding foreign glycoproteins. We validate the system using glycoproteins from different viral families, including arenaviruses, coronaviruses, and hantaviruses, as well as highlight their utility for studying the effects of mutations on viral fitness. Overall, the methods described herein can facilitate the study of both native and recombinant VSV encoding foreign glycoproteins and can serve as the basis for the production of VSV-based vaccines.

The Triumph of HUMINT: The GDR Foreign Intelligence Services’ Collection of Defense Intelligence, 1951–1989

During the Cold War, the German Democratic Republic (GDR)’s foreign intelligence agencies collected much defense intelligence from human sources in West Germany. By the mid-1960s, the two services had created agent networks in their principal targets in West Germany’s government, armed forces, industry and universities. For the next 25 years, these agent networks supplied a wealth of varied and valuable military intelligence and scientific and technological intelligence. At their heart was a small number of outstanding human sources. The GDR’s intelligence agencies significantly strengthened the Soviet Bloc’s intelligence collection. The intelligence they obtained was more valuable to the Warsaw Pact than the GDR’s armed forces and would have been of great benefit to the Pact if war had broken out. Their success ran counter to the trend of military intelligence collection at that time, which was to rely increasingly heavily on technical collection.

Transcriptomics-based Exploration of the Mechanism of Kaempferol in the Treatment of Osteoporosis in Ovariectomized Rats

Objective: We explore the pharmacodynamic targets of kaempferol in treating osteoporosis using transcriptomics and animal experiment. Methods: Firstly, we constructed an ovariectomized rat model (OVX). The anti-osteoporosis effect of kaempferol was evaluated by bone mineral density (BMD) and hematoxylin-eosin staining comprehensively. Moreover, differential genes between groups were screened by RNA sequencing technology (RNA-seq) for transcriptomics and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways enrichment analysis were performed. Finally, partial results of transcriptomics were verified to detect the expression of the relevant gene expression by immunohistochemistry. Results: The pharmacodynamic findings indicated that the administration of kaempferol resulted in an elevation in BMD ( P < .01) and a notable enhancement in tibia microstructural indices in the experimental rats ( P < .01). The transcriptomic analysis revealed that NTN1, LTBP4, GSN, and EBF1 were identified as the principal targets for therapeutic intervention in osteoporosis. The results of animal experiments showed that kaempferol promoted osteogenesis and inhibited bone resorption by downregulating the protein expression of NTN1, LTBP4, GSN, and EBF1 ( P < .01). Conclusion: Kaempferol enhances BMD levels, retards tibial bone loss with structural deterioration in OVX model rats, and promotes bone formation while curbing bone resorption through NTN1, LTBP4, GSN, and EBF1 protein down-regulation.

Physiological role for S-nitrosylation of RyR1 in skeletal muscle function and development

Excitation-contraction coupling in skeletal muscle myofibers depends upon Ca2+ release from the sarcoplasmic reticulum through the ryanodine receptor/Ca2+-release channel RyR1. The RyR1 contains ∼100 Cys thiols of which ∼30 comprise an allosteric network subject to posttranslational modification by S-nitrosylation, S-palmitoylation and S-oxidation. However, the role and function of these modifications is not understood. Although aberrant S-nitrosylation of multiple unidentified sites has been associated with dystrophic diseases, malignant hyperthermia and other myopathic syndromes, S-nitrosylation in physiological situations is reportedly specific to a single (1 of ∼100) Cys in RyR1, Cys3636 in a manner gated by pO2. Using mice expressing a form of RyR1 with a Cys3636→Ala point mutation to prevent S-nitrosylation at this site, we showed that Cys3636 was the principal target of endogenous S-nitrosylation during normal muscle function. The absence of Cys3636 S-nitrosylation suppressed stimulus-evoked Ca2+ release at physiological pO2 (at least in part by altering the regulation of RyR1 by Ca2+/calmodulin), eliminated pO2 coupling, and diminished skeletal myocyte contractility in vitro and measures of muscle strength in vivo. Furthermore, we found that abrogation of Cys3636 S-nitrosylation resulted in a developmental defect reflected in diminished myofiber diameter, altered fiber subtypes, and altered expression of genes implicated in muscle development and atrophy. Thus, our findings establish a physiological role for pO2-coupled S-nitrosylation of RyR1 in skeletal muscle contractility and development and provide foundation for future studies of RyR1 modifications in physiology and disease.

Desensitizing the autonomic nervous system to mitigate anti-GD2 monoclonal antibody side effects.

Anti-GD2 monoclonal antibodies (mAbs) have shown to improve the overall survival of patients with high-risk neuroblastoma (HR-NB). Serious adverse events (AEs), including pain, within hours of antibody infusion, have limited the development of these therapies. In this study, we provide evidence of Autonomic Nervous System (ANS) activation as the mechanism to explain the main side effects of anti-GD2 mAbs. Through confocal microscopy and computational super-resolution microscopy experiments we explored GD2 expression in postnatal nerves of infants. In patients we assessed the ANS using the Sympathetic Skin Response (SSR) test. To exploit tachyphylaxis, a novel infusion protocol (the Step-Up) was mathematically modelled and tested. Through confocal microscopy, GD2 expression is clearly visible in the perineurium surrounding the nuclei of nerve cells. By computational super-resolution microscopy experiments we showed the selective expression of GD2 on the cell membranes of human Schwann cells in peripheral nerves (PNs) significantly lower than on NB. In patients, changes in the SSR were observed 4 minutes into the anti-GD2 mAb naxitamab infusion. SSR latency quickly shortened followed by gradual decrease in the amplitude before disappearance. SSR response did not recover for 24 hours consistent with tachyphylaxis and absence of side effects in the clinic. The Step-Up protocol dissociated on-target off-tumor side effects while maintaining serum drug exposure. We provide first evidence of the ANS as the principal non-tumor target of anti-GD2 mAbs in humans. We describe the development and modeling of the Step-Up protocol exploiting the tachyphylaxis phenomenon we demonstrate in patients using the SSR test.

Proof of stability of an RSV Controlled Human Infection Model challenge agent

In 2018, SGS Belgium NV developed RSV-NICA (Respiratory Syncytial Virus-Nasobronchial Infective Challenge Agent), an RSV type A challenge agent for use in RSV Controlled Human Infection Model (CHIM) studies.It is widely recognized that the stability of RSV can be influenced by a variety of environmental parameters, such as temperature and pH. Consequently, our objective was to evaluate the stability of the viral titer of RSV-NICA following five years of controlled storage and to determine the uniformity of the viral titers across different vials of a GMP-qualified batch of RSV-NICA. In addition, we examined the capacity of RSV-NICA to infect human primary airway epithelial cells (MucilAir™), the principal target cells of RSV, and evaluated the influence of single and recurrent freeze–thaw cycles on the infectious viral titer of the challenge agent.The aliquoted RSV-NICA virus stock was subjected to standard virological and molecular methods to gather data on the titer and consistency of the viral titer contained within 24 representative vials of the stock. Our findings illustrate that over a span of five years of cryo-storage, the infectious viral titer in 75% of the tested vials exhibited a comparable average infectious viral titer (4.75 ± 0.06 vs 4.99 ± 0.11; p-value = 0.14). A considerable reduction down to an undetectable level of infectious virus was observed in the remaining vials. RSV-NICA demonstrated its capacity to effectively infect differentiated human airway epithelial cells, with active virus replication detected in these cells through increasing RSV genome copy number over time. Virus tropism for ciliated cells was suggested by the inhibition of cilia beating coupled with an increase in viral RNA titers. No discernable impact on membrane barrier function of the epithelial lung tissues nor cytotoxicity was detected. Pooling of vials with infectious titers > 4.0 log10 TCID50/ml and freeze-thawing of these combined vials showed no deterioration of the infectious titer. Furthermore, pooling and re-aliquoting of vials spanning the entire range of viral titers (including vials with undetectable infectious virus) along with subjecting the vials to three repeated freeze–thaw cycles did not result in a decrease of the infectious titers in the tested vials.Taken together, our findings indicate that long-term cryo-storage of vials containing RSV-NICA challenge agent may influence the infectious viral titer of the virus, leading to a decrease in the homogeneity of this titer throughout the challenge stock. However, our study also demonstrates that when heterogeneity of the infectious titer of an RSV stock is observed, rounds of pooling, re-aliquoting and subsequent re-titration serve as an effective method not only to restore the homogeneity of the infectious titer of an RSV-A stock, but also to optimize patient-safety, scientific and operational aspects of viral inoculation of study participants during at least the period of one RSV CHIM trial. RSV-NICA is a stable, suitable CHIM challenge agent that can be utilized in efficacy trials for RSV vaccines and antiviral entities.

Therapeutic mechanism of Cornus Officinalis Fruit Coreon on ALI by AKT/Nrf2 pathway and gut microbiota

BackgroundAcute liver injury (ALI) often precipitates severe liver function impairment and is associated with high mortality rates. Traditional Chinese Medicine (TCM) has demonstrated efficacy in mitigating hepatic damage by exhibiting anti-inflammatory effects, enhancing antioxidant activity, and modulating gut microbiota (GM). Numerous studies have identified similar or identical bioactive compounds within the Cornus Officinalis Fruit Coreon(COFO) and its flesh. Notably, Cornus Officinalis has been shown to possess potent hepatoprotective properties. However, studies on the pharmacological effects and mechanism of action of COFO for hepatoprotection have received little attention. PurposeTo elucidate the mechanisms underlying the COFO effect in ALI by integrating GM gene sequencing, quantifying Short-Chain Fatty Acids (SCFAs), and examining relevant signaling pathways. Materials and MethodsA rat model for carbon tetrachloride (CCl4)-induced ALI was established, and the best liver protective components of COFO were selected by pathological observation and biochemical determination. The therapeutic efficacy of COFO in mitigating liver injury was elucidated through an integrated approach that included network pharmacology, biochemical indexes, 16S rDNA sequencing analyses, short-chain fatty acids, Western blotting analysis of protein levels, and immunohistochemical evaluations. ResultsPharmacological evaluation established that the n-butanol fraction (CNBP) provided optimal hepatoprotective effects. Firstly, the chemical constituents of CNBP were characterized, and its principal anti-ALI targets, such as ALI, AKT1, TNF, and IL-6, were identified through network pharmacology analysis. Secondly, experimental validation revealed that CNBP may enhance the genetic diversity of the GM, augmenting the diversity of the microbial community, increasing the levels of three SCFAs, and activating key proteins in the AKT/Nrf2 signaling pathway (AKT1, TNF-α, IL-6, NF-κB p65, Nrf2, and HO-1). Consequently, CNBP exhibited hepatoprotective effects, with antioxidative and anti-inflammatory properties. ConclusionCNBP may mitigate GM-induced disturbances, augment the levels of three SCFAs, activate the AKT/Nrf2 signaling pathway, and exhibit antioxidant and anti-inflammatory effects, thereby conferring hepatoprotective benefits.

PLAG1 interacts with GPX4 to conquer vulnerability to sorafenib induced ferroptosis through a PVT1/miR-195-5p axis-dependent manner in hepatocellular carcinoma

BackgroundSorafenib is a standard first-line treatment for advanced hepatocellular carcinoma (HCC), yet its effectiveness is often constrained. Emerging studies reveal that sorafenib triggers ferroptosis, an iron-dependent regulated cell death (RCD) mechanism characterized by lipid peroxidation. Our findings isolate the principal target responsible for ferroptosis in HCC cells and outline an approach to potentially augment sorafenib's therapeutic impact on HCC.MethodsWe investigated the gene expression alterations following sgRNA-mediated knockdown induced by erastin and sorafenib in HCC cells using CRISPR screening-based bioinformatics analysis. Gene set enrichment analysis (GSEA) and the "GDCRNATools" package facilitated the correlation studies. We employed tissue microarrays and cDNA microarrays for validation. Ubiquitination assay, Chromatin immunoprecipitation (ChIP) assay, RNA immunoprecipitation (RIP) assay, and dual-luciferase reporter assay were utilized to delineate the specific mechanisms underlying ferroptosis in HCC cells.ResultsOur study has revealed that pleiomorphic adenoma gene 1 (PLAG1), a gene implicated in pleomorphic adenoma, confers resistance to ferroptosis in HCC cells treated with sorafenib. Sorafenib leads to the opposite trend of protein and mRNA levels of PLAG1, which is not caused by affecting the stability or ubiquitination of PLAG1 protein, but by the regulation of PLAG1 at the transcriptional level by its upstream competitive endogenous long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1). Data from 139 HCC patients showed a significant positive correlation between PLAG1 and GPX4 levels in tumor samples, and PLAG1 is instrumental in redox homeostasis by driving the expression of glutathione peroxidase 4 (GPX4), the enzyme that reduces lipid peroxides (LPOs), which further leads to ferroptosis inhibition.ConclusionsFerroptosis is a promising target for cancer therapy, especially for patients resistant to standard chemotherapy or immunotherapy. Our findings indicate that PLAG1 holds therapeutic promise and may enhance the efficacy of sorafenib in treating HCC.

Effects of social environments on male primate HPG and HPA axis developmental programming.

Developmental plasticity is particularly important for humans and other primates because of our extended period of growth and maturation, during which our phenotypes adaptively respond to environmental cues. The hypothalamus-pituitary-gonadal (HPG) and hypothalamus-pituitary-adrenal (HPA) axes are likely to be principal targets of developmental "programming" given their roles in coordinating fitness-relevant aspects of the phenotype, including sexual development, adult reproductive and social strategies, and internal responses to the external environment. In social animals, including humans, the social environment is believed to be an important source of cues to which these axes may adaptively respond. The effects of early social environments on the HPA axis have been widely studied in humans, and to some extent, in other primates, but there are still major gaps in knowledge specifically relating to males. There has also been relatively little research examining the role that social environments play in developmental programming of the HPG axis or the HPA/HPG interface, and what does exist disproportionately focuses on females. These topics are likely understudied in males in part due to the difficulty of identifying developmental milestones in males relative to females and the general quiescence of the HPG axis prior to maturation. However, there are clear indicators that early life social environments matter for both sexes. In this review, we examine what is known about the impact of social environments on HPG and HPA axis programming during male development in humans and nonhuman primates, including the role that epigenetic mechanisms may play in this programming. We conclude by highlighting important next steps in this research area.

Neuroinflammation generated by HIV-infected microglia promotes dysfunction and death of neurons in human brain organoids.

Despite the success of combination antiretroviral therapy (ART) for individuals living with HIV, mild forms of HIV-associated neurocognitive disorder (HAND) continue to occur. Brain microglia form the principal target for HIV infection in the brain. It remains unknown how infection of these cells leads to neuroinflammation, neuronal dysfunction, and/or death observed in HAND. Utilizing two different inducible pluripotent stem cell-derived brain organoid models (cerebral and choroid plexus [ChP] organoids) containing microglia, we investigated the pathogenic changes associated with HIV infection. Infection of microglia was associated with a sharp increase in CCL2 and CXCL10 chemokine gene expression and the activation of many type I interferon stimulated genes (MX1, ISG15, ISG20, IFI27, IFITM3 and others). Production of the proinflammatory chemokines persisted at low levels after treatment of the cell cultures with ART, consistent with the persistence of mild HAND following clinical introduction of ART. Expression of multiple members of the S100 family of inflammatory genes sharply increased following HIV infection of microglia measured by single-cell RNA-seq. However, S100 gene expression was not limited to microglia but was also detected more broadly in uninfected stromal cells, mature and immature ChP cells, neural progenitor cells and importantly in bystander neurons suggesting propagation of the inflammatory response to bystander cells. Neurotransmitter transporter expression declined in uninfected neurons, accompanied by increased expression of genes promoting cellular senescence and cell death. Together, these studies underscore how an inflammatory response generated in HIV-infected microglia is propagated to multiple uninfected bystander cells ultimately resulting in the dysfunction and death of bystander neurons.