Viable Target Research Articles

Kdm2a inhibition in skeletal muscle improves metabolic flexibility in obesity.

Skeletal muscle is a critical organ in maintaining homoeostasis against metabolic stress, and histone post-translational modifications are pivotal in those processes. However, the intricate nature of histone methylation in skeletal muscle and its impact on metabolic homoeostasis have yet to be elucidated. Here, we report that mitochondria-rich slow-twitch myofibers are characterized by significantly higher levels of H3K36me2 along with repressed expression of Kdm2a, an enzyme that specifically catalyses H3K36me2 demethylation. Deletion or inhibition of Kdm2a shifts fuel use from glucose under cold challenge to lipids under obese conditions by increasing the proportion of mitochondria-rich slow-twitch myofibers. This protects mice against cold insults and high-fat-diet-induced obesity and insulin resistance. Mechanistically, Kdm2a deficiency leads to a marked increase in H3K36me2 levels, which then promotes the recruitment of Mrg15 to the Esrrg locus to process its precursor messenger RNA splicing, thereby reshaping skeletal muscle metabolic profiles to induce slow-twitch myofiber transition. Collectively, our data support the role of Kdm2a as a viable target against metabolic stress.

Genetic and peripheral biomarkers of comorbid posttraumatic stress disorder and traumatic brain injury: a systematic review

BackgroundPosttraumatic stress disorder (PTSD) commonly cooccurs with traumatic brain injury (TBI) in military populations and is a significant predictor of poor long-term outcomes; however, it is unclear to what extent specific biological variables are associated with comorbidity. This PROSPERO-registered systematic review evaluates the current body of literature on genetic and peripheral biomarkers associated with comorbid TBI and PTSD.MethodsSearches were conducted in four databases (PubMed, PsycInfo, PTSDPubs, Scopus). We included published studies examining differences in peripheral biomarkers among civilian, military, and veteran participants with both TBI and PTSD compared to those with TBI alone as well as, in some cases, PTSD alone and healthy controls. Data were extracted from included studies and evidence quality was assessed.ResultsOur final analysis included 16 studies, the majority of which were based on data from active duty military and veteran participants. The results suggest that multiple gene variants are likely to contribute to the cumulative risk of PTSD comorbid with TBI. An elevated circulating level of the pro-inflammatory cytokine IL-6 was the most consistently replicated blood-based indicator of comorbid illness, compared to mTBI alone.ConclusionSeveral genetic and protein markers of cellular injury and inflammation appear to be promising indicators of chronic pathology in comorbid TBI and PTSD. Additional research is needed to determine how such factors indicate, predict, and contribute to comorbidity and to what extent they represent viable targets for the development of novel diagnostic tools and therapeutic interventions.

Impact and mechanism of the TBX-22 gene mutation G874A on the epithelial-mesenchymal transition in medial edge epithelial cells.

Cleft lip and palate (CL/P) are prevalent congenital anomalies with complex genetic causes. The G874A mutation of T-box transcription factor 22 (TBX-22) gene is notably associated with CL/P, while the underlying mechanism remains to be clarified. Studies have shown that the restriction of epithelial-mesenchymal transformation (EMT) process in medial edge epithelial cells (MEEs) is crucial for CL/P development. In our current research, primary MEEs, isolated and cultured from mouse embryos, were genetically introduced the TBX-22 G874A mutation and subsequently treated with TGF-β1. They were then utilized to test the hypothesis that the G874A mutation in TBX22 plays a role in the regulation of the EMT in MEEs. Our findings reveal that TBX22 reduces miR140-5p transcription by binding to its promoter, while miR140-5p downregulates TGFBR1 protein expression by targeting its mRNA 3'-UTR. In other words, TBX22 could indirectly positively regulates TGFBR1 expression post-transcriptionally. Functional cellular assays showed that the G874A mutation of TBX-22 counteracted TGF-β1-induced decrease in proliferation and migration. Western blotting results showed that the G874A mutation of TBX-22 inhibited EMT protein expression (α-SMA and Vimentin) and promoted E-cadherin in TGF-β1-induced MEEs. To summarize, our research reveals that the G874A mutation of TBX22 impedes the progression of EMT in MEEs through the upregulation of miR140-5p and the downregulation of TGFBR1. This highlights TGFBR1 as a viable target for the prevention of CL/P.

In silico development of a multi-epitope-based vaccine against Burkholderia cepacia complex using reverse vaccinology

BackgroundMultidrug-resistant Burkholderia cenocepacia and Burkholderia multivorans have emerged as significant pathogens, particularly in patients with cystic fibrosis (CF) and chronic granulomatous disease (CGD).ObjectiveGiven the absence of approved vaccines, this study aimed to identify potential vaccine candidates against these pathogens.MethodsThe complete genomes of B. cenocepacia and B. multivorans were retrieved from the GenBank. Surface-exposed proteins that were antigenic, non-allergenic, and non-homologous to human proteins were selected for further analysis. The conserved domains of the selected proteins were analyzed, and their presence was examined across 68 genomes. Subsequently, linear and conformational B-cell epitopes and human MHC II binding sites were identified. Highly conserved and immunogenic B-cell epitopes from outer membrane proteins (OMPs) were incorporated into a multi-epitope vaccine (MEV). Molecular docking analysis was performed to assess the interaction of the selected proteins. Finally, molecular dynamics (MD) simulations were conducted using GROMACS 2019 to evaluate the feasibility and dynamics of the interactions between the chimeric MEV and Toll-like receptor complexes, TLR2 and TLR4.ResultsOf 16,723 proteins identified in B. multivorans and B. cenocepacia strains, nine proteins (six OMPs and three extracellular) were selected as ideal candidates based on established criteria. These proteins had a molecular weight of 110 kDa and were present in ≥ 75% of the dataset of B. multivorans and B. cenocepacia genomes. In addition, molecular docking and MD indicated stable and feasible interactions between MEV and TLRs. The MEV-TLR4 system demonstrates the greatest stability and tightly bound interaction, with minimal fluctuations and high structural integrity. In contrast, the MEV-only system exhibits significant flexibility and dynamic behavior as a free ligand, while the MEV-TLR2 system balances stability and flexibility, showing a dynamic but stable interaction.ConclusionNine potential immunogenic proteins were identified as viable targets for vaccine development. An optimized MEV was explicitly designed for B. multivorans and B. cenocepacia. The novel MEV platform exhibited high binding affinity to immune receptors and favorable molecular docking characteristics. Although these findings are encouraging, additional in vitro and in vivo testing is necessary to validate the vaccine’s effects.

Evaluation of the transmission-blocking potential of Plasmodium vivax antigen Pvg37 using transgenic rodent parasites and clinical isolates

BackgroundPlasmodium vivax is a major cause of malaria, particularly outside Africa, necessitating effective strategies for public health management. Transmission-blocking vaccines (TBVs) have shown the potential to inhibit malaria transmission by targeting antigens expressed in sexual-stage parasites. Pbg37, a conserved protein expressed in sexual stages from gametocyte to ookinete in the rodent parasite P. berghei, is a viable target for TBV development.Methods and findingsIn this study, we constructed a transgenic strain, TrPvg37Pb, expressing Pvg37 using the P. berghei ΔPbg37 strain. Initial findings demonstrated that the replacement of Pbg37 with the exogenous Pvg37 did not impact parasite growth or development. Notably, Pvg37 was expressed during the gametocyte to ookinete development and was associated with the plasmic membrane, similar to Pbg37. To evaluate the potential of Pvg37 as a TBV candidate, we synthesized two Pvg37 polypeptides and immunized rabbits to generate antibodies. In vitro experiments demonstrated that anti-Pvg37-P2 antibodies significantly inhibited the formation of male gametes and ookinetes in the transgenic TrPvg37Pb parasite. Additionally, in mosquito feeding assays, mosquitos feeding on TrPvg37Pb-infected mice passively transferred with anti-Pvg37-P2 antibodies showed a significant 80.2% decrease in oocyst density compared to the control group. Furthermore, in direct membrane feeding experiments using four clinical P. vivax isolates, the anti-Pvg37 antibodies significantly reduced oocyst density by 28.6–50.4%.ConclusionPvg37 is a promising candidate for P. vivax TBV development, deserving further research and optimization to enhance its immunogenicity and transmission-blocking activity.

Exploring the potential active components and mechanisms of Tetrastigma hemsleyanum against ulcerative colitis based on network pharmacology in LPS-induced RAW264.7 cells.

Ulcerative colitis (UC) is a chronic form of inflammatory bowel disease, which current treatments often show limited effectiveness. Ferroptosis, a newly recognized form of programmed cell death has been implicated in UC pathogenesis, suggesting that it may be viable therapeutic target. Tetrastigma hemsleyanum (TH) has shown potential anti-UC effects, though it is unclear whether its therapeutic benefits are mediated by ferroptosis. This study investigated the involvement of ferroptosis in the therapeutic effects of TH and identified key active components and pathways of TH against UC. The ethyl acetate extract of TH (TH_E) was found to be the most effective anti-inflammatory extract compared with the petroleum ether extract (TH_P), n-butanol extract (TH_N), and water-soluble extract (TH_W). TH_E's components were identified using UHPLC-MS/MS, ADME parameters, and network pharmacology. Additionally, TH_E's effects on ferroptosis were evaluated in an LPS-induced RAW264.7 cell model. TH_E exhibited the strongest anti-inflammatory activity among four extracts. 10 compounds (Linolenic acid; Apigenin; Protocatechualdehyde; Asiatic acid; Quercetin; Isorhamnetin; Kaempferol; Azelaic acid; Oleic Acid; Palmitic acid) were selected from SwissADME database. Then a total of 281 targets for these 10 compounds and 1,330 UC-related targets were identified from different database. Isorhamnetin was selected as the most promising anti-inflammatory component among 10 components. Furthermore, enrichment analysis revealed that ferroptosis was involved in UC development, with both TH_E and isorhamnetin exhibited inhibition of ferroptosis. Finally, isorhamnetin's anti-ferroptosis effects were linked to the Keap1/Nrf2/HO-1 pathway. The results demonstrate that TH_E and isorhamnetin alleviate LPS-induced UC through restraining ferroptosis. Moreover, isorhamnetin's anti-UC properties are mediated by inhibiting ferroptosis via activation of the Keap1/Nrf2/HO-1 axis.

PCDH17 induces colorectal cancer metastasis by destroying the vascular endothelial barrier

Compromised vascular integrity facilitates the cancer cells extravasation and metastasis. However, the mechanisms leading to a disruption in vascular integrity in colorectal cancer (CRC) remain unclear. In this study, PCDH17 expression was higher in the vascular endothelial cells of colon cancer with distant metastasis, and the rates of PCDH17+ endothelial cells (ECs) was associated with the M stage in clinical pathological characteristics analysis and correlated with a poor survival prognosis. The liver and lung metastatic dissemination of MC-38 was significantly decreased in PCDH17–/–mice. The ubiquitination and degradation of VEGFR2 was prevented by the interaction between PCDH17 and the E3 ubiquitin ligase MARCH5, which causing the separation of internalized VE-cadherin, and increased the vascular permeability and metastasis of CRC. These results highlight the importance of PCDH17 in maintaining vascular integrity, which has emphasis for endothelial barrier function in metastatic cancer. PCDH17 has the potential to be a marker for predicting tumor metastasis as well as a viable treatment target for CRC.

Modulation of Intestinal Inflammation and Protection of Dopaminergic Neurons in Parkinson’s Disease Mice through a Probiotic Formulation Targeting NLRP3 Inflammasome

Emerging evidence highlights the significance of peripheral inflammation in the pathogenesis of Parkinson’s disease (PD) and suggests the gut as a viable therapeutic target. This study aimed to explore the neuroprotective effects of the probiotic formulation VSL#3® and its underlying mechanism in a PD mouse model induced by MPTP. Following MPTP administration, the striatal levels of dopamine and its metabolites, as along with the survival rate of dopaminergic neurons in the substantia nigra, were significantly reduced in PD mice. MPTP also significantly increased the mRNA expression of pro-inflammatory cytokines TNF-α and IL-1β, while reducing anti-inflammation mediators, like glia cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) in the striatum. These pathological changes were notably mitigated by VSL#3® treatment, suggesting its neuroprotective and anti-inflammatory effects in the brain. Additionally, VSL#3® significantly lowered the circulating levels of pro-inflammatory cytokines, and reduced TNF-α and IL-1β mRNA expression in the liver, indicating an inhibition of cytokine transfer. In the intestine, the probiotic treatment markedly decreased the mRNA expression of pro-inflammatory cytokines, (TNF-α, IL-1β, IL-6 and IL-17), and the other two key components of the NLRP3 inflammasome, caspase-1 and NLRP3, demonstrating an inhibition of VSL#3® on gut NLRP3 inflammasome. VSL#3® exerts neuroprotective effects in PD mice through the suppression of intestinal inflammation, particularly inhibiting the intestinal NLRP3 inflammasome. This study supports the therapeutic potential of targeting intestinal inflammation and utilizing probiotics in PD treatment.

Molecular mechanism of osteoclast differentiation of PBMC in patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune condition that causes severe joint deformities and impaired functionality, affecting the well-being and daily life of individuals. Consequently, there is a pressing demand for identifying viable therapeutic targets for treating RA. This study aimed to explore the molecular mechanisms of osteoclast differentiation in PBMC from patients with RA through transcriptome sequencing and bioinformatics analysis. Blood samples were collected from 20 patients with RA, including 15 females and 5 males. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. Osteoclast differentiation was induced using a medium containing RANKL and M-CSF for 14 days, with medium changes every 2 days. After 14 days, osteoclasts were identified by TRAP staining, and multinucleated TRAP-positive cells were counted as osteoclasts. Subsequently, transcriptome sequencing was performed using the Illumina Novaseq 6000 platform, and differential expression analysis was conducted using the DESeq2 package in R. Differentially expressed genes were selected with a significance threshold of p < 0.05 and a fold change ≥ 2 (|Log2FC|≥ 1). Bioinformatics analysis was performed using R, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. TRAP staining showed successful induction of PBMCs into osteoclasts. Transcriptome sequencing revealed a significant number of differentially expressed genes (DEGs) in the induced groups compared with the control group. GO analysis showed that these DEGs were predominantly associated with biological processes related to the transmission of chemokine signals, reactions to living organisms, and bolstering neutrophil-driven defense mechanisms. KEGG analysis showed that these DEGs were enriched by primary signaling pathways, including interactions between cytokines and their receptors, chemokine signaling pathway, cell cycle regulation, neutrophil extracellular trap formation, and TNF signaling pathway. Osteoclast differentiation of PBMC from patients with RA involves various gene alterations, multiple biological processes, and signaling pathways, providing insight into the potential mechanism of PBMC osteoclast differentiation in RA. Key Points • A total of 1841 DEGs were obtained between the induced group and the normal group. • These DEGs were involved in multiple biological processes and signaling pathways.

Staphylococcus aureus Proteins Implicated in the Reduced Virulence of sarA and sarA/agr Mutants in Osteomyelitis.

Using a murine osteomyelitis model, we recently demonstrated that Staphylococcus aureus sarA and sarA/agr mutants generated in the USA300 strain LAC are attenuated to a greater extent than an isogenic agr mutant and that this can be attributed to a significant extent to the increased production of extracellular proteases in both mutants. Based on this, we used a mass-based proteomics approach to compare the proteomes of LAC, its isogenic agr, sarA, and sarA/agr mutants, and isogenic derivatives of all four of these strains unable to produce the extracellular proteases aureolysin, SspA, SspB, ScpA, or SplA-F. This allowed us to identify proteins that were present in reduced amounts in sarA, and sarA/agr mutants owing to the increased production of extracellular proteases. A total of 1039 proteins were detected in conditioned media (CM) from overnight cultures of LAC, and protease-mediated degradation was shown to contribute to the reduced abundance of 224 of these (21.6%) in CM from the sarA and sarA/agr mutants. Among these were specific proteins previously implicated in the pathogenesis and therapeutic recalcitrance of S. aureus osteomyelitis. This demonstrates that the ability of sarA to limit protease production plays a key role in post-translational remodeling of the S. aureus proteome to a degree that can be correlated with reduced virulence in our osteomyelitis model, and that it does so irrespective of the functional status of agr. This also suggests that at least some of these 224 proteins may be viable targets for prophylactic or therapeutic intervention.

Systematical identification of regulatory GPCRs by single-cell trajectory inference reveals the role of ADGRD1 and GPR39 in adipogenesis.

Adipogenesis is the healthy expansion of white adipose tissue (WAT), serving as a compensatory response to maintain metabolic homeostasis in the presence of excess energy in the body. Therefore, the identification of novel regulatory molecules in adipogenesis, specifically membrane receptors such as G protein-coupled receptors (GPCRs), holds significant clinical promise. These receptors can serve as viable targets for pharmaceuticals, offering potential for restoring metabolic homeostasis in individuals with obesity. We utilized trajectory inference methods to analyze three distinct single-nucleus sequencing (sNuc-seq) datasets of adipose tissue and systematically identified GPCRs with the potential to regulate adipogenesis. Through verification in primary adipose progenitor cells (APCs) of mice, we discovered that ADGRD1 promoted the differentiation of APCs, while GPR39 inhibits this process. In the obese mouse model induced by a high-fat diet (HFD), both gain-of-function and loss-of-function studies validated that ADGRD1 promoted adipogenesis, thereby improving metabolic homeostasis, while GPR39 inhibited adipogenesis, leading to metabolic dysfunction. Additionally, through the analysis of 2,400 ChIP-seq data and 1,204 bulk RNA-seq data, we found that the transcription factors (TFs) MEF2D and TCF12 regulated the expression of ADGRD1 and GPR39, respectively. Our study revealed the regulatory role of GPCRs in adipogenesis, providing novel targets for clinical intervention of metabolic dysfunction in obese patients.

Methylglyoxal-induced glycation stress promotes aortic stiffening: Putative mechanistic roles of oxidative stress and cellular senescence.

Here, we assessed the role of the advanced glycation end-product (AGE) precursor methylglyoxal (MGO) and its non-crosslinking AGE MGO-derived hydroimidazolone (MGH)-1 in aortic stiffening and explored the potential of a glycation stress-lowering compound (Gly-Low) to mitigate these effects. Young (3-6 month) C57BL/6 mice were supplemented with MGO (in water) and Gly-Low (in chow). Aortic stiffness was assessed in vivo via pulse wave velocity (PWV) and ex vivo through elastic modulus. Putative mechanisms underlying MGO- and MGH-1-induced aortic stiffening were explored using complementary experimental approaches in aortic tissue and cultured human aortic endothelial cells (HAECs). Moreover, aortic stiffness was assessed in old (24 month) mice after consumption of Gly-Low-enriched chow. MGO-induced glycation stress increased PWV in young mice by 21% (P<0.05 vs. control), which was prevented with Gly-Low (P=0.93 vs. control). Ex vivo, MGO increased aortic elastic modulus 2-fold (P<0.05), superoxide production by ∼40% (P<0.05), and MGH-1 expression by 50% (P<0.05), which were all mitigated by Gly-Low. Chronic MGO exposure elevated biomarkers of cellular senescence in HAECs, comparable to a known senescence inducer Doxorubicin, an effect partially blocked by Gly-Low. Moreover, elevated aortic elastic modulus induced by Doxorubicin (P<0.05 vs. control) was prevented with Gly-Low (P=0.71 vs. control). Aortic RNA sequencing implicated preservation of endogenous cellular detoxification pathways with Gly-Low following exposure to MGH-1. Old mice supplemented with Gly-Low had lower PWV (P<0.05) relative to old control mice. MGO-induced glycation stress contributes to aortic stiffening and glycation stress lowering compounds hold promise for mitigating these effects. This study provides the first comprehensive line of evidence that methylglyoxal (MGO)-induced glycation stress directly contributes to aortic stiffening and does so through mechanisms involving oxidative stress and cellular senescence. Using complementary in vivo , ex vivo , and in vitro experimental models, we establish that MGO-mediated glycation stress independently induces aortic stiffening. Furthermore, we demonstrate that the glycation-lowering compound, Gly-Low, mitigates MGO-induced aortic stiffening by mitigating excessive oxidative stress and cellular senescence, and can lower aortic stiffness in old mice. Mechanistically, activation of the detoxification enzyme, glyoxalase-1 (Glo-1), is a novel pathway by which Gly-Low mediates its therapeutic effects on aortic stiffening. Lastly, we show that Gly-Low holds promise for lowering aortic stiffness in old age. Aortic stiffening is a major risk factor for cardiovascular diseases (CVD) and a significant predictor of CV-related morbidity and mortality. Yet, the underlying mechanisms driving this process remain incompletely understood. This study identifies MGO-derived glycation stress as a critical and modifiable factor contributing to aortic stiffening through pathways involving excessive oxidative stress and cellular senescence. By establishing the efficacy of Gly-Low in mitigating these effects, our findings underscore the importance of targeting glycation stress in the context of aging, and likely in other settings of glycation stress, to improve arterial health and reduce CVD risk. These findings have significant clinical implications, as they demonstrate that glycation stress is a viable and modifiable therapeutic target for the prevention and treatment of aortic stiffening. Gly-Low offers a promising therapeutic approach to ameliorate glycation stress- and age-related aortic stiffening, by directly targeting excess glycation stress, oxidative stress, and cellular senescence. Additionally, the involvement of the Glo-1 detoxification pathway suggests a specific molecular target for future interventions aimed at improving arterial health and mitigating the progression of CVD.

Sirt6, Deubiquitinated and Stabilised by USP9X, Takes Essential Actions on the Pathogenesis of Experimental Autoimmune Myasthenia Gravis by Regulating CD4+ T Cells.

Myasthenia gravis (MG) presents with symptoms that significantly affect patients' daily lives. Long-term MG therapies may lead to substantial side effects, predominantly due to prolonged immune suppression. Sirt6, which plays a vital role in maintaining cellular homeostasis and is recognised for its involvement in cytokine production in immune cells, has not yet been explored in relation to MG. PBMCs and CD4+ T cells were isolated from blood samples. RT-qPCR, western blot and ELISA were used to assess the expression of target genes and proteins. Flow cytometry was used to identify the subsets of T helper cells. Co-IP was conducted to investigate the interaction between USP9X and Sirt6. Finally, the experimental autoimmune myasthenia gravis (EAMG) model was established. In MG patients, Sirt6 levels were downregulated compared to healthy controls. Sirt6 overexpression led to a reduction in Th1 and Th17 cell populations while augmenting Treg cells in PBMCs. USP9X interacted with Sirt6, leading to its deubiquitination and stabilisation. Elevated Sirt6 levels subsequently mitigated symptoms in the EAMG model. The stabilisation of Sirt6, mediated by USP9X, has been found to relieve symptoms of EAMG by influencing the subtypes of T helper cells. This highlights the promising potential of Sirt6 as a viable therapeutic target in the treatment of MG.

Prediction of Pathologic Response in Unresectable Hepatocellular Carcinoma After Downstaging with Locoregional and Systemic Combination Therapy.

The combination of locoregional and systemic therapy may achieve remarkable tumor response for unresectable hepatocellular carcinoma (HCC). We aimed to investigate the correlation between radiologic and pathologic responses following combination therapy, evaluate their prognostic values, and to establish a non-invasive prediction system for pathologic response. This single-center retrospective study included 112 consecutive patients with HCC who underwent locoregional and systemic combination therapy followed by liver resection or transplantation. Radiologic response was assessed with Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 and modified RECIST (mRECIST). Pathologic necrosis percentage was assessed to determine major pathologic response (MPR, ≥90% tumor necrosis) and pathologic complete response (100% tumor necrosis). Performance of the response criteria in predicting pathologic response was assessed with the area under the receiver operator characteristic curve (AUC). Among all radiologic and pathologic response criteria, MPR was the only independent predictor of post-resection recurrence-free survival (RFS) (adjusted hazard ratio 0.34, 95% CI 0.16-0.72, p=0.004). In addition, mRECIST showed stronger correlation with pathologic response than RECIST 1.1 (spearman r values: 0.76 vs 0.42, p<0.001). A prediction system for MPR was developed that included a combination of mRECIST response (ie, >70% decrease of viable target lesions) with either >90% decrease in AFP (for AFP-positive group, n=75) or >80% decrease in PIVKA-II (for AFP-negative group, n=37), which yielded a respective AUC of 0.905 and 0.887. Furthermore, the system-defined dual-positive responders showed improved median RFS (not reached) than non-responders (7.1 months for AFP-positive group [p=0.043] and 13.3 months for AFP-negative group [p=0.099]). mRECIST was more indicative of pathologic response after combination therapy than RECIST 1.1. Integration of mRECIST with AFP or PIVKA-II responses allowed for accurate prediction of MPR and could support decision-making on subsequent curative-intent treatment.

Enhancement of anti-sarcoma immunity by NK cells engineered with mRNA for expression of a EphA2-targeted CAR.

Paediatric sarcomas, including rhabdomyosarcoma, Ewing sarcoma and osteosarcoma, represent a group of malignancies that significantly contribute to cancer-related morbidity and mortality in children and young adults. These cancers share common challenges, including high rates of metastasis, recurrence or treatment resistance, leading to a 5-year survival rate of approximately 20% for patients with advanced disease stages. Despite the critical need, therapeutic advancements have been limited over the past three decades. The advent of chimeric antigen receptor (CAR)-based immunotherapies offers a promising avenue for novel treatments. However, CAR-T cells have faced significant challenges and limited success in treating solid tumours due to issues such as poor tumour infiltration, immunosuppressive tumour microenvironments and off-target effects. In contrast, the adaptation of CAR technology for natural killer (NK) cells has demonstrated potential in both haematological and solid tumours, suggesting a new therapeutic strategy for paediatric sarcomas. This study developed and validated a novel CAR-NK cell therapy targeting the ephrin type-A receptor-2 (EphA2) antigen, which is highly expressed in various paediatric sarcomas. CAR expression was successfully detected on the surface of NK cells post-electroporation, indicating successful transfection. Significantly, EphA2-specific CAR-NK cells demonstrated enhanced cytotoxic activity against several paediatric sarcoma cell lines in vitro, including those of rhabdomyosarcoma, Ewing sarcoma and osteosarcoma, compared to unmodified NK cells. Transient messenger RNA (mRNA) transfection of NK cells is a safe approach in genetic engineering, with further chemical modifications to mRNA enhancing stability of temporal EphA2-CAR expression in NK cells, thereby promoting prolonged protein expression. Additionally, in vivo EphA2-CAR-NK cells showed promising anti-cancer activity in rhabdomyosarcoma and osteosarcoma mouse models. The study provides a foundational basis for the clinical evaluation of EphA2-targeted CAR-NK cell therapy across a spectrum of paediatric sarcomas. The enhanced anti-tumour effects observed in vitro/vivo suggests potential for improved therapeutic outcomes in hard-to-cure paediatric sarcomas. Addressing unmet clinical needs in paediatric Sarcomas. Paediatric sarcomas, including rhabdomyosarcoma, Ewing sarcoma, and osteosarcoma, exhibit poor survival rates in advanced disease stages. The lack of significant therapeutic progress over the past three decades necessitates innovative treatment approaches. Advancing immunotherapy with CAR-NK cells. Natural killer (NK) cells modified with chimeric antigen receptors (CARs) represent a promising strategy to overcome the limitations of CAR-T cells, particularly in solid tumours. CAR-NK cells are associated with enhanced tumour targeting, reduced off-target effects, and improved safety profiles. EphA2 as a therapeutic target. EphA2, a receptor overexpressed in multiple paediatric sarcomas, is identified as a viable target for CAR-based immunotherapy due to its critical role in tumour progression and angiogenesis. Innovations in mRNA-based engineering. This study demonstrates the feasibility of transient mRNA transfection to engineer NK cells for CAR expression, offering a non-integrative and safer alternative to viral transduction. Enhancements in mRNA stability through chemical modifications, can further optimise protein expression. Preclinical efficacy of EphA2-CAR NK cells. EphA2-specific CAR-NK cells exhibit superior cytotoxicity against sarcoma cell lines in vitro and demonstrate significant anti-tumour activity in in vivo mouse models of rhabdomyosarcoma and osteosarcoma. Clinical translation potential. The findings establish a strong preclinical rationale for the clinical evaluation of EphA2-targeted CAR-NK therapy as a novel immunotherapeutic option for paediatric sarcomas. Future research directions: Combining EphA2-CAR NK cells with immune checkpoint inhibitors or other immunomodulatory agents could further enhance therapeutic outcomes and durability. Advanced preclinical models mimicking human tumour microenvironments are needed to refine and optimise this therapeutic approach.

Acidic pH of Early Endosomes Governs SARS-CoV-2 Transport in Host Cells.

Endocytosis is a prominent mechanism for SARS-CoV-2 entry into host cells. Upon internalization into early endosomes (EEs), the virus is transported to late endosomes (LEs), where acidic conditions facilitate spike protein processing and viral genome release. Dynein and kinesin motors drive EE transport along microtubules; dynein moves EEs to the perinuclear region, while kinesins direct them towards the plasma membrane, creating a tug-of-war over the direction of transport. Here, we identify that luminal pH of EEs is a key factor regulating the outcome of this tug-of-war. Among the known endosomal pH regulators, only the sodium-proton exchanger NHE9 has so far been genetically linked to severe COVID-19 risk. NHE9 functions as a proton leak pathway specifically on endosomes. We show that limiting acidification of EEs by increasing the expression of NHE9 leads to decreased infectivity of the SARS-CoV-2 spike-bearing virus in host cells. Our investigation identified the EE membrane lipid phosphatidylinositol-3-phosphate (PI3P) as a link between luminal pH changes and EE transport. Normally, as EEs mature, PI3P depletes. However, in cells with high NHE9 expression, PI3P persists longer on EEs. PI3P plays a pivotal role in the recruitment of motor proteins and the subsequent movement of EEs. Consistently, we observed NHE9 mediated alkalization of EEs hindered perinuclear movement. Specifically, EE speed and run-length were negatively impacted, ultimately leading to EEs falling off microtubules and impairing the delivery of viral cargo to LEs. NHE9 thus offers a unique opportunity as a viable therapeutic target to impede SARS-CoV-2 host cell entry.

Impact of Patient Factors on Robotic-Assisted Partial Nephrectomy Operating Room Times: The Case for Fixed Operating Room Time as a Viable Efficiency Target.

Introduction: To analyze the association of patient characteristics on operating room (OR) times for robotic-assisted partial nephrectomies (RAPNs). Methods: In total, 248 consecutive RAPNs were performed by a single surgeon from October 2018 to August 2022. Data were collected on the following patient factors: sex, age, race, weight, body mass index (BMI), diabetes, hypertension (HTN), tumor side, tumor mass, and American Society of Anesthesiologists (ASA) score. Total fixed OR times were evaluated as the sum of in-room time to anesthesia-release time (IRAT), anesthesia-release time to cut time (ARCT), close time to wheels out time (CTWO), and in-room time to cut time (IRAT + ARCT). Total variable OR times were defined as cut time to close time (CTCT). Total operative time was defined as wheels into wheel out (WIWO). Results: Median OR time was 265 minutes (interquartile range [IQR]: 247-298 minutes) for WIWO, 191 minutes (IQR: 170-225 minutes) for CTCT, and 75 minutes (IQR: 68-83 minutes) for total fixed time. There was a significant increase in WIWO with the following patient variables: male sex (18.0 minutes, 95% confidence interval [CI]: 6.3-29.7 minutes), weight (5.6 minutes, 95% CI: 2.8-8.4 minutes), BMI (14.3 minutes, 95% CI: 4.2-24.4 minutes), HTN (15.1 minutes, 95% CI: 3.7-26.5 minutes), pathology tumor size (cm) (6.4 minutes, 95% CI: 2.2-10.6 minutes), and ASA score (13.8 minutes, 95% CI: 3.1-24.4 minutes). Sex, age, BMI, weight, HTN, and pathology tumor size significantly impacted CTCT (P < 0.05). Total fixed OR time was only affected by ASA score (P = 0.02). Conclusions: Patient variables significantly affect total OR time and variable OR time. These variables do not impact total fixed OR times, confirming fixed OR time as a viable OR efficiency point.

Inhibition of Mitochondrial Fission Protein Drp1 Ameliorates Myopathy in the D2-mdx Model of Duchenne Muscular Dystrophy.

Although current treatments for Duchenne Muscular Dystrophy (DMD) have proven to be effective in delaying myopathy, there remains a strong need to identify novel targets to develop additional therapies. Mitochondrial dysfunction is an early pathological feature of DMD. A fine balance of mitochondrial dynamics (fission and fusion) is crucial to maintain mitochondrial function and skeletal muscle health. Excessive activation of Dynamin-Related Protein 1 (Drp1)-mediated mitochondrial fission was reported in animal models of DMD. However, whether Drp1-mediated mitochondrial fission is a viable target for treating myopathy in DMD remains unknown. Here, we treated a D2-mdx model of DMD (9-10 weeks old) with Mdivi-1, a selective Drp1 inhibitor, every other day (i.p. injection) for 5 weeks. We demonstrated that Mdivi-1 effectively improved skeletal muscle strength and reduced serum creatine kinase concentration. Mdivi-1 treatment also effectively inhibited mitochondrial fission regulatory protein markers, Drp1(Ser616) phosphorylation and Fis1 in skeletal muscles from D2-mdx mice, which resulted in reduced content of damaged and fragmented mitochondria. Furthermore, Mdivi-1 treatment attenuated lipid peroxidation product, 4-HNE, in skeletal muscle from D2-mdx mice, which was inversely correlated with muscle grip strength. Finally, we revealed that Mdivi-1 treatment downregulated Alpha 1 Type I Collagen (Col1a1) protein expression, a marker of fibrosis, and Interleukin-6 (IL-6) mRNA expression, a marker of inflammation. In summary, these results demonstrate that inhibition of Drp1-mediated mitochondrial fission by Mdivi-1 is effective in improving muscle strength and alleviating muscle damage in D2-mdx mice. These improvements are associated with improved skeletal muscle mitochondrial integrity, leading to attenuated lipid peroxidation.

CircMETTL9 targets CCAR2 to induce neuronal oxidative stress and apoptosis via mitochondria-mediated pathways following traumatic brain injury.

Traumatic brain injury (TBI) remains a principal factor in neurological disorders, often resulting in significant morbidity due to secondary neuroinflammatory and oxidative stress responses. While circular RNAs are recognized for their high expression levels in the nervous system and play crucial roles in various neurological processes, their specific contributions to the pathophysiology of TBI remain underexplored. In this study, the possible molecular mechanisms through which circMETTL9 modulated oxidative stress and neurological outcomes following TBI were investigated. In vitro model of oxidative stress utilizing SH-SY5Y cells revealed that circMETTL9 knockdown significantly attenuated H₂O₂-induced reactive oxygen species production, reduced apoptosis, and preserved mitochondrial function. Additionally, CCAR2 has been identified as a circMETTL9-binding protein by mass spectrometry and RNA immunoprecipitation, with circMETTL9 positively regulating CCAR2 expression. Meanwhile, on the basis of silencing CCAR2, it was verified that the regulation of oxidative stress in SH- SY5Y cells by circMETTL9 was mediated by CCAR2. In vivo experiments using a TBI rat model further confirmed that CCAR2 knockdown alleviated central nervous system (CNS) injury, reduced oxidative stress and apoptosis, and protected mitochondrial integrity following TBI. These findings suggest a novel mechanism by which circMETTL9 targets CCAR2 via mitochondria-mediated Bax/Bcl-2/caspase-3 signaling to regulate apoptosis. CircMETTL9 may provide a viable therapeutic target for mitigating neurological dysfunction following TBI, offering new insights into potential interventions aimed at reducing secondary brain injury.

Prognostic significance of NUAK1 and its association with immune infiltration in stomach adenocarcinoma

BackgroundStomach adenocarcinoma (STAD) represents a significant global health burden, accounting for a considerable proportion of cancer-related mortalities, and NUAK1, a protein kinase, plays a crucial role in cellular metabolism, cell cycle regulation, migration, and tumor progression. However, its relationship with prognosis and immune infiltration in STAD has not been thoroughly investigated.MethodsRNA sequencing data from the Cancer Genome Atlas (TCGA) and Genotypic Tissue Expression Project (GTEx) databases were employed to assess disparities in NUAK1 expression between STAD tumour and normal tissues. Additionally, we investigated the correlation between NUAK1 expression and patient prognosis, in addition to the level of immune cell infiltration. The potential functions were elucidated through an examination of the Gene Ontology (GO) Encyclopedia, the Kyoto Encyclopedia of Genes and Genomes (KEGG), and an enrichment analysis (GSEA). The GeneMANIA was used to validate the functions of nuak1-related genes.ResultsOur analysis demonstrated that NUAK1 expression in tumour tissues exhibited a notable disparity from that observed in normal tissues, with elevated levels detected in STAD tissues. We used the GeneMANIA database to identify functionally similar genes with significantly higher expression for some genes in the unpaired group samples. An elevated NUAK1 expression level was found to correlate with a poorer overall survival (OS), disease-specific survival (DSS), and progression-free intervals (PFI). Additionally, immune infiltration analysis indicated a significant positive correlation between NUAK1 expression and various tumor-infiltrating immune cells, while a negative correlation was observed with T helper cell 17(Th17) cells. Furthermore, enrichment analysis was conducted to identify relevant biological features and pathways.ConclusionThe expression levels of NUAK1 are significantly increased in STAD, and this heightened expression correlates with diminished OS, DSS, and PFI among affected patients. These observations indicate that NUAK1 has the potential to function as a prognostic biomarker for STAD and may represent a viable therapeutic target for intervention in its management.