Objective To investigate the effect of ATP synthase inhibitory factor 1 (ATPIF1) on the antitumor activity of chimeric antigen receptor (CAR)-NK92 cells. Methods HER2-targeted CAR-NK92 cells with ATPIF1 overexpression or knockdown were constructed. CAR-positive expression rate was detected by flow cytometry. Cell proliferation capacity was measured using CCK-8 assay. Glycolytic capacity was analyzed by Seahorse metabolic analyzer. Mitochondrial membrane potential levels were detected using JC-1 probe. Target cell lysis rate was evaluated by firefly luciferase reporter assay. Expression levels of CD107a, natural-killer group 2 member D (NKG2D), granzyme B (GzmB), perforin, and interleukin 2 (IL-2) were detected via flow cytometry. Quantitative real-time PCR was used to measure the expression of interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), tumor necrosis factor α (TNF-α), ATPIF1, and hexokinase 1 (HK1). The impact of glycolytic inhibition by 2-Deoxy-D-glucose (2-DG) on CAR-NK92 antitumor capacity was examined. Results Successfully generated HER2-targeting control CAR-NK92 cells, as well as ATPIF1-overexpressing and ATPIF1 knockdown CAR-NK92 cells. The ATPIF1-overexpressing CAR-NK92 cells showed significantly enhanced target cell lysis rate, elevated expression levels of NKG2D and CD107a, increased secretion capacities of Granzyme B (GzmB) and IL-2, and upregulated mRNA expression levels of IFIT1 and TNF-α, while ATPIF1-knockdown cells exhibited opposite effects. ATPIF1 overexpression induced metabolic reprogramming in CAR-NK92 cells, manifested by significantly decreased mitochondrial membrane potential (δpsim), markedly upregulated HK1 mRNA expression, and enhanced basal glycolysis and glycolytic capacity. After glycolysis inhibition with 2-DG (5 μmol/L), both ATPIF1-overexpressing and knockdown CAR-NK92 cells showed no significant differences in NKG2D and CD107a expression levels compared to control cells. Conclusion ATPIF1 regulates the antitumor activity of CAR-NK92 cells through modulating glycolytic metabolism. Overexpression of ATPIF1 can enhance the antitumor efficacy of CAR-NK92 cells.
Humans ; Glycolysis ; Killer Cells, Natural/metabolism* ; Receptors, Chimeric Antigen/immunology* ; Granzymes/genetics* ; Hexokinase/metabolism* ; Cell Line, Tumor ; Interleukin-2/genetics* ; Cell Proliferation ; NK Cell Lectin-Like Receptor Subfamily K/genetics* ; Membrane Potential, Mitochondrial

This study investigated the specific immune response of BALB/c mice that was induced by a circular RNA (circRNA) vaccine expressing the herpes simplex virus type II (HSV-2) glycoprotein D (gD). The aim was to evaluate the immunological potential of this vaccine and lay a foundation for developing an mRNA vaccine against HSV-2. PCR and homologous recombination were employed to integrate the gD gene obtained from the pT7AMP-gD ectodomain plasmid into pUC57 to generate the recombinant plasmid pUC57-circ-gD, which was then sequenced and characterized. In vitro transcription and cyclization were performed on the template DNA to generate pUC57-circ-gD mRNA. To validate the formation of circular RNA, we cleaved the pUC57-circ-gD mRNA with RNase R and employed RT-PCR to validate the cyclization. The pUC57-circ-gD mRNA was then transfected into 293T cells. After 72 h, the cell supernatant was collected, and Western blotting was employed to measure the protein level of gD. Subsequently, the mRNA was encapsulated in lipid nanoparticles (LNPs) by microfluidic encapsulation. BALB/c mice were administrated with the encapsulated mRNA, and blood was collected from the fundus venous plexus after 21 and 35 days, and from the enucleated eyeballs after 49 days. Enzyme-linked immunosorbent assay was employed to measure the titers of antibodies, including virus-neutralizing antibodies. After 49 days, spleens were harvested and assessed for secretion of interferon-gamma (IFN-γ) by solid-phase enzyme-linked immunospot. The results showed successful construction and sequencing of the recombinant plasmid. RNase R digestion confirmed the presence of circular RNAs. Western blotting of the 293T cells transfected with the mRNA showed clear specific bands. The quality of the vaccine was tested by size exclusion chromatography-high performance liquid chromatography, which showed that the purity of the vaccine was about 90%. The mRNA-LNP showcased the particle size of 82.76 nm and an encapsulation rate of approximately 98%. Following three-dose vaccination, all immunized mice exhibited steady weight gain with 100% survival rate throughout the 28-day observation period, indicating no significant acute toxicity associated with the vaccine formulation. The immunized mice showed dose-dependent increases in serum IgG antibody titer and IFN-γ secretion by splenocytes and they were resistant to virus attacks. These findings indicate good immunogenicity and persistence of the pUC57-circ-gD mRNA vaccine, providing a reference for further studies on circRNA vaccines.
Animals ; Mice, Inbred BALB C ; RNA, Circular ; Mice ; Humans ; Herpesvirus 2, Human/genetics* ; Viral Envelope Proteins/genetics* ; Antibodies, Viral/blood* ; HEK293 Cells ; Female ; Nanoparticles ; Plasmids

In 2022， Hainan provincial government launched the project for the prevention and control of viral hepatitis with the goals of a hepatitis B screening rate of 90%， a diagnostic rate of 90%， and a treatment rate of 80% among people aged 18 years and above by the year 2025， and the main intervention measures include population-based prevention， case screening， antiviral therapy， and health management. As of December 31， 2024， a total of 6.875 million individuals in the general population had been screened for hepatitis B， with a screening rate of 95.6%. A total of 184 710 individuals with positive HBsAg were identified， among whom 156 772 were diagnosed through serological reexamination， resulting in a diagnostic rate of 84.9%. A total of 50 742 patients with chronic hepatitis B were identified， among whom 42 921 had hepatitis B-specific health records established for health management， with a file establishment rate of 84.6%. A total of 31 553 individuals received antiviral therapy， with a treatment rate of 62.2%. A total of 2.503 million individuals at a high risk of hepatitis C were screened， among whom 4 870 tested positive for HCV antibody and 3 858 underwent HCV RNA testing， resulting in a diagnostic rate of 79.2%， and 1 824 individuals with positive HCV RNA were identified， among whom 1 194 received antiviral therapy， with a treatment rate of 65.5%. In addition， 159 301 individuals with negative HBsAg and anti-HBs and an age of 20 — 40 years were inoculated with hepatitis B vaccine free of charge. Through the implementation of the project for the prevention and control of viral hepatitis， a large number of hepatitis patients have been identified， treated， and managed in the province within a short period of time， which significantly accelerates the efforts to eliminate the crisis of viral hepatitis.

Insulin is an important protein hormone that participates in multiple metabolic pathways.Biosynthetic insulin has been widely used in the treatment of type 1 and type 2 diabetes.Currently,the number of reported cases of insulin overdose both at home and abroad is gradually increasing,and insulin homicide is no longer a means of"committing murder without leaving a trace".At present,there are no systematic protocols for the identification of insulin overdose in the field of forensic medi-cine in China.This article introduces the causes,toxicological characteristics,forensic examination,labo-ratory testing methods and indicator reference of insulin overdose.Based on the identification practice and research results and referring to relevant studies on insulin overdose at home and abroad,this pa-per aims to provide recommendations and references for the formulation of forensic identification guide-lines for insulin overdose cases.

The diagnostic value of Mycobacterium tuberculosis(MTB)complex and rifampicin resistance test kits(fluorescence PCR method)in detecting for MTB complex and rifampicin resistancein sputum samples was evaluated.A total of 271 patients with suspected tuberculosis were prospectively and consecutively enrolled at Hunan Chest Hospital between April 1,2024,and November 30,2024.Of these,229 patients were confirmed to have tuberculosis,whereas 42 patients were not-tuberculosis samples were col-lected from all patients and subjected to fluorescence PCR,Xpert MTB/RIF(abbreviated as Xpert),and MGIT 960 culture and drug sensitivity testing.Clinical diagnosis and MTB culture results served as reference standards for TB diagnosis,whereas phenotypic drug susceptibility testing and Xpert served as reference standards to for assessment of rifampicin resistance.The sensitivity,specificity,positive predictive value,and negative predictive value of the fluorescence PCR method were analyzed.Kappa tests were performed to analyze the concordance between detection techniques.With clinical diagnosis as the gold standard,the sensitivity and specificity of the fluorescence PCR method for the diagnosis of TB were 65.1%(149/229)and 97.6%(41/42),and the consistency test for the fluo-rescence PCR and Xpert methods showed high consistency(Kappa value=0.993).With the MGIT 960 liquid culture as the reference standard,the positive detection rate of the fluorescence PCR method for the detection of patients with positive cultures was 91.9%(102/111,95%CI:85.2%-96.2%),and the positive detection rate for 147 patients with sputum culture-negative TB was 27.9%(41/147,95%CI:21.3%-35.6%).With the phenotypic drug sensitivity test as the gold standard,the sensitivity and specificity of the fluo-rescence PCR method for the detection of rifampicin resistance were 100.0%(31/31)and 96.6%(28/29)respectively,and the consis-tency between tests was high(Kappa value=0.967).With Xpert as the gold standard,the sensitivity and specificity of fluorescence PCR for the detection of rifampicin resistance were 95.8%(46/48)and 99.0%(99/100),respectively,and the consistency between tests was high(Kappa value=0.953).Finally,samples with rifampicin resistance detected with the fluorescence PCR method had a clear rpoB gene mutation type according to one-generation sequencing.In conclusion,the fluorescence PCR method showed high sen-sitivity in detecting MTB complex groups and rifampicin resistance,and had high concordance with Xpert.Therefore,this technique can serve a rapid test for TB diagnosis to increase the rate of positive TB pathology and detection of rifampicin resistance.This method is particularly suitable for use in lower-income countries and economically disadvantaged grassroots communities.

Wound repair and infection have long posed significant challenges in clinical medicine.Am-phibians,adapting to complex environments through long-term natural selection,have evolved skin sys-tems with remarkable abilities to resist external damage and promote rapid wound healing,making them an important source for discovering candidate molecules for wound healing.In this study,a novel peptide composed of 22 amino acids,with the sequence"VGKAGLETAACKATNSCFNIDW"and a molecular weight of 2299.6 D,was screened from the cDNA library of Odorrana tormota and named PN-VW22.The peptide was synthesized by solid-phase synthesis,and its effects on cell proliferation were evaluated using the CCK-8 assay and scratch wound healing assay in human keratinocytes(HaCaT)and mouse em-bryonic fibroblasts(NIH3T3).Antibacterial activity was assessed by determining the minimum inhibitory concentration(MIC).Results showed that PN-VW22 at various concentrations had no significant effect on the cell viability in NIH3T3 cells(P＞0.05),but significantly enhanced HaCaT cell viability at 0.5μmol/L(P＜0.001).Meanwhile,PN-VW22 induced cell proliferation and promoted wound healing in HaCaT cells,with a healing rate of 64.44％after 24 h incubation at 0.5 μmol/L.Furthermore,PN-VW22 exhibited antibacterial activity against Staphylococcus aureus with an MIC value of 13.92 μmol/L.In sum,this study identified PN-VW22 as a novel bifunctional peptide with both wound repair and anti-infective properties,providing a new toxin peptide template for the development of wound healing drugs.

Wound repair and infection have long posed significant challenges in clinical medicine.Am-phibians,adapting to complex environments through long-term natural selection,have evolved skin sys-tems with remarkable abilities to resist external damage and promote rapid wound healing,making them an important source for discovering candidate molecules for wound healing.In this study,a novel peptide composed of 22 amino acids,with the sequence"VGKAGLETAACKATNSCFNIDW"and a molecular weight of 2299.6 D,was screened from the cDNA library of Odorrana tormota and named PN-VW22.The peptide was synthesized by solid-phase synthesis,and its effects on cell proliferation were evaluated using the CCK-8 assay and scratch wound healing assay in human keratinocytes(HaCaT)and mouse em-bryonic fibroblasts(NIH3T3).Antibacterial activity was assessed by determining the minimum inhibitory concentration(MIC).Results showed that PN-VW22 at various concentrations had no significant effect on the cell viability in NIH3T3 cells(P＞0.05),but significantly enhanced HaCaT cell viability at 0.5μmol/L(P＜0.001).Meanwhile,PN-VW22 induced cell proliferation and promoted wound healing in HaCaT cells,with a healing rate of 64.44％after 24 h incubation at 0.5 μmol/L.Furthermore,PN-VW22 exhibited antibacterial activity against Staphylococcus aureus with an MIC value of 13.92 μmol/L.In sum,this study identified PN-VW22 as a novel bifunctional peptide with both wound repair and anti-infective properties,providing a new toxin peptide template for the development of wound healing drugs.

The diagnostic value of Mycobacterium tuberculosis(MTB)complex and rifampicin resistance test kits(fluorescence PCR method)in detecting for MTB complex and rifampicin resistancein sputum samples was evaluated.A total of 271 patients with suspected tuberculosis were prospectively and consecutively enrolled at Hunan Chest Hospital between April 1,2024,and November 30,2024.Of these,229 patients were confirmed to have tuberculosis,whereas 42 patients were not-tuberculosis samples were col-lected from all patients and subjected to fluorescence PCR,Xpert MTB/RIF(abbreviated as Xpert),and MGIT 960 culture and drug sensitivity testing.Clinical diagnosis and MTB culture results served as reference standards for TB diagnosis,whereas phenotypic drug susceptibility testing and Xpert served as reference standards to for assessment of rifampicin resistance.The sensitivity,specificity,positive predictive value,and negative predictive value of the fluorescence PCR method were analyzed.Kappa tests were performed to analyze the concordance between detection techniques.With clinical diagnosis as the gold standard,the sensitivity and specificity of the fluorescence PCR method for the diagnosis of TB were 65.1%(149/229)and 97.6%(41/42),and the consistency test for the fluo-rescence PCR and Xpert methods showed high consistency(Kappa value=0.993).With the MGIT 960 liquid culture as the reference standard,the positive detection rate of the fluorescence PCR method for the detection of patients with positive cultures was 91.9%(102/111,95%CI:85.2%-96.2%),and the positive detection rate for 147 patients with sputum culture-negative TB was 27.9%(41/147,95%CI:21.3%-35.6%).With the phenotypic drug sensitivity test as the gold standard,the sensitivity and specificity of the fluo-rescence PCR method for the detection of rifampicin resistance were 100.0%(31/31)and 96.6%(28/29)respectively,and the consis-tency between tests was high(Kappa value=0.967).With Xpert as the gold standard,the sensitivity and specificity of fluorescence PCR for the detection of rifampicin resistance were 95.8%(46/48)and 99.0%(99/100),respectively,and the consistency between tests was high(Kappa value=0.953).Finally,samples with rifampicin resistance detected with the fluorescence PCR method had a clear rpoB gene mutation type according to one-generation sequencing.In conclusion,the fluorescence PCR method showed high sen-sitivity in detecting MTB complex groups and rifampicin resistance,and had high concordance with Xpert.Therefore,this technique can serve a rapid test for TB diagnosis to increase the rate of positive TB pathology and detection of rifampicin resistance.This method is particularly suitable for use in lower-income countries and economically disadvantaged grassroots communities.

Mitophagy, a highly precise form of autophagy, plays a pivotal role in maintaining cellular homeostasis by selectively targeting and eliminating damaged mitochondria through a process known as mitophagy. Within this tightly regulated mechanism, dysfunctional mitochondria are specifically delivered to lysosomes for degradation. Disruptions in mitophagy have been implicated in a diverse range of pathological conditions, spanning diseases of the nervous system, cardiovascular system, cancer, aging, and metabolic syndrome. The elucidation of mitophagy’s impact on cardiovascular disorders, liver diseases, metabolic syndromes, immune dysfunctions, inflammatory conditions, and cancer has significantly advanced our understanding of the complex pathogenesis underlying these conditions. These studies have shed light on the intricate connections between dysfunctional mitophagy and disease progression. Among the disorders associated with mitochondrial dysfunction, insulin resistance (IR) stands out as a prominent condition linked to metabolic disorders. IR is characterized by a diminished response to normal levels of insulin, necessitating higher insulin levels to trigger a typical physiological reaction. Hyperinsulinemia and metabolic disturbances often coexist with IR, primarily due to defects in insulin signal transduction. Oxidative stress, stemming from mitochondrial dysfunction, exerts dual effects in the context of IR. Initially, it disrupts insulin signaling pathways and subtly contributes to the development of IR. Additionally, by inducing mitochondrial damage and autophagy, oxidative stress indirectly impedes insulin signaling pathways. Consequently, mitophagy acts as a protective mechanism, encapsulating damaged or dysfunctional mitochondria through the autophagy-lysosome pathway. This efficient process eliminates excessive oxidative stress reactive. The intricate interplay between mitochondrial function, oxidative stress, mitophagy, and IR represents a captivating field of investigation in the realm of metabolic disorders. By unraveling the underlying complexities and comprehending the intricate relationships between these intertwined processes, researchers strive toward uncovering novel therapeutic strategies. With a particular focus on mitochondrial quality control and the maintenance of redox homeostasis, these interventions hold tremendous potential in mitigating IR and enhancing overall metabolic health. Emerging evidence from a myriad of studies has shed light on the active involvement of mitophagy in the pathogenesis of metabolic disorders. Notably, interventions such as exercise, drug therapies, and natural products have been documented to induce mitophagy, thereby exerting beneficial effects on metabolic health through the activation of diverse signaling pathways. Several pivotal signaling molecules, including AMPK, PINK1/Parkin, BNIP3/Nix, and FUNDC1, have been identified as key regulators of mitophagy and have been implicated in the favorable outcomes observed in metabolic disorders. Of particular interest is the unique role of PINK1/Parkin in mitophagy compared to other proteins involved in this process. PINK1/Parkin exerts influence on mitophagy through the ubiquitination of outer mitochondrial membrane proteins. Conversely, BNIP3/Nix and FUNDC1 modulate mitophagy through their interaction with LC3, while also displaying certain interrelationships with each other. In this comprehensive review, our objective is to investigate the intricate interplay between mitophagy and IR, elucidating the relevant signaling pathways and exploring the treatment strategies that have garnered attention in recent years. By assimilating and integrating these findings, we aim to establish a comprehensive understanding of the multifaceted roles and intricate mechanisms by which mitophagy influences IR. This endeavor, in turn, seeks to provide novel insights and serve as a catalyst for further research in the pursuit of innovative treatments targeting IR.