ObjectiveTo clarify whether METTL14 mediates the core role of acupuncture at Neiguan (PC6) in promoting myelination and improving behavior in young autistic rats through gene intervention technology. MethodsThe ASD model was established by intraperitoneal injection of valproic acid (VPA) in pregnant rats. Male offspring were intracerebroventricularly injected with adenovirus-packaged METTL14 shRNA (sh-METTL14) or its control (sh-NC) on postnatal day 1, with a model group set as well. Subsequently, the juvenile rats were divided into model group, acupuncture group, acupuncture+sh-NC group, and acupuncture+sh-METTL14 group. The acupuncture group received acupuncture at Neiguan (PC6) from postnatal day 7, once daily for 21 consecutive days. Neurobehavioral changes were evaluated by behavioral tests; METTL14 knockdown efficiency and the expression of METTL14, METTL3, and PTEN were detected by quantitative real-time PCR (qRT-PCR) and Western blot (WB); PTEN m⁶A levels were measured by RNA immunoprecipitation-qPCR (RIP-qPCR); myelin ultrastructure, expression of myelin basic protein (MBP) and neurofascin 155 (NF155), and dendritic spine density were observed using transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), immunofluorescence, qRT-PCR, and primary neuron culture. ResultsBehaviorally, knockdown of METTL14 significantly counteracted the beneficial effects of acupuncture in improving self-grooming, open field exploration, three-chamber social interaction, and Morris water maze learning and memory (P<0.05, P<0.01). Compared with the acupuncture+sh-NC group, the acupuncture+sh-METTL14 group showed significantly decreased mRNA and protein expression of hippocampal METTL14 (P<0.01), and the upregulating effects of acupuncture on METTL3 and PTEN expression were reversed (P<0.01). Meanwhile, knockdown of METTL14 significantly inhibited the acupuncture-induced increase in PTEN m⁶A levels (P<0.01). Morphologically, knockdown of METTL14 attenuated the improvement of myelin structure by acupuncture, reversed the downregulation of MBP and upregulation of NF155 induced by acupuncture, and blocked the increase in dendritic spine density (P<0.05, P<0.01). ConclusionMETTL14 is a key molecule mediating the therapeutic effect of acupuncture at Neiguan. Acupuncture at Neiguan upregulates METTL14, thereby enhancing m⁶A methylation modification of PTEN mRNA to stabilize its expression, ultimately promoting myelin development and improving behavioral symptoms in ASD juvenile rats. This preliminarily reveals the modern biological connotation of “opening Xuanfu and dredging myelin”.

ObjectiveTo clarify whether METTL14 mediates the core role of acupuncture at Neiguan (PC6) in promoting myelination and improving behavior in young autistic rats through gene intervention technology. MethodsThe ASD model was established by intraperitoneal injection of valproic acid (VPA) in pregnant rats. Male offspring were intracerebroventricularly injected with adenovirus-packaged METTL14 shRNA (sh-METTL14) or its control (sh-NC) on postnatal day 1, with a model group set as well. Subsequently, the juvenile rats were divided into model group, acupuncture group, acupuncture+sh-NC group, and acupuncture+sh-METTL14 group. The acupuncture group received acupuncture at Neiguan (PC6) from postnatal day 7, once daily for 21 consecutive days. Neurobehavioral changes were evaluated by behavioral tests; METTL14 knockdown efficiency and the expression of METTL14, METTL3, and PTEN were detected by quantitative real-time PCR (qRT-PCR) and Western blot (WB); PTEN m⁶A levels were measured by RNA immunoprecipitation-qPCR (RIP-qPCR); myelin ultrastructure, expression of myelin basic protein (MBP) and neurofascin 155 (NF155), and dendritic spine density were observed using transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), immunofluorescence, qRT-PCR, and primary neuron culture. ResultsBehaviorally, knockdown of METTL14 significantly counteracted the beneficial effects of acupuncture in improving self-grooming, open field exploration, three-chamber social interaction, and Morris water maze learning and memory (P<0.05, P<0.01). Compared with the acupuncture+sh-NC group, the acupuncture+sh-METTL14 group showed significantly decreased mRNA and protein expression of hippocampal METTL14 (P<0.01), and the upregulating effects of acupuncture on METTL3 and PTEN expression were reversed (P<0.01). Meanwhile, knockdown of METTL14 significantly inhibited the acupuncture-induced increase in PTEN m⁶A levels (P<0.01). Morphologically, knockdown of METTL14 attenuated the improvement of myelin structure by acupuncture, reversed the downregulation of MBP and upregulation of NF155 induced by acupuncture, and blocked the increase in dendritic spine density (P<0.05, P<0.01). ConclusionMETTL14 is a key molecule mediating the therapeutic effect of acupuncture at Neiguan. Acupuncture at Neiguan upregulates METTL14, thereby enhancing m⁶A methylation modification of PTEN mRNA to stabilize its expression, ultimately promoting myelin development and improving behavioral symptoms in ASD juvenile rats. This preliminarily reveals the modern biological connotation of “opening Xuanfu and dredging myelin”.

ObjectiveHepatocellular carcinoma (HCC) represents 90% of all primary liver cancers. The main risk factors associated with HCC include viral hepatitis (B and/or C), alcohol abuse, and metabolic dysfunction-associated steatotic liver disease (MASLD), which progressively advance to liver fibrosis, cirrhosis, and ultimately evolve into HCC. Surgical resection represents the most effective treatment for HCC, while recent advances in immunotherapy, including immune checkpoint inhibitors and adoptive cell therapies, have provided improved treatment prospects for patients with unresectable HCC. However, the complex metabolic heterogeneity of HCC limits the therapeutic efficacy. Metabolic intermediates acyl-CoA not only provide energy and substrates for numerous biochemical reactions but also serve as donors for protein lysine acylation, a major class of post-translational modification (PTM). Therefore, a deeper understanding of the molecular mechanisms underlying protein lysine acylation and hepatocarcinogenesis is urgently needed. MethodsThe levels of protein lysine acylation and silence information regulator 5 (SIRT5) expression levels in clinical HCC samples were analyzed by Western blot. Quantitative malonylome and succinylome of HCC samples were analyzed by antibody-based affinity enrichment coupled with tandem mass spectrometry. The proliferation of HCC cells was analyzed with Cell Counting Kit-8 (CCK-8) assays, the apoptosis was quantified by Annexin V-FITC/propidium iodide (PI) staining coupled with flow cytometry, and the ability of cells to migrate was assayed by Transwell assays. The enzymatic activity of glutathione S-transferase Mu 1 (GSTM1) was quantified. Transgenic mice with hepatic overexpression of SIRT5 were constructed using CRISPR-Cas9, and primary hepatocarcinogenesis was induced by administration of diethylnitrosamine. ResultsWestern blot analysis indicated that the expression level of SIRT5 was elevated in clinical samples from HCC patients, and the levels of lysine malonylation, glutarylation, and succinylation were significantly reduced in HCC tissues. Knockout of SIRT5 in MHCC-97H and MHCC-97L hepatoma cells suppressed cell proliferation, and increased the percentage of apoptotic cells significantly. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the differentially malonylome and succinylome of HCC samples revealed significant enrichment in two major classes of biological processes: core energy metabolism (e.g., glycolysis/gluconeogenesis, tricarboxylic acid metabolic process, fatty acid beta oxidation) and detoxification and oxidative stress response (e.g., response to toxic substance, chemical carcinogenesis, reactive oxygen species (ROS)). SIRT5 removes malonylation from lysine residues in GSTM1 and restores its detoxification activity, which is crucial for the survival of hepatocytes under stressed conditions. More importantly, in vivo experiment indicated that hepatic-specific overexpression of SIRT5 in mice accelerated diethylnitrosamine-induced liver fibrosis and hepatocarcinogenesis, indicating the critical role of SIRT5 in HCC progression. ConclusionThis study highlights the previously unrecognized SIRT5-GSTM1 axis as a key regulator in hepatocarcinogenesis, and suggests a potential target for the treatment of patients with HCC.

Owing to its inherent advantages—such as being non-destructive, rapid, highly molecule-specific, and minimally interfered with by moisture—Raman spectroscopy has been widely adopted in the fields of skin barrier function assessment, monitoring the transdermal penetration of active cosmetic ingredients, and the identification and quality control of cosmetic products. Despite these strengths, the practical application of this technique faces a significant bottleneck: the strong fluorescence background generated by endogenous skin components and exogenous cosmetic additives. Endogenous skin substances, such as structural proteins (e.g., collagen and elastin), metabolic coenzymes (e.g., nicotinamide adenine dinucleotide), and pigments (e.g., melanin), together with exogenous cosmetic constituents like organic colorants, chemical sunscreens, and fragrances, often possess strong absorption and emission characteristics. When excited by lasers, these components produce a fluorescence background that can be 10⁶ to 10⁸ times stronger than the Raman scattering signals, effectively masking the inherently weak vibrational fingerprint information. In recent years, driven by the rapid development of optoelectronic hardware and artificial intelligence algorithms, fluorescence suppression strategies have evolved from isolated, single-method approaches into comprehensive, multi-level synergistic systems. These systems are categorized into three distinct tiers: sample preparation, signal acquisition, and data processing. At the sample preparation level, techniques such as photobleaching and surface-enhanced Raman spectroscopy (SERS) are employed to eliminate or bypass the generation of fluorescence at the source. At the signal acquisition level, instrumental improvements—including the use of long-wavelength near-infrared excitation (typically 785 nm or 1 064 nm), confocal spatial filtering, and shifted excitation Raman difference spectroscopy (SERDS)— are utilized to physically isolate Raman signals from the fluorescence background. Furthermore, at the data processing level, numerical baseline correction methods such as polynomial fitting, penalized least squares (e.g., airPLS, arPLS), wavelet transform, and derivative algorithms are increasingly integrated into the analytical pipeline to extract Raman spectral features from mixed signals without increasing hardware costs or acquisition time. This review provides a systematic categorization and critical evaluation of these fluorescence suppression methods, detailing their underlying principles, technical advantages, and inherent limitations in diverse experimental setups. By focusing on critical application scenarios—including skin barrier assessment, percutaneous absorption monitoring, the routine quality control of cosmetics, and the emerging field of portable on-site detection—this paper explores the current state of technique selection and optimization. Finally, the article discusses future development trends, emphasizing the necessity of constructing adaptive, tiered suppression strategies, developing intelligent and automated data processing algorithms, and promoting the integration of portable, multi-modal diagnostic devices. The objective of this review is to provide a comprehensive technical reference to facilitate the transition of Raman spectroscopy from a specialized laboratory tool into a routine, robust analytical platform for advancements in skin science and cosmetic research.

Objective To verify the efficacy of a domestic human DNA quantification kit based on real-time fluorescence quantitative PCR in detecting the total human DNA concentration,male DNA concen-tration in mixed male/female DNA samples,the degree of DNA degradation and inhibitor tolerance.Methods Samples with different concentrations,different male/female ratios,different concentrations of inhibitors,and different degradation degrees were tested using the domestic human DNA quantification kit based on real-time fluorescence quantitative PCR.This kit was compared with a similar product on the market and was applied to the detection of DNA from real cases.Results This human DNA quan-tification kit can effectively detect human DNA as low as 0.001 65 ng/μL,and 6.25 pg/μL of male DNA in mixed samples with a male-to-female ratio of 1∶15 000.Even when the sample contains as high as 400 ng/μL of humic acid or 1 000 μmol/L of hemin alone,the DNA concentration can still be accurately detected.The degradation index can effectively characterize the degradation degree of the sample.This kit has been successfully applied in forensic practice.Conclusion This human DNA quan-tification kit is accurate and reliable in detection.It can accurately reflect the degradation of DNA and inhibitor tolerance.It has good performance in quantitative accuracy,determination of the male/female ratio in mixed samples,and inhibitor tolerance.It has application potential in forensic case examination.

Objective To construct an aptamer-functionalized carboxylated graphene oxide(CGO)fluo-rescent sensor to achieve highly sensitive and specific detection of ketamine(KET)and its metabolite norketamine(NK)using an aptamer capable of simultaneously recognizing KET and NK.Methods A specific aptamer for simultaneous recognition of KET and NK was screened using graphene oxide-sys-tematic evolution of ligand by exponential enrichment(GO-SELEX)and molecular docking tech-niques.The aptamer,labeled with Cy5 fluorescence,was chemically conjugated to CGO to construct an aptamer-functionalized CGO fluorescent sensor.By optimizing detection conditions,including the mass concentration of CGO,aptamer concentration,reaction temperature,and incubation time,quantita-tive analysis of the target analytes was achieved using the ratio of fluorescence intensity changes be-fore and after target addition.The stability of the sensor in biological matrices was evaluated by moni-toring fluorescence intensity changes over incubation time in blank blood and urine,in comparison with the traditional physical adsorption-based CGO fluorescent sensor.Spiked recovery experiments in blank blood and urine were conducted to compare performance with that of HPLC-MS/MS.Results A specific aptamer A5 was selected and chemically conjugated with CGO to construct the aptamer-functionalized CGO fluorescent sensor.Under optimized conditions,the proposed fluorescent sensor ex-hibited a linear detection range of 1.0-5.0 ng/mL for KET,with a limit of detection(LOD)of 0.86 ng/mL;while for NK,the linear detection range was 1.0-5.0 ng/mL,with an LOD of 0.70 ng/mL.Com-pared with the CGO fluorescent sensor constructed via physical adsorption,this sensor demonstrated greater stability in blood and urine.The spiked recovery rates of KET and NK in blank blood and urine ranged from 81.50%to 110.03%,exhibiting detection performance comparable to that of HPLC-MS/MS.Conclusion The aptamer screening method offers a novel approach for selecting aptamers tar-geting drugs and their metabolites.The constructed aptamer-functionalized CGO fluorescent sensor pro-vides an efficient and reliable strategy for the high-performance detection of KET and NK.

Aim To perform high-throughput screen-ing of immunomodulatory traditional Chinese medicine(TCMs)based on an in vitro B cell differentiation-antibody secretion model,identifying active herbal candidates with immune-enhancing properties to pro-vide novel therapeutic options and theoretical support for influenza virus treatment in immunocompromised in-dividuals.Methods B cells were stimulated with dif-ferent concentrations of cytosine-phosphate-guanine oli-godeoxynucleotide 2006(CpG)and nterleukin-2(IL-2)to promote proliferation,differentiation,and anti-body secretion,and the effects of varying concentra-tions of the solvent DMSO were also evaluated.The op-timal conditions for the B cell differentiation-anti-body secretion model were determined based on the se-cretion levels of three antibody isotypes.The feasibility of the model was further validated using rapamycin,a known B cell function inhibitor.On this basis,a high-throughput screening platform for immunomodulatory a-gents was optimized and established.Subsequently,the immune-enhancing activity of 465 polarity extract from TCMs was evaluated.Results The optimal con-ditions for the model were determined as 2 mg·L-1 CpG,1.67 × 106 nkat·L-1 IL-2,and DMSO with a volume fraction of 0.1％.Rapamycin effectively inhib-ited B cell differentiation into plasmablast and signifi-cantly reduced antibody production,indicating the reli-ability of the model.Multiple rounds of screening re-vealed that the dichloromethane extract of licorice,the dichloromethane extract of Vinegar-processed Curcumae Rhizoma,the cyclohexane extract of Honey-prepared Radix Asteris,and the aqueous extract of Siphonostegia chinensis Benth were identified to significantly promote both B cell proliferation and differentiation and anti-body secretion at a concentration of 600 μg·L-1.Conclusion This study successfully optimizes an in vitro B cell differentiation-antibody secretion model and identifies several TCM extracts,including licorice,with potential immune-enhancing activity.

Aim To investigate the therapeutic effects of a newly developed Tadalafil tablet on pulmonary fi-brosis induced by paraquat(PQ)in rats,as well as its impact on histone acetylation levels in epithelial cells.Methods SD rats were randomly divided into four groups:the control group(control),the model group(PQ),the Tadalafil new tablet treatment group(N-Tad,1 mg·kg-1),and the positive control drug treatment group(Cialis,5 mg·kg-1).The model group and treatment group rats were intraperitoneally injected with PQ(30 mg·kg-1).Two hours after the initial treatment,the rats in the treatment group re-ceived N-Tad or Cialis via gavage,while the control and model groups were administered an equal volume of physiological saline by gavage once daily for 28 days.The weight gain rate and lung tissue index for each group of rats were calculated.Additionally,the effects of N-Tad treatment on lung tissue structural damage and collagen deposition in rats with PQ-in-duced pulmonary fibrosis were observed using HE stai-ning,Masson trichrome staining,and immunohisto-chemical techniques.By employing the Western blot technique,the effects of Tadalafil intervention on the expression of the epithelial marker E-cadherin(E-Cad),the stromal marker fibronectin(Fn),and the histone acetylation marker acetylated histones(Ac-his-tones)in A549 cells were observed.Results Com-pared to the control group,rats with PQ-induced pul-monary fibrosis exhibited a significant decrease in the rate of body weight growth,an increase in lung tissue index(P＜0.05),and a notable increase in the expression and distribution of the fibrosis marker alpha-smooth muscle actin(α-SMA)in lung tissue.The structure of the lung tissue was disrupted,accompanied by the deposition of interstitial collagen fibers.Both N-Tad and Cialis treatments could significantly enhance the rate of weight gain,decrease the lung tissue index,inhibit the expression of α-SMA,and reduce the depo-sition of interstitial collagen in the lung tissue of rats with pulmonary fibrosis.Notably,low-dose N-Tad treatment was comparable to high-dose Cialis treat-ment.At the cellular level,Tadalafil significantly in-hibited the high expression of Fn induced by transfor-ming growth factor beta 1(TGF-β1)in A549 cells.It also upregulated the expression of E-cadherin and sig-nificantly increased the levels of acetylated histones(P＜0.05).Conclusions N-Tad promotes histone acetylation in alveolar epithelial cells,significantly in-hibits epithelial-mesenchymal transition,increases E-cadherin expression,and improves lung tissue structur-al damage and collagen deposition caused by PQ.Ad-ditionally,it offers the advantage of a lower effective dose compared to Cialis,providing a new option for the treatment of pulmonary fibrosis.

CRM 197(cross-reacting material 197),a naturally occurring mutant of diphtheria toxin,is a safe and effective vaccine vector and extensively used on developing conjugate or combined vaccines.The mutant loses its enzymatic activity,but fully retains its receptor-binding ability and immunogenicity.In current work,the diphtheria toxin mutant CRM 197 and its fusion proteins with the receptor-binding do-main of botulinum neurotoxin serotype E(EHc)were developed using genetic engineering technology.These recombinant proteins were confirmed by Western blotting and SDS-PAGE.BALB/c mice were im-munized with the CRM197-EHc and EHc-CRM197 fusion proteins,and their immunogenicity was evalua-ted.These two fusion protein molecules,CRM197-EHc and EHc-CRM197,as subunit vaccines,elicited a robust humoral immune response targeting both CRM197 and EHc antigens in the immunized mice.Compared to the mixture of CRM197 and EHc,the mice vaccinated with the fusion proteins(CRM197-EHc and EHc-CRM197)induced higher levels of anti-CRM197 antibodies,and the mice vaccinated with EHc-CRM197 also generated strongest anti-EHc antibodies.Consequently,as a carrier molecule in the fusion protein vaccine,EHc enhances the immunogenicity of CRM197 molecules.Likewise,CRM197 boosts the immunogenicity of EHc in the EHc-CRM197 fusion protein.

Aim To perform high-throughput screen-ing of immunomodulatory traditional Chinese medicine(TCMs)based on an in vitro B cell differentiation-antibody secretion model,identifying active herbal candidates with immune-enhancing properties to pro-vide novel therapeutic options and theoretical support for influenza virus treatment in immunocompromised in-dividuals.Methods B cells were stimulated with dif-ferent concentrations of cytosine-phosphate-guanine oli-godeoxynucleotide 2006(CpG)and nterleukin-2(IL-2)to promote proliferation,differentiation,and anti-body secretion,and the effects of varying concentra-tions of the solvent DMSO were also evaluated.The op-timal conditions for the B cell differentiation-anti-body secretion model were determined based on the se-cretion levels of three antibody isotypes.The feasibility of the model was further validated using rapamycin,a known B cell function inhibitor.On this basis,a high-throughput screening platform for immunomodulatory a-gents was optimized and established.Subsequently,the immune-enhancing activity of 465 polarity extract from TCMs was evaluated.Results The optimal con-ditions for the model were determined as 2 mg·L-1 CpG,1.67 × 106 nkat·L-1 IL-2,and DMSO with a volume fraction of 0.1％.Rapamycin effectively inhib-ited B cell differentiation into plasmablast and signifi-cantly reduced antibody production,indicating the reli-ability of the model.Multiple rounds of screening re-vealed that the dichloromethane extract of licorice,the dichloromethane extract of Vinegar-processed Curcumae Rhizoma,the cyclohexane extract of Honey-prepared Radix Asteris,and the aqueous extract of Siphonostegia chinensis Benth were identified to significantly promote both B cell proliferation and differentiation and anti-body secretion at a concentration of 600 μg·L-1.Conclusion This study successfully optimizes an in vitro B cell differentiation-antibody secretion model and identifies several TCM extracts,including licorice,with potential immune-enhancing activity.