Oral squamous cell carcinoma (OSCC) is a common head and neck malignancy. Approximately 50% to 60% of patients with OSCC are diagnosed at a locally advanced stage (clinical staging III-IVa). Even with comprehensive and sequential treatment primarily based on surgery, the 5-year overall survival rate remains below 50%, and patients often suffer from postoperative functional impairments such as difficulties with speaking and swallowing. Programmed death receptor-1 (PD-1) inhibitors are increasingly used in the neoadjuvant treatment of locally advanced OSCC and have shown encouraging efficacy. However, clinical practice still faces key challenges, including the definition of indications, optimization of combination regimens, and standards for efficacy evaluation. Based on the latest research advances worldwide and the clinical experience of the expert group, this expert consensus systematically evaluates the application of PD-1 inhibitors in the neoadjuvant treatment of locally advanced OSCC, covering combination strategies, treatment cycles and surgical timing, efficacy assessment, use of biomarkers, management of special populations and immune related adverse events, principles for immunotherapy rechallenge, and function preservation strategies. After multiple rounds of panel discussion and through anonymous voting using the Delphi method, the following consensus statements have been formulated: 1) Neoadjuvant therapy with PD-1 inhibitors can be used preoperatively in patients with locally advanced OSCC. The preferred regimen is a PD-1 inhibitor combined with platinum based chemotherapy, administered for 2-3 cycles. 2) During the efficacy evaluation of neoadjuvant therapy, radiographic assessment should follow the dual criteria of Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 and immune RECIST (iRECIST). After surgery, systematic pathological evaluation of both the primary lesion and regional lymph nodes is required. For combination chemotherapy regimens, PD-L1 expression and combined positive score need not be used as mandatory inclusion or exclusion criteria. 3) For special populations such as the elderly (≥ 70 years), individuals with stable HIV viral load, and carriers of chronic HBV/HCV, PD-1 inhibitors may be used cautiously under the guidance of a multidisciplinary team (MDT), with close monitoring for adverse events. 4) For patients with a poor response to neoadjuvant therapy, continuation of the original treatment regimen is not recommended; the subsequent treatment plan should be adjusted promptly after MDT assessment. Organ transplant recipients and patients with active autoimmune diseases are not recommended to receive neoadjuvant PD-1 inhibitor therapy due to the high risk of immune related activation. Rechallenge is generally not advised for patients who have experienced high risk immune related adverse events such as immune mediated myocarditis, neurotoxicity, or pneumonitis. 5) For patients with a good pathological response, individualized de escalation surgery and function preservation strategies can be explored. This consensus aims to promote the standardized, safe, and precise application of neoadjuvant PD-1 inhibitor strategies in the management of locally advanced OSCC patients.

Triple-negative breast cancer is therapeutically challenging due to the low expression of tumor markers and 'cold' tumor immunosuppressive microenvironment. Here, we present a dual-targeting peptide-drug conjugate (PDC) for tumor inhibition. Our PDC efficiently and selectively delivers cytotoxic Monomethyl Auristatin E (MMAE) into tumor cells via C-X-C chemokine receptor type 4 (CXCR4) and folate receptor 1 (FOLR1) for synergistic inhibition of growth and metastasis. Our results show that the dual-targeting PDC has potent antitumor activity in cultured human cells and several murine transplanted tumor models without apparent toxicity. The combination of dual-targeting PDC and radiotherapy modulates the tumor immunosuppressive microenvironment by increasing CD8⁺ T cell infiltration and attenuating the proportion of myeloid-derived suppressor and regulatory T cells. Therefore, our dual-targeting PDC represents a promising new strategy for cancer therapy that rebalances the immune system and promotes tumor regression.

DNA genetic markers have always played important roles in individual identification, kinship analysis, ancestry inference and phenotype characterization in the field of forensic medicine. DNA methylation has unique advantages in biological age inference, body fluid identification and prediction of phenotypes. The majority of current studies independently examine DNA and DNA methylation markers using various workflows, and they use various analytical procedures to interpret the biological information these two markers present. Integrated methods detect DNA and DNA methylation markers simultaneously through a single experimental workflow using the same preparation of sample. Therefore, they can effectively reduce consumption of time and cost, streamline experimental procedures, and preserve valuable DNA samples taken from crime scenes. In this paper, the integrated detection approaches of DNA and DNA methylation markers on different detection platforms were reviewed. In order to convert methylation modifications to detectable forms, several options were available for pretreatment of genomic DNA, including digestion with methylation-sensitive restriction enzyme, affinity enrichment of methylated fragments, conversion of methylated or unmethylated cytosine. Multiplexed primers can be designed for DNA markers and converted DNA methylation markers for co-amplification. The schemes of using capillary electrophoresis platform for integrated detection add the pretreatment of genomic DNA on the basis of detecting DNA genetic markers. DNA and DNA methylation markers are then integrated by co-amplification. But the limited number of fluorescent options available and the length of amplicons restrict the type and quantity of markers that can be integrated into a panel. Pyrophosphate sequencing also supports integrated detection of DNA and DNA methylation markers. On this platform, due to the conversion of unmethylated cytosine to thymine after treatment with bisulfite, the methylation level of CpG site can be directly calculated using the peak height ratio of cytosine bases and thymine bases. Therefore, the methylation levels and SNP typing can be simultaneously obtained. However, due to the limited read length of sequencing, the detection of markers with longer amplicons is restricted. It is not conducive to fully interpret the complete information of the target sequence. Next-generation sequencing also supports integrated detection of DNA and DNA methylation markers. A preliminary experimental process including DNA extraction, pretreatment of genomic DNA, co-preparation of DNA and DNA methylation library and co-sequencing, has been formed based on the next-generation sequencing platform. It confirmed the feasibility of next-generation sequencing technology for integrated detection of DNA and DNA methylation markers. In field of biomedicine, various integrated detection schemes and corresponding data analysis approaches of DNA and DNA genetic markers developed based on the above detection process.Co-analysis can simultaneously obtain the genomic genetic and epigenetic information through a single analytic process. These schemes suggest that next-generation sequencing may be an effective method for achieving more accurate and highly integrated detection, helping to explore the potential for application in forensic biological samples. We finally explore the impact of interactions between sites and different pretreatment methods on the integrated detection of DNA and DNA methylation markers, and also propose the challenge of applying third-generation sequencing for integrated detection in forensic samples.

Objective:To investigate the application value of optical coherence tomography (OCT) and intravascular ultrasound (IVUS) in evaluating flow diverter (FD) apposition and endothelialization in aneurysm animal models, and analyze the effect of incomplete stent apposition (ISA) on aneurysm lumen healing and stent endothelialization.Methods:Lateral common carotid artery aneurysm models in swines were established by surgical method and then FD was implanted. Immediately after surgery, OCT and IVUS were used to evaluate the locations and degrees of ISA, and difference between these 2 methods in evaluating FD apposition was compared. DSA was performed at 12 weeks after surgery to evaluate the aneurysm occlusion (Kamran grading) and stent patency. OCT and IVUS were used again to observe the stent endothelial situation; by comparing with histopathologic results, effect of ISA on aneurysm healing and stent endothelialization was analyzed.Results:Lateral common carotid artery aneurysm models in 6 swines were established, and 6 Tubridge FDs were successfully implanted. Compared with IVUS (3 stents, 4 locus), OCT could detect more ISA (6 stents, 14 locus); and the vascular diameter change area (7 locus), aneurysm neck area (4 locus) and the head and tail of FD (3 locus) were the main sites of FD malapposition; average distance between stent wire and vessel wall was (560.14±101.48) μm. At 12 weeks after surgery, DSA showed that 1 patient had a little residual contrast agent at the aneurysm neck (Kamran grading 3), and the remaining 5 had complete aneurysm occlusion (Kamran grading 4). One FD had moderate lumen stenosis, and the other 5 FDs had lumen patency. OCT indicated mostly disappeared acute ISA; ISA proportion decreased to 21.4 % (3/14), including 2 in the aneurysm neck and 1 in the partial stent. Histopathological results showed bare stent woven silk, without obvious endothelial coverage; in one FD with luminal stenosis, intimal hyperplasia was mainly composed of vascular smooth muscle cells.Conclusion:In carotid artery aneurysm model with FD implantation, OCT can detect more ISA than IVUS; most acute ISA have good outcome at 12 th week of follow-up, while severe ISA can cause delayed FD endothelialization and delayed aneurysm occlusion.

Objective To evaluate the efficacy and safety of brexpiprazole in treating acute schizophrenia.Methods Patients with schizophrenia were randomly divided into treatment group and control group.The treatment group was given brexpiprozole 2-4 mg·d-1 orally and the control group was given aripiprazole 10-20 mg·d-1orally,both were treated for 6 weeks.Clinical efficacy of the two groups,the response rate at endpoint,the changes from baseline to endpoint of Positive and Negative Syndrome Scale(PANSS),Clinical Global Impression-Improvement(CGI-S),Personal and Social Performance scale(PSP),PANSS Positive syndrome subscale,PANSS negative syndrome subscale were compared.The incidence of treatment-related adverse events in two groups were compared.Results There were 184 patients in treatment group and 186 patients in control group.After treatment,the response rates of treatment group and control group were 79.50％(140 cases/184 cases)and 82.40％(150 cases/186 cases),the scores of CGI-I of treatment group and control group were(2.00±1.20)and(1.90±1.01),with no significant difference(all P＞0.05).From baseline to Week 6,the mean change of PANSS total score wese(-30.70±16.96)points in treatment group and(-32.20±17.00)points in control group,with no significant difference(P＞0.05).The changes of CGI-S scores in treatment group and control group were(-2.00±1.27)and(-1.90±1.22)points,PSP scores were(18.80±14.77)and(19.20±14.55)points,PANSS positive syndrome scores were(-10.30±5.93)and(-10.80±5.81)points,PANSS negative syndrome scores were(-6.80±5.98)and(-7.30±5.15)points,with no significant difference(P＞0.05).There was no significant difference in the incidence of treatment-related adverse events between the two group(69.00％vs.64.50％,P＞0.05).Conclusion The non-inferiority of Brexpiprazole to aripiprazole was established,with comparable efficacy and acceptability.

Objective To investigate the clinical features and genetic characteristics of patients with late-onset Pompe disease(LOPD).Methods A total of 13 patients diagnosed with LOPD in the First Affilia-ted Hospital of Zhejiang University School of Medicine from September 2020 to December 2023 were selected,and all patients were subjected to clinical investigation,GAA activity detection and GAA gene testing.Results Among the 13 patients,7 were males and 6 were females;5 were family patients and 8 were sporadic patients;and the median age of onset was 17 years(8-52 years),the median age of presentation was 24 years(10-52 years),and the median age of diagnosis was 31 years(14-58 years).In terms of the first symptoms,10 pa-tients presented with limb weakness and 3 patients presented with dyspnea.The average serum creatine kinase level was 552 U/L(55-1084 U/L),and the serum creatine kinase level was normal in one patient.All pa-tients had scoliosis and different degrees of restrictive ventilatory dysfunction.Neuroelectrophysiological exami-nations of 9 patients showed myogenic damage,and 8 of them had muscle tonic discharge.The mean value of GAA activity was 0.3 μmol/(L·h)[0.17-0.5 μmol/(L·h)].A total of 13 mutations were detected in GAA gene,and the most common mutation was c.2238G＞C(p.W746C).There were five new variant sites:c.543del(p.F181Lfs*40),c.839_840insCC(p.R281Pfs*34),c.1800_1823del(p.S601_R608del),c.2296T＞C(p.Y766H)and c.995C＞A(p.S332*).Conclusions LOPD is a rare disease that tends to delay diagnosis.Proximal limb weakness,decreased respiratory function,mild-to-moderate elevation of creatine kinase,scoliosis,and clinical inferior tonic discharge on electromyography are high-risk images of LOPD.c.2238G＞C(p.W746C)is a hotspot mutation,and the discovery of five new mutations enriches the GAA gene mutations lineage.

ObjectiveBody fluid stains left at crime scenes are frequently trace amounts, while the identification of body fluids through real time fluorogenic quantitative technique often necessitates the repeated detection within the limited sample, as multiple miRNA markers are the basis for the identification. Based on the goal of both the throughput and efficiency improvement of miRNA analysis in trace samples, a duplex real time fluorogenic quantitative PCR assay system was designed to accurately quantify two miRNAs simultaneously, and the system should be further verified by actual sample for the body fluid identification. MethodsThe duplex real time fluorogenic quantitative PCR system of miR-451a to miR-21-5p was established with specially designed primers and probes, and the concentrations of the primers and probes were both optimized. The specificity, sensitivity and reproducibility of the system were validated, while its capability for body fluid identification was assessed using the miR-451a to miR-21-5p ratio. ResultsThe optimized assay system exhibited excellent specificity and repeatability, with coefficients of variation consistently below 8% for both intra- and inter-batch variability. The amplification efficiency of miR-451a and miR-21-5p reached 71.77% and 74.81%, respectively, with high and relatively consistent results. By utilizing this duplex real time fluorogenic quantitative PCR assay system, a total of 58 body fluid samples were analyzed, exhibiting a discrimination rate of 100% between blood and non-blood samples, as well as between peripheral blood and menstrual blood samples. Moreover, the results, obtained from single real time fluorogenic quantitative PCR assay system and duplex real time fluorogenic quantitative PCR assay system, showed no statistically significant difference with randomly selected blood samples (n=20). Compared to previous single real time fluorogenic quantitative PCR assay system, the sensitivity of duplex real time fluorogenic quantitative PCR assay system exhibited remarkable improvement. A minimum input of only 0.1 ng total RNA was sufficient for accurate detection of peripheral blood and menstrual blood samples, while saliva, semen, and vaginal secretion required only 1 ng total RNA for precise identification purposes. Additionally, the duplex real time fluorogenic quantitative PCR assay system successfully differentiated between different types of body fluids in simulated samples under natural outdoor conditions. ConclusionThe duplex real time fluorogenic quantitative PCR assay system effectively reduced both the time and material costs by half compared to the single system, especially suitable for the examination of body fluid stains left at crime scenes, solving the contradiction between the trace amount and the multiple sample volumes demand of repeated real time fluorogenic quantitative PCR. The duplex real time fluorogenic quantitative PCR assay successfully distinguished blood and other body fluid, as well as peripheral blood and menstrual blood samples, which maintains an equivalent capability for body fluid identification with half sample, time and reagent consumption. This system provides an efficient tool for identifying suspicious body fluids, as well as a foundation for more multiplexed real time fluorogenic quantitative PCR assay system research.

AIM To establish the HPLC characteristic chromatogram of Baqi Rougan Decoction reference sample,and to investigate its quantitative transmission laws.METHODS The contents of calycosin 7-O-glucoside,hesperidin,rosmarinic acid,curcumenol and nystose were determined.The transfer rates of decoction piece-aqueous decoction-reference sample were calculated,after which the paste-forming rate and pH value were recorded.RESULTS There were sixteen characteristic peaks in fifteen batches of reference samples with the similarities of 0.90,nine of which were identified.The average transfer rates of nystose and calycosin 7-O-glucoside in the reference sample were(83.14±6.25)%and(77.81±8.31)%,while those of rosmarinic acid and curcumenol in the aqueous decoction-reference sample were(81.71±6.27)%and(72.16±5.91)%,along with the average paste-forming rate and pH value of(38.91%±1.46%)and 5.13±0.08,respectively.CONCLUSION This stable and feasible method can provide a reference for the selection of preparation process and evaluation of key chemical properties for Baqi Rougan Decoction.

OBJECTIVE Optimizing the water extraction technology of Xiangqin jiere granules. METHODS The orthogonal test of 3 factors and 3 levels was designed， and comprehensive scoring was conducted for the above indexes by using G1-entropy weight to obtain the optimized water extraction technology of Xiangqin jiere granules with water addition ratio， extraction time and extraction times as factors， using the contents of forsythoside A， baicalin， phillyrin， oroxylin A-7-O-β-D-glycoside， wogonoside， baicalein and wogonin， and extraction rate as evaluation indexes. BP neural network modeling was used to optimize the network model and water extraction process using the results of 9 groups of orthogonal tests as test and training data， the water addition multiple， decocting time and extraction times as input nodes， and the comprehensive score as output nodes. Then the two analysis methods were compared by verification test to find the best water extraction process parameters. RESULTS The water extraction technology optimized by the orthogonal test was 8-fold water， extracting 3 times， extracting for 1 h each time. Comprehensive score was 96.84 （RSD＝0.90%）. The optimal water extraction technology obtained by BP neural network modeling included 12-fold water， extracting 4 times， extracting for 0.5 h each time. The comprehensive score was 92.72 （RSD＝0.77%）， which was slightly lower than that of the orthogonal test. CONCLUSIONS The water extraction technology of Xiangqin jiere granules is optimized successfully in the study， which includes adding 8-fold water， extracting 3 times， and extracting for 1 hour each time.