Objective: To explore the potential application of PAK4-PROTAC targeted degradation drug (PpD) in renal cancer immunotherapy. Methods: TIMER 2.0 and TISIDB databases were used to analyze the relationship among PAK4 expression, tumor purity and abundance of immune cell infiltration in renal tumor microenvironment (TEM).Renal cancer cell lines OS-RC-2, 786-O and ACHN were treated with 0, 125 and 250 nmol/L PpD, and the effects of Jurkat cell co-culture on the results were investigated.The cell apoptosis was detected with flow cytometry, and the expression of programmed cell death 1 ligand 1 (PD-L1) in renal cancer cells was detected with immunoblotting. Results: The high expression of PAK4 was positively related to immune purity, and inhibited the abundance of immune killer cells in TEM, such as CD8 T cells, CD4 T cells, natural killer cells and dendritic cells.With 250 nmol/L PpD treatment, there were 21.02% apoptotic cells in OS-RC-2, 29.67% apoptotic cells in 786-O, and 15.39% apoptotic cells in ACHN, respectively.However, with the same concentration of 250 nmol/L PpD treatment, cell apoptotic rate was sharply increased to 70.13% in OS-RC-2/Jurkat, 70.68% in 780-O/Jurkat, and 60.27% in ACHN/Jurkat co-culture models, respectively. Conclusion: PpD can promote apoptosis of renal cancer cells by reducing the expression of PAK4 protein, and enhance the killing effects of immune cells on tumor cells.

Arginine methylation is a critical post-translational modification that plays multifaceted biological functions. However, the manipulation of protein arginine methylation largely depends on genetic or pharmaceutic inhibition of the regulatory enzymes, protein arginine methyltransferases (PRMTs), or non-methylation substitution of corresponding arginine residue to lysine or alanine of protein of interest (POI), which inevitably affects other substrates, or disrupts the structure of POI. Thus, it urges an approach to specifically modulate the arginine methylation of a POI under physiological conditions. To this end, we report the discovery of a methylation tagging system (MeTAG), that enables targeted modification of protein arginine methylation. Through bridging the methyltransferase PRMT5 proximity to a POI, MeTAG facilitates the arginine methylation of POIs, including known arginine methylated proteins, androgen receptor (AR) and protein kinase B (AKT), as well as a neo-substrate E1A binding protein (p300), in a reversible and PRMT5-dependent manner. Moreover, MeTAG can regulate downstream signaling in a methylation dependent manner, leading to downregulation of PSMA mRNA level and activation of AKT. Therefore, MeTAG represents a feasible approach to modulate protein methylation and thereby perturbs protein function in biological and therapeutic contexts.

The seventy-two grid palm technique is an important theoretical source of traditional Chinese medicine hand diagnosis. Starting from the ecological medical model， we analyse the seventy-two grid palm technique， and believe that its diagnosis of human body integrates biological， ecological， psychological， social and other factors， and each factor is based on physiological and pathological theories， and its external social interpretation of the nature of the human body is inseparable from health state. It is proposed that the seventy-two grid palm technique should be integrated with the ecological and natural viewpoints based on the biomedical models or bio-psycho-social medical models， and the research should be conducted from the perspective of the ecological medical model， in order to promote the development of hand diagnosis.

OBJECTIVES: Nasopharyngeal cracinoma is a kind of head and neck malignant tumor with high incidence and high mortality. Due to the characteristics of local recurrence, distant metastasis, and drug resistance, the survival rate of patients after treatment is not high. Paclitaxel (PTX) is used as a chemotherapy drug in treating nasopharyngeal carcinoma, but nasopharyngeal carcinoma cells are easy to develop resistance to PTX. Inhibition of heat shock protein 90 (Hsp90) can overcome common signal redundancy and resistance in many cancers. This study aims to investigate the anti-tumor effect of ginkgolic acids C15꞉1 (C15:1) combined with PTX on nasopharyngeal carcinoma CNE-2Z cells and the mechanisms. METHODS: This experiment was divided into a control group (without drug), a C15:1 group (10, 30, 50, 70 μmol/L), a PTX group (5, 10, 20, 40 nmol/L), and a combination group. CNE-2Z cells were treated with the corresponding drugs in each group. The proliferation of CNE-2Z cells was evaluated by methyl thiazolyl tetrazolium (MTT). Wound-healing assay and transwell chamber assay were used to determine the migration of CNE-2Z cells. Transwell chamber was applied to the impact of CNE-2Z cell invasion. Annexin V-FITC/PI staining was used to observe the effect on apoptosis of CNE-2Z cells. The changes of proteins involved in cell invasion, migration, and apoptosis after the combination of C15꞉1 and PTX treatment were analyzed by Western blotting. RESULTS: Compared with the control group, the C15꞉1 group and the PTX group could inhibit the proliferation of CNE-2Z cells (all P<0.05). The cell survival rates of the C15꞉1 50 μmol/L combined with 5, 10, 20, or 40 nmol/L PTX group were lower than those of the single PTX group (all P<0.05), the combination index (CI) value was less than 1, suggesting that the combined treatment group had a synergistic effect. Compared with the 50 μmol/L C15꞉1 group and the 10 nmol/L PTX group, the combination group significantly inhibited the invasion and migration of CNE-2Z cells (all P<0.05). The results of Western blotting demonstrated that the combination group could significantly down-regulate Hsp90 client protein matrix metalloproteinase (MMP)-2 and MMP-9. The results of double staining showed that compared with the 50 μmol/L C15꞉1 group and the 10 nmol/L PTX group, the apoptosis ratio of CNE-2Z cells in the combination group was higher (both P<0.05). The results of Western blotting suggested that the combination group could decrease the Hsp90 client proteins [Akt and B-cell lymphoma-2 (Bcl-2)] and increase the Bcl-2-associated X protein (Bax). CONCLUSIONS The combination of C15꞉1 and PTX has a synergistic effect which can inhibit cell proliferation, invasion, and migration, and induce cell apoptosis. This effect may be related to the inhibition of Hsp90 activity by C15꞉1.
Humans ; Nasopharyngeal Carcinoma ; Paclitaxel/therapeutic use* ; Nasopharyngeal Neoplasms/metabolism* ; Antineoplastic Agents/therapeutic use* ; Apoptosis ; Cell Proliferation ; Cell Line, Tumor

Background: Long coronavirus disease 2019 (COVID-19) in recovered patients (RPs) is gradually recognized by more people. However, how long it will last and the underlining mechanism remains unclear. Methods: We conducted a prospective follow-up study to evaluate the long-term symptoms and clinical indices of RPs at one-year after discharge from Union Hospital, Wuhan, China between December 2020 to May 2021. We also performed the 16S rRNA sequencing of stool samples from RPs and healthy controls (HCs) and analyzed the correlation between the gut microbiota and long COVID-19. Results: In total, 187 RPs were enrolled, among them, 84 (44.9%) RPs reported long COVID-19 symptoms at one-year after discharge. The most common long-term symptoms were cardiopulmonary symptoms, including chest tightness after activity (39/187, 20.9%), palpitations on exercise (27/187, 14.4%), sputum (21/187, 11.2%), cough (15/187, 8.0%) and chest pain (13/187, 7.0%), followed by systemic symptoms including fatigue (34/187, 18.2%) and myalgia (20/187, 10.7%), and digestive symptoms including constipation (14/187, 7.5%), anorexia (13/187, 7.0%), and diarrhea (8/187, 4.3%). Sixty-six (35.9%) RPs presented either anxiety or depression (42/187 [22.8%] and 53/187 [28.8%] respectively), and the proportion of anxiety or depression in the long symptomatic group was significantly higher than that in the asymptomatic group (41/187 [50.6%] vs. 25/187 [24.3%]). Compared with the asymptomatic group, scores of all nine 36-Item Short Form General Health Survey domains were lower in the symptomatic group (all P < 0.05). One hundred thirty RPs and 32 HCs (non-severe acute respiratory syndrome coronavirus 2 infected subjects) performed fecal sample sequencing.Compared with HCs, symptomatic RPs had obvious gut microbiota dysbiosis including significantly reduced bacterial diversities and lower relative abundance of short-chain fatty acids (SCFAs)-producing salutary symbionts such as Eubacterium_hallii_group, Subdoligranulum, Ruminococcus, Dorea, Coprococcus, and Eubacterium_ventriosum_group. Meanwhile, the relative abundance of Eubacterium_hallii_group, Subdoligranulum, and Ruminococcus showed decreasing tendencies between HCs, the asymptomatic group, and the symptomatic group. Conclusion This study demonstrated the presence of long COVID-19 which correlates with gut microbiota dysbiosis in RPs at one-year after discharge, indicating gut microbiota may play an important role in long COVID-19.

Objective:To screen the HIV standard strains with typical biological characteristics of HIV strains circulating in China through the isolation, culture, genotype and phenotype identification of HIV from the whole blood samples of HIV-infected persons, confirm genetic characteristics, traceability, and in line with the Standard Strains of Pathogenic Microorganism-technical Specifications for Establishment of HIV Strains (T/CPMA 027—2023).Methods:Whole blood samples were collected from 48 HIV infected patients. Peripheral blood mononuclear cells (PBMCs) were isolated from the samples and co-cultured with PBMCs isolated from healthy persons′ whole blood samples to isolate and culture HIV from infected persons. We determined concentration of p24 antigen and the virus titer in the culture supernatant. The viral RNA was extracted from the successfully isolated strains, and the gag, pol genes and env C2V3 fragments of the viral genome were amplified and sequenced. The genotype, gene recombination and drug resistance sites were determined according to the viral gene sequences. Virus infection and replication were monitored by inoculating the virus culture supernatant into Ghost cells expressing CCR5 or CXCR4 to determine the viral tropism.The formation of syncytium was observed by inoculating the virus culture supernatant into MT-2 cells to determine whether was a syncytium-induced phenotype. Results:Fourteen strains with p24 antigen concentration > 1 ng/ml in culture supernatant were isolated and cultured from 48 fresh EDTA anticoagulated whole blood samples of HIV infected persons. Of the 14 strains, only one strain with a titer≥10 5 TCID 50/ml, 8 strains with titers ≥10 4 TCID 50/ml, and the other 5 strains with titers≥10 3 TCID 50/ml. Phylogenetic analysis showed that the genotypes of the strains were 9 strains of subtype B, 3 strains of CRF01_AE and 2 strains of CRF07_BC recombinant. Genotypic resistance analysis showed that 11 strains contained drug resistance sites. Ghost cells were used to verify the tropism of the strains, and it was found that 8 strains were CCR5 tropism, 6 strains were CXCR4 & CCR5 dual tropism. Only 2 of the 14 strains could induce MT-2 cytopathic effect, which was syncytium-inducing phenotype. Conclusions:Fourteen HIV strains with typical biological and genetic characteristics were isolated to screen the standard HIV strains. Among which, 1 strain was evaluated as a standard HIV strain that meets the Standard Strains of Pathogenic Microorganism-technical Specifications for Establishment of HIV Strains (T/CPMA 027—2023). This study can also provide technical guidance for the screening of the HIV standard strains. Next step is to complete the application and reserve database construction according to the sharing mechanism of the HIV standard strains, to provide resources for the researches of HIV vaccines and drugs.

Background: Most patients including health care workers (HCWs) survived the coronavirus disease 2019 (COVID-19), however, knowledge about the sequelae of COVID-19 after discharge remains limited. Methods: A prospectively observational 3-month follow-up study evaluated symptoms, dynamic changes of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) IgG and IgM, lung function, and high resolution computed tomography (HRCT) of survivors of COVID-19 after discharge at Wuhan Union Hospital, China. Results: Seventy-six survivors (55 females) with a mean age of 41.3 ± 13.8 years were enrolled, and 65 (86%) were HCWs. A total of 69 (91%) patients had returned to their original work at 3-months after discharge. Most of the survivors had symptoms including fever, sputum production, fatigue, diarrhea, dyspnea, cough, chest tightness on exertion and palpitations in the three months after discharge. The serum troponin-I levels during the acute illness showed high correlation with the symptom of fatigue after hospital discharge (r = 0.782; P = 0.008) and lymphopenia was correlated with the symptoms of chest tightness and palpitations on exertion of patients after hospital discharge (r = −0.285, P = 0.027; r = −0.363, P = 0.004, respectively). The mean values of forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), FEV1/FVC, total lung capacity and diffusion capacity were all normal (> 80% predicted) and lung HRCTs returned to normal in most of the patients (82%), however, 42% of survivors had mild pulmonary function abnormalities at 3-months after discharge. SARS-CoV-2 IgG turned negative in 11% (6 of 57 patients), 8% (4 of 52 patients) and 13% (7 of 55 patients), and SARS-CoV-2 IgM turned negative in 72% (41 of 57 patients), 85% (44 of 52 patients) and 87% (48 of 55 patients) at 1-month, 2-months and 3-months after discharge, respectively. Conclusion Infection by SARS-CoV-2 caused some mild impairments of survivors within the first three months of their discharge and the duration of SARS-CoV-2 antibody was limited, which indicates the necessity of long-term follow-up of survivors of COVID-19.

OBJECTIVE: To investigate the effects of methanol-ethyl acetate partitioned fractions from (MEDS) on the proliferation and apoptosis of human non-small cell lung cancer H1975 cells. METHODS: The systemic solvent extraction method was used to preliminary separation of the effective fractions in the methanol extract of . The cytotoxicity of each extract (5, 10, 20, 40, and 80 μg/mL) was tested using MTT assay. Colony cloning method was used to assess the effect of different concentrations of methanol-ethyl acetate partitioned fractions from MEDS (5, 10, 20, 40, and 80 μg/ mL) on the proliferation of H1975 cells. Flow cytometric analysis with Annexin V-FITC/PI staining was performed to detect the apoptosis of the cells after treatment with different concentrations of MEDS fractions (10, 20, and 40 μg/mL). Western blotting was used to evaluate the effects of MEDS fractions on the expressions of apoptosis-related proteins Akt, Bax, and Bcl-2. The anti-tumor activity of 100 mg/kg MEDS fractions was tested in a nude mouse model bearing H1975 cell xenografts. RESULTS: MTT assay and colony forming experiment showed that MEDS fractions significantly inhibited the proliferation of H1975 cells in a dose-and time-dependent manner ( < 0.05). The results of flow cytometry showed that MEDS fractions induced obvious apoptosis of H1975 cells in a concentration-dependent manner ( < 0.05). MEDS fractions also significantly decreased the expressions of Bcl-2 and Akt protein and increased the protein expression of Bax ( < 0.05). In the tumor-bearing nude mouse model, MEDS fractions showed potent anti-tumor effects with a low toxicity to affect the body weight and organs of the mice. CONCLUSIONS The methanol-ethyl acetate partitioned fractions from MEDS show potent anti-tumor activity both and , suggesting their value as promising therapeutic agents against lung cancer.
Acetates ; Animals ; Antineoplastic Agents, Phytogenic ; isolation & purification ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Non-Small-Cell Lung ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Heterografts ; Humans ; Lung Neoplasms ; pathology ; Methanol ; Mice ; Mice, Nude ; Plant Extracts ; isolation & purification ; pharmacology