Cells not only contain membrane-bound organelles (MBOs), but also membraneless organelles (MLOs) formed by condensation of many biomacromolecules. Examples include RNA-protein granules such as nucleoli and PML nuclear bodies (PML-NBs) in the nucleus, as well as stress granules and P-bodies in the cytoplasm. Phase separation is the basic organizing principle of the form of the condensates or membraneless organelles (MLOs) of biomacromolecules including proteins and nucleic acids. In particular, liquid-liquid phase separation (LLPS) compartmentalises and concentrates biological macromolecules into liquid condensates. It has been found that phase separation of biomacromolecules requires some typical intrinsic characteristics, such as intrinsically disordered regions, modular domains and multivalent interactions. The phase separation of biomacromolecules plays a key role in many important cell activities. In recent years, the phase separation of biomacromolecules phase has become a focus of research in gene transcriptional regulation. Transcriptional regulatory elements such as RNA polymerases, transcription factors (TFs), and super enhancers (SEs) all play important roles through phase separation. Our group has previously reported for the first time that long-term inactivation or absence of assembly factors leads to the formation of condensates of RNA polymerase II (RNAPII) subunits in the cytoplasm, and this process is reversible, suggesting a novel regulatory model of eukaryotic transcription machinery. The phase separation of biomacromolecules provides a biophysical understanding for the rapid transmission of transcriptional signals by a large number of TFs. Moreover, phase separation during transcriptional regulation is closely related to the occurrence of cancer. For example, the activation of oncogenes is usually associated with the formation of phase separation condensates at the SEs. In this review, the intrinsic characteristics of the formation of biomacromolecules phase separation and the important role of phase separation in transcriptional regulation are reviewed, which will provide reference for understanding basic cell activities and gene regulation in cancer.

Objective To monitor the susceptibility of clinical isolates to antimicrobial agents in tertiary hospitals in major regions of China in 2022.Methods Clinical isolates from 58 hospitals in China were tested for antimicrobial susceptibility using a unified protocol based on disc diffusion method or automated testing systems.Results were interpreted using the 2022 Clinical &Laboratory Standards Institute(CLSI)breakpoints.Results A total of 318 013 clinical isolates were collected from January 1,2022 to December 31,2022,of which 29.5％were gram-positive and 70.5％were gram-negative.The prevalence of methicillin-resistant strains in Staphylococcus aureus,Staphylococcus epidermidis and other coagulase-negative Staphylococcus species(excluding Staphylococcus pseudintermedius and Staphylococcus schleiferi)was 28.3％,76.7％and 77.9％,respectively.Overall,94.0％of MRSA strains were susceptible to trimethoprim-sulfamethoxazole and 90.8％of MRSE strains were susceptible to rifampicin.No vancomycin-resistant strains were found.Enterococcus faecalis showed significantly lower resistance rates to most antimicrobial agents tested than Enterococcus faecium.A few vancomycin-resistant strains were identified in both E.faecalis and E.faecium.The prevalence of penicillin-susceptible Streptococcus pneumoniae was 94.2％in the isolates from children and 95.7％in the isolates from adults.The resistance rate to carbapenems was lower than 13.1％in most Enterobacterales species except for Klebsiella,21.7％-23.1％of which were resistant to carbapenems.Most Enterobacterales isolates were highly susceptible to tigecycline,colistin and polymyxin B,with resistance rates ranging from 0.1％to 13.3％.The prevalence of meropenem-resistant strains decreased from 23.5％in 2019 to 18.0％in 2022 in Pseudomonas aeruginosa,and decreased from 79.0％in 2019 to 72.5％in 2022 in Acinetobacter baumannii.Conclusions The resistance of clinical isolates to the commonly used antimicrobial agents is still increasing in tertiary hospitals.However,the prevalence of important carbapenem-resistant organisms such as carbapenem-resistant K.pneumoniae,P.aeruginosa,and A.baumannii showed a downward trend in recent years.This finding suggests that the strategy of combining antimicrobial resistance surveillance with multidisciplinary concerted action works well in curbing the spread of resistant bacteria.

Objective:To investigate the effect of progression of disease within 24 months(POD24)on overall survival(OS)in patients with mantle cell lymphoma(MCL),and compare the clinical characteristics between POD24 and non-POD24 patients.Methods:A retrospective analysis was performed on 50 MCL patients with treatment indications and regular treatment who were admitted to the Affiliated Hospital of Xuzhou Medical University from January 2010 to August 2020.According to the occurrence of POD24,the patients were grouped for prognostic evaluation and clinical characteristics comparison.Results:Univariate Cox regression analysis showed that POD24,PLT,albumin,MIPI score,ECOG PS score,LDH were the factors influencing OS in newly diagnosed MCL patients(all P＜0.05).The results of multivariate Cox regression analysis showed that POD24[HR=16.797(95％CI:3.671-76.861),P＜0.001],albumin＜40 g/L[HR=3.238(95％CI:1.095-9.572),P=0.034]and ECOG PS score≥2[HR=4.005(95％CI:1.033-15.521),P=0.045]were independent risk factors influencing OS in MCL patients.The incidence of PLT＜100 × 109/L(33.3％vs 5.9％,P=0.033)and ECOG PS score≥2(45.5％vs 5.9％,P=0.040)were significantly higher in POD24 patients than those in non-POD24 patients.Conclusion:POD24 is an independent poor prognostic factor affecting the OS of MCL patients,and the patients with PLT＜100 × 109/L and ECOG PS score ≥2 at diagnosis have a higher probability of POD24.

Objective:To investigate the effect of endothelial activation and stress index(EASIX)on the prognosis of patients with angioimmunoblastic T-cell lymphoma(AITL)and peripheral T-cell lymphoma,not otherwise specified(PTCL-NOS),and to compare the clinical characteristics of patients in the low EASIX and high EASIX groups.Methods:The clinical data of 59 newly diagnosed AITL and PTCL-NOS patients admitted to the Affiliated Hospital of Xuzhou Medical University from January 2010 to September 2021 were retrospectively analyzed.The optimal cut-off value of EASIX was determined by receiver operating characteristic(ROC)curve;The chi-square test was used to analyze the correlation between EASIX and clinical features of patients with AITL and PTCL-NOS;The Kaplan-Meier survival curve was used to analyze the overall survival(OS)and progression-free survival(PFS)of the patients;Univariate and multivariate analyses were performed by using Cox proportional hazards model.Results:The optimal cut-off value of EASIX was 0.95,based on which the patients were divided into a low EASIX(＜0.95)group and a high EASIX(≥ 0.95)group.Compared with the low EASIX group,the high EASIX group had a higher proportion of patients with advanced Ann Arbor stage,higher risk according to IPI,elevated LDH,hypoproteinemia,anemia,B symptoms,extranodal involvement,and bone marrow involvement.Survival analysis showed that the OS and PFS of patients in the high EASIX group were significantly shorter than those in the lower EASIX group(P＜0.001).The multivariate analysis showed that EASIX was an independent risk factor for OS[HR=7.217(95％CI:1.959-26.587),P=0.003]and PFS[HR=2.718(95％CI:1.032-7.161),P=0.043]of PTCL patients.Conclusion:High EASIX in newly diagnosed patients with AITL and PTCL-NOS suggests a poor prognosis,and high EASIX is a risk factor affecting prognosis of the patients.

Refractory prolactinoma is the most common pituitary neuroendocrine tumor.Dopamine receptor agonists(DA)are the primary choice for drug treatment.Most patients with prolactinomas respond well to DA.However,a minority of prolactinomas patients still show resistance to DA.Although drug-resistant and refractory prolactinomas are rare in clinical practice,their treatment is extremely challenging.Even a combination of drug therapy,multiple surgeries,and radiotherapy may not yield satisfactory outcomes.Therefore,standardizing the diagnosis and treatment process and pathway for refractory prolactionmas and exploring more effective multidisciplinary collaborative treatment strategies are urgent problems to be solved.In the clinical management of refractory prolactinomas,it is often necessary to consider the patient's condition comprehensively,replace other types of DA,or consider surgery,radiotherapy,and immunotherapy,which requires multidisciplinary diagnosis and treatment.This review synthesizes the latest literature at home and abroad to systematically discuss the latest advances in drug therapy,surgery,and radiotherapy treatments for refractory prolactionmas,aiming to provide new ideas for basic research,clinical diagnosis and treatment.

Network pharmacology, molecular docking, and in vivo and in vitro experiments were employed to study the molecular mechanism of Blaps rynchopetera Fairmaire in the treatment of non-small cell lung cancer(NSCLC). The components of B. rynchopetera were collected by literature review, and the active components were screened out through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). PharmMapper was used to obtain the targets of the active components. The targets of NSCLC were obtained from DrugBank, GeneCards, OMIM, TTD, and PharmGKB. The Venn diagram was drawn to identify the common targets shared by the active components of B. rynchopetera and NSCLC. The "drug component-target" network and protein-protein interaction(PPI) network were constructed by Cytoscape, and the key targets were screened by Centiscape. Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment of the above key targets were performed by DAVID. AutoDock and PyMOL were used for the molecular docking between the key targets and corresponding active components. A total of 31 active components, 72 potential targets, and 11 key targets of B. rynchopetera against NSCLC were obtained. The active components of B. rynchopetera had good binding activity with key targets. Further, the serum containing B. rynchopetera was prepared and used to culture human lung adenocarcinoma A549 cells. The CCK-8 assay was employed to determine the inhibition rates on the growth of A549 cells in blank control group and those exposed to different concentrations of B. rynchopetera-containing serum, cisplatin, and drug combination(B. rynchopetera-containing serum+cisplatin) for different time periods. The cell migration and invasion of A549 cells were detected by cell scratch assay and Transwell assay, respectively. Western blot was employed to determine the expression levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X(Bax), caspase-3, cell division cycle 42(CDC42), proto-oncogene tyrosine-protein kinase SRC, and vascular endothelial growth factor(VEGF) in A549 cells. C57BL/6 mice were inoculated with Lewis cells and randomly assigned into a model control group, a B. rynchopetera group, a cisplatin group, and a drug combination(B. rynchopetera+cisplatin) group, with 12 mice per group. The body weight and the long diameter(a) and short diameter(b) of the tumor were monitored every other day during treatment, and the tumor volume(mm~3) was calculated as 0.52ab~2. After 14 days of continuous medication, the mice were sacrificed for the collection of tumor, spleen, and thymus, and the tumor inhibition rate and immune organ indexes were calculated. The tissue morphology of tumors was observed by hematoxylin-eosin(HE) staining, and the positive expression of Bax, Bcl-2, caspase-3, CDC42, SRC, and VEGF in the tumor tissue was detected by immunohistochemistry. The results indicated that B. rynchopetera and the drug combination regulated the expression levels of Bax, Bcl-2, caspase-3, CDC42, SRC, and VEGF to inhibit the proliferation, migration, and invasion of A549 cells and Lewis cells, thus playing a role in the treatment of NSCLC via multiple ways.
Humans ; Animals ; Mice ; Mice, Inbred C57BL ; Carcinoma, Non-Small-Cell Lung/genetics* ; Caspase 3 ; Network Pharmacology ; Vascular Endothelial Growth Factor A ; Cisplatin ; Molecular Docking Simulation ; bcl-2-Associated X Protein ; Lung Neoplasms/genetics* ; Cell Proliferation ; Drugs, Chinese Herbal/pharmacology* ; Medicine, Chinese Traditional

Objective: Retrospective analysis of the efficacy and influencing factors of bladder preservation integrated therapy for unresectable invasive bladder cancer confined to the pelvis was done, also including the bladder function preservation and adverse effects analysis. Methods: Sixty-nine patients with unresectable locally invasive bladder cancer who received radiotherapy-based combination therapy from March 1999 to December 2021 at our hospital were selected. Among them, 42 patients received concurrent chemoradiotherapy, 32 underwent neoadjuvant chemotherapyand 43 with transurethral resection of bladder tumors (TURBT) prior to radiotherapy. The late adverse effect of radiotherapy, preservation of bladder function, replase and metastasis and survival were followed-up. Cox proportional hazards models were applied for the multifactorial analysis. Results: The median age was 69 years. There were 63 cases (91.3%) of uroepithelial carcinoma, 64 of stage Ⅲ and 4 of stage Ⅳ. The median duration of follow-up was 76 months. There were 7 grade 2 late genito urinary toxicities, 2 grade 2 gastrointestinal toxicities, no grade 3 or higher adverse events occurred. All patients maintained normal bladder function, except for 8 cases who lost bladder function due to uncontrolled tumor in the bladder. Seventeen cases recurred locally. There were 11 cases in the concurrent chemoradiotherapy group with a local recurrence rate of 26.2% (11/42) and 6 cases in the non-concurrent chemoradiotherapy group with a local recurrence rate of 22.2% (6/27), and the difference in local recurrence rate between the two groups was not statistically significant (P=0.709). There were 23 cases of distant metastasis (including 2 cases of local recurrence with distant metastasis), including 10 cases in the concurrent chemoradiotherapy group with a distant metastasis rate of 23.8% (10/42) and 13 cases in the non-concurrent chemoradiotherapy group with a distant metastasis rate of 48.1% (13/27), and the distant metastasis rate in the non-concurrent chemoradiotherapy group was higher than that in the concurrent chemoradiotherapy group (P=0.036). The median 5-year overall survival (OS) time was 59 months and the OS rate was 47.8%. The 5-year progression-free survival (PFS) time was 20 months and the PFS rate was 34.4%. The 5-year OS rates of concurrent and non-concurrent chemoradiotherapy group were 62.9% and 27.6% (P<0.001), and 5-year PFS rates were 45.4% and 20.0%, respectively (P=0.022). The 5-year OS rates of with or without neoadjuvant chemotherapy were 78.4% and 30.1% (P=0.002), and the 5-year PFS rates were 49.1% and 25.1% (P=0.087), respectively. The 5-year OS rates with or without TURBT before radiotherapy were 45.5% and 51.9% (P=0.233) and the 5-year PFS rates were 30.8% and 39.9% (P=0.198), respectively. Multivariate Cox regression analysis results showed that the clinical stage (HR=0.422, 95% CI: 0.205-0.869) was independent prognostic factor for PFS of invasive bladder cancer. The multivariate analysis showed that clinical stages (HR=0.278, 95% CI: 0.114-0.678), concurrent chemoradiotherapy (HR=0.391, 95% CI: 0.165-0.930), neoadjuvant chemotherapy (HR=0.188, 95% CI: 0.058-0.611), and recurrences (HR=10.855, 95% CI: 3.655-32.638) were independent prognostic factors for OS of invasive bladder cancer. Conclusion: Unresectable localized invasive bladder cancer can achieve satisfactory long-term outcomes with bladder-preserving combination therapy based on radiotherapy, most patients can retain normal bladder function with acceptable late adverse effects and improved survival particularly evident in patients with early, concurrent chemoradiotherapy and neoadjuvant chemotherapy.
Humans ; Aged ; Treatment Outcome ; Retrospective Studies ; Combined Modality Therapy ; Chemoradiotherapy/methods* ; Urinary Bladder Neoplasms/radiotherapy* ; Antineoplastic Combined Chemotherapy Protocols/therapeutic use* ; Neoplasm Staging

Abstract：Objective To prepare human monoclonal antibody against spike protein（S protein）of severe acute respiratory syndrome coronavirus 2（SARS⁃CoV⁃2）by using single B cell，and determine its neutralizing activity. Methods Venous blood with high antibody level was collected from people immunized with inactivated SARS⁃CoV⁃2 vaccine（Vero cells） twice，of which peripheral blood mononuclear cells（PBMCs）were isolated by lymphocyte stratified fluid and used to isolate single B cell expressing S protein antibody by magnetic beads coupled with S1 protein. Variable region genes of IgG heavy chain and light chain were amplified by nested PCR after reverse transcription of single B cell，which were connected with CMV promoter，IgG leader sequence，IgG constant region and polyA sequence by overlapping PCR to construct antibody linear expression cassette. Linear expression cassette of the heavy chain and light chain from the same B cell was transfected to HEK293T cells to express human monoclonal antibody of SARS⁃CoV⁃2 S protein. Immunoreactivity was detected by immuno⁃ fluorescence while neutralizing activity by pseudovirus neutralization test. Results A total of 26 monoclonal antibodies against SARS⁃CoV⁃2 S protein were expressed，which showed heavy chain and light chain protein bands of IgG antibody at

Abstract： Objective To develop and validate a reverse phase⁃high performance liquid chromatography（RP⁃HPLC） method for determination of residual N⁃hydroxy succinimide（NHS）content in semaglutide. Methods A RP⁃HPLC method was developed based on the screening of chromatographic column and optimization of mobile phase（phosphate concentration and the ratio of acetonitrile），validated for specificity，suitability，accuracy，reproducibility and stability， and determined for linear range，limit of quantitation（LOQ）and limit of detection（LOD）. The NHS contents in three batches of semaglutide were determined by the developed method. Results The optimal condition for RP⁃HPLC was as follows：CAPCELL PAK ADME column（4. 6 mm × 150 mm，3 μm）was adopted，serving 0. 05 mol／L potassium dihy⁃ drogen phosphate solution⁃acetonitrile（98∶2）as mobile phase A，and 70% acetonitrile as mobile phase B with gradient elution（0 min，0% B；10 min，0% B；19 min，90% B；19. 1 min，0% B；25 min，0% B）at a flow rate of 0. 8 mL／min. The detection wave length was set at 260 nm，while the column temperature was 30 ℃. The developed method showed good specificity and systemic suitability，of which the linear range was 0. 2 ~ 3. 0 μg／mL（R2 = 1. 000 0），while the LOD and LOQ were 4. 8 and 9. 6 ng respectively. The RSD of recovery rates of NHS samples at three concentrations was 0. 58%， indicating a high accuracy. The RSD of NHS contents in six test samples was 0. 16%，indicating a high reproducibility. The RSD of peak areas of NHS after storage at room temperature for 0，4，8，12，16，20 and 24 h was 0. 34%，indicating a high stability. No NHS was detected in three batches of semaglutide by the developed method. Conclusion The developed RP⁃HPLC method is simple and sensitive，which may be used for the determination of NHS content in semaglutide.

The rol genes on pRiA4 plasmid of Agrobacterium rhizogenes are potent genes that promote secondary metabolism. Molecular breeding of Atropa belladonna can be conducted by introducing rol genes to increase tropane alkaloids (TAs) content in A. belladonna. In this study, the rolB gene was overexpressed in A. belladonna plants to study the effect of rolB gene on the biosynthesis of TAs. The phenotype, TAs content and expression levels of key enzyme genes in the pathway of TAs biosynthesis of transgenic A. belladonna were analyzed. The results showed that transgenic A. belladonna had developed root system, enlarged leaves, increased leaf fresh weight, deepened leaf color, enlarged flowers, changed flower shape, reduced pistil height and decreased pollen vitality. The content of TAs in the stems of transgenic A. belladonna was significantly higher than that of the control, and the contents of scopolamine, anisodamine, hyoscyamine can reach 2.11-2.91, 1.23-2.37 and 4.88-5.20 times of the control, respectively. Compared with the control group, the expressions of key enzymes putrescine N-methyltransferase (PMT), type III polyketide synthase (PYKS), tropinone reductase I (TRI), aromatic amino acid aminotransferase 4 (ArAT4), UDP-glycosyltransferase 1 (UGT1) and hyoscyamine 6-β-hydroxylase (H6H) in the TAs biosynthesis pathway were up-regulated, and the expression of tropinone reductase II (TRII) as a metabolic shunting gene was down-regulated. The results indicated that rolB gene enhanced TAs synthesis ability in roots and accumulation in stems of A. belladonna by enhancing metabolic flow of TAs synthesis pathway and weakening the metabolic shunt of competing pathway. This study laid a foundation for molecular breeding of A. belladonna with high-yield TAs content using rolB gene.