Identification of suitable reference genes for quantitative gene expression analysis in clam Cyclina sinensis under salinity stress and Vibrio infection

The appropriate reference gene is a prerequisite for accurate normalization of gene expression level,and research on suitable reference genes in clam Cyclina sinensis is scarce.To improve the situation,we selected five commonly used housekeeping genes,including β-actin,Elongation factor 1-α(EF1-α),Glyceraldehyde-3-pho sphate dehydrogenase(GAPDH),40S ribosomal protein S18(RPS18),and Tubulin a(TUB-α),then evaluated their expression stability in different adult tissues and under different experimental treatments(salinity stress and Vibrio parahaemolyticus infection).Their expression stability was analyzed by three frequently used programs,geNorm,NormFinder,and BestKeeper.This analysis indicated that multiple genes should be used for normalization,and we concluded that the reference gene combination GAPDH-RPS18-β-actin,should be used for qRT-PCR analysis in different tissues of C.sinensis under normal physiological conditions.For the clams under salinity stress and Vibrio infection,EF1-α-GAPDHRPS18 was recommended as the gene combination for qRT-PCR normalization.TUB-αwas generally poorly ranked by all programs,and should not be used in future studies.This study should provide fundamental support for accurate quantitative gene expression analysis of this species.

Effect of Salinity on the Growth Performance, Body Composition, Antioxidant Indexes of Perinereis aibuhitensis and Total Nitrogen in the Substrate

In this study, the effects of different salinity levels (9, 12, 15, 18, 21, 24, 27, 30, and 33) on the growth performance, body composition, antioxidant indexes of Perinereis aibuhitensis (initial average mass, 20.4 ± 0.3 mg) and total nitrogen in the substrate were investigated. The survival rate, specific growth rate, feed coefficient, and protein efficiency ratio under different salinity levels were measured. The results showed that the survival rate of P. aibuhitensis at the salinity level of 9 was significantly lower than that of P. aibuhitensis at other salinity levels (P P. aibuhitensis at other salinity levels was not significant (P > 0.05). On the basis of quadratic polynomial fitting of the relationship between salinity levels and the specific growth rate, feed coefficient, and protein efficiency ratio, it was concluded that 25.36 - 25.9 is the most suitable salinity range for the growth performance of P. aibuhitensis. The main body composition (moisture, crude fat, crude protein, and ash content) was measured at different salinity levels. The results indicated that, with the increase in salinity, the moisture content of P. aibuhitensis decreased gradually;in contrast, the ash content increased gradually, as the salinity level increased. However, in the salinity range of 18 to 33, the difference in ash content was not significant (P > 0.05). Salinity had a significant influence on the crude protein content (P P. aibuhitensis specimens were cultured for 60 days was higher than the total nitrogen in the soil. With an increase in salinity, the total nitrogen content first decreased and then increased, and the lowest value was observed at the salinity level of about 24.

Accuracy of glomerular filtration rate estimation equations in patients with hematopathy

Renal dysfunction is a common side-effect of chemotherapeutic agents in patients with hematopathy. Although broadly used, glomerular filtration rate(GFR) estimation equations were not fully validated in this specific population. Thus, this study was designed to further assess the accuracy of various GFR equations, including the newly 2012 CKD-EPI equations. Referring to ^(99m)Tc-DTPA clearance method, three Scr-based(MDRD, Peking, and CKD-EPI_(Scr)), three Scys C-based(Steven 1, Steven 2, and CKD-EPI_(Scys C)), and three Scr-Scys C combination based(Ma, Steven 3, and CKD-EPI_(Scr-Scys C)) equations were included. Bias, P_(30), and misclassification rate were applied to compare the applicability of the selected equations. A total of 180 Chinese hematological patients were enrolled.Mean bias, absolute mean bias, P_(30), misclassification rate and Bland-Altman plots of the CKD-EPI_(Scr-Scys C) equation were 7.90 mL/minute/1.73 m^2, 17.77 mL/minute/1.73 m^2, 73.3%, 38% and 79.7 mL/minute/1.73 m^2, respectively.CKD-EPI_(Scr-Scys C) predicted the most precise eGFR both in lymphoma and leukemia subgroups. Additionally, CKDEPI_(Scys C) equation in the rGFR■90 mL/minute/1.73 m^2 subgroup and Steven 2 equation in the rGFR<90 mL/minute/1.73 m^2 subgroup provided more accurate estimates in each subgroup. The CKD-EPI_(Scr-Scys C) equation could be recommended to monitor kidney function in hematopathy patients. The accuracy of GFR equations may be closely related with GFR level and kidney function markers, but not the primary cause of hematopathy.

Constructing and packaging scAAV vectors and detecting their transduction efficiency

Gene therapy is a form of molecular medicine that promises to provide new treatments for a large number of inherited and acquired diseases.One of the biggest stumbling blocks to successful widespread application of genetic treatments is the development of safe and effective vectors with which to ferry genetic material into cells.Adeno-associated virus(AAV),a nonpathogenic human parvovirus,has gained attention as a potentially safe vector for gene transfer and gene therapy.The AAV genome is a single-stranded DNA,which is transcriptionally inactive.It has been reported that viral second-strand DNA synthesis is the rate-limiting step in AAV-mediated transgene expression.Self-complementary adeno-associated viral(scAAV) vectors bypass the requirement for viral second-strand DNA synthesis,and lead to a significant increase in transduction efficiency.scAAV vectors have been successfully constructed by Xiao and Smulski in the USA in 2003.Recently,we have developed and packaged scAAV,and reported as following.Several endonuclease were used to remove the D-sequence,terminal resolution site(Trs),Rep and Cap gene of pSub201 to develop plasmid of scAAV.The plasmid of scAAV constructed by us,which contain 2.4 kb exogenous gene including CMV promoter driving hrGFP gene(CMVp-hrGFP),named pJQ2.Plasmids of pJQ2 and pAAV-hrGFP were respectively transfected into adenovirus trans-formed 293 cells,using the calcium phosphate triple-plasmid transfection protocol.Low relative molecular mass(low-Mr) DNA samples were isolated 72 h post-transfection by the procedure described by Hirt,digested extensively with DpnI and analyzed on Southern blots with a 32P-labeled hrGFP-specific DNA probe.It is evident that efficient rescue and replication of the recombinant AAV genome,represented by the characteristic monomeric(m) and dimeric(d) replicative DNA intermediates,occurred from both of pJQ2 and pAAV-hrGFP.Then,scAAV vectors were produced by the calcium phosphate triple-plasmid transfection protocol.The ratios of double-stranded(ds) and single-stranded(ss) AAV vectors were determined by electrophoresis on 1% alkaline agarose gels followed by South-ern blot analysis with a 32P-labeled hrGFP DNA probe.The results suggested that most of pJQ2 was successfully packaged into scAAV vectors,while pAAV-hrGFP was packaged only in single stranded AAV vectors.The titers of vJQ2 are similar to those of vAAV-hrGFP packaged at same time.In addition,the transduction efficiency in 293 and HeLa cells were measured in 48 h post-infection by fluores-cence microscope.The transduction efficiency of vJQ2 vectors was increased to 5-fold and 14-fold in 293 and HeLa cells,compared with conventional AAV.In summary,we have constructed and packaged double-stranded AAV,which has implications in the optimal use of recombinant AAV vectors in human gene therapy,and will promote the study in AAV mediated gene transfer.

Clinical characteristics of malignant germ cell tumors in adolescents:A multicenter 10-year retrospective study in Beijing

Background:The aim of this study was to review clinical features of adolescent malignant germ cell tumors(MGCTs)in Beijing and analyze the peculiar characteristics of this age group.Methods:Clinical characteristics,pathological presentations,and survival outcomes of 34 patients were analyzed retrospectively.Results:Of 34 patients,12 girls and 22 boys,18(52.9%)had an extra-cranial tumor,including one testicular tumor,five ovarian tumors,one sacrococcygeal tumor,and 11 mediastinal tumors.Histologically,we found immature teratomas(n=6),yolk sac tumors(n=5),mixed malignant tumors(n=5),an embryonic carcinoma(n=1),and seminoma(n=1).Three-year event-free survival(EFS)and overall survival(OS)were 48.8%and 62.9%,respectively.Another 16(47.1%)patients had an intracranial tumor,including nine in the pineal region,five in the suprasellar region,one in basal ganglia,and one in cerebellopontine.All patients had localized disease and an excellent outcome with 3-year EFS and OS of 93.7%and 100%,respectively.Conclusions:Adolescent MGCTs are rare with a strong dependence on gender,and the mediastina and pineal region are the most common tumor locations.The prognosis is promising compared with that of other adolescent tumors and MGCTs in other age groups.MGCTs in mediastina have a tendency to companion with other hematological malignancies,and the prognosis is extremely poor in these patients.

A real-world study of immune checkpoint inhibitors in advanced triple-negative breast cancer

Background:Triple-negative breast cancer(TNBC)is the most aggressive type of breast cancer.Immune checkpoint inhibitors(ICIs)have been widely used to treat various tumors and have changed the landscape of tumor management,but the data from real-world studies of ICIs for TNBC treatment remain limited.The aim of this study was to evaluate the efficacy of ICIs in the treatment of patients with advanced TNBC in a real-world setting and to explore possible correlates.Methods:The clinical data of advanced TNBC patients who received ICI treatment in the Chinese People's Liberation Army(PLA)General Hospital were collected.Treatment responses,outcomes and adverse events(AEs)were assessed.Results:Eighty-one patients were included in the study.The confirmed objective response rate(ORR)was 32.1%,and the disease control rate(DCR)was 64.2%.The median progression-free survival(PFS)was 4.2 months,and the median overall survival(OS)was 11.0 months.PFS and OS were longer in patients who achieved clinical benefit from ICIs and shorter in patients who received later-line ICIs and higher levels of inflammation;specifically,patients with higher TILs had longer PFS.Overall AEs were tolerable.Conclusions:ICIs are effective in the treatment of advanced TNBC,and the adverse reactions are tolerable.A panel of biomarkers including LDH,ALP,and bNLR were identified to predict the efficacies of ICIs in TNBC treatment.

Diagnosis and treatment of infantile malignant solid tumors in Beijing,China:A multicenter 10-year retrospective study

Importance:Cancer is the main cause of death by disease in children.Children experience the highest incidence of cancer in the first year of life.However,there is no comprehensive registration system for children with tumors in China.Objective:To summarize the diagnosis and treatment of infant cancer and analyze the status of standardized diagnosis and management among several treatment centers in Beijing,China,thereby providing evidence to guide further clinical research.Methods:From January 1,2010 to December 31,2019,patients with newly diagnosed infantile malignant solid tumors were admitted to six large tertiary pediatric solid tumor diagnosis and treatment centers in Beijing.The epidemiology,clinical features,and therapeutic effects of tumors in these patients were analyzed retrospectively.All patients were followed up until March 31,2020.Results:In total,938 patients were enrolled in this study.There were 530 boys(56.5%)and 408 girls(43.5%);the median age was 6.0 months(range,0-12.0 months).The three most common tumors were retinoblastoma in 366 patients(39.0%),neuroblastoma in 266 patients(28.4%),hepatoblastoma in 133 patients(14.2%),and central nervous system tumors in 52 patients(5.5%).The estimated 5-year overall survival rate was 81.3%±1.8%,and the 5-year event-free survival rate was 71.8%±2.9%.The 5-year overall survival rates of non-rhabdomyosarcoma soft tissue sarcoma,neuroblastoma,and retinoblastoma were 100%,88%±2.2%,and 86.9%±2.1%,respectively.The 5-year event-free survival rates were 81.1%±2.7%for neuroblastoma,81.6%±9.8%for non-rhabdomyosarcoma soft tissue sarcoma,and 72.7%±14.1%for extracranial malignant germ cell tumors.Interpretation:The three most common infantile malignant solid tumors were retinoblastoma,neuroblastoma,and hepatoblastoma.Multidisciplinary combined diagnosis and treatment is needed for infantile tumors.

竹叶椒抗炎内生真菌的分离

内生真菌富含具有多种生物活性的次生代谢产物。本研究从竹叶椒中分离鉴定了8种内生真菌,并通过体外实验对其抗菌活性进行了筛选。结果发现4种内生真菌提取物(Fusarium oxysporum Schlecht.(Z-1)、Alternaria arborescens E.G Simmons(Z-3)、Mucor fragilis Bainier(Z-4)和Aspergillus terreus Thom(Z-6))对LPS诱导的RAW264.7细胞一氧化氮过量生成具有显著抑制作用。其中,真菌Fusarium oxysporum Schlecht提取物具有最好的抗炎活性,其IC50值为174.0μg/m L。真菌Fusarium oxysporum Schlecht的抗炎活性与其抑制多种细胞炎症因子的释放有关。实时荧光定量PCR结果显示,真菌Fusarium oxysporum Schlecht对LPS诱导的RAW264.7细胞中Il-1β、Il-6、iNOS等炎症因子的m RNA表达具有显著抑制作用。总之,竹叶椒中多种内生真菌具有抗炎活性,其中Fusarium oxysporum Schlecht有望发展成为新的抗炎活性药物。

Fluorescence imaging mitochondrial copper(Ⅱ) via photocontrollable fluorogenic probe in live cells

Monitoring mitochondrial derived copper(Ⅱ) in live cells is highly demanded, but accurately detecting is unmet due to the interference with cytoplasmic copper(Ⅱ). Herein, we have reported the design,synthesis and characterization of photocontrollable fluorogenic probe, M_(Cu)^(-3), which is equipped with a photo-labile group(nitrobenzyl group) and mitochondria targeting unit(triphenylphosphonium salt).This novel probe showed an intense fluorescence enhancement in response to copper(Ⅱ) without interference from other metal cations in the biological condition(p H 6–9). The detection limit is 1.7 ×10^(-7) mol/L in HEPES buffer. The confocal fluorescence imaging results demonstrated M_(Cu)^(-3) can visualize mitochondrial copper(Ⅱ) in live mammalian cells. The clear advantage of our photocontrollable method is successful to avoid the influence of cytoplasmic copper(Ⅱ) during mitochondria specific detection.

Paclitaxel liposome for injection (Lipusu) plus cisplatin versus gemcitabine plus cisplatin in the first-line treatment of locally advanced or metastatic lung squamous cell carcinoma: A multicenter, randomized, open-label, parallel controlled clinical study

Background:Lipusu is the first commercialized liposomal formulation of pacli-taxel and has demonstrated promising efficacy against locally advanced lung squamous cell carcinoma(LSCC)in a small-scale study.Here,we conducted a multicenter,randomized,phase 3 study to compare the efficacy and safety of cis-platin plus Lipusu(LP)versus cisplatin plus gemcitabine(GP)as first-line treat-ment in locally advanced or metastatic LSCC.Methods:Patients enrolled were aged between 18 to 75 years,had locally advanced(clinical stage IIIB,ineligible for concurrent chemoradiation or surgery)or metastatic(Stage IV)LSCC,had no previous systemic chemother-apy and at least one measurable lesion as per the Response Evaluation Criteria in Solid Tumors(version 1.1)before administration of the trial drug.The primary endpoint was progression-free survival(PFS).The secondary endpoints included objective response rate(ORR),disease control rate(DCR),overall survival(OS),and safety profiles.To explore the possible predictive value of plasma cytokines for LP treatment,plasma samples were collected from the LP group at baseline and first efficacy evaluation time and were then subjected to analysis by 45-Plex ProcartaPlex Panel 1 to detect the presence of 45 cytokines using the Luminex xMAP technology.The correlation between treatment outcomes and dynamic changes in the levels of cytokines were evaluated in preliminary analyses.Results:The median duration of follow-up was 15.4 months.237 patients in the LP group and 253 patients in the GP group were included in the per protocol set(PPS).In the PPS,the median PFS was 5.2 months versus 5.5 months in the LP and GP group(hazard rtio[HR]:1.03,P=0.742)respectively.The median OS was 14.6 months versus 12.5 months in the LP and GP group(HR:0.83,P=0.215).The ORR(41.8%versus 45.9%,P=0.412)and DCR(90.3%versus 88.1%,P=0.443)were also similar between the LP and GP group.A significantly lower proportion of patients in the LP group experienced adverse events(AEs)leading to treatment interruptions(10.9%versus 26.4%,P<0.001)or treatment termination(14.3%versus 23.1%,P=0.011).The analysis of cytokine levels in the LP group showed that low baseline levels of 27 cytokines were associated with an increased ORR,and 15 cytokines were associated with improved PFS,with 14 cytokines,including TNF-a,IFN-y,IL-6,and IL-8,demonstrating an overlapping trend.Conclusion:The LP regimen demonstrated similar PFS,OS,ORR and DCR as the GP regimen for patients with locally advanced or metastatic LSCC but had more favorable toxicity profiles.The study also identified a spectrum of different cytokines that could be potentially associated with the clinical benefit in patients who received the LP regimen.

Workload-based randomization byzantine fault tolerance consensus protocol

This paper introduces a new Byzantine fault tolerance protocol called workload-based randomization Byzantine fault tolerance protocol(WRBFT).Improvements are made to the Practical Byzantine Fault Tolerance(PBFT),which has an important position in the Byzantine Fault consensus algorithm.Although PBFT has numerous ad-vantages,its primary node selection mechanism is overly fixed,the communication overhead of the consensus process is also high,and nodes cannot join and exit dynamically.To solve these problems,the WRBFT proposed in this paper combines node consensus workload and verifiable random function(VRF)to randomly select the more reliable primary node that dominates the consensus.The selection of the nodes involved in the consensus is based on the node workload,and the optimization of the agreement protocol of the PBFT is also based on this.Simulation results show that the WRBFT has higher throughput,lower consensus latency,and higher algorithmic efficiency compared to the PBFT.