Correlation analysis of liver tumor-associated genes with liver regeneration

AIM: To study at transcriptional level the similarities and differences of the physiological and biochemical activities between liver tumor (LT) and regenerating liver cells. METHODS: LT-associated genes and their expression changes in LT were obtained from databases and scientific articles, and their expression profiles in rat liver regeneration (LR) were detected using Rat Genome 230 2.0 array. Subsequently their expression changes in LT and LR were compared and analyzed. RESULTS: One hundred and twenty one LT-associated genes were found to be LR-associated. Thirty four genes were up-regulated, and 14 genes were down-regulated in both LT and regenerating liver; 20 genes up-regulated in LT were down-regulated in regenerating liver; 21 up-regulated genes and 16 down-regulated genes in LT were up-regulated at some time points and down-regulated at others during LR. CONCLUSION: Results suggested that apoptosis activity suppressed in LT was still active in regenerating liver, and there are lots of similarities and differences between the LT and regenerating liver at the aspects of cell growth, proliferation, differentiation, migration and angiogenesis.

Expression pattern and action analysis of genes associated with the responses to chemical stimuli during rat liver regeneration

AIM: To study the genes associated with the responses to chemokines, nutrients, inorganic substances, organic substances and xenobiotics after rat partial hepatectomy (PH) at transcriptional level. METHODS: The associated genes involved in the five kinds of responses were obtained from database and literature, and the gene expression changes during liver regeneration in rats were checked by the Rat Genome 230 2.0 array. RESULTS: It was found that 60, 10, 9, 6, 26 genes respectively participating in the above five kinds of responses were associated with liver regeneration. The numbers of initially and totally expressed genes occurring in the initial phase of liver regeneration (0.5-4 h after PH), G0/G1 transition (4-6 h after PH), cell proliferation (6-66 h after PH), cell differentiation and structure- functional reconstruction (66-168 h after PH) were 51, 19, 52, 6 and 51, 43, 98, 68 respectively, illustrating that the associated genes were mainly triggered in the initiation and transition stages, and functioned at different phases. According to their expression similarity, these genes were classified into 5 groups: only up- regulated (47), predominantly up-regulated (18), only down-regulated (24), predominantly down-regulated (10), and up- and down-regulated (8). The total times of their up-regulated and down-regulated expression were 441 and 221, demonstrating that the number of up- regulated genes is more than that of the down-regulated genes. Their time relevance and gene expression patterns were classifi ed into 14 and 26 groups, showing that the cell physiological and biochemical activitieswere staggered, diversifi ed and complicated during liver regeneration in rats. CONCLUSION: The chemotaxis was enhanced mainly in the forepart and metaphase of LR. The response of regenerating liver to nutrients and chemical substances was increased, whereas that to xenobiotics was not strong. One hundred and seven genes associated with LR play important roles in the responses to chemical substances.

Expression patterns and action analysis of genes associated with physiological responses during rat liver regeneration:Cellular immune response

AIM： To study the cellular immune response during rat liver regeneration （LR） at a transcriptional level. METHODS： Genes associated with the cellular immune response were obtained by collecting the data from databases and retrieving articles. Gene expression changes during LR were detected by rat genome 230 2.0 array. RESULTS： A total of 127 genes were found to be associated with LR. The number of initially and totally expressing genes in the initial phase of LR [0.5-4 h after partial hepatectomy （PH）], transition from Go-G, （4-6 h after PH）, cell proliferation （6-66 h after PH）, cell differentiation and structure-function reconstruction （66-168 h after PH） was 54, 11, 34, 3 and 54, 49, 70, 49 respectively, illustrating that the associated genes were mainly triggered at the initiation of LR, and worked at different phases. According to their expression similarity, these genes were classified into 41 up-regulated, 21 predominantly up-regulated, 41 down-regulated, 14 predominantly down-regulated, 10 similarly up-regulated and down-regulated genes, respectively. The total up- and down-regulated expression times were 419 and 274, respectively, demonstrating that the expression of most genes was increased while the expression of a small number of genes was decreased. Their time relevance was classified into 14 groups, showing that the cellular physiological and biochemical activities were staggered during LR. According to the gene expression patterns, they were classified into 21 types, showing the activities were diverse and complicated during LR.CONCLUSION： Antigen processing and presentation are enhanced mainly in the forepart, prophase and anaphase of LR. T-cell activation and antigen elimination are enhanced mainly in the forepart and prophase of LR. A total of 127 genes associated with LR play an important role in cellular immunity.

Mosquito densovirus signifcantly reduces the vector susceptibility to dengue virus serotype 2 in Aedes albopictus mosquitoes(Diptera:Culicidae)

Background Dengue virus(DENV)is a major public health threat,with Aedes albopictus being the confrmed vector responsible for dengue epidemics in Guangzhou,China.Mosquito densoviruses(MDVs)are pathogenic mosquitospecifc viruses,and a novel MDV was previously isolated from Ae.albopictus in Guangzhou.This study aims to determine the prevalence of MDVs in wild Ae.albopictus populations and investigate their potential interactions with DENV and impact on vector susceptibility for DENV.Methods The prevalence of MDV in wild mosquitoes in China was investigated using open access sequencing data and PCR detection in Ae.albopictus in Guangzhou.The viral infection rate and titers in MDV-persistent C6/36 cells were evaluated at 12,24,48,72,96,and 120 h post infection(hpi)by indirect immunofuorescence assay(IFA)and real time quantitative PCR(RT-qPCR).The midgut infection rate(MIR),dissemination rate(DR),and salivary gland infection rate(SGIR)in various tissues of MDV-infected mosquitoes were detected and quantifed at 0,5,10,and 15 days post infection(dpi)by RT-PCR and RT-qPCR.The chi-square test evaluated dengue virus serotype 2(DENV-2)and Aedes aegypti densovirus(AaeDV)infection rates and related indices in mosquitoes,while Tukey’s LSD and t-tests compared viral titers in C6/36 cells and tissues over time.Results The results revealed a relatively wide distribution of MDVs in Aedes,Culex,and Anopheles mosquitoes in China and an over 68%positive rate.In vitro,signifcant reductions in DENV-2 titers in supernatant at 120 hpi,and an appar‑ent decrease in DENV-2-positive cells at 96 and 120 hpi were observed.In vivo,DENV-2 in the ovaries and salivary glands was frst detected at 10 dpi in both monoinfected and superinfected Ae.albopictus females,while MDV super‑infection with DENV-2 suppressed the salivary gland infection rate at 15 dpi.DENV-2 titer in the ovary and salivary glands of Ae.albopictus was reduced in superinfected mosquitoes at 15 dpi.Conclusions MDVs is widespread in natural mosquito populations,and replication of DENV-2 is suppressed in MDVinfected Ae.albopictus,thus reducing vector susceptibility to DENV-2.Our study supports the hypothesis that MDVs may contribute to reducing transmission of DENV and provides an alternative strategy for mosquito-transmitted disease control.

Integrated analysis of miRNAs and transcriptomes in Aedes albopictus midgut reveals the differential expression profiles of immune-related genes during dengue virus serotype-2 infection

Mosquito microRNAs （miRNAs） are involved in host-virus interaction, and have been reported to be altered by dengue virus （DENV） infection in Aedes albopictus （Diptera： Culicidae）. However, little is known about the molecular mechanisms of Aedes albopictus midgut--the first organ to interact with DENV--involved in its resistance to DENV. Here we used high-throughput sequencing to characterize miRNA and messenger RNA （mRNA） expression patterns in Aedes albopictus midgut in response to dengue virus serotype 2. A total of three miRNAs and 777 mRNAs were identified to be differentially expressed upon DENV infection. For the mRNAs, we identified 198 immune-related genes and 31 of them were differentially expressed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses also showed that the differentially expressed immune-related genes were involved in immune response. Then the differential expression patterns of six immune-related genes and three miRNAs were confirmed by real-time reverse transcription polymerase chain reaction. Furthermore, seven known miRNA-mRNA interaction pairs were identified by aligning our two datasets. These analyses of miRNA and mRNA transcriptomes provide valuable information for uncovering the DENV response genes and provide a basis for future study of the resistance mechanisms in Aedes albopictus midgut.

Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus

Aedes (Stegomyia) albopictus, also known as the Asian tiger mosquito, is a mosquito which originated in Asia. In recent years, it has become increasingly rampant throughout the world. This mosquito can transmit several arboviruses, including dengue, Zika and chikungunya viruses, and is considered a public health threat. Despite the urgent need of genome engineering to analyze specific gene functions, progress in genetical manipulation of Ae. albopictus has been slow due to a lack of efficient methods and genetic markers. In the present study, we established targeted disruptions in two genes, kynurenine hydroxylase (kh) and dopachrome conversion enzyme (yellow), to analyze the feasibility of generating visible phenotypes with genome editing by the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) system in Ae. albopictus. Following Cas9 single guide RNA ribonucleoprotein injection into the posterior end of pre-blastoderm embryos, 30%-50% of fertile survivors produced alleles that failed to complement existing kh and yellow mutations. Complete eye and body pigmentation defects were readily observed in GI pupae and adults, indicating successful generation of highly heritable mutations. We conclude that the CRISPR/Cas9-mediated gene editing system can be used mAe. albopictus and that it can be adopted as an efficient tool for genome-scale analysis and biological study.

Nicousamide, a potent inhibitor of phosphorylation by TGF-b receptor II

Renal fibrogenesis is related to the development of diabetic nephropathy.TGF-b receptor II(TGF-b RII)plays a vital role during renal fibrogenesis by phosphorylation and activation of type I receptors and downstream regulators.Nicousamide is a 1.1 class of drug,which can inhibit renal fibrosis in animal models of diabetic nephropathy.After cloning and purification of TGF-b RII,an in vitro substrate phosphorylation assay was set up to investigate the ability of nicousamide to inhibit phosphorylation and formation of cellular downstream proteins.Our results validate nicousamide as a potent inhibitor of phosphorylation of TGF-b RII.

Photoperiodic diapause in a subtropical population of Aedes albopictus in Guangzhou,China:optimized fieldlaboratory-based study and statistical models for comprehensive characterization

Background:Aedes albopictus is among the 100 most invasive species worldwide and poses a major risk to public health.Photoperiodic diapause provides a crucial ecological basis for the adaptation of this species to adverse environments.Ae.albopictus is the vital vector transmitting dengue virus in Guangzhou,but its diapause activities herein remain obscure.Methods:In the laboratory,yeast powder and food slurry were compared for a proper diapause determination method,and the critical photoperiod(CPP)was tested at illumination times of 11,11.5,12,12.5,13,and 13.5 h.A 4-parameter logistic(4PL)regression model was selected to estimate the CPP.In the field,the seasonal dynamics of the Ae.albopictus population,egg diapause,and hatching of overwintering eggs were investigated monthly,weekly,and daily,respectively.A distributed lag non-linear model(DLNM)was used to assess the associations of diapause with meteorological factors.Results:In the laboratory,both the wild population and the Foshan strain of Ae.albopictus were induced to diapause at an incidence greater than 80%,and no significant difference(P>0.1)was observed between the two methods for identifying diapause.The CPP of this population was estimated to be 12.312 h of light.In the field,all of the indexes of the wild population were at the lowest levels from December to February,and the Route Index was the first to increase in March.Diapause incidence displayed pronounced seasonal dynamics.It was estimated that the day lengths of 12.111 h at week2016,43 and 12.373 h at week2017,41 contributed to diapause in 50%of the eggs.Day length was estimated to be the main meteorological factor related to diapause.Conclusions:Photoperiodic diapause of Ae.albopictus in Guangzhou of China was confirmed and comprehensively elucidated in both the laboratory and the field.Diapause eggs are the main form for overwintering and begin to hatch in large quantities in March in Guangzhou.Furthermore,this study also established an optimized investigation system and statistical models for the study of Ae.albopictus diapause.These findings will contribute to the prevention and control of Ae.albopictus and mosquito-borne diseases.

Alternative splicing patterns of doublesex reveal a missing link between Nix and doublesex in the sex determination cascade of Aedes albopictus

Sexual development in insects is regulated by a complicated hierarchical cascade of sex determination.The primary signals are diverse,whereas the central nexus doublesex(dsx)gene is relatively conserved within the pathway.Aedes(Stegomyia)albopictus is an important vector with an extensive worldwide distribution.We previously reported that Ae.albopictus dsx(Aalbdsx)yields one male-(Aalbdsx^(M))and three female-specific isoforms(Aalbdsx^(F1-3));however,the spatiotemporal expression profiles and mechanisms regulating sex-specific alternative splicing require further investigation.In this study,we demonstrated that the Aalbdsx^(M) messenger RNA(mRNA)represents the default pattern when analyzed in human foreskin fibroblasts and HeLa cells.We combined reverse transcription polymerase chain reaction with RNA immunoprecipitation using specific antibodies against tagged Ae.albopictus male-determining factor AalNix and confirmed that AalNix indirectly regulates dsx pre-mRNA and regulates its alternative splicing.During the early embryo stage(0-2 and 4-8 h),maternal dsx^(F) and default splicing dsx^(M) were detected in both sexes;the expression of dsx^(M) then decreased until sufficient AalNix transcripts accumulated in male embryos at 20-24 h.These findings suggest that one or more potential dsx splicing enhancers can shift dsx^(M) to dsx^(F) in both sexes;however,the presence of Nix influences the function of this unknown splicing enhancer and ultimately leads to the formation of dsx^(M) in males.Finally,our results provide important insight into the regulatory mechanism of dsx alternative splicing in the mosquito.

XLN306 induces apoptosis in human breast carcinoma MX-1 cells

XLN306 is a novel synthetic quinazoline derivative with potentially useful anticancer activity.In previous research,we showed that XLN306 is highly cytotoxic to many tumor cell lines.This paper reports an investigation of this cytotoxicity in a number of human carcinoma cell lines.The results show that human breast carcinoma MX-1 cells are extremely sensitive to XLN306 and that the cytotoxicity is due to dose-and time-dependent apoptosis as confirmed by DAPI stain and DNA fragmentation analysis.Both extrinsic and intrinsic pathways are involved in the apoptosis process.The findings indicate that XLN306 has apoptotic induction activity and may be useful for the management of various cancers,especially breast carcinoma.

Armigeres subalbatus is a potential vector for Zika virus but not dengue virus

Background:Zika virus (ZIKV) and dengue virus (DENV) are closely related flaviviruses primarily transmitted by Aedes mosquitoes. Armigeres subalbatus is an emerging and widely distributed mosquito, and ZIKV has been detected and isolated from it. However, it is not clear whether Ar. subalbatus could be a vector for ZIKV and DENV or not. In this study, we investigated the infection and transmission of Ar. subalbatus to ZIKV and DENV.Methods:A line of Ar. subalbatus was isolated from Guangdong, China, and further identified by the mitochondrial cytochrome oxidase subunit 1 (COI) gene. The adults of Ar. subalbatus were fed with blood meal containing ZIKV or DENV-2. At 4, 7, 10, 14, and 21 days post-inoculation (dpi), the infections of ZIKV or DENV-2 in the midguts, ovaries and salivary glands were detected and quantified by RT-PCR and RT-qPCR. To assess the transmissibility, suckling mice were exposed to bites of ZIKV-infected mosquitoes, and ZIKV was detected in brain tissue by RT-qPCR and plaque assays. Furthermore, the larvae of Ar. subalbatus were reared in artificial urine containing ZIKV or DENV-2. The infection rates and viral titers of larvae and adults were analyzed by RT-PCR and RT-qPCR, and the viral distribution in larval tissues was observed by immunohistochemistry. Chi-square test and one-way ANOVA analysis were used for assessing the infection rate and viral titer in varied tissues and different time points, respectively.Results:Following oral inoculation, ZIKV but not DENV-2 could be detected in Ar. subalbatus midguts at 4 dpi, ovaries at 7 dpi and salivary glands at 10 dpi. The highest infection rate (IR) of ZIKV was 27.8% in midgut at 7 dpi, 9.7% in ovary and 5.6% in salivary gland at 21 dpi. Eight days after being bitten by ZIKV-positive mosquitoes, ZIKV was detected in three brain tissues out of four suckling mice exposed to bites. ZIKV could be detected in the larvae reared in artificial urine contained ZIKV at a high concentration of 105 pfu/ml and various tissues of adults with a low infection rate (0.70–1.35%). ZIKV could be observed in anal papillae and midgut of larvae at 4 dpi under laboratory conditions.Conclusions:ZIKV but not DENV-2 can infect Ar. subalbatus by blood meal and artificial urine, and the infected mosquitoes can transmit ZIKV to suckling mice by bite. From these findings, we can conclude that the Ar. subalbatus isolated from Guangdong province, China, is a potential vector for ZIKV and should therefore be considered in vector control programs to prevent and control of Zika virus disease.

Synthesis and in vitro cytotoxic activities of sorafenib derivatives

A series of novel sorafenib derivatives have been designed and synthesized.The cytotoxic activities of these compounds were tested in three tumor cell lines.Most of the compounds showed potent antiproliferative activity against the tested cell lines with IC50= 0–20 mmol/L.Some compounds demonstrated competitive antiproliferative activities to sorafenib against all three cancer cell lines.Among them,compound 5g demonstrated significant inhibitory activity against A549,ACHN and MDAMB-231 cell lines with IC50values of 1.29,1.99,3.11 mmol/L,respectively.

Susceptibility and interactions between Aedes mosquitoes and Zika viruses

Zika virus disease is caused by Zika virus infection,as transmitted by Aedes spp.mosquitoes.Many of the Zika virus strains isolated from patients display different pathogenicities toward humans.The vector mosquitoes for Zika virus are mainly of the Aedes genus,especially Aedes aegypti and Aedes albopictus.However,susceptibility and interactions between Aedes spp.mosquitoes and Zika viruses remain unclear.In this study,we chose two Zika virus strains(FSS 13025 and PRVABC59)with different abilities to infect the primary vector mosquitoes Ae.aegypti and Ae.albopictus.The transcriptomes and small RNA profiles of infected and uninfected mosquitoes were comparatively analyzed,and differentially expressed genes were functionally examined using RNA interference.According to the results,the susceptibility of PRVABC59 was higher than that of FSS13025 in Aedes vector mosquitoes,and Ae.aegypti was more susceptible to Zika virus than was Ae.albopictus.For PRVABC59 infection,specific differential expression profiles correlated with Ae.aegypti and Ae.albopictus,and susceptibility was significantly affected when three targeted genes were successfully knocked down.Compared with PRVABC59,infection of Ae.albopictus with FSS 13025 generated more 21-nt virus small interference RNA.It can be concluded that the susceptibility of vector Aedes spp.mosquitoes to Zika viruses varies and that the interactions between mosquitoes and Zika virus correlate with susceptibility.

Bed expansion behavior and sensitivity analysis for super-high-rate anaerobic bioreactor

Bed expansion behavior and sensitivity analysis for super-high-rate anaerobic bioreactor （SAB） were performed based on bed expansion ratio （E）, maximum bed sludge content （Vpmax）, and maximum bed contact time between sludge and liquid （Tmax）. Bed expansion behavior models were established under bed unfluidization, fluidization, and transportation states. Under unfluidization state, Ewas 0, Vprnax was 4867 ml, and rmax was 844-3800 s. Under fluidization state, E, Vpmax, and Tmax were 5.28%-255.69%, 1368-4559 ml, and 104-732 s, respectively. Under transportation state, washout of granular sludge occurred and destabilized the SAB. During stable running of SAB under fluidization state, E correlated positively with superficial gas and liquid velocities （Ug and ul）, while Vpmax and Tmax correlated negatively. For E and Vpmax, the sensitivities of ug and ul were close to each other, while for Tmax, the sensitivity of ur was greater than that of Ug. The prediction from these models was a close match to the experimental data.

4,5,6,7-Tetrahydro-[1,2,3]triazolo[1,5-a]pyrazine as a new scaffold for heat shock protein 90 inhibitors

Heat shock protein 90（hsp90） is a promising anticancer drug target. A library of 2,4-dihydroxyphenyl（resorcinol） substituted 4,5,6,7-tetrahydro-[1,2,3]triazolo[1,5-a]pyrazine compounds that target this protein were designed and prepared based on our earlier study. The compounds were tested in five cancer cell lines and seven of them showed notable anticancer activity（IC_（50）2-10 mmol/L）. The active subset compounds were further subjected to a polarized fluorescent assay and exhibited high binding affinity toward purified hsp90（IC_（50）60-100 nmol/L）. These results indicated that the tetrahydrotriazolopyrazine motif of the molecules may represent a novel scaffold for the development of hsp90 inhibitors.

Defervescent dengue patients might be a potential source of infection for vector mosquitoes

Background:Dengue is a re-emerging public health problem and mosquito-borne infectious disease that is transmitted mainly by A edes aegypti and Ae.albopictus.Early diagnosis,isolation,and treatment of patients are critical steps for dengue epidemic control,especially to prevent secondary transmission of dengue virus(DENV).However,little is known about defervescent dengue patients as a source of infection.Methods:This case study describes 1268 dengue patients hospitalized at Guangzhou Eighth People's Hospital from June 2013 to December 2014.The viral loads of each individual were measured using real-time reverse transcription-polymerase chain reaction.Ae.aegypti and Ae.albopictus were exposed to blood meal with gradated dengue viral loads to characterize the relationship between viremia in dengue patients and the vector competence of vector mosquitoes.Results:The viral numbers in the blood were measured,ranging from 108 to 103 copies/ml from day 1 to day 12 after fever onset Vector competence analysis o fA e.aegypti and Ae.albopictus indicated that viremia>104 copies/ml can still infect vector mosquitoes,which implied that the defervescent dengue patients might be a source of infection.Conclusions:The results of this study indicate that some defervescent dengue patients still have sufficient viral load to infect vector mosquitoes.Therefore,the protection against mosquito biting for these people should be extended to prevent secondary transmission events.