Transcriptase-polymerase Chain Reaction Amplification Research Articles

Genotype classification and pathogenicity of infectious bursal disease virus circulating in vaccinated broiler chicken farms

This study investigated the genotype classification and pathogenicity of infectious bursal disease virus (IBDV) circulating in vaccinated broiler chicken farms in Egypt. A total of 150 samples were collected from 30 vaccinated commercial broiler chicken farms and pooled into 30 working samples. IBDV was tested using reverse transcriptase polymerase chain reaction (RT-PCR) amplification of the hypervariable region of the viral protein 2 (hvVP2) and the VP1 gene 5’ extremity. Both RT-PCR fragments were sequenced from six samples, and then the obtained nucleotide sequences were analyzed. The IBDV genotypes were identified using nucleotide sequences. Five sequences of the six strains examined were classified as genotype A3B2 for the highly virulent segments A and B (vv-A/vv-B IBDV). Interestingly, this study identified and classified a novel segment-reassortant strain as the A1B2 genotype. Specifically, it involved the segment reassortment of classical virulent segment A (cv-A) with vv-B producing cv-A/vv-B reassortant IBDV. Subsequently, we compared the pathogenicity of reassortant (cv-A/vv-B) IBDV and vvIBDV strains identified in this study. Both strains developed typical IBD clinical signs, postmortem lesions, histopathology, immunohistochemistry, and lesion scores, which were more severe in vvIBDV than reassortant IBDV. In conclusion, this is the first report of the genotype classification based on both genome segments (hvVP2 and VP1) with pathogenicity of IBDV circulating in vaccinated broiler chicken farms and this pathogenicity is more severe in vvIBDV strain than a novel reassortant IBDV strain.

Do established infection prevention and control measures prevent spread of SARS-CoV-2 to the hospital environment beyond the patient room?

SummaryThe role of the hospital environment in the transmission of infection is well described. With an emerging infection whose mode of transmission is under investigation, strict infection prevention and control measures, including patient isolation, hand hygiene, personal protective equipment that is doffed on exiting the patient room, and environmental cleaning should be implemented to prevent spread. Environmental testing demonstrated that COVID-19 patients contaminated the patient area (11/26, 42.3% of tests) but contamination of general ward areas was minimal (1/30, 3%) and the virus was detected after cleaning on one item only (1/25, 4%) which was noted to be in disrepair.

Clinical and computed tomographic (CT) images characteristics in the patients with COVID-19 infection: What should radiologists need to know?

OBJECTIVE:To evaluate the clinical and computed tomographic (CT) features in the patients with COVID-19 pneumonia confirmed by the real-time reverse transcriptase polymerase chain reaction (rRT-PCR) amplification of the viral DNA from a sputum sample.MATERIAL AND METHODS:Clinical information and CT findings of a total of 14 patients with COVID-19 infection (age range, 12–83 years; females 6) were analyzed retrospectively. The clinical information includes the history of exposure, laboratory results, and the symptoms (such as fever, cough, headache, etc.); CT findings of chest include the extension and distribution of lesion, the ground-glass opacity (GGO), consolidation, bronchovascular enlarged, irregular linear appearances, pleural effusion, and lymphadenopathy.RESULTS:Eight patients had the exposure history for recent travel to Wuhan of Hubei province (8/14, 57%), 6 had the exposure to patients with COVID-19 infection. Significant statistical differences were observed in lymphocyte percentage decreased and C-reactive protein elevated (p = 0.015). Seven patients had fever, 7 had cough, 2 had headache, 3 had fatigue, 1 had body soreness, 3 had diarrhea, and 2 had no obvious symptoms. In chest CT examination, 10 patients were positive (10/14, 71.43%). Among these patients, 9 had lesions involving both lungs (9/10, 90%), 8 had lesions involving 4 to 5 lobes (8/10, 80%). Most of lesions were distributed peripherally and the most significant lesions were observed in the right lower lobe in 9 patients (9/10, 90%). Nodules were observed in 5 patients (5/10, 50%); GGO, consolidation, and bronchovascular enlarged were shown in 9 patients (9/10, 90%); irregular linear appearances were revealed in 7 patients (7/10, 70%); and pleural effusions were exhibited in 2 patients (2/10, 20%). Last, no patients showed lymphadenopathy.CONCLUSION:There were some typical CT features for diagnosis of COVID-19 pneumonia. The radiologists should know these CT findings and clinical information, which could help for accurate analysis in the patients with 2019 novel coronavirus infection.

Transcriptional re-programming in rat central nervous system two weeks after burn trauma: the impact of nephrilin treatment on the expression of oxidative stress-related genes.

Introduction:Survivors of severe burns suffer lifetime neuroinflammatory consequences manifested by higher incidence of major depression and neurodegenerative disease. In a scald model, nephrilin peptide has previously been shown to protect rats from loss of lean body mass, kidney function and glycaemic control, complications that have also been shown to endure in burn patient populations. Nephrilin’s mechanism of action has been suggested to involve protection from excessive oxidative stress.Methods:Using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) amplification of transcripts in total RNA extracted from dorsal root ganglia of male rats 14 days after exposure to thermal insult, we query the relative levels of expression of 34 genes believed to be associated with oxidative stress biology in the central nervous system (CNS). We use these data to explore the central role of oxidative stress in astrogliosis, immunosuppression and mitochondrial homeostasis.Results and Discussion:Rats that received nephrilin treatment (4 mg/kg by subcutaneous bolus injection once daily for seven days after scald injury) showed significantly reduced elevations in gene expression of some key genes such as NOX2, GFAP, AQP4 and RAC1, but not of others such as NOX4, STEAP4, ARG1 and CCL2.Conclusion:The implications of these data with reference to nephrilin’s potential clinical utility for mitigating the enduring effects of burn trauma on the CNS are discussed. Nephrilin reduces the expression of some genes implicated in neurodegeneration after burn insult.Lay SummaryNephrilin peptide is a novel treatment for short- and long-term systemic effects of burn trauma. This study measures the capability of nephrilin to address post-traumatic neurodegenerative disease by looking at the expression of genes in the central nervous system, in a rat scald model. Nephrilin appears to have beneficial effects by reducing the expression of some key genes known to be relevant in neurodegenerative processes, but not others.

Eukaryotic elongation factor 2 is involved in the anticoccidial action of diclazuril in the second-generation merozoites of Eimeria tenella

Eimeria tenella, an obligate intracellular parasite, can actively invade the cecal epithelial cells of chickens and cause severe enteric disease. Eukaryotic elongation factor 2 (eEF2) plays a major role in protein synthesis and cell survival. This study aims to explore the exact mechanisms underlying diclazuril inhibition in second-generation merozoites of E. tenella. The eEF2 cDNA of the second-generation merozoites of E. tenella (EtEF2) was cloned by reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends. Diclazuril-induced expression profiles of EtEF2 were also analyzed. The cloned full-length cDNA (2893 bp) of the EtEF2 nucleotide sequence encompassed a 2499 bp open reading frame (ORF) that encoded a polypeptide of 832 residues with an estimated molecular mass of 93.12 kDa and a theoretical isoelectric point of 5.99. The EtEF2 nucleotide sequence was submitted to the GenBank database with the accession number KF188423. The EtEF2 protein sequence shared 99 % homology with the eEF2 sequence of Toxoplasma gondii (GenBank XP_002367778.1). The GTPase activity domain and ADP-ribosylation domain were conserved signature sequences of the eEF2 gene family. The changes in the transcriptional and translational levels of EtEF2 were detected through quantitative real-time PCR and Western blot analyses. The mRNA expression level of EtEF2 was 2.706 fold increases and the protein level of EtEF2 was increased 67.31 % under diclazuril treatment. In addition, the localization of EtEF2 was investigated through immunofluorescence assay. Experimental results demonstrated that EtEF2 was distributed primarily in the cytoplasm of second-generation merozoites, and its fluorescence intensity was enhanced after diclazuril treatment. These findings indicated that EtEF2 may have an important role in understanding the signaling mechanism underlying the anticoccidial action of diclazuril and could be a promising target for novel drug exploration.

Phylogenetic analysis of Classical swine fever virus from archival formalin fixed clinical tissues reveals vietnamese origin of the isolates

Detection of Classical swine fever virus (CSFV) nucleic acid in archival formalin fixed tissue samples and their use for phylogenetic analysis was investigated. Ten samples were examined for the presence of CSFV nucleic acid by reverse transcriptase polymerase chain reaction (RT-PCR) amplification of 5'UTR and E2 gene. RT-PCR was found positive for 5'UTR fragment in eight samples while only one tissue samples showed amplification for E2 gene target fragment. For molecular epidemiology of the disease, 5'UTR PCR product of sample from Darbhanga (Bihar), was cloned and sequenced. The sequence was compared with the sequences available in database. The phylogenetic analysis reveals that the isolate belongs to subgroup 2.2 sharing 98.7% nucleotide identities with Vietnamese isolate (CaTh05-1, AB252170), indicating towards the possible origin of genogroup 2.2 CSFV isolates involved in the outbreak from Vietnam. From the study, it can be concluded that the tissue samples collected and stored in buffer formalin for years can be used to detect CSFV nucleic acid. Results are also suggestive of that the 5'UTR region of genome is more suitable target for RT-PCR based detection of CSFV in archival formalin fixed specimens. The study also indicates the potential of archival formalin fixed tissues for molecular epidemiology and genotyping of the CSF virus.

Molecular characterization and expression pattern of three GABA receptor-like subunits in the small brown planthopper Laodelphax striatellus (Hemiptera: Delphacidae)

Ionotropic γ-aminobutyric acid (GABA)-gated chloride channel receptors mediate rapid inhibitory neurotransmission in vertebrates and invertebrates. GABA receptors are well known to be the molecular targets of synthetic insecticides or parasiticides. Three GABA receptor-like subunits, LsLCCH3, LsGRD and LS8916, of the small brown planthopper, Laodelphax striatellus (Fallén), a major insect pest of crop systems in East Asia, had been identified and characterized in this study. All three genes were cloned using the reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). They shared common structural features with known Cys-loop ligand-gated ion channels (LGICs): the well-conserved dicysteine-loop structures, an extracellular N-terminal domain containing six distinct regions (loops A–F) that form the ligand binding sites and four transmembrane regions (TM1–4). Additionally, temporal and spatial transcriptional profiling analysis indicated that Lslcch3 was significantly higher than the other two genes. All of them were expressed at higher levels in fifth-instar nymph and adults than in eggs and from first- to fourth-instar nymph. They were predominantly expressed in the heads of 2-d old female adults. These findings enhanced our understanding of cys-loop LGIC functional characterization in Hemiptera and provided a useful basis for the development of improved insecticides that targeting this important agricultural pest.

CDNA cloning, expression, and immunolocalization of gonad-inhibiting hormone (GIH) in Litopenaeus vannamei

In this study, the full-length GIH cDNA sequence from Litopenaeus vannamei was cloned from the eyestalk by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. The full-length GIH cDNA was 865 bp with a 288 bp open-reading frame, which encoded a 96 amino acid prepro-GIH with 17 amino acid signal peptide. L. vannamei GIH (LvGIH) can be classified as a member of type-II crustacean hyperglycemic hormone polypeptide family. LvGIH shares 93.8 and 66.7 % amino acid sequence identity with GIH from Penaeus monodon, and the molt-inhibiting hormone from Marsupenaeus japonicas, respectively. By quantitative real-time PCR (qPCR), LvGIH mRNA transcripts were detected in fertilized eggs, nauplius, zoea, mysis and juveniles of 25, 35 and 40 days old. LvGIH transcript levels increased significantly with the development from fertilized eggs to juveniles. LvGIH transcript levels were highest in juveniles at 35 days old. By RT-PCR, LvGIH mRNA transcripts were detected only in the eyestalks and brains but not in the muscles, intestines, gills, heart, hepatopancreas, ovaries and testes of adults, and there was no difference in the expression level of LvGIH between males and females. Using the P. monodon anti-GIH antibody, we showed that LvGIH was located mainly in the XO-SG and slightly in axon, with similar fluorescence intensity found in XO and SG. To summarize, we have cloned and characterized the GIH of the shrimp L. vannamei. In addition to the GIH properties described in other crustaceans, a peak of LvGIH expression was identified at the time of sexual differentiation (i.e., day 35 larvae) suggesting that LvGIH may also be involved in the control of this process.

Expression Profile of Two HSP70 Chaperone Proteins in Response to Extreme Thermal Acclimation inXestia c-nigrum(Lepidoptera: Noctuidae)

Heat shock proteins 70 (HSP70s) are highly evolutionarily conserved and play important roles in helping insects survive under extreme temperatures. In this study, Xestia c-nigrum (L.) (Lepidoptera: Noctuidae) was stressed at various temperatures, and the impacts on thermotolerance and cold endurance were examined. Two complementary deoxyribonucleic acid (cDNA) clones encoding heat shock cognate 70 (HSC70) and inducible heat shock protein 70 (HSP70), named Xc-HSC70 and Xc-HSP70, respectively, were isolated from X. c-nigrum using reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Amino acid sequence analysis indicated that Xc-HSC70 and Xc-HSP70 contained the signature sequences of the HSP70 family, and phylogenetic analysis showed that Xc-HSP70s were consistent with the known insect classification. Genomic DNA structure analysis revealed that the Xc-HSC70 gene contained 8 introns that all conformed to the GT/AG rule, whereas the Xc-HSP70 gene did not contain any introns in the coding region. Fluorescent real-time quantitative RT-PCR revealed that both genes were significantly up-regulated under thermal stress and cold stress. The expression patterns indicated that Xc-HSP70 was heat inducible and Xc-HSC70 was constitutively expressed. The temperature response was more intense for Xc-HSP70 than for Xc-HSC70, regardless of the temperature or developmental stage; however, the expression of Xc-HSC70 and Xc-HSP70 was not altered in the pupa. Taken together, the expression profiles of both genes (Xc-HSP70s) provide good support to the insect for coping with adverse conditions.

IDENTIFICATION AND EXPRESSION ANALYSIS OF VITELLOGENIN RECEPTOR FROM THE WILD SILKWORM, Bombyx mandarina.

The vitellogenin receptor (VgR) plays a key role on embryonic development in oviparous animals. Here, we cloned a VgR gene, which was identified from the wild silkworm Bombyx mandarina (BmaVgR) using reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Sequence analysis revealed that BmaVgR is 5,861 bp long with an open reading frame encoded by 1,811 amino acid residues. The predicted amino acid sequence has 99.7 and 98.2% identity with the VgRs of Actias selene and Bombyx mori, respectively. The class B domain sequence of BmaVgR was cloned and expressed in Escherichia coli, and purified by a Ni-NTA column. Polyclonal antibodies were produced against the purified recombinant protein, and titer of the antibody was about 1:12,800 measured by enzyme-linked immunosorbent assay (ELISA). Western blot and RT-qPCR showed that BmaVgR was expressed in the ovary and fat body of female larvae and the ovary of moth, and the expression level was highest at the third day and then declined from third day to seventh in fat body of pupa. After knockdown of the BmaVgR gene through RNA interference (RNAi), other three BmaVgR-related genes (Vg, egg-specific protein, and low molecular weight lipoprotein LP gene) were all downregulated significantly.

Cloning and characterization of ADP-glucose pyrophosphorylase small subunit gene in Cyperus esculentus (yellow nutsedge).

ADP-glucose pyrophosphorylase (ADPGlcPPase) controls the first committed step of starch synthesis by catalyzing the biosynthesis of ADP-glucose from glucose-phosphate and ATP. It is a tetrameric protein consisting of two small and two large subunits. The small subunits have a catalytic function, while the large subunits regulate the enzyme activity. Cyperus esculentus (yellow nutsedge) is a perennial C4 plant grown from rhizomes and tubers. Previous studies on yellow nutsedge have mostly focused on the morphology and cultivation of tubers, their application in food, and biochemical analyses of the tubers. In this study, the gene encoding the ADPGlcPPase small subunit (CeAGPS) in yellow nutsedge was cloned and characterized. The full-length CeAGPS cDNA sequence contained an 81-bp 5'-untranslated region (UTR), a 188-bp 3'-UTR, and a 1539-bp open reading frame encoding 512-amino acid residues. The genomic sequence of CeAGPS comprises a nine exon-eight intron structure similar to the previously reported cotton and Arabidopsis thaliana AGPS genes. The deduced translation product of the CeAGPS gene contained a well-conserved catalytic domain and regulatory elements typical of plant AGPS. Reverse transcriptase polymerase chain reaction amplification of the target gene in various plant parts using gene-specific primers indicated that the expression of CeAGPS was most abundant in the tuber, and relatively lower in nutsedge roots.

Hypolipidemic and Hepatic Steatosis Preventing Activities of the Wood Ear Medicinal Mushroom Auricularia auricula-judae (Higher Basidiomycetes) Ethanol Extract In Vivo and In Vitro.

Obesity, a rapidly growing threat to human health worldwide, is responsible for a large proportion of the total burden of disease. Therefore, obesity control could be a vital scheme to prevent many diseases. The aim of this study was to examine the activities and mechanism of Auricularia auricula-judae 70% ethanol extract (AAE) in preventing hypolipidemic and hepatic steatosis. A normal diet (ND) and a high-fat diet (HFD) with or without 0.1% (w/w), 0.3% (w/w), and 1% (w/w) AAE were given to male C57BL/6 mice. Plasma lipids and liver enzymes were measured and tissue sections of liver were examined. Further mechanistic studies of mouse 3T3-L1 adipocytes were performed in vitro by verifying triglyceride, glycerol, and glycerol-3-phosphate dehydrogenase activity and messenger RNA expression of adipogenic and lipogenic genes using reverse transcriptase polymerase chain reaction amplification. Body weight and adipose tissue mass were significantly reduced in mice fed an ND and a HFD plus AAE compared with mice fed an HFD. In AAE-supplemented groups, plasma lipids and liver enzymes decreased dose-dependently. AAE suppressed the expression of adipogenic/lipogenic genes (PPARγ, C/EBPα, FAS) in 3T3-L1 cells without cytotoxicity. These findings suggest that AAE may reduce the risk of hepatic steatosis by modulating plasma lipids via the regulation of adipogenic/lipogenic transcriptional factors. AAE may have interesting applications to improve plasma lipids and liver enzymes.

Molecular cloning, characterization and expression analysis of Melanotransferrin from the sea cucumber Apostichopus japonicus

Melanotransferrin (MTf), a member of the transferrin families, plays an important role in immune response. But the research about MTf in sea cucumber is limited till now. In this study, the Melanotransferrin (Aj-MTf) gene was firstly cloned and characterized from the sea cucumber Apostichoupus japonicus by reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. The full-length cDNA of Aj-MTf is 2,840 bp in length and contains a 2,184 bp open reading frame that encodes a polypeptide of 727 amino acids. An iron-responsive element-like structure is located at the 5'-UTR of Aj-MTf cDNA. Sequence analysis shows that the Aj-MTf contains two conserved domains, and the binding-iron (III) sites, including eight amino acid residues (D81,Y109,Y215,H283,D425,Y454,Y565 and H634) and three N-linked glycosylation sites (N121V122S123,N173A174S175 and N673S674T675). Quantitative real-time polymerase chain reaction (qRT-PCR) analyses suggested that the Aj-MTf expressions in the coelomic fluid, body cavity wall and respiratory trees were significantly changed from 4 to 24 h post lipopolysaccharide (LPS) injection. The mRNA levels of Aj-MTf in coelomic fluid was significantly up-regulated at 12 and 24 h in treatment group, and Aj-MTf shared a similar expression pattern with C-type lectin in coelomic fluid, while both genes appears to gradually increase after 4 h of LPS injection. These results indicate that the Aj-MTf plays a pivotal role in immune responses to the LPS challenge in sea cucumber, and provide new information that it is complementary to the sea cucumber immune genes and initiate new researches concerning the genetic basis of the holothurian immune response.

Molecular cloning, characterization and expression analysis of the retinoid X receptor in the swimming crab, Portunus trituberculatus (Miers, 1876) (Decapoda, Portunidae)

To elucidate the role of the retinoid X receptor (RXR) in moulting and ovarian development of crustaceans, the full-length cDNA of RXR (PtRXR) in Portunus trituberculatus (Miers, 1876) was cloned by nested reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA sequence of PtRXR was determined to be 1365 bp in length and contained an open reading frame (ORF) of 1140 bp encoding a 379-amino-acid residue protein. The deduced amino-acid sequence of PtRXR shared high identities with other known RXRs. Phylogenetic analysis showed that PtRXR was clustered among crustacean RXRs and located closer to the vertebrate RXRs than the insect ultraspiracle (USP, an orthologue of RXR). Quantitative real-time PCR (qRT-PCR) was used to analyse the tissue distribution of PtRXR and its expression patterns during the moulting cycle and the second ovarian development of P. trituberculatus. The results showed that PtRXR was widely distributed in the tested tissues. PtRXR mRNA levels were significantly high in ovary and Y-organs (YO) of intermoult crabs. The mRNA levels of PtRXR in YO and mandibular organs (MO) decreased significantly from intermoult to premoult. In addition, PtRXR was expressed at each stage of the second ovarian development in ovary, hepatopancreas, YO and MO, and the expression levels reached maximal values when the ovary reached the final stage of maturation. These results indicate that PtRXR might have an important role in regulating the moulting and ovarian development of P. trituberculatus.

Persistent human herpesvirus‐6 infection in patients with an inherited form of the virus

Human herpesvirus-6 (HHV-6)A and 6B are ubiquitous betaherpesviruses viruses with lymphotropic and neurotropic potential. As reported earlier, these viruses establish latency by integration into the telomeres of host chromosomes. Chromosomally integrated HHV-6 (CIHHV-6) can be transmitted vertically from parent to child. Some CIHHV-6 patients are suffering from neurological symptoms, while others remain asymptomatic. Four patients with CIHHV-6 and CNS dysfunction were treated with valganciclovir or foscarnet. HHV-6 replication was detected by reverse transcriptase polymerase chain reaction amplification of a late envelope glycoprotein. In this study we also compared the inherited and persistent HHV-6 viruses by DNA sequencing. The prevalence of CIHHV-6 in this cohort of adult patients from the USA suffering from a wide range of neurological symptoms including long-term fatigue were found significantly greater than the reported 0.8% in the general population. Long-term antiviral therapy inhibited HHV-6 replication as documented by loss of viral mRNA production. Sequence comparison of the mRNA and the inherited viral genome revealed that the transcript is produced by an exogenous virus. In conclusion, the data presented here document that some individuals with CIHHV-6 are infected persistently with exogenous HHV-6 strains that lead to a wide range of neurological symptoms; the proposed name for this condition is inherited herpesvirus 6 syndrome or IHS.

CLONING AND PROKARYOTIC EXPRESSION PEROXIREDOXIN 6 GENE IN CRISTARIA PLICATA

Peroxiredoxin 6 has glutathione peroxidase and phospholipid enzyme A2 double activity and plays an important role in antioxidant protection and metabolism of lung surface-active material.Cristaria plicata is one of the most important freshwater mussels for pearl production in China.In this study,a Prx6 homologue-CpPrx6 in Cristaria plicata,was cloned by utilizing reverse transcriptase polymerase chain reaction and rapid amplification of the cDNA ends.A phylogenetic tree was constructed based on the amino acid sequence of CpPrx6 using neighbor-joining method and MEGA 4.1 software.The three-dimensional structure of CpPrx6 was constructed using the Swiss-model.The expression patterns,both in tissues and towards microorganism A.hydrophila stimulation,were then characterized by real-time fluorescence quantitative PCR.The results showed that the full length cDNA of CpPrx6 contained a 5'-untranslated region(UTR) of 71 bp,an open reading frame of 657 bp that encoded 218 amino acids,and a 3'-UTR of 889 bp with a 29 bp polyadenylic acid tail.The predicted molecular mass and estimated isoelectric point(pI) of CpPrx6 were 24.24 kD and 6.33,respectively.No signal peptide,the transmembrane region,mitochondria,lysosomes,peroxisomes and nuclei localization sequence were found and contained 1 potential N-linked glycosylation site(N9FTA) in CpPrx6,which suggested that it is a cytosolic protein.The deduced amino acid sequence of CpPrx6 shared 70%—72% overall similarity with known Molluscan Prx6.The conserved peroxidase catalytic center "PVCTTE" and a lipase motif "GKSWA" were observed in the sequence,the tertiary structures of CpPrx6 contained 6 α-helixes and 12 β-sheets,the catalytic center "PVCTTE" located in fifth α-helix,three conserved residues His22,Asp134 and Ser28 together formed the catalytic center of the phospholipase A2,indicating that it was a member of 1-Cys Prx.Phylogenetic analyses showed that CpPrx6 sequence clustered together with the other Molluscan Prx6 sequences.The mRNA transcript of CpPrx6 was detected in most C.plicata organs,such as haemocyte,mantle,adductor muscle,hepatopancreas and gill,with the highest expression level found in gill.After injection of Aeromonas hydrophilai,the expression levels of CpPrx6 significantly increased in hepatopancreas at 6h and 12h,in haemocytes and gills at 12h(6h,P0.05;12h,P0.01),respectively.The expression levels then returned to normal at 24h.This suggested that CpPrx6 perhaps played an important role in resistance to oxidative stress and basic immunization of C.plicata.According to the ORF,designed site(KpnI and EcoRI) primers,the CpPrx6 gene was subcloned into a vector pET-32a(+) and transformed into Escherichia coli BL21(DE3),the constructed recombinant expression plasmids were induced by the chemical inducer IPTG.The expression products were analyzed by SDS-PAGE.The results indicated that recombination CpPrx6 was successfully expressed in E.coli.The expressed protein had a molecular weight of 40 kD.

Identification of New and UnusualrevandnefTranscripts Expressed by an HIV Type 1 Primary Isolate

We analyzed RNA splice site usage in three HIV-1 subtype B primary isolates through reverse transcriptase polymerase chain reaction (RT-PCR) amplification of spliced RNAs using a fluorescently labeled primer, with computerized size determination and quantification of PCR products, which were also identified by clone sequencing. In one isolate, P2149-3, unusual and unreported spliced transcripts were detected. This isolate preferentially used for rev RNA generation a 3' splice site (3'ss) located five nucleotides upstream of A4a, previously identified only in a T cell line-adapted virus and in a group O isolate, and designated A4d. P2149-3 also used an unreported 3'ss for rev RNA generation, designated A4h, located 20 nucleotides upstream of 3'ss A4c. Additionally, unusual nef RNAs using 3'ss A5a and A7a and with exon composition 1.3.7 were identified. The identification of several unusual and unreported spliced transcripts in an HIV-1 primary isolate suggests a greater diversity of splice site usage in HIV-1 than previously appreciated.

A novel serine/threonine protein phosphatase type 5 from second-generation merozoite of Eimeria tenella is associated with diclazuril-induced apoptosis

Screening the anticoccidial drug targets is very important for developing novel drugs and revealing the molecular basis of drug resistance in coccidia. Due to high effectivity and safety, diclazuril was used widely in the poultry industry. To assess the roles of the serine/threonine protein phosphatase type 5 of second-generation merozoites in Eimeria tenella (EtPP5) in the anticoccidial activity of diclazuril against chicken coccidiosis, EtPP5 was cloned using reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends. Ultrastructural changes in second-generation merozoites and mRNA expression level of EtPP5 were monitored by transmission electron microscopy (TEM) and quantitative real-time PCR, respectively. The results showed that the full length of the cloned EtPP5 cDNA (2,495 bp) encompassed a 1,647-bp open reading frame encoding a polypeptide of 548 residues with an estimated molecular mass of 60.82 kDa and a theoretical isoelectric point of 5.89. Molecular analysis of EtPP5 reveals the presence of a C-terminal phosphatase domain and an extended N-terminal tetratricopeptide repeat motif, a typical feature of protein phosphatases. The cDNA sequence has been submitted to the GenBank database with accession number JX987508. EtPP5 shared 89% homology with the published sequence of a PP5 ortholog of Toxoplasma gondii at the amino acid level (GenBank XP_002364442.1). TEM observed that diclazuril induced ultrastructural changes in second-generation merozoites. Quantitative real-time PCR analysis showed that compared with the control group, the level of EtPP5 mRNA expression was significantly downregulated by 51.4% by diclazuril treatment. The high similarity of EtPP5 to previously described PP5 of other organisms, as well as its downregulated expression and connection with apoptosis in the second-generation merozoites induced by diclazuril, suggests that it could act an important role in understanding the signaling mechanism underlining the diclazuril-induced merozoites apoptosis.

Systemic Delivery of Thiol-Specific Antioxidant hPRDX6 Gene by AAV2/8 Inhibits Atherogenesis in LDLR KO Mice on HCD

Atherosclerosis is an inflammatory disorder of arteries and reactive oxygen species (ROS) have been implicated as a major etiology. Various anti-ROS genes, such as superoxide dismutates and catalase, have been studied by gene transfer for their abilities to limit a variety of ROS-related cardiovascular injuries such as balloon-induced and ischemia-reperfusion injury. However the thiol-ROS compartment has never been explored by gene therapy/ gene delivery for potential therapeutic intervention. Human peroxiredoxin 6 (hPRDX6, AOL2) is fully unique in that in addition to its thiol-specific anti-oxidant ability it has a coupled phospholipase A2 catalytic domain. Here we delivered hPRDX6, expressed by the cytomegalovirus immediate early promoter (CMV), by systemic pseudotyped adeno-associated virus 2/8 (AAV2/8) gene transfer into low density lipoprotein receptor knockout mice on high cholesterol diet (LDLR KO HCD). It was found that AAV2/8/hPRDX6 gene delivery and expression inhibited systolic blood velocity, aortic cross sectional area, and aortic wall thickness compared to Neo-HCD control, consistent with reduced atherosclerosis. Markers of macrophages/foam cells, CD68, ITGAM, EMR, verified lower atherosclerosis, as all were lower in the AAV8/hPRDX6-HCD-treated animals compared to Neo-HCD controls, analyzed by either quantitative reverse transcriptase polymerase chain reaction amplification or by immuno-histochemistry, or both. Analysis of the immune state of the aortas (Th1 or Th2 cytokine expression) revealed nothing significant, with only IL-10 expression being lower in PRDX6-treated animals. This study, for the first time, demonstrated that hPRDX6 gene delivery, augmenting endogenous mouse (m)PRDX6 expression, giving therapeutic benefit against HCDdriven atherosclerosis and suggests further studies of PRDX6, and related anti-thiol-ROS approaches, is warranted.

Receptor for activated C kinase ortholog of second-generation merozoite in Eimeria tenella: clone, characterization, and diclazuril-induced mRNA expression

The receptor for activated C kinase (RACK) cDNA of second-generation merozoites of Eimeria tenella was cloned using reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends, compared with other species, and then successfully expressed using the pET-28a vector in Escherichia coli BL21 (DE3) (EtRACK). Nucleotide sequence analysis revealed that the full length of the cloned cDNA (1,264 bp) encompassed a 957-bp open reading frame encoding a polypeptide of 318 residues with an estimated molecular mass of 34.94 kDa and a theoretical isoelectric point of 5.97. Molecular analysis of EtRACK reveals the presence of seven WD40 repeat motifs. EtRACK localizes to the cytoplasm and nucleus in second-generation merozoites of E. tenella. The cDNA sequence has been submitted to the GenBank Database with accession number JQ292804. EtRACK shared 98% homology with the published sequence of a RACK protein from Toxoplasma gondii at the amino acid level (GenBank XP_002370996.1). Recombinant protein expression was induced using 1 mM of isopropyl β-D-1-thiogalactopyranoside in vitro at 30 °C. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that the 39.79-kDa fusion protein existed in unsolvable form. Quantitative real-time PCR analysis showed that compared with the control group, the level of EtRACK mRNA expression in the treatment group was downregulated by 81.3% by diclazuril treatment. The high similarity of EtRACK to previously described RACKs of other organisms, as well as its downregulated expression in second-generation merozoites induced by diclazuril, suggests that it could play a key role in the signaling event that precedes protein secretion and parasite invasion. Moreover, the downregulation of EtRACK mRNA expression also enriches studies on the mechanism of action of diclazuril on E. tenella.