Oligo dT Research Articles

131 RNA-Seq TRANSCRIPTOME PROFILING OF INDIVIDUAL RHESUS MACAQUE OOCYTES AND PRE-IMPLANTATION EMBRYOS

The objective of this study was to perform whole transcriptome sequencing (RNA-Seq) on individual rhesus macaque oocytes and intracytoplasmic sperm injection-derived embryos. Nine developmental stages were assayed (n = 2–3 per stage): germinal vesicle (GV), meiosis I (MI), and meiosis II (MII) oocytes and pronuclear (PN), 2-cell (2C), 4-cell (4C), 8-cell (8C), morula (MO), and blastocyst (BL) stage embryos. Rhesus females were superovulated with hrFSH and oocytes collected by follicular aspiration of ovaries recovered at necropsy 35 h after hCG treatment. Based on GV and polar body (PB) presence/absence, oocytes were classified as GV, MI, and MII. Embryos were produced by intracytoplasmic sperm injection of MII oocytes and cultured in HECM-9 media. Individual oocytes and embryos were collected at different developmental stages and frozen until use. The Clontech SMARTer Ultra Low Input RNA Kit for Illumina Sequencing was used for cDNA synthesis (oligo dT) and amplification. Sequencing libraries were created from fragmented cDNA with the Illumina TruSeq DNA kit. Single 100-bp reads were produced with a HiSEqn 2000 apparatus. Read alignment was done in CLC Genomics Workbench software to the RhesusBase genome annotation (53 788 genes). A gene was considered expressed if reads per kilobase of transcript model per million mapped reads (RPKM) was >0.4. Principal component analysis (PCA), hierarchical clustering, and differential gene expression analyses were performed with the DESEqn 2 package in R. On average, 22 311 310 reads were produced per sample with 63% aligning to the rhesus transcriptome. A total of 14 527 genes were detected in all replicates of at least one stage, with an average of 8855 genes per stage. The PCA and hierarchical clustering of all expressed genes discriminated between samples of different stages, except for 2C and PN, which were indistinguishable. All oocytes grouped to the same subcluster and close to PN-4C embryos, forming another subcluster. The 8C embryos constituted their own subcluster, which was closer to MO and BL than to other pre-implantation stages. Of genes expressed exclusively in embryo samples (n = 2823), 42% were expressed beginning at the 8C stage, 39% began expression at MO or BL, and 18% started expression between PN to 4C stage. The highest numbers of differentially expressed (DE) genes between consecutive stages were for MI-MII, MII-PN, 4C-8C, and 8C-MO, with an average of 5306 DE genes, whereas only 1060 were found in other comparisons. When gene expression was compared relative to the MII stage, 8C, MO, and BL had the highest numbers of DE genes (8965, 9635, and 9100, respectively, v. an average of 4713 for all other embryo stages). The high proportion of embryo-specific genes beginning expression at the 8C stage along with large numbers of DE genes observed between MII and 8C and the isolation of 8C samples to a unique expression cluster with PCA/hierarchical clustering indicates that major embryonic genome activation occurs at the 8C stage in the rhesus macaque. Overall, the dataset represents a comprehensive resource for analysis of polyadenylated transcript levels throughout early development in a nonhuman primate species.

Construction and identification of a cDNA library for use in the yeast two-hybrid system from duck embryonic fibroblast cells post-infected with duck enteritis virus

To explore and isolate genes related to duck embryonic fibroblast cells (DEFs) post-infected with duck enteritis virus (DEV), a cDNA library was established using SMART (Switching Mechanism At 5' end of the RNA Transcript) technique coupling with a homologous recombination method. The cells were harvested and total RNA was extracted at 48 h post infection. Then the mRNAs were purified and reverse transcribed to first-strand cDNAs using oligo (dT) primers (CDS III). Subsequently, long distance-PCR was performed, the double-stranded cDNAs were purified, and a transformation assay was carried out in that order. Eventually, a high qualitative library was successfully established according to an evaluation on quality. The transformation efficiency was about 2.33 × 10(6) transformants/4.34 μg pGADT7-Rec (>1.0 × 10(6)). The cell density of the library was 1.75 × 10(9) cells/mL (>2 × 10(7) cells/mL). The titer of the primary cDNA library and amplified cDNA library was 6.75 × 10(5) and 2.33 × 10(7) CFU/mL respectively. The numbers for the primary cDNA library and amplified cDNA library were 1.01 × 10(7) and 1.14 × 10(9), respectively, and the recombinant rate was 97.14 %. The sequence results of 27 randomly picked independent clones revealed the insert ranged from 0.323 to 2.017 kb with an average insert size of 0.807 kb. Full-length transcripts of DEV-CHv LORF3, UL26 and UL35 genes were acquired through sequence similarity analysis from the non-redundant nucleic acid or protein database. Five polyA sites were identified in the DEV-CHv genome. Also, a new transcript of 668 bp was found between the IRS gene and US1 gene of the DEV-CHv genome. Thus, we concluded that the constructed cDNA library will be a useful tool in proteomic analysis of interactions between the DEV and host DEFs, and discovery of biomarkers studies on the mechanism of DEV and subsequently exploitation original vaccines and antiviral drugs to prevent or cure diseases.

Introduction of antioxidant-loaded liposomes into endothelial cell surfaces through DNA hybridization

Ischemia–reperfusion damage is a problem in organ transplantation. Reactive oxygen species are produced in cells by blood-mediated reactions at the time of blood reperfusion. In this study, we developed a method to immobilize and internalize antioxidants in endothelial cells, using vitamin E-loaded liposomes. The liposomes loaded with vitamin E and human umbilical vein endothelial cells (HUVECs) were modified with poly(ethylene glycol)–phospholipid conjugates carrying 20-mer of deoxyadenylic acid (oligo(dA)20) and 20-mer of complementary deoxythymidylic acid (oligo(dT)20), respectively. The liposomes were effectively immobilized on HUVECs through DNA hybridization between oligo(dA)20 and oligo(dT)20. The liposomes loaded with vitamin E were gradually internalized into HUVECs. Then, the cells were treated with antimycin A to induce oxidative stress. We found the amount of reactive oxygen species was greatly reduced in HUVECs carrying vitamin E-loaded liposomes.

A Role For MMP-3 In The Modulation Of Early T Cell Responses In Gvhd

IntroductionDuring graft-versus-host disease (GVHD), activated alloreactive T cells recirculate from the secondary lymphoid organs and extravasate via the blood vessels into target tissues, causing pathology which if severe enough can ultimately lead to mortality. Extensive work from our laboratory has shown that lethal GVHD can be induced between MHC-matched murine strains expressing multiple minor histocompatibility antigen (miHA) differences. In the C57BL/6y (B6)–>BALB.B model, both CD4+ and CD8+ donor T cells can mediate lethal GVHD, whereas in the B6 –>CXB-2 model, only CD8+ T cells are lethal. CXB-2 mice are a recombinant inbred strain which expresses a subset of the same miHA as BALB.B mice, and B6 CD4+ T cells do initially respond by proliferation in both recipients, yet tissue pathology, particularly in the intestinal tract is much more limited in CXB-2 mice. We therefore hypothesized that differences at the level of the host issue may have an influence on GVHD development. In order for alloreactive T cells to infiltrate target tissues, they must traverse the extracellular matrix (ECM), which makes up the majority of the intercellular space in solid tissues. While the role of various T cell subsets has been studied extensively in GVHD, the involvement of ECM structural and regulatory proteins, specifically matrix metalloproteinases (MMP), is much less defined. MMP not only participate in ECM turnover, but also are also capable of influencing immune responses through the cleavage and activation of inflammatory mediators. Specifically, MMP-3 has been demonstrated to not only cleave ECM structural proteins such as collagens, fibronectin and laminin, but also can activate other MMP, TNF-α precursors and IL-1β. We therefore examined the expression patterns and potential function of MMP-3 in the development of GVHD. MethodsSmall intestine tissue samples were collected from either lethally irradiated (9 Gy, split dose) BALB.B or CXB-2 mice receiving only 2 X 106 anti-Thy 1 mAb + C' treated (T cell- depleted) B6 bone marrow cells (ATBM) for hematopoietic reconstitution, or from BALB.B and MMP3-KO mice (on a BALB.B background) exposed to irradiation (9 Gy, split dose) and transplanted with 1 X 107 BALB.B presensitized B6 T cells along with 2 X 106ATBM. Samples were collected on days 8, 20 and 30 post-irradiation and homogenized using a mechanical dissociator (Miltenyi Biotec). Total RNA was converted into cDNA using oligo(dT) as a primer for reverse transcription. Expression of MMP-3 was determined using real time PCR analysis. IFN-γ production by donor T cells was evaluated using ELISPOT assays where responder presensitized B6 enriched T cells were cocultured with either BALB.B or MMP-3 KO stimulator splenocytes (exposed to 15 Gy irradiation). ResultsRelative gene expression of MMP-3 using GAPDH as an endogenous control was significantly lower (39.9% decrease, p<0.05) at day 8 following irradiation of hosts in BALB.B mice when compared to CXB2 mice. No significant differences in expression levels were observed at later timepoints (20 and 30 days post-irradiation). GVHD experiments comparing MMP-3 knockout (KO) mice on a BALB.B background with BALB.B controls, revealed that a lack of MMP-3 caused a decrease in the median survival time (MST) of recipient mice from day 40 to day 31(p=0.06). ELISPOT assays studying B6 T cell alloresponses against BALB.B or MMP-3 KO splenocytes showed an increase in mean IFNγ+ spots in MMP-3 KO-stimulated wells from 205.7 to 241.7 (p=0.098) when compared to BALB.B controls, Taken together, these data suggest: 1) a protective role for MMP-3 in the early stages of GVHD development in the B6–> BALB.B model; and 2) a potential involvement of both tissue and immune associated MMP-3 in the development of GVHD. Disclosures:No relevant conflicts of interest to declare.

New Variant of Rabbit Hemorrhagic Disease Virus, Portugal, 2012–2013

New Variant of Rabbit Hemorrhagic Disease Virus, Portugal, 2012–2013

Codon Optimization Enhances Protein Expression of Bombyx mori Nucleopolyhedrovirus DNA Polymerase in E. coli

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major viral agent that causes deadly grasserie disease in silkworms, while BmNPV DNA polymerase (BmNPV-pol), encoded by ORF53 gene, plays a central role in viral DNA replication. Efficacy studies of BmNPV-POL are limited because of poor heterologous protein expression in E. coli. Here, we redesigned the BmNPV-pol to preferentially match codon frequencies of E. coli without altering the amino acid sequence. Following de novo synthesis, codon-optimized BmNPV-pol (co-BmNPV-pol) gene was cloned into pET32a and pGEX-4T-2 vector. The expression of co-BmNPV-POL in E. coli was significantly increased when BmNPV-POL was fused with GST protein rather than a His-tag. The co-BmNPV-POL fusion proteins were isolated using GST affinity chromatography and Mono Q iron exchange chromatography. Protein purity and identity were confirmed by western blot and MALDI-TOF analyses. The biological activity of purified proteins was measured on a poly(dA)/oligo(dT) primer/template. The specific polymerasing activity of the recombinant BmNPV-POL was 6,329 units/mg at optimal conditions. Thus, a large amount of purified protein as a soluble form with high activity would provide many benefits for the functional research and application of BmNPV-POL.

A Human Proteome Microarray Identifies that the Heterogeneous Nuclear Ribonucleoprotein K (hnRNP K) Recognizes the 5′ Terminal Sequence of the Hepatitis C Virus RNA

Stem-loop I (SL1) located in the 5' untranslated region of the hepatitis C virus (HCV) genome initiates binding to miR-122, a microRNA required for hepatitis HCV replication. However, proteins that bind SL1 remain elusive. In this study, we employed a human proteome microarray, comprised of ∼17,000 individually purified human proteins in full-length, and identified 313 proteins that recognize HCV SL1. Eighty-three of the identified proteins were annotated as liver-expressing proteins, and twelve of which were known to be associated with hepatitis virus. siRNA-induced silencing of eight out of 12 candidate genes led to at least 25% decrease in HCV replication efficiency. In particular, knockdown of heterogeneous nuclear ribonucleoprotein K (hnRNP K) reduced HCV replication in a concentration-dependent manner. Ultra-violet-crosslinking assay also showed that hnRNP K, which functions in pre-mRNA processing and transport, showed the strongest binding to the HCV SL1. We observed that hnRNP K, a nuclear protein, is relocated in the cytoplasm in HCV-expressing cells. Immunoprecipitation of the hnRNP K from Huh7.5 cells stably expressing HCV replicon resulted in the co-immunoprecipitation of SL1. This work identifies a cellular protein that could have an important role in the regulation of HCV RNA gene expression and metabolism.

Trichosanthes kirilowii: A New Host of Cucurbit mild mosaic virus in China.

Chinese cucumber (Trichosanthes kirilowii Maxim.) is a type of perennial liana plant of the Cucurbitaceae family that is mainly distributed in East Asia and northern Australia. It is an important medicinal plant and commonly used in Chinese herbalism, where it is considered to be one of the 50 fundamental herbs (2). During the summer and autumn of 2012, T. kirilowii plants showing symptoms of mild mosaic on the upper leaves and bright yellow color on the lower leaves were observed in the Haidian district of Beijing, China. Recently similar symptoms induced by Cucurbit mild mosaic virus (CuMMV) on squash have been reported. CuMMV is a new member of the genus Fabavirus in the Comovirinae subfamily, discovered in China in 2006 (1). Total RNA was extracted from five leaf samples of independent plants and used for reverse transcription with an oligo (dT)18 primer, followed by PCR with a pair of CuMMV virus-specific primers FaR13012F (5'-CGAGTGCGAGTTAGAAATTGGGATG-3') and FaR15783R (5'-TCACTTTGAGGTGATAAAACAATCC-3') to amplify a 2,772-bp fragment including RNA-dependent RNA polymerase (RdRp) coding region. The expected target fragment was obtained in all symptomatic plant samples but not from an asymptomatic plant. Nucleotide sequence comparison analysis showed that the virus isolated from T. kirilowii (GenBank Accession No. KC959843) had 95.33% nucleotide identity and 99.15% amino acid identity in the RdRp sequence with a CuMMV isolate from squash (GenBank Accession No. FJ194941) (1). In addition, symptomatic samples tested positive for CuMMV by Western blot using CuMMV small coat protein (SCP) specific polyclonal antibody (1). To our knowledge, this is the first report of T. kirilowii as natural host of CuMMV in China. The impact of CuMMV on T. kirilowii production remains to be determined; however, the extended host range for this virus suggests a potential threat of CuMMV to cucurbit crops in China.

Utilization of paramagnetic microparticles for automated isolation of free circulating mRNA as a new tool in prostate cancer diagnostics

Determination of serum mRNA gained a lot of attention in recent years, particularly from the perspective of disease markers. Streptavidin-modified paramagnetic particles (SMPs) seem an interesting technique, mainly due to possible automated isolation and high efficiency. The aim of this study was to optimize serum isolation protocol to reduce the consumption of chemicals and sample volume. The following factors were optimized: amounts of (i) paramagnetic particles, (ii) oligo(dT)20 probe, (iii) serum, and (iv) the binding sequence (SMPs, oligo(dT)20 , serum vs. oligo(dT)20 , serum and SMPs). RNA content was measured, and the expression of metallothionein-2A as possible prostate cancer marker was analyzed to demonstrate measurable RNA content with ability for RT-PCR detection. Isolation is possible on serum volume range (10-200 μL) without altering of efficiency or purity. Amount of SMPs can be reduced up to 5 μL, with optimal results within 10-30 μL SMPs. Volume of oligo(dT)20 does not affect efficiency, when used within 0.1-0.4 μL. This optimized protocol was also modified to fit needs of automated one-step single-tube analysis with identical efficiency compared to conventional setup. One-step analysis protocol is considered a promising simplification, making RNA isolation suitable for automatable process.

Link Protein N-terminal Peptide Binds to Bone Morphogenetic Protein (BMP) Type II Receptor and Drives Matrix Protein Expression in Rabbit Intervertebral Disc Cells

Intervertebral disc (IVD) degeneration and associated spinal disorders are leading sources of morbidity, and they can be responsible for chronic low back pain. Treatments for degenerative disc diseases continue to be a challenge. Intensive research is now focusing on promoting regeneration of degenerated discs by stimulating production of the disc matrix. Link protein N-terminal peptide (LPP) is a proteolytic fragment of link protein, an important cross-linker and stabilizer of the major structural components of cartilage, aggrecan and hyaluronan. In this study we investigated LPP action in rabbit primary intervertebral disc cells cultured ex vivo in a three-dimensional alginate matrix. Our data reveal that LPP promotes disc matrix production, which was evidenced by increased expression of the chondrocyte-specific transcription factor SOX9 and the extracellular matrix macromolecules aggrecan and collagen II. Using colocalization and pulldown studies we further document a noggin-insensitive direct peptide-protein association between LPP and BMP-RII. This association mediated Smad signaling that converges on BMP genes leading to expression of BMP-4 and BMP-7. Furthermore, through a cell-autonomous loop BMP-4 and BMP-7 intensified Smad1/5 signaling though a feedforward circuit involving BMP-RI, ultimately promoting expression of SOX9 and downstream aggrecan and collagen II genes. Our data define a complex regulatory signaling cascade initiated by LPP and suggest that LPP may be a useful therapeutic substitute for direct BMP administration to treat IVD degeneration and to ameliorate IVD-associated chronic low back pain.

Construction of cDNA library and partial ESTs analysis of Strongylo-centrotus intermedius

Sea urchin(Strongylocentrotus intermedius) is a species of commercial and ecological significance,and as a economic species,sea urchin has been demanded steadily in Europe and Southeastern area for a long time.Recent studies focused on the development of its genomic resources,which is a key step towards further investi-gation,identification of gene and gene networks involved in its economic characters.Efforts have been focused on genes that can affect expression of important economic traits,such as growth and nutritional traits related genes.In this study,a cDNA library of sea urchin has been constructed from gonad using Gubler-Hoffman cDNA library technique.Total RNA was extracted from the grounded frozen powder of gonad tissue by using acid-guanidine-phenol-chloroform(AGPC).Poly(A)+ RNA was isolated and purified by oligo(dT)18 anchor primer containing NotⅠsite.Both ends of the newly generated and polished double-stranded(ds) cDNA were attached by EcoR Ⅰadaptors and the dscDNA was then restricted by NotⅠ.The short cDNA(400 bp) was removed by Spin Col-umn.The library consisted of 2.10×106 PFU/mL colony forming units with average insert size of 1.4 kb,ranging from 0.5 kb to 4.2 kb and was quantified to construct a cDNA library.Eight hundred and fourteen ESTs were com-pared with sequences in GenBank database of NCBI using Blast search,among which 65 ESTs and cDNA clones had been identified.These identified ESTs were submitted to GenBank with accession number of GO448010-GO448016 and GR410172-GR410229.The cDNA library provides rich source for better understanding of func-tional genes in sea urchin,and thus will play an important role in further development of sea urchin research.

The RNA-binding protein repertoire of embryonic stem cells

RNA-binding proteins (RBPs) have essential roles in RNA-mediated gene regulation, and yet annotation of RBPs is limited mainly to those with known RNA-binding domains. To systematically identify the RBPs of embryonic stem cells (ESCs), we here employ interactome capture, which combines UV cross-linking of RBP to RNA in living cells, oligo(dT) capture and MS. From mouse ESCs (mESCs), we have defined 555 proteins constituting the mESC mRNA interactome, including 283 proteins not previously annotated as RBPs. Of these, 68 new RBP candidates are highly expressed in ESCs compared to differentiated cells, implicating a role in stem-cell physiology. Two well-known E3 ubiquitin ligases, Trim25 (also called Efp) and Trim71 (also called Lin41), are validated as RBPs, revealing a potential link between RNA biology and protein-modification pathways. Our study confirms and expands the atlas of RBPs, providing a useful resource for the study of the RNA-RBP network in stem cells.

Development of a Magnetic Electrochemical Bar Code Array for Point Mutation Detection in the H5N1 Neuraminidase Gene

Since its first official detection in the Guangdong province of China in 1996, the highly pathogenic avian influenza virus of H5N1 subtype (HPAI H5N1) has reportedly been the cause of outbreaks in birds in more than 60 countries, 24 of which were European. The main issue is still to develop effective antiviral drugs. In this case, single point mutation in the neuraminidase gene, which causes resistance to antiviral drug and is, therefore, subjected to many studies including ours, was observed. In this study, we developed magnetic electrochemical bar code array for detection of single point mutations (mismatches in up to four nucleotides) in H5N1 neuraminidase gene. Paramagnetic particles Dynabeads® with covalently bound oligo (dT)25 were used as a tool for isolation of complementary H5N1 chains (H5N1 Zhejin, China and Aichi). For detection of H5N1 chains, oligonucleotide chains of lengths of 12 (+5 adenine) or 28 (+5 adenine) bp labeled with quantum dots (CdS, ZnS and/or PbS) were used. Individual probes hybridized to target molecules specifically with efficiency higher than 60%. The obtained signals identified mutations present in the sequence. Suggested experimental procedure allows obtaining further information from the redox signals of nucleic acids. Moreover, the used biosensor exhibits sequence specificity and low limits of detection of subnanogram quantities of target nucleic acids.

First Report of Banana bract mosaic virus in 'Cavendish' Banana in Ecuador.

Banana bract mosaic virus (BBrMV), a member of the genus Potyvirus, family Potyviridae, is the causal agent of bract mosaic disease. The disorder has been considered a serious constraint to banana and plantain production in India and the Philippines, where the virus was first identified (3). To date, the presence of BBrMV has been reported only in a few banana-growing countries in Asia (3). In the Americas, BBrMV has been detected by ELISA tests in Colombia only (1). The efficient spread of BBrMV through aphids and vegetative material increases the quarantine risk and requires strict measures to prevent entrance of the virus to new areas. In Ecuador-the world's number one banana exporter-the banana industry represents the main agricultural income source. Thus, early detection of banana pathogens is a priority. In June of 2012, mosaic symptoms in bracts and bunch distortion of 'Cavendish' banana were observed in a commercial field in the province of Guayas, Ecuador. Leaves from 35 symptomatic plants were tested for Cucumber mosaic virus (CMV), Banana streak virus (BSV), and BBrMV using double antibody sandwich ELISA kits from Adgen (Scotland, UK). Twenty-one plants tested positive for BBrMV but not for CMV or BSV. In order to confirm the ELISA results, fresh or lyophilized leaf extracts were used for immunocapture reverse transcription (IC-RT)-PCR. In addition, total RNA was extracted from the ELISA-positive samples and subjected to RT-PCR. The RT reactions were done using both random and oligo dT primers. Several sets of primers, flanking conserved regions of the virus coat protein (CP), have been used for PCR-detection of BBrMV (2,3,4). The Ecuadorian BBrMV isolate was successfully detected by three primer sets with reported amplification products of 324, 280, and 260 nucleotides long, respectively (3,4). Amplification products of the expected size were purified and sequenced. All the nucleotide sequences obtained from 20 PCR-positive symptomatic plants were 100% identical between each other. However, 99% identity was observed when PCR products from the Ecuadorian isolate were compared with the corresponding fragment of a BBrMV isolate from the Philippines (NCBI Accession No. DQ851496.1). PCR products of the Ecuadorian isolate, amplified by the different CP primers described above, were assembled into a 408-bp fragment and deposited in the NCBI GenBank (KC247746). Further testing confirmed the presence of BBrMV in symptomatic plants from four different provinces. To our knowledge, this is the first report of BBrMV in Ecuador and the first BBrMV partial nucleotide sequence reported from the Americas. It is worth mentioning that primer set Bract 1/Bract 2, which amplifies a 604-bp product (2), was not effective in detecting the Ecuadorian isolate. It is hypothesized that nucleotide variation at the reverse primer site is the cause of the lack of amplification with this primer set, since the forward primer is part of the sequenced product and no variation was found. Sequencing of the entire CP region is underway to conduct phylogenetic analysis and determine genetic relationships across several other BBrMV isolates.

Molecular cloning and characterization of arginine kinase gene of Toxocara canis.

Toxocara canis is an important gastrointestinal nematode of dogs and also a causative agent of visceral larva migrans in humans. Arginine kinase (AK) gene is one of the important biomolecule of phosphagen kinase of T. canis which is emerging as an exciting novel diagnostic target in toxocarosis. The present study was carried out to clone and characterize AK gene of T. canis for future utilization as a diagnostic molecule. Total RNA was extracted from intact adult worms and reverse transcription was done with oligo dT primers to obtain complementary DNA (cDNA). Polymerase chain reaction (PCR) was carried out using cDNA as template with specific primers which amplified a product of 1,202bp. The amplicon was cloned into pDrive cloning vector and clone was confirmed by colony PCR and restriction endonuclease analysis. Sequence analysis of the gene showed 99.8 and 77.9% homology with the published AK gene of T. canis (EF015466.1) and Ascaris suum respectively. Structural analysis shown that the mature AK protein consist of 400 amino acids with a molecular wt of 45360.73Da. Further expression studies are required for producing the recombinant protein for its evaluation in the diagnosis of T. canis infection in humans as well as in adult dogs.

Expression, purification, and enzymatic characterization of Bombyx mori nucleopolyhedrovirus DNA polymerase

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major viral agent that causes deadly grasserie disease in silkworms. BmNPV DNA polymerase (Bm-DNAPOL), encoded by the ORF53 gene, plays a central role in viral DNA replication. In this work, a His-tagged Bm-DNAPOL fusion protein, constructed using a novel MultiBac expression system, was overexpressed in Sf-9 insect cells, purified to near homogeneity on Ni-NTA agarose beads and further purified by ion-exchange chromatography. About 0.4 mg of enzyme was obtained from about 1 × 10(9) infected Sf-9 cells in suspension culture. Characterization of the highly purified enzyme indicated that Bm-DNAPOL is a monomer with an apparent molecular mass of approximately 110,000 Da. It possessed a specific activity of 15,126.3 U/mg under optimal in vitro reaction conditions and behaved in the manner of a proliferating cell nuclear antigen (PCNA)-independent DNA polymerase on both poly(dA)/oligo(dT) primer/template and singly premiered M13 DNA. BmNPV viral replication may be independent of replication factor C and a PCNA complex, while single-stranded DNA binding protein might play an important role in BmNPV DNA replication. These findings will be significant in studies on BmNPV-based disease in silkworms and for using silkworms as a bioreactor for the production of biomolecules of commercial importance.

Insulin receptor substrate-1 (IRS-1) forms a ribonucleoprotein complex associated with polysomes

Insulin receptor substrates (IRSs) are known to play important roles in mediating intracellular insulin-like growth factors (IGFs)/insulin signaling. In this study, we identified components of messenger ribonucleoprotein (mRNP) as IRS-1-associated proteins. IRS-1 complex formation analysis revealed that IRS-1 is incorporated into the complexes of molecular mass more than 1000kDa, which were disrupted by treatment with RNase. Furthermore, oligo(dT) beads precipitated IRS-1 from cell lysates, showing that the IRS-1 complexes contained messenger RNA. Taken together with the data that IRS-1 was fractionated into the polysome-containing high-density fractions, we concluded that IRS-1 forms the novel complexes with mRNPs.

Assessment of the viability of Cryptosporidium parvum oocysts with the induction ratio of hsp70 mRNA production in manure

Determining the presence of viable Cryptosporidium parvum oocysts in complex environmental matrices in hygiene control can prevent the contamination of water resources and food with this pathogen. This study assessed the induction ratio of hsp70 mRNA production by heat shock in different oocysts as a marker of viability. Using different procedures for (m)RNA extraction directly from manure and reverse transcription real-time qPCR, this study found slightly increased hsp70 mRNA contents in viable oocysts that were heat shock induced at 45°C for 20min compared to not induced oocysts (1.6 fold induction in average). Prolonging the heat shock treatment to 2h did not further increase the copy numbers. Heat shock by consecutive stimuli, such as freezing and then heating, did not yield significantly higher copy numbers than the 45°C treatment. There was a certain background level of hsp70 mRNA in viable oocysts that were not exposed to heat shock, indicating a constitutive production of the transcripts in the oocysts. The production of hsp70 mRNA induced by heat shock in oocysts aged for 9months that exhibited reduced viability was lower than in fresher oocysts (induction ratio<1.2). No production of hsp70 mRNA by heat shock was detected in 12months old oocysts that were not viable in the excystation test. Oocysts inactivated at 75°C for 30min were not able to respond to heat shock, and low amount of copies were occasionally measured only in total RNA extracts, but not in mRNA extracts that were purified directly with an oligo (dT)25 based system. The induction ratio of hsp70 mRNA varied according to the viability of the organisms in a sample. Copy numbers of β-tubulin mRNA in viable oocysts were lower than hsp70 mRNA, therefore the latter is more suitable to detect low numbers of oocysts by RT-qPCR.

Transcriptional Regulation by the Wilms Tumor Protein, Wt1, Suggests a Role of the Metalloproteinase Adamts16 in Murine Genitourinary Development

ADAMTS16 (a disintegrin and metalloproteinase with thrombospondin motifs) is a secreted mammalian metalloproteinase with unknown function. We report here that murine Adamts16 is co-expressed with the Wilms tumor protein, Wt1, in the developing glomeruli of embryonic kidneys. Adamts16 mRNA levels were significantly reduced upon transfection of embryonic murine kidney explants with Wt1 antisense vivo-morpholinos. Antisense knockdown of Adamts16 inhibited branching morphogenesis in kidney organ cultures. Adamts16 was detected by in situ mRNA hybridization and/or immunohistochemistry also in embryonic gonads and in spermatids and granulosa cells of adult testes and ovaries, respectively. Silencing of Wt1 by transfection with antisense vivo-morpholinos significantly increased Adamts16 mRNA in cultured embryonic XY gonads (11.5 and 12.5 days postconception), and reduced Adamts16 transcripts in XX gonads (12.5 and 13.5 days postconception). Three predicted Wt1 consensus motifs could be identified in the promoter and the 5'-untranslated region of the murine Adamts16 gene. Binding of Wt1 protein to these elements was verified by EMSA and ChIP. A firefly luciferase reporter gene under control of the Adamts16 promoter was activated ∼8-fold by transient co-transfection of human granulosa cells with a Wt1 expression construct. Gradual shortening of the 5'-flanking sequence successively reduced and eventually abrogated Adamts16 promoter activation by Wt1. These findings demonstrate that Wt1 differentially regulates the Adamts16 gene in XX and XY embryonic gonads. It is suggested that Adamts16 acts immediately downstream of Wt1 during murine urogenital development. We propose that Adamts16 is involved in branching morphogenesis of the kidneys in mice.

Simultaneous detection and identification of four cherry viruses by two step multiplex RT-PCR with an internal control of plant nad5 mRNA

A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed and standardized for the simultaneous detection of four cherry viruses: Cherry virus A (CVA, Genus; Capillovirus), Cherry necrotic rusty mottle virus (CNRMV, unassigned species of the Betaflexiviridae), Little cherry virus 1 (LChV-1, Genus; Closterovirus) and Prunus necrotic ringspot virus (PNRSV, Genus; Ilarvirus) with nad5 as plant internal control. A reliable and quick method for total plant RNA extraction from pome and stone fruit trees was also developed. To minimize primer dimer formation, a single antisense primer for CVA and CNRMV was used. A mixture of random hexamer and oligo (dT) primer was used for cDNA synthesis, which was highly suited and economic for multiplexing. All four viruses were detected successfully by mRT-PCR in artificially created viral RNA mixture and field samples of sweet cherry. The identity of the viruses was confirmed by sequencing. The assay could detect above viruses in diluted cDNA (10−4) and RNA (10−3, except PNRSV which was detected only till ten times lesser dilution). The developed mRT-PCR will not only be useful for the detection of viruses from single or multiple infections of sweet cherry plants but also for other stone and pome fruits. The developed method will be therefore quite helpful for virus indexing, plant quarantine and certification programs. This is the first report for the simultaneous detection of four cherry viruses by mRT-PCR.