Publications by authors named "Shihai Huang"

24 Publications

  • Page 1 of 1

AQP8 participates in oestrogen-mediated buffalo follicular development by regulating apoptosis of granulosa cells.

Reprod Domest Anim 2021 Feb 27. Epub 2021 Feb 27.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, China.

Aquaporins (AQPs), a family of small membrane-spanning proteins, are involved in fluid transport, cell signalling and reproduction. Regulating AQP8 expression influences apoptosis of granulosa cells (GCs), ovarian folliculogenesis, oogenesis and early embryonic development in mice, but its role has never been investigated in other species. The aim of the present study was to characterize the AQP8 function in buffalo follicular development. The expression pattern of AQP8 in buffalo follicle was analysed by immunohistochemistry method. 17β-Estradiol (E2) or oestrogen receptor antagonist ICI182780 was used to treat GCs cultured in vitro, and the expression of AQP8 was detected using qRT-PCR. Its roles in apoptosis of buffalo GCs were investigated by shRNA technology. AQP8 was found to be expressed higher in secondary follicles (p < .05), and its mRNA level in GCs was upregulated by E2 via receptor-mediated mechanism in a dose-dependent manner. A 732-bp buffalo AQP8 coding region was obtained, which was highly conserved at the amino acid level among different species. AQP8-shRNA2 had more effective inhibition on target gene than AQP8-shRNA1 (66.49% vs. 58.31%) (p < .05). Knockdown of AQP8 induced GCs arrested at G2/M stage and occurred apoptosis. Compared with the control group, higher Caspase9 expression were observed in AQP8-shRNA2 lentivirus infected GCs (p < .05), while Bcl-2 and Bax expression levels had no obvious change (p > .05). Altogether, the above results indicate that AQP8 is involved in oestrogen-mediated regulation of buffalo follicular development by regulating cell cycle progression and apoptosis of GCs.
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http://dx.doi.org/10.1111/rda.13921DOI Listing
February 2021

Expression patterns of ZO-1/2 and their effects on porcine oocyte in vitro maturation and early embryonic development.

Theriogenology 2021 Feb 13;161:262-270. Epub 2020 Dec 13.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, 530005, PR China. Electronic address:

Zonula occludens (ZO)-1 and ZO-2 are involved in epithelial polarity maintenance, gene transcription, cell proliferation and tumor cell metastasis. Regulating ZO-1/2 expression influences the early embryonic development of mice, but whether they are involved in oocyte maturation is still poorly understood. In the present study, the expression patterns of ZO-1 and ZO-2 in porcine cumulus cells and oocytes matured in vitro and early embryos from parthenogenetic activation were detected by qRT-PCR or Western blot, and then their roles in porcine oocyte maturation and early embryo development were investigated by shRNA technology. ZO-1 and ZO-2 were found to be expressed in cumulus cells, oocytes and early embryos, while ZO-1α was expressed only in cumulus cells, morula and blastocysts. During in vitro maturation (IVM), the abundance of ZO-1 and ZO-2 in oocytes was significantly higher than that in cumulus cells at 0 h (P < 0.01), and their mRNA and protein levels displayed relatively higher expression at 0 and 18 h, respectively. Compared with the control groups, cumulus cell expansion, oocyte nucleus maturation, and subsequent cleavage were not influenced by treatment of the cumulus-oocyte complexes (COCs) with ZO-1-shRNA1, ZO-2-shRNA2 or combined ZO-1-shRNA1 and ZO-2-shRNA2 lentivirus (P > 0.05). However, the blastocyst rate was reduced by treatment of COCs with ZO-1-shRNA1 but not ZO-2-shRNA2. The total cell number of blastocysts was decreased by downregulation of ZO-1 and ZO-2 (P < 0.05). Downregulation of ZO-1 and ZO-2 also resulted in a significant decrease (P < 0.05) in the expression of Cx43, Cx45, PTX3 and PTGS2 in cumulus cells, Cx45, BMP15, ZP3 and C-KIT in MII oocytes, and Nanog in blastocysts, with the exception of HAS2 expression in cumulus cells and Oct4 expression in blastocysts (P > 0.05). Altogether, the above results indicate that ZO-1 and ZO-2 display similar expression patterns during porcine oocyte IVM and are critical to porcine oocyte maturation and early embryonic development.
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http://dx.doi.org/10.1016/j.theriogenology.2020.12.009DOI Listing
February 2021

Clinical characteristics and treatment of different types of cesarean scar pregnancy.

Ginekol Pol 2020;91(7):406-411

Affiliated Chaohu Hospital of Anhui Medical University, Hefei, Anhui, Department of Ultrasonography, PR China.

Objectives: The purpose of this study was to evaluate the clinical characteristics and compare the treatment efficacy of different types of cesarean scar pregnancy (CSP).

Material And Methods: We performed a retrospective chart review of 66 women (69 cases) with CSP who received treatment with mifepristone/methotrexate (MTX) plus curettage, uterine artery embolization (UAE) plus curettage, additional MTX, or laparotomy, and compared the clinical characteristics, treatment efficacy, and occurrence of complications among 3 types of CSP (partial, complete, and mass type).

Results: Review of the 69 cases revealed a considerable increase of gestational duration(p < 0.001), sac/lesion size(p < 0.001) and vaginal bleeding (p < 0.05) in patients with mass-type CSP compared to that of other types. All CSP cases were successfully treated, 4 cases of mass-type received laparotomy and none of the cases required a hysterectomy. Severe bleeding was observed in 2 cases of partial-type and complete-type, respectively, and 3 cases for mass-type. Moreover, bleeding occurred during initial treatment with mifepristone plus curettage in partial-type cases, but not with UAE plus curettage.

Conclusions: UAE plus curettage is a more effective treatment option for partial- and complete-type of CSP than mifepristone plus curettage. The cases of mass-type often need surgery and are prone to have longer gestational duration, larger lesions, and more vaginal bleeding.
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http://dx.doi.org/10.5603/GP.2020.0065DOI Listing
January 2020

Multicenter clinical evaluation of alinity m HCV assay performance.

J Clin Virol 2020 08 3;129:104531. Epub 2020 Jul 3.

West of Scotland Specialist Virology Centre, Glasgow, UK.

Background: Nucleic acid testing is essential for the detection and quantification of HCV RNA in the diagnosis of HCV infection and treatment monitoring. The Alinity m HCV assay was recently developed by Abbott Molecular for rapid detection and quantification of HCV RNA on the fully automated, continuous, random-access Alinity m analyzer.

Objectives: Our study assessed the performance of the new Alinity m HCV assay for detection and quantification of HCV RNA in a large series of patient samples of various genotypes. This international, multicentric study evaluated the linearity, precision, and reproducibility of the Alinity m HCV assay and its performance in comparison to three other HCV assays currently used in clinical practice.

Results: The Alinity m HCV assay demonstrated high linearity (correlation coefficient r = 1.00), precision (coefficients of variation [CV] 6.6-13.5 %) and reproducibility (CV 1.7-4.3 % across three control lots). At a concentration near the lower limit of detection, the Alinity m HCV assay exhibited >98 % detectability. The Alinity m HCV assay showed excellent correlation with comparator HCV assays in serum (n = 406) and plasma (n = 1401) samples (correlation coefficients ≥0.96, bias 0.01 to 0.14 Log IU/mL). More than 95 % of the quantified results with the Alinity m HCV assay were less than mean bias ± 1.96 SD different from those of the comparator assays.

Conclusions: The newly developed Alinity m HCV assay is sensitive, reproducible, and accurately quantifies HCV RNA levels in serum and plasma samples from patients with chronic HCV infection, with no impact of HCV genotype on assay performance.
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http://dx.doi.org/10.1016/j.jcv.2020.104531DOI Listing
August 2020

Cloning and functional characterization of STAT5a and STAT5b genes in buffalo mammary epithelial cells.

Anim Biotechnol 2020 Feb 15;31(1):59-66. Epub 2018 Nov 15.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning 530004, China.

Signal transducer and activator 5 (STAT5) plays important roles in regulating mammary glandular cell proliferation and milk-protein gene expression. However, the functions of STAT5a and STAT5b genes in lactation of buffalo remain uninvestigated. In this study, full-length STAT5a (2502 bp) and STAT5b (2515 bp) coding sequences were isolated for the first time. The highest STAT5a gene expression was found in buffalo mammary glands and the highest STAT5b gene expression was found in buffalo brains and mammary glands. H&E staining indicated that STAT5a and STAT5b are mainly expressed in epithelial cells of buffalo mammary glands. Next, we investigated the functions of STAT5 by knocking down and overexpressing STAT5 in buffalo mammary epithelial cells (BuMECs). According to our results, STAT5 knockdown resulted in inhibited G1/S transition of BuMECs and significantly lower expression of milk-protein genes, whereas overexpression of STAT5 resulted in significantly higher expression of milk-protein genes. In summary, our results demonstrate that STAT5 can regulate the cell cycle transition of BuMECs and affect the expression of milk-protein genes. Our research lays a foundation for further study of the role of in mammary gland development and lactation.
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http://dx.doi.org/10.1080/10495398.2018.1538014DOI Listing
February 2020

Inhibition of Suv39H1 enhances transgenic IFNα-2b gene expression in Bcap-37 cells.

Anim Biotechnol 2019 Oct 4;30(4):358-365. Epub 2018 Sep 4.

State Key Laboratory of Subtropical Bioresource Conservation and Utilization at Guangxi University , Guangxi , China.

The low expression of exogenous transferred gene limited the application of transgenic animal technology. Suppressor of variegation 3 ∼ 9 homolog 1(SUV39H1) gene plays a prominent role on repressive heterochromatin and transcription. To understand if exogenous transgenic gene expression was affected by SUV39H1 epigenetic modification, in this paper, the effective shRNA fragments targeting SUV39H1 gene were first screened, their roles on expression of exogenous transgenic genes were determined by using Bcap-37 cell line with stable expressing IFNα-2b gene as a model, the preliminary regulation mechanism of SUV39H1 gene was investigated. The results showed that the designed shRNA1/2 fragments of SUV39H1 gene had an obvious inhibition effect on the expression of SUV39H1 gene, reached 53.07 and 31.28%, respectively by qRT-PCR analysis. Compared with the control group, the expression of IFNα-2b gene in transgenic Bcap-37 cells infected with shRNA1 and 2 viruses significantly increased by 96.25 and 121.08%, respectively ( 0.05). In addition, the expression of DNMT1, HDAC1 and G9a gene in the shRNA infected cells reduced significantly, and the expression of the HAT1 gene increased significantly ( 0.05). The above results indicated that the expression of exogenous transgenic gene could be promoted by suppressing SUV39H1 gene at the cell level.
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http://dx.doi.org/10.1080/10495398.2018.1500373DOI Listing
October 2019

Production of D-alanine from DL-alanine using immobilized cells of Bacillus subtilis HLZ-68.

World J Microbiol Biotechnol 2017 Sep 13;33(9):176. Epub 2017 Sep 13.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning, 530004, Guangxi, China.

Immobilized cells of Bacillus subtilis HLZ-68 were used to produce D-alanine from DL-alanine by asymmetric degradation. Different compounds such as polyvinyl alcohol and calcium alginate were employed for immobilizing the B. subtilis HLZ-68 cells, and the results showed that cells immobilized using a mixture of these two compounds presented higher L-alanine degradation activity, when compared with free cells. Subsequently, the effects of different concentrations of polyvinyl alcohol and calcium alginate on L-alanine consumption were examined. Maximum L-alanine degradation was exhibited by cells immobilized with 8% (w/v) polyvinyl alcohol and 2% (w/v) calcium alginate. Addition of 400 g of DL-alanine (200 g at the beginning of the reaction and 200 g after 30 h of incubation) into the reaction solution at 30 °C, pH 6.0, aeration of 1.0 vvm, and agitation of 400 rpm resulted in complete L-alanine degradation within 60 h, leaving 185 g of D-alanine in the reaction solution. The immobilized cells were applied for more than 15 cycles of degradation and a maximum utilization rate was achieved at the third cycle. D-alanine was easily extracted from the reaction solution using cation-exchange resin, and the chemical and optical purity of the extracted D-alanine was 99.1 and 99.6%, respectively.
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http://dx.doi.org/10.1007/s11274-017-2341-3DOI Listing
September 2017

MicroRNA-148a overexpression improves the early development of porcine somatic cell nuclear transfer embryos.

PLoS One 2017 30;12(6):e0180535. Epub 2017 Jun 30.

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, Guangxi Zhuang Autonomous Region, China.

Incomplete epigenetic reprogramming of donor cell nuclei is one of the main contributors to the low efficiency of somatic cell nuclear transfer (SCNT). To improve the success of SCNT, somatic cell DNA methylation levels must be reduced to those levels found in totipotent embryonic cells. Recent studies have demonstrated that miR-148a can affect DNA methylation via DNMT1 modulation in various cancers. Therefore, the focus of this study was to examine the influence of miR-148a on DNA methylation in donor cells and in SCNT embryo development. Thus, a stable cell line overexpressing miR-148a was established and used to produce SCNT embryos. Upon examination, DNMT1 was found to be a miR-148a target in porcine fetal fibroblasts (PFF). Furthermore, miR-148a overexpression in PFFs significantly decreased DNMT1 expression and global DNA methylation levels (P < 0.05). Moreover, miRNA-148a expression levels in SCNT embryos were significantly lower at the 2-cell and 4-cell stages when compared to IVF and parthenogenetic embryos. The group overexpressing miRNA-148a also showed a significant increase in blastocyst formation and total cell numbers (P < 0.05). Additionally, miR-148a overexpression altered the immunofluorescence signal of 5-mC and H3K9ac, and enhanced pluripotent gene (Oct4 and Nanog) expression levels during embryo development. These results indicate that miR-148a overexpression enhances the developmental potential of SCNT embryos and modifies epigenetic status.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0180535PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5493425PMC
October 2017

HIV-1 viral load measurement in venous blood and fingerprick blood using Abbott RealTime HIV-1 DBS assay.

J Clin Virol 2017 07 13;92:56-61. Epub 2017 May 13.

BARC-SA and Lancet Laboratories, Johannesburg, South Africa.

Background: HIV RNA suppression is a key indicator for monitoring success of antiretroviral therapy. From a logistical perspective, viral load (VL) testing using Dried Blood Spots (DBS) is a promising alternative to plasma based VL testing in resource-limited settings.

Objectives: To evaluate the analytical and clinical performance of the Abbott RealTime HIV-1 assay using a fully automated one-spot DBS sample protocol.

Study Design: Limit of detection (LOD), linearity, lower limit of quantitation (LLQ), upper limit of quantitation (ULQ), and precision were determined using serial dilutions of HIV-1 Virology Quality Assurance stock (VQA Rush University), or HIV-1-containing armored RNA, made in venous blood. To evaluate correlation, bias, and agreement, 497 HIV-1 positive adult clinical samples were collected from Ivory Coast, Uganda and South Africa. For each HIV-1 participant, DBS-fingerprick, DBS-venous and plasma sample results were compared. Correlation and bias values were obtained. The sensitivity and specificity were analyzed at a threshold of 1000 HIV-1 copies/mL generated using the standard plasma protocol.

Results: The Abbott HIV-1 DBS protocol had an LOD of 839 copies/mL, a linear range from 500 to 1×10 copies/mL, an LLQ of 839 copies/mL, a ULQ of 1×10 copies/mL, and an inter-assay SD of ≤0.30 log copies/mL for all tested levels within this range. With clinical samples, the correlation coefficient (r value) was 0.896 between DBS-fingerprick and plasma and 0.901 between DBS-venous and plasma, and the bias was -0.07 log copies/mL between DBS-fingerprick and plasma and -0.02 log copies/mL between DBS-venous and plasma. The sensitivity of DBS-fingerprick and DBS-venous was 93%, while the specificity of both DBS methods was 95%.

Conclusion: The results demonstrated that the Abbott RealTime HIV-1 assay with DBS sample protocol is highly sensitive, specific and precise across a wide dynamic range and correlates well with plasma values. The Abbott RealTime HIV-1 assay with DBS sample protocol provides an alternative sample collection and transfer option in resource-limited settings and expands the utility of a viral load test to monitor HIV-1 ART treatment for infected patients.
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http://dx.doi.org/10.1016/j.jcv.2017.05.002DOI Listing
July 2017

Structural insights into cardiolipin transfer from the Inner membrane to the outer membrane by PbgA in Gram-negative bacteria.

Sci Rep 2016 08 4;6:30815. Epub 2016 Aug 4.

Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.

The outer membrane (OM) of Gram-negative bacteria is a unique asymmetric lipid bilayer in which the outer leaflet is composed of lipopolysaccharide (LPS) and the inner leaflet is formed by glycerophospholipid (GPL). The OM plays a fundamental role in protecting Gram-negative bacteria from harsh environments and toxic compounds. The transport and assembly pathways for phospholipids of bacterial OM are unknown. Cardiolipin (CL) plays an important role in OM biogenesis and pathogenesis, and the inner membrane (IM) protein PbgA, containing five transmembrane domains and a globular domain in periplasm has been recently identified as a CL transporter from the IM to the OM with an unknown mechanism. Here we present the first two crystal structures of soluble periplasmic globular domain of PbgA from S. typhimurium and E. coli, which revealed that the globular domains of PbgA resemble the structures of the arylsulfatase protein family and contains a novel core hydrophobic pocket that may be responsible for binding and transporting CLs. Our structural and functional studies shed an important light on the mechanism of CL transport in Gram-negative bacteria from the IM to the OM, which offers great potential for the development of novel antibiotics against multi-drug resistant bacterial infections.
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http://dx.doi.org/10.1038/srep30815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4973235PMC
August 2016

Complete genome sequence of Bacillus amyloliquefaciens B15 isolated from grape skin, a strain of strong inhibitory activity against fungi.

J Biotechnol 2016 Jun 22;228:28-29. Epub 2016 Apr 22.

The Department of Traditional Fermentation Engineering (Brewing), China National Research Institute of Food and Fermentation Industries, 100015 Beijing, China. Electronic address:

Bacillus amyloliquefaciens B15 is a Gram-positive, plant-associated bacterium which shows strong antifungal activity, isolated from grape skin in Xinjiang, China. The genome of B. amyloliquefaciens B15 comprises a 4,006,754bp long circular chromosome containing 3991 protein coding genes and 109 RNA genes. Based on genomic analysis, we identified the giant gene clusters, nonribosomal peptidesynthetases (NRPS), and polyketide synthases (PKS), responsible for the biosynthesis of numerous bioactive metabolites. In addition, several functionally related genes, such as TasA, were also been identified for the antagonistic effect on pathogenic fungi but has no effect on the growth of itself.
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http://dx.doi.org/10.1016/j.jbiotec.2016.04.036DOI Listing
June 2016

Establishment of a mouse model to express bovine CD14 short hairpin RNA.

BMC Vet Res 2015 Feb 15;11:36. Epub 2015 Feb 15.

State Key Laboratory of Subtropical Bioresource Conservation and Utilization at Guangxi University, Nanning, Guangxi, China.

Background: Cluster of differentiation 14 (CD14) functions as a co-receptor for Toll-like receptor (TLR)-4 and myeloid differentiation factor (MD)-2 in detecting bacterial lipopolysaccharide. Together, these complexes promote the phagocytosis and digestion of Gram-negative bacteria, and initiate immune responses. To date, much of our understanding of CD14 function during Gram-negative bacterial inflammation comes from studies on mouse knockout models and cell transfection. To identify the effect of CD14 knockdown in this process in large livestock animals, we established a mouse model expressing bovine CD14 short hairpin (sh) RNA. shRNA fragments targeting bovine CD14 were screened by co-transfection in HEK 293 cells, and the most effective CD14 shRNA fragment was cloned into the eukaryotic expression vector pSilencer4.1-CD14 shRNA-IRES (internal ribosome entry site) and transferred into mouse zygotes by pronuclear microinjection to obtain transgenic mice. Expression of the enhanced green fluorescent protein (EGFP) reporter and genes related to the TLR4 signaling pathway was detected by immunohistochemistry (IHC) and quantitative polymerase chain reaction (PCR), respectively.

Results: One effective shRNA fragment (shRNA-674) targeting bovine CD14 was obtained, the sequence of which was shown to be conserved between cows, buffalos, sheep, and humans. Thirty-seven founder pups were obtained by pronuclear microinjection, of which three were positive for the transgene. In the F(1) generation, 11 of 33 mice (33%) were positive for the transgene as detected by PCR. IHC analysis detected exogenous EGFP expression in the liver, kidney, and spleen of transgenic F(1) mice, indicating that they were chimeric. The expression of endogenous CD14 mRNA in the heart, liver, spleen, lung, and kidney of transgenic F(1) mice was decreased 8-, 3-, 19.5-, 6-, and 11-fold, respectively. The expression patterns of endogenous MD-2, TLR4, interleukin-6 and tumor necrosis factor-α genes in transgenic mice also varied.

Conclusions: This study confirms that transgenic mice expressing bovine CD14 shRNA can be generated by pronuclear microinjection, and demonstrates inhibited endogenous mouse CD14 expression that alters gene expression related to the TLR4 signaling pathway.
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http://dx.doi.org/10.1186/s12917-015-0353-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4332730PMC
February 2015

The effect of buffalo CD14 shRNA on the gene expression of TLR4 signal pathway in buffalo monocyte/macrophages.

Cell Mol Biol Lett 2014 Dec 29;19(4):623-37. Epub 2014 Oct 29.

State Key Laboratory of Subtropical Bioresource Conservation and Utilization at Guangxi University, Nanning, Guangxi, China,

CD14 plays a crucial role in the inflammatory response to lipopolysaccharide (LPS), which interacts with TLR4 and MD-2 to enable cell activation, resulting in inflammation. Upstream inhibition of the inflammation pathway mediated by bacterial LPS, toll-like receptor 4 (TLR4) and cluster of differentiation antigen 14 (CD14) was proven to be an effective therapeutic approach for attenuating harmful immune activation. To explore the effect of CD14 downregulation on the expression of TLR4 signaling pathway-related genes after LPS stimulation in buffalo (Bubalus bubalis) monocyte/macrophages, effective CD14 shRNA sequences were screened using qRT-PCR and FACS analysis with buffalo CD14 shRNA lentiviral recombinant plasmids (pSicoRGFP-shRNA) and buffalo CD14 fusion expression plasmids (pDsRed-N1-buffalo CD14) co-transfected into HEK293T cells via liposomes. Of the tested shRNAs, shRNA-1041 revealed the highest knockdown efficiency (p < 0.01). When buffalo peripheral blood monocyte/macrophages were infected with shRNA-1041 lentivirus and stimulated with LPS, the expression of endogenous CD14 was significantly decreased by CD14 shRNA (p < 0.01), and the mRNA expression levels of TLR4, IL-6 and TNF-α were also significantly downregulated compared to the control groups (p < 0.01). These results demonstrated that the knockdown of endogenous CD14 had clear regulatory effects on the signal transduction of TLR4 after stimulation with LPS. These results may provide a better understanding of the molecular mechanisms of CD14 regulation in the development of several buffalo diseases.
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http://dx.doi.org/10.2478/s11658-014-0217-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6275898PMC
December 2014

Biochemical characteristics of a fibrinolytic enzyme purified from a marine bacterium, Bacillus subtilis HQS-3.

Int J Biol Macromol 2013 Nov 1;62:124-30. Epub 2013 Sep 1.

College of Life Science and Technology, Guangxi University, Nanning 530004, China.

A fibrinolytic enzyme isolated from marine Bacillus subtilis HQS-3 was purified to electrophoretic homogeneity using ammonium sulphate precipitation, alkaline solution treatment, membrane concentration, dialysis, ion exchange, and gel filtration chromatography. SDS-PAGE and gel filtration chromatography showed that it was a monomeric protein with an apparent molecular weight of 26 kDa. The purified enzyme was active at pH 6.0-10.0 with an optimum pH of 8.0. It was stable at temperatures ranging from 25 to 37 °C, exhibiting maximum activity between 45 °C and 50 °C. The isoelectric point of the enzyme was 9.0-9.2, which was higher than those of other known fibrinolytic enzymes from Bacillus species. PMSF, EDTA, Cu(2+), Zn(2+), and Co(2+) inhibited the enzyme activity significantly. This enzyme did not cause hemolysis in vitro and preferred direct degradation of fibrin in the following order: α, β, and γ-γ chains. Thus, these results suggest that the marine-derived enzyme is a plasmin-like serine metalloprotease, which is distinct from other fibrinolytic enzymes from genus Bacillus.
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http://dx.doi.org/10.1016/j.ijbiomac.2013.08.048DOI Listing
November 2013

A novel RealTime HIV-1 Qualitative assay for the detection of HIV-1 nucleic acids in dried blood spots and plasma.

J Virol Methods 2011 Dec 24;178(1-2):216-24. Epub 2011 Sep 24.

Abbott Molecular Inc., 1300 E Touhy Avenue, Des Plaines, IL 60018-3315, USA.

Abbott RealTime HIV-1 Qualitative is an in vitro real-time PCR assay for detecting HIV-1 nucleic acids in human plasma and dried blood spots (DBS). The assay was designed to be used in diagnosis of HIV-1 infections in pediatric and adult patients, with an emphasis on the applicability in resource-limited settings. Use of DBS facilitates specimen collection from remote areas and transportation to testing laboratories. Small sample input requirement facilitates testing of specimens with limited collection volume. The Abbott RealTime HIV-1 Qualitative assay is capable of detecting HIV-1 group M subtypes A-H, group O and group N samples. HIV-1 virus concentrations detected with 95% probability were 80 copies/mL of plasma using the plasma protocol, and 2469 copies/mL of whole blood using the DBS protocol. The assay detected HIV-1 infection in 13 seroconversion panels an average 10.5 days earlier than an HIV-1 antibody test and 4.9 days earlier than a p24 antigen test. For specimens collected from 6 weeks to 18 months old infants born to HIV-1 positive mothers, assay results using both the DBS and plasma protocols agreed well with the Roche Amplicor HIV-1 DNA Test version 1.5 (95.5% agreement for DBS and 97.8% agreement for plasma).
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http://dx.doi.org/10.1016/j.jviromet.2011.09.015DOI Listing
December 2011

High-risk HPV detection and concurrent HPV 16 and 18 typing with Abbott RealTime High Risk HPV test.

J Clin Virol 2009 Jul;45 Suppl 1:S25-8

Abbott Molecular Inc., Des Plaines, IL 60018-3315, USA.

Background: High-risk human papillomavirus (HPV) is the causative agent of cervical cancer. Among the high-risk types, infection with HPV 16 and 18 is associated with significantly higher risk of disease progression, and consequently these two types together cause approximately 70% of invasive cervical cancer worldwide. Identification of HPV 16 and HPV 18 can provide valuable information for risk stratification and clinical management of patients infected with these two types in both ASC-US triage and primary screening in women over age 30. It may also be valuable in the assessment of HPV vaccine efficacy. Abbott RealTime High Risk (HR) HPV is a recently developed test for the detection of 14 high-risk HPV types with the ability to concurrently identify HPV 16 and 18.

Objective: To evaluate the clinical performance of Abbott RealTime HR HPV test.

Study Design: Abbott RealTime HR HPV was evaluated with 253 cervical specimens obtained from patients with CIN 3 and 340 specimens from patients with cervical cancer to determine clinical sensitivity of the test and the prevalence of types 16 and 18. Additionally, 757 cervical specimens obtained from women 30 years of age or older with normal cytology in a general screening population were tested to determine high-risk HPV positivity rate.

Results: The Abbott RealTime HR HPV test detected 97.2% (246/253) of CIN 3 specimens and 98.5% (335/340) of cancer specimens. HPV 16 was the most prevalent type in both CIN 3 (72.8%) and cancer specimens (64.5%). HPV 16 and 18 combined were detected in 78.9% of high-risk HPV positive CIN 3 and 84.8% of high-risk HPV positive cancer specimens. In specimens from women 30 years of age or older with normal cytology in a screening population, the HPV positivity rate was 6.5% (49/757).

Conclusions: Abbott RealTime HR HPV is a highly sensitive test for detection of high-grade cervical disease and cancer. The HPV 16 and HPV 18 typing capability of the test offers the advantage of stratifying patients at greater risk of progression and may thus aid in better patient care and management.
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http://dx.doi.org/10.1016/S1386-6532(09)70005-8DOI Listing
July 2009

Clinical performance of Abbott RealTime High Risk HPV test for detection of high-grade cervical intraepithelial neoplasia in women with abnormal cytology.

J Clin Virol 2009 Jul;45 Suppl 1:S19-23

Abbott Molecular Inc., Des Plaines, Illinois 60018-3315, USA.

Background: Abbott RealTime High Risk (HR) HPV is a recently developed test for the detection of 14 high-risk oncogenic HPV types combined with the ability to concurrently identify genotypes 16 and 18.

Objectives: The clinical performance of the Abbott RealTime HR HPV test was evaluated in comparison with the Hybrid Capture 2 (HC2) test for the detection of cervical intraepithelial neoplasia 2 or worse (CIN2+). The relative accuracy of the Abbott RealTime HR HPV to detect high-risk HPV was also determined.

Study Design: Cervical specimens were collected from 702 patients with abnormal cytology who were referred for colposcopy, and were tested with liquid based cytology (LBC), Abbott RealTime HR HPV and HC2. Genotyping was done using the Linear Array (LA) method. Histological assessment was used as the gold standard for disease status. Clinical performance for detection of disease was evaluated for Abbott RealTime HR HPV in comparison with HC2 in the overall population and in each cytological grade. The relative accuracy for detection of high-risk HPV was assessed by concordance between the two tests and based on LA genotyping.

Results And Conclusions: The Abbott RealTime HR HPV showed similar clinical performance for detection of CIN2+ when compared with HC2, for both the overall population and those with a cytological grade of atypical squamous cells of undetermined significance (ASC-US). The accuracy for detection of high-risk HPV was significantly higher with Abbott RealTime HR HPV than with HC2.
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http://dx.doi.org/10.1016/S1386-6532(09)70004-6DOI Listing
July 2009

Principles and analytical performance of Abbott RealTime High Risk HPV test.

J Clin Virol 2009 Jul;45 Suppl 1:S13-7

Abbott Molecular Inc., Des Plaines, Illinois 60018-3315, USA.

Background: Abbott RealTime High Risk (HR) HPV is a new automated, qualitative real-time PCR test for detection of DNA from 14 high-risk human papillomavirus (HPV) types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) in cervical specimens. The test can also differentiate between HPV 16, HPV 18 and non-HPV 16/18 types in a single reaction.

Objectives: This article describes the principles of assay design and the analytical performance of Abbott RealTime HR HPV.

Study Design: The analytical performance characteristics of Abbott RealTime HR HPV were evaluated in terms of its sensitivity for each of the 14 high-risk types included in the test, specificity (cross-reactivity), potential for interference by substances that may be present in cervical specimens, and reproducibility.

Results: Abbott RealTime HR HPV provided sensitive detection of the 14 high-risk HPV types included in the test. It was also highly specific to the HPV types targeted by the test and did not show cross-reactivity with 15 low-risk HPV types tested, or non-specific reactivity with other common microorganisms that may be present in the female anogenital tract. Test results were not impacted by potential interfering substances evaluated in the study. The test generated highly reproducible results in an in-house study and in studies carried out at 13 external evaluation sites.

Conclusions: Abbott RealTime HR HPV demonstrated a robust analytical performance with reproducible and reliable results.
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http://dx.doi.org/10.1016/S1386-6532(09)70003-4DOI Listing
July 2009

A RealTime HIV-1 viral load assay for automated quantitation of HIV-1 RNA in genetically diverse group M subtypes A-H, group O and group N samples.

J Virol Methods 2007 Dec 17;146(1-2):236-45. Epub 2007 Aug 17.

Abbott Molecular Inc., D-9ND, Bldg. DP1, 1300 E Touhy Avenue, Des Plaines, IL 60018-3315, USA.

The Abbott RealTime HIV-1 assay is an automated test for monitoring HIV-1 viral load in plasma samples. The assay uses reverse transcription polymerase chain reaction (RT-PCR) technology with homogeneous real-time fluorescent detection. Automated sample preparation is performed on the m2000sp instrument where RNA is isolated using magnetic microparticle technology and dispensed to a PCR tray together with the amplification reagents. The PCR tray is then transferred to the Abbott m2000rt instrument for amplification and real-time detection. The assay utilizes two distinct sets of primers and probes for HIV-1 and for internal control (IC). The IC is processed along with each sample to control for sample recovery and inhibition. The HIV-1 primer and probe sequences are targeted to the integrase (IN) region of the polymerase (pol) gene. Due to the selection of a highly conserved target region and a novel, mismatch tolerant probe design, the assay can quantitate HIV-1 group M subtypes A-H, group O, and group N isolates. The assay provides high reproducibility and a wide dynamic range, allowing quantitation from 40 copies to 10 million copies of HIV-1 RNA per milliliter of plasma. HIV-1 RNA concentrations detected with 95% probability were 25copies/mL with 1.0mL of plasma, 39copies/mL with 0.6mL of plasma, 65copies/mL with 0.5mL of plasma, and 119copies/mL with 0.2mL of plasma.
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http://dx.doi.org/10.1016/j.jviromet.2007.07.003DOI Listing
December 2007

Thermodynamically modulated partially double-stranded linear DNA probe design for homogeneous real-time PCR.

Nucleic Acids Res 2007 9;35(16):e101. Epub 2007 Aug 9.

Abbott Molecular Inc., Des Plaines, IL, USA.

Real-time PCR assays have recently been developed for diagnostic and research purposes. Signal generation in real-time PCR is achieved with probe designs that usually depend on exonuclease activity of DNA polymerase (e.g. TaqMan probe) or oligonucleotide hybridization (e.g. molecular beacon). Probe design often needs to be specifically tailored either to tolerate or to differentiate between sequence variations. The conventional probe technologies offer limited flexibility to meet these diverse requirements. Here, we introduce a novel partially double-stranded linear DNA probe design. It consists of a hybridization probe 5'-labeled with a fluorophore and a shorter quencher oligo of complementary sequence 3'-labeled with a quencher. Fluorescent signal is generated when the hybridization probe preferentially binds to amplified targets during PCR. This novel class of probe can be thermodynamically modulated by adjusting (i) the length of hybridization probe, (ii) the length of quencher oligo, (iii) the molar ratio between the two strands and (iv) signal detection temperature. As a result, pre-amplification signal, signal gain and the extent of mismatch discrimination can be reliably controlled and optimized. The applicability of this design strategy was demonstrated in the Abbott RealTime HIV-1 assay.
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http://dx.doi.org/10.1093/nar/gkm551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2018630PMC
September 2007

Evaluation of performance across the dynamic range of the Abbott RealTime HIV-1 assay as compared to VERSANT HIV-1 RNA 3.0 and AMPLICOR HIV-1 MONITOR v1.5 using serial dilutions of 39 group M and O viruses.

J Virol Methods 2007 Apr 20;141(1):49-57. Epub 2006 Dec 20.

Abbott Diagnostics, AIDS Research and Retrovirus Discovery, D-09NG, Bldg. AP20, 100 Abbott Park Road, Abbott Park, IL 60064-6015, USA.

Performance of the Abbott m2000 instrument system and the Abbott RealTime HIV-1 assay was evaluated using a panel of 37 group M (subtypes A-D, F, G, CRF01_AE, CRF02_AG and unique recombinant forms) and 2 group O virus isolates. Testing was performed on 273 sample dilutions and compared to VERSANT HIV-1 RNA 3.0 (bDNA) and AMPLICOR HIV-1 MONITOR v1.5 (Monitor v1.5) test results. RealTime HIV-1, bDNA, and Monitor v1.5 tests quantified 87%, 78%, and 81% of samples, respectively. RealTime HIV-1 detected an additional 31 samples at < 40 copies/mL. For group M, RealTime HIV-1 dilution profiles and viral loads were highly correlated with bDNA and Monitor v1.5 values; 87% and 89% of values were within 0.5 log(10) copies/mL. In contrast, the group O viruses were not detected by Monitor v1.5 and were substantially underquantified by approximately 2 log(10) copies/mL in bDNA relative to the RealTime HIV-1 assay. Sequence analysis revealed that RealTime HIV-1 primer/probe binding sites are highly conserved and exhibit fewer nucleotide mismatches relative to Monitor v1.5. The automated m2000 system and RealTime HIV-1 assay offer the advantages of efficient sample processing and throughput with reduced "hands-on" time while providing improved sensitivity, expanded dynamic range and reliable quantification of genetically diverse HIV-1 strains.
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http://dx.doi.org/10.1016/j.jviromet.2006.11.026DOI Listing
April 2007

Performance of the automated Abbott RealTime HIV-1 assay on a genetically diverse panel of specimens from London: comparison to VERSANT HIV-1 RNA 3.0, AMPLICOR HIV-1 MONITOR v1.5, and LCx HIV RNA Quantitative assays.

J Virol Methods 2006 Nov 28;137(2):184-92. Epub 2006 Jul 28.

Abbott Laboratories, AIDS Research and Retrovirus Discovery, D-09NG, Bldg. AP20, 100 Abbott Park Road, Abbott Park, IL 60064-6015, USA.

Automated RNA extraction and quantitation of HIV-1 by real-time PCR offer potential advantages of efficient sample processing, improved sensitivity, expanded dynamic range and reduced contamination risk. In this study, plasma was collected from 100 HIV-1 infected patients visiting The Courtyard Clinic of St. George's Hospital in London, United Kingdom (UK). Viral loads measured using the automated Abbott RealTime HIV-1 assay (m2000sp sample preparation and m2000rt amplification and detection instruments) were compared to results obtained with Versant HIV-1 RNA 3.0 (bDNA), AMPLICOR HIV-1 MONITOR v1.5 (Monitor v1.5) and LCx HIV RNA Quantitative (LCx HIV) assays. Based on gag p24, pol integrase, and env gp41 sequences, the panel included 26 subtype A, 20 B, 27 C, 10 D, 1 CRF01_AE, 3 CRF02_AG and 13 recombinant viruses. RealTime HIV-1, bDNA, Monitor v1.5 and LCx HIV quantitated 82, 74, 82, and 83% of samples, respectively, with 82, 71, 69 and 80 of the 100 samples measured within the dynamic ranges. Viral loads were highly correlated with 99% of values within 1 log(10) copies/ml between tests. The automated m2000 system and RealTime HIV-1 assay can increase laboratory throughput, enhance overall efficiency and reduce operator-associated errors while providing reliable quantitation of genetically diverse strains of HIV-1.
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http://dx.doi.org/10.1016/j.jviromet.2006.06.010DOI Listing
November 2006

Performance of the automated Abbott RealTime HIV-1 assay on a genetically diverse panel of specimens from Brazil.

J Virol Methods 2006 Jun 28;134(1-2):237-43. Epub 2006 Feb 28.

Abbott Laboratories, AIDS Research and Retrovirus Discovery, Abbott Park, IL 60064, USA.

The combination of automated sample preparation and real-time RT-PCR for measurement of HIV-1 viral load has the potential to significantly enhance throughput, reduce operator-associated error, and increase assay sensitivity and dynamic range. In this study, RNA was extracted from the plasma of 91 HIV-1 seropositive Brazilian blood donors using the Abbott m2000sp automated sample preparation system. Viral loads measured using the RealTime HIV-1 (RealTime HIV-1) assay and the Abbott m2000rt instrument were compared to values obtained in the LCx HIV RNA quantitative assay. Subtype was determined for 89 of 91 specimens by sequence/phylogenetic analysis of three genomic regions: gag p24, pol integrase, and env gp41. The panel included 69 subtype B, 1 C, 2 F, and 17 recombinant strains. Eighty-seven specimens were quantified by both assays. Two specimens were quantified only in RealTime HIV-1. Two additional specimens below the detection limit of both assays were also negative on PCR amplification. Viral load results were highly correlated, and good agreement was observed between assays with 90% of values within 0.5 log(10)copies/ml. The RealTime HIV-1 assay and m2000 system offer the advantages of automation while providing reliable quantification of diverse HIV strains.
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http://dx.doi.org/10.1016/j.jviromet.2006.01.012DOI Listing
June 2006

Protein unfolding by the mitochondrial membrane potential.

Nat Struct Biol 2002 Apr;9(4):301-7

Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500, USA.

Mitochondria can unfold importing precursor proteins by unraveling them from their N-termini. However, how this unraveling is induced is not known. Two candidates for the unfolding activity are the electrical potential across the inner mitochondrial membrane and mitochondrial Hsp70 in the matrix. Here, we propose that many precursors are unfolded by the electrical potential acting directly on positively charged amino acid side chains in the targeting sequences. Only precursor proteins with targeting sequences that are long enough to reach the matrix at the initial interaction with the import machinery are unfolded by mitochondrial Hsp70, and this unfolding occurs even in the absence of a membrane potential.
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http://dx.doi.org/10.1038/nsb772DOI Listing
April 2002