scholarly journals PSXIII-18 Conceptus stimuli in peripheral blood mono and polymorphonuclear cells at the beginning of pregnancy

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 372-372
Author(s):  
Gabriela Dalmaso de Melo ◽  
Igor Garcia Motta ◽  
Cecilia Constantino Rocha ◽  
Angela Maria Gonella Diaza ◽  
Juliano Coelho da Silveira ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Fixed Effects ◽  
Target Genes ◽  
Rna Extraction ◽  
Relative Increase ◽  
Polymorphonuclear Cells ◽  
Interferon Tau ◽  
Pregnant Females ◽  
Similar Expression Profile ◽  
Zymo Research

Abstract The objective of this experiment was to compare the expression of Interferon-tau Stimulated Genes (ISGs) in peripheral blood mono and polymorphonuclear cells (PBMCs and PMNs) in heifers following insemination. Twenty-nine Nelore heifers had estrous cycle synchronized, and FTAI occurred on D0. Pregnancy diagnosis was performed by ultrasonography on D28 post FTAI. On D0, 10, 14, 16, 18 and 20, blood (25mL) from the jugular vein was collected in heparinized tubes for isolation of PMNs and PBMCs. The isolation was performed using Ficoll®Paque Plus (GE Healthcare). PMNs and PBMCs samples from 8 pregnant and 9 non-pregnant heifers were subjected to RNA extraction using the DirectZol-RNA kit (Zymo-Research) and Trizol (Invitrogen), respectively. The expression of the target genes (ISG15, OAS-1, MX1 and MX2) was normalized in relation to the two reference genes (GAPDH/ACTB for PMNs and GAPDH/PPIA for PBMCs). The abundance of transcripts was evaluated by analysis of variance considering fixed effects of group, day and group by day interaction using the PROC MIXED procedure in SAS. PMNs and PBMCs had a similar expression profile of ISG15 and OAS-1, showing a relative increase (P < 0.05) from D18, and a significant increase in (P < 0.05) expression in pregnant compared to non-pregnant females on D18 and D20. These results were also observed for MX1 in PBMCs. In PMNs, no significant effects for MX1 were found. For MX2, in both cells types, only a group effect (P < 0.05) was observed, indicating a higher expression in pregnant heifers (0.57±0.11 vs. 0.21±0.03) on the days evaluated. When comparing the relative expression of the target genes to D0, no significant (P > 0.1) differences were found between PMNs and PBMCs. In summary, ISG expression is similar in PMNs and PBMCs, specifically for ISG15 and OAS-1, which both seem to be suitable biomarkers for potential early pregnancy determination in heifers.

scholarly journals Towards the validation of high-throughput sequencing (HTS) for routine plant virus diagnostics: measurement of variation linked to HTS detection of citrus viruses and viroids

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Rachelle Bester ◽  
Glynnis Cook ◽  
Johannes H. J. Breytenbach ◽  
Chanel Steyn ◽  
Rochelle De Bruyn ◽  
...  
Keyword(s):  
High Throughput ◽  
Extraction Method ◽  
Pathogen Detection ◽  
High Throughput Sequencing ◽  
Expression Profiles ◽  
Rna Extraction ◽  
Healthy Plant ◽  
Ion Torrent ◽  
Extraction Protocol ◽  
Zymo Research

Abstract Background High-throughput sequencing (HTS) has been applied successfully for virus and viroid discovery in many agricultural crops leading to the current drive to apply this technology in routine pathogen detection. The validation of HTS-based pathogen detection is therefore paramount. Methods Plant infections were established by graft inoculating a suite of viruses and viroids from established sources for further study. Four plants (one healthy plant and three infected) were sampled in triplicate and total RNA was extracted using two different methods (CTAB extraction protocol and the Zymo Research Quick-RNA Plant Miniprep Kit) and sent for Illumina HTS. One replicate sample of each plant for each RNA extraction method was also sent for HTS on an Ion Torrent platform. The data were evaluated for biological and technical variation focussing on RNA extraction method, platform used and bioinformatic analysis. Results The study evaluated the influence of different HTS protocols on the sensitivity, specificity and repeatability of HTS as a detection tool. Both extraction methods and sequencing platforms resulted in significant differences between the data sets. Using a de novo assembly approach, complemented with read mapping, the Illumina data allowed a greater proportion of the expected pathogen scaffolds to be inferred, and an accurate virome profile was constructed. The complete virome profile was also constructed using the Ion Torrent data but analyses showed that more sequencing depth is required to be comparative to the Illumina protocol and produce consistent results. The CTAB extraction protocol lowered the proportion of viroid sequences recovered with HTS, and the Zymo Research kit resulted in more variation in the read counts obtained per pathogen sequence. The expression profiles of reference genes were also investigated to assess the suitability of these genes as internal controls to allow for the comparison between samples across different protocols. Conclusions This study highlights the need to measure the level of variation that can arise from the different variables of an HTS protocol, from sample preparation to data analysis. HTS is more comprehensive than any assay previously used, but with the necessary validations and standard operating procedures, the implementation of HTS as part of routine pathogen screening practices is possible.

scholarly journals Identification of a C3bi-specific membrane complement receptor that is expressed on lymphocytes, monocytes, neutrophils, and erythrocytes

1982 ◽  
Vol 155 (1) ◽  
pp. 96-110 ◽  
Author(s):  
GD Ross ◽  
JD Lambris
Keyword(s):  
Peripheral Blood ◽  
Myeloid Cell ◽  
Fluid Phase ◽  
Cell Types ◽  
Peripheral Blood Lymphocytes ◽  
Polymorphonuclear Cells ◽  
Coated Particles ◽  
Blood Lymphocytes ◽  
T Cell Antigens ◽  
D Region

Cells expressing a membrane C receptor (CR(3)) specific for C3b-inactivator- cleaved C3b (C3bi) were identified by rosette assay with C3bi-coated sheep erythrocytes (EC3bi) or C3bi-coated fluorescent microspheres (C3bi-ms). C3bi- ms, probably because of their smaller size, bound to a higher proportion of cells than did EC3bi. C3bi-ms bound to greater than 90 percent of mature neutrophils, 85 percent of monocytes, 92 percent of erythrocytes, and 12 percent of peripheral blood lymphocytes. Binding of C3bi-ms to neutrophils, monocytes, and erythrocytes was inhibited by fluid-phase C3bi, Fab anti-C3c, or Fab anti-C3d but was not inhibited by F(ab')(2) anti-CR(1) (C3b receptor) or F(ab')(2) anti-CR(2) (C3d receptor) nor by fluid-phase C3b, C3c, or C3d. This indicated that monocytes, neutrophils, and erythrocytes expressed C3bi receptors (CR(3)) that were separate and distinct from CR(1) and CR(2) and specific for a site in the C3 molecule that was only exposed subsequently to cleavage of C3b by C3b inactivator and that was either destroyed, covered, or liberated by cleavage of C3bi into C3c and C3d fragments. Lymphocytes differed from these other cell types in that they expressed CR2 in addition to CRa. Lymphocyte C3bi-ms rosettes were inhibited from 50 to 84 percent by F(ab')(2)-anti-CR(2) or fluid-phase C3d, whereas C3d-ms rosettes were inhibited completely by F(ab')(2) anti-CR(2), fluid-phase C3bi, or fluid- phase C3d. Thus, with lymphocytes, C3bi was bound to CR(3), and in addition was bound to CR(2) by way of the intact d region of the C3bi molecule. In studies of the acquisition of C receptors occurring during myeloid cell maturation, the ability to rosette with C3bi-coated particles was detected readily with immature low-density cells, whereas this ability was nearly undetectable with high density mature polymorphonuclear cells. This absence of C3bi binding to polymorphs was not due to a loss of the CR(3) but instead was due to the maturation-linked acquisition of the abiity to secrete elastase that cleaved reagent particle-bound C3bi into CR(3)-unreactive C3d. Neither neutrophils nor monocytes bound C3d-coated particles at any stage of maturation. Assay of CR(3) with mature neutrophils required inhibition of neutrophil elastase with either soybean trypsin inhibitor or anti-elastase antibodies, and the amounts of these elastase inhibitors required to allow EC3bi rosette formation increased with neutrophil maturation. Because lymphocytes bound C3bi to CR(2) as well as to CR(3), specific assay of lymphocyte CR(3) required saturation of membrane CR(2) with Fab' anti-CR(2) before assay for rosettes with C3bi-ms. Only 3.5 percent of anti-CR(2)- treated peripheral blood lymphocytes bound C3bi-ms. Therefore, among normal blood lymphocytes the majority of the 12 percent C3bi-ms-binding cells expressed only CR(2) (8.5 percent), and the small proportion of C3bi-ms- binding cells that expressed CR(3) (3.5 percent) represented a distinct subset from the CR2(+) cells. Double-label assay indicated that 3.0 percent out of 3.5 percent of these CR(3)-bearing lymphocytes were B cells because they expressed membrane immunoglobulins. Of the remaining CR(3)(+) cells, 0.2 percent expressed either Leu-1 or 3A1 T cell antigens, and 0.6 percent expressed the OKM-1 monocyte-null lymphocyte determinant.

scholarly journals Wide application of minimally processed saliva on multiple RT-PCR kits for SARS-CoV-2 detection in Indonesia

2021 ◽  
Author(s):  
Caroline Mahendra ◽  
Maria M. M. Kaisar ◽  
Suraj R. Vasandani ◽  
Sem Samuel Surja ◽  
Enty Tjoa ◽  
...  
Keyword(s):  
Target Genes ◽  
Local Population ◽  
Rna Extraction ◽  
Human Saliva ◽  
Attractive Alternative ◽  
Rt Pcr ◽  
Sampling Device ◽  
Agreement Rate ◽  
Extraction Step ◽  
Oropharyngeal Swab

Saliva as a sample matrix has been an attractive alternative for the detection of SARS-CoV-2. However, due to potential variability in collection and processing steps, it is recommended to evaluate a proposed workflow amongst the local population. Here, we aim to validate collection and treatment of human saliva as a direct specimen for RT-qPCR based detection of SARS-CoV-2 in Indonesia. We demonstrated that SARS-CoV-2 target genes were detected in saliva specimen and remained stable for five days refrigerated or room temperature storage. The method of processing saliva specimen described in this report is free from RNA-extraction step, thereby reduces cost, time, and manpower required for processing samples. The developed method was validated for use on three COVID-19 RT-PCR kits commercially available. Our developed method achieved 85% agreement rate when compared to paired nasopharyngeal and oropharyngeal swab specimens (NPOP). With the assistance of a specimen sampling device, QuickSpit(TM), collection was found to be more convenient for individuals and improved agreement rate to 90%.

Elevation of MicroRNA-126 Levels in Intracranial Aneurysm and Bioinformatic Analysis of Potential Molecular Mechanisms

2021 ◽  
Vol 11 (4) ◽  
pp. 573-579
Author(s):  
Pan Huang ◽  
Min Xu ◽  
Xiao-Ying He
Keyword(s):  
Intracranial Aneurysm ◽  
Peripheral Blood ◽  
Target Gene ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Kegg Pathway ◽  
Bioinformatic Analysis ◽  
Control Group ◽  
Potential Target ◽  
Fas Signaling

The study is to investigation of microRNA-126 levels in patients with intracranial aneurysm and bioinformatic analysis of the molecular mechanisms involved. A total of 166 patients with ICA who were hospitalized or examined in our hospital from September 2015 to December 2017 were used as the experimental group (ICA group). This group included 120 patients with unruptured intracranial aneurysm (UICA; UICA group) and 46 patients with ruptured intracranial aneurysm (RICA); RICA group). The UICA group was further subdivided into 42 surgical groups (S group) and 78 nonsurgical groups (NS group). Sixty-three normal people without intracranial aneurysms were selected as the control group. RT-PCR was used to quantitatively detect the relative expression of microRNA- 126 in peripheral blood mononuclear cells at the time of admission and immediately after surgery. The UCSC database was used to analyze the gene locus and homology of microRNA-126. The TargetScan database and CoMeTa database were used to predict the potential target genes of microRNA-126. The DAVID database was used to enrich the function of potential target genes of microRNA-126 (GO enrichment) and KEGG pathway enrichment for analysis. The expression level of microRNA-126 in peripheral blood was significantly higher in the ICA group than in the control group (P <0.01), significantly higher in the RICA group than in the UICA group (P <0.05). Expression was also higher in the NS group than in the S group but the difference was nonsignificant (P >0.05). A total of 15 potential target genes including ITGA6, CRK, PCDH7, and ADAM9 were identified through the target gene prediction software and GO analysis and KEGG pathway analysis showed that the function of the microRNA-126 target gene was mainly focused on protein binding and the FAS signaling pathway. In Conclusion the microRNA-126 is up-regulated in ICA patients and affects ICA by regulating multiple target genes in the FAS signaling pathway.

198 PROGESTERONE, ESTROGEN (ER-α AND ER-β), AND OXYTOCIN RECEPTOR GENE EXPRESSION IN CANINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 248
Author(s):  
A. A. P. Derussi ◽  
A. C. S. Castilho ◽  
R. W. A. Souza ◽  
R. Volpato ◽  
C. R. F. Guaitolini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Relative Abundance ◽  
Hormone Receptors ◽  
Target Genes ◽  
Receptor Gene ◽  
Rna Extraction ◽  
Amplification Efficiency ◽  
Mrna Levels ◽  
Lh Surge ◽  
Total Rna

The aim of this study was to compare the mRNA levels of hormone receptor for progesterone (PR), oestrogen α (ER-α), oestrogen β (ER-β), and oxytocin (OTR) in canine morulae and blastocysts. Ten healthy mature bitches were inseminated based on monitoring vaginal cytology and progesterone concentration. The first insemination was performed on Day 2 after the preovulatory LH surge (progesterone 4 ng mL–1), and the second was performed 48 h later. All females were submitted to ovariohysterectomy (OVH), and the oviduct as well the uterurs were flushed with PBS solution to obtain the embryos. The females were divided into two groups: Group A (n = 5), morulae were collected 8 days after the LH surge and Group B (n = 5), blastocysts were collected 12 days after the LH surge. The pools (n = 10) of embryos (5 embryos/pool) were stored in RNAlater® (Ambion, Life Technologies, USA) at –80°C. The samples were analysed together. The RNA later was removed used PBS calcium free and the total RNA extraction was performed using the Qiagen RNeasy micro-kit (Hildesheim, Germany). Before reverse-transcription (RT) reaction, the total RNA was treated with DNase I Amplification Grade (Invitrogen Life Technologies, Carlsbad, CA, USA). The gene expression of target genes was assessed by real-time RT-qPCR, using SuperScript III for RT and power SYBR Green PCR Master Mix (Applied Biosystems, USA) for cDNA for PCR. The primers for target genes were designed using the software Primer Express® (Applied Biosystems, USA). The gene expression of target genes was normalized by HPRT gene and the relative abundance of mRNA was determined by the ΔΔct method corrected by amplification efficiency using Pffafl’s equation. The means of mRNA relative abundance were compared by t-test. The PR mRNA expression only in blastocysts is similar to the results obtained by Hou et al. (1997) in rat embryos. It is believed that the absence of PR in the early stages of cleavage is due to the indirect action of progesterone by growth factors produced by the maternal reproductive tract (2). Apparently, ER-β action does not occur in the embryo canine phases analysed; however, the action of ER-α seems related to the deployment signal as seen by Hou et al. (1996) in rats. Similarly to findings in the literature, OTR expression decreased in canine embryonic development. This receptor was produced by blastocysts while present in the uterus, which may represent an incidental mechanism to the embryo control of endometrial receptivity, such as also to prevent the development of endometrial luteolytic mechanism. The variation in hormone receptors gene expression in canine embryos can be influencing the transition from morula to blastocyst. In addition, a hormonal influence on these structures can occur in different ways.

Cyclical changes in the uterine endometrium and peripheral plasma concentrations of progesterone in the marsupial Trichosurus vulepcula

1973 ◽  
Vol 21 (1) ◽  
pp. 1 ◽  
Author(s):  
CD Shorey ◽  
RL Hughes
Keyword(s):  
Blood Plasma ◽  
Peripheral Blood ◽  
Plasma Concentrations ◽  
Secretory Activity ◽  
Oestrous Cycle ◽  
Uterine Endometrium ◽  
Pregnant Females ◽  
Peripheral Plasma ◽  
Functional Differences ◽  
Peripheral Blood Plasma

The proliferation and secretory activity of the uterine endometrium in the marsupial T. vulpecula is examined at the cellular and subcellular levels throughout the 26-day oestrous cycle. The observations described are correlated with measured concentrations of progesterone in the peripheral blood plasma. Evidence cited indicates that there are no significant functional differences in the uterine endometrial secretory activity during the 17.5-day gestation period in pregnant females, compared with those in a normal oestrous cycle. Progesterone assays carried out on blood plasma taken from 20 staged animals throughout the oestrous cycle, five of which were at known stages of gestation, also supports the view that pregnancy does not significantly alter the physiological pattern of the reproductive cycle in this marsupial.

Determinants of Gender Differences in Change in Pay among Job-Switching Executives

ILR Review ◽  
2020 ◽  
pp. 001979392093071
Author(s):  
Boris Groysberg ◽  
Paul Healy ◽  
Eric Lin
Keyword(s):  
Gender Differences ◽  
Women Executives ◽  
Fixed Effects ◽  
Supply And Demand ◽  
Relative Increase ◽  
Global Search ◽  
Men And Women ◽  
Explanatory Factors ◽  
Pay Differences ◽  
Performance Based Compensation

The authors investigate what determines differences in change in pay between men and women executives who move to new employers. Using proprietary data of 2,034 executive placements from a global search firm, the authors observe narrower pay differences between men and women after job moves. The unconditional gap shrinks from 21.5% in the prior employer to 15% in the new employer. After controlling for typical explanatory factors, the residual gap falls by almost 30%, from 8.5% at the prior employer to 6.1% in the new placement. This change reflects a relative increase in performance-based compensation for women and a lower level of unexplained pay inequality generally in external placements. Controlling for individual fixed effects, observed women have higher pay raises than do men. Finally, the authors find suggestive evidence that pay differences may also be moderated by differences in the supply and demand for women executives.

Applied use of interferon-tau stimulated genes expression in polymorphonuclear cells to detect pregnancy compared to other early predictors in beef cattle

2020 ◽  
Vol 152 ◽  
pp. 94-105
Author(s):  
Gabriela Dalmaso de Melo ◽  
Barbara Piffero Mello ◽  
Catia Aparecida Ferreira ◽  
Carlos Alberto Souto Godoy Filho ◽  
Cecilia Constantino Rocha ◽  
...  
Keyword(s):  
Beef Cattle ◽  
Polymorphonuclear Cells ◽  
Interferon Tau ◽  
Early Predictors ◽  
Genes Expression
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