scholarly journals Molecular detection of Mycobacterium tuberculosis in pulmonary and extrapulmonary samples in a hospital-based study

2020 ◽  
Vol 20 (4) ◽  
pp. 1617-23
Author(s):  
Kalal Iravathy Goud ◽  
Matam Kavitha ◽  
Adi Mahalakshmi ◽  
Ravi Vempati ◽  
Abdulaziz A Alodhayani ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Pcr Analysis ◽  
Sequencing Analysis ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Non Invasive ◽  
Invasive Test ◽  
Keywords Tuberculosis ◽  
Bronchoalveolar Fluid

Objective: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a deadly infectious disease. India contributes to one-third of the global TB burden. However, no studies have been carried out in the Telangana (Hyderabad) population using real-time polymerase chain reaction (RT-PCR). Therefore, the current study evaluated the role of RT-PCR as a rapid and non-invasive test to diagnose TB by testing for pulmonary tuberculosis (PTB) and extrapulmonary tubercu- losis (EPTB). Materials and methods: This hospital-based study examined 1670 samples (900 EPTB; 770 PTB) comprising tissue (n = 537), peritoneal fluid (n = 420), sputum (n = 166), bronchial fluid (n = 126), cerebrospinal fluid (n = 145), ascetic fluid (n = 76), sputum pus (n = 78), urine (n = 79), and bronchoalveolar fluid (n = 43) samples. DNA from samples was separated using specific isolation kits and subjected to RT-PCR. Results: In this study, we enrolled 1670 subjects and categorized 54.4% as females and 45.6% as males. The collected sam- ples were categorized as 48.5% of fluid samples, followed by tissue (32.2%), sputum (9.9%), urine (4.7%), and pus-swab (4.6%). RT-PCR analysis revealed that 4.7% patients were positive for Mtb. Our results revealed that 61% of the affected patients were male and 39% were female. Among the specimen types, tissue samples gave the highest proportion of positive results (36.3%). Conclusion: The results showed that RT-PCR should be implemented and given top priority in TB diagnosis to save time and facilitate a definitive diagnosis. Tissue samples are highly recommended to screen the Mtb through the technique RT- PCR. Future studies should extend the technique to the global population and exome sequencing analysis should be per- formed to identify TB risk markers. Keywords: Tuberculosis (TB); EPTB; PTB; Mycobacterium tuberculosis (Mtb).

Islet Co-Expression of CD133 and ABCB5 in Human Retinoblastoma Specimens

2021 ◽  
Author(s):  
Marco Zschoche ◽  
Sergej Skosyrski ◽  
Neele Babst ◽  
Mahdy Ranjbar ◽  
Felix Rommel ◽  
...  
Keyword(s):  
Treatment Resistance ◽  
Sphingosine Kinase ◽  
Immunohistochemical Analysis ◽  
Pcr Analysis ◽  
Sphingosine Kinase 1 ◽  
Rt Pcr ◽  
Beam Radiation ◽  
Sphingosine Kinase 2 ◽  
Human Retinoblastoma

Abstract Background The role of CD133 und ABCB5 is discussed in treatment resistance in several types of cancer. The objective of this study was to evaluate whether CD133+/ABCB5+ colocalization differs in untreated, in beam radiation treated, and in chemotherapy treated retinoblastoma specimens. Additionally, CD133, ABCB5, sphingosine kinase 1, and sphingosine kinase 2 gene expression was analyzed in WERI-RB1 (WERI RB1) and etoposide-resistant WERI RB1 subclones (WERI ETOR). Methods Active human untreated retinoblastoma specimens (n = 12), active human retinoblastoma specimens pretreated with beam radiation before enucleation (n = 8), and active human retinoblastoma specimens pretreated with chemotherapy before enucleation (n = 7) were investigated for localization and expression of CD133 and ABCB5 by immunohistochemistry. Only specimens with IIRC D, but not E, were included in this study. Furthermore, WERI RB1 and WERI ETOR cell lines were analyzed for CD133, ABCB5, sphingosine kinase 1, and sphingosine kinase 2 by the real-time polymerase chain reaction (RT-PCR). Results Immunohistochemical analysis revealed the same amount of CD133+/ABCB5+ colocalization islets in untreated and treated human retinoblastoma specimens. Quantitative RT-PCR analysis showed a statistically significant upregulation of CD133 in WERI ETOR (p = 0.002). No ABCB5 expression was detected in WERI RB1 and WERI ETOR. On the other hand, SPHK1 (p = 0.0027) and SPHK2 (p = 0.017) showed significant downregulation in WERI ETOR compared to WERI RB1. Conclusions CD133+/ABCB5+ co-localization islets were noted in untreated and treated human retinoblastoma specimens. Therefore, we assume that CD133+/ABCB5+ islets might play a role in retinoblastoma genesis, but not in retinoblastoma treatment resistance.

scholarly journals Environmental reservoirs of Mycobacterium bovis and Mycobacterium tuberculosis in the Ruaha region, Tanzania

2019 ◽  
Author(s):  
Emma R. Travis ◽  
Yujiun Hung ◽  
David Porter ◽  
Goodluck Paul ◽  
Robert James ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Bacterial Species ◽  
Sample Type ◽  
Household Dust ◽  
Non Invasive ◽  
Temporal Correlations ◽  
Invasive Test ◽  
Cattle Faeces ◽  
Ruaha National Park

ABSTRACTThis study was designed to investigate the prevalence of members of the Mycobacterium tuberculosis complex (MTBC) in the environment of pastoralists and villagers in the Iringa district, adjacent to the Ruaha National Park in Tanzania. Utilising specific qPCR assays, both Mycobacterium bovis and Mycobacterium tuberculosis were detected in cattle faeces, boma soil, water and household dust. M. bovis was also found in goat faeces and goat boma soil. This is the first report of faecal shedding of M. bovis in goats and the first molecular survey of faecal shedding in cattle. The prevalence of both bacterial species varied by village, area, season and sample type. Geographical and temporal correlations across sample types were suggestive of cross species transmission. This non-invasive test has previously been rigorously validated for screening other mammals; in this study it has successfully been applied to detect M. bovis and M. tuberculosis in livestock faeces and the environment.

A Second Promoter for the Human KCl Cotransporter 1 (KCC1) Gene Drives Differential Expression of a Variant Isoform in Sickle Versus Normal Reticulocytes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3592-3592
Author(s):  
Kathleen Anderson ◽  
Scott C. Crable ◽  
Suzan M. Hammond ◽  
Clinton H. Joiner ◽  
Patrick G. Gallagher
Keyword(s):  
Promoter Activity ◽  
Regulatory Elements ◽  
Dominant Negative ◽  
Jurkat Cells ◽  
Pcr Analysis ◽  
Luciferase Expression ◽  
Alternative Transcript ◽  
Red Cell ◽  
Rt Pcr ◽  
Tissue Samples

Abstract The K+Cl- cotransporter plays a significant role in the maintenance of red cell volume. During cellular maturation, this cotransporter actively moves K+ and Cl- out of the cell. The accompanying movement of water results in dehydration and shrinking of the red cell. Because KCl cotransporter activity is higher in sickle compared to normal reticulocytes, it has been considered a potential modifier gene for sickle cell disease. We have evidence for expression of three KCC genes in human reticulocytes and have investigated the promoter for KCC1. While the expression of the principal KCC1 transcript did not differ in SS compared to normal reticulocytes, we now describe an alternative transcript of the KCC1 gene emanating from a second promoter and exhibiting a restricted tissue distribution. Investigation of the EST databases revealed spliced ESTs corresponding to the use of four distinct N-terminal exons in the KCC1 gene, each reported multiple times in the dataset. Primers were developed for these 5′ regions (exon1, 1a, 1b, and 1c) and used in an RT-PCR reaction with human reticulocyte RNA. The exon1 form and the exon1b variant were expressed. When the relative levels of these forms were compared, expression of the exon1 transcript was unchanged, while significantly higher levels of the exon1b variant was evident in the AA reticulocyte RNA compared to numerous SS samples. In an analysis of seven other human tissue samples, the exon1b isoform was highly expressed in kidney, lung, and heart, while the KCC1ex1 transcript was expressed at a constant level in all tissues. Although the transcript for this variant could arise from the KCC1 promoter we have previously characterized, the pattern of expression suggested control from a second promoter. A 915bp region corresponding to −787 to +128 was isolated and cloned into a reporter construct to test for promoter activity. This clone was compared with KCC1 promoter constructs in transient transfection assays. The exon1b construct not only exhibited promoter activity by directing high levels of luciferase expression in K562 cells, it also demonstrated tissue-specificity with a low level of activity in Jurkat cells. This recapitulates the endogenous levels detected by RT-PCR analysis of these cell lines. The −787/+128 exon1b construct is also 3-fold more active than the ubiquitously expressed exon1 promoter. To identify the control elements for this promoter, we produced a series of deletion constructs; the smallest construct contained 181bp. No reduction in reporter gene activity was evident, indicating the major regulatory elements lie very close to this promoter. Since exon1 encodes 39aa and exon1b only 7aa, the use of this smaller first exon effectively produces an N-terminal truncation in the protein. Studies with the mouse KCC1 cDNA have demonstrated that proteins produced by an N-terminal truncation are not only inactive for K+Cl- cotransport, but also function as dominant negative regulators of a full-length KCC1 protein. High level expression of this variant in AA cells compared to SS cells would therefore be consistent with the low reported activity in the AA reticulocytes. Induction or modulation of the expression of the KCC1ex1b variant may be an key factor in the control of red cell hydration.

The Critical Role of Peptidyl-Prolyl cis/trans Isomerase, Pin1 in Acute Myeloid Leukemia with C/EBPalpha Mutations.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2213-2213
Author(s):  
J. Pulikkan ◽  
A. Peer Zada ◽  
M. Geletu ◽  
V. Dengler ◽  
Daniel G. Tenen ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Dominant Negative ◽  
Pcr Analysis ◽  
Wild Type ◽  
Rt Pcr ◽  
Promoter Assay ◽  
Type C ◽  
Acute Myeloid

Abstract CCAAT enhancer binding protein alpha (C/EBPα) is a myeloid specific transcription factor that coordinates cellular differentiation and cell cycle arrest. Loss of C/EBPα expression or function in leukemic blasts contributes to a block in myeloid cell differentiation. C/EBPα is mutated in around 9% of acute myeloid leukemia (AML). The mutations reported in C/EBPα are frame shift mutations and point mutations at basic region Leucine zipper. The mutant form of C/EBPα ie C/EBPα-p30 exhibits dominant negative function over the wild type protein. The role of peptidyl-prolyl cis/trans isomerase, Pin1 in tumorogenesis and its overexpression in many cancers led us to investigate its role in acute myeloid leukemia with C/EBPα mutation. Here we show that Pin1 is upregulated in patients with acute myeloid leukemia by affymetrix analysis. By quantitative Real-Time RT-PCR analysis, we show C/EBPα-p30 could induce Pin1 transcription, while the wild type C/EBPα downregulates Pin1 expression. Luciferase promoter assay for the Pin1 promoter shows that wild type C/EBPα is able to block Pin1 promoter activity. Mean while, C/EBPα-p30 couldn’t block Pin1 promotor activity. By silencing Pin1 by RNA Interference as well as with inhibitor against Pin1 (PiB) we could show myeloid differentiation in human CD34+ cord blood cells as well as in Kasumi-6 cells as assessed by FACS analysis with granulocytic markers. We investigated the mechanism underlying the dominant negative action of C/EBPα-p30 over the wild type protein. We report that Pin1 increases the transcriptional activity of the oncogene c-jun. We also show that c-jun blocks the DNA binding and transactivation of C/EBPα protein as assessed by gel shift assay and promoter assay respectively. We have previously shown that c-jun expression is high in AML patients with C/EBPα mutation and c-jun could block C/EBPα function by protein-protein interaction. Quantitative Real-Time RT-PCR analysis shows that inhibition of Pin1 by the inhibitor PiB downregulates c-jun mRNA expression. In conclusion, inhibition of Pin1 leads to granulocytic differentiation. Our results show Pin1 as a novel target in treating AML patients with C/EBPα mutation.

scholarly journals Complex N-Glycans Are Important for Normal Fruit Ripening and Seed Development in Tomato

2021 ◽  
Vol 12 ◽  
Author(s):  
Heidi Kaulfürst-Soboll ◽  
Melanie Mertens-Beer ◽  
Randolf Brehler ◽  
Markus Albert ◽  
Antje von Schaewen
Keyword(s):  
Rna Interference ◽  
Fruit Ripening ◽  
Reproductive Stage ◽  
Pcr Analysis ◽  
Plant Responses ◽  
Rt Pcr ◽  
Signaling Crosstalk ◽  
Long Time ◽  
Glycan Modification

Complex N-glycan modification of secretory glycoproteins in plants is still not well understood. Essential in animals, where a lack of complex N-glycans is embryo-lethal, their presence in plants seemed less relevant for a long time mostly because Arabidopsis thaliana cgl1 mutants lacking N-acetyl-glucosaminyltransferase I (GNTI, the enzyme initiating complex N-glycan maturation in the Golgi apparatus) are viable and showed only minor impairments regarding stress tolerance or development. A different picture emerged when a rice (Oryza sativa) gntI T-DNA mutant was found to be unable to reach the reproductive stage. Here, we report on tomato (Solanum lycopersicum) lines that showed severe impairments upon two RNA interference (RNAi) approaches. Originally created to shed light on the role of core α1,3-fucose and β1,2-xylose residues in food allergy, plants with strongly reduced GNTI activity developed necrotic fruit-attached stalks and early fruit drop combined with patchy incomplete ripening. Correspondingly, semiquantitative RT-PCR of the abscission zone (az) revealed an increase of abscission markers. Also, GNTI-RNA interference (RNAi) plants were more susceptible to sporadic infection. To obtain vital tomatoes with comparable low allergenic potential, Golgi α-mannosidase II (MANII) was chosen as the second target. The resulting phenotypes were oppositional: MANII-reduced plants carried normal-looking fruits that remained attached for extended time without signs of necrosis. Fruits contained no or only few, but enlarged, seeds. Furthermore, leaves developed rolled-up rims simultaneously during the reproductive stage. Trials to cross MANII-reduced plants failed, while GNTI-reduced plants could be (back-)crossed, retaining their characteristic phenotype. This phenotype could not be overcome by ethephon or indole-3-acetic acid (IAA) application, but the latter was able to mimic patchy fruit ripening in wild-type. Phytohormones measured in leaves and 1-aminocyclopropane-1-carboxylic acid (ACC) contents in fruits showed no significant differences. Together, the findings hint at altered liberation/perception of protein-bound N-glycans, known to trigger auxin-like effects. Concomitantly, semiquantitative RT-PCR analysis revealed differences in auxin-responsive genes, indicating the importance of complex N-glycan modification for hormone signaling/crosstalk. Another possible role of altered glycoprotein life span seems subordinate, as concluded from transient expression of Arabidopsis KORRIGAN KOR1-GFP fusion proteins in RNAi plants of Nicotiana benthamiana. In summary, our analyses stress the importance of complex N-glycan maturation for normal plant responses, especially in fruit-bearing crops like tomato.

scholarly journals Rapid detection of SARS-CoV-2 infection by multicapillary column coupled ion mobility spectrometry (MCC-IMS) of breath. A proof of concept study

2020 ◽  
Author(s):  
Claus Steppert ◽  
Isabel Steppert ◽  
Gunther Becher ◽  
William Sterlacci ◽  
Thomas Bollinger
Keyword(s):  
Discriminant Analysis ◽  
Ion Mobility ◽  
Influenza A ◽  
Cross Validation ◽  
Viral Disease ◽  
Pcr Analysis ◽  
Proof Of Concept ◽  
Influenza Epidemic ◽  
Rt Pcr ◽  
Non Invasive

AbstractThere is an urgent need for screening patients of having a communicable viral disease to cut infection chains.We could recently demonstrate that MCC-IMS of breath is able to identify Influenza-A infected patients. With decreasing Influenza epidemic and upcoming SARS-CoV-2 infections we extended our study to the analysis of patients with suspected SARS-CoV-2 infections.51 patients, 23m, 28f, aged 64 ± 16 years, were included in this study.Besides RT-PCR analysis of nasopharyngeal swabs all patients underwent MCC-IMS analysis of breath. 16 patients, 7m, 9f, were positive for SARS-CoV-2 by RT-PCR. There was no difference in gender or age according to the groups.Stepwise canonical discriminant analysis could correctly classify the infected and non-infected subjects in 98% by cross-validation. Afterwards we combined the Influenza-A sub study and the SARS-CoV-2-sub study for a total of 75 patients, 34m, 41f, aged 64.8 ± 1.8 years, 14 positive for Influenza-A, 16 positive for SARS-CoV-2, the remaining 44 patients were used as controls. In one patient RT-PCR was highly suspicious of SARS-CoV-2 but inconclusive.There was no imbalance between the groups for age or gender.97.3% of the patients could be correctly classified to the respective group by discriminant analysis. Even the inconclusive patient could be mapped to the SARS-CoV-2 group applying the discrimination function.ConclusionMCC-IMS is able to detect SARS-CoV-2 infection and Influenza-A infection in breath. As this method provides exact, fast non-invasive diagnosis it should be further developed for screening of communicable viral diseases.Study registration: NCT04282135

scholarly journals Biomarker Potential of Plasma MicroRNA-150-5p in Prostate Cancer

Medicina ◽  
2019 ◽  
Vol 55 (9) ◽  
pp. 564 ◽  
Author(s):  
Ionut Andrei Paunescu ◽  
Razvan Bardan ◽  
Anca Marcu ◽  
Diana Nitusca ◽  
Alis Dema ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Area Under The Curve ◽  
Plasma Samples ◽  
Tissue Samples ◽  
New Class ◽  
Non Invasive ◽  
Plasma Microrna ◽  
Cancer Tissues ◽  
Tissue Cancer

Background and Objectives: Over decades, prostate cancer (PCa) has become one of the leading causes of cancer mortality in men. Extensive evidence exists that microRNAs (miRNAs or miRs) are key players in PCa and a new class of non-invasive cancer biomarkers. Materials and Methods: We performed miRNA profiling in plasma and tissues of PCa patients and attempted the validation of candidate individual miRs as biomarkers. Results: The comparison of tissue and plasma profiling results revealed five commonly dysregulated miRs, namely, miR-130a-3p, miR-145-5p, miR-148a-3p, miR-150-5p, and miR-365a-3p, of which only three show concordant changes—miR-130a-3p and miR-150-5p were downregulated and miR-148a-3p was upregulated in both tissue and plasma samples, respectively. MiR-150-5p was validated as significantly downregulated in both plasma and tissue cancer samples, with a fold change of −2.697 (p < 0.001), and −1.693 (p = 0.035), respectively. ROC analysis showed an area under the curve (AUC) of 0.817 (95% CI: 0.680–0.995) for plasma samples and 0.809 (95% CI: 0.616–1.001) for tissue samples. Conclusions: We provide data indicating that miR-150-5p plasma variations in PCa patients are associated with concordant changes in prostate cancer tissues; however, given the heterogeneous nature of previous findings of miR-150-5p expression in PCa cells, additional future studies of a larger sample size are warranted in order to confirm the biomarker potential and role of miRNA-150-5p in PCa biology.

scholarly journals Rapid competitive PCR determination of relative gene expression in limiting tissue samples

1996 ◽  
Vol 42 (2) ◽  
pp. 227-231 ◽  
Author(s):  
Y H Jiang ◽  
L A Davidson ◽  
J R Lupton ◽  
R S Chapkin
Keyword(s):  
Gene Expression ◽  
Mrna Level ◽  
Internal Standard ◽  
Pcr Amplification ◽  
Pcr Analysis ◽  
Relative Gene Expression ◽  
Competitive Pcr ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Relative Gene

Abstract Reverse transcriptase (RT)-PCR is widely used to study gene transcription in many biological systems. Despite the development of a variety of procedures, quantification of RT-PCR products remains difficult, particularly when processing a large number of samples. Therefore, we developed a novel alternative PCR technique that we term "rapid competitive PCR" (RC-PCR), designed to study the relative expression of specific genes in a large number of small tissue biopsies. RC-PCR is characterized by measuring relative gene expression at the mRNA level of two or more samples with a nonradioactive assay based on competitive PCR amplification between identical sequences of internal standard and target cDNA. Only a single reaction tube per sample is used in this technique, and it was validated by comparing RC-PCR of protein kinase C zeta and alpha expression in rat colonic mucosa samples with competitive RT-PCR analysis (requiring 6-8 reaction tubes per sample). We conclude that RC-PCR is a simple, rapid, highly sensitive technique that is capable of detecting less than twofold differences in mRNA expression.

scholarly journals Histochemical Expression of Mast Cell Chymase in Chronic Periodontitis and Cyclosporine-Induced Gingival Overgrowth

2013 ◽  
Vol 2013 ◽  
pp. 1-5
Author(s):  
Tamilselvan Subramani ◽  
Kamatchiammal Senthilkumar ◽  
Soundararajan Periasamy
Keyword(s):  
Mast Cell ◽  
Chronic Periodontitis ◽  
Vital Role ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Drug Induced ◽  
Tissue Samples ◽  
Gingival Overgrowth ◽  
Significant Expression ◽  
Mast Cell Chymase

Mast cell (MC) mediators play a vital role in fibrosis. The purpose of this study was to investigate the MCs and their enzyme chymase in gingival tissues showing drug-induced gingival overgrowth (DIGO) and also to evaluate the correlation of MC counting and expression with the chronic periodontitis. In this study, 30 samples, including cyclosporine-induced gingival overgrowth, chronic periodontitis (10 for each), and ten normal gingival tissues, were collected. We analyzed the histochemical expression of MC chymase in all the collected tissues. In addition, the number of MCs was counted for each deparaffinized section stained with toluidine blue. Furthermore, total RNA was extracted from tissue samples, and RT-PCR was performed for MC chymase. The numbers of MCs were found to be increased in relative lesions compared to normal gingival tissues (). Moreover, chymase-containing MCs in DIGO tissues showed striking differences from those of control subjects and chronic periodontitis (). The RT-PCR analysis further revealed that MC chymase mRNA increased significantly in DIGO tissues. In conclusion, although the MCs were less numerous in numbers, the cells exhibited significant expression of chymase enzyme suggesting the involvement of MCs in DIGO.

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