Clinacanthus Nutans Induces Antiproliferative and Apoptosis in Human Breast Cancer Cells Through Targeted Apoptosis Pathway

Clinacanthus nutans dichloromethane fraction (CN-Dcm) extract has previously been proven to suppress breast cancer (MCF7) cell proliferation. Despite this, the molecular mechanisms involved in C. nutans extract-treated MCF7 cells are unknown. Hence, the molecular mechanism of apoptosis in treated MCF7 was investigated in this current study. This study was intended to subfractionate CN-Dcm extract using column chromatography and analysed the treated MCF7 cells using the CellTiter 96® AQueous One Solution Cell Proliferation (MTS) assay, Annexin V/propidium iodide (PI) assay, western blot and reverse transcription-qualitative polymerase chain reaction (RT-qPCR). Out of nine subfraction extracts (SF1 to SF9), SF2 extract strongly inhibited MCF7 cells with the lowest IC50 value (23.51 ± 0.99 µg/mL) and substantially induced apoptosis in the MCF7 cells. SF2 extract significantly downregulated BCL-2 expression and upregulated P53, BAX, BID, BCL-2, caspase-8, caspase-9 and caspase-3 expressions in treated MCF7 cells. Therefore, SF2 extract was analysed using liquid chromatography coupled to quadrupole time–of–flight mass spectrometry (LC-QTOF-MS), which confirmed the presence of bioactive chemical compounds. Thus, it can be concluded that the compounds found in SF2 extract may potentially cause apoptosis in MCF7 cells through intrinsic and extrinsic pathways.

Phenotypic manifestations of arterial hypertension according to polymorphism of vitamin D receptor gene

Objective. To determine the phenotypic manifestations of essential arterial hypertension (EAH) according to the vitamin D receptor gene polymorphism (VDR rs10735810, rs2228570).Material and methods. The case-control study involved 100 patients with EAH stage II, 1-3 degrees of blood pressure (BP), high and very high cardiovascular risk, 21% (21) men, 79% (79) women. The mean age of patients was 59.86 ± 6.22 y.o. The control group consisted of 60 healthy individuals, comparable in age and gender. To study the VDR gene polymorphism (rs10735810, rs2228570) performed a qualitative polymerase chain reaction in real-time. Results. Almost half of the patients with elevated normal BP (44.4%) and 34% of patients with EAH 2-3 d. there is concomitant diabetes mellitus (DM) type 2, while for EAH 1 d. it is only 19%. Obesity of 1-3 degrees was shown in 53% of patients with EAH: average EAH of 1 d. - 21%, among the EAH 2-3 d. - 25%. In the control group, 16% suffered from obesity. The distribution of VDR gene polymorphism genotypes according to the presence of DM showed that it was present in 35% of patients with AA-genotype, which is 1.6 times more often than in patients with GG-genotype (22%). Most smokers among patients with GG-genotype (26%), which is twice as common as those with AA- and AG-genotype (13% and 14%, respectively). Obesity of 1-3 degrees most often met among carriers of GG-genotype - 74%, and in the control group 14%. An elevated level of waist-hip ratio (WHR) among women with EAH was in 80% of the AA-genotype carriers, in the control group, all women had normal values. In 76% of the AG-genotype carriers and in 81% of the GG-genotype carriers, the WHR was increased by 2.3 and 2.8 times, respectively, that in the control group. Deviations of systolic and diastolic BP according to the VDR gene polymorphic variants have not been established.Conclusions. The AA-genotype is associated with DM 2 and with elevated WHR in women; GG-genotype - with elevated BMI, especially in men.

Event-specific quantitative polymerase chain reaction methods for detection of double-herbicide-resistant genetically modified corn MON 87419 based on the 3′-junction of the insertion site

ABSTRACT MON 87419 was one of the new transgenic corn events developed in US with the trait of herbicide resistance to both dicamba and glyphosate. To monitor unintended release of genetically modified organism in the future, as well as to meet GM-labeling requirements, it is requisite to develop a reliable method for the detection and quantification of MON 87419, an event-specific primer pair was designed to amplify the 3′-junction site between the endogenous genome sequence and the transferred DNA of GM event MON 87419, amplicons of desired size were produced by qualitative polymerase chain reaction (PCR) assay. For the validation of this quantitative method, the mixed samples containing 10%, 1%, and 0.1% MON 87419 ingredient were quantified. The precisions were expressed as relative standard deviations, deviated by 7.87%, 12.94%, and 19.98%, respectively. These results clearly demonstrate that the PCR methods we developed herein can be used for event-specific quantitative testing of the double-herbicide-resistant corn MON 87419.

Efficacy of Sovodak in the Management of Patients Co-infected with HIV/HCV

Purpose : Sofosbuvir (SOF) and daclatasvir (DOC) are suggested for the treatment of hepatitis C virus (HCV) in patients with concomitant HCV and human immunodeficiency virus (HIV). In 2016, Sovodak tablet a combination of SOF and DOC was introduced. In the present study we assessed the effectiveness of SOF in the treatment of HCV in patients co-infected with HIV. Methods: A total of 26 HCV patients co-infected with HIV received SOF for 3 months. One patient did not adhere to the drug protocol and was removed from the final analysis. The blood sample for qualitative polymerase chain reaction (PCR) was obtained after treatment and sustained virological response (SVR) was calculated. Results: Twenty five patients finished the study. The mean patients’ age was 44.16±6.21 years. About 72% of participants had HCV genotype 1a, 8% genotype 1b, and 20% genotype 3a. After 3 months of intervention with Sovodak, the SVR12 was about 96%. None of the patients reported any adverse events. Conclusion: For the first time, the results of the present study showed that Sovodak had high SVR12 in HCV patients co-infected with HIV. However, for a precise conclusion, there is a need for larger studies and an equal number of patients with different virus genotypes.

Event-specific qualitative polymerase chain reaction analysis for two T-DNA copies in genetically modified orange Petunia

In 2017, various orange coloured petunia on the market turned out to be genetically modified (GM) without an official authorization for commercialization. Sequence analysis suggested these undeclared plants most probably originated from a plant transformation experiment performed in the 1980s. For a deeper understanding how GM petunia entered classical breeding programmes worldwide, and whether they originated from a single source or not, we undertook a molecular genetic characterization of the T-DNA integration sites in different GM petunia cultivars and breeding lines. By means of genome walking, we isolated different T-DNA sequences, which are located at the junctions between the T-DNA(s) and the petunia DNA. Based on the results obtained we conclude that there are at least two T-DNA copies of different lengths. This is supported by Southern blot analysis. For T-DNA1, the 3′-junction sequence was isolated, whereas the 5′-junction remained unclear. In contrast, for T-DNA2, the 5′-junction sequence was isolated, whereas the sequence isolated from the 3′-region consists only of T-DNA, but did not include the junction from the T-DNA to the petunia DNA. We developed primers for event-specific PCRs and screened a set of three orange GM petunia cultivars and 126 GM offspring from a commercial breeding program. We show that both T-DNA copies are present in all our tested GM petunia samples, which underpins the assumption of a single transgenic origin of the undeclared GM petunia. Most likely, the two T-DNAs are integrated in close proximity into the petunia genome.

Exosomal Small RNA Sequencing Uncovers Dose-Specific MiRNA Markers for Ionizing Radiation Exposure

Introduction: Acute exposure to ionizing radiation (IR) is hazardous or even lethal. Accurate estimation of the doses of IR exposure is critical to wisely determining the following treatments. Exosomes are nanoscale vesicles harboring biomolecules and mediate the communications among cells and tissues to influence biological processes. Screening out the microRNAs (miRNAs) contained in exosomes as biomarkers can be useful for estimating the IR exposure doses and exploring the correlation between these miRNAs and the occurrence of disease. Methods: We treated mice with 2.0, 6.5, and 8.0 Gy doses of IR and collected the mice sera at 0, 24, 48, and 72 hours after exposure. Then, the serum exosomes were isolated by ultracentrifuge and the small RNA portion was extracted for sequencing and the following bioinformatics analysis. Qualitative polymerase chain reaction was performed to validate the potential dose-specific markers. Results: Fifty-six miRNAs (31 upregulated, 25 downregulated) were differentially expressed after exposure of the above 3 IR doses and may act as common IR exposure miRNA markers. Bioinformatic analysis also identified several dosage-specific responsive miRNAs. Importantly, IR-induced miR-151-3p and miR-128-3p were significantly and stably increased at 24 hours in different mouse strains with distinct genetic background after exposed to 8.0 Gy of IR. Conclusion: Our study shows that miR-151-3p and miR-128-3p can be used as dose-specific biomarkers of 8.0 Gy IR exposure, which can be used to determine the exposure dose by detecting the amount of the 2 miRNAs in serum exosomes.

Combination of Direct Antiviral Therapy in Hepatitis C Patients, Population of Karachi

Background: Pakistan has approximately eight million Hepatitis C Virus infected patients. Initial regimen of interferon-based along with ribavirin showed SVR (Sustained Virological Response Rate) of up to 50%. The new standard ‘DAA therapy’ with improved response rates raised SVR rates to as high as 90%. This study was conducted to determine the outcome of the novel combined DAA regimen in hepatitis C infected patients in Karachi, Pakistan. Methods: Fifty patients with infected with HCV were participants of this study. They were from the gastroenterology ward and OPD Jinnah Medical Hospital (JMCH), Karachi. Initial investigations included blood samples for complete picture (CP) and liver function test (LFT). After performing qualitative Polymerase Chain Reaction (PCR), patients diagnosed with hepatitis C (pangenotypes) were prescribed direct antiviral therapy. Results: Out of total fifty patients diagnosed with HCV infection, forty compensated patients of hepatitis C were prescribed combination of Sofosbuvir and Velpatasvir, of these thirty five patients (100%) had shown to be PCR negative after three months of therapy and negative PCR after 3 months follow-up, five patients were loss to follow. Ten patients decompensated (with ascites, cirrhosis or hepatic encephalopathy) were prescribed Sofosbuvir + Velpatasvir along with ribavirin, seven (100%) had shown to be PCR negative and three were loss to follow. Conclusion: Sofosbuvir and Velpatasvir was most effective combination of direct antiviral regimen in treatment of HCV pan-genotype patients, with least adverse-effects and much better outcome in both compensated and decompensated (with ascites and cirrhosis) hepatitis C infected patients.

Quantitative PCR in diagnosing infectious urogenital pathology

This article describes the contemporary methods of diagnosing sexually transmitted infections, their advantages and disadvantages, indications for use. The authors describe application of quantitative polymerase chain reaction in diagnosing inflammatory diseases and dysbiotic conditions in men and women. This method, which is currently the “golden standard” in urogenital pathology diagnostics, has undeniable advantages over microbiological methods and qualitative polymerase chain reaction: the preanalytical stage requirements (preservation of quantitative ratios between microorganisms or nucleic acids of microorganisms) are not as strict, the risk of contamination from outside environment and subsequent corruption of the results is significantly smaller, the conditions for all microorganisms, including those impossible and hard to cultivate, are the same sensitivity and specificity-wise, it is possible to sample materials and evaluate microbiota (ratios of microorganisms and their groups) and also possible to collect samples non-invasively, the speed of testing is high.

Evaluation of HBV-Like Circulation in Wild and Farm Animals from Brazil and Uruguay

The origin of the hepatitis B virus is a subject of wide deliberation among researchers. As a result, increasing academic interest has focused on the spread of the virus in different animal species. However, the sources of viral infection for many of these animals are unknown since transmission may occur from animal to animal, human to human, animal to human, and human to animal. The aim of this study was to evaluate hepadnavirus circulation in wild and farm animals (including animals raised under wild or free conditions) from different sites in Brazil and Uruguay using serological and molecular tools. A total of 487 domestic wild and farm animals were screened for hepatitis B virus (HBV) serological markers and tested via quantitative and qualitative polymerase chain reaction (PCR) to detect viral DNA. We report evidence of HBsAg (surface antigen of HBV) and total anti-HBc (HBV core antigen) markers as well as low-copy hepadnavirus DNA among domestic and wild animals. According to our results, which were confirmed by partial genome sequencing, as the proximity between humans and animals increases, the potential for pathogen dispersal also increases. A wider knowledge and understanding of reverse zoonoses should be sought for an effective One Health response.