Ribonucleic Acid Extraction Research Articles

Detection of Human Papillomavirus among Cervical Cancer Patients by Qualitative Polymerase Chain Reaction on Formalin- fixed, Paraffin-embedded Tissue: A Retrospective Observational Analysis

Introduction: Cervical cancer is second most commonly diagnosed and third most common cause for cancer death among women in the developing countries. It is now-established that Human Papillomavirus (HPV) infection is responsible for pathogenesis of cervical cancer. HPV Deoxyribonucleic Acid (DNA) detection is generally done on cytology specimens to triage women undergoing cervical cancer screening, but testing of Formalin-fixed, Paraffin- embedded tissue (FFPE) is not yet widely used. Aim: To study the detection of HPV by Qualitative PCR by extraction of DNA from FFPE. Materials and Methods: This retrospective observational analysis was carried out at Department of Pathology, Armed Forces Medical College, Pune, Maharashtra, India, managed at the centre from 2008 to 2015. The data analysis was done Aug 2013-Oct 2015 on 35 patients of cervical cancer which were reconfirmed by histopatholgical study of sections. Tissue blocks were obtained from the selected subjects and 3-5 micron sections were taken and prepared for Haematoxylin and Eosin (H&E) staining. Qualitative PCR was run on DNA extracted from FFPE tissue for evaluation of HPV. The amplified DNA varied between 230-270 base pairs (bp) and was analysed for oncogenic HPV type 16, 18, 31, 33, 35, 45, 52b and 58 by gel electrophoresis. Data was tabulated in Microsoft excel and mean, frequency and percentages were calculated. Pearson’s Chi-square test was used to calculate the significance. Results: Out of the total 35 samples analysed (mean age: 51.08±10.6 years),15 cases were large cell non keratinising carcinoma, 12 cases of keratinising squamous cell carcinoma, 5 cases of carcinoma in-situ and 3 cases were adenocarcinoma. A total of 13 cases out of 35 showed the bands of HPV genomes, indicating either of the HPV strain. Conclusion: Although molecular diagnostics on FFPE tissue is need of hour, stringent protocols for timing of fixation, technical expertise for extraction of DNA or Ribonucleic Acid (RNA), careful handling of the sample and quality control is of paramount importance. Fragmentation is a problem in DNA extracted from FFPE tissue, so primers having small base pairs should be used to maximise the yield.

Local monitoring of SARS-CoV-2 variants in two large California counties in 2021

Coronavirus Disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to persist due to mutations resulting in newer, more infectious variants of concern. We aimed to leverage an ongoing private SARS-CoV-2 testing laboratory’s infrastructure to monitor SARS-CoV-2 variants in two large California counties. Study enrollment was offered to adults aged 18 years or older in Los Angeles County and Riverside County who recently tested positive for SARS-CoV-2 with a polymerase chain reaction (PCR) assay. A cycle threshold value less than or equal to 30 cycles was considered a positive test for sequencing purposes. Within 5 days of study enrollment, clinician-monitored, self-collected oral fluid and anterior nares swab specimens were obtained from participants. Specimens were transported and stored at 8 °C or cooler. Samples underwent ribonucleic acid extraction, library preparation, and sequencing. SARS-CoV-2 lineages were identified using sequencing data. Participant and genomic data were analyzed using statistical tools and visualized with toolkits. The study was approved by Advarra Institutional Review Board (Pro00053729). From May 27, 2021 to September 9, 2021, 503 individuals were enrolled and underwent specimen collection. Of the 503 participants, 238 (47.3%) participants were women, 329 (63.6%) participants were vaccinated, and 221 (43.9%) participants were of Hispanic or Spanish origin. Of the cohort, 496 (98.6%) participants had symptoms at the time of collection. Among the 503 samples, 443 (88.1%) nasal specimens and 353 (70.2%) oral specimens yielded positive sequencing results. Over our study period, the prevalence of the Alpha variant of SARS-CoV-2 decreased (initially 23.1% [95% confidence interval (95% CI): 0–0.49%] to 0% [95% CI 0.0–0.0%]) as the prevalence of the Delta variant of SARS-CoV-2 increased (initially 33.3% [95% CI 0.0–100.0%] to 100.0% [95% CI 100.0–100.0%]). A strain that carried mutations of both Delta and Mu was identified. We found that outpatient SARS-CoV-2 variant surveillance could be conducted in a timely and accurate manner. The prevalence of different variants changed over time. A higher proportion of nasal specimens yielded results versus oral specimens. Timely and regional outpatient SARS-CoV-2 variant surveillance could be used for public health efforts to identify changes in SARS-CoV-2 strain epidemiology.

Medical laboratory staff labor cost analysis and modeling for SARS-CoV-2 RNA PCR-testing: A time-and-motion study

BACKGROUND: Reverse transcription polymerase chain reaction (PCR) is the main method for detecting the viral agent of coronavirus disease-2019 (COVID-19). This analysis is performed in a non-automated mode in many medical laboratories, which implies a significant labor intensity of a test. However, its magnitude, as well as the workload on staff in the context of variability in testing volumes, has not yet been quantified due to the lack of systematic approaches to studying the labor costs of laboratory staff.
 AIM: This study aimed to investigate the structure and magnitude of the labor cost components of laboratory staff when performing manual PCR for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ribonucleic acid (RNA) and obtain stratified data that characterize the labor intensity of the testing, which are suitable for workload modeling.
 MATERIALS AND METHODS: The study of the labor costs (as labor time) of the PCR laboratory staff was conducted based on the Clinical Diagnostic Laboratory of the Multidisciplinary Medical Center of the Bank of Russia using a time-and-motion study methodology. Hence, the authors method for researching the labor costs of medical laboratory staff was applied.
 RESULTS: The lists of standard manipulations of the time-studied labor process were formed workplaces, the manipulations were classified according to the labor cost components, and the duration of each component was statistically determined. The labor intensity per test under current working conditions was 110.2 s for the reception and registration, 144.6 s for RNA extraction, and 79.4 s for reaction mixture preparation/amplification workplaces. The workload magnitude for the personnel of these workplaces corresponded to 47%, 62%, and 34% of a lab specialists annual working rate budget. The stratification of labor costs into fixed and variable allowed us to model the workload depending on the change in the testing volume and the number of analytical runs, as well as develop nomograms for the annual total labor time calculation.
 CONCLUSION: The proposed approach to labor cost accounting and analysis makes it possible to obtain suitable stratified data for modeling the workload of personnel during manual PCR-testing for SARS-CoV-2 RNA. The developed nomograms based on them can serve the purposes of planning the laboratory work and increasing its efficiency by choosing the optimal schemes for organizing the labor process. The obtained data can be used in other laboratories that perform real-time PCR tests for molecular genetic markers.

Diversity of mosquito species and potential arbovirus transmission in long-tailed macaque (Macaca fascicularis) breeding facilities

Background and Aim:Mosquito-borne viral infections are diseases that reduce human and animal health levels. Their transmission involves wildlife animals as reservoirs and amplifying hosts, including long-tailed macaques (Macaca fascicularis), and potentially transmits to humans and vice versa. This study aimed to determine the species diversity, richness, and biting activity of mosquitoes in a long-tailed macaque breeding area facility and discover the presence of Flavivirus and Alphavirus as the two main arboviruses reported to infect macaques.Materials and Methods:Human landing catch, light trap, and sweep net methods were used for mosquito collection around long-tailed macaques cages at parallel times for 12 h (18:00–06:00) for 12 nights. Mosquito species were identified to the species level based on the morphological identification key for Indonesian mosquitoes. Mosquito diversity was analyzed by several diversity indices. Mosquitoes caught using the human landing catch method were pooled based on mosquito species for viral ribonucleic acid extraction. Reverse transcription-polymerase chain reaction (RT-PCR) detected the non-structural protein 5 of the Flavivirus region and the non-structural protein 4 of the Alphavirus region. This study used the man-hour density and man-biting rate formulas for mosquito density.Results:Ten mosquito species were collected, namely, Aedes albopictus, Anopheles aconitus, Anopheles minimus, Anopheles vagus, Armigeres foliatus, Armigeres subalbatus, Culex gelidus, Culex hutchinsoni, Culex tritaeniorhynchus, and Culex quinquefasciatus. The number of mosquitoes caught using the light trap method had the highest abundance. In contrast, the number of mosquito species caught using the sweep net method had lower diversity than the other two methods. Seven mosquito species were obtained using the human landing catch method. The mosquito species with the highest density was Cx. quinquefasciatus within the observed densest period from 20:00 to 21:00. Negative results were obtained from RT-PCR testing on five species detected using universal Flavivirus and Alphavirus primers.Conclusion:The occurrence of mosquitoes in long-tailed macaque breeding facilities can be a source of transmission of zoonotic vector-borne diseases between animals and humans and vice versa.

Effects of Cyclosporine A and Adalimumab on the expression profiles histaminergic system-associated genes and microRNAs regulating these genes in HaCaT cells

ABSTRACT Previous studies have not completely elucidated the role of the histaminergic system in the pathogenesis of psoriasis. This study aimed to evaluate the effects of adalimumab and cyclosporine A on the expression of histaminergic system-related genes and miRNAs regulating these genes in bacterial lipopolysaccharide A (LPS)-stimulated human keratinocyte (HaCaT) cells. HaCaT cells were treated with 1 µg/mL LPS for 8 h, followed by treatment with 8 µg/mL adalimumab or 100 ng/mL cyclosporine A for 2, 8, or 24 h. Untreated cells served as controls. The cells were subjected to ribonucleic acid (RNA) extraction and microarray, quantitative real-time polymerase chain reaction, and enzyme-linked immunosorbent assay analyses. Statistical analysis was performed using the Statistica 13.0 PL (StatSoft, Cracow, Poland) and the Transcriptome Analysis Console programs (Affymetrix, Santa Clara, CA, USA) (p < 0.05). The differential expression of the following two miRNAs was not affected in LPS-stimulated cells upon treatment with cyclosporine A or adalimumab: hsa-miR-583 (downregulated expression), involved in the regulation of histamine receptor 1 – HRH1 (overexpression); has-miR-1275 (downregulated expression), involved in the regulation of histamine receptor 1 – HRH3 (overexpression) and Solute carrier family 22 member 3 – SLC23A2 (downregulated expression)). Adalimumab and cyclosporine A modulated the histaminergic system in HaCaT cells in vitro. However, further studies are needed to elucidate the underlying mechanisms. Abbreviations: (-) – downregulated in comparison to the control, (+) – overexpression in comparison to the control, ACTB - β-actine, ADA - Adenosine deaminase, ADCYAP1 - Adenylate Cyclase Activating Polypeptide 1, BMP - bone morphogenetic protein, bp - base pair, cAMP - adenosine 3’ 5’-cyclic monophosphate, CBX7 - Chromobox protein homolog 7, cDNA - double-stranded complementary DNA, CSA - cyclosporine A DAG – diacylglycerol, DIAPH - Diaphanous related formin 1, DNMT - DNA methyltransferases, DRD2 - Dopamine receptor D2, EDN1 - Endothelin 1, EDNRA - Endothelin receptor type A, ELISA - Enzyme-linked immunosorbent assay, EZH2 - Enhancer of zeste homolog 2, FC - fold change, GABRB1 - Gamma-aminobutyric acid (GABA) A receptor, alpha 1, GABRB2 - Gamma-aminobutyric acid (GABA) A receptor, alpha 2, GABRB3 - Gamma-aminobutyric acid (GABA) A receptor, alpha 3, HaCaT - Human adult, low-calcium, high-temperature keratinocytes, HIS - Human Histamine, HLAs - human leukocyte antigens, HNMT - Histamine N-methyltransferase, HNMT - Histamine N-Methyltransferase, HRH1 – histamine receptor 1, HRH2 – histamine receptor 2, HRH3 – histamine receptor 3, HRH4 – histamine receptor 4, HTR6 - 5-Hydroxytryptamine Receptor 6, IGF1 - Insulin-like growth factor 1, IL10 -interleukin 10, IL12 -interleukin 12, IL6 - interleukin 6, IP3 - inositol 1,4,5-triphosphate, LPS - bacterial lipopolysaccharide A, LYN - LYN Proto-Oncogene, Src Family Tyrosine Kinase, MAPKs -mitogen-activated protein kinases, miRNA - micro RNA, MMP2 - matrix metalloproteinase-2, NHDF – Normal Human Dermal Fibroblasts, NHEK - Normal Human Epidermal Keratinocytes, OCT3 - organic cation transporter 3, PANTHER - Protein ANalysis THrough Evolutionary Relationships Classification, PBS - phosphate-buffered saline, PI3K-AKT - phosphatidylinositol 3-kinase-protein kinase B, PIP2 - phosphatidylinositol 4,5 bisphosphate, PMSF - phenylmethylsulfonyl fluoride, PSORS1- psoriasis susceptibility gene 1, qRT-PCR - quantitative Reverse Transcription Polymerase Chain Reaction, RNA - ribonucleic acid, RNAi - RNA interference, RTqPCR - Real-Time Quantitative Reverse Transcription Reaction, SLC223A2 - Solute carrier family 22 member 3, SNX -Sorting nexin, SOX9 - SRY-Box Transcription Factor 9, TGF-α - transforming growth factor α, TGF-β - transforming growth factor beta, TNF-α - tumor necrosis factor alpha, TP53 - tumor protein 5 z, VAMP2 - Vesicle associated membrane protein 2.

Investigation of the Effect of Lactobacillus Brevis Bacteria on the Expression of Rel A, IKB, and Casp3 Genes in HT29 Colon Cancer Cells

Aims Studies have shown that probiotic bacteria inhibit the onset and progression of carcinogenesis through different pathways. Our objective in this study was to determine the effect of probiotic bacteria on the expression of growth-related genes Rel A, IKB, and Casp3 in HT29 colon cancer cells Methods &amp; Materials In this study, the Lactobacillus brevis probiotic bacteria were first cultured, and after the supply of media conditioning, they were treated on HT29 cancer cells. The bacterium’s cytotoxic effect (bacterial T cells) was investigated using a microculture tetrazolium test (MTT). DNA was extracted from the treated cells, and a DNA Ladder assay was performed. Also, the 4′,6-diamidino-2-phenylindole (DAPI) test was performed to show cell apoptosis. After ribonucleic acid (RNA) extraction and complementary DNA (cDNA) preparation to determine the mechanism of the effect of this bacterium on cellular signaling, the expression of growth-related genes Rel A, IKB, and Cas3 was measured using a real-time polymerase chain reaction (PCR) method. Findings The microculture tetrazolium (MTT) test showed that L. b bacteria inhibit HT29 cells’ proliferation, induce apoptosis in these cells, and inhibit Rel A gene proliferation by increasing IKB gene expression. Also, 4′,6-diamidino-2-phenylindole (DAPI), and DNA ladder assay following the treatment of HT29 cells regarding the mentioned bacteria showed qualitative changes in cell apoptosis. In addition, real-time polymerase chain reaction (PCR) results showed that L. b increased Casp3 gene expression in HT29 colon cancer cells (P=0.038). Conclusion Our findings indicate that L. b stimulates the apoptotic cell signaling pathway in HT29 colon cancer cells. It can be used as a new treatment strategy or therapy for colon cancer treatment.

Assessment of RNA extraction protocols from cladocerans.

The usage of cladocerans as non-model organisms in ecotoxicological and risk assessment studies has intensified in recent years due to their ecological importance in aquatic ecosystems. The molecular assessment such as gene expression analysis has been introduced in ecotoxicological and risk assessment to link the expression of specific genes to a biological process in the cladocerans. The validity and accuracy of gene expression analysis depends on the quantity, quality and integrity of extracted ribonucleic acid (RNA) of the sample. However, the standard methods of RNA extraction from the cladocerans are still lacking. This study evaluates the extraction of RNA from tropical freshwater cladocerans Moina micrura using two methods: the phenol-chloroform extraction method (QIAzol) and a column-based kit (Qiagen Micro Kit). Glycogen was introduced in both approaches to enhance the recovery of extracted RNA and the extracted RNA was characterised using spectrophotometric analysis (NanoDrop), capillary electrophoresis (Bioanalyzer). Then, the extracted RNA was analysed with reverse transcription polymerase chain reaction (RT-PCR) to validate the RNA extraction method towards downstream gene expression analysis. The results indicate that the column-based kit is most suitable for the extraction of RNA from M. micrura, with the quantity (RNA concentration = 26.90 ± 6.89 ng/μl), quality (A260:230 = 1.95 ± 0.15, A280:230 = 1.85 ± 0.09) and integrity (RNA integrity number, RIN = 7.20 ± 0.16). The RT-PCR analysis shows that the method successfully amplified both alpha tubulin and actin gene at 33-35 cycles (i.e. Ct = 32.64 to 33.48). The results demonstrate that the addition of glycogen is only suitable for the phenol-chloroform extraction method. RNA extraction with high and comprehensive quality control assessment will increase the accuracy and reliability of downstream gene expression, thus providing more ecotoxicological data at the molecular biological level on other freshwater zooplankton species.

Detection and Phylogenetic Analysis of Hepatitis A Virus in the Wastewater Treatment Plant of Ekbatan Town in Tehran, Iran

Background: Limited sources of fresh water necessitate the application of health policies for treatment and decontamination of human sewage for further use. A wide variety of infectious agents, including bacteria, fungi, parasites, and viruses, can be found in sewage. Enteric viruses such as hepatitis A virus (HAV) can survive the current treatments and infect susceptible hosts. Objectives: This study aimed to evaluate HAV contamination in human sewage before and after treatment in the wastewater treatment plant of Ekbatan town in Tehran, Iran, and analyze the phylogenetic properties of the identified viruses. Methods: Over a 12-month period, we collected the wastewater samples including influent, before chlorination, and effluent, from the wastewater treatment plant of Ekbatan town in Tehran, Iran. Ribonucleic acid (RNA) extraction, complementary deoxyribonucleic acid (cDNA) synthesis, and semi-nested polymerase chain reaction (PCR) were performed to identify HAV contamination. Phylogenetic analysis was performed to investigate subgenotypes of the virus. Results: HAV was detected in all influents and samples before chlorination, while the virus was detected in 50% of the effluent samples. All detected viruses belonged to subgenotype IB. Conclusions: Investigating the presence of HAV in sewage provides a general picture of the virus spread in the population of interest. HAV was detected in all influent samples, indicating that the infection is endemic in this area all year round. This also indicates the inability of the current treatment protocols in virus removal, which can be a threat to the public health.

Efficacy of Nucleic Acid Extraction by Manual versus Automated Magnetic Bead-based Method to Detect SARS-CoV-2

Introduction: In December 2019, a rapid spread of highly infectious, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), was reported in Wuhan, China. The gold standard for diagnosis of SARS-CoV-2 infection is nucleic acid amplification technology by detecting its viral Ribonucleic Acid (RNA) from respiratory swabs (oropharyngeal and nasopharyngeal) by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) whose specificity is approximately 95%. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Aim: To compare the efficacy of nucleic acid extraction by manual method and automated magnetic bead-based method to detect SARS-CoV-2. Materials and Methods: The present observational comparative study was conducted in the Department of Microbiology, Autonomous State Medical College, Firozabad, Uttar Pradesh, India. Duration of the study was from February to March 2022. A total of 470 oropharyngeal and nasopharyngeal samples were included in this study to observe the efficacy of nucleic acid extraction by manual extraction and automated extraction for SARS-CoV-2. Data were entered in Microsoft Excel software and analysed using Statistical Package for the Social Sciences (SPSS) version 26.0. Results: During this study period, a total of 470 individual samples were tested in 94 pools. Out of these 470 individuals, 331 were males (70.5%) and 139 were females (29.5%). All 94 pools were found negative by both automatic and manual extraction methods. Envelope (E) gene was found in one pool (1.06%) by the manual RNA extraction method. E gene was absent in 93 pools (98.94%) by manual method. Internal control was found highest in 88 pools (93.62%) by the automated extraction method. Conclusion: Automated workflows avoid human error from the sample processing pipeline and also ensure as well as enhance the meaningful output, diagnostic precision, and testing capacity. Automated instruments are in wide usage because of their capability of processing thousands of samples per day with the support of minimal staff.

Severe Acute Respiratory Syndrome Coronavirus-2 - A Surge of CoronaVirus Disease-2019: An Epidemiological Study in Coimbatore District.

Context:Coronavirus disease-2019 (COVID-19) is an ongoing pneumonia-like cluster syndrome which originated in Wuhan city of China and is still now on escalation, causing severe outbreaks all over the world. Being a ribonucleic acid (RNA) virus which has the low proofreading RNA-dependent RNA polymerase leads to many mutations and that serves as the major cause for the progress of the disease. As per the recent research works done, 99% of COVID-19 severe acute respiratory syndrome coronavirus (SARS-CoV-2) are due to pangolin-associated coronavirus which causes the super spreading events of coronavirus. SARS-CoV-2 was identified in the nasopharyngeal swabs received in the viral transport medium at optimum temperature.Materials and Methods:The tests were conducted for a time period of 1 year from July 2020 to June 2021. A total of 77,824 samples were tested in the laboratory as per ICMR guidelines using approved RNA extraction kits and polymerase chain reaction kits.Results:In the total of 77,824 samples tested in our laboratory, 14174 positives were identified. In that, about seven positive cases (0.004%) were identified in the month of July 2020 which increased to the maximum in September 2020 to about 865 positive cases (6%) which is the peak of first wave COVID-19 in Coimbatore district, Tamil Nadu. Out of 77,824 samples tested, the actual cumulative laboratory-confirmed positive cases of about 14174 were identified. In that, 7731 (55%) male positive cases were identified, 6171 (43%) female positive cases were identified, and 270 (2%) children who were below 12 years of age also were tested positive.Conclusions:The findings of the study indicated a high predominance of SARS-CoV-2 infection in the male gender population when compared to females and children below 12 years of age in Coimbatore district as of June 2021. The surge of cases was high in September 2020 as well as in May 2021, indicating the first and second wave of COVID-19.

Identification of peste des petits ruminants virus along with co-infecting diseases of goats in Bangladesh.

Peste des petits ruminants (PPR) virus is the main infectious cause of goat mortality in Bangladesh, and co-infection may make diseases more severe. This study aimed to detect PPR and co-infecting diseases in goats. One hundred goats suspected to be infected with the PPR virus were collected from various areas of Mymensingh district, Bangladesh. A systemic post-mortem examination was carried out on PPR-suspected goats. Lungs, spleen, and lymph nodes (pre-scapular) were used for ribonucleic acid extraction, whereas lungs and mesenteric lymph nodes were used for deoxyribonucleic acid extraction. Seven-pair primer sets were used for molecular detection of pathogens specific for PPR, goat pox, contagious ecthyma (Orf), foot and mouth disease (FMD) virus, Klebsiella sp., and Mycobacterium sp. Reverse transcriptase-polymerase chain reaction (RT-PCR) or polymerase chain reaction (PCR) were used to find the exact cause. Out of 100 PPR-suspected goats examined, 55 goats were confirmed as PPR-detected by RT-PCR. Among the 55 PPR-positive goats, 2 were co-infected with goat pox, 2 with tuberculosis, 10 with Klebsiella sp. infection, and 6 with FMD as detected by PCR and RT-PCR. Moreover, 12 goats were co-infected with PPRV and fascioliasis. About 58% of PPR virus-infected goats were co-infected with other organisms. There is a need to design technology to detect the state of co-infectivity at its early onset and future preventive and therapeutic strategies for co-infecting diseases. This is the first study in Bangladesh to describe co-infecting diseases of goats along with PPR.

Accumulation of radio-iron and plutonium, alone and in combination, in Pseudomonas putida grown in liquid cultures

The impact of low doses of ionising radiation on biological and environmental systems have been historically difficult to study. Modern biological tools have provided new methods for studying these mechanisms but applying these tools to a dose–response relationship may require refinement of dosimetric techniques that incorporate a detailed understand of radionuclide accumulation in biological cells, particularly when assessing the impact of low doses of ionising radiation. In this work Pseudomonas putida (KT2440) grown in liquid culture was exposed to low dose rates (10–20 mGy d−1) of 239Pu and 55Fe, both alone and in combination, for a period of 20 days, and the accumulation of 239Pu and 55Fe in cell pellets was analysed via liquid scintillation counting. The study also considered of cells grown with 239Pu and stable Fe (primarily 56Fe). In addition to the analysis of cell pellet and media samples, this work includes analysis of the radiological content of ribonucleic acid extraction samples to examine uptake of radionuclides. Results indicate that 239Pu inhibited the uptake of 55Fe, and that the presence of stable and radioactive isotopes of Fe in cultures may promote pathways for Fe accumulation that are used by 239Pu. The work herein provides foundational insight into future dosimetric models for our work with environmental bacteria.

A Pilot Study: Composition and Diversity of 16S rRNA Based Skin Bacterial Microbiome in Indonesian Atopic Dermatitis Population.

BackgroundAtopic dermatitis (AD) interferes with quality of life and is influenced by important factors like skin microbiome. The results of the skin microbiome composition and diversity in AD varied in some studies.PurposeThis study aims to determine the composition and diversity of the skin microbiome in Indonesian AD patients.Patients and MethodsGenomic deoxyribonucleic acid (DNA) preparations were obtained from skin swabs of the cubital fossa of 16 subjects, nine of which were having mild AD, three moderate AD, and four healthy individuals. DNA extraction and sequencing of the 16S ribosomal ribonucleic acid (rRNA) gene using next-generation sequencing and bioinformatics analysis were further performed.ResultsFirmicutes (p), Bacilli (c), Bacillales (o), Staphylococcaceae (f), and Staphylococcus (g) were dominant in moderate AD. On the contrary, Proteobacteria (p), Gammaproteobacteria (c), Pseudomonadales (o), Moraxellaceae (f), and Acinetobacter (g) were dominant in mild AD. Staphylococcus aureus was found in the highest number in individuals with moderate AD. Interestingly, Ensifer adhaerens was found in mild AD. Microbial diversity was decreased in moderate AD.ConclusionMetagenomic analysis in this study identified microbes in moderate and mild AD and showed a low diversity of skin microbiomes in moderate AD. Interestingly, this is the first time that the bacteria Ensifer adhaerens was detected on the human skin.

Differential Expression of MMP2 and TIMP2 in Peripheral Blood Mononuclear Cells After Roux-en-Y Gastric Bypass.

Matrix metalloproteinases (MMP) and their endogenous inhibitor, the tissue inhibitor of metalloproteinases (TIMP), are expressed in many different cell types and play an important role in physiologic and pathological degradation of extracellular matrix (ECM). Starting from these observations and considering the activation state of peripheral blood mononuclear cells (PBMCs) in obesity, we investigated the gene expression of metalloproteinases before and after Roux-en-Y gastric bypass (RYBG). The study was performed in the Ribeirão Preto Medical School University Hospital. Seventy-three women were divided into a study group (SG), composed of 53 individuals with severe obesity before and after 6 months of RYGB, and a control group (CG), composed of 20 normal-weight individuals. Anthropometric and body composition data were collected, and peripheral blood for ribonucleic acid (RNA) extraction. The biological samples were submitted to a quantitative real-time polymerase chain reaction to evaluate the expression of MMP2 and TIMP2 genes. Alterations in weight loss, body mass index (BMI), and fat mass (FM) were observed after 6 months of RYGB (p < 0.05). A reduction of gene expression of TIMP2 was observed after 6 months of RYGB, contributing positively to the weight loss (R2 = 0.33 p = 0.04). The enrichment analyses highlighted the interaction between TIMP2 and MMP2 genes and the molecular pathways involving the ECM remodeling in the obesity condition. RYGB contributes significantly to weight loss, improved BMI, reduced FM, and reduced TIMP2 expression in PBMCs, which might contribute to the ECM remodeling in the obesity and could be useful as a circulating biomarker.

Evaluation of Anticancer Effects and Molecular Mechanisms of Pomegranate Peel Extract in the Mouse Cellular Model (4T1) of Breast Cancer

Background: Breast cancer is the most common female cancer that, despite recent progress, its existing therapies do not have sufficient efficiency, and scientists are trying to find complementary therapies. Medicinal plants, such as pomegranate (Punica granatum L.), attract a great deal of attention in this regard. Objectives: The present study aimed to assess the anticancer effects of pomegranate peel alcoholic extract (PPE) in the mouse cellular model (4T1) of breast cancer and investigate the related molecular mechanism of antiproliferative effects of the extract through the evaluation of the expression level of apoptosis genes (ie, BAX and BCL2). Methods: For the accomplishment of the study objectives, after the preparation of PPE, an evaluation of cellular cytotoxicity was carried out using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. According to the MTT assay, the cells were treated for 24 h with selective doses of the extract. Then, ribonucleic acid extraction, complementary deoxyribonucleic acid synthesis, and gene expression analysis of BAX and BCL2 using real-time polymerase chain reaction were performed in this study. Results: The results showed that PPE reduced cancer cell viability in a dose- and time-dependent manner. The molecular analysis indicated that observed cell death could be due to an increase in the BAX/BCL2 ratio. Conclusions: Overall, PPE can be proposed as a potential complementary therapy for breast cancer. However, further studies on animal models and clinical trials are needed to verify the clinical usage of such complementary drugs.

Optimized method for RNA extraction from leaves of forest tree species

Extraction of ribonucleic acid (RNA) from woody plants is a difficult task due to the peculiarities of plant material rich in polysaccharides and starch. The available techniques are often ineffective, since they result in the absence/reduced quality/reduced amount of RNA in the final preparation. The method we have optimized is based on the use of cethyltrimethyl ammonium bromide (CTAB), purification by phenol-chloroform extraction, use of lithium chloride and ammonium acetate. The method showed high efficiency for the extraction of RNA from the leaves of birch and poplar samples, in vitro and mature plants, in comparison with previously used methods (extraction using NucleoSpin® RNA Plant (Macherey-Nagel, Germany) columns, Su (2009) method, standard guanidine thiocyanate method). Electropherograms of RNA preparations showed its high integrity and concentration (up to 85 ng/μl), significantly higher purity of the preparation (up to 2.7 times). Purification of the preparation in the process of extraction can significantly reduce the yield of desoxyribonucleic acid (DNA). The optimized method is highly reproducible and can be used for further research, complementary DNA (cDNA) synthesis, qualitative and quantitative PCR analysis. The method allows obtaining high-quality RNA from other objects of agricultural and forest plants.

Multicenter study evaluating novel multi-specimen pooling assay for the detection of SARS-CoV-2: High sensitivity and high throughput testing.

Background/purposeMass screening for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is important to prevent the spread of coronavirus disease 2019 (COVID-19). Pooling samples can increase the number of tests processed. LabTurbo AIO 48 is an automated platform that allows ribonucleic acid extraction and sample analysis on the same instrument. We created a novel pooling assay on this platform for SARS-CoV-2 detection and demonstrated that the pooling strategy increases testing capacity without affecting accuracy and sensitivity.MethodsComparative limit of detection (LoD) assessment was performed on the LabTurbo AIO 48 platform and the current standard detection system based on real-time reverse transcription polymerase chain reaction (rRT-PCR) using 55 clinically positive samples. An additional 330 primary clinical samples were assessed.ResultsSix samples pooled into one reaction tube were detected in approximately 2.5 h using the World Health Organization rRT-PCR protocol. LabTurbo AIO 48 also demonstrated a higher throughput than our reference rRT-PCR assay, with an LoD of 1000 copies/mL. The overall percentage agreement between the methods for the 330 samples was 100%.ConclusionWe created a novel multi-specimen pooling assay using LabTurbo AIO 48 for the robust detection of SARS-CoV-2, allowing high-throughput results; this assay will aid in better control and prevention of COVID-19. The diagnostic assay was cost-effective and time-efficient; thus, the pooling strategy is a practical and effective method for diagnosing large quantities of specimens without compromising precision.

Investigation of the role of IL18, IL-1β and NLRP3 inflammasome in reducing expression of FLG-2 protein in Psoriasis vulgaris skin lesions

ABSTRACT We investigated the effects of the NACHT leucine-rich repeat- and PYD-containing proteins (NLRP3) inflammasome, interleukin −18 (IL-18) and interleukin-1 beta (IL-1β) cytokines on the expression of filaggrin-2 (FLG-2) protein in psoriasis patients. Peripheral blood mononuclear cells (PBMC), including T cells, were isolated from psoriasis patients and healthy donors. Ribonucleic acid (RNA) extraction and reverse transcription-polymerase chain reaction (RT-PCR) were performed for all specimens. Immunohistochemical analysis for FLG-2 in normal and psoriatic epidermal tissue also was performed. Western blot was used to separate and identify FLG-2 protein, and immunohistochemical staining was performed to assess FLG-2 expression for psoriasis skin lesions and normal skin. RT-PCR analysis indicated that NLRP3 inflammasome, IL-18 cytokine and IL‐1β cytokine expression were increased in psoriatic epidermis compared to normal skin. We found that the expression of FLG-2 was decreased in psoriatic epidermis compared to normal skin. Higher levels of NLRP3 help decrease the FLG-2 level.

High Efficiency RNA Extraction From Sperm Cells Using Guanidinium Thiocyanate Supplemented With Tris(2-Carboxyethyl)Phosphine.

The extraction of high-quality ribonucleic acid (RNA) from tissues and cells is a key step in many biological assays. Guanidinium thiocyanate-phenol-chloroform (AGPC) is a widely used and efficient method to obtain pure RNA from most tissues and cells. However, it is not efficient with some cells like sperm cells because they are resistant to chaotropic lysis solutions containing guanidinium thiocyanate such as Buffer RLT+ and Trizol. Here, we show that disulfide bonds are responsible for the chemical resistance of sperm cells to RNA extraction reagents. We show that while β-mercaptoethanol (βME) can increase sperm lysis in Buffer RLT+, it has no effect in Trizol and leaves sperm cells intact. We measured the reduction of disulfide bonds in 2,2′-dithiodipyridine (DTDP) and observed that βME has a pH-dependent activity in chaotropic solutions, suggesting that pH is a limiting factor. We identified tris(2-carboxyethyl)phosphine (TCEP) as an efficient lysis enhancer of AGPC solutions that can retain reducing activity even at acidic pH. Trizol supplemented with TCEP allows the complete and rapid lysis of sperm cells, increasing RNA yield by 100-fold and resulting in RNA with optimal quality for reverse transcription and polymerase chain reaction. Our findings highlight the importance of efficient cell lysis and extraction of various macromolecules for bulk and single-cell assays, and can be applied to other lysis-resistant cells and vesicles, thereby optimizing the amount of required starting material and animals.

Direct Saliva versus Conventional Nasopharyngeal Swab qRT-PCR to Diagnose SARS – CoV2: Validity Study

Background: Saliva has been demonstrated as a feasible alternative specimen to nasopharyngeal swab for the detection of SARS-CoV-2 using real-time or quantitative reverse transcription polymerase chain reaction (qRT-PCR) method that bypasses the need for explicit viral ribonucleic acid (RNA) extraction.&#x0D; Aim: To assess the diagnostic validity of direct saliva-to-qRT-PCR in the detection of SARS-CoV-2 compared to conventional nasopharyngeal swab qRT-PCR.&#x0D; Methodology: Self-collected saliva samples were processed by heating at 95oC for 30 minutes followed by addition of buffer and detergent while viral RNA from nasopharyngeal swabs were extracted using the Sansure Biotech sample release reagent. Paired samples were used as templates for qRT-PCR using the Sansure Novel Coronavirus (COVID-19) Nucleic Acid Diagnostic Kit and Sansure Biotech MA6000 Real-Time Quantitative PCR System. Direct saliva-to-qRT-PCR was compared to nasopharyngeal swab qRT-PCR in terms of diagnostic validity and agreement parameters, and both platforms were compared separately in terms of similar parameters with a composite reference standard (CRS) wherein the criteria for a positive result is SARS-CoV-2 detection in at least either nasopharyngeal swab or saliva.&#x0D; Results: Of the 238 nasopharyngeal swab-saliva pairs tested, 20 (8.4%) nasopharyngeal swab and 24 (10.1%) saliva specimens tested positive. We documented a sensitivity of 85.0% (95% CI: 62.1%, 96.8%), specificity of 96.8% (95% CI: 93.5%, 98.7%), accuracy of 95.8% (95% CI: 92.4%, 98.0%) and Cohen Kappa of 0.75 (95% CI: 0.60, 0.90) when direct saliva-to-qRT-PCR was compared to the conventional platform. When the two platforms were individually compared to the CRS, numerically higher but not statistically significant sensitivity and accuracy were noted for direct saliva-to-qRT-PCR than for nasopharyngeal swab qRT-PCR.&#x0D; Conclusion: Direct saliva-to-qRT-PCR is non-inferior to nasopharyngeal swab qRT-PCR for detecting SARS-CoV-2 using the Sansure Novel Coronavirus Nucleic Acid Diagnostic Kit.