cDNA Synthesis Research Articles

FOXA1, FOXA2, SOX10 and GAS2 Gene Expression in Oral Squamous Cell Carcinoma and Their Relationship with Clinicopathological Indices

Background: The use of molecular methods in cancer diagnosis has led to a better prognosis. One of the important gene families in the carcinogenic pathways of various cancers is the forkhead box (FOX) family genes. Moreover, developmental transcription factors and proapoptotic proteins play critical roles in cell function and carcinogenesis. Objectives: The current study aimed to evaluate the expression of A1 FOXA1, FOXA2, SOX10, and growth arrest specific 2 (GAS2) genes in oral squamous cell carcinoma (OSCC) tumors due to biomarker discovery and early diagnosis of cancer. Methods: To evaluate the expression of FOXA1, FOXA2, SOX10, and GAS2 genes, 30 OSCC samples and 30 normal specimens were obtained from Imam Khomeini Hospital Cancer Institute. RNA extraction and cDNA synthesis were done by relevant kits. After a specific primer design for FOXA1, FOXA2, SOX10, and GAS2 genes, real-time PCR was done to evaluate the genes’ expression for molecular biomarker discovery and validation. ANOVA and independent t-test were used to analyze the data. Results: Significant differences were observed in the expression of the studied genes in tumor and control tissues (P < 0.001). The results showed that FOXA1, GAS2, and SOX10 expressions in tumor and normal cells have significant differences (P < 0.001). Regardless of FOXA1, FOXA2 and SOX10, there was a significant difference in the expression of GAS2 genes in term patients’ age (P < 0.05) and overexpressed in patients over 55 years. SOX10 gene is upregulated in grade II OSCC tumors but there is no significant difference in expression of FOXA1, FOXA2, and GAS2 in different stages and grades. The ROC curve analysis, FOXA1, and FOXA2 showed AUC = 0.66 and AUC = 0.57 respectively. Meanwhile, SOX10 and GAS2 showed AUC = 0.9 and AUC = 1 respectively. Conclusions: In general, the expression of FOXA1, GAS2, and SOX10 genes in cancer and control tissues were different, and therefore the role of these genes in OSCC is confirmed. Also, in the present study, the biomarker potential of SOX10 and GAS2 genes for OSCC diagnosis was demonstrated. In the current study, the important role of the studied genes in OSCC diagnosis was shown. However, further studies are needed to confirm this.

Molecular epidemiological surveillance of viral agents of acute lower respiratory tract infections in children in Accra, Ghana

BackgroundAcute lower respiratory tract infection (ALRTI) in children under 5 years is known to be predominantly caused by respiratory syncytial virus (RSV). In recent times, however, human metapneumovirus (HMPV) has also been implicated. This study sought to investigate and genotype respiratory syncytial virus and human metapneumovirus in children presenting with ALRTIs infection at the Princess Marie Louis Children’s Hospital in Accra, Ghana.MethodsChildren below 5 years who were clinically diagnosed of ALRTI and on admission at the study site were recruited between September 2015 and November 2016 for this study. Demographic data information was obtained by means of a standardized questionnaire; and relevant clinical information was obtained from medical records. Nasopharyngeal swabs were collected from 176 children recruited for the study. Ribonucleic acid was extracted from swabs and cDNA syntheses were performed by RT-PCR. RSV-positive amplicons were sequenced and analyzed for genotype assignment.ResultsRSV and HMPV prevalence among the sampled subjects were 11.4 and 1.7% respectively. Of the RSV positives, 8/20 (40%) were RSV-A and 12/20 (60%) were RSV-B. The highest prevalence was observed in children less than 12 months old. Phylogenetic analysis of the second hypervariable region of the RSV G-gene revealed that all RSV group A viruses belonged to the “novel” ON1 genotype containing the 72-nucleotide duplication; and RSV group B viruses belong to the BA IX genotype.ConclusionRSV is frequently detected in children aged under 5 years admitted with ALRTI in Ghana. Continued surveillance of viral aetiological agents is warranted to elucidate the prevalence and transmission patterns of viral pathogens that cause respiratory tract infections among children. This will help inform appropriate intervention approaches.

Residual Feed Intake in Beef Cattle Is Associated With Differences in Hepatic mRNA Expression of Fatty Acid, Amino Acid, and Mitochondrial Energy Metabolism Genes

We evaluated the mRNA expression of genes involved in hepatic fatty acid, amino acid, and mitochondrial energy metabolism in crossbred beef steers with divergent low and high residual feed intake (RFI). Low-RFI beef steers (n = 8; RFI = - 1.93 kg/d) and high-RFI beef steers (n = 8; RFI = + 2.01kg/d) were selected from a group of 56 growing crossbred beef steers (average BW = 261 ± 18.5 kg) fed a high-forage total mixed ration after a 49-d performance testing period. At the end of the 49-d performance testing period, liver biopsies were collected from the low-RFI and high-RFI beef steers for RNA extraction and cDNA synthesis. The mRNA expression of 84 genes each related to fatty acid metabolism, amino acid metabolism, and mitochondrial energy metabolism were analyzed using pathway-focused PCR-based arrays. The mRNA expression of 8 genes (CRAT, SLC27A5, SLC27A2, ACSBG2, ACADL, ACADSB, ACAA1, and ACAA2) involved fatty acid transport and β-oxidation were upregulated (FC ≥ 2.0, FDR ≤ 0.05) in low-RFI, compared to high-RFI steers. Among those involved in amino acid metabolism, hepatic mRNA expression of a gene encoding for aminoadipate aminotransferase, an enzyme related to lysine degradation, was downregulated (FC = -5.45, FDR = 0.01) in low-RFI steers, whereas those of methionine adenosyltransferase I and aspartate aminotransferase 2, which both link amino acid and lipid metabolism, were upregulated (FC ≥ 2.0, FDR ≤ 0.05). Two mitochondrial energy metabolism genes (UQCRC1 and ATP5G1) involved in ATP synthesis via oxidative phosphorylation were upregulated (FC ≥ 2.0, FDR ≤ 0.05) in low-RFI beef steers, compared to high-RFI beef steers. The results of this study demonstrated that low-RFI beef steers exhibit upregulation of molecular mechanisms related to fatty acid transport, fatty acid β-oxidation, and mitochondrial ATP synthesis, which suggest that low-RFI beef steers have enhanced metabolic capacity to maximize capture of energy and nutrients from feeds consumed.

Evaluating changes in the expression of BCL-2 gene, lncRNA SRA, and miR-361-3p in unexplained recurrent pregnancy loss

Unexplained recurrent pregnancy loss (RPL) composed almost half of all diagnosed miscarriage cases. As the apoptosis pathway is involved in the pregnancy process the present investigation aimed to assess the differential expression of the BCL-2 gene, SRA lncRNA, miR-361-3p in unexplained RPL patients. In this study, RNA was isolated from 50 blood samples of people with a history of RPL, and 50 blood samples of people with healthy fertility. After cDNA synthesis from these samples, alterations in the expression levels of the above-mentioned genes were examined by Real-Time PCR. Our results showed that the expression of BCL-2 and lncRNA SRA was significantly higher in the blood samples of RPL patients than in controls, while the expression of miR-361-3p was significantly downregulated. Besides, there were significant correlations between the changes in the expression of lncRNA SRA and miR-361-3p with BCL-2, in positive and negative directions, respectively. Also, miR-361-3p presented as a good diagnostic marker with the highest AUC value to discriminate between RPL and the healthy control subjects. These results proposed that ncRNAs may have a significant role in the regulation of apoptosis relates genes expression in RPL.

PB1791: ON THE ISSUE OF HOUSEKEEPING GENE PRIMER DESIGN FOR ADEQUATE ASSESSMENT OF THE EXPRESSION OF THE CHIMERIC TRANSCRIPTS.

Background: Chimeric transcripts, which are the markers of hematological malignancies, is often assessed by expression level relative to the control housekeeping gene. The critical step for the analysis is RNA isolation and subsequent cDNA synthesis. Routine RNA isolation methods do not guarantee the absence of genomic DNA impurities, which may interfere with cDNA amplification. This results in the bias in control gene expression and underestimation of the relative expression. The use of sophisticated RNA isolation procedures or treatment with RNase-free DNAses significantly complicates and increases the cost of analysis, and can also lead to degradation of target sequences by endogenous nucleases. Here we describe a simple and effective solution to this problem. Aims: To optimize the RT-PCR approach for quantitative assessment of the chimeric transcripts gene expression relative to the control ABL1 gene, for the insensitivity to genomic DNA impurities. Methods: Total RNA was isolated from peripheral blood leukocytes or bone marrow cells as described by Chomczynski (1987). RT-PCR was performed using reagents and primers from Synthol (Moscow, Russia). Results: For ABL1 mRNA amplification primers flanking 2 gene introns were designed (see Table 1). This precludes PCR amplification of the corresponding (139708bp long) genomic sequence. At the same time, the cDNA of this fragment is only 100 nucleotides long. The primers recommended by Europe Against Cancer Association (EAC) and widely used (including as part of commercial diagnostic kits) flank 687bp ABL1 gene DNA fragment. In this case, the admixture of genomic DNA is co-amplified with the cDNA transcript in PCR. We have compared CBFB::MYH11 chimeric transcript relative expression levels in patients with AML, obtained with ABL-EAC primers and those proposed by us (ABL-N). We have tested 40 cDNA samples obtained from diverse RNA isolation routines, including 8 archival samples stored in the freezer for more than 6 months. The threshold cycles of amplification (ΔCt) for the samples from freshly isolated RNA were mostly the same with ABL-EAC and ABL-N primers. For archival RNA, cDNA, and RNA isolated from frozen cells, ΔCt with ABL-N primers drops significantly while ΔCt with ABL-EAC primers is preserved. This may indicate degradation in archival RNA and cDNA samples, while the level of genomic DNA decreases slightly. Thus, usage of ABL-EAC primers may lead in some circumstances to a significant bias in chimeric transcript evaluation. While primers we offer (ABL-N) are still effective even in the cases with compromised sample quality. Image:Summary/Conclusion: Genomic DNA amplification does not tamper with fusion gene transcripts RT-PCR measurement due to the absence of an appropriate target in the genomic DNA. While primers for measuring housekeeping genes expression (even those widely used) may have these targets and therefore amplify genomic DNA along with cDNA. Here we propose primers for measuring ABL1 control gene expression allowing reliable calculation of chimeric transcripts relative expression, regardless of RNA sample purity, in contrast to primers recommended by the EAC.

Expression of Cytotoxicity, ROS, MMP and Quantitative PCR of Barleria lupulina Lindl. on THP-1 Cells

Barleria lupulina Lindl. (Hop-headed) is a popular medicinal plant that has been used as drug plant since the ancient time. It is a large genus comprising of over 300 species of shrubs, and many of which are known for their ornamental and or medicinal values. The plant is commonly known as Sornomukhi, Hophead Philipine violet, Vishalyakarni, etc. It is native to India and widely distributed in Southern and Western India. The plant is a small shrub, possess potent anti-inflammatory, analgesic, anti-leukemic, antitumor, anti- hyperglycemic, anti-amoebic, virucidal, diuretic, bactericidal and antibiotic properties. Cytotoxicity, bioactive assay and genetic analysis of Barleria lupulina Lindl. were investigated in the present communication. The leaf extract of B. lupulina were investigated by MTT, NRU, DNA fragment, reactive oxygen species generation, mitochondrial membrane potential assay, gene expression analysis and cDNA synthesis to evaluate anti-cancerous potency using cancerous THP-1 cell lines in vitro and in vivo. HPTLC analysis reveals four spots and GC-MS analysis displayed the presence of eleven bioactive compounds among which benzofuranon, hexadecanoic acid, ethyl 9,12,15-octadecatrienoate, and 3,7,11,15-tetramethyl-2-hexadecanoic acid were the most prominent compounds. The ethanolic extract showed significant cytotoxicity (P<0.5) against THP-1 cell line at a concentration of 1mg/ml. The cells were also observed for apoptosis through DNA fragmentation in B. lupulina treated cells. It can be concluded that if the dose range was further refined within the range of 100-1000 μg/ml there could be dose at which the entire population of the THP-1 cell line would be apoptosis induced. The extract induced ROS in the cells after 30 minutes of exposure displaying cytotoxic effects and DNA fragmentation assay. The B. lupulina contain anti-cancerous activity so that it can be used as an alternative drug.

Abstract 437: Characterization of SARS-CoV-2 using the Ion AmpliSeq SARS-CoV-2 research panel

Abstract Background: The rapid spread of COVID-19 has resulted in an urgent need for effective diagnostic and therapeutic strategies against SARS-CoV-2. Next-generation sequencing (NGS) is a powerful tool in the identification and characterization of this pathogen and genomic information may aid in understanding the mechanisms of therapeutic resistance, vaccine escape, virulence, and pathogenicity. The Ion AmpliSeq SARS-CoV-2 Research Panel is a targeted NGS solution that facilitates sequence analysis of the SARS-CoV-2 genome. Paired with a bioinformatics assembly and variant calling pipelines, this assay allows for accurate characterization of the dominant SARS-CoV-2 variant. This assay’s performance was analytically validated for the detection of mutations (substitutions, insertions, and deletions) in RNA derived from nasopharyngeal (NP) swabs. Method: The Ion AmpliSeq SARS-CoV-2 Research panel consists of two primer pair pools generating 237 amplicons specific to the SARS-CoV-2 virus. Reverse transcription of the RNA was performed using the SuperScript VILO cDNA Synthesis kit. Library preparation was then completed using the Ion AmpliSeq Library Kit Plus kit. The final library was quantified, normalized, pooled, and sequenced. Raw sequencing data was aligned to the AmpliSeq SARS-CoV-2 Research panel, using the MN908947.3 reference genome. Variants were called using the Torrent Variant Caller and annotated using the COVID19AnnotateSnpEff plugin. The reference-guided iterative assembler IRMA was used to produce a single consensus sequence consisting of the reference genome sequence modified to include sequence variations supported by the reads. The Pangolin COVID-19 lineage assigner software tool was used to assign SARS-CoV-2 lineage. Analytical validation was completed using controls (Twist Biosciences, BEI Resources, ATCC) and RNA derived from NP swabs. Accuracy and specificity were examined by evaluating the correctness of calling true negative variants compared to false positive and all other variant calls, respectively. Precision and limit of detection (LoD) were examined by evaluating the concordance of variants across replicate samples. Limit of Blank (LoB) was calculated as the 95th percentile of reads per amplicon in the negative samples. Results: Accuracy of base calling, specificity, and precision were 100% for SNVs, insertions, and deletions above 25% allele frequency. LoD was determined to be 576 viral copies/mL. LoB was determined to be 202 reads per amplicon. Pangolin lineage assignment was 100% for all samples. Conclusions: This panel accurately characterizes SARS-CoV-2 variants, allowing for accurate consensus sequence generation, mutation annotation, and lineage assignment. Citation Format: Desiree Unselt, Katherine Knudsen, Christopher Rounds, Janet Doolittle-Hall, Fernando Torres, Jennifer Sims, Jennifer Mason. Characterization of SARS-CoV-2 using the Ion AmpliSeq SARS-CoV-2 research panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 437.

Targeted Virome Sequencing Enhances Unbiased Detection and Genome Assembly of Known and Emerging Viruses-The Example of SARS-CoV-2.

Targeted virome enrichment and sequencing (VirCapSeq-VERT) utilizes a pool of oligos (baits) to enrich all known—up to 2015—vertebrate-infecting viruses, increasing their detection sensitivity. The hybridisation of the baits to the target sequences can be partial, thus enabling the detection and genomic reconstruction of novel pathogens with <40% genetic diversity compared to the strains used for the baits’ design. In this study, we deploy this method in multiplexed mixes of viral extracts, and we assess its performance in the unbiased detection of DNA and RNA viruses after cDNA synthesis. We further assess its efficiency in depleting various background genomic material. Finally, as a proof-of-concept, we explore the potential usage of the method for the characterization of unknown, emerging human viruses, such as SARS-CoV-2, which may not be included in the baits’ panel. We mixed positive samples of equimolar DNA/RNA viral extracts from SARS-CoV-2, coronavirus OC43, cytomegalovirus, influenza A virus H3N2, parvovirus B19, respiratory syncytial virus, adenovirus C and coxsackievirus A16. Targeted virome enrichment was performed on a dsDNA mix, followed by sequencing on the NextSeq500 (Illumina) and the portable MinION sequencer, to evaluate its usability as a point-of-care (PoC) application. Genome mapping assembly was performed using viral reference sequences. The untargeted libraries contained less than 1% of total reads mapped on most viral genomes, while RNA viruses remained undetected. In the targeted libraries, the percentage of viral-mapped reads were substantially increased, allowing full genome assembly in most cases. Targeted virome sequencing can enrich a broad range of viruses, potentially enabling the discovery of emerging viruses.

Ovarian expression of follicle stimulating hormone and activin receptors genes in a prenatally-androgenized rat model of polycystic ovary syndrome in adulthood.

The expression of genes involved in basic pathways, such as folliculogenesis and steroidogenesis may be affected following prenatal androgen exposure. Besides, exposure to androgens during prenatal life plays a central role in developing polycystic ovary syndrome (PCOS) in females in later life. In the present study, we aimed to examine the expression of the follicle stimulating hormone receptor (FSHR) and activin receptor (actR) genes in ovarian granulosa cells (GCs) of a prenatally-androgenized rat model of PCOS in adulthood. In the adult rat model of PCOS and their controls (n = 8 in each group), different phases of the estrous cycle were determined by vaginal smear. Total RNA was extracted from the ovarian GCs using the TRIzol protocol, a reverse transcription kit was used for complementary DNA (cDNA) synthesis, and the expression of FSHR and actR genes was measured by SYBR-Green Real-Time PCR. GraphPad Prism was used for statistical analysis of data, and the t-Student'stestwasused to compare the results between the two groups. PCOS rats had longer and irregular estrous cycles compared to controls. The expression of FSHR and actR genes were significantly decreased in the rat model of PCOS compared to control rats. In PCOS rats, genes expression ratios for FSHR and actR were 0.91 ± 0.11 times (P = 0.008) and 0.42 ± 0.13 times (P = 0.048) less than controls, respectively. Reduced expression of the FSHR and actR genes in ovarian GCs may be one of the mechanisms mediating PCOS-related disorders, especially abnormal ovarian folliculogenesis and ovulation dysfunction, following exposure to androgens during fetal life.

Redundancy of cDNA synthesis in RNA virus direct detection using real-time qPCR

Redundancy of cDNA synthesis in RNA virus direct detection using real-time qPCR

Accelerated Evolution by Diversity-Generating Retroelements.

Diversity-generating retroelements (DGRs) create vast amounts of targeted, functional diversity by facilitating the rapid evolution of ligand-binding protein domains. Thousands of DGRs have been identified in bacteria, archaea, and their respective viruses. They are broadly distributed throughout the microbial world, with enrichment observed in certain taxa and environments. The diversification machinery works through a novel mechanism termed mutagenic retrohoming, whereby nucleotide sequence information is copied from an invariant DNA template repeat (TR) into an RNA intermediate, selectively mutagenized at TR adenines during cDNA synthesis by a DGR-encoded reverse transcriptase, and transferred to a variable repeat (VR) region within a variable-protein gene (54). This unidirectional flow of information leaves TR-DNA sequences unmodified, allowing for repeated rounds of mutagenic retrohoming to optimize variable-protein function. DGR target genes are often modular and can encode one or more of a wide variety of discrete functional domains appended to a diversifiable ligand-binding motif. Bacterial variable proteins often localize to cellsurfaces, although a subset appear to be cytoplasmic, while phage-encoded DGRs commonly diversify tail fiber-associated receptor-binding proteins. Here, we provide a comprehensive review of the mechanism and consequences of accelerated protein evolution by these unique and beneficial genetic elements.

228-OR: Differential Expression of miRNA in Human Subcutaneous and Visceral Adipose Tissue Is Related to Insulin Resistance

Introduction: Obesity is related to insulin resistance (IR) but not all obese individuals are IR. Differences in regional fat distribution and function contribute to the metabolic heterogeneity of obesity. miRNA is known to regulate mRNA and may play a role in mediating IR by modifying adipose tissue function. We tested the hypothesis that miRNA in human visceral (VAT) and subcutaneous (SAT) fat would be differentially expressed in individuals who were classified as insulin sensitive (IS) or IR. Methods: 12 participants, classified as IR or IS (top 40 percentile) via steady-state plasma glucose (SSPG) test, were recruited from the Stanford Bariatric Surgery Clinic. Tissue samples (SAT and VAT) were harvested from the greater momentum during bariatric surgery. Total RNA was extracted with QIAGEN RNeasy® Lipid Tissue Mini Kit. The miRNA experiment used QuantStudio™ 12K Flex OpenArray® microRNA Starter Kit with modifications by adding Ath-miR-159a spike-in, and use of SuperScript VILO cDNA Synthesis Kit. Data analysis done via Applied Biosystems™ Relative Quantitation Analysis. miRNA comparisons with fold-change &amp;lt;0.5 or &amp;gt;2 and p&amp;lt;0.were considered statistically significant. Known target genes of significant miRNA were quantified via RT-PCR from tissue samples. Results: Differential expression in VAT vs. SAT showed one upregulated (miR-885-5p) and 12 downregulated miRNAs. In SAT, for IR vs. IS individuals showed 12 upregulated miRNA, while in VAT, IR vs. IS included one upregulated (miR-30c) and downregulated miRNA. Conclusion: In VAT relative to SAT, miR-885-5p was a significant predictor of fatty liver and majority of downregulated miRNA (miR-335, 196b, 497, 22, 106b, 340) reduced lipogenesis and increased insulin signaling. In IR vs. IS of SAT, majority of miRNA reduced insulin sensitivity (miR-146b, 29c, 502-3p) . For IR vs. IS of VAT, miR-30c promoted adipocyte differentiation and majority of downregulated miRNA increased insulin sensitivity (miR-106b, 24, 143, 340) . Disclosure N.Saini: None. A.Deng: None. H.Chen: None. Y.Xu: None. P.S.Tsao: None. T.Mclaughlin: Board Member; January, Inc., Research Support; Merck &amp; Co., Inc., Novo Nordisk, Stock/Shareholder; Eiger BioPharmaceuticals.

Developing novel liquid biopsy by selective capture of viral RNA on magnetic beads to detect COVID-19.

Objective(s):Early, specific, and sensitive detection methods of COVID-19 are essential for force stopping its worldwide infection. Although CT images of the lung and/or viral RNA extraction followed by real-time reverse-transcriptase-polymerase chain reaction (rRT-PCR) are widely used; they have some limitations. Here, we developed a highly sensitive magnetic bead-based viral RNA extraction assay followed by rRT-PCR. Materials and Methods:Case group included oropharyngeal/nasopharyngeal and blood samples from 30 patients diagnosed positive by PCR test for COVID-19 and control group included 30 same samples from COVID-19 negative PCR test individuals. RNA was extracted, using viral RNA extraction kit as well as using our hand-made capture bead-based technique. A one-step cDNA synthesis and Real Time PCR was conducted. A two-step comparison of the different viral RNA extraction methods for oropharyngeal/nasopharyngeal and blood samples was performed. Student t-test was applied with a P<0.05 considered statistically significant. Results:In the case group, all 30 mucosal samples extracted either with viral RNA extraction kit or with beads-based assay were COVID-19 positive although in the latter category, Cqs were much lower. Although 43% of plasma samples extracted by bead-based method were found to be positive but no plasma samples extracted with column-based kit were detected positive by Real Time PCR. Conclusion:Bead-based RNA extraction method can reduce RNA loss by its single-tube performance and enhance the test sensitivity. It is also more sensitive to lower viral loads as shown in the detection of blood samples and the lower Cqs of mucosal samples.

Prospective quantitative gene expression analysis of kallikrein-related peptidase KLK10 as a diagnostic biomarker for childhood acute lymphoblastic leukemia.

BackgroundThe most common malignancy in children is acute lymphoblastic leukemia (ALL). This study aimed to explore KLK10 mRNA expression as a potential diagnostic biomarker for ALL in children and to examine the effect of chemotherapy on KLK10 mRNA expression following the induction and after three months of receiving chemotherapy.MethodsIn this prospective study, total RNA was extracted from blood samples of 23 pediatric ALL patients on diagnosis, after one month and three months of receiving chemotherapy. Healthy pediatric volunteers (n = 12) were selected as control individuals. After cDNA synthesis, KLK10 mRNA gene expression levels were quantified using quantitative real-time PCR (qRT-PCR).ResultsKLK10 mRNA expression levels were significantly decreased in leukemic cells compared to their levels in cells of normal blood samples (p = 0.0001). KLK10 expression levels in ALL patients after one month and three months of receiving chemotherapy decreased compared to normal blood samples (p < 0.0001 and p = 0.0175 respectively). The expression level of KLK10 mRNA in ALL patients after one month of chemotherapy was decreased compared to their level on diagnosis (p = 0.4413). KLK10 mRNA expression levels in ALL patients after three months of chemotherapy were increased compared to their level on diagnosis (p = 0.0602). The ROC curve illustrated that KLK10 mRNA expression could very efficiently discriminate ALL patients from normal counterparts (AUC=0.886, 95% CI [0.7720–1.000], SE = 0.0582, p = 0.0004).ConclusionKLK10 mRNA expression could serve as a potential diagnostic molecular biomarker for ALL in children.

Investigation of the Toll-like receptors expression profile in peripheral blood mononuclear cells in COVID-19 patients in Iran

Introduction: Coronavirus disease 2019 (COVID-19) spreads all around the world and leads to several new infection cases and mortality. A better understanding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis could lead to more efficient therapeutic approaches. Objectives: The current study aimed to investigate the Toll-like receptors expression profile in peripheral blood mononuclear cells in COVID-19 patients in Iran. Patients and Methods: In this cross-sectional study, we evaluated 32 COVID-19 patients. At the admission time based on the disease severity patients were divided into two groups of severe and mild COVID-19. A group of 16 normal people was evaluated as the healthy control. Blood samples were collected before any treatment from patients at the admission time. Peripheral blood mononuclear cell isolation preformed on blood samples. RNA is extracted from human peripheral blood mononuclear cells and use for the cDNA synthesis. The expression level of TLR-3, 4, and 7 were evaluated by using semi-quantitative real-time polymerase chain reaction (PCR). Results: The patient’s mean age was 57.12±3.08 years and 15 (47%) were male. The background disorders represent two patients in the severe group with respiratory disorders, four patients with cardiovascular disease, and seven patients with diabetes. The TLRs expression levels represent a higher expression of TLR-4 in COVID-19 in comparison with controls for TLR-4 (P=0.007), TLR-3 and 7 also represent up-regulation however there were no statistically significant differences in the relative fold changes (RFCs) between groups for all other evaluated TLRs (P&gt;0.05). Conclusion: The study highlights the importance of the TLR-4 in COVID-19 patients in expression level. Further studies for a clear conclusion about TLRs expression levels are recommended.

Evaluation of a Quantitative Taqman Real-Time PCR Assay to Measure Proviral load from Human Immunodeficiency Virus Type 1 individuals

Human Immunodeficiency Virus (HIV) is a virus belonging to the family Retroviridae. HIV – 1 is found to be predominant in India and many parts of Africa. The intention of this study was to quantify the HIV Proviral Deoxyribonucleic Acid (DNA) from newly infected HIV-1 individuals. Fifty patients who were tested positive for HIV were included in this study. Proviral Ribo Nucleic Acid (RNA) was extracted by QIAmp® RNA Mini Kit (QIAGEN, Germany) method. Complementary Deoxyribo Nucleic Acid (cDNA) was synthesized by using Invitrogen Superscript III cDNA synthesis Kit (USA). This cDNA was subjected to Polymerase Chain Reaction (PCR) and Gene cloning by transformation method. The quantification of Real time PCR was done by Applied Bio-System (ABI)-Prism 7700. A linear standard curve was obtained 10 copies to 106 copies per reaction. The assay had good analytic sensitivity and linear dynamic range greater than 6 logs. From the results obtained in this study, It was concluded that Taqman Real-Time PCR Assay plays a major role in monitoring the HIV infected patients in routine diagnostics and clinical practice.

Taper up-off treatment of opium may reverse some dysregulations of gene expression patterns caused by four type of leukemia, AML, ALL, CML and CLL; a pilot animal model study

Leukemia is a group of lethal diseases characterized by hematological neoplasm with complex and abnormalities in proliferation of bone marrow stem cells, that cause sever loss of functional capacity of hematopoietic tissue. Four most common types of leukemia are included acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML). It has been reported that opium besides of pain killer effects, may have positive effects on cardiovascular disease treatments. Opium may consider as a potential treatment method in cancer. The present study aimed to assess the gene expression pattern alterations in the rat models of four types of Leukemia (ALL, AML, CLL, and CML) under taper up-off treatment method with of opium. Present study was included twelve group, referred to four type of Leukemia (ALL, AML, CLL, and CML) that each type studied in three group. Group one contented normal rats, group two rat model of each type of leukemia with no treatment and group three the rat model of that type of leukemia with 26 days period of opium taper up-off treatment. After the scarification of rats, the bone marrow tissue were extracted. Then, RNA extraction and cDNA synthesis had been conducted on bone marrow tissues. Whole genome expression profiling was conducted by using the Affymetrix GeneChip Array Platform. Microarray results were confirmed by Real time PCR. Microarray analysis detected that hundreds of genes which were involved in Ontogenesis and apoptosis, humoral immune system, tool like receptors, natural killer cells regulators and metabolic pathways and were deregulated in leukemic models, were partially reregulated in treatment groups. Results showed that opium may have positive effects on reregulation of affected gene expression patterns in all types of leukemia. It may suggest that opium taper up-off treatment could be consider as re-regulator of immune system and a novel potential treatment for leukemia with low side effects and high viability.

Increasing the Sensitivity of Leishmania RNA Virus 2 (LRV2) Detection with a Modification in cDNA Synthesis

Leishmania RNA virus was detected the first time in the New World Leishmania species. Recent studies were also showed the presence of Leishmania RNA virus 2 (LRV2) in Old Word Leishmania species including Turkish L. major and L. tropica isolates. This study aimed to increase the sensitivity of qPCR with a modification in the denaturation step of cDNA preparation protocol. In this study, LRV2+ three L. major, two L. tropica strains and L. major control strain (MHOM/SU/73/5-ASKH) were included. Total RNA isolation was done using different numbers of Leishmania promastigotes (108, 105 and 103). Before cDNA synthesis, samples were denatured at 95 °C for 2 min, as a modification of the kit procedure. qPCR was undertaken using 0.5 mM primers (LRV F-HR/LRV R-HR) diluted in SYBR Green Master mix. We observed lower Ct values in amplicons with the modified version than with the classical kit protocol for cDNA synthesis, in all of the strains used in the study. The addition of pre-denaturation step at 95 °C showed lower Ct values meaning the sensitivity increased. Different parasite dilutions showed similar results. It is important to increase the sensitivity especially with the aim for detecting LRV in clinical samples obtained from patients probably have less number of parasites. The presence and burden of the virus can help to understand the relationship between the clinical findings and the pathogenicity of the parasite which may lead to changes in the course of treatment.

Natural Infection of Cichorium intybus (Asteraceae) by Groundnut Ringspot Virus (Genus Orthotospovirus) Isolates in Brazil.

Leaf chicory (Cichorium intybus L.) is a nutritionally rich vegetable used in regional cuisine in Brazil. Plants of C. intybus displaying symptoms (viz. chlorotic and necrotic ringspots, mosaic, and leaf deformation) similar to that induced by orthotospoviruses (genus Orthotospovirus, family Tospoviridae) were observed in three fields (≈ 0.2 ha each) in Gama County, in the Federal District, Brazil, from September 2016 to January 2020 in plants of the cultivars 'Folha-Larga' and 'Spadona' (Fig. 1). Incidence of symptomatic plants was nearly 10% in each field. Transmission electron microscopic examination of thin sections from symptomatic leaf samples showed typical membrane-bounded orthotospovirus particles within cisternae of spongy parenchymal cells (Fig 2). Two individual leaf samples per field were collected and submitted to dot enzyme-linked immunosorbent assay with polyclonal antisera against N protein of tomato spotted wilt virus (TSWV), groundnut ringspot virus (GRSV) and tomato chlorotic spot virus (TCSV). Symptomatic samples strongly reacted only against GRSV antibodies. Total RNA was extracted (Trizol®, Sigma) from all six samples and used as template in RT-PCR assays. The primer J13 (5'-CCCGGATCCAGAGCAAT-3') was employed for cDNA synthesis using M-MLV reverse transcriptase. PCR assays were done with the primer pair BR60/BR65 (Eiras et al., 2001) to obtain ≈ 500 bp fragment of untranslated region and partial N gene in the S RNA segment from each sample. Purified RT-PCR products of two randomly selected individual samples were directly sequenced (GenBank MW467981 and MZ126602) and their BLASTn analyses displayed 99 to 100% nucleotide identity to GRSV isolates previously reported infecting C. endivia L. in Brazil (Jorge et al., 2021). Our analyses combining N protein serology and N-gene sequencing (both directed to the S RNA segment) allowed us to confirm the GRSV infection of C. intybus, but the potential reassortant nature of these isolates (Webster et al., 2015; Silva et al., 2019) are unknown since their M RNA segments were not characterized. Individual leaf extracts (in phosphate buffer, pH 7.0) of the sequenced isolates were mechanically inoculated onto ten seedlings of two C. intybus cultivars ('Folha Larga' and 'Pão-de-Açúcar') and three plants each of the indicator hosts Capsicum chinense PI 159236, Nicandra physalodes; Nicotiana rustica; Datura stramonium; and tomato cv. Santa Clara. Systemic chlorotic and necrotic ringspots, mosaic, and leaf deformation developed in the indicator hosts and infection by GRSV was confirmed via serological assays 20 days after inoculation. However, no symptoms and no serological reaction to GRSV antibodies were observed on the C. intybus cultivars even after two successive mechanical inoculations. This transmission failure might be due to factors such as the requirement of the thrips vector(s), physicochemical barriers in the foliage or the presence of non-mechanically transmissible helper agent(s) necessary to ensure GRSV infection of C. intybus. The natural infection of C. intybus by a not fully characterized orthotospovirus (mostly likely TSWV) has been observed since 1938 in Brazil (Kitajima, 2020). Our report of GRSV infecting C. intybus is thus confirming previous speculations that similar symptoms in this vegetable crop were induced by orthotospovirus infection in Brazil.

MiR-203a-3p, ABL1 and TP63 gene expression is altered in the endometrium of women with endometriosis

Objective Many genes and miRNAs have been shown to be associated with the pathogenesis of endometriosis. TP63 (p63) is implicated in lineage specification, proliferative potential, differentiation, cell death and survival. The ABL1 proto-oncogene encodes a cytoplasmic and nuclear protein tyrosine kinase implicated in cell differentiation, cell division, and cell adhesion. Moreover, hsa-miR-203a-3p was reported to play pivotal roles in tumor progression by targeting multiple genes, including ABL1 and TP63. The aim of this study was to investigate the expression of ABL1, TP63, and miR-203a-3p in endometriosis. Methods This study included 30 women with endometriosis (stage III: n = 12 and stage IV: n = 18) and 30 age-matched controls. Total RNA extraction and cDNA synthesis were performed, and a quantitative polymerase chain reaction technique was used to determine the expression of miR-203a-3p, TP63, and ABL1. Results TP63 and ABL1 were significantly overexpressed in stages III and IV endometriosis as compared to controls (p < .0001). Moreover, overexpression of ABL1 and TP63 was observed in stage IV compared to stage III (p = .0006 and p = .0002, respectively). Furthermore, significant increase miR-203a-3p expression has been seen in stage IV endometriosis compared to controls (p = .006). The expression of miR-203a-3p in stage III was not significant compared to stage IV and control (p = .33 and p = .43, respectively). Conclusion It is concluded that miR-203a-3p, ABL1 and TP63 gene expression is altered in the endometrium of patients with endometriosis. It is also suggested that miR-203a-3p, ABL1, and TP63 might be candidate factors for the pathogenesis of endometriosis and suggesting its therapeutic potential in endometriosis.