Total Ribonucleic Acid Research Articles

Total RNA Extraction from Rice Vegetative Tissues Using Magnetic Beads.

Ribonucleic acid (RNA) extraction is the first critical step in gene expression analysis. In this chapter, we describe a high-throughput RNA extraction method using guanidine thiocyanate and isopropyl alcohol (HighGI). The use of carboxyl-coated paramagnetic beads, instead of silica membrane columns, enables semi-automation using a liquid handling system and high-throughput RNA extraction for large-scale transcriptome studies. Homemade mixes of paramagnetic beads and buffers make HighGI inexpensive. In addition, HighGI-extracted RNA retains low molecular weight RNA molecules less than 200bp, which is typically lost in commercial column-based kits.

Comparative analysis of high-throughput RNA extraction kits in Naïve Non-Human Primate (NHP) tissues for downstream applications utilizing Xeno Internal Positive Control (IPC)

Ribonucleic acid (RNA) extraction and purification play pivotal roles in molecular biology and cell and gene therapy, where the quality and integrity of RNA are critical for downstream applications. Automated high-throughput systems have gained interest due to their potential for scalability and reduced labor requirements compared to manual methods. However, ensuring high-throughput capabilities, reproducibility, and reliability while maintaining RNA yield and purity remains challenging.This study evaluated and compared the performance of four commercially available high-throughput magnetic bead-based RNA extraction kits across six types of naïve non-human primate (NHP) tissue matrices: brain, heart, kidney, liver, lung, and spleen. The assessment focused on RNA purity, yield, and extraction efficiency (EE) using Xeno Internal Positive Control (IPC) spiking.Samples (∼50 mg) were homogenized via bead-beating and processed according to the manufacturer's protocol on the KingFisher Flex platform in eight replicates. RNA purity and yield were measured using a NanoDrop® spectrophotometer, while EE was evaluated via real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR).The findings indicate consistent high RNA purity across all tested extraction kits, yet substantial variation in RNA yield. Extraction efficiency exhibited variations across tissue types, with decreasing trends observed from brain to lung tissues. These results underscore the importance of careful kit selection and method optimization for achieving reliable downstream applications. The MagMAX™ mirVana™ Total RNA Isolation Kit stands out as the most accurate and reproducible, making it the preferred choice for applications requiring high RNA quality and consistency. Other kits, such as the Maxwell® HT simplyRNA Kit, offer a good balance between cost and performance, though with some trade-offs in precision. These findings highlight the importance of selecting the appropriate RNA isolation method based on the specific needs of the research, underscoring the critical role of accurate nucleic acid extraction in gene and cell therapy research.In conclusion, this study highlights the critical factors influencing RNA extraction performance, emphasizing the need for researchers and practitioners to consider both kit performance and tissue characteristics when designing experimental protocols. These insights contribute to the ongoing efforts to enhance the reproducibility and reliability of RNA extraction methods in molecular biology and cell/gene therapy applications.

Initial Evaluation of lncRNA A2M-AS1 Gene Expression in Multiple Sclerosis Patients

Background: Multiple sclerosis (MS) is one of the three leading neurodegenerative diseases worldwide. Gene expression profile studies play an important role in recognizing and preventing disease. Considering the inherent ability of biomarkers to diagnose and prognose the occurrence of a disease, with the aim of gene therapy and changing gene expression, it can be helped to treat it. In this study, by examining the gene interaction and expression of non-coding genes in patients with MS, using bioinformatics analyses, laboratory research and potential non-coding diagnostic biomarkers of MS were selected for further investigations. Materials and Methods: First, by using micro-array data analysis of the GEO database, the expression status of the long non-coding ribonucleic acid (RNA) (lncRNA) A2M-AS1 gene was investigated in patients with MS. lncRNA–mRNA interaction analysis was performed in the lncRRisearch database. After sample collection, the total RNA extracted using the RNA extraction kit from 20 patient samples and 20 healthy samples was synthesized into cDNA with the synthesis kit. The quantitative reverse transcriptase polymerase chain reaction experiment was performed for the final validation of expression change. Results: Based on bioinformatic and laboratory analysis, the expression of the A2M-AS1 gene in MS samples showed a significant decrease in expression compared to healthy samples. Also, based on the receiver operating characteristic analysis, lncRNA A2M-AS1 can be introduced as an acceptable diagnostic biomarker to distinguish MS samples from healthy samples. Conclusion: lncRNA A2M-AS1, by reducing its expression as an acceptable diagnostic biomarker, can increase the risk of developing MS.

Correlation between Fatty Acid Transport Proteins and Phosphoinositide 3-Kinase Pathway in Breast Cancer

Abstract Background: Breast cancer (BC) is currently the fifth largest cause of mortality worldwide and has become the most frequent type of cancer. Fatty acid transport proteins (FATPs) assist cancer cells in meeting their higher metabolic needs by increasing fatty acid uptake, which is a significant source of energy for cancer cells. The phosphoinositide 3-kinase/protein kinase B/mammalian target of the rapamycin (PI3K/Akt/mTOR) pathway is an important signaling pathway that coordinates the uptake and utilization of various nutrients, including fatty acids. The current study aimed to correlate the FATPs with the PI3K/Akt/mTOR pathway in BC. Methods: Eighty serum samples were collected from BC and control subjects after obtaining an informed consent form. Total ribonucleic acid (RNA) was isolated, and the relative messenger RNA (mRNA) expression of PI3K, Akt, and mTOR was analyzed by a reverse transcriptase-quantitative polymerase chain reaction. Serum FATPs were estimated using commercially available enzyme-linked immunoassay kits. P < 0.05 was indicated as statistically significant. Results: The serum FATPs in subjects with BC differed significantly compared to the control. Relative mRNA expression of PI3K, Akt, and mTOR differed significantly between the groups. Further, on correlating the serum FATPs with PI3K-related signaling molecules showed a significant positive correlation within BC subjects. Conclusion: Significant positive correlation between FATPs and the PI3K, Akt, and mTOR pathway suggests a crucial role of FATPs in promoting BC.

Mefloquine-curcumin combinations improve host mitochondrial respiration and decrease mitotoxic effects of mefloquine in Plasmodium berghei-infected mice

Plasmodium infection is a health challenge. Although, antiplasmodial drugs kill the parasites, information on the effects of infection and drugs on the expression of some genes is limited.Malaria was induced in two different studies using NK65 (chloroquine-susceptible, study 1), and ANKA (chloroquine-resistant, study 2) strains of Plasmodium berghei in 30 male Swiss mice (n = 5) in each study. Mice orally received 10 mL/kg distilled water, (infected control), Mefloquine (MF) (10 mg/kg), MF and Curcumin (CM) (25 mg/kg), MF and CM (50 mg/kg), CM (25 mg/kg) and CM (50 mg/kg). Five mice (un-infected) were used as the control. After treatment, total Ribonucleic acid (RNA) was isolated from liver and erythrocytes while Deoxyribonucleic acid (DNA)-free RNA were converted to cDNA. Polymerase Chain Reaction (PCR) amplification was performed and relative expressions of FIKK12, AQP3, P38 MAPK, NADH oxidoreductase, and cytochrome oxidase expressions were determined. Markers of glycolysis, toxicity and antioxidants were determined using ELISA assays. While the expression of FIKK12 was blunted by MF in the susceptible study, co-treatment with curcumin (25 mg/kg) yielded the same results in the chloroquine-resistant study. Similar results were obtained on AQP3 in both studies. Curcumin decreased P38 MAPK in both studies. Plasmodium infection decreased NADH oxidoreductase and cytochrome oxidase but mefloquine-curcumin restored the expression of these genes. While glycolysis and toxicity were inhibited, antioxidant systems improved in the treated groups. Curcumin is needed for effective therapeutic efficacy and prevention of toxicity. Plasmodium infection and treatment modulate the expressions of some genes in the host. Curcumin combination with mefloquine modulates the expression of some genes in the host.

Chitosan bioactive glass scaffolds for in vivo subcutaneous implantation, toxicity assessment, and diabetic wound healing upon animal model

This study aims to develop chitosan-bioactive glass (BG) scaffolds for diabetic wound healing, toxicity valuation, and subcutaneous implantation in animals for biocompatibility assessment. The scaffolds were prepared by lyophilization technique. In specific BG without sodium (Na), composited with chitosan for better biological activities. The equipped scaffolds were studied for their physiochemical, biological, in vitro and in vivo performances. The chitosan and chitosan-BG (Na free) scaffolds show reliable biocompatibility, cytocompatibility, anti-oxidant, and tissue regeneration. The biocompatibility, toxicity assessments, and diabetic skin wound healing experiments were examined through in vivo studies using Sprague Dawley rats. The extracted tissue samples were analyzed using hematoxylin-eosin- (H and E) and Masson's trichrome staining. Further, tissue excised after scaffold implantation declared non-toxic, non-allergic, and anti-inflammatory nature of chitosan scaffolds. Moreover, the total ribonucleic acid (RNA) expression levels were measured using reverse transcription-polymerase chain reaction (RT-PCR) for the scaffolds against vascular endothelial growth factor (VEGF), and collagen type one (Col-1) primers. Admirably, the scaffolds achieved the best level of skin wound healing via tissue regeneration by increasing epithetical cell formation and collagen deposition. Thus, the biocompatibility, non-toxicity, anti-inflammatory, and wound healing efficiency proved that the chitosan-BG (Na free) scaffold can be readily substantial for wound healing.

Effect of endometrial biopsy method on ribonucleic acid quality and gene expression analysis in patients with leiomyoma

To compare ribonucleic acid (RNA) quantity and purity in tissue collected with different endometrial sampling methods to establish the optimal tool for use in endometrial gene expression studies. Observational study. University hospital. Fourteen patients with submucosal leiomyomas. Unguided biopsies were obtained using a low-pressure suction device before hysteroscopy from 14 patients with submucosal leiomyomas followed by guided biopsy with a resectoscope loop. Fifty-seven samples were collected: 25 obtained using a suction device and 32 with a loop. Total biopsy weight, RNA purity, and RNA yield for each collection method. After complementary deoxyribonucleic acid synthesis, HOXA10 expression was measured by quantitative polymerase chain reaction in the endometrium overlying and remote from the leiomyoma, as similar expression throughout the cavity was a prerequisite for the use of unguided biopsy method. The median weight of the samples was significantly larger when obtained with the low-pressure suction device than with the resectoscope loop (153 vs. 20 mg). The RNA yield was similar (suction curette, 1,625 ng/mg; resectoscope loop, 1,779 ng/mg). The A260-to-A280 ratio was satisfactory for 94.7 % of the samples, with no difference between the groups. The endometrial expression of HOXA10 was similar in areas overlying the leiomyoma compared with that in remote endometrial sites (2-ΔCt = 0.0224 vs. 0.0225). Low-pressure endometrial suction devices provide tissue samples with acceptable RNA purity and quantity for gene expression studies. The expression of HOXA10 did not differ between endometrial sampling sites even in the presence of leiomyomas.

EXTH-11. ADOPTIVE CELLULAR THERAPY OVERCOMES GLIOMA IMMUNE ESCAPE BY DISRUPTING MDSC PROLIFERATION AND RECRUITMENT

Abstract INTRODUCTION Our group has previously shown that adoptive cellular therapy (ACT) significantly extends survival in models of medulloblastoma, brain stem glioma, and glioblastoma. Further, ACT significantly decreases myeloid-derived suppressor cells (MDSC) in the tumor microenvironment (TME), suggesting that ACT may ameliorate glioma-mediated immunosuppression. We hypothesize that ACT-mediated reduction of MDSCs is driven by cell proliferation and recruitment mechanisms, but these mechanisms have not been defined. In this study, we quantified the proportions and cell cycle states of MDSCs in the TME and peripheral lymphoid tissues as well as chemokines in serum to determine how ACT affects MDSC recruitment. METHODS ACT was performed in C57BL/6J mice bearing a syngeneic high-grade glioma derived from KR158B-luciferase cell line. The treatment platform included total body irradiation, hematopoietic stem cell rescue, total tumor ribonucleic acid (ttRNA) electroporated dendritic cells, and tumor-reactive T cells. Following ACT completion, we evaluated the proportions and cell cycle states of MDSCs using flow cytometry. Bromodeoxyuridine (BRDU) and 7-aminoactinomycin (7-AAD) defined cell cycle phases. Serum chemokines were measured using a membrane-based sandwich immunoassay. RESULTS We replicated our previous findings that ACT reduces the level of MDSCs in the TME. Additionally, ACT significantly decreased the proportion of MDSCs in S-phase in the TME. Conversely, levels of MDSCs in peripheral lymphoid tissue were significantly increased in mice treated with ACT, with a significant increase of MDSCs in S-phase. Analysis of serum chemokines revealed significant decreases in multiple chemokines implicated in MDSC recruitment such as CXCL13 and CXCL16. CONCLUSION These findings suggest that ACT decreases proliferation and chemotactic recruitment of MDSCs to the TME, which may also explain MDSC accumulation peripheral lymphoid tissue in ACT-treated mice. These immune mechanisms likely contribute to ACT’s efficacy against brain cancer, preventing tumor immune escape, and offer new therapeutic targets for optimizing cellular immunotherapies.

RNA-seq analysis of human trabecular endothelial cells after treatment with timolol maleate.

Timolol maleate (timolol), a β-receptor blocker, reduces intraocular pressure by decreasing aqueous humor production. Timolol reportedly also protects ganglion cells, decreases aqueous humor outflow facility, and destroys the extracellular matrix in the trabecular meshwork. In this study, we investigated the effects of timolol on cultured human trabecular endothelial cells purchased from ScienCell using next-generation sequencing. Experimental investigation. Total ribonucleic acid (RNA) was extracted after 24 h. More than 100 million RNAs in control and timolol-treated group were sequenced using a next-generation sequencer. The expression of 55,778 RNAs was analyzed. A total of 2,105 genes were significantly upregulated and 2,125 genes were downregulated, after the addition of timolol. VGF nerve growth factor inducible (VGF) (388-fold) had the maximum increase in expression, followed by amphiregulin (333-fold), a member of the epidermal growth factor family. Moreover, the expression of extracellular matrix-degrading enzymes, matrix metalloproteinases (MMPs) 1, 2, 3, 10, 12, and 14, increased. Timolol exerts various effects on human trabecular endothelial cells. The increase in MMP expression may contribute to the decrease in the aqueous humor outflow facility.

Precise isolation of high-quality RNA from leaves and storage roots of cassava (Manihot esculenta Crantz) for gene expression studies

BackgroundThe isolation of Ribonucleic Acid (RNA) from leaves and storage roots of cassava (Manihot esculenta Crantz) is a challenging one, due to the presence of large amounts of polyphenolic compounds, polysaccharides, and tuber proteins. RNA with high quality and intact integrity is vital for gene expression studies. We hereby report a precise, reproducible, and less cumbersome technique for isolating high-quality RNA from leaves and storage roots of cassava with minimal contamination from polyphenols, polysaccharides, and other secondary metabolites, using affordable reagents. This protocol functions without guanidinium salts in the extraction buffer. The presence of guanidinium salts usually leads to the formation of agglomerates during the extraction of RNA from plant tissues with high starch contents.ResultsThe isolated RNA from leaves and storage roots of the ten cassava genotypes yielded between 1576.1 and 2861.9 µg/ml for RNA isolated from the leaf tissues and 2761.2–3873.5 µg/ml for RNA isolated from the storage roots. The A260:A280 ratios of the total RNA were more than 2.0 for both leaf and storage root samples, indicating minimal contamination from polysaccharides and polyphenols. The RNA samples recorded intact integrity, as demonstrated by clear 28 S and 18 S rRNA bands observed on agarose gel electrophoresis. The RNA integrity number (RIN) values ranged between 7.2 and 8.0. Also, the RNA samples were successfully used for transcriptome sequencing.ConclusionThe present method which yielded high-quality and transcriptionally competent RNA samples is suitable for use in gene expression studies and downstream applications in the molecular breeding of cassava and related root/tuber crops.

IL-18 receptor-α signalling pathway contributes to autoantibody-induced arthritis via neutrophil recruitment and mast cell activation.

The interleukin (IL)-18 signalling pathway is involved in animal models of collagen-induced arthritis, but the role of this pathway in autoantibody-induced arthritis is poorly understood. An autoantibody-induced arthritis model, K/BxN serum transfer arthritis, reflects the effector phase of arthritis and is important in innate immunity including neutrophils and mast cells. This study aimed to investigate the role of the IL-18 signalling pathway in autoantibody-induced arthritis using IL-18 receptor (IL-18R) α-deficient mice. K/BxN serum transfer arthritis was induced in IL-18Rα-/- and wild-type B6 (controls) mice. The severity of arthritis was graded, and histological and immunohistochemical examinations were performed on paraffin-embedded ankle sections. Total Ribonucleic acid (RNA) isolated from mouse ankle joints was analysed by real-time reverse transcriptase-polymerase chain reaction. IL-18 Rα-/- mice had significantly lower arthritis clinical scores, neutrophil infiltration, and numbers of activated, degranulated mast cells in the arthritic synovium than in controls. IL-1β, which is indispensable for the progression of arthritis, was significantly downregulated in inflamed ankle tissue in IL-18 Rα-/- mice. IL-18/IL-18Rα signalling contributes to the development of autoantibody-induced arthritis by enhancing synovial tissue expression of IL-1β and inducing neutrophil recruitment and mast cell activation. Therefore, inhibition of the IL-18Rα signalling pathway might be a new therapeutic strategy for rheumatoid arthritis.

Promotion of the resistance of human oral epithelial cells to herpes simplex virus type I infection via N6-methyladenosine modification

ObjectiveThis study aimed to explore the mechanism behind N6-methyladenosine (m6A) modification of the total ribonucleic acid (RNA) involved in the resistance to herpes simplex virus type I (HSV-1) infection in oral epithelial cells.MethodThe variation in m6A modification level on messenger RNA following HSV-1 infection was determined using the RNA dot blot method. The expression levels of alpha-ketoglutarate-dependent dioxygenase lab homolog 5 (ALKBH5) protein and fatty mass and obesity-associated genes (FTO) were determined using real-time fluorescence quantification polymerase chain reaction and the western blot technique, respectively. Next, after suppressing the expression of ALKBH5 or FTO via small interfering RNA, human immortalised oral epithelial cells (HIOECs) were infected with HSV-1, followed by measurement of the viral load or expression level of type I interferon (I-IFN) and interferon-stimulated genes (ISGs).ResultsThe m6A modification level was significantly increased following HSV-1 infection of the HIOECs (P < 0.05), while the expression of ALKBH5 and FTO genes was reduced (P < 0.01). Moreover, the suppression of ALKBH5 or FTO increased the production of I-IFN and ISGs during the HSV-1 infection of the HIOECs (P < 0.01), and the viral load was significantly reduced (P < 0.01).ConclusionDuring oral HSV-1 infection, the m6A level was increased through the down-regulation of ALBHK5 and FTO expression, increasing I-IFN production and the promotion of HSV-1 clearing in HIOECs.

Sequence analysis of SARS-CoV-2 Delta variant isolated from Makassar, South Sulawesi, Indonesia

IntroductionThis study aimed to perform mutation and phylogenetic analyses of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Delta variants and analyze the characteristic signs and symptoms of patients infected with SARS-CoV-2 Delta variant originated from Makassar during the Delta outbreak.Methods: We collected samples from patients who were infected with coronavirus disease 2019 (COVID-19) between June and October 2021. We selected the Quantitative Reverse Transcription-Polymerase Chain Reaction (PCR)-positive samples with a cycle threshold value of <30 for whole genome sequencing. Total viral ribonucleic acid (RNA) was isolated from 34 PCR-positive nasopharyngeal swab samples, and whole genome sequencing was performed using the Oxford Nanopore GridlON sequencer. Phylogenetic and maximum clade credibility analyses were performed using the Bayesian Markov chain Monte Carlo method. ResultsIt was found that 33 patients were infected with the SARS-CoV-2 Delta variant in this cohort study, among whom 63.6% (21) patients were female. According to the clinical data, 24 (72.7%), 7 (21.2%), and 2 (6.1%) patients had mild, moderate, and severe COVID-19 infections. Phylogenetic analysis based on the spike and RNA-dependent RNA polymerase (RdRp) genes showed that the collected samples were clustered in the main lineage of B.1.617.2 (Delta variant). The Delta variants had a high frequency of distinct mutations in the spike protein region, including T19R (94.12%), L452R (88.23%), T478K (91.17%), D614G (97%), P681R (97%), and D950 N (97%). Other unique mutations found in a smaller frequency in our samples were present in the N-terminal domain, including A27T (2.94%) and A222V (14.70%), and in the receptor-binding domain, including Q414K (5.88%), G446V (2.94%), and T470 N (2.94%). ConclusionThis study revealed the unique mutations in the S protein region of Delta variants. T19R, L452R, T478K/T478R, D614G, P681R, and D950 N were the most common substitutions in Makassar's Delta variant.

Immune Biomarkers in Blood from Sarcoma Patients: A Pilot Study.

The main role of the host immune system is to identify and eliminate cancer cells, which is a complex process, but it is not a fail-safe mechanism. Many sarcoma patients succumb to this disease despite treatments rendered. The aim of this pilot study was to compare the levels of CD4+ T-cells, T-regulatory (Treg) cells, and cytokines such as tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-17A (IL-17A), and transforming growth factor-beta-1 (TGF-β1) in peripheral blood leukocytes of sarcoma patients and healthy controls. For gene expression studies, total ribonucleic acid (RNA) was extracted from peripheral blood leukocytes and genes that were differentially regulated in peripheral blood leukocytes of sarcoma patients compared with healthy controls were determined using a commercial T-helper cell differentiation quantitative polymerase chain reaction (qPCR) array. Flow cytometer analysis was performed on blood samples from 26 sarcoma patients and 10 healthy controls to identify the levels of CD4+ T-cells and T-reg cells. The level of cytokines in plasma and culture supernatant were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits. A marked reduction in the percentage of CD4+ T-cells (p = 0.037) and levels of TNF-α (p = 0.004) and IFN-γ (0.010) was observed in sarcoma patients. Gene expression analysis showed five genes (homeobox A10 (HOXA10), GATA binding protein 3 (GATA3), prostaglandin D2 receptor 2 (PTGDR2), thymocyte selection associated high mobility group box (TOX), and C-C motif chemokine receptor 3 (CCR3)) were dysregulated (p < 0.05) in sarcoma patients. This study suggests that T-helper-1 immune responses are reduced in sarcoma patients.

Relation between Serological Findings and Expression of IL-1β and IL-6 genes in Patients Infected with Mycobacterium Tuberculosis in Iraq.

Mycobacterium Tuberculosis (TB) is one of the serious bacterial infections that cause diseases and may lead to death. In this study, 178 individuals were examined for TB infection at Baghdad TB center during the period from 15th January to 1st October 2021. Out of 178 participants, 73 were shown to be positive for TB infection, while 105 showed negative results. According to the results, there was no significant variation between infected males and females with TB in comparison to the control group (P>0.05). The results showed that the mean age of the patients for both males and females was in the range of 2-65 years. Additionally, there were significant differences in patients with TB compared to the control group in terms of the weight loss of 8.82 ± 6.75 Kg, red blood cell (RBC) count (3.43 ± 0.56) × 106/μl, white blood cell (WBC) count (3.12 ± 1.57) × 106/μl, platelet count (1.03 ± 0.56) × 106/μl, and hemoglobin level (6.66 ± 1.34) g/dl. A total of 30 TB patients and 50 normal individuals were genotyped to detect the IL-1β rs 114534 gene. The polymerase chain reaction (PCR) was used for exon amplification in region 5 of the ILB1 gene in the TB patients by using specific primers. The finding showed that there was an amplified product of 249bp located in chromosome 2q13-14. A total of 30 TB patients and 50 normal individuals were also genotyped to detect the IL-6 rs 1800795 gene. The PCR was used for amplification of the IL-6 gene in TB patients by using specific primers. The finding showed that there was an amplified product of 431 bp located in chromosome 7p15-p2. The expression of the ILB1 gene was investigated in TB patients and healthy controls by using qPT-PCR. Results showed that there was a high Ct value for patients and controls with a high Ct value of templates, preoperational to the total ribonucleic acid (RNA) concentration and gene expression. The expression of the IL-6 gene was investigated in TB patients and healthy controls by using qPT-PCR. Our findings revealed a high Ct value for patients and controls with a high Ct value of templates, preoperational to the total RNA concentration and gene expression.

SARS-CoV-2 transmission from infected owner to household dogs and cats is associated with food sharing

ObjectivesSeveral cases of reverse transmission of SARS-CoV-2 from human to pets were reported during the first year of the COVID-19 pandemic. Accordingly, the World Organization for Animal Health has recommended to improve SARS-CoV-2 surveillance on household animals to assess the risk of transmission between species. After such recommendation, we studied the potential SARS-CoV-2 infection in household dogs and cats in the city of Guayaquil, the most populated city in Ecuador. MethodsOral and nasal swab samples were collected from dogs and cats within 10 days of a positive SARS-CoV-2 test result of their owners. Total ribonucleic acid was extracted and detection of viral gene targets N and ORF1ab was performed by quantitative reverse transcription polymerase chain reaction. ResultsFrom the 50 cats and dogs tested, 12 were SARS-CoV-2 positive, giving a total positivity rate of 24%. A total of 1 of 8 cats tested positive, whereas 11 of 42 dogs were positive, yielding a positivity rate of 12.5% and 26.2%, respectively. SARS-CoV-2 was confirmed by whole genome sequencing. In addition, we also found a statistically significant association between SARS-CoV-2 pet positivity and food sharing with infected owners. ConclusionThis study is the second active surveillance of SARS-CoV-2 in household dogs and cats in Latin America. Moreover, it is the first study to address the risk factors associated with potential anthropogenic SARS-CoV-2 transmission to domestic cats and dogs. Given the high presence of free-roaming dogs and cats in rural and urban areas in Latin American countries and the high capacity shown by coronaviruses for interspecies transmission, our findings support the view that SARS-CoV-2 surveillance in pets is necessary to better understand the role that pet-human interaction plays in the COVID-19 spread.

Analysis of HIV-1 genetic subtype and pretreatment drug resistance among men who have sex with men infected with HIV-1 from 19 cities of 6 provinces in China

Objective: To investigate the distribution of HIV-1 genetic subtypes and pretreatment drug resistance (PDR) among men who have sex with men (MSM) from 19 cities of 6 provinces in China. Methods: From April to November 2019, 574 plasma samples of ART-naive HIV-1 infected MSM were collected from 19 cities in Hebei, Shandong, Jiangsu, Zhejiang, Fujian, and Guangdong provinces, total ribonucleic acid (RNA) was extracted and amplified the HIV-1 pol gene region by nested polymerase chain reaction (PCR) after reverse transcription. Then sequences were used to construct a phylogenetic tree to determine genetic subtypes and submitted to the Stanford drug resistance database for drug resistance analysis. Results: A total of 479 samples were successfully amplified by PCR. The HIV-1 genetic subtypes included CRF01_AE, CRF07_BC, B, CRF55_01B, CRF59_01B, CRF65_cpx, CRF103_01B, CRF67_01B, CRF68_01B and unrecognized subtype, which accounted for 43.4%, 36.3%, 6.3%, 5.9%, 0.8%, 0.8%, 0.4%, 0.4%, 0.2% and 5.5%, respectively. The distribution of genetic subtypes among provinces is statistically different (χ2=44.141, P<0.001). The overall PDR rate was 4.6% (22/479), the drug resistance rate of non-nucleoside reverse transcriptase inhibitors, nucleoside reverse transcriptase inhibitors, and protease inhibitors were 3.5% (17/479), 0.8% (4/479) and 0.2% (1/479), respectively. The PDR rate of recent infections was significantly higher than that of long-term infections (χ2=4.634, P=0.031). Conclusions: The HIV-1 genetic subtypes among MSM infected with HIV-1 from 19 cities of 6 provinces in China are diverse, and the distribution of subtypes is different among provinces. The overall PDR rate is low, while the PDR rate of recent infections was significantly higher than that of long-term infections, suggesting the surveillance of PDR in recent infections should be strengthened.

Association between forkhead box P3 expression level and gender of Iranian juvenile idiopathic arthritis patients

ObjectivesJuvenile idiopathic arthritis (JIA) is a childhood autoimmune rheumatoid disease. Past studies have confirmed that JIA is a complex disease, which means that genes and environmental factors affect the aetiology of the disease. In this study, we analysed the expression of interleukin 32, forkhead box P3 (FOXP3), methyl-CpG binding domain protein 1 (MBD1), and methyl-CpG-binding protein 2 (MECP2) in peripheral blood mononuclear cells of children with JIA in comparison with the expression of those in healthy children. Interleukin 32 is an inflammatory factor, FOXP3 is a transcription factor, and MBD1 and MECP2 are binding proteins that bind to the methylated deoxyribonucleic acid (DNA).Material and methodsWe collected blood from JIA patients who had been diagnosed and classified into clinical subtypes by a rheumatologist from the division of paediatric rheumatology. Healthy children, whose clinical and preclinical analysis confirmed they had no disease and just came to the hospital for a check-up or minor surgical procedures were considered as a control group. Age and gender were matched in patients and the control group. Total ribonucleic acid was extracted from blood, and cDNA was synthesized. Eventually, the transcript levels were analysed by quantitative polymerase chain reaction, and statistical analysis was carried out.ResultsStatistical analysis of gene expressions in young females affected by JIA demonstrated that MECP2 and FOXP3 were increased significantly (p-value = 0.002 and 0.05, respectively). Interleukin 32 gene expression was also increased (p-value = 0.14), whereas MBD1 gene expression was decreased (p-value = 0.06); however, these changes in the expression of all 4 genes were not significant in young males.ConclusionsDifferent expression levels of the mentioned genes between affected young females and males result from hormones in both gender and also methotrexate (MTX) drug. Also, the reason affected young females are more prone to JIA than males can be the lower level of FOXP3 expression in healthy females than healthy males.

Investigating the Production of Antimicrobial Nanoparticles by Chlorella vulgaris and the Link to Its Loss of Viability.

Chlorella vulgaris from Al-Ahsa, KSA was proved to be an active silver and gold nanoparticle producer. Nanogold and nanosilver particles were characterized using UV-visible spectroscopy, Fourier-transform infrared spectroscopy, and scanning electronmicroscopy. Both nanoparticles were used in the antimicrobial bioassay. The two nanoparticles showed antibacterial activities, with the silver nanoparticles being the most effective. To investigate the argumentative nature of their biosynthesis (i.e., whether it is a biotic or abiotic process), we isolated total ribonucleic acid RNA as an indicator of vitality. RNA was completely absent in samples taken after one week of incubation with silver nitrate and even after one or two days. However, successful extraction was only achievable in samples taken after incubation for one and four hours with silver nitrate. Most importantly, the gel image showed recognizable shearing of the nucleic acid after 4 h as compared to the control. An assumption can be drawn that the synthesis of nanoparticles may start biotically by the action of enzyme(s) and abiotically by action of reducing entities. Nonetheless, with prolonged incubation, excessive nanoparticle accumulation can be deadly. Hence, their synthesis continues abiotically. From the RNA banding profile, we suggest that nanosilver production starts both biotically and abiotically in the first few hours of incubation and then continues abiotically. Nanosilver particles proved to have more of an antimicrobial impact than nanogold and hence are recommended for different applications as antibacterial agents.

Lemongrass essential oil and its major constituent citral isomers modulate adipogenic gene expression in 3T3-L1 cells.

Obesity is a predisposing factor to diseases such as diabetes mellitus, hypertension, and coronary artery disease. Lemongrass essential oil (LEO), from Cymbopogon flexuosus, possesses numerous therapeutic properties including modulation of obesity in vivo. This experiment investigated the effect of LEO and its major components citral (3,7-dimethyl-2,6-octadienal), citral dimethyl acetal (1,1-dimethoxy-3,7-dimethylocta-2,6-diene), and citral diethyl acetal (1,1-diethoxy-3,7-dimethylocta-2,6-diene) in modulation of adipogenesis and genetic expression in adipocytes. Adipogenesis was induced from murine 3T3-L1 preadipocytes procured from ATCC and maintained in Dulbecco's modified Eagle's medium (DMEM) enriched with calf serum. Differentiation was conducted using DMEM enriched with 10% fetal bovine serum, Dexamethasone 0.25µM, 3-isobutyl-methylxanthine 0.5mM, and insulin 10mg/ml for 2days, followed by 5days of insulin 10mg/ml alone. Samples were subjected to experimental treatments at a concentration of 2.5×10-3 . Intracellular triglycerides were quantified and photomicrographs were obtained following Oil red O (ORO) staining procedure. Total ribonucleic acid was extracted and expression of genes effecting in lipid metabolism were quantitated using real-time polymerase chain reaction. ORO staining procedure and spectrophotometric analysis demonstrated decreased lipid accumulation following treatments. LEO and its major constituents significantly inhibited expression of sterol response binding protein 2, cluster of differentiation 36, fatty acid binding protein 4, and peripilin. These results indicate modulation of lipid accumulation through decreased lipid uptake, increased lipolysis, decreased differentiation, and downregulated lipid biosynthesis. This investigation suggests that LEO and its constituents exert effects on adipocyte metabolism and are important for understanding metabolic disease. Further investigation is required to elucidate the degree that each mechanism implicated contributes to the observed effect.