Real-time Quantitative Reverse Transcriptase-polymerase Research Articles

Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

BackgroundCoronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient.MethodIn this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients.ResultThe limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively.ConclusionIn conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

Expression of microRNA-181a and microRNA-196b in Egyptian Pediatric acute Lymphoblastic Leukemia.

Background:Differential expression of miRNA provides important insights into pathogenesis of cancer including leukemia. Deregulation of microRNA may contribute to hematopoietic malignancies. In this study, we aimed to evaluate the role of miR-181a and miR-196b in acute lymphoblastic leukemia (ALL) and correlate their expression with clinical and laboratory data. Methods:The study was performed on bone marrow samples of 70 consecutive newly diagnosed pediatric (ALL) patients, of which 56 were evaluated for both miR-181a and miR-196b (all 70 for miR-181a) by real-time quantitative reverse transcriptase polymerase chain reaction (RT-qPCR). In addition, bone marrow from seven age and sex matched healthy controls derived from donors of bone marrow transplantation were assessed. Results: miR-181a expression was significantly up-regulated in ALL patients compared with healthy controls (p<0.001). However, miR-196b expression was significantly down-regulated in patients compared with healthy controls (p=0.038). Conclusion:Our results suggest that miR-181a has an oncogenic, while miR-196b has a tumor suppressive role in pediatric ALL patients. A finding which demonstrate the potential role of these microRNAs in pathogenesis of pediatric ALL. Also, estimation of their expression level may provide a tool for confirmation of a diagnosis of childhood ALL and could be a possible predictor of early relapse.

To Study The Role Of Pre-treatment MicroRNA Expression As A Predictor Of Response To Chemoradiation In Locally Advanced Carcinoma Cervix

Cervical cancer is the fourth most common cancer worldwide. External beam radiotherapy with concurrent cisplatin followed by brachytherapy is considered standard of care in locally advanced carcinoma cervix, with 50% of patients presenting with recurrence or persistent disease. At present there is no prognostic factor to predict the outcome of disease in locally advanced carcinoma cervix patients treated with standard therapy. MicroRNAs(miRNAs)are small, single stranded noncoding RNA constituting 18-22 nucleotides. It has been shown that miRNAs are differentially expressed (upregulated or downregulated) in many cancer cells. Expression of miRNAs can be used as molecular biomarkers to predict clinical response in locally advanced carcinoma cervix patients. In an observational study, we enrolled 32 patients of locally advanced carcinoma cervix (Stage IB- IVA) after pathological confirmation of biopsy sample from 2017-2018. Expression of six microRNA (miRNA-9 5p, miRNA-31 3p, miRNA-100 5p, miRNA-125a 5p, miRNA-125b-5p, miRNA–200a 5p) in formalin fixed paraffin embedded (FFPE) biopsied tissue were analyzed by real time quantitative reverse transcriptase polymerase chain reaction (RT qPCR). Pre-treatment evaluation of disease status was done with clinical examination and MRI pelvis imaging. All patients received external beam radiotherapy with concurrent chemoradiotherapy followed by brachytherapy as standard treatment. Patients were evaluated for clinical response after 3 months of completion of treatment, with clinical examination and MRI pelvis scan using RECIST 1.1 criteria. Responses were classified as complete response (CR) as disappearance of all disease in response to treatment and Non-response (NR) as patients with partial response, stable or progressive disease. Results were statistically analyzed using Mann Whiney U test to examine significant difference between expression of microRNA between patients with complete clinical response (CR) and those with non-response (NR). Out of total 32 patients, 24 patients (75%) had Complete Response and 8 patients (25%) had Non-Response to standard therapy. Out of six miRNAs, expression of miRNA-100 5p was upregulated in complete responders (CR) and downregulated in Nonresponders (NR), which showed a trend towards statistical significance (p value = 0.05). Other microRNAs (miRNA-9 5p, miRNA-31 3p, miRNA-125a 5p, miRNA-125b 5p, miRNA–200a 5p) showed differential expression in complete responders compared to non-responders but their expression did not reach statistical significance level (p value > 0.05). miRNA-100 5p can serve as a potential molecular biomarker in predicting clinical response to chemoradiation in locally advanced Carcinoma cervix patients. Its role should be further investigated in larger study population.

Prevalence and factors influencing the distribution of influenza viruses in Kenya: Seven-year hospital-based surveillance of influenza-like illness (2007-2013).

BackgroundInfluenza viruses remain a global threat with the potential to trigger outbreaks and pandemics. Globally, seasonal influenza viruses’ mortality range from 291 243–645 832 annually, of which 17% occurs in Sub-Saharan Africa. We sought to estimate the overall prevalence of influenza infections in Kenya, identifying factors influencing the distribution of these infections, and describe trends in occurrence from 2007 to 2013.MethodsSurveillance was conducted at eight district hospital sites countrywide. Participants who met the case definition for influenza-like illness were enrolled in the surveillance program. The nasopharyngeal specimens were collected from all participants. We tested all specimens for influenza viruses with quantitative reverse transcriptase real-time polymerase chain reaction (RT-qPCR) assay. Bivariate and multivariate log-binomial regression was performed with a statistically significant level of p<0.005. An administrative map of Kenya was used to locate the geographical distribution of surveillance sites in counties. We visualized the monthly trend of influenza viruses with a graph and chart using exponential smoothing at a damping factor of 0.5 over the study period (2007–2013).ResultsA total of 17446 participants enrolled in the program. The overall prevalence of influenza viruses was 19% (n = 3230), of which 76% (n = 2449) were type A, 21% (n = 669) type B and 3% (n = 112) A/ B coinfection. Of those with type A, 59% (n = 1451) were not subtyped. Seasonal influenza A/H3N2 was found in 48% (n = 475), influenza A/H1N1/pdm 2009 in 43% (n = 434), and seasonal influenza A/ H1N1 in 9% (n = 88) participants. Both genders were represented, whereas a large proportion of participants 55% were ≤1year age. Influenza prevalence was high, 2 times more in other age categories compared to ≤1year age. Category of occupation other than children and school attendees had a high prevalence of influenza virus (p< <0.001). The monthly trends of influenza viruses’ positivity showed no seasonal pattern. Influenza types A and B co-circulated throughout the annual calendar during seven years of the surveillance.ConclusionsInfluenza viruses circulate year-round and occur among children as well as the adult population in Kenya. Occupational and school-based settings showed a higher prevalence of influenza viruses. There were no regular seasonal patterns for influenza viruses.

Abstract B41: Piritramide analgesia reduces CEA mRNA-positive circulating tumor cells’ presence compared to morphine and epidural analgesia following radical colon cancer surgery

Abstract Purpose: Piritramide is a strong opioid widely used for postoperative analgesia (PA) in Europe while morphine and epidural PA are well established worldwide. Our retrospective analysis revealed that compared to morphine, piritramide improved cancer-specific survival in patients undergoing radical colon cancer surgery. Since circulating tumor cells’ (CTCs) presence has negative effect on colon cancer survival, we hypothesized that different types of PA could affect postoperative CTCs levels. Patients and Methods: In total, 60 patients undergoing radical colon cancer surgery were enrolled in this prospective, randomized, two-center pilot study. Patients were randomized to receive either piritramide, morphine, or epidural PA. The presence of carcinoembryonic antigen (CEA) and cytokeratin 20 (CK-20) mRNA positive CTCs in peripheral blood was measured at four points (before and immediately after surgery, on postoperative day 2, and one month after surgery) using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). Perioperative treatment was standardized. Results: In piritramide-treated patients, the levels of CEA mRNA positive cells were significantly lower on postoperative day 2 compared to other patients (p=0.04). CK-20 mRNA positive cells levels remained similar. In all groups, CTCs levels returned to preoperative baseline one month after surgery. Groups were similar regarding age, sex, ASA status, TNM, and grading. Conclusion: Compared to morphine and epidural analgesia, piritramide reduces postoperative CEA mRNA-positive circulating tumor cells’ presence following radical colon cancer surgery. Further trials enrolling larger numbers of patients are needed. Acknowledgments: This study was supported by Ministry of Health of the Czech Republic (NV18-03-00470), Ministry of Education, Youth and Sport of the Czech Republic (LO1304, LM2015089), European Regional Development Fund (ENOCH CZ.02.1.01/0.0/0.0/16_019/0000868) and Cancer Research Czech Republic. Citation Format: Josef Srovnal, Emil Berta, Alona Rehulkova, Monika Vidlarova, Petr Prasil, Lubomir Vecera, Petr Stourac, Pavla Kourilova, Marian Hajduch. Piritramide analgesia reduces CEA mRNA-positive circulating tumor cells’ presence compared to morphine and epidural analgesia following radical colon cancer surgery [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B41.

Identification of Novel Candidate Biomarkers for Oral Squamous Cell Carcinoma Based on Whole Gene Expression Profiling.

This study aimed to determine the whole gene expression profiles and to ascertain potential biomarkers for 22 oral squamous cell carcinoma (OSCC) among Thai patients using the Illumina Human HT-12, V4.0 Expression BeadChip array. Result indicated 2,724 differential expressed genes composed of 1,560 up-regulated and 1,164 down-regulated genes (unpaired t-test, p-value <0.05; fold change ≥2.0 and ≤2.0). The top 9 up-regulated genes were validated in 39 OSCC cases using TaqMan real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay. Among these, the up-regulation of peptidase inhibitor 3 (PI3) and keratin 17 (KRT17) genes was harbored in all 39 OSCC patients (100%). Likewise, statistical analysis indicated that gene expression in 8 selective genes including keratin 16 (KRT16), keratin 14 (KRT14), keratinocyte differentiation-associated protein (KRTDAP), keratin 6B (KRT6B), PI3, S100 calcium binding protein A7 (S100A7), stratifin (SFN) and keratin 5 (KRT5) was significantly associated with well differentiated OSCC (p-value <0.05). Moreover, high level of KRT17 protein was significantly associated with well differentiated OSCC compared to moderately OSCC (p-value = 0.041). Notably, using nested-PCR analysis indicated all OSCC cases in this study were HPV-free. Especially, KRTDAP, PI3, SFN mRNA expression were first reported among patients with OSCC. Conclusion, the whole transcript expression study and TaqMan real-time qRT-PCR assay were relevant regarding the increase in gene expression in OSCC. In addition, the up-regulation of PI3 and KRT17 might constitute potential candidate molecular biomarkers to diagnose patients with OSCC.

The effect of spleen tyrosine kinase inhibitor R406 on diabetic retinopathy in experimental diabetic rats.

To investigate the effect of spleen tyrosine kinase (Syk) inhibitor R406 on diabetic retinopathy (DR) in diabetic mellitus (DM) rats. Rats were randomized into Normal, DM, DM + 5mg/kg R406 and DM + 10mg/kg R406 groups. DM rats were established via injection of streptozotocin (STZ). One week after model establishment, rats in treatment groups received 5mg/kg or 10mg/kg R406 by gavage administration for 12weeks consecutively, followed by the detection with hematoxylin-eosin (HE) staining, Evans blue angiography, retinal trypsin digestion assay, Western blotting, immunohistochemistry, TUNEL assay, immunofluorescence assay and quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR). The retina of DM rats presented different degree of edema, disordered and loose structure, swollen cells with enlarged intercellular space, and dilated and congested capillaries. Besides, the retinal vessels of DM rats showed high fluorescence leakage. However, R406 alleviated the above-mentioned conditions, which was much better with high concentration of R406 (10mg/kg). R406 also reversed the down-regulations of occludin, claudin-5, ZO-1 and the up-regulation of and VEGF in retinal tissues of DM rats; inhibited retinal cell apoptosis; strengthened retinal cell proliferation; and reduced expressions of IL-1β, IL-6, TNF-α and nuclear p65 NF-κB in retinal tissues. The improvement in all these indexes was much more significant in rats of DM + 10mg/kg R406 group than in rats of DM + 5mg/kg R406 group. Syk inhibitor R406 could attenuate retinal inflammation in DR rats via the repression of NF-κB activation.

Gastric juice piR-1245: A promising prognostic biomarker for gastric cancer.

e16574 Background: Emerging reports demonstrated that PIWI-interacting RNAs (piRNAs) played an indispensable role in tumorigenesis. However, it still remains elusive whether piR-1245 in gastric juice specific in stomach could be employed as a biomarker for gastric cancer (GC). The present work is aiming at exploring the possibility of piR-1245 in gastric juice as a potential marker to judge for diagnosis and prognosis of gastric cancer. Methods: Gastric juice was collected from 66 GC patients and 66 healthy individuals. Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) was employed to measure the levels of piR-1245 expression. Then, the pattern of piR-1245 expression in gastric juice was determined between GC patients and healthy individuals. A receiver operating characteristic (ROC) curve was constructed for distinguishing GC from healthy individuals. Results: Gastric juice piR-1245 levels in GC were higher than those of controls (P &lt; 0.0001). The value of area under ROC (AUC) was 0.885 (sensitivity, 90.9%; specificity, 74.2%; 95% confidence interval, 0.8286 to 0.9414). High gastric juice piR-1245 expression was signally correlated with tumor size (P = 0.013) and TNM stage (P = 0.001). GC patients with high piR-1245 expression in gastric juice exerted a poorer overall survival (OS) (P = 0.0152) and progression-free survival (PFS) (P = 0.013). COX regression analysis verified that gastric juice piR-1245 expression was an independent prognostic risk variable for OS (P &lt; 0.05). Conclusions: The current study suggested that piR-1245 in gastric juice had the potential to be a useful biomarker for GC detection and prognosis prediction.

Bacterial cellulose membrane conjugated with plant-derived osteopontin: Preparation and its potential for bone tissue regeneration

Bacterial cellulose membrane (BCM) has been recently recognized as a new generation of carbohydrate-based nanomaterial that possesses a great potential in tissue engineering applications. This research aims to develop an active non-resorbable guided tissue regeneration (GTR) membrane from BCM by conjugating with plant-derived recombinant human osteopontin (p-rhOPN), an economically produced and RGD-containing biomolecule. The BCM was initially grafted with poly(acrylic acid) (PAA) brushes to form poly(acrylic acid)-grafted BCM. Multiple carboxyl groups introduced to the BCM by PAA can serve as active anchoring points for p-rhOPN conjugation and yielded p-rhOPN-BCM. All chemically modified BCMs were characterized by attenuated total reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, while their surface morphology was evaluated by field emission-scanning electron microscopy and atomic force microscopy analyses. The amount of p-rhOPN adhered on the membrane was quantified by enzyme-linked immunosorbent assay. The immunocytochemistry, two-stage quantitative real-time reverse transcriptase polymerase chain reaction and in vitro mineralization analyses strongly suggested that p-rhOPN-BCM could elicit biological functions leading to the enhancement of osteogenic differentiation of human periodontal ligament stem cells as effective as BCM conjugated with commercially available rhOPN from mammalian cells (rhOPN-BCM), suggesting its potential to be used as GTR membrane to promote bone tissue regeneration.

Over-expression of arginine vasopressin in magnocellular neurosecretory cells of hypothalamus and its potential relationship with development of diabetic nephropathy

IntroductionWe aimed to assess our hypothesis that the expression changes of arginine vasopressin (AVP) in the magnocellular neurosecretory cells (MNCs) of hypothalamus and V2 receptor for AVP (RVP) in kidney may contribute to the pathogenesis of diabetic nephropathy (DN).Material and methodsTwenty-five male Wistar rats were randomly assigned to the control group and the diabetes mellitus (DM) group. Periodic acid-Schiff (PAS) staining and electron microscopy were used for morphological studies. Immunohistochemical staining for glial fibrillary acidic protein (GFAP) is standard for visualization of reactive astrocytes in the hypothalamus. Hypothalamus was used for immunofluorescence of AVP. Kidney was used for immunohistochemical staining of RVP. Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) was used for quantitative determinations of AVP mRNA in hypothalamus and RVP mRNA in kidney. Western blot was used to measure the protein expression of AVP in hypothalamus and RVP in kidney.ResultsMorphological studies showed abnormalities in kidney and hypothalamus in the DM group. The number of neurons and the gray value of astrocytes in hypothalamus in the DM group were markedly decreased. The expression level of AVP in hypothalamus and the expression level of RVP in kidney of DM rats were significantly increased. The positive correlations between the proteinuria and expression (mRNA and protein) of AVP, proteinuria and expression (mRNA and protein) of RVP, and the expression of AVP and RVP levels were found.ConclusionsAVP was upregulated in the MNCs of hypothalamus and RVP was upregulated in kidney in streptozotocin-induced DM rats, indicating their potential roles in the development of DN.

PolyI:C Upregulated CCR5 and Promoted THP-1-Derived Macrophage Chemotaxis via TLR3/JMJD1A Signalling.

Objective This study aimed to evaluate the specific roles of polyinosinic:polycytidylic acid (polyI:C) in macrophage chemotaxis and reveal the potential regulatory mechanisms related to chemokine receptor 5 (CCR5). Materials and Methods In this experimental study, THP-1-derived macrophages (THP1-Mφs) induced from THP- 1 monocytes were treated with 25 μg/mL polyI:C. Toll-like receptor 3 (TLR3), Jumonji domain-containing protein (JMJD)1A, and JMJD1C small interfering RNA (siRNAs) were transfected into THP1-Mφs. Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) was used to detect the expression levels of TLR3, CCR5, 23 Jumonji C domain-containing histone demethylase family members, JMJD1A, and JMJD1C in THP1-Mφs with different siRNAs transfections. Western blot was performed to detect JMJD1A, JMJD1C, H3K9me2, and H3K9me3 expressions. A transwell migration assay was conducted to detect THP1-Mφ chemotaxis toward chemokine ligand 3 (CCL3). A chromatin immunoprecipitation (ChIP) assay was performed to detect H3K9me2-CCR5 complexes in THP1- Mφs. Results PolyI:C significantly upregulated CCR5 in THP1-Mφs and promoted chemotaxis toward CCL3 (P<0.05); these effects were significantly inhibited by TLR3 siRNA (P<0.01). JMJD1A and JMJD1C expression was significantly upregulated in polyI:C-stimulated THP1-Mφs, while only JMJD1A siRNA decreased CCR5 expression (P<0.05). JMJD1A siRNA significantly increased H3K9me2 expression in THP1-Mφs but not in polyI:C-stimulated THP1-Mφs. The ChIP result revealed that polyI:C significantly downregulated H3K9me2 in the promoter region of CCR5 in THP1- Mφs. ConclusionPolyI:C can enhance THP1-Mφ chemotaxis toward CCL3 regulated by TLR3/JMJD1A signalling and activate CCR5 expression by reducing H3K9me2 in the promoter region of CCR5.

Toxoplasma ROP16I/III ameliorated inflammatory bowel diseases via inducing M2 phenotype of macrophages.

BACKGROUNDInflammatory bowel disease (IBD) is characterized by chronic and non-specific inflammation of the intestinal mucosa and mainly includes ulcerative colitis and Crohn's disease.AIMTo explore the beneficial effect of ToxoROP16I/III-induced M2 phynotype macrophages in homeostasis of IBDs through downregulation of M1 inflammatory cells.METHODSRAW264.7 macrophages stimulated by lipopolysaccharide (LPS) (M1 cells) were co-cultured with Caco-2 cells as an inflammatory model of IBD in vitro. The expression of ToxoROP16I/III was observed in RAW264.7 macrophages that were transfected with pEGFP-rop16I/III. The phenotypes of M2 and M1 macrophage cells were assessed by quantitative real-time reverse transcriptase polymerase chain reaction and the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, transforming growth factor (TGF)-β1, IL-10, inducible nitric oxide synthase (iNOS), and arginase-1 (Arg-1) was detected. The expression of iNOS, Arg-1, signal transducer and activator of transcription 3 (Stat3), p-Stat3, Stat6, p-Stat6, programmed death ligand-2 (PD-L2), caspase-3, -8, and -9 was analyzed by Western blotting, and Griess assays were performed to detect nitric oxide (NO). TNF-α, IL-1β, IL-6, TGF-β1, and IL-10 expression in the supernatants was detected by enzyme-linked immunosorbent assay, and Caco-2 cell apoptosis was determined by flow cytometry after mixing M1 cells with M2 cells in a Caco-2 cell co-culture system.RESULTSM1 cells exhibited significantly increased production of iNOS, NO, TNF-α, IL-1β, and IL-6, while ToxoROP16I/III induced macrophage bias to M2 cells in vitro, showing increased expression of Arg-1, IL-10 and TGF-β1 and elevated production of p-Stat3 and p-Stat6. The mixed M1 and M2 cell culture induced by ToxoROP16I/III exhibited decreased production of NO and iNOS and upregulated expression of Arg-1 and PD-L2. Accordingly, Caco-2 cells became apoptotic, and apoptosis-associated proteins such as caspase-3, -8 and -9 were dampened during co-culture of M1 and M2 cells. Flow cytometry analysis showed that co-culture of M1 cells with Caco-2 cells facilitated the apoptosis of Caco-2 cells, but co-culture of M1 and M2 cells alleviated Caco-2 cell apoptosis.CONCLUSIONToxoROP16I/III-induced M2 macrophages inhibited apoptosis of Caco-2 cells caused by M1 macrophages. This finding may help gain a better understanding of the underlying mechanism and represent a promising therapeutic strategy for IBDs.

Development of a high-efficient concentrated pretreatment method for noroviruses detection in independent oysters:An extension of the ISO/TS 15216-2:2013 standard method

Noroviruses are the primary cause of gastroenteritis and foodborne diseases, affecting millions of individuals annually worldwide. Oysters are frequently associated with norovirus outbreaks. Hence, inexpensive and simple norovirus detection methods are important to detect and contain foodborne illness outbreaks. The objective of this study is to develop a high-efficient concentrated pretreatment method for reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) detection. Based on the existing ISO/TS 15216-2:2013 standard (ISO/TS 15216-2., 2013), four methods are compared for recovery of norovirus from spiked digestive tissue of oysters. A method is found to be the most efficient based on protease K method of increasing buffer volume and PEG precipitation method. The recovery rate and amplification efficiency approached 11.07 ± 0.09% and 124.12 ± 5.99%, respectively, being 7-fold that of the original ISO/TS 15216-2:2013 method. This method serves as a rapid (1.5h) sample concentration tool with a limit of detection as low as 7.05 × 103 copies. Thirty-eight oyster samples from an aquatic products market in Guangzhou are tested using this method, and noroviruses are detected in 13 samples. This method is an effective extension of the existing ISO/TS 15216-2:2013 standard and it is potentially applicable for detecting norovirus contamination in oysters.

Human Umbilical Cord-Derived Mesenchymal Stem Cell Therapy Ameliorates Nonalcoholic Fatty Liver Disease in Obese Type 2 Diabetic Mice.

Nonalcoholic fatty liver disease (NAFLD) is increasingly common among patients with type 2 diabetes mellitus (T2DM). The two conditions can act synergistically to produce adverse outcomes. However, the therapeutic options for patients with NAFLD and T2DM are currently limited. Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) have shown therapeutic potential for diabetes and hepatic disorders such as liver cirrhosis and fulminant hepatic failure. The present study is aimed at investigating the effect of human UC-MSCs on a mouse model of NAFLD and T2DM, characterized by obesity-induced hyperglycaemia, dyslipidaemia, hepatic steatosis, and liver dysfunction. Thirty-week-old male C57BL/6 db/db mice were infused with human UC-MSCs or phosphate-buffered saline (PBS) via the tail vein once a week for six weeks. Age-matched male C57BL/6 wild-type db/+ mice were used as controls. Body weight and random blood glucose were measured every week. One week after the sixth infusion, intraperitoneal glucose tolerance tests and insulin tolerance tests were performed and the blood and liver were harvested for biochemical and histopathological examinations. Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), immunofluorescence staining, and western blot were performed to monitor the expression of the lipid metabolism- and regulatory pathway-related genes. UC-MSC infusions significantly ameliorated hyperglycaemia, attenuated the elevation of hepatic transaminases, and decreased lipid contents, including triglyceride, total cholesterol, and low-density lipoprotein cholesterol. Moreover, histological lesions in the liver diminished markedly, as evidenced by reduced lipid accumulation and attenuated hepatic steatosis. Mechanistically, UC-MSCs were found to regulate lipid metabolism by increasing the expression of fatty acid oxidation-related genes and inhibiting the expression of lipogenesis-related genes, which were associated with the upregulation of the HNF4α-CES2 pathway. Our results demonstrate that human UC-MSCs can ameliorate NAFLD and reverse metabolic syndrome in db/db mice. Thus, UC-MSCs may serve as a novel therapeutic agent for T2DM patients with NAFLD.

EFEMP2 Inhibits Breast Cancer Invasion And Metastasis In Vitro And In Vivo.

BackgroundEGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) is an extracellular matrix (ECM) glycoprotein, which is regarded as potential prognostic biomarkers in some carcinoma. Little is known about the association of EFEMP2 and breast cancer.MethodsEFEMP2 expressions in normal breast tissue, benign fibroadenoma, breast cancer, the normal mammary epithelial cell line, and 4 different invasive breast cancer cell lines were evaluated by immunohistochemistry (IHC) or immunocytochemistry (ICC) and real time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR). Expression and prognostic value of EFEMP2 in breast cancer were verified by the Public databases (Oncomine and Kaplan-Meier plotter database). Lentiviral vector with EFEMP2 cDNA was constructed and used to infect breast cancer cell lines to investigate the effects of EFEMP2 on the biological behavior of breast cancer cells by functional in vitro and in vivo assays.ResultsDown-regulated EFEMP2 expression was found in breast cancer tissues and cells, and low expression of EFEMP2 was associated with poor prognosis in patients with breast cancer. Analysis by the Public database leaded to the same conclusion. Up-regulated EFEMP2 expression significantly hampered the invasion and metastasis abilities of breast cancer cells and the process of epithelial interstitial transformation (EMT) via the Wnt/β-catenin pathway.ConclusionEFEMP2 expression was lower in breast cancer and closely related to the prognosis of patients, its anti-oncogenic roles indicated the underlying therapeutic target for the future treatment of breast cancer.

Hypothermia and nutrient deprivation alter viability of human adipose-derived mesenchymal stem cells

PurposeAdipose-derived mesenchymal stem cells (MSCs) are attractive biological agents in regenerative medicine. To optimize cell therapies, it is necessary to determine the most effective delivery method for MSCs. Therefore, we evaluated the biological properties of MSCs after exposure to various temperatures to define optimal storage conditions prior to therapeutic delivery of MSCs. DesignProspective observational study. Methods and materialsAdherent and non-adherent MSCs were incubated at multiple temperatures (i.e., 4, 23 and 37 °C) in Lactated Ringers (LR) solution lacking essential cell growth ingredients, or in culture media which is optimized for cell growth. Cells were assessed either after the temperature changes (4 h) or after recovery (24 h). Metabolic activity of MSCs, cell number and expression of representative mRNA biomarkers were evaluated to assess the biological effects of temperature. We monitored changes in mRNAs expression related to cytoprotective- or stress-related responses (e.g., FOS, JUN, ATF1, ATF4, EGR1, EGR2, MYC), proliferation (e.g., HIST2H4, CCNB2), and extracellular matrix production (ECM; e.g., COL3A1, COL1A1) by quantitative real time reverse-transcriptase polymerase chain reaction (RT-qPCR) analysis. ResultsOur study demonstrates that storing MSCs in Lactated Ringers (LR) solution for 4 h decreases cell number and metabolic activity. The number of viable MSCs decreased significantly when cultured at physiological temperature (37 °C) and severe hypothermia (4 °C), while cells grown at ambient temperature (23 °C) exhibited the least detrimental effects. There were no appreciable biological differences in mRNA markers for proliferation or ECM deposition at any of the temperatures. However, biomarkers related to cytoprotective- or stress-responses were selectively elevated depending on temperature or media type (i.e., LR versus standard media). ConclusionThe biological impact of nutrient-free media and temperature changes after 4 h exposure persists after a 24 h recovery period. Hence, storage temperature and media conditions should be optimized to improve effective dosing of MSCs.

The prognostic significance of BRCA1 gene expression in patients with breast cancer

Background. Nowadays, BRCA-associated breast cancer occupies a special position in the choice of treatment strategy. Although, the drug treatment of such patients was almost the same as the treatment of sporadic disease. Of particular interest is the assessment of the deficit of homologous recombination, which may include various dysfunctions of the BRCA1 gene. Moreover, it has been shown that a decrease in the functional activity of BRCA1 is reflected in the sensitivity of the tumor to DNA damaging agents, and the prognosis of the disease becomes more favorable.Objective: assessment of the association of BRCA1 gene expression in a breast tumor with the prognosis of the disease.Materials and methods. The study included 111 patients with breast cancer T1–4N0–3M0 (stage IIA–IIIB), with a morphologically verified diagnosis. Initial and postoperative expression of BRCA1 in tumor material was evaluated. RNA was isolated from the tumor material before treatment and after neoadjuvant chemotherapy. The level of BRCA1 expression was assessed using reverse transcriptase quantitative real-time polymerase chain reaction.Results. As a result of the study, long-term results of treatment of patients were evaluated depending on the level of BRCA1 gene expression. The presence of BRCA1 hypoexpression (level less than 1) in tumor tissue after neoadjuvant chemotherapy was found to be a favorable prognostic factor and is associated with high rates of metastasis-free survival (p = 0.0002). In addition, a similar result is shown for patients treated with the FAC (fluorouracil, doxorubicin, cyclophosphamide) neoadjuvant chemotherapy regimen (p = 0.005). Despite the absence of statistically significant differences, in 8 patients who underwent chemotherapy with the inclusion of platinum drugs, there was a low level of BRCA1 expression in a residual tumor and 100 % non-metastatic and overall survival.Conclusion. Based on the data obtained, it can be assumed that the aberrant state of the BRCA1 gene (low expression) in a breast tumor may also be a promising marker for the prognosis of the disease, which confirms the undoubted relevance of the study, but also requires further detailed study.

Respiratory virus associated with surgery in children patients

BackgroundViral respiratory infection (VRI) is a common contraindication to elective surgery. Asymptomatic shedding among pediatric surgery patients (PSPs) could potentially lead to progression of symptomatic diseases and cause outbreaks of respiratory diseases. The aim of this study is to investigate the incidence of infection among mild symptomatic PSP group and asymptomatic PSP group after surgical procedure.MethodsWe collected the induced sputum from enrolled 1629 children (under 18 years of age) with no respiratory symptom prior to pediatric surgery between March 2017 and February 2019. We tested 16 different respiratory virus infections in post-surgery mild symptomatic PSP group and asymptomatic PSP group using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) assay panel. We analyzed symptom data and quantitative viral load to investigate the association between viruses, symptoms and viral quantity in qRT-PCR-positive PSPs.ResultsOut of 1629 children enrolled, a total of 204 respiratory viruses were present in 171 (10.50%) PSPs including 47 patients with mild symptoms and 124 with no symptoms after surgery. Commonly detected viruses were human rhino/enterovirus (HRV/EV, 42.19%), parainfluenza virus 3 (PIV3, 24.48%), coronavirus (CoV NL63, OC43, HKU1, 11.46%), and respiratory syncytial virus (RSV, 9.9%). PIV3 infection with a higher viral load was frequently found in PSPs presenting with mild symptoms, progressing to pneumonia with radiographic evidence after surgery. HRV/EV were the most commonly detected pathogens in both asymptomatic and mild symptomatic PSPs. CoV (OC43, HKU1) infections with a higher viral load were mostly observed in asymptomatic PSPs progressing to alveolar or interstitial infiltration.ConclusionsOur study suggested that PIV3 is a new risk factor for VRI in PSPs. Employing a more comprehensive, sensitive and quantitative method should be considered for preoperative testing of respiratory viruses in order to guide optimal surgical timing.

Selection of Suitable Reference Genes for RT-qPCR Gene Expression Analysis in Siberian Wild Rye (Elymus sibiricus) under Different Experimental Conditions.

Elymus sibiricus, which is a perennial and self-pollinated grass, is the typical species of the genus Elymus, which plays an important role in forage production and ecological restoration. No reports have, so far, systematically described the selection of optimal reference genes for reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) analysis in E. sibiricus. The goals of this study were to evaluate the expression stability of 13 candidate reference genes in different experimental conditions, and to determine the appropriate reference genes for gene expression analysis in E. sibiricus. Five methods including Delta Ct (ΔCt), BestKeeper, NormFinder, geNorm, and RefFinder were used to assess the expression stability of 13 potential reference genes. The results of the RefFinder analysis showed that TBP2 and HIS3 were the most stable reference genes in different genotypes. TUA2 and PP2A had the most stable expression in different developmental stages. TBP2 and PP2A were suitable reference genes in different tissues. Under salt stress, ACT2 and TBP2 were identified as the most stable reference genes. ACT2 and TUA2 showed the most stability under heat stress. For cold stress, PP2A and ACT2 presented the highest degree of expression stability. DNAJ and U2AF were considered as the most stable reference genes under osmotic stress. The optimal reference genes were selected to investigate the expression pattern of target gene CSLE6 in different conditions. This study provides suitable reference genes for further gene expression analysis using RT-qPCR in E. sibiricus.

PS-04-006 Immunomodulatory effects of Dihydrotestosterone (DHT) in rat vaginal smooth muscle cells

Androgens, in particular Dihydrotestosterone (DHT), show immunomodulatory protective effects in experimental models of chronic inflammation. We aimed to evaluate the possible immunomodulatory effects of DHT in smooth muscle cells (SMCs) isolated from vagina of mature intact Sprague-Dawley rats. SMCs derived from distal vagina were isolated and characterized with α-smooth muscle actin (a-SMA) and myosin heavy chain 11 (MHCII). Then they were left untreated (control) or treated in vitro with different stimuli: Lipopolysaccharides (LPS; 100 ng/ml), the strongest immunogenic ligand for TLR; DHT (30nM); and DHT+LPS (30nM, 100ng/ml) for 24 hours. With Real-Time Quantitative Reverse Transcriptase Polymerase Chain Reaction, we evaluated mRNA expression of the main pro-inflammatory markers: interleukin-6 (IL-6), IL-1b, chemokine (C-X-C motif) ligand (1CXCL1), cyclooxygenase-2 (COX-2), CD4, RAR-related orphan receptor C (RORC), chemokine for recruitment of macrophages [monocyte chemotactic protein 1 (MCP1) and membrane receptors which mediate the cell inflammatory response, such as Toll-Like Receptors (TLRs). In addition, immunofluorescence studies were performed to analyze the nuclear translocation of NF-kB, an important transcriptional mediator of the inflammatory response.