Qualitative Reverse Transcription-polymerase Chain Reaction Research Articles

Prevalence of COVID-19 in End Stage Renal Disease Patients on Hemodialysis & Dialysis Providers During SARs-Cov-19 spike in Pakistan.

The global pandemic of coronavirus disease 2019 (COVID-19) poses a significant threat to both hemodialysis staff and patients. However, the prevalence of COVID-19 among asymptomatic end-stage renal disease (ESRD) patients on chronic hemodialysis (MHD) and healthcare staff including nurses and technicians in dialysis unit remains uncertain. Given that hemodialysis patients and dialysis nurses are particularly prone in congregate settings, this research aimed to examine the prevalence of COVID-19 in two hemodialysis units at Jinnah Hospital, Lahore, Pakistan. This cross-sectional study involved the participation of 198 maintenance hemodialysis patients with end-stage renal disease (ESRD) and 83 dialysis staff members. These patients or staff members did not have any symptoms at the time of testing. Testing was conducted using qualitative reverse transcription-polymerase chain reaction (RT-PCR) on nasal & pharyngeal swabs collected from June 18th to June 23rd, 2020. 
 Out of the total participants, 45 out of 198 dialysis patients (22.7%) and 10 out of 83 dialysis staff members (12%) were found to be positive for COVID-19. The overall prevalence of COVID-19, combining patients and staff, was 19.5% (55 out of 281). The mortality rate among the infected patients was 6.6% (3 out of 45). To conclude, the findings of this study demonstrate a significant prevalence of SARS-CoV-2 among hemodialysis staff and patients in Lahore, Pakistan.

Investigation of Blood Plasma Viral Nucleocapsid Antigen as a Marker of Active Severe Acute Respiratory Syndrome Coronavirus 2 Omicron Variant Infection.

Nasopharyngeal qualitative reverse-transcription polymerase chain reaction (RT-PCR) is the gold standard for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but it is not practical or sufficient in every clinical scenario due to its inability to distinguish active from resolved infection. Alternative or adjunct testing may be needed to guide isolation precautions and treatment in patients admitted to the hospital. We performed a single-center, retrospective analysis of residual clinical specimens and medical record data to examine blood plasma nucleocapsid antigen as a candidate biomarker of active SARS-CoV-2. Adult patients admitted to the hospital or presenting to the emergency department with SARS-CoV-2 ribonucleic acid (RNA) detected by RT-PCR from a nasopharyngeal swab specimen were included. Both nasopharyngeal swab and a paired whole blood sample were required to be available for analysis. Fifty-four patients were included. Eight patients had positive nasopharyngeal swab virus cultures, 7 of whom (87.5%) had concurrent antigenemia. Nineteen (79.2%) of 24 patients with detectable subgenomic RNA and 20 (80.0%) of 25 patients with N2 RT-PCR cycle threshold ≤ 33 had antigenemia. Most individuals with active SARS-CoV-2 infection are likely to have concurrent antigenemia, but there may be some individuals with active infection in whom antigenemia is not detectable. The potential for high sensitivity and convenience of a blood test prompts interest in further investigation as a screening tool to reduce reliance on nasopharyngeal swab sampling and as an adjunct diagnostic test to aid in clinical decision making during the period after acute coronavirus disease 2019.

Oligosaccharides from Sargassum thunbergii inhibit osteoclast differentiation via regulation of IRF-8 signaling

Osteoporosis (OP) is a systemic bone degenerative disease characterized by low bone mass and deteriorated microarchitecture of bone tissue, causing high morbidity and mortality rates. Bone resorption by overactivated osteoclasts (OCs) is the main cause of osteoporosis. Glucuronomannan and its oligomers (Gs) and their sulfated derivatives (SGs) were previously prepared. The anti-osteoporosis activities of these glycans were evaluated. Firstly, we determined the viability of RAW264.7 by CCK-8 test. Nextly, we investigated the inhibitory effects of Gs and SGs on the differentiation of RAW264.7 cells into OCs using tartrate-resistant acid phosphatase (TRAP) staining, F-actin ring staining, qualitative reverse-transcription polymerase chain reaction(qRT-PCR) and western blotting. TRAP staining revealed that Gs significantly blocked RANKL-induced OC generation while SGs did not exhibit this ability. F-actin staining assays demonstrated that Gs inhibits RANKL-induced actin ring formation. qRT-PCR analyses indicated that Gs dose-dependently inhibited the expression of OCs marker genes including Trap, NFATc1, c-Fos, DC-Stamp and ATP60 during the differentiation process, while SGs did not suppress. Regarding the mechanism of Gs, it was found that Gs suppressed osteoclastogenesis via inhibiting the degradation of IRF-8 and interfering with NF-κB pathway activation. Together, these results suggest that Gs have the ability to inhibit osteoclastogenesis by modulating IRF-8 signaling.

Long noncoding RNA WT1-AS regulates trophoblast proliferation, migration, and invasion via the microRNA-186-5p/CADM2 axis.

This study aimed to determine the role of long noncoding RNA (lncRNA) WT1 antisense RNA (WT1-AS) in the occurrence and progression of preeclampsia (PE) and to determine the underlying molecular mechanisms. The associations between WT1-AS and microRNA (miR)-186-5p, and miR-186-5p and cell adhesion molecule 2 (CADM2) were predicted using StarBase software and verified via dual-luciferase assays. To explore the role of the human chorionic trophoblast line HTR-8/SVneo, gene (WT1-AS/miR-186-5p) gain/loss of function experiments were performed. Qualitative reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to evaluate transfection efficiency. Cell proliferation, apoptosis, cell migration, and invasion were assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), flow cytometry, and transwell analysis, respectively. Moreover, CADM2 protein expression was measured by western blotting. The results indicated that overexpression of WT1-AS inhibited cell viability, migration, and invasion, and induced apoptosis in HTR-8/SVneo cells. We observed that miR-186a-5p directly targeted WT1-AS, and miR-186a-5p knockdown reversed the effects of WT1-AS knockdown in HTR-8/SVneo cells. Binding sites were found between miR-186-5p and CADM2, and CADM2-overexpression reversed the influence of miR-186-5p mimic on HTR-8/SVneo cells. In summary, our findings demonstrated that lncRNA WT1-AS participates in PE by regulating the proliferation and invasion of placental trophoblasts, through the miR-186-5p/CADM2 axis.

MiRNA-16-1 Suppresses Mcl-1 and Bcl-2 and Sensitizes Chronic Lymphocytic Leukemia Cells to BH3 Mimetic ABT-199.

ObjectiveChronic lymphoid leukemia (CLL) is the most common type of leukemia among adults. Increased levels of Mcl-1 and Bcl-xL is linked to resistance to Bcl-2 inhibitors including ABT-199. In this study, we investigated the effect of miRNA-16-1 on apoptosis and sensitivity of the CLL cells to ABT-199. Materials and Methods In this experimental study, the Mcl-1 and Bcl-2 expression were measured using qualitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting. The effect of treatments on cell survival and growth were explored with MTT assay and Trypan blue assay, respectively. The drug interaction was evaluated using combination index analysis. Apoptosis was assessed by ELISA cell death and caspase-3 activity assays. Results MiRNA-16-1 markedly inhibited the expression of Mcl-1 and Bcl-2 in a time dependent manner (P<0.05, relative to blank control). Pretreatment with miRNA-16-1 synergistically suppressed the cell growth and survival and reduced the half-maximal inhibitory concentration (IC50) value of ABT-199. Moreover, miRNA-16-1 markedly augmented the apoptotic effect of ABT-199 in CLL cells (P<0.05). ConclusionOur findings propose that miRNA-16-1 act in concert with ABT-199 to exert synergistic anticancer efficacy against CLL, which is attributed to the inhibition of Bcl-2 and Mcl-1. This may propose a promising strategy for CLL resistant patients.

Development of a Rapid Reverse Transcription-Recombinase Polymerase Amplification Couple Nucleic Acid Lateral Flow Method for Detecting Porcine Epidemic Diarrhoea Virus.

Simple SummaryPorcine epidemic diarrhea virus infection is an important acute diarrheal disease of swine especially in infected piglets can caused severe diarrhea, dehydration with difficulty in digesting milk curd, leading to death. The diagnosis of this viral infection is essential for monitoring and managing the disease. There is surprisingly little evidence such as easy rapid detection in the field. In this study, we developed rapid the reverse transcription-recombinase polymerase amplification couple nucleic acid lateral flow for Porcine epidemic diarrhea virus detection targeted the membrane gene in the genome sequence of the virus. Herein, the results shown that the established assay is simple and rapid, increases high sensitivity and specificity, and can be applied in the field.Porcine epidemic diarrhea virus (PEDV) infection is an important acute diarrheal disease of swine that results in economic and industrial losses worldwide. The clinical manifestations in infected piglets are severe diarrhea, dehydration with milk curd indigestion, leading to death. The diagnosis of PEDV is essential for monitoring and managing the disease. PEDV can be detected and identified by serology and the nucleic acid of the virus in clinical samples. Therefore, a novel isothermal amplification and detection technique, reverse transcription-recombinase polymerase amplification couple nucleic acid lateral flow (RT-RPA-NALF) was developed for the rapid detection of PEDV. Qualitative reverse transcription-polymerase chain reaction (RT-qPCR) was established as the gold standard assay to compare results. Specific primer pairs and probes were designed, and RT-RPA conditions were optimized to amplify the M gene of PEDV. The established RT-RPA-NALF assay could finish in 25 min at a temperature of 42 °C and the amplicon interpreted by visual detection. The developed RT-RPA-NALF assay was specific to the M gene of PEDV, did not detect other common swine diarrhea pathogens, and showed minimal detection at 102 TCID50/mL PEDV. The RT-RPA-NALF assay can detect PEDV in 5 simulated fecal samples. Furthermore, in 60 clinical fecal samples, the results of RT-RPA-NALF correlated with RT-qPCR assay, which provides sensitivity of 95.65% and specificity of 100%, with a coincident rate of 98.33%. The rapid RT-RPA-NALF is simple and rapid, increases high sensitivity, and can be used in the field.

Detection of SARS-CoV-2 in Clinical Samples: Target-specific Analysis of Qualitative Reverse Transcription–Polymerase Chain Reaction(RT-PCR) Diagnostic Kits

INTRODUCTIONThe Coronavirus disease 2019 pandemic caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the rise of many available modalities for diagnosis. One such modality is the Reverse Transcription–Polymerase Chain Reaction (RT-PCR) kits which require evaluation amongst the many available commercial kits in the market. METHODSWe conducted a performance evaluation of twelve RT‐PCR SARS‐CoV‐2 commercial kits. A total of 75 nasopharyngeal and oropharyngeal clinical samples were selected with their cycling threshold (Ct) values. Inclusion of 5 gene targets: E gene, N gene, S gene, RdRp and ORF1ab were assessed. Data was analyzed using R software version 4.1.1 and Microsoft Excel RESULTSWe observe that, the positive sample's Ct values differs significantly across the 12 diagnostic kits. However, for gene-specific analysis, we observe that, positive sample's Ct values does not differ significantly across gene targets. There is significant difference in Ct values in Commercial kits targeting all genes except S-gene. All the commercial kits Altona (E and S genes), Thermo (ORF1ab and N genes), Multiplex (E, ORF1ab, RdRdp genes), Meril (N and ORF1ab genes), S D Biosensor (E and ORF1ab genes), Lab Gun (RdRp and N genes) and Lab systems (ORF1ab and E genes) scored a sensitivity of 100%. All other kits scored sensitivity above 95% and lowest sensitivity with the Genes2me (E gene) and Genes2me (RdRp) at 95.08% each. All kits were 100% specific. CONCLUSIONThis study provides an accurate comprehensive assessment of the different kits in the detection of SARS-CoV-2 which may promote standardization of testing across laboratories.

Christia vespertilionis extract induced antiproliferation and apoptosis in breast cancer (MCF7) cells.

C. vespertiliomis extracts were evaluated for antiproliferative and apoptosis effect on breast cancer (MCF7) cells. The leaves extracts were analysed for its antiproliferative effect on breast cancer (MCF7) cells and normal epithelial breast (MCF 10A) cells using Sulforhodamine B (SRB) assay. The selective extract was evaluated for its ability to induce apoptosis using Annexin V-FITC apoptosis staining and the expression of molecular genes using qualitative reverse transcription-polymerase chain reaction (RT-PCR) against MCF7 cells. Gas chromatography-mass spectrometry (GC-MS) was used to identify the compounds from the selective extract. The findings showed that dichloromethane fraction (CV-Dcm) extract had high antiproliferative effect against MCF7 cells (IC50 = 24µg/mL, selective index (SI) = 8.17). The percentages of apoptosis cells in CV-Dcm-treated MCF7 cells was 58.8%. The CV-Dcm extract induced downregulation of PCNA level. The apoptotic genes were also triggered in both extrinsic and intrinsic signaling pathways, affecting a 1.5-fold increase in BAX, 1.4-fold increase in cytochrome c, 1.3-fold increase in caspase-8, 1.7-fold increase in caspase-3 and 0.5-fold-decrease in BCL-2. Treated MCF7 cells also activated P53-dependent apoptotic death pathway. The present work strongly suggests that high efficacy of CV-Dcm extract was attributed to its antiproliferative and apoptosis-inducing activation in MCF7 cells, most likely due to its favourable compounds.

SARS-CoV-2 Infections and Hospitalizations Among Persons Aged ≥16 Years, by Vaccination Status - Los Angeles County, California, May 1-July 25, 2021.

COVID-19 vaccines fully approved or currently authorized for use through Emergency Use Authorization from the Food and Drug Administration are critical tools for controlling the COVID-19 pandemic; however, even with highly effective vaccines, a proportion of fully vaccinated persons will become infected with SARS-CoV-2, the virus that causes COVID-19 (1). To characterize postvaccination infections, the Los Angeles County Department of Public Health (LACDPH) used COVID-19 surveillance and California Immunization Registry 2 (CAIR2) data to describe age-adjusted infection and hospitalization rates during May 1-July 25, 2021, by vaccination status. Whole genome sequencing (WGS)-based SARS-CoV-2 lineages and cycle threshold (Ct) values from qualitative reverse transcription-polymerase chain reaction (RT-PCR) for two SARS-CoV-2 gene targets, including the nucleocapsid (N) protein gene region and the open reading frame 1 ab (ORF1ab) polyprotein gene region,* were reported for a convenience sample of specimens. Among 43,127 reported SARS-CoV-2 infections in Los Angeles County residents aged ≥16 years, 10,895 (25.3%) were in fully vaccinated persons, 1,431 (3.3%) were in partially vaccinated persons, and 30,801 (71.4%) were in unvaccinated persons. Much lower percentages of fully vaccinated persons infected with SARS-CoV-2 were hospitalized (3.2%), were admitted to an intensive care unit (0.5%), and required mechanical ventilation (0.2%) compared with partially vaccinated persons (6.2%, 1.0%, and 0.3%, respectively) and unvaccinated persons (7.6%, 1.5%, and 0.5%, respectively) (p<0.001 for all comparisons). On July 25, the SARS-CoV-2 infection rate among unvaccinated persons was 4.9 times and the hospitalization rate was 29.2 times the rates among fully vaccinated persons. During May 1-July 25, the percentages of B.1.617.2 (Delta) variant infections estimated from 6,752 samples with lineage data increased among fully vaccinated persons (from 8.6% to 91.2%), partially vaccinated persons (from 0% to 88.1%), and unvaccinated persons (from 8.2% to 87.1%). In May, there were differences in median Ct values by vaccination status; however, by July, no differences were detected among specimens from fully vaccinated, partially vaccinated, and unvaccinated persons by gene targets. These infection and hospitalization rate data indicate that authorized vaccines were protective against SARS-CoV-2 infection and severe COVID-19 during a period when transmission of the Delta variant was increasing. Efforts to increase COVID-19 vaccination, in coordination with other prevention strategies, are critical to preventing COVID-19-related hospitalizations and deaths.

Femtomolar SARS-CoV-2 Antigen Detection Using the Microbubbling Digital Assay with Smartphone Readout Enables Antigen Burden Quantitation and Tracking.

High-sensitivity severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen assays are desirable to mitigate false negative results. Limited data are available to quantify and track SARS-CoV-2 antigen burden in respiratory samples from different populations. We developed the Microbubbling SARS-CoV-2 Antigen Assay (MSAA) with smartphone readout, with a limit of detection of 0.5 pg/mL (10.6 fmol/L) nucleocapsid antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs. We developed a computer vision and machine learning-based automatic microbubble image classifier to accurately identify positives and negatives and quantified and tracked antigen dynamics in intensive care unit coronavirus disease 2019 (COVID-19) inpatients and immunocompromised COVID-19 patients. Compared to qualitative reverse transcription-polymerase chain reaction methods, the MSAA demonstrated a positive percentage agreement of 97% (95% CI 92%-99%) and a negative percentage agreement of 97% (95% CI 94%-100%) in a clinical validation study with 372 residual clinical NP swabs. In immunocompetent individuals, the antigen positivity rate in swabs decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity. Antigen was detected for longer and variable periods of time in immunocompromised patients with hematologic malignancies. Total microbubble volume, a quantitative marker of antigen burden, correlated inversely with cycle threshold values and days-after-symptom-onset. Viral sequence variations were detected in patients with long duration of high antigen burden. The MSAA enables sensitive and specific detection of acute infections and quantification and tracking of antigen burden and may serve as a screening method in longitudinal studies to identify patients who are likely experiencing active rounds of ongoing replication and warrant close viral sequence monitoring.

Humoral immune responses in hospitalized COVID-19 patients.

BackgroundThe emerging coronavirus 2019 (COVID-19) disease, caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a worldwide public health crisis. Antibody analysis is an important procedure for the diagnosis of COVID-19 patients. We investigated the IgG, IgM, and IgA responses against the SARS-CoV-2 spike (S) protein among hospitalized COVID-19 patients.Materials and methodsHospitalized COVID-19 patients (n = 178) in the Al Madinah region, Saudi Arabia, participated in this study. Of the 178 patients, 72 (40%) were categorized as severe, including 50 (69%) males and 22 (31%) females. The remaining106 (60%) patients were categorized as non-severe, including 85 (80%) males and 21 (20%) females. Qualitative reverse transcription-polymerase chain reaction (RT-PCR) to detect the presence of SARS-CoV-2 RNA was used to confirm the diagnosis of each patient. The specific anti-SARS-CoV-2 S protein IgG, IgM, and IgA antibodies in patients’ sera were measured using enzyme-linked immunosorbent assay (ELISA) and compared between case presentations.ResultsThe current study showed that all severe hospitalized patients presented significantly (p < 0.0001) increased anti-S IgG and IgM antibody accumulation compared with non-severe patients. Additionally, the results also showed that 50% of severe males were positive to anti-S IgG, IgM, and IgA antibodies, whereas only 40% positivity for all three-antibody isotypes was observed in severe females. The study also showed that 86% of males and 81% of females categorized as severe were positive for both IgG and IgM antibodies but negative for the IgA antibody against the S protein.ConclusionThe humoral immune response against SARS-CoV-2 proteins commonly results in the production of antibodies against viral proteins. Specific anti-SARS-CoV-2 S protein IgG class antibodies were detected at significantly higher levels than IgM class antibodies, and both IgG and IgM antibodies were detected at significantly higher levels than the IgA antibody among all patients. The variations of the humoral immune responses among hospitalized patients reflect the association between disease presentations and immunity against the virus. Collectively, these findings afford new insights into the different antibody isotypes in responses to COVID-19 hospitalized patients with dissimilar disease severity.

Performance of Unobserved Self-Collected Nasal Swabs for Detection of SARS-CoV-2 by RT-PCR Utilizing a Remote Specimen Collection Strategy.

BackgroundThe use of a remote specimen collection strategy employing a kit designed for unobserved self-collection for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription polymerase chain reaction (RT-PCR) can decrease the use of personal protective equipment (PPE) and exposure risk. To assess the impact of unobserved specimen self-collection on test performance, we examined results from a SARS-CoV-2 qualitative RT-PCR test for self-collected specimens from participants in a return-to-work screening program and assessed the impact of a pooled testing strategy in this cohort.MethodsSelf-collected anterior nasal swabs from employee return-to-work programs were tested using the Quest Diagnostics Emergency Use Authorization SARS-CoV-2 RT-PCR. The cycle threshold (Ct) values for the N1 and N3 N-gene targets and a human RNase P (RP) gene control target were tabulated. For comparison, we utilized Ct values from a cohort of health care provider–collected specimens from patients with and without coronavirus disease 2019 symptoms.ResultsAmong 47 923 participants, 1.8% were positive. RP failed to amplify for 13/115 435 (0.011%) specimens. The median (interquartile range) Cts were 32.7 (25.0–35.7) for N1 and 31.3 (23.8–34.2) for N3. Median Ct values in the self-collected cohort were significantly higher than those of symptomatic but not asymptomatic patients. Based on Ct values, pooled testing with 4 specimens would have yielded inconclusive results in 67/1268 (5.2%) specimens but only a single false-negative result.ConclusionsUnobserved self-collection of nasal swabs provides adequate sampling for SARS-CoV-2 RT-PCR testing. These findings alleviate concerns of increased false negatives in this context. Specimen pooling could be used for this population, as the likelihood of false-negative results is very low when using a sensitive, dual-target methodology.

High SARS-CoV-2 IgG/IGM seroprevalence in asymptomatic Congolese in Brazzaville, the Republic of Congo

IntroductionThe Republic of the Congo detected its first case of coronavirus disease 2019 (COVID-19) on March 14, 2020, and within several weeks, the country had introduced protective measures that were still in force in July 2020. Over the course of time, the progression in the number of clinical cases has appeared to be lower than expected, although reverse transcription polymerase chain reaction (RT-PCR) testing has been somewhat limited. In order to evaluate the incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within the Congolese population, a seroprevalence study was conducted on healthy individuals from different districts of Brazzaville who were willing to know their COVID-19 infection status. MethodsOropharyngeal swab and blood samples were collected from 754 healthy volunteers between April 2020 and July 2020. The samples were analyzed for SARS-CoV-2 using a qualitative RT-PCR assay, and Immunoglobulin G (IgG) and Immunoglobulin M (IgM) antibodies were detected using two different rapid tests. ResultsA total of 56 participants (7.4%) tested positive for SARS-CoV-2. The remaining 698 participants (92.6%) had negative RT-PCR results; of these, 117 were found to have anti-SARS-CoV-2 antibodies using serological tests. For these RT-PCR-negative subjects, the seroprevalence of IgG and IgM was found to increase over time: from 1.7% and 2.5% in April, up to 14.2% and 17.6% in July, respectively. In April 2020, 5% of the women were found to have IgG or IgM antibodies, whereas the antibodies were not detected in any of the men. The seroprevalence in RT-PCR negative subjects was significantly higher in women within IgG (P = 0.012) and IgM (P = 0.045) over the first three months. ConclusionThe proportion of the population who seroconvert over the course of the first wave is an important data to predict the risk of future COVID-19 waves and this will facilitate the efficient use of limited resources in a low income country like the Republic of the Congo.

Human placental hydrolysate promotes the long-term culture of hepatocyte-like cells derived from canine bone marrow.

Long-term culture of canine artificial hepatocytes has not been established. We hypothesized that human placental hydrolysate (hPH) may support the long-term culture of differentiated hepatocyte-like cells. Canine bone marrow cells were cultured using modified hepatocyte growth medium supplemented with hPH. Quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemical analysis for albumin, qualitative RT-PCR for cytochrome P450 1A1 (CYP1A1), hepatocyte growth factor (HGF), Cytokeratin 7 (CK7), CD90, CD44, and CD34, and functional analyses of CYP450 activity and low-density lipoprotein (LDL) uptake were performed. Cultured hepatocyte-like cells were able to maintain hepatocyte characteristics, including morphology, albumin synthesis, CYP450 activity, and LDL uptake for 80 days. Thus, hPH may be a potential facilitator for the long-term culture of hepatocyte-like cells. Clinicopathologically, this culture protocol of artificial hepatocytes will contribute to liver function evaluation.

Retinol/inflammation affect stemness and differentiation potential of gingival stem/progenitor cells via Wnt/β-catenin.

Inflammatory cytokines impact the course of periodontal disease, repair, and regeneration. Vitamin A and its metabolites are inflammation-modulatory biomolecules, affecting cellular pluripotency. The aim of this study was to investigate the effect of retinol and periodontal inflammatory cytokines (IL-1β/TNF-α/IFN-γ) on pluripotency and proliferative properties of gingival mesenchymal stem/progenitor cells (G-MSCs), for the first time. Human G-MSCs (n=5) were STRO-1 immuno-magnetically sorted, characterized and expanded in basic medium (control group), in basic medium with IL-1β (1ng/mL), TNF-α (10ng/mL), and IFN-γ (100ng/mL) (inflammatory group), in basic medium with retinol (20μmol/L) (retinol group) and with retinol added to the inflammatory group (inflammatory/retinol group). β-catenin levels at 1hour, cellular proliferation over 14days, and colony-forming units (CFUs) at 14days were investigated. Pluripotency gene expressions were examined at 1, 3, and 5days via reverse transcription-polymerase chain reaction (RT-PCR). Multilineage differentiation potential was evaluated, following 5days priming, using qualitative and quantitative histochemistry and RT-PCR. G-MSCs were CD14- , CD34- , CD45- , CD73+ , CD90+ , CD105+ , and showed mesenchymal stem/progenitor cells' hallmarks, CFUs, and multilineage differentiation potential. Intracellular β-catenin significantly declined in the stimulated groups (P<0.001, Friedman test). Cellular proliferation at 72hours was most prominent in the control and inflammatory group [Median cell numbers (Q25/Q75); 6806 (4983/7312) and 5414 (4457/7230), respectively], followed by an upsurge in the retinol group. At 14days, the retinol group exhibited the highest CFUs [Median CFUs (Q25/Q75); 35 (20/58), P=0.043, Wilcoxon signed-rank]. Nanog was most expressed in the inflammatory and retinol group [Median gene expression/PGK1 (Q25/Q75); 0.0006 (0.0002/0.0014) and 0.0005 (0.0003/0.0008)]. Inflammation significantly upregulated Sox2 expression [0.0002 (0.0008/0.0005)], while its expression was diminished in the retinol and inflammatory/retinol group (P<0.001, Friedman test). Inflammatory/retinol group exhibited the highest multilineage differentiation potential. Controlled short-term inflammatory/retinol stimuli activate the Wnt/β-catenin pathway, affecting G-MSCs' pluripotency, proliferation, and differentiation. The present findings provide further insights into the inflammatory-regenerative interactions and their modulation potential for G-MSCs-mediated periodontal repair/regeneration.

A role for dystroglycan in the pathophysiology of acute leukemic cells

AimsPrevious reports have demonstrated that alterations or reduced expression of Dystroglycan (Dg) complex (αDg and βDg subunits) are related to progression and severity of neoplastic solid tissues. Therefore we determined the expression pattern and subcellular distribution of Dg complex in Acute Myeloid Leukemia (AML) primary blasts (M1, M2, and M3 phenotypes), as well as HL-60 and Kasumi-1 leukemia cell lines. Additionally, we evaluated the relative expression of the main enzymes controlling α-Dg glycosylation to ascertain the post-translational modifications in the leukemia cell phenotype. Main methodsPrimary leukemia blasts and leukemia cell lines were processed by confocal analysis to determine the subcellular distribution of α-Dg, β-Dg, and phosphorylated β-Dg (Y892), to evaluate the expression pattern of the different Dg species we performed Western Blot (WB) assays, while the messenger RNA (mRNA) expression of enzymes involved in α-Dg glycosylation, such as POMGnT1, POMT1, POMT2, LARGE, FKTN, and FKRP, were evaluated by qualitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Finally, in an attempt to ameliorate the leukemia cell phenotype, we transfected leukemia cells with a plasmid expressing the Dg complex. Key findingsThe Dg complex was altered in leukemia cells, including decreased mRNA, protein, and α-Dg glycosylated levels, mislocalization of β-Dg, and a diminution of mRNA expression of LARGE in patients leukemia blasts and in cell lines. Interestingly, the exogenous expression of Dg complex promoted filopodial formation, differentiation, and diminished proliferation, attenuating some HL-60 and Kasumi cells characteristics. SignificanceDg complex integrity and balance are required for a proper hematopoietic cell function, in that its disruption might contribute to leukemia pathophysiology.

Ectopic Hard Tissue Formation by Odonto/Osteogenically In Vitro Differentiated Human Deciduous Teeth Pulp Stem Cells.

There have been many attempts to use the pulp tissue from human deciduous teeth for dentin or bone regeneration. The objective of this study was to determine the effects of odonto/osteogenic in vitro differentiation of deciduous teeth pulp stem cells (DTSCs) on their in vivo hard tissue-forming potential. DTSCs were isolated from extracted deciduous teeth using the outgrowth method. These cells were exposed to odonto/osteogenic stimuli for 4 and 8 days (Day 4 and Day 8 groups, respectively), while cells in the control group were cultured in normal medium. The in vitro differentiated DTSCs and the control DTSCs were transplanted subcutaneously into immunocompromised mice with macroporous biphasic calcium phosphate and sacrificed at 8 weeks post-implantation. The effect of odonto/osteogenic in vitro differentiation was evaluated using alkaline phosphatase (ALP) staining and quantitative reverse transcription polymerase chain reaction (RT-PCR). The in vivo effect was evaluated by qualitative RT-PCR, assessment of ALP activity, histologic analysis, and immunohistochemical staining. The amount of hard tissue was greater in Day 4 group than Day 8 group (p = 0.014). However, Day 8 group generated lamellar bone-like structure, which was immunonegative to anti-human dentin sialoprotein with significantly low expression level of DSPP compared with the control group (p = 0.008). This study demonstrates that odonto/osteogenic in vitro differentiation of DTSCs enhances the formation of bone-like tissue, instead of dentin-like tissue, when transplanted subcutaneously using MBCP as a carrier. The odonto/osteogenic in vitro differentiation of DTSCs may be an effective modification that enhances in vivo bone formation by DTSCs.

Evaluation of Xist expression in preattachment equine embryos

Until now, sex determination in equine embryos has been performed by detection of Y-chromosome-specific sequences only. In the present study, expression of a Barr-body-specific marker, the X-inactivated-specific transcript (Xist) gene, whose gene product consists of RNA which coats and thereby inactivates one of the X chromosomes, was investigated in equine embryos produced in vivo. Preattachment embryos at different times after ovulation (Day 8: n = 9; Day 10: n = 12; Day 12: n = 15) were analyzed for Xist RNA expression using quantitative and qualitative reverse transcription-polymerase chain reaction (RT-PCR). Female and male primary equine dermal cell cultures were used as positive and negative controls, respectively. Embryos tested negative for Xist were evaluated for expression of the male-specific eSRY gene by qualitative PCR at the DNA level. From 36 embryos assessed by qualitative RT-PCR, 18 showed positive Xist expression (50%). From 29 embryos tested by quantitative RT-PCR, 16 showed positive Xist expression (55%). All of the Xist-negative equine embryos tested by quantitative PCR were positive for eSRY. We also demonstrated by strand-specific RT-PCR that in the horse, as in humans, the counter transcript Tsix seems to be truncated not reaching Exon 1. In contrast to many other species, neither Xist nor Tsix was expressed in equine male testicular tissue. The results demonstrate that expression of Xist is restricted to female equine embryos. Xist can thus be considered an X-inactivation-specific marker which can be used in concert with Y-specific markers for sex determination.

Reciprocal expression of survivin and SMAC/DIABLO in primary breast cancer

There is an intimate relation between disturbance in survivin and SMAC/DIABLO expressions and cancer formation in many tissues; however, this was not confirmed in the primary breast cancer. The aim of this study was to evaluate the relationship between survivin and SMAC/DIABLO mRNA expressions in primary breast cancer using RT-PCR and their relationship to some of the risk factors (age, family history, breast-feeding, use of contraceptive pills and hormonal receptors). Breast cancer tissues were studied for the detection of the mRNA expression of survivin and SMAC/DIABLO. This was done by qualitative reverse transcription-polymerase chain reaction (RT-PCR). SMAC/DIABLO mRNA was detected by RT-PCR only in 1 sample in breast cancer tissues (6.25%). However, it was expressed in 16 out of 25 (64%) of the benign tumor tissues (P=0.00). In contrast, survivin mRNA was highly expressed in breast cancer tissues, 36 out of 48 (75%) (P=0.00), while its expression was very low in benign tumor tissues, only 1 out of 37 (2.7%) (P=0.00). Expressions of SMAC/DIABLO and survivin were significantly reciprocal in breast cancer and benign tumor tissues. SMAC/DIABLO inhibits apoptosis of breast cancer cells by suppression of survivin. These two genes probably form an important link in the signaling pathway of formation of breast cancer cells, which may be chosen as therapeutic targets in the primary breast cancer.

A randomized trial of 24 versus 48 weeks of peginterferon α-2a in patients infected with chronic hepatitis C virus genotype 2 or low viral load genotype 1: a multicenter national study in Japan

In a country such as Japan with the average age of patients with chronic hepatitis C treated with antivirals sometimes well above 60years, the standard combination therapy is not well tolerated. In this randomized, prospective, controlled trial, we investigated the efficacy of 24-week peginterferon α monotherapy for easy-to-treat patients. A total of 132 patients chronically infected with hepatitis C virus (HCV) genotype 2 (n=115) or low viral load HCV genotype 1 (<100kIU/ml, n=17) were treated with peginterferon α-2a (180μg/week). Patients with a rapid virological response (RVR, HCV RNA negative or <500IU/ml at week 4) were randomized for a total treatment duration of 24 (group A) or 48 (group B) weeks. Patients who did not show RVR (group C) were treated for 48weeks. Sustained virological response (SVR) was assessed by qualitative reverse-transcription polymerase chain reaction. One hundred eight of 132 (82%) patients with RVR were randomized. SVR rates were 60% (group A), 79% (group B), and 27% (group C), respectively. Similar SVR rates were achieved in patients infected with HCV genotype 2 with low pretreatment viral load (<1000kIU/ml) in group A (81%) and group B (79%) (P=0.801), whereas in those with higher viral load (≥1000kIU/ml), a lower SVR rate was identified in group A (26%) than in group B (67%) (P=0.041). In conclusion, in patients infected with HCV genotype 2 and pretreatment viral load below 1000kIU/ml who achieve RVR, 24-week treatment with peginterferon α-2a alone is clinically sufficient. Those who show no RVR or have higher baseline viral load, require alternative therapies.