Development of a reverse transcription loop-mediated isothermal amplification assay with novel quantitative pH biosensor readout method for SARS-CoV-2 detection.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a molecular amplification method that can detect SARS-CoV-2 in a shorter time than the current gold-standard molecular diagnostic reverse transcription-polymerase chain reaction (RT-PCR). However, previously developed RT-LAMP assays have mostly relied on highly subjective visual colorimetric interpretation. In this study, an RT-LAMP assay was developed with quantitative measurement of reaction pH using a novel portable pH biosensor compared to qualitative colorimetric interpretation and gel electrophoresis, with 57 clinical COVID-19 samples used for validation of the test. The LoD of the assay is 103 copies/µL. The highest sensitivity was found in the qualitative methods (93.75%), while the highest specificity and likelihood ratio was found in the pH sensor (87.5% and 6.72). On the sensor measurement, a significant difference (p < 0.0001) was observed between the average pH of the RT-PCR (+) COVID-19 (6.15 ± 0.27), while the average pH of the RT-PCR (-) samples (6.72 ± 0.22). Correlation analysis revealed a strong correlation (r = 0.78, p < 0.0001) between the Ct values obtained from RT-PCR with the biosensor pH readout. RT-LAMP with the quantitative pH sensor readout method has the potential to be further developed as an objective molecular assay for rapid and simple detection of SARS-CoV-2.

Secretome of Hypoxia-Preconditioned Mesenchymal Stem Cells Promotes Liver Regeneration and Anti-Fibrotic Effect in Liver Fibrosis Animal Model.

<b>Background and Objective:</b> Liver fibrosis (LF) is a most common pathological process characterized by the activation of hepatocytes leading to the accumulation of extracellular matrix (ECM). Hypoxia precondition treated in MSCs (H-MSCs) could enhance their immunomodulatory and regeneration capability, through expressing robust anti-inflammatory cytokines and growth factors, known as H-MSCs secretome (SH-MSCs) that are critical for the improvement of liver fibrosis. However, the study regarding the efficacy and mechanism of action of SH-MSCs in ameliorating liver fibrosis is still inconclusive. In this study, the therapeutic potential and underlying mechanism for SH-MSCs in the treatment of liver fibrosis were investigated. <b>Materials and Methods:</b> A rat model with liver fibrosis induced by CCl₄ was created and maintained for 8 weeks. The rats received intravenous doses of SH-MSCs and secretome derived from normoxia MSCs (SN-MSCs), filtered using a tangential flow filtration (TFF) system with different molecular weight cut-off categories, both at a dosage of 0.5 mL. The ELISA assay was employed to examine the cytokines and growth factors present in both SH-MSCs and SN-MSCs. On the ninth day, the rats were euthanized and liver tissues were collected for subsequent histological examination and analysis of mRNA expression. <b>Results:</b> The ELISA test revealed that SH-MSCs exhibited higher levels of VEGF, PDGF, bFGF, IL-10, TGF-ß and IL-6 compared to SN-MSCs. <i>In vivo</i>, administration of SH-MSCs notably decreased mortality rates. It also demonstrated a reduction in liver fibrosis, collagen fiber areas, α-SMA positive staining and relative mRNA expression of TGF-ß. Conversely, SN-MSCs also contributed to liver fibrosis improvement, although SH-MSCs demonstrated more favorable outcomes. <b>Conclusion:</b> Current findings suggested that SH-MSCs could improve CCl₄-induced liver fibrosis and decrease α-SMA and TGF-ß expression.

GLUT5, GLUT7, and GLUT11 expression and Bcl-2/Bax ratio on Breast Cancer Cell Line MCF-7 Treated with Fructose and Glucose.

OBJECTIVE: Fructose and glucose are types of sugars commonly found in the diet that have been linked to cancer development. Glucose transporters (GLUTs) are facilitating the uptake of these hexoses. Expression of GLUT5 is higher in cancer cells than in healthy tissue. GLUT7 and GLUT11 facilitate the transport of glucose and fructose; however, their expression in breast cancer has not been extensively studied. The Bcl-2 family has been known as a regulator of the cell's survival and death. Here, we investigated the effect of the fructose-glucose combination in MCF-7 breast cancer cells on the viability, migration, and expression of GLUT5, GLUT7, GLUT11, and Bcl-2/Bax ratio. METHODS: Breast cancer cells MCF-7 were treated with fructose, glucose, and combinations of fructose:glucose (75%:25%, 50%:50%, 25%:75%). Cell viability was assessed using an MTT test. Cell migration was examined with a wound-healing assay. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the mRNA expression of GLUT5, GLUT7, GLUT11, and Bcl-2/Bax. RESULTS: The viability and migration of MCF-7 breast cancer cells elevated when treated with a combination of fructose and glucose, and glucose alone, compared to fructose alone. The expression levels of GLUT5 and GLUT7 were highest in combination of fructose:glucose (75%:25%). Conversely, the expression of GLUT11 was consistently low across all treated media. The highest Bcl-2/Bax ratio was shown in fructose:glucose combination (25%:75%). CONCLUSION: The viability, migration, and Bcl-2/Bax ratio are enhanced in the combination media with higher glucose. In contrast, when the fructose composition was higher in the media, expression of GLUT5 and GLUT7 increased.

Point-of-Care Saliva Osmolarity Testing for the Screening of Hydration in Older Adults With Hypertension.

OBJECTIVES: Older adults have an elevated risk of dehydration, a state with proven detrimental cognitive and physical effects. Furthermore, the use of diuretics by hypertensive patients further compounds this risk. This prospective study investigated the diagnostic accuracy of point-of-care (POC) salivary osmolarity (SOSM) measurement for the detection of dehydration in hypertensive adults with and without diuretic pharmacotherapy. DESIGN: Prospective diagnostic accuracy study. SETTING: Home visits to patients recruited from 4 community health centers in West Sulawesi, Indonesia. PARTICIPANTS: A total of 148 hypertensive older adults (57 men, 91 women). The mean ages of male and female patients were 69.4 ± 11.4 and 68.1 ± 7.8 years, respectively. METHODS: Hypertensive adults were divided into 2 groups based on the presence of diuretics in their pharmacotherapeutic regimen. First-morning mid-stream urine samples were used to perform urine specific gravity (USG) testing. Same-day SOSM measurements were obtained using a POC saliva testing system. RESULTS: Both USG (P = .0002) and SOSM (P < .0001) were significantly elevated in hypertensive patients with diuretic pharmacotherapy. At a USG threshold of ≥1.030, 86% of diuretic users were classified as dehydrated compared with 55% of non-using participants. A strong correlation was observed between USG and SOSM measurements (r = 0.78, P < .0001). Using a USG threshold of ≥1.030 as a hydration classifier, an SOSM threshold of ≥93 mOsm had a sensitivity of 78.6% and a specificity of 91.1% for detecting dehydration. CONCLUSIONS AND IMPLICATIONS: Hypertensive patients on diuretics have significantly higher first-morning USG and SOSM values, indicating a higher likelihood of dehydration relative to those on other classes of antihypertensive medication. POC SOSM assessment correlates strongly with first-morning USG assessment, and represents a rapid and noninvasive alternative to urinary hydration assessment that may be applicable for routine use in populations with elevated risk of dehydration.

Benefits and challenges in the implementation of virtual laboratory simulations (vLABs) for medical biochemistry in Indonesia.

The pandemic caused major shifts in the delivery of education worldwide. In the teaching of medical biochemistry, the greatest impact was towards the delivery of traditional laboratory simulations. In this study, we highlight the benefits and barriers encountered in the use of virtual laboratories (vLABs) to substitute traditional laboratory practicals. The subjects were a class of 271 medical students at the Faculty of Medicine, Hasanuddin University, all freshman undergoing the Biomedicine Block. The study assessed the use of a commercial vLAB on antibodies and blood typing procedures, which were implemented using our four-step model of vLAB implementation. Collected data include the lecturer-assigned pre- and post-test result, built-in vLAB assessment result of the student first and best attempts, a student perception questionnaire based on a 5-point Likert scale, and an open ended questionnaire regarding student perceptions of the advantages and disadvantages of the vLAB. We observed a remarkable increase of lecturer assigned pre- and post-test scores and built-in first and best attempt scores (p < 0.0001, Wilcoxon signed rank test). A majority of students reported increased motivation when using the vLABs, and favored the ability of mastery through repetition. However, technical and language barriers were highlighted by students during the vLAB implementation. We demonstrate a successful implementation of commercial vLABs in a cohort of non-native English speakers using our four-step approach. Implementation requires strong support from faculty to address technical and language barriers that arise during use of vLABs.

Detection of voluntary dehydration in paediatric populations using non-invasive point-of-care saliva and urine testing.

AIM: Voluntary dehydration, or lack of fluid intake despite water availability, is common in otherwise healthy children, and can lead to adverse effects. Most dehydration biomarkers are impractical for routine assessment in paediatric populations. This study aimed to assess two non-invasive hydration assessment tools, urine specific gravity (USG ) and a novel point-of-care (POC) salivary osmolarity (SOSM) sensor, in healthy children. METHODS: Volunteers were tested by colorimetric USG and a handheld SOSM system. Observed values were compared against previous studies to determine hydration status, as was the concordance between parameters. RESULTS: At the common USG threshold of 1.020, 42.4% of the 139 healthy children were dehydrated. The same prevalence was found using the 70-mOSM cut-off value. Comparative analysis of SOSM at varying USG thresholds demonstrated significantly higher SOSM in dehydrated children with a USG  ≥ 1.030 (P = 0.002). CONCLUSION: At the USG threshold of 1.020 and SOSM threshold of 70 mOSM, 42.4% of healthy children were found to be voluntarily dehydrated. Significantly higher SOSM was observed in dehydrated children (USG ≥ 1.030). As the first study on the utility of POC SOSM measurements for detecting dehydration, these results provide a foundation for future POC characterisation of SOSM in other populations and clinical contexts.

Suppression of carbon tetrachloride-induced liver fibrosis by transplantation of a clonal mesenchymal stem cell line derived from rat bone marrow.

Transplantation of hepatocytes or bone marrow-derived cells has been shown to ameliorate liver fibrosis in animal models, but no direct comparison of relative efficiency has been made. The aim of this study was to compare the efficiency of a bone marrow-derived clonal mesenchymal stem cell line established by us (rBM25/S3) with that of its adipogenic or hepatogenic differentiation derivative for suppression of rat liver fibrosis. After induction of differentiation of rBM25/S3 cells into adipogenic or hepatogenic cells in culture, we intrasplenically transplanted the three types of cells into rats (3 x 10(7) cells/rat) before and 4 weeks after initiation of carbon tetrachloride treatment (1 ml/kg body weight twice a week for 8 weeks) to induce liver fibrosis. Undifferentiated rBM25/S3 cells were the most effective for suppression of liver fibrosis, followed by the adipogenic cells and hepatogenic cells. Expression levels of MMP-2 and MMP-9 were also highest in undifferentiated rBM25/S3 cells. These results indicate that bone marrow-derived clonal mesenchymal stem cell lines are useful for further mechanistic studies on cell-mediated suppression of liver fibrosis and that such cell lines will provide information on an appropriate cell source for transplantation therapy for cirrhosis.

Derivation of hepato-pancreatic intermediate progenitor cells from a clonal mesenchymal stem cell line of rat bone marrow origin.

We have recently established a clonal mesenchymal stem cell line (rBM25/S3) from adult rat bone marrow. The cells have practically unlimited proliferation capacity (over 300 PDL), maintaining multipotency for differentiation. In the present study, we examined the potential for rBM25/S3 cells to differentiate into insulin-secreting cells. When cultured in the presence of HGF and FGF-4 on Matrigel, rBM25/S3 cells expressed genes specific to pancreatic beta-cells as well as those specific to hepatocytes. They still maintained proliferation capacity with a doubling time of approximately 30 h. These hepato-pancreatic intermediate progenitor cells, but not the original undifferentiated rBM25/S3 cells, were induced by the overexpression of PDX-1 to produce significant amounts of insulin in a manner responding to glucose concentration in medium. The present culture system indicates a direction for further studies aimed at the realization of cell transplantation therapy for type I diabetes mellitus.

Isolation of a bone marrow-derived stem cell line with high proliferation potential and its application for preventing acute fatal liver failure.

Transplantation of hepatocytes or hepatocyte-like cells of extrahepatic origin is a promising strategy for treatment of acute and chronic liver failure. We examined possible utility of hepatocyte-like cells induced from bone marrow cells for such a purpose. Clonal cell lines were established from the bone marrow of two different rat strains. One of these cell lines, rBM25/S3 cells, grew rapidly (doubling time, approximately 24 hours) without any appreciable changes in cell properties for at least 300 population doubling levels over a period of 300 days, keeping normal diploid karyotype. The cells expressed CD29, CD44, CD49b, CD90, vimentin, and fibronectin but not CD45, indicating that they are of mesenchymal cell origin. When plated on Matrigel with hepatocyte growth factor and fibroblast growth factor-4, the cells efficiently differentiated into hepatocyte-like cells that expressed albumin, cytochrome P450 (CYP) 1A1, CYP1A2, glucose 6-phosphatase, tryptophane-2,3-dioxygenase, tyrosine aminotransferase, hepatocyte nuclear factor (HNF)1 alpha, and HNF4alpha. Intrasplenic transplantation of the differentiated cells prevented fatal liver failure in 90%-hepatectomized rats. In conclusion, a clonal stem cell line derived from adult rat bone marrow could differentiate into hepatocyte-like cells, and transplantation of the differentiated cells could prevent fatal liver failure in 90%-hepatectomized rats. The present results indicate a promising strategy for treating human fatal liver diseases.