Evaluating the efficiency of specimen pooling for PCR-based detection of COVID-19.

In the age of a pandemic, such as the ongoing one caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the world faces a limited supply of tests, personal protective equipment, and factories and supply chains are struggling to meet the growing demands. This study aimed to evaluate the efficacy of specimen pooling for testing of SARS-CoV-2 virus, to determine whether costs and resource savings could be achieved without impacting the sensitivity of the testing. Ten previously tested nasopharyngeal and throat swab specimens by real-time polymerase chain reaction (PCR), were pooled for testing, containing either one or two known positive specimens of varying viral concentrations. Specimen pooling did not affect the sensitivity of detecting SARS-CoV-2 when the PCR cycle threshold (Ct) of original specimen was lower than 35. In specimens with low viral load (Ct > 35), 2 of 15 pools (13.3%) were false negative. Pooling specimens to test for Coronavirus Disease 2019 infection in low prevalence (≤1%) areas or in low risk populations can dramatically decrease the resource burden on laboratory operations by up to 80%. This paves the way for large-scale population screening, allowing for assured policy decisions by governmental bodies to ease lockdown restrictions in areas with a low incidence of infection, or with lower-risk populations.

Galactomannan Pentasaccharide Produced from Copra Meal Enhances Tight Junction Integration of Epithelial Tissue through Activation of AMPK.

Mannan oligosaccharide (MOS) is well-known as an effective fed supplement for livestock to increase their nutrients absorption and health status. Pentasaccharide of mannan (MOS5) was reported as a molecule that possesses the ability to increase tight junction of epithelial tissue, but the structure and mechanism of action remains undetermined. In this study, the mechanism of action and structure of MOS5 were investigated. T84 cells were cultured and treated with MOS5 compared with vehicle and compound C, a 5'-adenosine monophosphate-activated protein kinase (AMPK) inhibitor. The results demonstrated that the ability of MOS5 to increase tight junction integration was inhibited in the presence of dorsomorphine (compound C). Phosphorylation level of AMPK was elevated in MOS5 treated group as determined by Western blot analysis. Determination of MOS5 structure was performed using enzymatic mapping together with 1H, 13C NMR, and 2D-NMR analysis. The results demonstrated that the structure of MOS5 is a ß-(1,4)-mannotetraose with α-(1,6)-galactose attached at the second mannose unit from non-reducing end.

Production and purification of mannan oligosaccharide with epithelial tight junction enhancing activity.

BACKGROUND: Mannanan oligosaccharide (MOS) is well-known as effective supplement food for livestock to increase their nutrients absorption and health status, but the structure and identification of bioactive MOS remain unclear. In this study, MOS production was accomplished, using enzymatic hydrolysis of pretreated coconut meal substrate with recombinant mannanase. METHODS: The mannanase gene was cloned from Bacillus subtilis cAE24, then expressed in BL21. Purified Mannanase exhibit stability over a wide range of pH and temperature from pH 6-8 and 4 °C to 70 °C, respectively. SEM analysis revealed that sonication could change the surface characteristic of copra meal, which gave better MOS yield, compared to untreated substrates. The separation and purification of each MOS were achieved using Biogel-P2 column chromatography. Determination of biological active MOS species was also investigated. T84 cells were cultured and treated with each of the purified MOS species to determine their tight junction enhancing activity. RESULTS: Scanning electron microscope imaging showed that pretreatment using sonication could disrupt the surface of copra meal better than grinding alone, which can improve the production of MOS. Pentamer of MOS (M5) significantly increased tight junction integration of T84 cells measured with TEER (p < 0.0001).