Icarust, a real-time simulator for Oxford Nanopore adaptive sampling.

MOTIVATION: Oxford Nanopore Technologies (ONT) sequencers enable real-time generation of sequence data, which allows for concurrent analysis during a run. Adaptive sampling leverages this real-time capability in extremis, rejecting or accepting reads for sequencing based on assessment of the sequence from the start of each read. This functionality is provided by ONT's software, MinKNOW (Oxford Nanopore Technologies). Designing and developing software to take advantage of adaptive sampling can be costly in terms of sequencing consumables, using precious samples and preparing sequencing libraries. MinKNOW addresses this in part by allowing the replay of previously sequenced runs for testing. However, as we show, the sequencing output only partially changes in response to adaptive sampling instructions. Here we present Icarust, a tool enabling more accurate approximations of sequencing runs. Icarust recreates all the required endpoints of MinKNOW to perform adaptive sampling and writes output compatible with current base-callers and analysis pipelines. Icarust serves nanopore signal simulating a MinION or PromethION flow cell experiment from any reference genome using either R9 or R10 pore models. We show that simulating sequencing runs with Icarust provides a realistic testing and development environment for software exploiting the real-time nature of Nanopore sequencing. AVAILABILITY AND IMPLEMENTATION: All code is open source and freely available here-https://github.com/LooseLab/Icarust. Icarust is implemented in Rust, with a docker container also available. The data underlying this article will be shared on reasonable request to the corresponding author.

Vitamin D3 induces stem cell activation via Lgr5-Bmi1 expression and improving mouse colitis histology index.

Conventional therapy for inflammatory bowel disease using long-term anti-inflammatory drugs does not seem to provide optimal results. Adjuvant therapy using vitamin D3 is believed to have an essential role in repairing the colonic mucosa through the activation of colonic stem cells. The aim of this study was to demonstrate the effect of vitamin D3 in mucosal repair through stem cell activation, marked by leucin-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and B lymphoma Mo-MLV insertion region 1 (Bmi1) expression and decrease the mouse colitis histology index (MCHI) score. In this study, 50 Mus musculus strain BALB/c were divided into five groups: negative control group, colitis group, and colitis groups with vitamin D3 administration of 0.2 mcg, 0.4 mcg, and 0.6 mcg per 25 g body weight for seven days. Dextran sulfate sodium (DSS) 5% was used to induce colitis. Lgr5-Bmi1 expression was measured using immunodoublestain fluorescent labeling method. Our data suggested that administration of vitamin D3 significantly increased expression of Lgr5-Bmi1 in the colonic mucosa. The colitis group treated with the highest dose of vitamin D3 (0.6 mcg/25 gram) showed the lowest MCHI score (3.60±0.64) while the lowest dose of vitamin D3 had the highest MCHI score (12.60±1.47). In conclusion, by stimulating stem cells, vitamin D3 administration stimulates mucosal regeneration, as demonstrated by upregulated expression of Lgr5-Bmi-1.

Genome Profiling of SARS-CoV-2 in Indonesia, ASEAN and the Neighbouring East Asian Countries: Features, Challenges and Achievements.

Whole-genome sequencing (WGS) has played a significant role in understanding the epidemiology and biology of SARS-CoV-2 virus. Here, we investigate the use of SARS-CoV-2 WGS in Southeast and East Asian countries as a genomic surveillance during the COVID-19 pandemic. Nottingham-Indonesia Collaboration for Clinical Research and Training (NICCRAT) initiative has facilitated collaboration between the University of Nottingham and a team in the Research Center for Biotechnology, National Research and Innovation Agency (BRIN), to carry out a small number of SARS-CoV-2 WGS in Indonesia using Oxford Nanopore Technology (ONT). Analyses of SARS- CoV-2 genomes deposited on GISAID reveal the importance of clinical and demographic metadata collection and the importance of open access and data sharing. Lineage and phylogenetic analyses of two periods defined by the Delta variant outbreak reveal that: (1) B.1.466.2 variants were the most predominant in Indonesia before the Delta variant outbreak, having a unique spike gene mutation N439K at more than 98% frequency, (2) Delta variants AY.23 sub-lineage took over after June 2021, and (3) the highest rate of virus transmissions between Indonesia and other countries was through interactions with Singapore and Japan, two neighbouring countries with a high degree of access and travels to and from Indonesia.

Molecular Analysis of Colorectal Cancers Suggests a High Frequency of Lynch Syndrome in Indonesia.

There is about three times higher incidence of young patients <50 years old with colorectal cancer, termed EOCRC, in Indonesia as compared to Europe, the UK and USA. The aim of this study was to investigate the frequency of Lynch Syndrome (LS) in Indonesian CRC patients. The previously described Nottingham Lynch Syndrome Test (N_LyST) was used in this project. N_LyST is a robust high-resolution melting (HRM)-based test that has shown 100% concordance with standard reference methods, including capillary electrophoresis and Sanger sequencing. The test consisted of five mononucleotide microsatellite markers (BAT25, BAT26, BCAT25, MYB, EWSR1), BRAF V600E mutation and MLH1 region C promoter for methylation (using bisulphite-modified DNA). A total of 231 archival (2016-2019) formalin-fixed, paraffin-embedded (FFPE) tumour tissues from CRC patients collected from Dr. Sardjito General Hospital Yogyakarta, Indonesia, were successfully tested and analysed. Among those, 44/231 (19.05%) were MSI, 25/231 (10.82%) were harbouring BRAF V600E mutation and 6/231 (2.60%) had MLH1 promoter methylation. Almost all-186/197 (99.45%)-MSS cases were MLH1 promoter unmethylated, while there were only 5/44 (11.36%) MSI cases with MLH1 promoter methylation. Similarly, only 9/44 (20.45%) of MSI cases were BRAF mutant. There were 50/231 (21.65%) EOCRC cases, with 15/50 (30%) regarded as MSI, as opposed to 29/181 (16.02%) within the older group. In total, 32/231 patients (13.85%) were classified as "Probable Lynch" (MSI, BRAF wildtype and MLH1 promoter unmethylated), which were enriched in EOCRC as compared to older patients (24% vs. 11.05%, p = 0.035). Nonetheless, 30/50 (76.00%) cases among the EOCRC cases were non-LS (sporadic) and were significantly associated with a left-sided tumour. The overall survival of both "Probable Lynch" and non-LS (sporadic) groups (n = 227) was comparable (p = 0.59), with follow up period of 0-1845 days/61.5 months. Stage, node status, histological grading and ECOG score were significantly associated with patient overall survival (p < 0.005), yet only ECOG was an independent factor for OS (HR: 4.38; 95% CI: 1.72-11.2; p = 0.002). In summary, this study is the first to reveal a potentially higher frequency of LS among CRC patients in Indonesia, which may partially contribute to the reported much higher number of EOCRC as compared to the incidence in the West.

Role of vitamin D in Wnt pathway activation for colonic epithelial cell differentiation.

OBJECTIVES: Inflammatory bowel disease (IBD) is a medical condition that represents a pathological form of inflammation, causing damage to the colonic mucosa. Adjunctive vitamin D therapy may activate the Wnt/ß-catenin pathway that results in cell differentiation and proliferation via stem cell signalling. This study aims to evaluate the effect of vitamin D on ß-catenin and cytokeratin 20 (KRT20) as markers of Wnt pathway activation for colonic cell repair. METHODS: For the experiment, we used 30 musculus mice strains of BALB/c, which were categorised into five groups; the control group (K-) and four other groups, where colitis was induced by dextran sulphate sodium (DSS) for seven days. On the seventh day, the remaining three groups were administered vitamin D with an initial dose of 0.2 µg/25.0 g, 0.4 µg/25.0 g and 0.6 µg/25.0 g until day 14. An objective index of disease activity and a histological score were required as markers of inflammation to evaluate the results of the clinical trials. RESULTS: ß-catenin and KRT20 showed a significant increase in the proliferation index of vitamin D at a dose of 0.6 µg/25.0 g (91.50 ± 4.09 and 48.75 ± 2.28, respectively; p < 0.05) compared to the colitis group. CONCLUSIONS: This study demonstrates that vitamin D could be used as an induction agent of Wnt activation for healing colonic mucosa via multipotent stem cells.

Anti-aging Effects of Mangosteen Peel Extract and Its Phytochemical Compounds: Antioxidant Activity, Enzyme Inhibition and Molecular Docking Simulation.

Skin aging is a complex natural process characterised by gradual diminishment of structural integrity and physiological imbalance of the skin tissue. Since the oxidative stress is tightly corelated to the skin aging process, the usage of antioxidant may serve as favourable strategies for slowing down the skin aging process. Mangosteen is an important fruit commodity and its extract had been extensively studied and revealing various biological activities. Present study aimed to assess the antioxidant and antiaging activity of mangosteen peel extract (MPE) and its phytochemical compounds. MPE and its compounds were subjected to ferric reducing antioxidant power (FRAP), hydroperoxide (H2O2) scavenging, anti-collagenase, anti-elastase, anti-hyaluronidase and anti-tyrosinase assay. MPE has the highest FRAP 116.31 ± 0.60 µM Fe(II) µg-1 extract, IC50 of MPE on H2O2 scavenging activity was 54.61 µg mL-1. MPE also has the highest anti elastase activity at IC50 7.40 µg mL-1. Alpha-mangostin showed potent anti-collagenase activity (IC50 9.75 µg mL-1). While gamma-mangostin showed potent anti-hyaluronidase (IC50 23.85 µg mL-1) and anti-tyrosinase (IC50 50.35 µg mL-1). MPE and its compounds were evaluated in vitro for antioxidant and antiaging activities. Current findings may provide scientific evidence for possible usage of mangosteen extract and its compounds as antioxidant and antiaging agent.

Differences in systemic humoral immune response among Balb/c mice administered with probiotic, LPS Escherichia coli, and probiotic-LPS E. coli.

BACKGROUND AND OBJECTIVES: The aim of this study was to compare the systemic humoral immune responses, including IgE, IgA, IgG and IgM levels in Balb/c mice administered a probiotic, LPS derived from Escherichia coli (E.coli), and probiotic-LPS derived from E. coli. MATERIALS AND METHODS: Thirty-two male Balb/c mice, 10-12 weeks of age with body weight ranging from 30-40 g were randomly divided into four experimental groups (n=8). The treatment regimens were as follows: Group 1, mice did not receive LPS or probiotic (control group); Group 2, mice received only LPS on the first day; Group 3, mice received probiotic for 7 days; Group 4, mice received LPS on the first day, and then continued, with probiotic for 7 days. The mice were observed for 8 days, and then, euthanized the next day (day 9). The serum was collected, and the levels of IgE, IgA, IgG and IgM were measured using ELISA. RESULTS: The humoral immune response was higher in the presence of a probiotic compared to that in the control; IgE (9.02 ± 0.58 units/ml, p=0.000), IgA (3.26 ± 0.99 units/ml, p=0.316), IgG (7.29 ± 0.24 units/ml, p=0.000), and IgM (4.01 ± 2.98 units/ml, p=0.505). When administered with LPS E. coli along with probiotic, the humoral immune response was the highest; IgE (10.68 ± 1.63 units/ml, p=0.000), IgA (8.34 ± 1.47 units/ml, p=0.000), IgG (9.96 ± 0.98 units/ml, p=0.000), and IgM (4.31 ± 1.05 units/ml, p=0.319) compared to the control group. CONCLUSION: Probiotic-LPS derived from E. coli treatment induced a higher humoral immune response (highest IgE, IgA, IgG and IgM levels) compared to treatment with probiotic only.