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.

A year after the World Health Organisation declared COVID-19 as a pandemic, much has been learned with respect to SARS-CoV-2 epidemiology, vaccine production and disease treatment. Whole-genome sequencing (WGS) has played a significant role in contributing to our understanding of the epidemiology and biology of this virus. In this paper, we investigate the use of SARS-CoV-2 WGS in Southeast and East Asia and the impact of technological development, access to resources, and demography of individual countries on its uptake. Facilitated by the Nottingham-Indonesia Collaboration for Clinical Research and Training (NICCRAT) initiative, we showcased a bilateral collaboration between the University of Nottingham and the Indonesian Institute of Sciences (LIPI/Lembaga Ilmu Pengetahuan Indonesia) to establish WGS of SARS-CoV-2 using Oxford Nanopore Technology® in Indonesia. Analyses of SARS-CoV-2 genomes deposited on GISAID from Southeast and East Asian countries reveals the importance of collecting clinical and demographic metadata and the importance of open access and data sharing. Lineage and phylogenetic analyses per 1 June 2021 found that: 1) B.1.466.2 variants were the most predominant in Indonesia, with mutations in the spike protein including D614G at 100%, N439K at 99.1%, and P681R at 69.7% frequency, 2) The variants of concern, B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta) were first detected in Indonesia in January 2021, 2) B.1.470 was first detected in Indonesia and spread to the neighbouring regions, and 3) The highest rate of virus transmissions between Indonesia and the rest of the world appears to be through interactions with Singapore and Japan, two neighbouring countries with high degree of access and travels to and from Indonesia. Overall, we conclude that WGS of SARS-CoV-2 using Oxford Nanopore Technology® platforms fits well with the Indonesian context and can catalyse the increase of sequencing rates in the country.

In the past ten years, diabetes prevalence has increased rapidly in low- and middle-income countries due to lifestyle changes. This increased number of diabetic patients leads to the escalation of recombinant insulin demand, which is creating a large global insulin market. Pichia pastoris has appeared as an alternative host to produce recombinant proteins. It has excellent qualifications as an expression host for large-scale production of recombinant proteins for therapeutic use. In this study, we attempted to express the insulin precursor (IP) in P. pastoris. We used a synthetic IP-encoding gene constructed in frame with the truncated α-factor secretory signal and a short C-peptide (DGK) linked A- and B-chain of human insulin in a pD902 expression vector. Several zeocin resistant clones were successfully obtained and verified with PCR using AOX1 specific primers for the integration of the expression cassette into the P. pastoris genome and for the identification of Mut phenotypes. The secretion of IP by the Pichia pastoris clone in the culture supernatant was confirmed using SDS-PAGE, where a single band of the secreted IP with a molecular mass above 6.5 kDa was found.

Pichia pastoris is an alternative yeast expression system to produce heterologous proteins. It has excellent characteristics for an industrial cell factory, such as its ability to reach high cell densities, high secretory capacity, and a low level of native proteins. In our previous study, we introduced a synthetic insulin precursor (IP)-encoding gene constructed in a pD902 expression vector into P. pastoris. However, the P. pastoris recombinant strains expressed a little amount of IP protein. Here, we modified the expression conditions, including inoculum density, methanol concentration, methanol induction time, pH, and temperature, to obtain a higher amount of secreted IP than our previous result. Protein analysis for studying the five parameters was conducted by SDS-PAGE, and the protein amount was estimated by ImageJ applying lysozyme as standard. We successfully enhanced the IP expression by modifying expression conditions. The highest increased of up to 100 folds was achieved when methanol concentration for induction was arranged at 3% (v/v), and the initial cell density for methanol induction was set at an optical density at 600 nm (OD600) of approximately 10 compared to the standard procedure, where the expression was set at 0.5% (v/v) methanol induction and initial cell density at OD600 = 1.

Background Human insulin was the first FDA-approved biopharmaceutical drug produced through recombinant DNA technology. The previous studies successfully expressed recombinant human insulin precursors (HIP) in Pichia pastoris truncated and full-length α-factor recombinant clones. The matting α-factor (Matα), a signal secretion, direct the HIP protein into the culture media. This study aimed to compare the HIP expression from full-length and truncated α-factor secretory signals clones that grown in two types of media, buffered methanol complex medium (BMMY) and methanol basal salt medium (BSMM). Results ImageJ analysis of the HIP’s SDS-PAGE shows that the average HIP expression level of the recombinant P. pastoris truncated α-factor clone (CL4) was significantly higher compared to the full-length (HF7) when expressed in both media. Western blot analysis showed that the expressed protein was the HIP. The α-factor protein structure was predicted using the AlphaFold and visualized using UCSF ChimeraX to confirm the secretion ability for both clones. Conclusions CL4 clone, which utilized a truncated α-factor in the P. pastoris HIP expression cassette, significantly expressed HIP 8.97 times (in BMMY) and 1.17 times (in BSMM) higher than HF7 clone, which used a full-length α-factor secretory signal. This research confirmed that deletion of some regions of the secretory signal sequence significantly improved the efficiency of HIP protein expression in P. pastoris.

Sand sea cucumber (Holothuria scabra) is an aquatic product that belongs to Echinodermata, a habitant in almost all Indonesian seas. The main component of the sea cucumber is protein, one of which is collagen. This study aimed to extract and characterize collagen from the species using the acid-base extraction method. The characterization of sea cucumber collagen includes molecular weight, amino acid components, Fourier transform infrared spectrophotometry, and scanning electron microscopy analysis. This study has successfully extracted collagen from the sample using an extraction system: NaOH 0.1 M; CH3COOH 0.1 M; and distilled water under 45°C treatments, gave 6% yield. The collagen has a molecular weight 110-130 kDa. Based on the infrared spectra, the specific functional groups of the collagen are amide A (3379.29 cm-1), amide B (2924.09 cm-1), amide I (1681.93 cm-1), amide II (1560.41 cm-1), and amide III (1249.87 cm-1). The collagen falls into type I. We suggest an alternative resource of collagen from sand sea cucumber, other than poultry and mammals.   Keywords: characterization, collagen, extraction, fishery, sand sea cucumber

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