Full-length versus truncated α-factor secretory signal sequences for expression of recombinant human insulin precursor in yeast Pichia pastoris: a comparison.

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.

Possible intraindividual evolution of SARS-CoV-2 in nasopharyngeal and anal swab in an octogenarian: a case report.

Introduction: COVID-19 is an emerging infectious disease that remains to be further investigated. Case report: Here, we describe a case of COVID-19 in an octogenarian woman with comorbidities who slowly recovered during hospitalization, but died due to sudden cardiac death after 2 weeks of hospitalization. Her nasopharyngeal and anal swabs returned positive for SARS-CoV-2 by RT-PCR on day 7 of hospitalization. The NGS showed possible intraindividual evolution of virus. The sample from the nasopharyngeal swab yielded a B.1470 variant classified as clade GH. This variant showed mutation in the spike gene D614G; N gene; NS3 gene; NSP2 gene and NSP12 gene. The sample from the anal swab showed similar mutation but with additional point mutation in spike gene S12F and was classified as B.1.465 variant. Conclusions: The possibility of the gastrointestinal tract that served as reservoir for virus mutation accumulation should also be considered and the potential impact of viral fecal transmission in the environment should be further investigated.

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.

The dynamics of circulating SARS-CoV-2 lineages in Bogor and surrounding areas reflect variant shifting during the first and second waves of COVID-19 in Indonesia.

Background: Indonesia is one of the Southeast Asian countries with high case numbers of COVID-19 with up to 4.2 million confirmed cases by 29 October 2021. Understanding the genome of SARS-CoV-2 is crucial for delivering public health intervention as certain variants may have different attributes that can potentially affect their transmissibility, as well as the performance of diagnostics, vaccines, and therapeutics. Objectives: We aimed to investigate the dynamics of circulating SARS-CoV-2 variants over a 15-month period in Bogor and its surrounding areas in correlation with the first and second wave of COVID-19 in Indonesia. Methods: Nasopharyngeal and oropharyngeal swab samples collected from suspected patients from Bogor, Jakarta and Tangerang were confirmed for SARS-CoV-2 infection with RT-PCR. RNA samples of those confirmed patients were subjected to whole genome sequencing using the ARTIC Network protocol and sequencer platform from Oxford Nanopore Technologies (ONT). Results: We successfully identified 16 lineages and six clades out of 202 samples (male n = 116, female n = 86). Genome analysis revealed that Indonesian lineage B.1.466.2 dominated during the first wave (n = 48, 23.8%) while Delta variants (AY.23, AY.24, AY.39, AY.42, AY.43 dan AY.79) were dominant during the second wave (n = 53, 26.2%) following the highest number of confirmed cases in Indonesia. In the spike protein gene, S_D614G and S_P681R changes were dominant in both B.1.466.2 and Delta variants, while N439K was only observed in B.1.466.2 (n = 44) and B.1.470 (n = 1). Additionally, the S_T19R, S_E156G, S_F157del, S_R158del, S_L452R, S_T478K, S_D950N and S_V1264L changes were only detected in Delta variants, consistent with those changes being characteristic of Delta variants in general. Conclusions: We demonstrated a shift in SARS-CoV-2 variants from the first wave of COVID-19 to Delta variants in the second wave, during which the number of confirmed cases surpassed those in the first wave of COVID-19 pandemic. Higher proportion of unique mutations detected in Delta variants compared to the first wave variants indicated potential mutational effects on viral transmissibility that correlated with a higher incidence of confirmed cases. Genomic surveillance of circulating variants, especially those with higher transmissibility, should be continuously conducted to rapidly inform decision making and support outbreak preparedness, prevention, and public health response.

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.

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

A year after the World Health Organisation (WHO) declared COVID-19 as a pandemic, much has been learned regarding 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. Using Oxford Nanopore Technology (ONT), Nottingham-Indonesia Collaboration for Clinical Research and Training (NICCRAT) initiative has facilitated collaboration between the University of Nottingham and a team in Research Centre for Biotechnology, Indonesian Institute of Sciences (Lembaga Ilmu Pengetahuan Indonesia/LIPI) to carry out a small number of SARS-CoV-2 WGS in Indonesia. The ONT offers sequencing advantages that fit within the Indonesian context. Analyses of SARS-CoV-2 genomes deposited on GISAID from Southeast and East Asian countries reveal 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 (VoCs) B.1.1.7 (Alpha), B.1.351 (Beta), and B.1.617.2 (Delta) were first detected in Indonesia in January 2021, 3) B.1.470 was first detected in Indonesia and spread to the neighbouring regions, and 4) 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 a high degree of access and travels to and from Indonesia.