Tuan-Nghia Phan

Fusion tags are widely employed to enhance recombinant protein expression, solubility, and purification. However, efficient tag removal is often necessary to obtain proteins in their native form. In this study, SARS-CoV-2 3C-like protease (3CLpro) was evaluated as a sequence-specific protease for fusion-tag removal using a recombinant fusion protein substrate under standard protein purification conditions. A substrate containing the canonical recognition sequence AVLQ↓SG was efficiently cleaved by 3CLpro, whereas no detectable cleavage was observed for a variant substrate harboring the sequence TVRLQ↓SG. Biochemical characterization demonstrated that 3CLpro maintained cleavage activity across a range of pH values and temperatures and in the presence of reagents commonly used for protein purification and stabilization. 3CLpro-mediated cleavage was successfully integrated into different fusion-protein processing workflows, including in-solution cleavage, on-column cleavage, and dialysis-coupled cleavage. To further evaluate the practical utility of 3CLpro, a modular expression vector (pET_RPLa) incorporating a 3CLpro cleavage site was constructed and validated using the light chain of botulinum neurotoxin serotype A (LC-BoNT/A) as a model protein. Following expression and purification, the fusion protein (6 × His-GST-AVLQSG-LC-BoNT/A) retained SNAP25-cleaving activity and was efficiently processed by 3CLpro. Together, these results support the potential use of SARS-CoV-2 3CLpro as a versatile sequence-specific protease for fusion-tag removal and recombinant protein processing.

Botulinum neurotoxins (BoNTs) comprise a highly diverse group of seven serotypes (from A-G) and over 40 subtypes worldwide. Previous primer- and probe-based nucleic acid amplification tests (NAATs) for detection of BoNT encoding genes are challenged by high levels of nucleotide polymorphism both across and within subtypes. In this study, multiple BoNT gene sequences were aligned to identify highly conserved regions for the design of new primers that enable the detection of all seven serotypes under the same conditions. Specific primer sets were designed and validated using in silico , conventional and real-time PCR with constructed plasmids carrying the target fragments and spiked food matrices. The established procedure achieved highly specific and sensitive detection of BoNT serotypes A–G with sensitivity of 10 copies/reaction and a total turnaround time of approximately 1.5 hours. The procedure also eliminated the carryover PCR product by using uracil-N-glycosylase in combination with dUTP in the assay reaction mix. This study provides an alternative NAAT with higher coverage and compliments the traditional mouse bioassays in enhancing global botulism surveillance capabilities.

This study aimed to characterize and determine the distribution of proteases in various anatomical parts of Penaeus monodon using mainly electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide gels impregnated with casein (casein-SDS-PAGE) and specific protease inhibitors. Twelve caseinolytic bands designated A-L, with estimated molecular masses of 11.4-49.6 kDa were detected by SDS-PAGE, and were most abundant in the hepatopancreas. Among the 12 bands, E, J, K and L were predominant, A, G and H were identified as chymotrypsin-like proteases, D, E, I, J, K and L were identified as trypsin-like proteases, and B, C and F were identified as serine proteases. The proteases were most active at 50°C and pH 7.0. The proteolytic and trypsin-like activities of Penaeus monodon were most abundant in hepatopancreas extracts and least abundant in extracts from the haemolymph. Trypsin-like proteases in the hepatopancreas extract were found to activate prophenoloxidase (proPO) to form phenoloxidase (PO).

Aims We investigated the potential cooperative effects of carotenoid‐producing Bacillus aquimaris SH6 and non‐pigmented B. subtilis SH23 on white‐leg shrimp growth and health. Methods and Results SH6, SH23, and a combination of both spores (1 × 10⁶ CFU g⁻¹ pellet) were administered in shrimp. The growth rate (2.36% day⁻¹), red‐colour score (25), and astaxanthin concentration (3.5 µg g⁻¹ shrimp) were maximum in two‐spore‐administered shrimp. Immune‐related Rho mRNA expression level and phenoloxidase and superoxidase dismutase activities were higher in two‐spore‐administered shrimp than in control shrimp, with Rho mRNA expression level being 55‐fold higher in two‐spore‐administered shrimp than in SH6‐administered shrimp and phenoloxidase activity being 1.2‐fold higher in two‐spore‐administered shrimp than in SH23‐administered shrimp. Although live SH6 count was 2.7‐fold lower, SH6 germination level was 3.5‐fold higher in the combination group than in SH6 group. Conclusions When both SH6 and SH23 spores were administered, SH6 spore germination was enhanced and cooperative improvement were seen in growth, astaxanthin level, and red‐colour score of white‐leg shrimp; however, immune‐related parameters were induced in a non‐cooperative manner. Significance and Impact of Study This is the first report showing the cooperative probiotic activities of Bacillus strains and their possible mechanisms in a shrimp model.

Leigh syndrome is a rare inherited, heterogeneous and progressive neurometabolic disorder that is mainly caused by specific mutations in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). The present study reported a case of childhood Leigh syndrome with a point mutation at bp 8,993 in the mitochondrial ATPase6 gene. A 21-month-old male child had developed epilepsy, muscular weakness and vomiting, which was accompanied by high fever. Magnetic resonance imaging indicated typical characteristics of Leigh syndrome, including a symmetric abnormal signal in the dorsal medulla oblongata and Sylvian fissure enlargement in association with an abnormal signal in the periventricular white matter and in the putamina and caudate heads. The diagnosis was further supported with genetic tests including polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), sequencing, and quantitative PCR. The patient was found to carry a mitochondrial T8993C (m.T8993C) mutation in peripheral blood with 94.00 +/- 1.34% heteroplasmy. Eight of his relatives were also subjected to quantification of the m.T8993C mutation. The percentages of heteroplasmy in samples taken from the grandmother, mother, aunt, cousin 1, and cousin 2 were 16.33 +/- 1.67, 66.81 +/- 0.85, 71.66 +/- 3.22, 87.00 +/- 1.79, and 91.24 +/- 2.50%, respectively. The mutation was not found in samples taken from the father, the husband of the aunt, or the grandfather of the patient. The obtained data showed that the mutation was maternally inherited and accumulated through generations. Even though the heteroplasmy levels of his mother, aunt, cousin 1, and cousin 2 were relatively high (66.81-91.24%), they remained asymptomatic, indicating that the threshold at which this mutation shows effects is high. To the best of our knowledge, this is the first report of a case of Leigh syndrome in a Vietnamese individual harboring a mtDNA mutation at the 8,993 bp site, and showing a correlation between the heteroplasmy and clinical phenotype. These findings may be useful in helping to improve the clinical diagnosis and treatment of Leigh syndrome.

In this study, Bacillus subtilis spores expressing a chimeric protein, CotB‐VP28, were used as a probiotic vaccine to protect black tiger shrimps (Penaeus monodon) against white spot syndrome virus (WSSV) infection. Oral administration of pellets coated with CotB‐VP28 spores (at ≥1 × 10⁹ CFU per g pellet) to shrimps induced immune‐relating phenoloxydase activity (PO) in shrimps after 14 days of feeding (prior challenge) and at day 3 post challenge (1·26 and 1·70 fold increase respectively). A 75% protection rate was obtained by continuous feeding of the spore‐coated pellets at ≥1 × 10⁹ CFU per g for 14 days prior to WSSV challenge and during all the postchallenge period. Even when the amount of CotB‐VP28 spores in feed pellets was reduced down to ≥5 × 10⁷ CFU per g and ≥1 × 10⁶ CFU per g, relatively high protection rates of 70 and 67·5%, respectively, were still obtained. By contrast, feeding pellets without spores (untreated group) and with naked spores (PY79 group) at ≥1 × 10⁹ CFU per g could not protect shrimps against WSSV. These data suggest that supplementation of CotB‐VP28 spores at low dose of ≥1 × 10⁶ CFU per g could be effective as a prophylactic treatment of WSS for black tiger shrimps. Significance and Impact of the Study This study reports the protective efficacy of Bacillus subtilis CotB‐VP28 spores on black tiger shrimps (Penaeus monodon) against white spot syndrome virus infection. Oral administration of pellets coated with CotB‐VP28 spores (≥1 × 10⁹ CFU per g) conferred 75% protection after white spot syndrome virus challenge. Even after reducing CotB‐VP28 spores in feed pellets to ≥1 × 10⁶ CFU per g, 67·5% protections was still obtained. These data indicate that supplementation of CotB‐VP28 spores at a low dose of ≥1 × 10⁶ CFU per g could be effective in prophylaxis against white spot syndrome in black tiger shrimps.