Intraspecific Mitochondrial DNA Comparison of Mycopathogen Mycogone perniciosa Provides Insight Into Mitochondrial Transfer RNA Introns.

Mycogone perniciosa is the main causative agent of wet bubble disease, which causes severe damage to the production of the cultivated mushroom Agaricus bisporus around the world. Whole-genome sequencing of 12 isolates of M. perniciosa was performed using the Illumina sequencing platform, and the obtained paired-end reads were used to assemble complete mitochondrial genomes. Intraspecific comparisons of conserved protein-coding genes, transfer RNA (tRNA) and ribosomal RNA (rRNA) genes, introns, and intergenic regions were conducted. Five different mitochondrial DNA (mtDNA) haplotypes were detected among the tested isolates, ranging from 89,080 to 93,199 bp in length. All of the mtDNAs contained the same set of 14 protein-coding genes and 2 rRNA and 27 tRNA genes, which shared high sequence similarity. In contrast, the number, insertion sites, and sequences of introns varied greatly among the mtDNAs. Eighteen of 43 intergenic regions differed among the isolates, reflecting 65 single nucleotide polymorphisms, 76 indels, and the gain/loss of nine long fragments. Intraspecific comparison revealed that two introns were located within tRNA genes, which is the first detailed description of mitochondrial tRNA introns. Intronic sequence comparison within the same insertion sites revealed the formation process of two introns, which also illustrated a fast evolutionary rate of introns among M. perniciosa isolates. Based on the intron distribution pattern, a pair of universal primers and four pairs of isolate-specific primers were designed and were used to identify the five mtDNA types. In summary, the rapid gain or loss of mitochondrial introns could be an ideal marker for population genetics analysis.

Brij-58, a potential injectable protein-stabilizer used in therapeutic protein formulation.

Polysorbates (PSs) are common protein stabilizers used in biotherapeutic formulations. However, PSs are heterogeneous and unstable in liquid protein formulations [1,2]. The purpose of this work is to explore possible alternatives for polysorbate replacements that demonstrate superior protein protection, superior self-stability, low toxicity, and wide applicability. For this purpose, 8 non-ionic surfactants that have not yet been used as excipients in marketed biotherapeutic products were investigated with PS20/80 as the benchmark. Compared with PS20/80, Brij-58 showed better protein protection ability in the mAb1 formulation under forced degradation conditions when examined by visual inspection, SEC, and dynamic lighting scanning. Additionally, Brij-58 has a better inherent stability than PS20/80 in the protein formulation when detected by UPLC-CAD. Moreover, Brij-58 is an inert excipient that does not affect protein bioactivity and conformation. In addition, the LD50 and hemolysis concentration of Brij-58 were determined, which is relatively safe when used as a parenteral injection. Furthermore, Brij-58 was also an effective protein stabilizer for the other two antibody products (IgG4 subtype and bispecific antibody) in the shaking study. In summary, Brij-58 stands out as a promising PS replacement in biotherapeutic formulations with a safe, stable and effective protein-protection profile among candidate surfactants.

In Vitro Expansion of Primary Human Hepatocytes with Efficient Liver Repopulation Capacity.

Transplantation of human hepatocytes (HHs) holds significant potential for treating liver diseases. However, the supply of transplantable HHs is severely constrained by limited donor availability and compromised capacity for in vitro expansion. In response to chronic injury, some HHs are reprogrammed into proliferative cells that express both hepatocyte and progenitor markers, suggesting exploitable strategies for expanding HHs in vitro. Here, we report defined medium conditions that allow 10,000-fold expansion of HHs. These proliferating HHs are bi-phenotypic, partially retaining hepatic features while gaining expression of progenitor-associated genes. Importantly, these cells engraft into injured mouse liver at a level comparable to primary HHs, and they undergo maturation following transplantation in vivo or differentiation in vitro. Thus, this study provides a protocol that enables large-scale expansion of transplantable HHs, which could be further developed for modeling and treating human liver disease.

High-throughput screening for Survivin and Borealin interaction inhibitors in hepatocellular carcinoma.

Survivin, a key member of the chromatin passenger complex (CPC), is often highly expressed in human cancers, making it a promising target for cancer treatment. Out of the numerous reported Survivin inhibitors, YM155 is only one entering clinical trial, but was recently failed in the Phase II trial. It is important to develop Survivin inhibitors with new strategies. We recently reported that both Survivin and its binding protein Borealin in the CPC complex are essential for the development of hepatocellular carcinoma, suggesting that disrupting the interaction between Survivin and Borealin would be a promising strategy. Here, we developed a high-throughput screening method based on bimolecular fluorescence complementation (BiFC) technology in cultured cells, which allowed the identification of small chemical inhibitors specifically blocking the Survivin and Borealin interaction. Primary hits from BiFC were further validated in an in vitro AlphaScreen system, which detects the direct interactions of Survivin and Borealin. Etoposide was identified as one of the effective hits. Direct interaction between Survivin and Etoposide was confirmed by surface plasmon resonance assay, and molecular docking analysis suggested the structural information on how Etoposide inhibits the Survivin and Borealin interaction. These results demonstrate a screening system to identify small molecule chemicals inhibiting Survivin and Borealin interaction. In future, an even larger scale screening may lead to identification of better Survivin and Borealin inhibitors.