Journal of bioscience and bioengineering最新文献

We have identified and characterized a circular bacteriocin, termed garvicin SC (GarSC), produced by Lactococcus garvieae ABG0038 isolated from pine cones. Genome analysis of L. garvieae ABG0038 revealed that GarSC was a variant of the circular bacteriocin, garvicin ML (GarML), caused by an amino acid substitution, and predicted that GarSC was produced through a biosynthetic mechanism very similar to that of GarML. The two circular bacteriocins were purified and characterized for activity, and several differences were observed in pH stability, enzyme sensitivity, and antimicrobial activity. In particular, GarSC showed excellent stability in the basic pH range, which might extend the range of garvicin's application to one broader than that of GarML.

The multi-attribute method (MAM) has been recognized as an optimal tool for quality control in biotherapeutics. New peak detection (NPD) is one of the functions of MAM for detecting unexpected differences in samples and is an essential feature required for replacing conventional methods with MAM. Not only used for release and stability testing, NPD is also considered valuable for evaluating comparability and identifying product quality attributes in the research phase. Although many researchers consider the processing parameter the key to NPD, the details of the decision-making process are unclear. Besides specific instruments and software packages has been used almost exclusively, yet the differences in NPD function between other choices have not been confirmed. Thus, this research aimed to confirm the applicability of our original decision-making approach for NPD processing parameters using two different systems. After optimization for each, under a condition that detected crucial differences and did not return false positives, they differed in the reproducibility of the results. To our knowledge, this was the first time the comparison of NPD results of different systems has been published, and the eligibility of processing methods was evaluated in light of the equivalency of conventional methods' detectability. The findings suggested that the capability of NPD is determined not only by the instrument's resolution but also by the software's capability. Our approach for optimizing the NPD processing parameter is deemed widely applicable and practical in developing therapeutic proteins. The revealed difference will help us select the fit-for-purpose system.

The main causes of high mortality in lung cancer patients are the malignant growth and migration of cancer cells. This study aims to investigate the underlying mechanisms of low-temperature plasma-activated medium (PAM) treating human lung cancer (HLC). Changes in the levels of reactive oxygen and nitrogen species both inside and outside the cells were evaluated. Our results showed that prolonged PAM exposure decreased cell viability, raised intracellular reactive oxygen species levels, and hindered cell migration while reducing mitochondrial membrane potential. Protein analysis revealed PAM increased GSK-3β and p-β-catenin expression but decreased PI3K, AKT, p-AKT, p-GSK-3β, Wnt, and β-catenin levels, thereby inhibiting the epithelial-mesenchymal transition. These findings suggest PAM suppresses HLC cells proliferation and migration by blocking the PI3K/AKT-Wnt pathway. The study will provide a valuable theoretical basis for future low-temperature plasma treatment, thereby improving the survival rates and prognosis of lung cancer.

Intestinal bacteria play a crucial role in human health, for example, by maintaining immune and metabolic homeostasis and protecting against pathogens. Survival in the human intestine depends on the bacterium's ability to utilize complex carbohydrates. Some species are known to use host-derived glycans; for example, Bifidobacteria can utilize O-glycan of mucin. However, there are few studies on intestinal bacteria utilizing host-derived N-glycan. Here, we identified the mechanism underlying the breakdown and utilization of complex-type N-glycan by the human intestinal bacterium Barnesiella intestinihominis. A growth assay showed that B. intestinihominis can utilize complex-type N-glycan as a carbon source, while RNA-seq analysis identified enzymes and transporters involved in the mechanism of N-glycan breakdown. In particular, the expression of three genes encoding glycoside hydrolase 85 endo-β-N-acetylglucosaminidase (endo-BIN1, endo-BIN2, and endo-BIN3) rose markedly in bacterial cells cultured in complex-type N-glycoprotein medium. We also found that the susC and susD genes, encoding the SusC/SusD membrane complex, form a gene cluster with endo-BIN genes, suggesting that SusC/SusD is involved in transportation of the glycan into the cell. Other genes encoding exo-type glycoside hydrolase enzymes showed elevated expression in cells grown in complex-type N-glycoprotein medium, suggesting that these enzymes function in further degradation of glycan for metabolism by the bacterium. Collectively, these findings suggest the survival strategy of an intestinal bacterium that has a unique metabolic pathway to use host-derived complex-type N-glycan as a nutrient.

Bioaugmentation with electrochemically active bacteria (EAB) has been suggested useful for improving the performance of bioelectrochemical systems (BESs) for sustainable energy generation, while its success is dependent on EAB introduced into the systems. Here we report on the isolation of a novel EAB, Geobacter sulfurreducens strain 60473, from microbes that colonized on an anode of a sediment microbial fuel cell. This strain is highly adhesive to graphite electrodes, forms dense biofilms on electrode surfaces, and generates high current densities in BESs. When microbial electrolysis cells (MECs) inoculated with paddy-field soil and fed starch as the major organic substrate were augmented with strain 60473, Geobacter bacteria predominantly colonized on anodes, and MEC performances, including current generation, hydrogen production and organics removal, were substantially improved compared to non-bioaugmented controls. Results suggest that bioaugmentation with electrode-adhesive EAB, such as strain 60473, is a promising approach for improving the performance of BESs, including MECs treating fermentable organics and biomass wastes.

Non-conventional yeasts are increasingly being used in the production of fermented beverages owing to their ability to create unique and high-quality products. The yeast Lachancea thermotolerans is of great industrial significance, particularly in the production of l(+)-lactic acid, which is beneficial for acidifying wine, beer, and potentially sake. To explore its potential in sake brewing, three L. thermotolerans strains were isolated from natural environments and their physiological and fermentative characteristics were examined. The isolates surpassed the L. thermotolerans type strain (NBRC 1985) in lactic acid production under various culture conditions and exhibited comparable growth rates to that of Saccharomyces cerevisiae at 15-20 °C. Sake brewing tests using these isolates yielded approximately 3500 ppm of lactic acid, with a slightly lower production of aroma components compared to that produced by sake yeast, and an ethanol content of approximately 11-12 % was obtained. Reverse transcription-quantitative polymerase chain reaction revealed variable expression in putative lactate dehydrogenase genes depending on the culture conditions. Our findings suggest that L. thermotolerans strains can be used in sake brewing to produce unique sake.

Antibiotic resistance genes (ARGs) present in urban rivers have the potential to disseminate antibiotic-resistant bacteria into other environments, posing significant threats to both ecological and public health. Although metagenomic analyses have been widely employed to detect ARGs in rivers, our understanding of their dynamics across different seasons in diverse watersheds remains limited. In this study, we performed a comprehensive genomic analysis of the Kanda River in Japan at 11 sites from upstream to estuary throughout the year to assess the spread of ARGs and their associations with bacterial communities. Analysis of 110 water samples using the 16S rRNA gene revealed variations in bacterial composition corresponding to seasonal changes in environmental parameters along the river. Shotgun metagenomics-based profiling of ARGs in 44 water samples indicated higher ARG abundance downstream, particularly during the summer. Weighted gene co-expression network analysis (WGCNA) linking bacterial lineages and ARGs revealed that 12 ARG subtypes co-occurred with 128 amplicon sequence variants (ASVs). WGCNA suggested potential hosts for ErmB, ErmF, ErmG, tetQ, tet (W/N/W), aadA2, and adeF, including gut-associated bacteria (e.g., Prevotella, Bacteroides, Arcobacter) and indigenous aquatic microbes (e.g., Limnohabitans and C39). In addition, Pseudarcobacter (a later synonym of Arcobater) was identified as a host for adeF, which was also confirmed by single cell genomics. This study shows that ARG distribution in urban rivers is affected by seasonal and geographical factors and demonstrates the importance of monitoring rivers using multiple types of genome sequencing, including 16S rRNA gene sequencing, metagenomics, and single cell genomics.

Banana is the fourth most consumed crop worldwide, and its high economic value and health benefits have made it very popular. Bananas are climacteric fruits that ripen after harvesting. It has been reported that the endogenous substances in bananas change significantly during the ripening process. This study focused on levels of gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD), an enzyme that catalyzes the synthesis of GABA, which reportedly fluctuates during the ripening stage. Previous studies have shown that GAD expression is associated with banana ripening; however, changes in its distribution during ripening have not been verified. This study aimed to clarify the relationship between GABA and GAD during ripening of ethylene-treated bananas. Visualization of the localization of endogenous GABA and GAD was performed using mass spectrometry imaging. To visualize GAD reaction, a glutamate-d₃ (labeled substrate) was supplied to the sample, and a GABA-d₃ (labeled product) was regarded as the localization of the enzymatic reaction. Liquid chromatography-mass spectrometry was also used to confirm the amount of GABA and activity of the GAD. This will allow us to clarify the direct relationship between GABA and GAD and to understand the role of the GAD reaction in phytohormones.

Single-stranded DNA-binding protein (SSB) is essential to DNA replication, DNA repair, and homologous genetic recombination. Our previous study on the crystal structure of a C-terminally truncated SSB from Helicobacter pylori, HpSSBc, in complex with single-stranded DNA (ssDNA) suggests that several aromatic residues, including Phe37, Phe50, Phe56, and Trp84, were involved in ssDNA binding. To investigate the importance of these aromatic residues, the binding activity of four site-directed HpSSB mutants, including F37A HpSSB, F50A HpSSB, F56A HpSSB, and W84A HpSSB, was compared to that of wild-type HpSSB and HpSSBc by means of electrophoresis mobility shift assay (EMSA), tryptophan quenching fluorescence titration, and surface plasmon resonance (SPR). Molecular docking and molecular dynamic (MD) simulation of a F37A and a quadruple mutation model of HpSSBc support that the ssDNA-HpSSBc complex was destabilized when either one or four of the aromatic residues were mutated. The findings of this study suggest that mutation of the phenylalanine and tryptophan residues within the oligonucleotide-binding domain significantly diminished the ssDNA binding capability of HpSSB, highlighting the crucial role these aromatic residues play in the binding of ssDNA by HpSSB.

Liver biobanking is a promising approach that saves the lives of patients with end-stage liver disease. Cryopreservation based on vitrification enables semi-permanent organ preservation, contributing to overcome the shortage of donors for liver transplants. A technical challenge in cryopreservation of transplantable organs lies in thawing methodology, and conventional convective warming cannot maintain the glassy state during thawing because of the large temperature gradient between the inner and outer parts of the organs, leading to ice formation and damage of cells in the organ. Nanowarming, in which magnetic nanoparticles are dispersed in a vitrification solution and heated by exposure of alternating magnetic field, can achieve uniform and rapid heating of organs. Herein, we report that amphiphilic phospholipid polymers composed of 2-methacryloyloxyethyl phosphorylcholine and n-butyl methacrylate can function as a cryoprotectant for nanowarming. The amphiphilic phospholipid polymers enhanced the viability of primary rat hepatocytes after vitrification. Moreover, the polymers enhanced the dispersion stability of magnetic nanoparticles in vitrification solution, and the perfusion of the vitrification solution with magnetic nanoparticles into rat livers through portal vein provided uniform distribution of the nanoparticles in the liver. After perfusion, the vitrified liver was successfully thawed rapidly and uniformly by nanowarming, which maintained tissue integrity and cell viability.