The production of recombinant proteins in Escherichia coli is often challenged by cytoplasmic expression due to proteolytic degradation and inclusion body formation. Extracellular expression can overcome these problems by simplifying downstream processing and improving protein yields. This study aims to compare the efficiency of two Bacillus subtilis chitosanase signal peptides in mediating extracellular secretion in E. coli. We identified a naturally occurring mutant signal peptide (mCsn2-SP) from B. subtilis CH2 chitosanase (CH2CSN), which is characterized by a deletion of six amino acids in the N-region relative to the signal peptide (Csn1-SP) from B. subtilis CH1 chitosanase (CH1CSN). The CH1CSN and CH2CSN genes were cloned into the pET-11a vector and protein secretion was evaluated in E. coli BL21(DE3) host cells. Expression was induced with 0.1 mM and 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 30 °C for one and three days. CH2CSN showed higher secretion levels compared to CH1CSN under all experimental conditions, especially with 0.1 mM IPTG induction for 3 days, which resulted in a 2.37-fold increase in secretion. Furthermore, it was demonstrated that mCsn2-SP is capable of secreting human Cu,Zn-superoxide dismutase (hSOD) in E. coli BL21(DE3) and successfully translocating it to the periplasmic region.
This study represents the inaugural investigation into the utilisation of a naturally modified signal peptide, thereby corroborating the assertion that signal peptide deletion variants can influence protein secretion efficiency.
Furthermore, the findings substantiate the proposition that such variants can serve as a viable alternative for the secretion of heterologous proteins in E.
coli.
Myocardial infarction (MI) is a type of cardiovascular disease that influences millions of human beings worldwide and has a great rate of mortality and morbidity. Spironolactone has been used as a critical drug for the treatment of cardiac failure and it ameliorates cardiac dysfunction post-MI. Despite these findings, whether there is a relationship between the therapeutic effects of spironolactone and the gut microorganism after MI has not been determined. In our research, we used male C57BL/6 J mice to explore whether the gut microbiota mediates the beneficial function of spironolactone after myocardial infarction.
We demonstrated that deletion of the gut microbiota eliminated the beneficial function of spironolactone in MI mice, displaying exacerbated cardiac dysfunction, cardiac infarct size. In addition, the gut microbiota was altered by spironolactone after sham or MI operation in mice. We also used male C57BL/6 J mice to investigate the function of a probiotic in the myocardial infarction. In summary, our findings reveal a precious role of the gut flora in the therapeutic function of spironolactone on MI.
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Poly-γ-glutamic acid (γ-PGA) is a promising biopolymer for various applications.
In this study, we isolated a novel γ-PGA-producing strain, Bacillus halotolerans F29. The one-factor-at-a-time method was used to investigate the influence of carbon sources, nitrogen sources, and culture parameters on γ-PGA production. The optimal carbon and nitrogen sources were sucrose and (NH4)2SO4, respectively. The optimal culture conditions for γ-PGA production were determined to be 37 °C and a pH of 5.5. Response surface methodology was used to determine the optimum medium components: 77.6 g/L sucrose, 43.0 g/L monosodium glutamate, and 2.2 g/L K2HPO4. The γ-PGA titer increased significantly from 8.5 ± 0.3 g/L to 20.7 ± 0.7 g/L when strain F29 was cultivated in the optimized medium. Furthermore, the γ-PGA titer reached 50.9 ± 1.5 g/L with a productivity of 1.33 g/L/h and a yield of 2.23 g of γ-PGA/g of L-glutamic acid with the optimized medium in fed-batch fermentation. The maximum γ-PGA titer reached 45.3 ± 1.1 g/L, with a productivity of 1.06 g/L/h when molasses was used as a carbon source. It should be noted that the γ-PGA yield in this study was the highest of all reported studies, indicating great potential for the industrial production of γ-PGA.
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Transcriptomics-guided rational engineering in Bacillus licheniformis for enhancing poly-γ-glutamic acid biosynthesis using untreated molasses Rui Han, Qian Zhong, Yifan Yan, Juan Wang, Yifan Zhu, Sha Li, Peng Lei, Rui Wang, Yibin Qiu, Zhengshan Luo, Hong Xu International Journal of Biological Macromolecules.2024; 282: 137514. CrossRef
To elucidate the function of proteorhodopsin in Candidatus Puniceispirillum marinum strain IMCC1322, a cultivated representative of SAR116, we produced RNA-seq data under laboratory conditions. We examined the transcriptomes of six different cultures, including sets of expression changes under constant dark (DD), constant light (LL), and diel-cycled (LD; 14 h light: 10 h dark) conditions at the exponential and stationary/death phases. Prepared mRNA extracted from the six samples was analyzed on the Solexa Genome Analyzer with 36 cycles.
Differentially expressed genes on the IMCC1322 genome were distinguished as four clusters by K-mean clustering and each CDS (n = 2546) was annotated based on the KEGG BRITE hierarchy. Cluster 0 (n = 1573) covered most constitutive genes including proteorhodopsin, retinoids, and glycolysis/TCA cycle. Cluster 1 genes (n = 754) were upregulated in stationary/death phase under constant dark conditions and included genes associated with bacterial defense, membrane transporters, nitrogen metabolism, and senescence signaling. Cluster 2 genes (n = 197) demonstrated upregulation in exponential phase cultures and included genes involved in genes for oxidative phosphorylation, translation factors, and transcription machinery. Cluster 3 (n = 22) contained light-stimulated upregulated genes expressed under stationary/phases. Stringent response genes belonged to cluster 2, but affected genes spanned various cellular processes such as amino acids, nucleotides, translation, transcription, glycolysis, fatty acids, and cell wall components. The coordinated expression of antagonistic stringent genes, including mazG, ppx/gppA, and spoT/relA may provide insight into the controlled cultural response observed between constant light and constant dark conditions in IMCC1322 cultures, regardless of cell numbers and biomass.
Human adenoviruses (HAdVs) can infect various epithelial mucosal cells, ultimately causing different symptoms in infected organ systems. With more than 110 types classified into seven species (A-G), HAdV-D species possess the highest number of viruses and are the fastest proliferating. The emergence of new adenovirus types and increased diversity are driven by homologous recombination (HR) between viral genes, primarily in structural elements such as the penton base, hexon and fiber proteins, and the E1 and E3 regions. A comprehensive analysis of the HAdV genome provides valuable insights into the evolution of human adenoviruses and identifies genes that display high variation across the entire genome to determine recombination patterns. Hypervariable regions within genetic sequences correlate with functional characteristics, thus allowing for adaptation to new environments and hosts. Proteotyping of newly emerging and already established adenoviruses allows for prediction of the characteristics of novel viruses. HAdV-D species evolved in a direction that increased diversity through gene recombination. Bioinformatics analysis across the genome, particularly in highly variable regions, allows for the verification or re-evaluation of recombination patterns in both newly introduced and pre-existing viruses, ultimately aiding in tracing various biological traits such as virus tropism and pathogenesis. Our research does not only assist in predicting the emergence of new adenoviruses but also offers critical guidance in regard to identifying potential regulatory factors of homologous recombination hotspots.
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Acinetobacter baumannii (A. baumannii) causes autophagy flux disorder by degrading STX17, resulting in a serious inflammatory response. It remains unclear whether STX17 can alter the inflammatory response process by controlling autolysosome function. This study aimed to explore the role of STX17 in the regulation of pyroptosis induced by A. baumannii. Our findings indicate that overexpression of STX17 enhances autophagosome degradation, increases LAMP1 expression, reduces Cathepsin B release, and improves lysosomal function.
Conversely, knockdown of STX17 suppresses autophagosome degradation, reduces LAMP1 expression, augments Cathepsin B release, and accelerates lysosomal dysfunction. In instances of A. baumannii infection, overexpression of STX17 was found to improve lysosomal function and reduce the expression of mature of GSDMD and IL-1β, along with the release of LDH, thus inhibiting pyroptosis caused by A.
baumannii. Conversely, knockdown of STX17 led to increased lysosomal dysfunction and further enhanced the expression of mature of GSDMD and IL-1β, and increased the release of LDH, exacerbating pyroptosis induced by A. baumannii. These findings suggest that STX17 regulates pyroptosis induced by A. baumannii by modulating lysosomal function.
Due to the ever-increasing demand for meat, it has become necessary to identify cheap and sustainable sources of protein for animal feed. Feathers are the major byproduct of poultry industry, which are rich in hard-to-degrade keratin protein.
Previously we found that intact feathers can be digested into free amino acids, short peptides, and nano-/micro-keratin particles by the strain Bacillus licheniformis WHU in water, and the resulting feather hydrolysates exhibit prebiotic effects on mice. To explore the potential utilization of feather hydrolysate in the feed industry, we investigated its effects on the gut microbiota of broilers and fish. Our results suggest that feather hydrolysates significantly decrease and increase the diversity of gut microbial communities in broilers and fish, respectively. The composition of the gut microbiota was markedly altered in both of the animals. The abundance of bacteria with potentially pathogenic phenotypes in the gut microbial community of the fish significantly decreased. Staphylococcus spp., Pseudomonas spp., Neisseria spp., Achromobacter spp. were significantly inhibited by the feather hydrolysates. In addition, feather hydrolysates significantly improved proteolytic activity in the guts of broilers and fish. In fish, the expression levels of ZO-1 and TGF-α significantly improved after administration of feather hydrolysates. The results presented here suggest that feather hydrolysates generated by B. licheniformis WHU could be an alternative protein source in aquaculture and could exert beneficial effects on fish.
Cisplatin resistance is the main cause of colorectal cancer (CRC) treatment failure, and the cause has been reported to be
related to Fusobacterium nucleatum (Fn) infection. In this study, we explored the role of Fn in regulating cisplatin resistance
of CRC cells and its underlying mechanism involved. The mRNA and protein expressions were examined by qRT-PCR
and western blot. Cell proliferation and cell apoptosis were assessed using CCK8 and flow cytometry assays, respectively.
Dual-luciferase reporter gene assay was adopted to analyze the molecular interactions. Herein, our results revealed that Fn
abundance and miR-135b expression were markedly elevated in CRC tissues, with a favorable association between the two.
Moreover, Fn infection could increase miR-135b expression via a concentration-dependent manner, and it also enhanced
cell proliferation but reduced apoptosis and cisplatin sensitivity by upregulating miR-135b. Moreover, KLF13 was proved
as a downstream target of miR-135b, of which overexpression greatly diminished the promoting effect of miR-135b or
Fn-mediated cisplatin resistance in CRC cells. In addition, it was observed that upstream 2.5 kb fragment of miR-135b
promoter could be interacted by β-catenin/TCF4 complex, which was proved as an effector signaling of Fn. LF3, a blocker
of β-catenin/TCF4 complex, was confirmed to diminish the promoting role of Fn on miR-135b expression. Thus, it could be
concluded that Fn activated miR-135b expression through TCF4/β-catenin complex, thereby inhibiting KLF13 expression
and promoting cisplatin resistance in CRC.
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acid bacteria (LAB) and identified twelve strains that exclusively produced D-lactic acid in high titers. Of these strains,
Lactobacillus saerimneri TBRC 5746 was selected for further development because of its homofermentative metabolism.
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observed a titer of 99.4 g/L and a yield of 0.90 g/g glucose (90% of the theoretical yield). However, we also observed L-lactic
acid production, which reduced the product’s optical purity. We then used CRISPR/dCas9-assisted transcriptional repression
to repress the two Lldh genes in the genome of L. saerimneri TBRC 5746, resulting in a 38% increase in D-lactic acid
production and an improvement in optical purity. This is the first demonstration of CRISPR/dCas9-assisted transcriptional
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using single-copy core genes for the Bacteria domain. In this study, we established up-to-date archaeal core gene (UACG),
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Two novel Gram-stain-positive, aerobic, non-motile, and yellow-pigmented, irregular rod-shaped bacteria (JY.X269 and
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30183
T (98.6–98.8% 16S rRNA gene sequence similarity), O. ciconiae H23M54T
(98.5–98.6%) and O. murale 01-Gi-040T
(98.3–98.5%). The phylogenetic and phylogenomic trees based on the 16S rRNA gene and 537 core gene sequences, respectively,
revealed that the two strains formed a distinct cluster with the above three species. The digital DNA-DNA hybridization
(dDDH) and average nucleotide identity (ANI) values between our two isolates (JY.X269 and JY.X270T) and other
Ornithinimicrobium species were within the ranges of 19.0–23.9% and 70.8–80.4%, respectively, all below the respective
recommended 70.0% and 95–96% cutoff point. Furthermore, the major cellular fatty acids (> 10.0%) of strains JY.X269 and
JY.X270T were iso-C15:0, iso-C16:0, and summed feature 9. Strain JY.X270T contained MK-8(H4) and ornithine as the predominant
menaquinone and diagnostic diamino acid component within the cell wall teichoic acids. β-cryptoxanthin (
C40H56O) can
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and phenotypic analyses, the two strains could be classified as a novel species of the genus Ornithinimicrobium, for which
the name Ornithinimicrobium cryptoxanthini sp. nov. is proposed (type strain JY.X270T = CGMCC 1.19147T = JCM 34882T).
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Aeromonas veronii is a pathogen which can induce diseases in
humans, animals and aquatic organisms, but its pathogenic
mechanism and virulence factors are still elusive. In this study,
we successfully constructed a mutant strain (ΔascP) by homologous
recombination. The results showed that the deletion
of the ascP gene significantly down-regulated the expression
of associated effector proteins in A. veronii compared
to its wild type. The adhesive and invasive abilities of ΔascP to
EPC cells were 0.82-fold lower in contrast to the wild strain.
The toxicity of ΔascP to cells was decreased by about 2.91-fold
(1 h) and 1.74-fold (2 h). Furthermore, the LD50 of the mutant
strain of crucian carp was reduced by 19.94-fold, and
the virulence was considerably attenuated. In contrast to the
wild strain, the ΔascP content in the liver and spleen was considerably
lower. The titers of serum cytokines (IL-8, TNF-α,
and IL-1β) in crucian carp after the infection of the ΔascP strain
were considerably lower in contrast to the wild strain. Hence,
the ascP gene is essential for the etiopathogenesis of A. veronii
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Aptamers are short single-stranded DNA or RNA oligonucleotides
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aptamers are used in a wide range of biomedical fields,
including the diagnosis of diseases and targeted delivery of
therapeutic agents. The aptamers were selected using a process
called systematic evolution of ligands by exponential enrichment
(SELEX), which has been improved for various research
purposes since its development in 1990. In this protocol,
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high aptamer screening yields using two types of magnetic
beads. Using this method, we isolated an aptamer that
specifically binds to an antimicrobial peptide. We suggest that
by conjugating a small therapeutic-specific aptamer to a gold
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hemorrhagic fever, and dengue shock syndrome. DENV is
transmitted by mosquitoes, Aedes aegypti and A. albopictus,
and is mainly observed in areas where vector mosquitoes live.
The number of dengue cases reported by the World Health
Organization increased more than 8-fold over the last two
decades from 505,430 in 2000 to over 2.4 million in 2010 to
5.2 million in 2019. Although vaccine is the most effective method against DENV, only one commercialized vaccine exists,
and it cannot be administered to children under 9 years of
age. Currently, many researchers are working to resolve the
various problems hindering the development of effective dengue
vaccines; understanding of the viral antigen configuration
would provide insight into the development of effective
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effective vaccine development against DENV is suggested.
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Genetic variation in eukaryotes is mediated during meiosis by
the exchange of genetic material between homologous chromosomes
to produce recombinant chromosomes. Cohesin is
essential to promote proper chromosome segregation, chromosome
morphogenesis, and recombination in meiotic cells.
Cohesin consists of three main subunits–Smc1, Smc3, and the
kleisin subunit Mcd1/Scc1 (Rec8 in meiosis)–and cohesin accessory
factors. In Saccharomyces cerevisiae, the cohesin regulatory
subunit Pds5 plays a role in homolog pairing, meiotic
axis formation, and interhomolog recombination. In this
study, we examine the prophase functions of Pds5 by performing
physical analysis of recombination and three-dimensional
high-resolution microscopy analysis to identify its roles in
meiosis-specific recombination and chromosome morphogenesis.
To investigate whether Pds5 plays a role in mitoticlike
recombination, we inhibited Mek1 kinase activity, which result ed in switching to sister template bias by Rad51-dependent
recombination. Reductions in double-strand breaks
and crossover products and defective interhomolog recombination
occurred in the absence of Pds5. Furthermore, recombination
intermediates, including single-end invasion
and double-Holliday junction, were reduced in the absence
of Pds5 with Mek1 kinase inactivation compared to Mek1
kinase inactivation cells. Interestingly, the absence of Pds5 result ed in increasing numbers of chromosomes with hypercompaction
of the chromosome axis. Thus, we suggest that
Pds5 plays an essential role in recombination by suppressing
the pairing of sister chromatids and abnormal compaction
of the chromosome axis.
Citations
Citations to this article as recorded by
RPA interacts with Rad52 to promote meiotic crossover and noncrossover recombination Jeong H Joo, Soogil Hong, Mika T Higashide, Eui-Hwan Choi, Seobin Yoon, Min-Su Lee, Hyun Ah Kang, Akira Shinohara, Nancy Kleckner, Keun P Kim Nucleic Acids Research.2024; 52(7): 3794. CrossRef
Cohesin is required for meiotic spindle assembly independent of its role in cohesion in C. elegans Karen P. McNally, Elizabeth A. Beath, Brennan M. Danlasky, Consuelo Barroso, Ting Gong, Wenzhe Li, Enrique Martinez-Perez, Francis J. McNally, Sarit Smolikove PLOS Genetics.2022; 18(10): e1010136. CrossRef
Yeast polyubiquitin unit regulates synaptonemal complex formation and recombination during meiosis Min-Kyung Jo, Kiwon Rhee, Keun Pil Kim, Soogil Hong Journal of Microbiology.2022; 60(7): 705. CrossRef