Search

Abstract Background Streptomyces transglutaminase (TGase) is naturally synthesized as zymogen (pro-TGase), which is then processed to produce active enzyme by the removal of its N-terminal pro-peptide. This pro-peptide is found to be essential for overexpression of soluble TGase in E. coli. However, expression of pro-TGase by E. coli requires protease-mediated activation in vitro. In this study, we developed a novel co- expression method for the direct production of active TGase in E. coli. Results A TGase from S. hygroscopicus was expressed in E. coli only after fusing with the pelB signal peptide, but fusion with the signal peptide induced insoluble enzyme. Therefore, alternative protocol was designed by co-expressing the TGase and its pro-peptide as independent polypeptides under a single T7 promoter using vector pET-22b(+). Although the pro-peptide was co-expressed, the TGase fused without the signal peptide was undetectable in both soluble and insoluble fractions of the recombinant cells. Similarly, when both genes were expressed in the order of the TGase and the pro-peptide, the solubility of TGase fused with the signal peptide was not improved by the co-expression with its pro-peptide. Interestingly, active TGase was only produced by the cells in which the pro-peptide and the TGase were fused with the signal peptide and sequentially expressed. The purified recombinant and native TGase shared the similar catalytic properties. Conclusions Our results indicated that the pro-peptide can assist correct folding of the TGase inter-molecularly in E. coli, and expression of pro-peptide prior to that of TGase was essential for the production of active TGase. The co-expression strategy based on optimizing the order of gene expression could be useful for the expression of other functional proteins that are synthesized as a precursor.

To enhance the applied value of transglutaminase (TGase), various methods have been employed to improve its catalytic properties. However, most modifications have targeted the N-terminus, while the role of the C-terminus in determining TGase properties has been overlooked. In this study, we focused on enhancing the thermal stability of Streptomyces hygroscopicus TGase by engineering its C-terminal region. Modeling revealed that the C-terminal loop interacts with the N-terminal loop through hydrogen bonds between Trp331 and N-terminal residues (Asp19, Ala20, Tyr21). Removing the last C-terminal residue (Ser322) had no significant effect on TGase stability, but deleting additional residues (Trp331, Gly330, Gln299) led to inactivation. Substituting Trp331 with Ala reduced TGase's half-life at 50 °C and specific activity by 50% and 70%, respectively, highlighting the importance of C-terminal interactions in TGase stability. We also attempted to fuse three self-assembling amphipathic peptides (SAPs) (EAK16, KL15, ELK16) and a C-terminal sequence (IGCIILT) from Sulfolobus tokodaii RNase HI to TGase. The fusion of IGCIILT increased TGase's half-life by 1.5-fold without affecting specific activity, while the three SAPs had little effect on stability. Structural analysis showed that the fusion of IGCIILT raised TGase's melting temperature by 5.2 °C and altered its tertiary structure. Our results indicate that the C-terminus is important for modulating TGase properties, and fusing "stabilization tags" like IGCIILT at the C-terminus is a promising strategy to enhance thermal stability. [ABSTRACT FROM AUTHOR]

Aim: Respiratory sarcopenia (RS) has been newly defined in a position paper by four professional organizations in Japan, and it is necessary to examine its incidence and influencing factors using this new definition. So far, little work has been undertaken; we therefore conducted a longitudinal study to fill this gap. Methods: Our data were extracted from the China Health and Retirement Longitudinal Study. A total of 4301 older adults with complete data and without RS were chosen in 2011, of whom 3065 were followed up until 2015. The presence of low respiratory muscle strength plus low appendicular skeletal muscles mass was defined as RS based on t. A logistic regression model was used to identify the influencing factors for incident RS. Results: After an average of 4 years of follow‐up, the cumulative incidence of RS was 5.2%, and it was particularly high in participants aged >80 (20.9%). Logistic regression analysis showed that being unmarried/divorced/widowed (odds ratio [OR] = 1.85, 95% confidence interval [CI] = 1.28–2.66, P = 0.001), not having dyslipidemia (OR = 0.35, 95% CI = 0.19–0.65, P = 0.001), having digestive disease (OR = 1.56, 95% CI = 1.11–2.19, P = 0.010), asthma (OR = 2.77, 95% CI = 1.55–4.94, P = 0.001), edentulism (OR = 1.73, 95% CI = 1.24–2.42, P = 0.001), low handgrip strength (OR = 2.82, 95% CI = 1.99–3.99, P < 0.001), or low 5‐m gait speed (OR = 1.92, 95% CI = 1.23–3.01, P = 0.004) were associated with a greater likelihood of developing RS. After further adjustment for age and body mass index, asthma, edentulism, and low handgrip strength remained significant. Conclusions: The identification and management of older adults with the influencing factors described above could be important in preventing RS. Geriatr Gerontol Int 2024; 24: 1015–1021. [ABSTRACT FROM AUTHOR]

Increasing evidence has shown that gut microbiota (GM) was involved in the pathophysiology of musculoskeletal disorders through multiple pathways such as protein anabolism, chronic inflammation and immunity, and imbalanced metabolism. We performed a systematic review and meta-analysis of human studies to evaluate GM diversity differences between individuals with and without sarcopenia, and explore bacteria with potential to become biomarkers. PubMed, Embase and Cochrane library were systematically searched from inception to February 16, 2024. Studies were included if they (1) sampled adults with sarcopenia, and (2) performed GM analysis and reported α-diversity, β-diversity or relative abundance. The methodological quality of included studies and the certainty of evidence were assessed through the Joanna Briggs Institute critical appraisal checklist for analytical cross-sectional studies and the Grades of Recommendation, Assessment, Development and Evaluation (GRADE) Working Group system, respectively. Weighted standardized mean differences (SMDs) and corresponding 95% confidence intervals (CIs) were estimated for α-diversity indices using a fixed-effects and a random-effects model. Beta diversity and the relative abundance of GM were summarized qualitatively. A total of 19 studies involving 6,565 participants were included in this study. Compared with controls, significantly moderate decrease in microbial richness in participants with sarcopenia were found (Chao1: SMD = -0.44; 95%CI, -0.64 to -0.23, I2 = 57.23%, 13 studies; observed species: SMD = -0.68; 95%CI, -1.00 to -0.37, I2 = 66.07%, 5 studies; ACE index: SMD = -0.30; 95%CI, -0.56 to -0.04, I2 = 8.12%, 4 studies), with very low certainty of evidence. Differences in β-diversity were consistently observed in 84.6% of studies and 97.3% of participants. The detailed analysis of the gut microbial differential abundance identified a loss of Prevotellaceae, Prevotella, and Megamonas in sarcopenia compared with non-sarcopenia. In conclusion, sarcopenia was found to be associated with reduced richness of GM, and supplementing intestinal bacteria described above may contribute to preventing and treating this muscle disease. The research protocol was registered and approved in PROSPERO (CRD42023412849). [ABSTRACT FROM AUTHOR]

Insects rely on their innate immune system to eliminate pathogenic microbes. As a system component, cytokines transmit intercellular signals to control immune responses. Growth‐blocking peptide (GBP) is a member of the stress‐responsive peptide family of cytokines found in several orders of insects, including Drosophila. However, the physiological role of GBP in defence against pathogens is not thoroughly understood. In this study, we explored the functions of GBP in a lepidopteran pest, Ostrinia furnacalis. Injection of recombinant O. furnacalis GBP (OfGBP) precursor (proGBP) and chemically synthesised GBP significantly induced the transcription of antimicrobial peptides (AMPs) and other immunity‐related genes including immune deficiency (IMD) and Dorsal. The level of OfGBP mRNA was upregulated after bacterial infection. Knockdown of OfGBP expression led to a decrease in IMD, Relish, MyD88 and Dorsal mRNA levels. OfGBP induced phenoloxidase activity and affected hemocyte behaviours in O. furnacalis larvae. In summary, GBP is a potent cytokine, effectively regulating AMP synthesis, melanization response and cellular immunity to eliminate invading pathogens. [ABSTRACT FROM AUTHOR]

The influence of grinding process on the flotation of copper-nickel sulfide ore was summarized. The results showed that the size, shape, ratio, material of grinding media, grinding environment and other related parameters mainly affected the flotation of copper-nickel sulfide ore by affecting the particle size composition of the grinding products and the electrochemical reaction in the grinding process. The grinding media with specific shape, size and proportion can affect the grinding behavior of sulfide minerals and gangue minerals in the ore, and the grinding efficiency can be improved by specific regulation and control combined with the mechanical properties of the ore, the particle size distribution of the sulfide minerals and the gangue minerals in the grinding products was improved, and the flotation index was improved. The material of the grinding medium mainly affected the flotation of minerals by affecting the electrochemical reaction in the grinding process. Due to the galvanic effect, a large number of hydrophilic compounds will be produced in the grinding with active media such as cast iron, which will cover the surface of sulfide minerals and hinder their flotation. Using inert media such as stainless steel can avoid this and reduce the wear loss. In addition, iron ions produced by cast iron and other media may activate gangue minerals and further deteriorate the flotation index. Grinding in different atmospheres can also affect mineral flotation. In nitrogen atmosphere, the electrochemical reaction of grinding medium was weak, and less hydrophilic compounds were produced. Sulfide minerals can generally obtain higher recovery than grinding in air atmosphere, while grinding in oxygen atmosphere will reduce the floatability of minerals. The difference of grinding methods such as dry grinding and wet grinding can also affect the floatability of sulfide minerals by affecting their surface composition. In general, the grinding process had various effects on the flotation of copper-nickel sulfide ore. When the relevant parameters were adjusted, detailed experimental study should be carried out in combination with the specific ore properties to obtain good flotation index. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinases (MMPs) are crucial for tissue remodeling and immune responses in insects, yet it remains unclear how MMPs affect the various immune processes against pathogenic infections and whether the responses vary among insects. In this study, we used the lepidopteran pest Ostrinia furnacalis larvae to address these questions by examining the changes of immune‐related gene expression and antimicrobial activity after the knockdown of MMP14 and bacterial infections. We identified MMP14 in O. furnacalis using the rapid amplification of complementary DNA ends (RACE), and found that it was conserved and belonged to the MMP1 subfamily. Our functional investigations revealed that MMP14 is an infection‐responsive gene, and its knockdown reduces phenoloxidase (PO) activity and Cecropin expression, while the expressions of Lysozyme, Attacin, Gloverin, and Moricin are enhanced after MMP14 knockdown. Further PO and lysozyme activity determinations showed consistent results with gene expression of these immune‐related genes. Finally, the knockdown of MMP14 decreased larvae survival to bacterial infections. Taken together, our data indicate that MMP14 selectively regulates the immune responses, and is required to defend against bacterial infections in O. furnacalis larvae. Conserved MMPs may serve as a potential target for pest control using a combination of double‐stranded RNA and bacterial infection. [ABSTRACT FROM AUTHOR]

The International GNSS Service analysis centers provide orbit products of GPS satellites with weekly, daily, and sub-daily latency. The most frequent ultra-rapid products, which include 24 h of orbits derived from observations and 24 h of orbit predictions, are vital for real-time applications. However, the predicted part of the ultra-rapid orbits is less accurate than the estimated part and has deviations of several decimeters with respect to the final products. In this study, we investigate the potential of applying machine-learning (ML) and deep-learning (DL) algorithms to further enhance physics-based orbit predictions. We employed multiple ML/DL algorithms and comprehensively compared the performances of different models. Since the prediction errors of the physics-based propagators accumulate with time and have sequential characteristics, specific sequential modeling algorithms, such as Long Short-Term Memory (LSTM), show superiority. Our approach shows promising results with average improvements of 47% in 3D RMS within the 24-h prediction interval of the ultra-rapid products. In the end, we applied the orbit predictions improved by LSTM to kinematic precise point positioning and demonstrated the benefits of LSTM-improved orbit predictions for positioning applications. The accuracy of the station coordinates estimated based on these products is improved by 16% on average compared to those using ultra-rapid orbit predictions. [ABSTRACT FROM AUTHOR]