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Abstract Cartilage damage caused by injuries or degenerative diseases remains a major challenge in the field of regenerative medicine. In this study, we developed a composite hydrogel system for the delivery of melatonin and menstrual blood stem cells (MenSCs) to treat a rat model of cartilage defect. The composite delivery system was produced by incorporation of melatonin into the gelatin fibers and dispersing these fibers into calcium alginate hydrogels. Various characterization methods including cell viability assay, microstructure studies, degradation rate measurement, drug release, anti-inflammatory assay, and radical scavenging assay were used to characterize the hydrogel system. MenSCs were encapsulated within the nanocomposite hydrogel and implanted into a rat model of full-thickness cartilage defect. A 1.3 mm diameter drilled in the femoral trochlea and used for the in vivo study. Results showed that the healing potential of nanocomposite hydrogels containing melatonin and MenSCs was significantly higher than polymer-only hydrogels. Our study introduces a novel composite hydrogel system, combining melatonin and MenSCs, demonstrating enhanced cartilage repair efficacy, offering a promising avenue for regenerative medicine. Graphical Abstract

Two-component room temperature vulcanizing silicone adhesive RTV566 with a lower elastic modulus has been widely used in precision optomechanical products such as remote sensors and aerospace infrared cameras. However, the silicone adhesive is of poor manufacturability due to its extremely high viscosity, and the bonded joint usually exhibits low bonding strength and requires a long curing time. This paper investigates a way to improve both the adhesion strength and curing efficiency of Invar alloy and optical glass with RTV566 through single-lap experiments. It is found that adding small amounts of acetone and water can significantly reduce the viscosity, enhance the bonding strength, and shorten the curing time. The viscosity can be reduced by 63.4 % and the bonding strength can be improved by 136.4 % with the weight ratio of adhesive to acetone being 20:2. Moreover, a little amount of water in the weight ratio of 100:10:0.1 (adhesive: acetone: water) can shorten the curing time from 7 days to 4 days without harms to the bonding strength and elastic modulus. As to the mechanism, the silicone adhesive can be dissolved by acetone and its sulfuration reaction can be enhanced by water, resulting in good manufacturability and high curing efficiency. This work contributes a novel and easy-to-use method to greatly improve the performances of the bonding process of precision optical structures.

Safe and eco-friendly preservatives are crucial to preventing food spoilage and illnesses, as foodborne diseases caused by pathogens result in approximately 600 million cases of illness and 420,000 deaths annually. ε-Poly-L-lysine (ε-PL) is a novel food preservative widely used in many countries. However, its commercial application has been hindered by high costs and low production. In this study, ε-PL’s biosynthetic capacity was enhanced in Streptomyces albulus WG608 through metabolic engineering guided by multi-omics techniques. Based on transcriptome and metabolome data, differentially expressed genes (fold change >2 or p < 0.05) and differentially expressed metabolites (fold change >1.2 or S. albulus WME10, achieved an ε-PL production rate of 77.16 g/L in a 5 L bioreactor, which is the highest reported ε-PL production to date. These results suggest that the integrative analysis of the transcriptome and metabolome can facilitate the identification of key pathways and genetic elements affecting ε-PL synthesis, guiding further metabolic engineering and thus significantly enhancing ε-PL production. The method presented in this study could be applicable to other valuable natural antibacterial agents.

Abstract Objective To investigate the efficacy of tyrosine kinase inhibitors (TKIs) in the treatment of metastatic renal cell carcinoma (mRCC) with rhabdoid (mRCC‐R) and sarcomatoid (mRCC‐S) differentiations. Materials and Methods In this single‐institutional cohort study, we included patients with RCC with rhabdoid (RCC‐R) and sarcomatoid (RCC‐S) differentiation, who were treated with TKIs after metastasis at our institute from 2013 to 2021. Patient characteristics, treatments, and clinical outcomes were recorded and analyzed. Results We identified 111 patients with RCC‐R or RCC‐S differentiations, of which 23 patients were included in the final analysis. Of the 23 patients, 10 (43.5%) were grouped as mRCC‐R and 13 (56.5%) as mRCC‐S. At a median follow‐up of 40 months, mRCC‐R and mRCC‐S progressed in 7 of 10 and 12 of 13 patients, respectively. In addition, four and eight patients died in the mRCC‐R and mRCC‐S groups, respectively. The median progression‐free survival (PFS) of the two groups was 19 months (mRCC‐R: 95% confidence interval [CI] 4.08–33.92) and 7 months (mRCC‐S: 95% CI 2.03–11.96), while the median overall survival (OS) was 32 months and 21 months, respectively. mRCC‐S had a worse prognosis than mRCC‐R. Based on the univariate Cox regression model, single metastasis or multiple metastasis of tumor, rhabdoid differentiation, and sarcomatoid differentiation were predictors of PFS but not OS. Conclusion The efficacy of TKIs in the treatment of mRCC‐R and mRCC‐S may be different.

Background: Fatty acid metabolism (FAM) is closely connected with tumorigenesis as well as disease progression and affects the efficacy of platinum-based drugs. Exploring biomarkers related to FAM in bladder cancer (BLCA) is essential to improve cancer prognosis. Methods: High-throughput sequencing data from The Cancer Genome Atlas (TCGA) were bioinformatically resolved to identify molecular subtypes of fatty acid metabolic profiles in BLCA using coherent clustering analysis. Based on fatty acid metabolic profile, a prognostic model was created using COX and LASSO COX models. CIBERSORT, Estimation of STromal and Immune cells in MAlignant Tumours using Expression (ESTIMATE), MCP-Count, and single sample gene set enrichment analysis (ssGSEA) were used to assess the differences in tumor microenvironment (TME) among different molecular subtypes, prognostic groups. Kaplan-Meier (K-M) survival curve was plotted to assess patients’ prognosis. Receiver operating characteristic curve (ROC) and the clinical prognostic value of prognostic models was evaluated by the Nomogram. Results: Three molecular subtypes (FAMC1, FAMC2, FAMC3) of fatty acid metabolic patterns were determined. FAMC1 showed significant prognostic advantage with immunoreactivity. Five key prognostic FAMGs were identified and RiskScore was developed. We found that patients with low RiskScore showed significantly better immune microenvironment status, survival and response to immunotherapy. Similarly, both Nomogram and RiskScore demonstrated excellent prognostic value. Conclusions: In conclusion, our study showed that the RiskScore was closely related to the clinical traits of BLCA patients. The RiskScore may provide essential clinical guidance for predicting prognosis and treatment response in bladder cancer.

Abstract Background ε-Poly-L-lysine (ε-PL) is a natural and safe food preservative that is mainly produced by filamentous and aerobic bacteria Streptomyces albulus. During ε-PL biosynthesis, a large amount of ATP is used for the polymerization of L-lysine. A shortage of intracellular ATP is one of the major factors limiting the increase in ε-PL production. In previous studies, researchers have mainly tried to increase the oxygen supply to enhance intracellular ATP levels to improve ε-PL production, which can be achieved through the use of two-stage dissolved oxygen control, oxygen carriers, heterologous expression of hemoglobin, and supplementation with auxiliary energy substrates. However, the enhancement of the intracellular ATP supply by constructing an ATP regeneration system has not yet been considered. Results In this study, a polyphosphate kinase (PPK)-mediated ATP regeneration system was developed and introduced into S. albulus to successfully improve ε-PL production. First, polyP:AMP phosphotransferase (PAP) from Acinetobacter johnsonii was selected for catalyzing the conversion of AMP into ADP through an in vivo test. Moreover, three PPKs from different microbes were compared by in vitro and in vivo studies with respect to catalytic activity and polyphosphate (polyP) preference, and PPK2Bcg from Corynebacterium glutamicum was used for catalyzing the conversion of ADP into ATP. As a result, a recombinant strain PL05 carrying coexpressed pap and ppk2B cg for catalyzing the conversion of AMP into ATP was constructed. ε-PL production of 2.34 g/L was achieved in shake-flask fermentation, which was an increase of 21.24% compared with S. albulus WG608; intracellular ATP was also increased by 71.56%. In addition, we attempted to develop a dynamic ATP regulation route, but the result was not as expected. Finally, the conditions of polyP6 addition were optimized in batch and fed-batch fermentations, and the maximum ε-PL production of strain PL05 in a 5-L fermenter was 59.25 g/L by fed-batch fermentation, which is the highest ε-PL production reported in genetically engineered strains. Conclusions In this study, we proposed and developed a PPK-mediated ATP regeneration system in S. albulus for the first time and significantly enhanced ε-PL production. The study provides an efficient approach to improve the production of not only ε-PL but also other ATP-driven metabolites.

Objective To investigate the effects of high-dose hyperbaric trioxygen autologous blood therapy (HOT) on oxygenation index (PaO2/FiO2) and serum inflammatory factors in dogs with acute respiratory distress syndrome (ARDS). Methods Twelve healthy adult beagles were randomly divided into 3 groups (n=4). The blank group was injected with normal saline intravenously. The ARDS model was established by intravenous injection of oleic acid (0.12 mL/kg) in the ARDS group and ARDS+ HOT group. The mark of a successful model is that the oxygen and index (PaO2/FiO2)

ε-Poly-L-lysine (ε-PL) is a natural and safe food preservative mainly produced by the aerobic, filamentous bacterium Streptomyces albulus. Therefore, it is crucial to breed superior ε-PL-producing strains to enhance fermentation efficiency to reduce production costs. Metabolic engineering is an effective measure for strain modification, but there are few reports on key genes for ε-PL biosynthesis. In this study, metabolic flux analysis was employed to identify potential key genes in ε-PL biosynthesis in S. albulus WG-608. A total of six potential key genes were identified. Three effective key genes (ppc, pyc and pls) were identified for the first time in ε-PL biosynthesis through overexpression experiments. It also presents the first demonstration of the promoting effects of ppc and pyc on ε-PL biosynthesis. Three genes were then co-expressed in S. albulus WG-608 to obtain OE-ppc-pyc-pls, which exhibited an 11.4% increase in ε-PL production compared to S. albulus WG-608, with a 25.5% increase in specific ε-PL production. Finally, the metabolic flux analysis of OE-ppc-pyc-pls compared to S. albulus WG-608 demonstrated that OE-ppc-pyc-pls successfully altered the metabolic flux as expected. This study not only provides a theoretical basis for the metabolic engineering of ε-PL-producing strains but also provides an effective approach for the metabolic engineering of other metabolites.

Introductionε-poly-L-lysine (ε-PL) is a high value, widely used natural antimicrobial peptide additive for foods and cosmetic products that is mainly produced by Streptomyces albulus. In previous work, we developed the high-yield industrial strain S. albulus WG-608 through successive rounds of engineering.MethodsHere, we use integrated physiological, transcriptomic, and proteomics association analysis to resolve the complex mechanisms underlying high ε-PL production by comparing WG-608 with the progenitor strain M-Z18.ResultsOur results show that key genes in the glycolysis, pentose phosphate pathway, glyoxylate pathway, oxidative phosphorylation, and L-lysine biosynthesis pathways are differentially upregulated in WG-608, while genes in the biosynthetic pathways for fatty acids, various branched amino acids, and secondary metabolite by-products are downregulated. This regulatory pattern results in the introduction of more carbon atoms into L-lysine biosynthesis and ε-PL production. In addition, significant changes in the regulation of DNA replication, transcription, and translation, two component systems, and quorum sensing may facilitate the adaptability to environmental pressure and the biosynthesis of ε-PL. Overexpression of ppk gene and addition of polyP6 further enhanced the ε-PL production.DiscussionThis study enables comprehensive understanding of the biosynthetic mechanisms of ε-PL in S. albulus WG-608, while providing some genetic modification and fermentation strategies to further improve the ε-PL production.

Objective To investigate the effect of electrolyte concentration on transfusion related complications in patients undergoing cardiac surgery after short-term massive transfusion (MT). Methods Seventy-six cases of adult cardiac surgery in our hospital from January 2018 to December 2019 were collected. According to their perioperative blood transfusion units, they were divided into the massive blood transfusion group (red blood cell units≥ 10 U/ person) and the control group[red blood cell units (0~1)U/ person]. The concentrations of Na+ , K+ and Ca2+ ions in serum were detected by ISE (ion selective electrode method) and NM-BAPTA before and after intraoperative blood transfusion. The changes of Na+ , K+ and Ca2+ ions in serum before and after intraoperative blood transfusion were analyzed and compared through T-test. Results There were 38 patients in the MT group and 38 in the control group, and the demographic variables and types of surgeries (number) of patients in the two groups were comparable (P>0.05). The serum K+ , Ca2+ and Na+ before operation were similar (P>0.05), and within the normal range. The mean values of serum K+ , Ca2+ and Na+ (mmol/l) in the massive blood transfusion group and the control after operation (blood transfusion) were 4.25±0.44 vs 4.01±0.53, 2.31±0.12 vs 2.45±0.43, 140.82±2.31 vs 146.44±4.35 (P0.05), and all were within the normal range. Conclusion Patients undergoing cardiac surgery are prone to suffer electrolyte disorders after massive blood transfusion. Electrolyte concentration should be monitored in time during and after perioperative blood transfusion.

ε-poly-l-lysine (ε-PL) has been routinely used as a natural and safe preservative for many years in the food industry. However, most existing production methods struggle to achieve low cost and high production simultaneously. In this work, we present a co-culture fermentation strategy to enhance ε-PL production. Specifically, we screened a strain from five different strains that could be co-cultured with Streptomyces albulus to raise the production of ε-PL. Subsequently, a single factor experiment and response surface design were used to optimize the conditions of co-culture fermentation to further improve the production of ε-PL. Moreover, the optimal fermentation process was successfully verified in a 2-L fermentor with fed batch fermentation. The production of ε-PL reached 27.07 ± 0.47 g/L by 144 h. Compared with single strain (S. albulus) fermentation, the production of ε-PL was increased by 31.47%. At the same time, the amount of bacteria increased by 19.62%, which means that the ε-PL synthesis ability of bacteria had been improved. All the obtained results showed great potential for co-culture fermentation in large-scale ε-PL production and provide a new fermentation strategy for ε-PL biosynthesis.

ε-Poly-L-lysine (ε-PL), a natural food preservative, has recently gained interest and mainly produced by Streptomyces albulus. Lacking of efficient breeding methods limit ε-PL production improving, knockout byproducts and increase of main product flux strategies as a logical solution to increase yield. However, removing byproduct formation and improving main product synthesis has seen limited success due to the genetic background of ε-PL producing organism is not clear. To overcome this limitation, random mutagenesis continues to be the best way towards improving strains for ε-PL production. Recent advances in Illumina sequencing opened new avenues to understand improved strains. In this work, we used genome shuffling on strains obtained by ribosome engineering to generate a better ε-PL producing strain. The mutant strain SG-86 produced 144.7% more ε-PL than the parent strain M-Z18. Except that SG-86 displayed obvious differences in morphology and ATP compared to parent strain M-Z18. Using Illumina sequencing, we mapped the genomic changes leading to the improved phenotype. Sequencing two strains showed that the genome of the mutant strain was about 2.1 M less than that of the parent strain, including a large number of metabolic pathways, secondary metabolic gene clusters, and gene deletions. In addition, there are many SNPs (single nucleotide polymorphisms) and InDels (insertions and deletions) in the mutant strain. Based on the results of data analysis, a mechanism of ε-PL overproduction in S. albulus SG-86 was preliminarily proposed. This study is of great significance for improving the fermentation performance and providing theoretical guidance for the metabolic engineering construction of ε-PL producing strains.

ε-poly-L-lysine (ε-PL) is a naturally occurring poly(amino acid) of varying polymerization degree, which possesses excellent antimicrobial activity and has been widely used in food and pharmaceutical industries. To provide new perspectives from recent advances, this review compares several conventional and advanced strategies for the discovery of wild strains and development of high-producing strains, including isolation and culture-based traditional methods as well as genome mining and directed evolution. We also summarize process engineering approaches for improving production, including optimization of environmental conditions and utilization of industrial waste. Then, efficient downstream purification methods are described, including their drawbacks, followed by the brief introductions of proposed antimicrobial mechanisms of ε-PL and its recent applications. Finally, we discuss persistent challenges and future perspectives for the commercialization of ε-PL.

Summary WAP‐8294A is a group of cyclic lipodepsipeptides and considered as the first‐in‐class new chemical entity with potent activity against methicillin‐resistant Staphylococcus aureus. One of the roadblocks in developing the WAP‐8294A antibiotics is the very low yield in Lysobacter. Here, we carried out a systematic investigation of the nutritional and environmental conditions in an engineered L. enzymogenes strain for the optimal production of WAP‐8294A. We developed an activity‐based simple method for quick screening of various factors, which enabled us to optimize the culture conditions. With the method, we were able to improve the WAP‐8294A yield by 10‐fold in small‐scale cultures and approximately 15‐fold in scale‐up fermentation. Additionally, we found the ratio of WAP‐8294A2 to WAP‐8294A1 in the strains could be manipulated through medium optimization. The development of a practical method for yield improvement in Lysobacter will facilitate the ongoing basic research and clinical studies to develop WAP‐8294A into true therapeutics.

Abstract ε‐Poly‐L‐lysine (ε‐PL) is a food additive produced by Streptomyces and is widely used in many countries. Working with Streptomyces albulus FEEL‐1, we established a method to activate ε‐PL synthesis by successive introduction of multiple antibiotic‐resistance mutations. Sextuple mutant R6 was finally developed by screening for resistance to six antibiotics and produced 4.41 g/L of ε‐PL in a shake flask, which is 2.75‐fold higher than the level produced by the parent strain. In a previous study, we constructed a double‐resistance mutant, SG‐31, with high ε‐PL production of 3.83 g/L and 59.50 g/L in a shake flask and 5‐L bioreactor, respectively. However, we found that R6 did not show obvious advantages in fed‐batch fermentation when compared with SG‐31. For further activation of ε‐PL synthesis ability, we optimized the fermentation process by using an effective acidic pH shock strategy, by which R6 synthetized 70.3 g/L of ε‐PL, 2.79‐fold and 1.18‐fold greater than that synthetized by FEEL‐1 and SG‐31, respectively. To the best of our knowledge, this is the highest reported ε‐PL production to date. This ε‐PL overproduction may be due to the result of R99P and Q856H mutations in ribosomal protein S12 and RNA polymerase, respectively, which may be responsible for the increased transcription of the ε‐poly‐lysine synthetase gene (pls) and key enzyme activities in the Lys synthesis metabolic pathway. Consequently, ε‐PL synthetase activity, intracellular ATP, and Lys concentrations were improved and directly contributed to ε‐PL overproduction. This study combined ribosome engineering, high‐throughput screening, and targeted strategy optimization to accelerate ε‐PL production and probe the fermentation characteristics of hyperyield mutants. The information presented here may be useful for other natural products produced by Streptomyces.

Representation-based Classification (RC) algorithms have been extensively applied in the domain of face recognition. This paper introduces a novel approach by incorporating arbitrary norms into both the objective and constraint functions of conventional RC algorithms, resulting in the development of the Generalized RC (GRC) algorithm. The primary goal of this enhancement is to fully leverage the unique advantages offered by various norms. Within the majorization-minimization framework, an iterative procedure was designed to solve the optimization problem of GRC. This approach ensures that a closed-form solution is obtained in each iteration and a locally optimal solution can be found upon convergence. Experimental results on four benchmark face databases demonstrated that GRC generally outperforms competing RC algorithms in face recognition. Data and source code of this study are publicly available on the GitHub repository at https://github.com/yuzhounh/GRC. [ABSTRACT FROM AUTHOR]