High-titer production of staurosporine by heterologous expression and process optimization.

Staurosporine is the most well-known member of the indolocarbazole alkaloid family; it can induce apoptosis of many types of cells as a strong protein kinase inhibitor, and is used as an important lead compound for the synthesis of the antitumor drugs. However, the low fermentation level of the native producer remains the bottleneck of staurosporine production. Herein, integration of multi-copy biosynthetic gene cluster (BGC) in well characterized heterologous host and optimization of the fermentation process were performed to enable high-level production of staurosporine. First, the 22.5 kb staurosporine BGC was captured by CRISPR/Cas9-mediated TAR (transformation-associated recombination) from the native producer (145 mg/L), and then introduced into three heterologous hosts Streptomyces avermitilis (ATCC 31267), Streptomyces lividans TK24 and Streptomyces albus J1074 to evaluate the staurosporine production capacity. The highest yield was achieved in S. albus J1074 (750 mg/L), which was used for further production improvement. Next, we integrated two additional staurosporine BGCs into the chromosome of strain S-STA via two different attB sites (vwb and TG1), leading to a double increase in the production of staurosporine. And finally, optimization of fermentation process by controlling the pH and glucose feeding could improve the yield of staurosporine to 4568 mg/L, which was approximately 30-fold higher than that of the native producer. This is the highest yield ever reported, paving the way for the industrial production of staurosporine. KEYPOINTS: • Streptomyces albus J1074 was the most suitable heterologous host to express the biosynthetic gene cluster of staurosporine. • Amplification of the biosynthetic gene cluster had obvious effect on improving the production of staurosporine. • The highest yield of staurosporine was achieved to 4568 mg/L by stepwise increase strategy.

Enhancement of acarbose production by genetic engineering and fed-batch fermentation strategy in Actinoplanes sp. SIPI12-34.

BACKGROUND: Acarbose, as an alpha-glucosidase inhibitor, is widely used clinically to treat type II diabetes. In its industrial production, Actinoplanes sp. SE50/110 is used as the production strain. Lack of research on its regulatory mechanisms and unexplored gene targets are major obstacles to rational strain design. Here, transcriptome sequencing was applied to uncover more gene targets and rational genetic engineering was performed to increase acarbose production. RESULTS: In this study, with the help of transcriptome information, a TetR family regulator (TetR1) was identified and confirmed to have a positive effect on the synthesis of acarbose by promoting the expression of acbB and acbD. Some genes with low expression levels in the acarbose biosynthesis gene cluster were overexpressed and this resulted in a significant increase in acarbose yield. In addition, the regulation of metabolic pathways was performed to retain more glucose-1-phosphate for acarbose synthesis by weakening the glycogen synthesis pathway and strengthening the glycogen degradation pathway. Eventually, with a combination of multiple strategies and fed-batch fermentation, the yield of acarbose in the engineered strain increased 58% compared to the parent strain, reaching 8.04 g/L, which is the highest fermentation titer reported. CONCLUSIONS: In our research, acarbose production had been effectively and steadily improved through genetic engineering based on transcriptome analysis and fed-batch culture strategy.

A Study of Type II ɛ-PL Degrading Enzyme (pldII) in Streptomyces albulus through the CRISPRi System.

ε-Poly-L-lysine (ε-PL) is a widely used antibacterial peptide polymerized of 25-35 L-lysine residues. The antibacterial effect of ε-PL is closely related to the polymerization degree. However, the mechanism of ε-PL degradation in S. albulus remains unclear. This study utilized the integrative plasmid pSET152-based CRISPRi system to transcriptionally repress the ε-PL degrading enzyme (pldII). The expression of pldII is regulated by changing the recognition site of dCas9. Through the ε-PL bacteriostatic experiments of repression strains, it was found that the repression of pldII improves the antibacterial effect of the ε-PL product. The consecutive MALDI-TOF-MS results confirmed that the molecular weight distribution of the ε-PL was changed after repression. The repression strain S1 showed a particular peak with a polymerization degree of 44, and other repression strains also generated ε-PL with a polymerization degree of over 40. Furthermore, the homology modeling and substrate docking of pldII, a typical endo-type metallopeptidase, were performed to resolve the degradation mechanism of ε-PL in S. albulus. The hydrolysis of ε-PL within pldII, initiated from the N-terminus by two amino acid-binding residues, Thr194 and Glu281, led to varying levels of polymerization of ε-PL.

The effects of extenders, cryoprotectants and conditions in two-step cooling method on Varicorhinus barbatulus sperm.

In this study, we developed an optimal cryopreservation procedure for Varicorhinus barbatulus sperm. To this end, we optimized (1) the types and dilution ratios of extenders; (2) types and final concentration of cryoprotectants; and (3) freezing conditions, including equilibration time, height above the surface of liquid nitrogen (LN), and the cooling times in the two-step cooling method. The optimum result was obtained when the sperm was diluted at a 1:9 ratio in D-17 with 10% methanol, equilibrated at 4 °C for 10 min, held at 7 cm above LN for 2 min, and finally stored in LN. After storage for 12 h in LN, the sperm was thawed in a water bath at 40 °C for 6s, the post-thaw sperm motility was 66.10 ± 7.12%, while the corresponding rate for fresh sperm was 87.08 ± 2.38%. Using computer-assisted sperm analysis, we found a significant decrease in the motility parameters of post-thaw sperm, especially the parameters related to velocity. To evaluate the effects of cryopreservation on the structural integrity of sperm, transmission electron microscopy and scanning electron microscopy were employed, which showed the defects in frozen sperm, including: abnormal heads, damaged plasma membranes, broken tails, and the disappearance of the mitochondrial internal crest. In addition, we determined the mitochondrial membrane potential to assess the functional integrity of frozen sperm. Our results showed a decrease in the mitochondrial function of frozen sperm. This procedure could be used alongside cryopreservation of V. barbatulus and supports its commercial-scale production and species conservation.

Dihydromyricetin Acts as a Potential Redox Balance Mediator in Cancer Chemoprevention.

Dihydromyricetin (DHM) is a flavonoid extracted from the leaves and stems of the edible plant Ampelopsis grossedentata that has been used for Chinese Traditional Medicine. It has attracted considerable attention from consumers due to its beneficial properties including anticancer, antioxidative, and anti-inflammatory activities. Continuous oxidative stress caused by intracellular redox imbalance can lead to chronic inflammation, which is intimately associated with the initiation, promotion, and progression of cancer. DHM is considered a potential redox regulator for chronic disease prevention, and its biological activities are abundantly evaluated by using diverse cell and animal models. However, clinical investigations are still scanty. This review summarizes the current potential chemopreventive effects of DHM, including its properties such as anticancer, antioxidative, and anti-inflammatory activities, and further discusses the underlying molecular mechanisms of DHM in cancer chemoprevention by targeting redox balance and influencing the gut microbiota.

aMSGE: advanced multiplex site-specific genome engineering with orthogonal modular recombinases in actinomycetes.

Chromosomal integration of genes and pathways is of particular importance for large-scale and long-term fermentation in industrial biotechnology. However, stable, multi-copy integration of long DNA segments (e.g., large gene clusters) remains challenging. Here, we describe a plug-and-play toolkit that allows for high-efficiency, single-step, multi-locus integration of natural product (NP) biosynthetic gene clusters (BGCs) in actinomycetes, based on the innovative concept of "multiple integrases-multiple attB sites". This toolkit consists of 27 synthetic modular plasmids, which contain single- or multi-integration modules (from two to four) derived from five orthogonal site-specific recombination (SSR) systems. The multi-integration modules can be readily ligated into plasmids containing large BGCs by Gibson assembly, which can be simultaneously inserted into multiple native attB sites in a single step. We demonstrated the applicability of this toolkit by performing stabilized amplification of acetyl-CoA carboxylase genes to facilitate actinorhodin biosynthesis in Streptomyces coelicolor. Furthermore, using this toolkit, we achieved a 185.6% increase in 5-oxomilbemycin titers (from 2.23 to 6.37 g/L) in Streptomyces hygroscopicus via the multi-locus integration of the entire 5-oxomilbemycin BGC (72 kb) (up to four copies). Compared with previously reported methods, the advanced multiplex site-specific genome engineering (aMSGE) method does not require the introduction of any modifications into host genomes before the amplification of target genes or BGCs, which will drastically simplify and accelerate efforts to improve NP production. Considering that SSR systems are widely distributed in a variety of industrial microbes, this novel technique also promises to be a valuable tool for the enhanced biosynthesis of other high-value bioproducts.

Enhancement of FK520 production in Streptomyces hygroscopicus by combining traditional mutagenesis with metabolic engineering.

FK520 (ascomycin), a 23-membered macrolide with immunosuppressive activity, is produced by Streptomyces hygroscopicus. The problem of low yield and high impurities (mainly FK523) limits the industrialized production of FK520. In this study, the FK520 yield was significantly improved by strain mutagenesis and genetic engineering. First, a FK520 high-producing strain SFK-6-33 (2432.2 mg/L) was obtained from SFK-36 (1588.4 mg/L) through ultraviolet radiation mutation coupled with streptomycin resistance screening. The endogenous crotonyl-CoA carboxylase/reductase (FkbS) was found to play an important role in FK520 biosynthesis, identified with CRISPR/dCas9 inhibition system. FkbS was overexpressed in SFK-6-33 to obtain the engineered strain SFK-OfkbS, which produced 2817.0 mg/L of FK520 resulting from an increase in intracellular ethylmalonyl-CoA levels. In addition, the FK520 levels could be further increased with supplementation of crotonic acid in SFK-OfkbS. Overexpression of acetyl-CoA carboxylase (ACCase), used for the synthesis of malonyl-CoA, was also investigated in SFK-6-33, which improved the FK520 yield to 3320.1 mg/L but showed no significant inhibition in FK523 production. To further enhance FK520 production, FkbS and ACCase combinatorial overexpression strain SFK-OASN was constructed; the FK520 production increased by 44.4% to 3511.4 mg/L, and the FK523/FK520 ratio was reduced from 9.6 to 5.6% compared with that in SFK-6-33. Finally, a fed-batch culture was carried out in a 5-L fermenter, and the FK520 yield reached 3913.9 mg/L at 168 h by feeding glycerol, representing the highest FK520 yield reported thus far. These results demonstrated that traditional mutagenesis combined with metabolic engineering was an effective strategy to improve FK520 production.

Novel Polysaccharide H-1-2 from Pseudostellaria Heterophylla Alleviates Type 2 Diabetes Mellitus.

BACKGROUND/AIMS: To investigate the potential therapeutic effect of novel polysaccharide H-1-2 from pseudostellaria heterophylla against type 2 Diabetes Mellitus (T2DM) and elucidate the underling molecular mechanisms. METHODS: Relative expression of HIF1α and Sirt1 in T2DM patients was determined via real-time PCR. The direct binding of HIF1α on Sirt1 promoter was validated by ChIP assay. The inhibitory regulation of Sirt1 by HIF1α was analyzed using luciferase reporter assay. The endogenous protein of HIF1α and Sirt1 in response to H-1-2 treatment was quantified by western blotting. The blood glucose, secreted insulin and serous lipid profiles were measured with ELISA kits. RESULTS: We consolidated that HIF1α and Sirt1 was dysregulated in T2DM patients and subjected to H-1-2 modulation. H-1-2 significantly inhibited hypoxia and up-regulated Sirt1 expression in EndoC-ßH1 cells. Accordingly, H-1-2 enhanced glucose-stimulation insulin secretion and improved blood glucose and lipid profiles in T2DM cells, and elevated the glucose and insulin tolerance simultaneously. Furthermore, we demonstrated that H-1-2 alleviated T2DM via inhibition of hypoxia and up-regulation of Sirt1 in isolated pancreatic ß-cells from T2DM rats. CONCLUSION: Our data unambiguously demonstrated H-1-2 administration alleviated T2DM by enhancing Sirt1 expression through inhibition of hypoxia.

MilR2, a novel TetR family regulator involved in 5-oxomilbemycin A3/A4 biosynthesis in Streptomyces hygroscopicus.

Milbemycins produced by several Streptomyces species are a group of 16-membered macrolides with potent insecticidal and anthelminthic activity. Milbemycin A3/A4, the main components of the milbemycins biosynthetic pathway, and 5-oxomilbemycin A3/A4, the analogs of milbemycin A3/A4 without the reduction of the C-5 keto group, have been developed as acaricides, insecticides, and anthelmintics. However, so far, little is known about the regulation of milbemycins biosynthesis, which has greatly hampered the generation of high producing strains by metabolic engineering. Herein, a TetR family regulator MilR2 (encoded by sbi_00792) was identified being involved in activation of 5-oxomilbemycin A3/A4 biosynthesis in a high 5-oxomilbemycins-producing strain Streptomyces hygroscopicus SIPI-KF. The ΔmilR2 mutant with an in-frame deletion of the MilR2 DNA-binding domain resulted in significantly reduced 5-oxomilbemycin A3/A4 production (approximately 36.9 and 39.7%) at tested two time points, and accordingly introduction of an extra copy of milR2 into SIPI-KF led to enhanced production by 12.6 and 34.4%. We further showed that MilR2 could directly repress the transcription of the gene sbi_00791 encoding a putative hydrolase, which is located divergently from milR2. The precise MilR2-binding site consisting of a 7-nt perfect inverted repeat separated by 10-nt (5'-ACCAACCAGCTGGTAAGGGTTGGT-3') was defined. In situ mutagenesis of the MilR2-binding site resulted in 19.7 and 13.5% decreases in 5-oxomilbemycin A3/A4 production, which is much lower than the decreased rates of ΔmilR2. Collectively, the results demonstrated that MilR2 serves as an activator for 5-oxomilbemycin A3/A4 production and the function of MilR2 is only partially mediated through its repression on the transcription of sbi_00791.

Enhancement of A82846B yield and proportion by overexpressing the halogenase gene in Amycolatopsis orientalis SIPI18099.

The glycopeptide antibiotic A82846B (chloroeremomycin) produced by Amycolatopsis orientalis is the precursor of the semi-synthetic antibiotic oritavancin. However, during the industrial production of A82846B, two major impurities, A82846A (63.6%) and A82846C (12%) which are structurally similar to A82846B (24.4%), are also produced. In this study, to improve the ratio of A82846B to A and C, the genes encoding halogenase in A82846B and vancomycin synthesis were integrated into A. orientalis SIPI18099 to test their halogenation ability, respectively. The results indicated that chal from the A82846B biosynthesis pathway was more efficient in reducing A and C factors. Moreover, by increasing the chal copy number, the proportion of A and C were gradually reduced while the titer and proportion of A82846B were improved. In a scaled-up industrial process, the proportion of A and C were decreased to 11.6% and 0.2% in the recombinant strain A.orientalis chal-3 with three gene copies of chal and the titers of A82846B (2.2 g/L) has increased by 2.8-folds compared to 780 mg/L produced by the parental strain, suggesting that the recombinant strain was suitable for the industrial production of A82846B with lower impurities.

Enhanced doxorubicin production by Streptomyces peucetius using a combination of classical strain mutation and medium optimization.

Doxorubicin (DXR), which is produced by Streptomyces peucetius, is an important anthracycline-type antibiotic used for the treatment of various cancers. However, due to the low DXR productivity of wild-type S. peucetius, it is difficult to produce DXR by one-step fermentation. In this study, a DXR-resistance screening method was developed to screen for DXR high-producing mutants. Then, S. peucetius SIPI-11 was treated several times with UV and ARTP (atmospheric and room temperature plasma) to induce mutations. Treated strains were screened by spreading on a DXR-containing plate, isolating a mutant (S. peucetius 33-24) with enhanced DXR yield (570 mg/L vs. 119 mg/L for the original strain). The components of the fermentation medium, including the carbon and nitrogen sources, were optimized to further enhance DXR yield (to 850 mg/L). The pH of the fermentation medium and culture temperature were also optimized for effective DXR production. Finally, DXR production by S. peucetius 33-24 was investigated in flask culture and a fermenter. The yield of DXR was as high as 1100 mg/L in a 5-L fermenter, which is the highest DXR productivity reported thus far, suggesting that S. peucetius 33-24 has the potential to produce DXR by direct fermentation.

Suppression of pancreatic beta cell apoptosis by Danzhi Jiangtang capsule contributes to the attenuation of type 1 diabetes in rats.

BACKGROUND: Danzhi Jiangtang Capsule (DJC), a Chinese medicinal formula, has been clinically used for treatment of diabetes for many years. Previous studies have demonstrated that DJC was able to improve pancreatic islet function in diabetes, but the underlying mechanisms remained unclear. METHODS: Streptozotocin (STZ) induced type 1 diabetic rats were treated with DJC for 6 weeks. Fasting plasma insulin and fasting plasma glucose were determined at the end of experiment. Antioxidant status was evaluated by measuring total antioxidant capacity, superoxide dismutase activity and malondialdehyde content in plasma and pancreas. Paraffin sections of pancreas were subjected to H&E staining, TUNEL staining and immunohistochemical examination. Protein levels of Bcl-2, Bax and pancreatic duodenal homeobox-1 (PDX-1) were measured by western blot analysis. Activities of Caspase-3 and Caspase-9 were determined with commercially available kits. RESULTS: Supplementation with DJC resulted in a significant amelioration of type 1 diabetes as manifested by reduced blood glucose, increased fasting plasma insulin and improved body weight gains. The atrophy and reduction of pancreatic islets were also alleviated in DJC supplemented groups. DJC markedly reduced pancreatic beta cell apoptosis, with Bax protein down-regulated and Bcl-2 protein up-regulated significantly. The activities of caspase-3 and caspase-9 in pancreas were decreased evidently by DJC treatment. DJC effectively ameliorated oxidative stress in type 1 diabetic rats, with the expression of PDX-1 protein increased markedly. CONCLUSIONS: DJC was capable of attenuating STZ induced type 1 diabetes in rats, which might be attributed to the suppression of pancreatic beta cell apoptosis. This study would provide further evidence for clinical use of DJC in the management of diabetes.

[The effect of Salvianolic Acid B on the Apoptosis of HUVECs Induced by Intermittent High Glucose].

Objective: To investigate the effect of Salvianolic acid B (Sal B) on the apoptosis of human umbilical vein endothelial cells (HUVECs) induced by intermittent high glucose and to explore the possible mechanisms. Methods: HUVECs were preincubated with Sal B for 24 h, followed by incubation with intermittent high glucose (IHG, 5.5 mmol/L 12 h, 33.3 mmol/L 12 h) for 72 h. The viability of the HUVECs was determined by MTT assay, and the cells apoptosis was measured flow cytometry, respectively. The levels of nitric oxide (NO), total antioxidant capacity (T-AOC), malondialdehyde (MDA), and Caspase-3 activity were determined by colorimetric method. Intracellular ROS was evaluated by fluorescent microscopy. The protein levels of NOX4, p-eNOS, BAX, and BCL-2 were determined by Western-blot. Results: Pretreatment with Sal B significantly ameliorated IHG-induced cells injury as was manifested by increased cell viability, up-regulated eNOS activation, and promoted the release of NO in HUVECs (P < 0.05 or P < 0.01). Sal B evidently suppressed IHG-induced cell apoptosis, down-regulated the expression of BAX protein and up-regulated the expression of BCL-2 protein. The activity of Capase-3 was also significantly reduced. Pre-incubation with Sal B led to a significant enhancement of antioxidant capacity and a reduction of NOX4 protein expression, accompanied by a remarkable decrease of intracellular ROS and MDA content (P < 0.05 or P < 0.01). Conclusion: Sal B is capable of suppressing IHG-induced injury and apoptosis in HUVECs, which might be attributed to the attenuation of oxidative stress, regulation of BCL-2/BAX protein expression, and subsequent suppression of Caspase-3 activity.

[Effect of Danzhi Jiangtang Capsule on Myocardial Fibrosis in Diabetic Rats].

OBJECTIVE: To investigate the effect of Danzhi Jiangtang Capsule on myocardial fibrosis in diabetic rats with fluctuated blood glucose and the possible mechanisms implicated. METHODS: Following induction of diabetes with intraperitoneal injection of streptozotocin (STZ), rats were administered with insulin or glucose at different time during a day to induce blood glucose fluctuation. After treatment with Danzhi Jiangtang Capsule for six weeks, rats were sacrificed and the hearts were collected for the determination of cardiac mass index. Cardiac levels of angiotensin II (Ang II), type I and type III collagens and transforming growth factor-ß1 (TGF-ß1) were assayed by ELISA. Levels of Smad3 phosphorylation and protein expression of matrix metalloproteinase-2 ( MMP-2) and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) were determined by Western blot analysis. Total antioxidant capacity and malondialdehyde (MDA) content in cardiac tissues were measured colorimetrically. RESULTS: Treatment with Danzhi Jiangtang Capsule for six weeks significantly reduced cardiac mass index and cardiac levels of type I and type III collagens (P < 0.05 or P < 0.01). Levels of Ang II, TGF-ß1 and Smad3 phosphorylation in cardiac tissues were also decreased markedly (P < 0.05 or P < 0.01). Supplementation with Danzhi Jiangtang Capsule resulted in an evident up-regulation of MMP-2 protein and down-regulation of TIMP-2 protein expression in cardiac tissues (P < 0.05 or P < 0.01). In addition, Danzhi Jiangtang Capsule significantly enhanced total antioxidant capacity in diabetic rats, while cardiac content of MDA was decreased markedly( P < 0.05 or P < 0.01). CONCLUSION: Danzhi Jiangtang Capsule significantly ameliorated myocardial fibrosis in diabetic rats with fluctuated blood glucose, which might be derived from enhancement of antioxidant capacity, suppression of RAS and TGF-ß1/Smad3 signaling pathway and regulation of MMP-2/TIMP-2 protein expression.

[Medication rule for treatment of functional dyspepsia: an analysis of traditional Chinese medicine literature based on China National Knowledge Internet].

By retrieving the clinical research literature of treatment functional dyspepsia by traditional Chinese medicine (TCM) from January 2004 to December 2014 based on China National Knowledge Internet (CNKI), we would establish a TCM decoction database for treating functional dyspepsia in this study. One hundred and sixty-four literature were included, involving 159 prescriptions, 377 medicines, in a total of 1 990 herbs. These herbs can be divided into 18 categories according to the effectiveness; and qi-regulating herbs, blood circulation herbs, and antipyretic herbs ranked top three ones according to the frequency of usage of the herbs, whose medicine usage frequency accounted for 51.81%. Usage frequency of 16 herbs was over 30, and Atractylodes, Radix, Poriaranked top three according to the usage frequency. Medicinal properties were divided into 9 kinds according to the frequency statistics, and the top three were warm, flat, and cold. Taste frequency statistics were classifiedinto 9 kinds, and the top three were acrid, sweet, and bitter. In frequency statistics of the meridian tropism of herbs, it was classifiedinto 11 kinds, and the top three were spleen, stomach, lung. The analysis can provide a reference for treatment and study of TCM of functional dyspepsia.

[Effect of danzhi jiangtang capsule combined exercise on the protein expression of NADPH oxidase p22phox in pancreatic tissues of diabetic rats].

OBJECTIVE: To explore effects of exercise combined Danzhi Jiangtang Capsule (DJC) on the protein expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase p22phox in pancreatic tissues of diabetic rats. METHODS: Sixty male Wistar rats were injected with low dose of streptozotocin and fed with high fat diet to establish a diabetic rat model. The levels of p22phox and 8-hydroxy-2-de-oxyguanosine (8-OHdG) protein in pancreatic tissues were detected by immunohistochemical method, and the level of p22phox protein was also detected by Western blot in the normal group, the model group, the excise group, the DJC group, and the DJC +excise group, respectively. RESULTS: The expression levels of p22phox and 8-OHdG protein in pancreatic tissues were significantly higher in the model group than in the normal group (P <0.01). p22phox and 8-OHdG were mainly expressed in the cytoplasm of pancreatic cells. After administration of exercise or DJC, the expression lev- els of p22phox or 8-OHdG protein in pancreatic tissues decreased significantly (P <0. 01). Exercise combined DJC had synergistic effects in decreasing expressions of p22phox and 8-OHdG (P <0.05). CONCLUSION: Exercise, DJC, and exercise combined DJC could protect islet beta cells by decreasing the expression of NADPH oxidase in beta cells and reducing sources of oxidative stress.

Effective separation of protein from Polygonatum cyrtonema crude polysaccharide utilizing ionic liquid tetrabutylammonium bromide.

Extraction of plant polysaccharides often results in a large amount of proteins, which is hard to eliminate from the crude extract, and conventional approaches for deproteinization are time-consuming and often involve hazardous organic solvents. In this study, ionic liquid tetrabutylammonium bromide (TBABr) was used to create an ionic liquid aqueous two-phase system (ILATPS) for the separation of the polysaccharide (PcP) and protein extracted from the rhizome of Polygonatum cyrtonema. Bovine serum albumin (BSA) was first applied to assess the feasibility of the ILATPS, and MgSO4 was determined to be the most suitable inorganic salt. By adopting the Taguchi experiment with an L9 (3^4) orthogonal array, it was found that the best condition for the efficient separation of crude PcP was at 25°C, with 1.5 g of TBABr, 15 mg of PcP, and 2.0 g of MgSO4, with the extraction efficiency for the protein and polysaccharide as 98.6% and 93.5%, respectively. The purified PcP was homogeneous, and its weight average molecular weight (Mw) was 7,554 Da. Monosaccharide composition analysis indicated the PcP comprised mannose, galactose, glucose, galacturonic acid, arabinose, and rhamnose at a molar ratio of 33:13:8:3.5:2:1. This approach offers a practical tactic to purify polysaccharides of plant origin.

Recommendation of SLM Process Parameters Based on Analytic Hierarchy Process and Weighted Particle Swarm Optimization for High-Temperature Alloys.

Selective laser melting (SLM) of high-temperature alloys involves intricate interdependencies among key process parameters, such as laser power and scanning speed, affecting properties such as density and tensile strength. However, relying solely on experiential knowledge for process parameter design often hampers the precise attainment of target requirements. To address this challenge, we propose an innovative approach that integrates the analytic hierarchy process (AHP) and weighted particle swarm optimization (WPSO) to recommend SLM process parameters for high-temperature alloy fabrication. Our proposed AHP-WPSO model consists of three main steps. First, a comprehensive historical database is established, capturing the process parameters and performance metrics of high-temperature alloy SLM parts. Utilizing an AHP framework, we compute the performance similarity between target and historical cases, applying rational thresholds to identify analogous cases. When suitable analogs are elusive, the model seamlessly transitions to the second step. Here, the WPSO model optimizes and recommends process parameters according to target specifications. Lastly, our experimental validation of the GH4169 high-temperature alloy through SLM experiments corroborates the effectiveness of our AHP-WPSO model in making process parameter recommendations. The outcomes underscore the model's high accuracy, attaining a recommendation precision of 99.81% and 96.32% when historical analogs are present and absent, respectively. This innovative approach offers a robust and reliable solution to the challenges posed in SLM process parameter optimization for high-temperature alloy applications.

[Effects of danzhi jiangtang Capsule combined exercise on pancreatic oxidative stress and islet beta-cell function in diabetic rats].

OBJECTIVE: To explore the effects of Danzhi Jiangtang Capsule (DJC) and exercise on islet beta-cell function index (HOMA-% beta), blood glucose, and oxidative stress of diabetic rats. METHODS: A diabetic rat model was established using low dose streptozotocin and high fat forage in 60 male Wistar rats. The effects of exercise, DJC, and DJC combined exercise on the serum levels of fasting blood glucose (FBG), fasting insulin (FINS), triglyceride (TG), total cholesterol (TC), as well as the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and malondialdehyde (MDA) in the pancreatic tissue were observed. The HOMA-%beta was also calculated. The main factors that affecting HOMA-%beta were explored using multi-factor regression analysis. RESULTS: Compared with model group, the levels of FBG, TG, TC, and pancreatic MDA were significantly reduced after intervention of exercise or DJC (P < 0.05, P < 0.01), while HOMA-% beta obviously increased (P < 0.01). The pancreatic GSH-Px activity significantly increased in the exercise group (P < 0.01). Exercise and DJC had synergistic effects on FBG, TG, HOMA-% beta, pancreatic SOD, and GSH-Px activities (P < 0.05). There was a negative and linear regression correlation between FBG and pancreatic SOD and GSHPx activities. HOMA-%beta was negatively correlated with FBG, TG, TC, and pancreatic MDA content, and positively correlated with SOD and GSH-Px activities. Besides, there was a linear regression correlation between HOMA-%beta and FBG. CONCLUSION: Exercise and DJC played synergistic effects, could improve the glucose and lipid metabolisms and enhance antioxidant activities, thus relieving the injury of pancreatic beta cells.

[Effects of Chinese herbal medicine Danzhi Jiangtang Capsule and exercise on JNK signaling pathway in pancreatic tissues of diabetic rats].

OBJECTIVE: To explore the effects of exercise and Danzhi Jiangtang Capsule (DJC), a compound traditional herbal medicine, on the JNK signaling pathway in pancreatic tissues of diabetic rats and to investigate the possible mechanisms of exercise and DJC in treating diabetes. METHODS: Seventy-eight male Wistar rats were injected with low dose of streptozotocin and fed a high-fat diet to establish a diabetic model in rats. Then 60 diabetic rats were divided into diabetes group, exercise group, DJC group and exercise combined with DJC group. Another twelve rats were used as normal control. After eight months of treatment, the expression levels of phosphor-c-Jun N-terminal kinase (p-JNK), pancreatic and duodenal homeobox-1 (PDX-1), and insulin protein in pancreatic tissues from rats were detected by immunohistochemical method and Western blotting. RESULTS: In pancreatic tissues of diabetes group, the expression level of p-JNK protein was significantly higher than that in the normal group (P<0.01), and the expression levels of PDX-1 and insulin protein were significantly decreased (P<0.01). After administration of exercise and DJC, the expression level of p-JNK protein in pancreatic tissues of the diabetes group was decreased significantly, while the expression levels of PDX-1 and insulin protein were increased significantly (P<0.05 or P<0.01). CONCLUSION: Exercise and DJC effectively protect isletß-cell function in diabetic rats, which might be due to a decreased JNK signaling pathway.