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Androstenedione (AD) is a vital intermediate in the synthesis of steroid drugs, making its efficient production critical in the steroid drug industry. Acetyl-CoA acetyltransferase (FadA5), a thiolase enzyme, plays an important role in the metabolic process of degrading phytosterol side chains in Mycolicibacterium to produce AD. This work is the first systematic analysis of the role of FadA5 in the transformation of phytosterols by Mycolicibacterium to produce AD. The relationship between redox potential and AD production was examined using resting cells, and it was confirmed that FadA5 is a key enzyme for AD production. Mutating the 87th cysteine of FadA5 to alanine reduced its redox effect, enhancing the substrate tolerance and biotransformation capacity of the strain. Co-expressing Vitreoscilla hemoglobin (VHb) and propionyl-CoA metabolized the transcription activator (PrpR), decreased intracellular reactive oxygen species levels, and improved cell viability. The AD yield of MSP-fA5C87A-VP/ΔfA5 was 2.541 g/L, an increase of 16.83% over the control strain. Using a repeated batch fermentation process, the production efficiency of the MSP-fA5C87A-VP/ΔfA5 strain was 0.658 g/L/d, which was 1.82 times higher than that of the control strain. These findings provide a theoretical basis for understanding and regulating steroid side-chain catabolism in Mycolicibacterium and offer support for the rational modification of industrial strains for steroidal drug precursor production.

Chitin oligosaccharide (CHOS) is a chitin derivative with excellent biological activities. Enzymatic hydrolysis of chitin-rich biomass into CHOS is a hot topic in research on the high-value utilization of chitin resources. The disadvantages of complex preparation and purification processes and the high cost of chitin-degrading enzymes limit large-scale enzymatic production and application of CHOS. In this study, the activity of chitinase increased from 1.8 U/mL to 3.52 U/mL by 94.4% after optimizing the carbon and nitrogen source of Chitiniphilus sp. LZ32 fermentation. An enzyme production-adsorption-enzymolysis-product separation (EAES) system was constructed using fermentation, an adsorption purification module, and a product ultrafiltration module of a chitin-degrading enzyme. CHOS production by continuous enzymatic hydrolysis was performed in an EAES system using housefly larval powder (HLP) as the substrate. After the C. sp. LZ32 fermentation broth was circulated in the adsorption module for 90 min, the adsorption rate of the chitin-degrading enzyme reached more than 90%. The ultrafiltration module effectively separated CHOS at an operating pressure of 2 bar. Four batches of CHOS were produced in the EAES system using repeated batch fermentation. The running time of a single batch decreased from 115 h in the first batch to 48 h, and the CHOS output of each batch gradually increased. The total CHOS output was 61 g, and the production efficiency was 0.24 g/h. The CHOS produced by the EAES system (ECHOS) has high antioxidant activity. In this study, the EAES system was used to simplify the purification and separation steps of CHOS preparation, and the continuous production of CHOS was realized, which has potential application prospects in the field of green CHOS production.

The conventional methods used to produce N-acetyl-β-D-glucosamine (GlcNAc) from seafood waste require pretreatment steps that use acids or bases to achieve the extraction and decrystallization of chitin prior to enzymatic conversion. The development of an enzymatic conversion method that does not require the pretreatment of seafood waste is essential for the efficient and clean production of GlcNAc. In this study, the annotated metagenomic assembly data of domesticated microbiota (XHQ10) were analyzed to identify carbohydrate-active enzymes (CAZymes), and an in-depth analysis of the high-quality genome FS13.1, which was obtained from metagenomic binning, was performed; this enabled us to elucidate the catabolic mechanism of XHQ10 by using shrimp shell chitin as a carbon and nitrogen source. The only β-N-acetylglucosaminidase (named XmGlcNAcase) was cloned from FS13.1 and biochemically characterized. The direct production of GlcNAc from shrimp shell powder (SSP) via the use of a chitin enzyme cocktail was evaluated. Under the action of a chitin enzyme cocktail containing 5% recombinant XmGlcNAcase and a crude XHQ10 enzyme solution, the yield and purity of the final conversion of SSP to GlcNAc were 2.57 g/L and 82%, respectively. This is the first time that metagene-derived GlcNAcase has been utilized to achieve the enzymatic conversion of untreated seafood waste, laying the foundation for the low-cost and sustainable production of GlcNAc.

Androstenedione (AD) is an important intermediate for the production of steroidal drugs. The process of transforming phytosterols into AD by Mycolicibacterium is mainly the degradation process of the phytosterol side chain, and the excessive accumulation of propionyl-CoA produced by Mycobacterium will produce toxic effects, which seriously restricts the transformation performance of strains. In this study, Mycolicibacterium sp. LZ2 (Msp) was used as the research object to study the transcription factor PccD of the TetR family, which has the role of propionyl-CoA metabolism regulation. By constructing overexpression and deletion strains of pccD, it was confirmed that pccD had an inhibitory effect on the transcription of propionyl-CoA carboxylase genes (pccA and pccB). Electrophoretic Mobility Shift Assay (EMSA) and DNase I footprint analysis demonstrated that PccD is directly involved in the transcriptional regulation of pccA and pccB and is a negative transcriptional regulator of the pcc operon. In the study of phytosterol transformation, the growth rate and bacterial viability of Msp-ΔpccD were higher than Msp, but the growth of Msp-pccD was inhibited. As a result of testing of intracellular propionyl-CoA levels and AD production yields, it was found that lower propionyl-CoA levels and higher AD production yields were observed in Msp-ΔpccD. The results expand the cognition of propionyl-CoA metabolism regulation and provide a theoretical basis and reference for the rational transformation of phytosterol transformation strains and secondary metabolite synthesis strains with propionyl-CoA as a substrate, which has important research significance.

The direct enzymatic conversion of untreated waste shrimp and crab shells has been a key problem that plagues the large-scale utilization of chitin biological resources. The microorganisms in soil samples were enriched in two stages with powdered chitin (CP) and shrimp shell powder (SSP) as substrates. The enrichment microbiota XHQ10 with SSP degradation ability was obtained. The activities of chitinase and lytic polysaccharide monooxygenase of XHQ10 were 1.46 and 54.62 U/mL. Metagenomic analysis showed that Chitinolyticbacter meiyuanensis, Chitiniphilus shinanonensis, and Chitinimonas koreensis, with excellent chitin degradation performance, were highly enriched in XHQ10. Chitin oligosaccharides (CHOSs) are produced by XHQ10 through enzyme induction and two-stage temperature control technology, which contains CHOSs with a degree of polymerization (DP) more significant than ten and has excellent antioxidant activity. This work is the first study on the direct enzymatic preparation of CHOSs from SSP using enrichment microbiota, which provides a new path for the large-scale utilization of chitin bioresources.

Lytic polysaccharide monooxygenases (LPMO) are expected to change the current status of chitin resource utilization. This study reports that targeted enrichment of the microbiota was performed with chitin by the selective gradient culture technique, and a novel LPMO (M2822) was identified from the enrichment microbiota metagenome. First, soil samples were screened based on soil bacterial species and chitinase biodiversity. Then gradient enrichment culture with different chitin concentrations was carried out. The efficiency of chitin powder degradation was increased by 10.67 times through enrichment, and chitin degradation species Chitiniphilus and Chitinolyticbacter were enriched significantly. A novel LPMO (M2822) was found in the metagenome of the enriched microbiota. Phylogenetic analysis showed that M2822 had a unique phylogenetic position in auxiliary activity (AA) 10 family. The analysis of enzymatic hydrolysate showed that M2822 had chitin activity. When M2822 synergized with commercial chitinase to degrade chitin, the yield of N-acetyl glycosamine was 83.6% higher than chitinase alone. The optimum temperature and pH for M2822 activity were 35°C and 6.0. The synergistic action of M2822 and chitin-degrading enzymes secreted by Chitiniphilus sp. LZ32 could efficiently hydrolyze shrimp shell powder. After 12 h of enzymatic hydrolysis, chitin oligosaccharides (COS) yield reached 4,724 μg/mL. To our knowledge, this work is the first study to mine chitin activity LPMO in the metagenome of enriched microbiota. The obtained M2822 showed application prospects in the efficient production of COS.

Abstract A new serine carboxypeptidase gene, capA, was identified in Aspergillus niger CBS 513.88 by reading genomic information and performing sequence alignment, and the gene was cloned and expressed in Pichia pastoris GS115. In a shake flask, the enzyme activity of the recombinant strain GS115 (pPIC9K-capA) reached 209.3 U mg−1. The optimal temperature and pH for enzyme activity were determined to be 45 °C and 6.0, respectively. After incubation at 40–50 °C or at pH 4.0–8.0 for 1 h, the enzyme retained more than 80% or 60% of its initial activity. The presence of 1–10 mmol L−1 Mg2+ enhanced the activity of CapA, whereas 1–10 mmol L−1 Cu2+, Fe2+, or Co2+, 10 mmol L−1 Mn2+, or 1–10 mmol L−1 phenylmethylsulfonyl fluoride (PMSF) significantly inhibited its activity. CapA had a broad substrate specificity and preferred the hydrophobic amino acids Leu and Lys at the C terminus of proteins, and N-benzyloxycarbonyl-l-phenylalanyl-l-leucine (Cbz-Phe-Leu) was the optimal substrate, for which CapA exhibited K m 0.063 mmol L−1 and k cat /K m 186.35 mmol L−1 s−1. The good thermostability, pH stability and hydrolysis characteristics of CapA provide a solid foundation for application in the food and biotechnology fields.

Abstract Objective To determine the value of shear wave elastography (SWE) in assessing abdominal wall muscles, including rectus abdominis (RA), external oblique muscle (EO), internal oblique muscle, and transversus abdominis (TrA) in patients with diastasis recti abdominis (DRA) and healthy controls. Methods From October 2018 to December 2019, 36 postpartum DRA patients and 24 nulliparous healthy women were identified. Inter-rectus distance (IRD) measurements were taken by B-mode ultrasound. Shear wave speed (SWS) values were acquired by one operator at ten specific locations. Clinical and ultrasound variables, including demographics, IRD, muscle thickness, and muscle SWS, were compared between the two groups using Student’s t test or Fisher's exact test. Pearson correlation analyses were conducted for the variables of IRD, muscle thickness, and SWS in the 36 DRA patients. Results The maximum diameter of recti abdominus separation was located at the umbilicus in DRA patients (4.59 ± 1.14 cm). The SWS value was significantly lower in the RA (p = 0.003) and higher in the TrA muscle (p

Every year, seafood waste produced globally contains about 10 million tons of wasted crab, shrimp and lobster shells, which are rich in chitin resources. The exploitation and utilization of chitin resources are of great significance to environmental protection, economic development and sustainable development. Lytic polysaccharide monooxygenases (LPMOs) can catalyze polysaccharides by oxidative breakage of glycosidic bonds and have catalytic activity for chitin and cellulose, so they play an important role in the transformation of refractory polysaccharides into biomass. Although there have been many studies related to LPMOs, the research related to lytic chitin monooxygenases (LCMs) is still very limited. The specific catalytic mechanism of LCMs has not been fully elucidated, which poses a challenge to their application in industrial biomass conversion. This review introduces the present situation of resource development and utilization in chitin, the origin and classification of different LCMs families, the structural characteristics of LCMs and the relationship between structure and function. The research results related to activity detection, screening, preparation and transformation of LCMs were summarized and discussed. Finally, the synergistic effect of LCMs and chitin enzyme on biomass degradation was reviewed, and the existing problems and future research directions were pointed out. This is the first review focusing on Chitin-Active LPMOs in recent years, intending to provide a reference for applying chitin degradation enzymes system in the industry.

Abstract The early hepatic fibrosis staging is very important for timely diagnosis, prognosis, and treatment of all chronic liver diseases. Diffraction-enhanced imaging, which can provide much more information on soft tissue morphology than conventional absorption radiography, might be a potential noninvasive technique to diagnose and stage hepatic fibrosis. This paper presents different feature extraction strategies and classification methods to automatically classify hepatic fibrosis using diffraction-enhanced imaging images. Texture features are obtained using a total of three methods including first order feature, gray level co-occurrence matrix, and grayscale gradient co-occurrence matrix. The fusion of these texture features is also studied. The principal component analysis is used to reduce the dimension of the features and redundant information among data. The features are classified using two popular classification techniques, namely, K-nearest neighbors and support vector machines. On the basis of the comparison of different feature strategies and classification methods, we can identify the suitable methods for grading hepatic fibrosis. The proposed approach efficiently classifies the hepatic fibrosis DEI images into four classes with the highest classification accuracy of 99.99%. We further demonstrate the potential of the DEI images in staging hepatic fibrosis.

Abstract Background Androstenedione (AD) is an important steroid medicine intermediate that is obtained via the degradation of phytosterols by mycobacteria. The production process of AD is mainly the degradation of the phytosterol aliphatic side chain, which is accompanied by the production of propionyl CoA. Excessive accumulation of intracellular propionyl-CoA produces a toxic effect in mycobacteria, which restricts the improvement of production efficiency. The 2-methylcitrate cycle pathway (MCC) plays a significant role in the detoxification of propionyl-CoA in bacterial. The effect of the MCC on phytosterol biotransformation in mycobacteria has not been elucidated in detail. Meanwhile, reducing fermentation cost has always been an important issue to be solved in the optimizing of the bioprocess. Results There is a complete MCC in Mycobacterium neoaurum (MNR), prpC, prpD and prpB in the prp operon encode methylcitrate synthase, methylcitrate dehydratase and methylisocitrate lyase involved in MCC, and PrpR is a specific transcriptional activator of prp operon. After the overexpression of prpDCB and prpR in MNR, the significantly improved transcription levels of prpC, prpD and prpB were observed. The highest conversion ratios of AD obtained by MNR-prpDBC and MNR-prpR increased from 72.3 ± 2.5% to 82.2 ± 2.2% and 90.6 ± 2.6%, respectively. Through enhanced the PrpR of MNR, the in intracellular propionyl-CoA levels decreased by 43 ± 3%, and the cell viability improved by 22 ± 1% compared to MNR at 96 h. The nitrogen transcription regulator GlnR repressed prp operon transcription in a nitrogen-limited medium. The glnR deletion enhanced the transcription level of prpDBC and the biotransformation ability of MNR. MNR-prpR/ΔglnR was constructed by the overexpression of prpR in the glnR-deleted strain showed adaptability to low nitrogen. The highest AD conversion ratio by MNR-prpR/ΔglnR was 92.8 ± 2.7% at low nitrogen level, which was 1.4 times higher than that of MNR. Conclusion Improvement in phytosterol biotransformation after the enhancement of propionyl-CoA metabolism through the combined modifications of the prp operon and glnR of mycobacteria was investigated for the first time. The overexpress of prpR in MNR can increase the transcription of essential genes (prpC, prpD and prpB) of MCC, reduce the intracellular propionyl-CoA level and improve bacterial viability. The knockout of glnR can enhance the adaptability of MNR to the nitrogen source. In the MNRΔglnR strain, overexpress of prpR can achieve efficient production of AD at low nitrogen levels, thus reducing the production cost. This strategy provides a reference for the economic and effective production of other valuable steroid metabolites from phytosterol in the pharmaceutical industry.

A predicted metalloproteinase gene, HypZn, was cloned from Aspergillus niger CGMCC 3.7193 and expressed in Pichia pastoris GS115, and the physicochemical characteristics of recombinant HypZn were investigated after separation and purification. The results showed that the specific activity of the purified HypZn reached 1859.2 U/mg, and the optimum temperature and pH value of HypZn were 35°C and 7.0, respectively. HypZn remained stable both at 40°C and at pH values between 5.0 and 8.0. The preferred substrate of HypZn was soybean protein isolates, and the Km and Vmax values were 21.5 μmol/mL and 4926.6 μmol/(mL∙min), respectively. HypZn was activated by Co2+ and Zn2+ and inhibited by Cu2+ and Fe2+. The degree of soybean protein isolate hydrolysis reached 14.7%, and the hydrolysates were of uniform molecular weight. HypZn could tolerate 5000 mM NaCl and completely lost its activity after 30 min at 50°C. The enzymological characterizations indicated that HypZn has great application potential in the food industry, especially in fermented food processing.

Objective This study investigated the synergistic repair effects of Prontosan hydrogel and carbon photon therapy in a rat full-thickness wound model. Methods The wavelength distribution of the photon source was determined. Dehydration of the Prontosan hydrogel and fibroblast viability were analyzed following exposure to different durations of light exposure at different distances from the source. Indexes of wound healing in a full-thickness rat wound model were then determined in groups ( n = 8 each) subjected to either no treatment, Prontosan treatment only, carbon photon therapy only, or a combination of the two treatments (synergistic group). Results Carbon photon exposure for 15 minutes at a distance of 20 cm from the wound was found to be optimal. Wound healing occurred faster in the synergistic group compared with the control and single-treatment groups. Growth factor secretion, granulation tissue formation, inflammation regulation, collagen deposition, and neovascularization were all higher in the synergistic group. Conclusions Prontosan hydrogel combined with carbon photon therapy may provide an optimal environment for wound healing and serve as a novel physical approach to the treatment of wounds. However, the number of animals included in this study was relatively small and a larger study is required to confirm these findings.

Abstract Background Many studies have combined sorafenib with transcatheter arterial chemoembolization (TACE) to treat patients with advanced hepatocellular carcinoma (HCC), but the results are disputable. Thus, we conducted this meta-analysis to assess the efficacy and safety of the combination treatment in patients with advanced HCC. Methods Clinical data were collected from a computer search of literature published from January 2009 to June 2016 in PubMed, Web of Science, the Cochrane Library, China National Knowledge Infrastructure (CNKI), Wan Fang and the China Science and Technology Journal Database (CSTJ). The final analysis included 14 studies and 1670 patients. The primary endpoints were overall survival (OS), the objective response rate (ORR) and the disease control rate (DCR). Results The combination group exhibited significantly more improvement than the group treated with TACE alone in ORR (RR =1.62, 95% confidence interval (CI) = 1.34–1.94, p

Abstract Small cell lung cancer (SCLC), as a proportion, makes up only 15–17% of lung cancer cases. The development of treatments for SCLC has remained stagnant for decades, and SCLC is expected to persist as a threat to human health. To date, no publications based on large populations have been reported. We calculated survival changes in patients with SCLC during each decade between 1983 and 2012 to determine the roles of race, sex, age, and socioeconomic status (SES) on survival rates based on the Surveillance, Epidemiology, and End Results (SEER) registries. In total, 106,296 patients with SCLC were identified, with the overall incidence per 100,000 decreasing each decade from 9.6 to 7.8 to 5.8. The median survival for SCLC remained 7 months, and the 12-month relative survival rates (RSRs) remained relatively stable at 32.9%, 33.2% and 33.2% during each decade. The 5-year RSRs significantly improved from 4.9% to 5.9% to 6.4% during each decade, but remained extremely low. In addition, a narrowing of the survival gaps among SES groups and stable survival gaps between sexes were observed. Although the incidence of SCLC decreased during each decade, the overall survival remained relatively stable, highlighting the urgency of developing novel treatments and the importance of prevention and early detection.

Non–small cell lung cancer is the most common malignancy in males; it constitutes the majority of lung cancer cases and requires massive medical resources. Despite improvements in managing non–small cell lung cancer, long-term survival remains very low. This study evaluated survival improvement in patients with non–small cell lung cancer in each decade between 1983 and 2012 to determine the impact of race, sex, age, and socioeconomic status on the survival rates in these patients. We extracted data on non–small cell lung cancer cases in each decade between 1983 and 2012 from the Surveillance, Epidemiology, and End Results registries. In total, 573,987 patients with non–small cell lung cancer were identified in 18 Surveillance, Epidemiology, and End Results registry regions during this period. The 12-month relative survival rates improved slightly across three decades, from 39.7% to 40.9% to 45.5%, with larger improvement in the last two decades. However, the 5-year-relative survival rates were very low, with 14.3%, 15.5%, and 18.4%, respectively, in three decades, indicating the urgency for novel comprehensive cancer care. In addition, our data demonstrated superiority in survival time among non–small cell lung cancer patients of lower socioeconomic status and White race. Although survival rates of non–small cell lung cancer patients have improved across the three decades, the 5-year-relative survival rates remain very poor. In addition, widening survival disparities among the race, the sex, and various socioeconomic status groups were confirmed. This study will help in predicting future tendencies of incidence and survival of non–small cell lung cancer, will contribute to better clinical trials by balancing survival disparities, and will eventually improve the clinical management of non–small cell lung cancer.

A study on room temperature Suzuki cross-coupling in an aqueous medium was carried out using a simple hydrophilic palladium (II) complex, trans-PdCl2(NH2CH2COOH)2 as catalyst in the presence of K2CO3 in air. This approach with a comparatively inexpensive and hydrophilic catalyst, mild reaction condition and aqueous media exhibits excellent catalytic activity towards the Suzuki coupling of aryl bromides and arylboronic acids, and good yields were obtained in the Suzuki coupling of activated aryl chlorides.

A new and efficient Pd(II)/AgNO3-cocatalyzed homocoupling of aromatic terminal alkynes is described. Various symmetrical 1,4-disubstituted-1,3-diynes are obtained in good to excellent yields. This protocol employs a loading with relatively low palladium(II) in aqueous media under aerobic conditions.