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Xiao-Xi Li,1,* Hong Li,2,* Li-Quan Jin,3 Yun-Bo Tan1,3 1Dali University of Clinical Medicine School, Dali, Yunnan, 671000, People’s Republic of China; 2Department of Radiology, Affiliated Renhe Hospital of China Three Gorges University, Hubei, 443001, People’s Republic of China; 3Department of General Surgery, The First of Affiliated Hospital of Dali University, Dali, Yunnan, 671000, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yun-Bo Tan, Department of General Surgery, the First of Affiliated Hospital of Dali University, No. 32 of Carlsberg Road, Dali, Yunnan, 671000, People’s Republic of China, Tel +86 15517162885, Email tanyunbo6365@163.comObjective: To find pancreatic cancer (PC)-related hub genes based on weighted gene co-expression network analysis (WGCNA) construction and immune infiltration score analysis and validate them immunohistochemically by clinical cases, to generate new concepts or therapeutic targets for the early diagnosis and treatment of PC.Material and Methods: In this study, WGCNA and immune infiltration score were utilized to identify the relevant core modules of PC and the hub genes within these core modules.Results: Using WGCNA analysis, data from PC and normal pancreas integrated with TCGA and GTEX were analyzed and brown modules were chosen from the six modules. Five hub genes, including DPYD, FXYD6, MAP6, FAM110B, and ANK2, were discovered to have differential survival significance via validation tests utilizing survival analysis curves and the GEPIA database. The DPYD gene was the only gene associated with PC survival side effects. Validation of the Human Protein Atlas (HPA) database and immunohistochemical testing of clinical samples showed positive results for DPYD expression in PC.Conclusion: In this study, we identified DPYD, FXYD6, MAP6, FAM110B, and ANK2, as immune-related candidate markers for PC. Only the DPYD gene had a negative impact on the survival of PC patients. Through validation of the HPA database and immunohistochemical testing of clinical cases, we believe that the DPYD gene brings novel ideas and therapeutic targets in the diagnosis and treatment of PC.Keywords: DPYD, pancreatic cancer, tumor immunity, WGCNA

D-Allulose 3-epimerase (DAE) is a vital biocatalyst for the industrial synthesis of D-allulose, an ultra-low calorie rare sugar. However, limited thermostability of DAEs hinders their use at high-temperature production. In this research, hyperthermophilic TI-DAE (Tm = 98.4 ± 0.7 ℃) from Thermotoga sp. was identified via in silico screening. A comparative study of the structure and function of site-directed saturation mutagenesis mutants pinpointed the residue I100 as pivotal in maintaining the high-temperature activity and thermostability of TI-DAE. Employing TI-DAE as a biocatalyst, D-allulose was produced from D-fructose with a conversion rate of 32.5%. Moreover, TI-DAE demonstrated excellent catalytic synergy with glucose isomerase CAGI, enabling the one-step conversion of D-glucose to D-allulose with a conversion rate of 21.6%. This study offers a promising resource for the enzyme engineering of DAEs and a high-performance biocatalyst for industrial D-allulose production., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In response to the persistent expansion of global resource demands, considerable attention has been directed toward the synthetic microbial consortia (SMC) within the domain of microbial engineering, aiming to address the sustainable management and valorization of biomass wastes. This comprehensive review systematically encapsulates the most recent advancements in research and technological applications concerning the utilization of SMC for biomass waste treatment. The construction strategies of SMC are briefly outlined, and the diverse applications of SMC in biomass wastes treatment are explored, with particular emphasis on its potential advantages in waste degradation, hazardous substances control, and high value-added products conversion. Finally, recommendations for the future development of SMC technology are proposed, and prospects for its sustainable application are discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Transaminase (TA) is a crucial biocatalyst for enantioselective production of the herbicide L-phosphinothricin (L-PPT). The use of enzymatic cascades has been shown to effectively overcome the unfavorable thermodynamic equilibrium of TA-catalyzed transamination reaction, also increasing demand for TA stability. In this work, a novel thermostable transaminase (PtTA) from Pseudomonas thermotolerans was mined and characterized. The PtTA showed a high specific activity (28.63 U/mg) towards 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO), with excellent thermostability and substrate tolerance. Two cascade systems driven by PtTA were developed for L-PPT biosynthesis, including asymmetric synthesis of L-PPT from PPO and deracemization of D, L-PPT. For the asymmetric synthesis of L-PPT from PPO, a three-enzyme cascade was constructed as a recombinant Escherichia coli (E. coli G), by co-expressing PtTA, glutamate dehydrogenase (GluDH) and D-glucose dehydrogenase (GDH). Complete conversion of 400 mM PPO was achieved using only 40 mM amino donor L-glutamate. Furthermore, by coupling D-amino acid aminotransferase (Ym DAAT) from Bacillus sp. YM-1 and PtTA, a two-transaminase cascade was developed for the one-pot deracemization of D, L-PPT. Under the highest reported substrate concentration (800 mM D, L-PPT), a 90.43% L-PPT yield was realized. The superior catalytic performance of the PtTA-driven cascade demonstrated that the thermodynamic limitation was overcome, highlighting its application prospect for L-PPT biosynthesis. KEY POINTS: • A novel thermostable transaminase was mined for L-phosphinothricin biosynthesis. • The asymmetric synthesis of L-phosphinothricin was achieved via a three-enzyme cascade. • Development of a two-transaminase cascade for D, L-phosphinothricin deracemization., (© 2024. The Author(s).)

Previously, we reported that RhoA knockdown by morpholino antisense oligonucleotides (MOs), and enzymatic digestion of chondroitin sulfate proteoglycans (CSPGs) at the site of injury with chondroitinase ABC (ChABC), each can reduce retrograde neuronal apoptosis after spinal cord transection in the lamprey. To elucidate the mechanisms in neuronal survival and axon regeneration, we have investigated whether these two effects are additive in vivo . We used lampreys as a spinal cord injury model. MOs were used to knockdown RhoA and Chondroitinase ABC (ChABC) was used to digest CSPGs in vivo . Retrograde labeling, fluorochrome-labeled inhibitor of caspase activity (FLICA), immunohistochemistry, and western blots were performed to assess axonal regeneration, neuronal apoptotic signaling and Akt activation. Four treatment combinations were evaluated at 2-, 4-, and 10-weeks post-transection: (1) Control MO plus enzyme buffer (Ctrl); (2) control MO plus ChABC; (3) RhoA MO plus enzyme buffer (RhoA MO); and (4) RhoA MO plus ChABC (RhoA MO + ChABC). Consistent with our previous findings, at 4-weeks post-transection, there was less caspase activation in the ChABC and RhoA MO groups than in the Ctrl group. Moreover, the RhoA MO plus ChABC group had the best protective effect on identified reticulospinal (RS) neurons among the four treatment combinations. At 2 weeks post-transection, when axons have retracted maximally in the rostral stump and are beginning to regenerate back toward the lesion, the axon tips in the three treatment groups each were closer to the transection than those in the Ctr MO plus enzyme buffer group. Long-term axon regeneration also was evaluated for the large, individually identified RS neurons at 10 weeks post-transection by retrograde labeling. The percent regenerated axons in the RhoA MO plus ChABC group was greater than that in any of the other groups. Akt phosphorylation levels at threonine 308 was quantified in the identified RS neurons by western blots and immunofluorescence. The RhoA MO plus ChABC treatment enhanced pAkt-308 phosphorylation more than any of the other treatment groups. Although some of the effects of CSPGs are mediated through RhoA activation, some growth-inhibiting mechanisms of RhoA and CSPGs are independent of each other, so combinatorial therapies may be warranted., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hu, Zhang, Rodemer, Jin and Selzer.)

2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) is the essential precursor keto acid for the asymmetric biosynthesis of herbicide l-phosphinothricin (l-PPT). Developing a biocatalytic cascade for PPO production with high efficiency and low cost is highly desired. Herein, a d-amino acid aminotransferase from Bacillus sp. YM-1 (Ym DAAT) with high activity (48.95 U/mg) and affinity (K m  = 27.49 mM) toward d-PPT was evaluated. To circumvent the inhibition of by-product d-glutamate (d-Glu), an amino acceptor (α-ketoglutarate) regeneration cascade was constructed as a recombinant Escherichia coli (E. coli D), by coupling Ym d-AAT, d-aspartate oxidase from Thermomyces dupontii (TdDDO) and catalase from Geobacillus sp. CHB1. Moreover, the regulation of the ribosome binding site was employed to overcome the limiting step of expression toxic protein TdDDO in E. coli BL21(DE3). The aminotransferase-driven whole-cell biocatalytic cascade (E. coli D) showed superior catalytic efficiency for the synthesis of PPO from d,l-phosphinothricin (d,l-PPT). It revealed the production of PPO exhibited high space-time yield (2.59 g L -1  h -1 ) with complete conversion of d-PPT to PPO at high substrate concentration (600 mM d,l-PPT) in 1.5 L reaction system. This study first provides the synthesis of PPO from d,l-PPT employing an aminotransferase-driven biocatalytic cascade., (© 2023 Wiley Periodicals LLC.)

Cellobiose 2-epimerase (CE) is ideally suited to synthesize lactulose from lactose, but the poor thermostability and catalytic efficiency restrict enzymatic application. Herein, a non-characterized CE originating from Caldicellulosiruptor morganii (CmCE) was discovered in the NCBI database. Then, a smart mutation library was constructed based on FoldX ΔΔG calculation and modeling structure analysis, from which a positive mutant D226G located within the α 8 /α 9 loop exhibited longer half-lives at 65-75 °C as well as lower K m and higher k cat /K m values compared with CmCE. Molecular modeling demonstrated that the improvement of D226G was largely attributed to the rigidification of the flexible loop, the compactness of the catalysis pocket and the increment of substrate-binding capability. Finally, the yield of synthesizing lactulose catalyzed by D226G reached 45.5%, higher than the 35.9% achieved with CmCE. The disclosed effect of the flexible loop on enzymatic stability and catalysis provides insight to redesign efficient CEs to biosynthesize lactulose., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

To investigate the changes in C, N, P, and K contents and ecological stoichiometry of desert oasis soils and to elucidate their ecological responses to environmental factors, 10 sample plots were selected in the Zhangye Linze desert oasis in the middle part of the Hexi Corridor, and surface soil samples were collected to determine the C, N, P and K contents of soils and to reveal the distribution characteristics of soil nutrient contents and stoichiometric ratios in different habitats and the correlation with other environmental factors. The results showed that:① the distribution of soil carbon was uneven and heterogeneous across sites ( R =0.761, P =0.06). The highest mean value was 12.85 g·kg -1 in the oasis, followed by 8.65 g·kg -1 in the transition zone and 4.1 g·kg -1 in the desert. ② The content of total soil potassium did not vary significantly and was high among deserts, transition zones, and oases and low in saline areas. ③ The mean soil C:N value was 12.92, the mean C:P value was 11.69, and the mean N:P value was 0.9, all of which were lower than the global average soil content (13.33, 72.0, and 5.9) and the Chinese soil average (12, 52.7, and 3.9). ④ Soil water content was the most influential factor affecting C, N, P, K, and ecological stoichiometry characteristics of desert oasis soils, with a contribution of 86.9%, followed by soil pH and soil porosity, with contributions of 9.2% and 3.9%, respectively. The results of this study provide basic information for the restoration and conservation of desert and oasis ecosystems and establish a basis for future study of the biodiversity maintenance mechanism in the region and its correlation with the environment.

MiSeq sequencing technology was used to investigate the bacterial compositions and diversities of active patch, non-active patch, recovered patch, and healthy alpine meadows so as to understand the changes in soil bacterial community diversity during altitude change and alpine meadow degradation. The relationship between bacterial diversity and environmental factors was analyzed using redundancy analysis (RDA). The results showed that the dominant bacterial phyla in the soil included Proteobacteria, Actinobacteriota, and Acidobacteriota in the study areas. The dominant bacterial genera that were identified via the MiSeq were RB41, Sphingomonas, and Bradyrhizobium. The relative abundance of these genera decreased with altitude increase and increased with the restoration progress of degraded patches but was significantly lower than that in the alpine meadow ( P <0.05). The abundance of functional bacteria related to carbon fixation in degraded patches was higher than that in the healthy alpine meadow. The bacterial Chao1 index and species number in different types of degraded patches were significantly higher than those in the alpine meadow ( P <0.05). The results of the RDA suggest that biological soil crust coverage and total nitrogen were the main influencing factors on dominant bacterial phyla at the altitude of 4013 m. Biomass, total nitrogen, and pH had a great influence on the dominant bacterial phyla at the altitude of 4224 m. Biomass and total potassium significantly affected the distribution of bacterial genera at the altitude of 4013 m. Sedge coverage and available nitrogen were the main influencing factors on bacterial dominant genera at the altitude of 4224 m. Biological soil crusts and pH had a great influence on bacterial diversities. The bacterial influence factors varied greatly at different altitude areas. Therefore, we should not only pay attention to the effect of alpine meadow degradation but also consider the effect of altitude in the study of bacterial diversity changes.

Competing Interests: None

Axotomy in the CNS activates retrograde signals that can trigger regeneration or cell death. Whether these outcomes use different injury signals is not known. Local protein synthesis in axon tips plays an important role in axon retraction and regeneration. Microarray and RNA-seq studies on cultured mammalian embryonic or early postnatal peripheral neurons showed that axon growth cones contain hundreds to thousands of mRNAs. In the lamprey, identified reticulospinal neurons vary in the probability that their axons will regenerate after axotomy. The bad regenerators undergo early severe axon retraction and very delayed apoptosis. We micro-aspirated axoplasms from 10 growing, 9 static and 5 retracting axon tips of spinal cord transected lampreys and performed single-cell RNA-seq, analyzing the results bioinformatically. Genes were identified that were upregulated selectively in growing ( n = 38), static (20) or retracting tips (18). Among them, map3k2 , csnk1e and gtf2h were expressed in growing tips, mapk8(1) was expressed in static tips and prkcq was expressed in retracting tips. Venn diagrams revealed more than 40 components of MAPK signaling pathways, including jnk and p38 isoforms, which were differentially distributed in growing, static and/or retracting tips. Real-time q-PCR and immunohistochemistry verified the colocalization of map3k2 and csnk1e in growing axon tips. Thus, differentially regulated MAPK and circadian rhythm signaling pathways may be involved in activating either programs for axon regeneration or axon retraction and apoptosis.

Axon regrowth after spinal cord injury (SCI) is inhibited by several types of inhibitory extracellular molecules in the central nervous system (CNS), including chondroitin sulfate proteoglycans (CSPGs), which also are components of perineuronal nets (PNNs). The axons of lampreys regenerate following SCI, even though their spinal cords contain CSPGs, and their neurons are enwrapped by PNNs. Previously, we showed that by 2 weeks after spinal cord transection in the lamprey, expression of CSPGs increased in the lesion site, and thereafter, decreased to pre-injury levels by 10 weeks. Enzymatic digestion of CSPGs in the lesion site with chondroitinase ABC (ChABC) enhanced axonal regeneration after SCI and reduced retrograde neuronal death. Lecticans (aggrecan, versican, neurocan, and brevican) are the major CSPG family in the CNS. Previously, we cloned a cDNA fragment that lies in the most conserved link-domain of the lamprey lecticans and found that lectican mRNAs are expressed widely in lamprey glia and neurons. Because of the lack of strict one-to-one orthology with the jawed vertebrate lecticans, the four lamprey lecticans were named simply A, B, C, and D. Using probes that distinguish these four lecticans, we now show that they all are expressed in glia and neurons but at different levels. Expression levels are relatively high in embryonic and early larval stages, gradually decrease, and are upregulated again in adults. Reductions of lecticans B and D are greater than those of A and C. Levels of mRNAs for lecticans B and D increased dramatically after SCI. Lectican D remained upregulated for at least 10 weeks. Multiple cells, including glia, neurons, ependymal cells and microglia/macrophages, expressed lectican mRNAs in the peripheral zone and lesion center after SCI. Thus, as in mammals, lamprey lecticans may be involved in axon guidance and neuroplasticity early in development. Moreover, neurons, glia, ependymal cells, and microglia/macrophages, are responsible for the increase in CSPGs during the formation of the glial scar after SCI., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhang, Jin, Rodemer, Hu, Root, Medeiros and Selzer.)

d-Alanine belongs to nonessential amino acids that have diverse applications in the fields of food and health care. ( R )-transaminase [( R )-TA]-catalyzed asymmetric amination of pyruvate is a feasible alternative for the synthesis of d-alanine, but low catalytic efficiency and thermostability limit enzymatic utilization. In this work, several potential ( R )-TAs were discovered using NCBI database mining synchronously with enzymatic structure-function analysis, among which Capronia epimyces TA (CeTA) showed the highest activity for amination of pyruvate using ( R )-α-methylbenzylamine as the donor. Furthermore, enzymatic residues surrounding a large catalysis pocket were subjected to saturation and combinatorial mutagenesis, and positive mutant F113T showed dramatic improvement in activity and thermostability. Molecular modeling indicated that the substitution of phenylalanine with threonine afforded alleviation of steric hindrance in the pocket and induced formation of additional hydrogen bonds with neighboring residues. Finally, using recombinant cells containing F113T as a biocatalyst, the conversion yield of amination of 100 mM pyruvate to d-alanine achieved up to 95.2%, which seemed to be the highest level in the literature regarding synthesis of d-alanine using TAs. The inherent characteristics rendered CeTA F113T a promising platform for efficient preparation of d-alanine operating with high productivity. IMPORTANCE d-Alanine is an important compound with many valuable applications. Its asymmetric synthesis employing ( R )-ω-TA is considered an attractive choice. According to the stereoselectivity, ω-TAs have either ( R )- or ( S )-enantiopreference. There has been a variety of literature regarding screening, characterizing, and molecular modification of ( S )-ω-TAs; in contrast, the research about ( R )-ω-TA has lagged behind. In this work, we identify several ( R )-ω-TAs and succeeded in creating mutant F113T, which showed not only better efficiency toward pyruvate but also higher thermostability compared with the original enzyme. The obtained original enzymes and positive mutants displayed important application value for pushing symmetric synthesis of d-alanine to a higher level.

The use of Fu Shen and Fu Shen Mu as medicines has had a long history. Today Fu Shen is still taken as bulk medicinal materials, whereas Fu Shen Mu had disappeared in the medical market. Fu Shen, Yun Fu Shen, Bai Fu Shen, and Bao Mu Fu Shen were used in clinical application in the Qing Royals. Bai Fu Shen and Fu Shen Mu are still kept as speciment in the Palace Museum today. It was found that Bai Fu Shen in the Qing Royals was the same as Fu Shen after peeling and pine roots recorded in the herbal literatures of the Ming and Qing dynasties, with their character tests and historical analysis. It can be inferred that Fu Shen, Yun Fu Shen and Bai Fu Shen recorded in the Qing Royals were actually Fu Shen, with pine roots in sclerotia and after peeling and pine roots removed in processing. Bao Mu Fu Shen and Bao Fu Shen should refer to Fu Shen with pine roots. Fu Shen Mu should mean Fu Shen without white sclerotia and peel during processing. Fu Shen, currently used clinically, is Bao Mu Fu Shen in the Qing Dynasty. Fu Shen distinguishes greatly from Fu Shen Mu in their effects. Such identification and analysis of herbs provides a way of thinking for further hurb studies of the Qing Dynasty.

Transaminases catalyze the transfer of an amino group from a donor to a keto group of an acceptor substrate and are applicable to the asymmetric synthesis of herbicide L-phosphinothricin (L-PPT). Here, the important residue sites (C390, I22, V52, R141, Y138 and D239) of transaminase from Salmonella enterica (SeTA) were modified at the adjacency of the substrate-binding pocket to improve the enzyme activity. Among the constructed mutant library, the SeTA-Y138F mutant displayed higher activity than the wild-type enzyme. Compared to the wild-type, SeTA-Y138F showed improved catalytic efficiency with a 4.36-fold increase. The K m and k cat of SeTA -Y138F toward 4-(hydroxy(methyl) phosphoryl)-2-oxobutanoic acid (PPO) were 26.39 mM and 34.28 s -1 , respectively. Subsequently, the three-enzyme co-expression system of E. coli BL21 (DE3)/pACYCDuet-SeTA-Y138F/pETDuet-AlaDH-BsGDH was developed by combining a alanine dehydrogenase (AlaDH) to recycle the byproduct of amino donor, a glucose dehydrogenase (BsGDH) for cofactor recycling. Under the optimized conditions, an excellent L-PPT yield of 90.8% was achieved by the whole-cell biotransformation with 500 mM PPO. It exhibited the tri-enzymatic coupling system was potential for effective production of target L-PPT., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Glucose isomerase (GI) is a key enzyme in the preparation of high fructose corn syrup (HFCS). In this study, a mutant TEGI-M-L38 M/V137 L (TEGI-M2) of glucose isomerase (TEGI-M) originated from Thermoanaerobacter ethanalicus CCSD1 was obtained by site-directed mutagenesis. The TEGI-M2 showed an optimal activity at 85 ℃ and pH 6.5 with the divalent cations Co 2+ and Mg 2+ . The structural differences between TEGI-M and TEGI-M2 were investigated based on the homology modeling and molecular docking, to elucidate the mechanism of improvement in the enzymatic properties. Compared with the original enzyme, the TEGI-M2 showed a 2.0-fold increased enzyme activity and a decreased K m from 234.2 mM to 85.9 mM. Finally, the application of mutant TEGI-M2 in HFCS one-step biosynthesis was attempted, resulting in a d-fructose yield of 67.3 %, which was 14.3 % higher than that of TEGI-M. This improved catalytic performance of TEGI-M2 was of great importance for the industrial preparation of d-fructose in one-step process., (Copyright © 2021 Elsevier Inc. All rights reserved.)

MiSeq sequencing technology was used to analyze the microbial community diversity of soil in alpine wetlands to understand the degradation processes and environmental factors in these areas. The results showed that the severity of soil degradation changed the species diversity of soil microorganisms at the level of OTUs, and grass patches contained more species than frozen-thawing patches. The soil fungi species of OTUs changed significantly. The diversity indexes of bacteria (between the frozen-thawing patches and the grass patches) were higher than that of fungi. The dominant microbial species were consistent among different degradation stages. The dominant species of bacteria and fungi were Proteobacteria and RB41, and Ascomycota and Mortierella , respectively. The abundance of dominant microorganisms was significantly between un-degraded and heavily degraded areas, except for RB41 ( P <0.05). The dominant microorganisms in the grass patches were more sensitive than those in the frozen-thawing patches. It was found that the main factors affecting the microbial community structure of soil were water content, organic carbon, microbial biomass carbon, microbial biomass nitrogen, and sedge coverage. Microbial diversity may decrease in heavily degraded alpine wetlands. Thus, the frozen-thawing patches and sedge species should be first protected, and the supplements of soil water content, soil organic carbon, microbial biomass carbon, and nitrogen should be strengthened for alpine wetland restoration.

To improve the operational stability of glucose isomerase in E. coli TEGI-W139F/V186T, the immobilized cells were prepared with modified diatomite as a carrier and 74.1% activity of free cells was recovered after immobilization. Results showed that the immobilized cells still retained 86.2% of the initial transformational activity after intermittent reused 40 cycles and the yield of D-fructose reached above 42% yield at 60 °C. Moreover, the immobilized cells were employed in the continuous production of High Fructose Corn Syrup (HFCS) in a recirculating packed bed reactor for 603 h at a constant flow rate. It showed that the immobilized cells exhibited good operational stability and the yield of D-fructose retained above 42% within 603 h. The space-time yield of high fructose corn syrup reached 3.84 kg L -1  day -1 . The investigation provided an efficient immobilization method for recombinant cells expressing glucose isomerase with higher stability, and the immobilized cells are a promising biocatalyst for HFCS production.

Objective: To identify the factors associated with long-term survival and guide the decision for primary surgery in patients with advanced high-grade serous ovarian cancer(HGSOC). Methods: In this case-control study, clinical parameters, including surgical and non-surgical associated factors, were collected and compared between the patients with short-term (<2 years) and long-term (>5 years) survival who all underwent primary debulking surgery (PDS) followed by carboplatin and paclitaxel chemotherapy from January 2004 to December 2016. Univariate analysis was examined by chi-square test and multivariate analysis was performed by logistic regression analysis. Results: There were 95 cases long-term survival (LTS group) and 77 cases short-term survival (STS group) in 698 newly diagnosed HGSOC patients with International Federation of Gynecology and Obstetrics (FIGO) stage Ⅲc and Ⅳ who met include and exclude criteria. (1) Univariate analysis showed that the proportion of complete cytoreduction with no visible residual disease (R0) at PDS and platinum sensitivity in LTS group were significantly higher than those in STS group ( P <0.01). The surgical complexity score (SCS), the preoperative serum CA 125 level and the ascites volume in the LTS group were significantly lower than those of the STS group (all P <0.05). In the LTS group, the preoperative incidence of lesions in retrograde peritoneum of the bladder, serosal and mesangial membrane of the small intestine, upper abdominal peritoneum and liver parenchyma were significantly lower than those in the STS group (all P <0.05). Multivariate logistic regression analysis showed that platinum sensitivity ( OR =0.016, 95% CI : 0.004-0.063, P <0.01), ascites volume >500 ml ( OR =3.193, 95% CI : 1.285-7.930, P =0.012), and SCS ≥8 ( OR =17.433, 95% CI : 2.281-133.25, P =0.003) were independent factors affecting long-term survival ( P> 0.05). (2) Totally 37 of 95 in long-term survival and 16 of 77 in short-term survival achieved R0 cytoreduction at PDS. Univariate analysis showed that preoperative serum CA 125 level, preoperative lesion score, preoperative lesion (DS) score, ascites volume, platinum sensitivity,and SCS were significantly correlated with the R0 PDS (all P <0.05). Multivariate analysis showed that ascites volume >500 ml ( OR =5.199, 95% CI : 2.015-13.409, P =0.001), DS >2 ( OR =15.264, 95% CI : 5.843-39.874, P <0.01) and SCS ≥4 ( OR =4.176, 95% CI : 1.618-10.777, P =0.003) were independent factors associated with R0 cytoreduction. In patients with DS ≤2 or SCS <4, but not those with DS >2 or SCS ≥4, R0 cytoreduction was significantly associated with long-term survival. Conclusion: The intrinsic biology of tumor is the factor influencing long-term survival of advanced HGSOC patients, and those who present with wide intraperitoneal metastases and need to remove multiple organs may not benefit from R0 cytoreduction.

Paralysis following spinal cord injury (SCI) is due to failure of axonal regeneration. It is believed that axon growth is inhibited by the presence of several types of inhibitory molecules in central nervous system (CNS), including the chondroitin sulfate proteoglycans (CSPGs). Many studies have shown that digestion of CSPGs with chondroitinase ABC (ChABC) can enhance axon growth and functional recovery after SCI. However, due to the complexity of the mammalian CNS, it is still unclear whether this involves true regeneration or only collateral sprouting by uninjured axons, whether it affects the expression of CSPG receptors such as protein tyrosine phosphatase sigma (PTPσ), and whether it influences retrograde neuronal apoptosis after SCI. In the present study, we assessed the roles of CSPGs in the regeneration of spinal-projecting axons from brainstem neurons, and in the process of retrograde neuronal apoptosis. Using the fluorochrome-labeled inhibitor of caspase activity (FLICA) method, apoptotic signaling was seen primarily in those large, individually identified reticulospinal (RS) neurons that are known to be "bad-regenerators." Compared to uninjured controls, the number of all RS neurons showing polycaspase activity increased significantly at 2, 4, 8, and 11 weeks post-transection (post-TX). ChABC application to a fresh TX site reduced the number of polycaspase-positive RS neurons at 2 and 11 weeks post-TX, and also reduced the number of active caspase 3-positive RS neurons at 4 weeks post-TX, which confirmed the beneficial role of ChABC treatment in retrograde apoptotic signaling. ChABC treatment also greatly promoted axonal regeneration at 10 weeks post-TX. Correspondingly, PTPσ mRNA expression was reduced in the perikaryon. Previously, PTPσ mRNA expression was shown to correlate with neuronal apoptotic signaling at 2 and 10 weeks post-TX. In the present study, this correlation persisted after ChABC treatment, which suggests that PTPσ may be involved more generally in signaling axotomy-induced retrograde neuronal apoptosis. Moreover, ChABC treatment caused Akt activation (pAkt-308) to be greatly enhanced in brain post-TX, which was further confirmed in individually identified RS neurons. Thus, CSPG digestion not only enhances axon regeneration after SCI, but also inhibits retrograde RS neuronal apoptosis signaling, possibly by reducing PTPσ expression and enhancing Akt activation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hu, Rodemer, Zhang, Jin, Li and Selzer.)

Nitrile hydratase (NHase), an excellent bio-catalyst for the synthesis of amide compounds, was composed of two heterologous subunits. A thermoalkaliphilic NHase NHCTA1 (Tm = 71.3°C) obtained by in silico screening in our study exhibited high flexibility of α-subunit but excellent thermostability, as opposed to previous examples. To gain a deeper structural insight into the thermostability of NHCTA1, comparative molecular dynamics simulation of NHCTA1 and reported NHases was carried out. By comparison, we speculated that β-subunit played a key role in adjusting the flexibility of α-subunit and the different conformations of linker in "α5-helix-coil ring" supersecondary structure of β-subunit can affect the interaction between β-subunit and α-subunit. Mutant NHCTA1-α 6 C with a random coil linker and mutant NHCTA1-αβγ with a truncated linker were therefore constructed to understand the impact on NHCTA1 thermostability by varying the supersecondary structure. The varied thermostability of NHCTA1-α 6 C and NHCTA1-αβγ (Tm α6C = 74.4°C, Tm αβγ = 65.6°C) verified that the flexibility of α-subunit adjusted by β-subunit was relevant to the stability of NHCTA1. This study gained an insight into the NNHCTA1 thermostability by virtual dynamics comparison and experimental studies without crystallization, and this approach could be applied to other industrial-important enzymes., (© 2021 Wiley Periodicals LLC.)

A single-transaminase-catalyzed biocatalytic cascade was developed by employing the desired biocatalyst, ATA-117-Rd11, that showed high activity toward 2-oxo-4-[(hydroxy)(methyl)phosphinoyl] butyric acid (PPO) and α-ketoglutarate, and low activity against pyruvate. The cascade successfully promotes a highly asymmetric amination reaction for the synthesis of l-phosphinothricin (l-PPT) with high conversion (>95 %) and>99 % ee. In a scale-up experiment, using 10 kg pre-frozen E. coli cells harboring ATA-117-Rd11 as catalyst, 80 kg PPO was converted to ≈70 kg l-PPT after 24 hours with a high ee value (>99 %)., (© 2020 Wiley-VCH GmbH.)

Background: Ophiocordyceps sinensis is an important traditional Chinese medicine for its comprehensive active ingredients, such as cordycepin, cordycepic acid, and Cordyceps polysaccharide. O. sinensis zjut, a special strain isolated from O. sinensis, has similar pharmacological functions to wild O. sinensis. Currently, O. sinensis with artificial cultivation has been widely studied, but systematic fundamental research at protein levels has not been determined., Results: Proteomes of O. sinensis zjut at different culture periods (growth period, 3rd day; pre-stable period, 6th day; and stable period, 9th day) were relatively quantified by relative isotope markers and absolute quantitative technology. In total, 4005 proteins were obtained and further annotated with Gene Ontology, Kyoto Encyclopedia of Genes and Genomes database. Based on the result of the annotations, metabolic pathways of active ingredients, amino acids and fatty acid were constructed, and the related enzymes were exhibited. Subsequently, comparative proteomics of O. sinensis zjut identified the differentially expressed proteins (DEPs) by growth in different culture periods, to find the important proteins involved in metabolic pathways of active ingredients. 605 DEPs between 6d-VS-3d, 1188 DEPs between 9d-VS-3d, and 428 DEPs between 9d-VS-6d were obtained, respectively., Conclusion: This work provided scientific basis to study protein profile and comparison of protein expression levels of O. sinensis zjut, and it will be helpful for metabolic engineering works to active ingredients for exploration, application and improvement of this fungus.

We previously reported that spinal cord transection (TX) in the lamprey causes mRNA to accumulate in the injured tips of large reticulospinal (RS) axons. We sought to determine whether this mRNA accumulation results from phosphorylation and transport of retrograde signals, similar to what has been reported in mammalian peripheral nerve. Extracellular signal-regulated protein kinase (Erk), mediates the neurite outgrowth-promoting effects of many neurotrophic factors. To assess the role of Erk in retrograde signaling of RS axon injury, we used immunoblot and immunohistochemistry to determine the changes in phosphorylated Erk (p-Erk) in the spinal cord after spinal cord TX. Immunostaining for p-Erk increased within axons and local cell bodies, most heavily within the 1-2 mm closest to the TX site, at between 3 and 6 h post-TX. In axons, p-Erk was concentrated in 3-5 μm granules that became less numerous with distance from the TX. The retrograde molecular motor dynein colocalized with p-Erk, but vimentin, which in peripheral nerve was reported to participate with p-Erk as part of a retrograde signal complex, did not colocalize with p-Erk, even though vimentin levels were elevated post-TX. The results suggest that p-Erk, but not vimentin, may function as a retrograde axotomy signal in lamprey central nervous system neurons, and that this signal may induce transcription of mRNA, which is then transported down the axon to its injured tip., (Copyright © 2020 Jin, John, Hu and Selzer.)

Sugar alcohols are the prominent alternatives of sugars in food, medical, and health industries. The ruthenium supported on multiwalled carbon nanotubes (Ru/MWCNTs) catalysts were prepared based on the Ru valence regulation strategy and applied for selective sugar hydrogenation to prepare various sugar alcohols including xylitol, arabinitol, sorbitol, mannitol, and galactitol for the first time, with high selectivity (>99.0%) and yield (>98.0%) under mild conditions (≤110 °C, 3.0 MPa H 2 pressure). The hydrogenation reaction of xylose was further optimized and under mild conditions (100 °C, 3.0 MPa H 2 pressure, and 500 rpm), which were lower than ever reported for high efficient synthesis of xylitol, 99.8% xylose conversion and 99.0% xylitol yield were achieved after 120 min of reaction.

Objective: To explore the optimal methods for the protoplast preparation and regeneration of Hirsutella sinensis by optimizing the limiting factors., Results: During the treatment of enzymatic protoplast preparation, mycelium cultured for 7 days was the optimal start material. The maximum protoplast preparation rate of 4.3 × 10 7 protoplasts/g fresh weight (FW) was obtained after 0.5 h treatment of 1 mg/ml mixed lytic enzymes in KH 2 PO 4 -K 2 HPO 4 buffer (pH 5.5) with 0.6 M KCl at 18 °C. As for the protoplast regeneration, the maximum protoplast regeneration rate reached 12.32% through 5 × 10 3 protoplasts mL -1 cultivated for 20 days in the regeneration medium with 0.6 M mannitol and 1.5% agar., Conclusions: The preparation and regeneration of H. sinensis protoplasts was firstly established based on process optimization and it provided a foundation for the study of H. sinensis mutagenesis.

Objective: To investigate the therapeutic effect of Ginseng Guipi pill on rats with spleen failing to control blood syndrome and its effect on liver. Methods: Forty SPF male Sprague-Dawley rats were randomly divided into normal control group ( n =10) and experimental group ( n =30). For the first 42 days, the experimental group swam for 30 minutes every day, eating for one day and fasting for two days (diet disorder). On the 43rd to 72nd day, the rats were injected with low-molecular-weight heparin calcium to induce hemorrhage on the basis of exhaustion of swimming and diet disorder to construct a rat model of spleen failing to control blood syndrome. Those thirty rat that successfully established the model were randomly divided into 3 groups ( n =10), model control group (MD), natural rehabilitation group (NR) and Guipi Pill group (GP). On the 73rd to 103rd day, the MD group continued to apply factors (exhausted swimming, eating disorder, and injection of low-molecular-weight heparin calcium), the NR group and the GP group stopped applying the factors , the GP group was daily administered with Ginseng Guipi Pill solution (0.2 g/ml, 10 ml/(kg·d)) , and the NR group was given an equal dose of saline. After the 103 days, blood and liver samples were collected, and the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil) and total protein (TP) content were detected, the number of red blood cells (RBC), hemoglobin (HGB) content and hematocrit (HCT) were detected, the expressions of IL-6 and TNF-α in serum were determined by ELISA kit, the levels of apoptosis-related proteins Bcl-2, Bax and Caspase3 in liver tissue were detected by Western blot. The expression levels of apoptosis-related proteins Bcl-2, Bax and Caspase3 mRNA in liver tissue were detected by real-time PCR. Results: Compared with the control group, the serum levels of ALT, AST and TBil in the model group were increased significantly, while the serum level of TP was reduced significantly ( P ＜0.01), the number of RBC, HGB content and HCT were decreased significantly ( P ＜0.01), the expressions of IL-6 and TNF-α in serum were increased ( P ＜0.05), the Bcl-2 protein and mRNA levels were reduced, Bax and Caspase3 protein and mRNA levels were increased significantly ( P ＜0.01); Compared with the model group, the serum levels of ALT, AST and TBil were significantly lower in the natural rehabilitation group and the Guipi Pill group, the level of TP was increased significantly ( P ＜0.01), the blood RBC, HGB and HCT were increased significantly ( P ＜ 0.01), the expressions of IL-6 and TNF-α were decreased ( P ＜0.05), the levels of Bcl-2 protein and mRNA were increased significantly, the Bax, Caspase3 protein and mRNA levels were decreased ( P ＜0.05); Compared with the natural rehabilitation group, the serum levels of ALT, AST and TBil were reduced , and the TP content was increased significantly in the Guipi Pill group ( P ＜ 0.01), the number of RBC was increased significantly in the Guipi Pill group ( P ＜0.05), the levels of Bcl-2 protein and mRNA were increased ( P ＜0.05),the level of Caspase3 protein was decreased ( P ＜0.05), the expression of Bax mRNA was reduced significantly ( P ＜0.05). Conclusion: Ginseng Guipi Pill has protective effect on liver injury in rats with spleen failing to control blood syndrome. The mechanism may be related to the inhibition the liver cell apoptosis and the release of pro-inflammatory cytokines.

Transaminase responsible for alienating prochiral ketone compound is applicable to asymmetric synthesis of herbicide L-phosphinothricin (L-PPT). In this work, the covalent immobilization of recombinant transaminase from Citrobacter koseri (CkTA) was investigated on different epoxy resins. Using optimum ES-105 support, a higher immobilized activity was obtained via optimizing immobilization process in terms of enzyme loading, coupling time and initial PLP concentration. Crucially, due to blocking unreacted epoxy groups on support surface with amino acids, the reaction temperature of blocked immobilized biocatalyst was enhanced from 37 to 57 °C. Its thermostability at 57 °C was also found to be superior to that of free CkTA. The K m value was shifted from 36.75 mM of free CkTA to 39.87 mM of blocked immobilized biocatalyst, demonstrating that the affinity of enzyme to the substrate has not been apparently altered. Accordingly, the biocatalyst performed the consecutive synthesis of L-PPT for 11 cycles (yields>91%) with retaining more than 91.13% of the initial activity. The seemingly the highest reusability demonstrates this biocatalyst has prospective for reducing the costs of consecutive synthesis of L-PPT with high conversion.

This retrospective study was to compare the image quality of right coronary artery (RCA) and effective radiation dose on prospective ECG-gated method between 320 row computed tomography (CT) and 2nd generation (128-slice) dual source CT. A total of 215 candidates underwent CT coronary angiography using prospective ECG-gated method, 120 patients enrolled in 320 row CT group, and 95 patients in dual source CT group. We divided RCA image quality scores as 1/2/3/4, which means excellent/good/adequate/not assessable and heart rates were considered, as well as the radiation dose. There is no statistically significant difference of RCA image quality of Score 1/2 between 320 row CT and 2nd generation dual source CT, but lower heart rate (<70/min) improved RCA image quality. Meanwhile, the 2nd generation dual source CT scan have significant lower radiation dose. For patients with high level heart rate variation, both prospective ECG-gated method of 320 row CT scan (Toshiba) and 2nd generation dual source CT scan (Siemens) basically provided good image quality on RCA. There is an advantage of effective radiation dose reduction in prospective ECG-gated method using the 2nd generation dual source CT scan. After the iodine contrast agent was injected into elbow vein, the threshold triggering method was used to carry out prospective gated scanning, and the acquired fault image was reconstructed by the standard post-processing software of each manufacturer. The radiation dose value is obtained through the dose report automatically generated after each scan., (© 2020 The Authors. Journal of Applied Clinical Medical Physics published by Wiley Periodicals, Inc. on behalf of American Association of Physicists in Medicine.)

Ophiocordyceps sinensis has been used as a traditional medicine or healthy food in China for thousands of years. Hirsutella sinensis was reported as the only correct anamorph of O. sinensis. It is reported that the laboratory-grown H. sinensis mycelium has similar clinical efficacy and less associated toxicity compared to the wild O. sinensis. The research of the H. sinensis is becoming more and more important and urgent. To gain deeper insight into the biological and pharmacological mechanisms, we sequenced the genome of H. sinensis. The genome of H. sinensis (102.72 Mb) was obtained for the first time, with > 99% coverage. 10,200 protein-encoding genes were predicted based on the genome sequence. A detailed secondary metabolism analysis and structure verification of the main ingredients were performed, and the biosynthesis pathways of seven ingredients (mannitol, cordycepin, purine nucleotides, pyrimidine nucleotides, unsaturated fatty acid, cordyceps polysaccharide and sphingolipid) were predicted and drawn. Furthermore, infection process and mechanism of H. sinensis were studied and elaborated in this article. The enzymes involved in the infection mechanism were also predicted, cloned and expressed to verify the mechanism. The genes and proteins were predicted and annotated based on the genome sequence. The pathways of several active components in H. sinensis were predicted and key enzymes were confirmed. The work presented here would improve the understanding of the genetic basis of this organism, and contribute to further research, production and application of H. sinensis.

Traumatic spinal cord injury (SCI) results in persistent functional deficits due to the lack of axon regeneration within the mammalian CNS. After SCI, chondroitin sulfate proteoglycans (CSPGs) inhibit axon regrowth via putative interactions with the LAR-family protein tyrosine phosphatases, PTPσ and LAR, localized on the injured axon tips. Unlike mammals, the sea lamprey, Petromyzon marinus , robustly recovers locomotion after complete spinal cord transection (TX). Behavioral recovery is accompanied by heterogeneous yet predictable anatomical regeneration of the lamprey's reticulospinal (RS) system. The identified RS neurons can be categorized as "good" or "bad" regenerators based on the likelihood that their axons will regenerate. Those neurons that fail to regenerate their axons undergo a delayed form of caspase-mediated cell death. Previously, this lab reported that lamprey PTPσ mRNA is selectively expressed in "bad regenerator" RS neurons, preceding SCI-induced caspase activation. Consequently, we hypothesized that PTPσ deletion would reduce retrograde cell death and promote axon regeneration. Using antisense morpholino oligomers (MOs), we knocked down PTPσ expression after TX and assessed the effects on axon regeneration, caspase activation, intracellular signaling, and behavioral recovery. Unexpectedly, PTPσ knockdown significantly impaired RS axon regeneration at 10 weeks post-TX, primarily due to reduced long-term neuron survival. Interestingly, cell loss was not preceded by an increase in caspase or p53 activation. Behavioral recovery was largely unaffected, although PTPσ knockdowns showed mild deficits in the recovery of swimming distance and latency to immobility during open field swim assays. Although the mechanism underlying the cell death following TX and PTPσ knockdown remains unknown, this study suggests that PTPσ is not a net negative regulator of long tract axon regeneration in lampreys., (Copyright © 2020 Rodemer, Zhang, Sinitsa, Hu, Jin, Li and Selzer.)

Iron is essential for a broad range of biochemical processes in the brain, but the mechanisms of iron metabolism in the brain remain elusive. Here we show that iron functionally translocates among brain regions along specific axonal projections. We identified two pathways for iron transport in the brain: a pathway from ventral hippocampus (vHip) to medial prefrontal cortex (mPFC) to substantia nigra; and a pathway from thalamus (Tha) to amygdala (AMG) to mPFC. While vHip-mPFC transport modulates anxiety-related behaviors, impairment of Tha-AMG-mPFC transport did not. Moreover, vHip-mPFC iron transport is necessary for the behavioral effects of diazepam, a well-known anxiolytic drug. By contrast, genetic or pharmacological promotion of vHip-mPFC transport produced anxiolytic-like effects and restored anxiety-like behaviors induced by repeated restraint stress. Taken together, these findings provide key insights into iron metabolism in the brain and identify the mechanisms underlying iron transport in the brain as a potential target for development of novel anxiety treatments.

Promoter engineering is an enabling technology in metabolic engineering and synthetic biology. As an indispensable part of synthetic biology, the promoter is a key factor in regulating genetic circuits and in coordinating multi-gene biosynthetic pathways. In this review, we summarized the recent progresses in promoter engineering in microbes. Specifically, the endogenous promoters are firstly discussed, followed by the statement of the influence of nucleotides exchange on the strength of promoters explored by site-selective mutagenesis. We then introduced the promoter libraries with a wide range of strength, which are constructed focusing on core promoter regions and upstream activating sequences by rational designs. Finally, the application of promoter libraries in the optimization of multi-gene metabolic pathways for high-yield production of metabolites was illustrated with a couple of recent examples.

α-Transaminase (α-TA) responsible for catalyzing the reversible transfer of amino groups between amine donors and amine acceptors, is applicable to enzymatic route for asymmetric synthesis of herbicide l-phosphinothricin (l-PPT). In the search for α-TAs with better catalysis performance, three α-TAs were discovered by genome mining approach using a known sequence encoding Escherichia coli tyrosine TA (TyrB) as probe. Through detailed comparison of their expression amount, activities and characteristics, Citrobacter koseri TA (CkTA) exhibited better activity and thermostability, which retain 65.9% of initial activity after incubation at 57 °C for 4 h. The K m and k cat /K m values of CkTA were 36.75 mM and 34.29 mM -1  min -1 , respectively. In addition, recombinant CkTA cells were immobilized onto Celite 545 using tris(hydroxymethyl)phosphine as crosslinker. During five repetitive asymmetric synthesis of l-PPT from 20 g/L prostereogenic ketone using l-Glu as amine donor, all the yields of l-PPT reached up to 91.2% (＞99% ee). These characteristics made CkTA a valuable addition to the currently scarce α-TA library for stereospecific synthesis of l-PPT., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In this study, recombinant E. coli BL21(DE3)/pCDFDuet-1-XR-GDH harboring xylose reductase (XR) and glucose dehydrogenase (GDH) were immobilized and applied for the production of xylitol from xylose mother liquor (XML). Various immobilization methods were screened and the cross-linking approach with diatomite and polyetherimide as the raw materials and glutaraldehyde as the cross-linking agent was the optimal one, and the recovery activity reached of 80.3% after immobilization. The half-life of immobilized cells was 1.52 times to that of free cells. Batch experiments showed that the enzyme activity of immobilized cells remained 70.5% of the initial activity after 10 batches and the space-time yield of xylitol reached of 11.5 g/(L h). The production of xylitol from xylose mother liquor by immobilized E. coli cells containing xylose reductase and glucose dehydrogenase was reported for the first time, which paved a foundation for industrial production of xylitol from waste xylose mother liquor., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Xylitol is an important functional sugar alcohol which is widely used in industries. To efficiently produce xylitol from xylose by xylose reductase in a single step, the coupled system and the coexpression system using xylose reductase (XR) from Rhizopus oryzae and glucose dehydrogenase (GDH) from Exiguobacterium sibiricum were constructed, the latter of which was used for cofactor regeneration. One hundred fifty grams/liter of xylose could be fully converted to xylitol by employing the two-enzyme coupled system with cell-free extract, and xylitol productivity of 21.2 g/L/h was reached by biotransformation in vitro. The whole-cell biocatalyst coexpressing xylose reductase and glucose dehydrogenase was successfully used to synthesize xylitol via enzymatic reduction of xylose. After optimization of the induction condition, the enzyme activity reached 1533 U/L. Two hundred grams/liter of xylose could be completely converted to xylitol, and the highest xylitol productivity of 6.37 g/L/h was obtained under the optimal transformation conditions. Thus, recombinant Escherichia coli coexpressing xylose reductase and glucose dehydrogenase is a promising candidate for xylitol production from xylose.

2-Chloronicotinic acid is a key intermediate of pharmaceuticals and pesticides. Amidase-catalyzed hydrolysis provides a promising enzymatic method for 2-chloronicotinic acid production from 2-chloronicotinamide. However, biocatalytic hydrolysis of 2-chloronicotinamide is difficult due to the strong steric and electronic effect caused by 2-position chlorine substituent of the pyridine ring. In this study, an amidase from a Pantoea sp. ( Pa -Ami) was designed and engineered to have improved catalytic properties. Single mutant G175A and double mutant G175A/A305T strains exhibited 3.2- and 3.7-fold improvements in their specific activity for 2-chloronicotinamide, and the catalytic efficiency was significantly increased, with k cat / K m values 3.1 and 10.0 times higher than that of the wild type, respectively. Structure-function analysis revealed that the distance between Oγ of Ser177 (involved in the catalytic triad) and the carbonyl carbon of 2-chloronicotinamide was shortened in the G175A mutant, making the nucleophilic attack on the Oγ of Ser177 easier by virtue of proper orientation. In addition, the A305T mutation contributed to a suitable tunnel formation to facilitate the substrate entry and product release, resulting in improved catalytic efficiency. With the G175A/A305T double mutant as a biocatalyst, a maximum of 1,220 mM 2-chloronicotinic acid was produced with a 94% conversion, and the space-time yield reached as high as 575 g product liter -1 day -1 These results provide not only a novel robust biocatalyst for the production of 2-chloronicotinic acid but also new insights into amidase structure-function relationships. IMPORTANCE In recent years, the demand for 2-chloronicotinic acid has been greatly increased. To date, several chemical methods have been used for the synthesis of 2-chloronicotinic acid, but all include tedious steps and/or drastic reaction conditions, resulting in both economic and environmental issues. It is requisite to develop an efficient and green synthesis route. We recently screened Pa -Ami and demonstrated its potential for synthesis of 2-chloronicotinic acid from 2-chloronicotinamide. However, chlorine substitution on the pyridine ring of nicotinamide significantly affected the activity of Pa -Ami. Especially for 2-chloronicotinamide, the enzyme activity and catalytic efficiency were relatively low. In this study, based on structure-function analysis, we succeeded in engineering the amidase by structure-guided saturation mutagenesis. The engineered Pa -Ami exhibited quite high catalytic activity toward 2-chloronicotinamide and could serve as a promising biocatalyst for the biosynthesis of 2-chloronicotinic acid., (Copyright © 2019 American Society for Microbiology.)

Objective: To investigate the therapeutic effects of the black buckwheat leaf (BBL) in type 2 diabetes mellitus mice and its effects on pancreas and spleen., Methods: Forty male C57 / B16 mice (SPF) were randomly divided into normal control (NC) group (n=10) and the experimental group (n=30), the experimental group were fed with high sugar and high fat, combined with intraperitoneal injection of streptozotocin in small dose to establish the model of type 2 diabetes mellitus (T2DM). Those thirty model mice were randomly divided into 3 groups (n=10), diabetes mellitus group (DM), low dose of BBL (DM+L) treated group, high dose of BBL (DM+H) treated group. The mice in the NC group and the DM group were given normal saline per day, and the DM+L group and DM+H group were treated with black tartary buckwheat at the doses of 0.21g/kg·d -1 and 0.42g/kg·d -1 respectively. After 14 days. All mice were executed by cervical dislocation, then blood samples were collected, pancreas and spleen were removed for subsequent experiments. The serum levels of fasting blood glucose (FBG), triglyceride (TG), total cholesterol (TCH) and insulin were detected. TNF-α protein in spleen tissue was detected by ELISA kit. The morphology of pancreas tissue was observed by HE staining, and the spleen coefficient was calculated. The expression levels of insulin receptor substrate-1(IRS-1) mRNA and IRS-1 protein in pancreatic tissue were detected., Results: Compared with the control group, the serum levels of FBG, TC and TCH in the model group were increased significantly, while the serum level of insulin was decreased significantly (P＜0.05), the expression of TNF-α protein in spleen tissues was obviously raised, the expressions of IRS-1 mRNA and IRS-1 protein in pancreatic tissue in model group were decreased significantly (P＜0.05). Compared with the model group, the serum levels of FBG, TC and TCH were decreased significantly in the BBL treated groups. The serum insulin level, spleen coefficient, TNF-α protein expression level in spleen tissue, IRS-1 mRNA expression and IRS-1 protein expression levels in pancreatic tissue in BBL treated group were increased significantly (P＜ 0.05). High-dose black tartary buckwheat leaves (0.42g/kg·d -1 ) exerted a more significant effect., Conclusion: Stem and leaf of black bitter buckwheat has significant therapeutic effects on reducing blood sugar and blood fat in type 2 diabetic mice, and has certain protective effects on pancreas and spleen of diabetic mice.

Iminodiacetic acid (IDA) is widely used as an intermediate in the manufacturing of chelating agents, glyphosate herbicides and surfactants. To improve activity and tolerance to the substrate for IDA production, Acidovorax facilis nitrilase was selected for further modification by the gene site saturation mutagenesis method. After screened by a two-step screening method, the best mutant (Mut-F168V/T201N/S192F/M191T/F192S) was selected. Compared to the wild-type nitrilase, Mut-F168V/T201N/S192F/M191T/F192S showed 136% improvement in specific activity. Co 2+ stimulated nitrilase activity, whereas Cu 2+ , Zn 2+ and Tween 80 showed a strong inhibitory effect. The V max and k cat of Mut-F168V/T201N/S192F/M191T/F192S were enhanced 1.23 and 1.23-fold, while the K m was decreased 1.53-fold. The yield of Mut-F168V/T201N/S192F/M191T/F192S with 453.2 mM of IDA reached 71.9% in 5 h when 630 mM iminodiacetonitrile was used as substrate. This study indicated that mutant nitrilase obtained in this study is promising in applications for the upscale production of IDAN., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Glucose isomerase (GI) responsible for catalyzing the isomerization from d-glucose to d-fructose, was an important enzyme for producing high fructose corn syrup (HFCS). In a quest to prepare HFCS at elevated temperature and facilitate enzymatic recovery, an effective procedure for whole cell immobilization of refractory Thermus oshimai glucose isomerase (ToGI) onto Celite 545 using tris(hydroxymethyl)phosphine (THP) as crosslinker was established. The immobilized biocatalyst showed an activity of approximate 127.3 U/(g·immobilized product) via optimization in terms of cells loading, crosslinker concentration and crosslinking time. The pH optimum of the immobilized biocatalyst was displaced from pH 8.0 of native enzyme to neutral pH 7.0. Compared with conventional glutaraldehyde (GLU)-immobilized cells, it possessed the enhanced thermostability with 70.1% residual activity retaining after incubation at 90°C for 72 h. Moreover, the THP-immobilized biocatalyst exhibited superior operational stability, in which it retained 85.8% of initial activity after 15 batches of bioconversion at 85°C. This study paved a way for reducing catalysis cost for upscale preparation of HFCS with higher d-fructose concentration., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

2-Chloronicotinic acid (2-CA) is an important building block for a series of agrochemicals and pharmaceuticals. Amidase-catalyzed hydrolysis of 2-chloronicotinamide is one of the most attractive approaches for 2-CA production. However, development of the bioprocess was plagued by low activity of amidase for 2-chloronicotinamide. In this work, an amidase signature (AS) family amidase from Pantoea sp. (Pa-Ami), with superior activity for nicotinamide and its chlorinated derivatives, was exploited and characterized. Kinetic analysis and molecular docking clearly indicated that chlorine substitution in the pyridine ring of nicotinamide, especially the substitution at 2-position led to a dramatic decrease of Pa-Ami activity. The productivity of the bioprocess was significantly improved using fed-batch mode at low reaction temperature and 2-CA was produced as high as 370 mM with a substrate conversion of 94.2%. These results imply that Pa-Ami is potentially promising biocatalyst for industrial production of 2-CA., (Copyright © 2017 Elsevier Inc. All rights reserved.)

tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is the key intermediate for synthesis of atorvastatin and rosuvastatin. Carbonyl reductase exhibits excellent activity toward tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH) to synthesize (3R,5S)-CDHH. In this study, a whole cell biosynthesis reaction system to produce (3R,5S)-CDHH was constructed in organic solvents. A solution of 10% (v/v) Tween-80 was introduced to the reaction system as a co-solvent, which greatly enhanced biotransformation process, giving 98.9% yield, >99% ee and 1.8-fold higher space time yield in 5 h bioconversion of 1 M (S)-CHOH, compared with 98.7% yield and >99% ee in 9 h bioconversion of a purely aqueous reaction system. Moreover, a water-octanol biphasic reaction system was built and 20% of octanol was added as reservoir of substrate resulting in 98% yield, >99% ee and 4.08 mmol L -1  h -1  g -1 (wet cell weight) space time yield. This study paved a way for the whole cell biosynthesis of (3R,5S)-CDHH in mono and biphasic media., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The acid-catalyzed treatment was a conventional process for xylose production from corncob. To increase the release of xylose and to reduce the by-products formation and water usage, the oxalic acid was used as catalyst to hydrolyze the corncob and the hydrolytic conditions were investigated. The highest xylose yield of 32.7 g L -1 , representing 96.1% of total theoretical xylose yield, was obtained using 1.2% oxalic acid after hydrolysis for 120 min at 130 °C, which was more than 10% higher than that of sulfuric acid-catalyzed hydrolysis. Mixed acids-catalyzed hydrolysis performed a synergistic effect for xylose production and 31.7 g L -1 of xylose was reached after reacting for 90 min with oxalic acid and sulfuric acid at a ratio of 1:4 (w/w). A kinetic model was developed to elucidate the competitive reaction between xylose formation and its degradation in the hydrolysis process, and the experimental data obtained in this study were perfectly in agreement with that of predicted from the model. Furthermore, the final xylose yield of 85% was achieved after purification and crystallization. It was demonstrated that xylose production from the corncob hydrolysis with oxalic acid as the catalyst was an effective alternative to the traditional sulfuric acid-based hydrolysis.

Glucose isomerase is the important enzyme for the production of high fructose corn syrup (HFCS). One-step production of HFCS containing more than 55% fructose (HFCS-55) is receiving much attention for its industrial applications. In this work, the Escherichia coli harboring glucose isomerase mutant TEGI-W139F/V186T was immobilized for efficient production of HFCS-55. The immobilization conditions were optimized, and the maximum enzyme activity recovery of 92% was obtained. The immobilized glucose isomerase showed higher pH, temperature, and operational stabilities with a K m value of 272 mM and maximum reaction rate of 23.8 mM min -1 . The fructose concentration still retained above 55% after the immobilized glucose isomerase was reused for 10 cycles, and more than 85% of its initial activity was reserved even after 15 recycles of usage at temperature of 90 °C. The results highlighted the immobilized glucose isomerase as a potential biocatalyst for HFCS-55 production.

In this paper, an optimal longitudinal slip ratio system for real-time identification of electric vehicle (EV) with motored wheels is proposed based on the adhesion between tire and road surface. First and foremost, the optimal longitudinal slip rate torque control can be identified in real time by calculating the derivative and slip rate of the adhesion coefficient. Secondly, the vehicle speed estimation method is also brought. Thirdly, an ideal vehicle simulation model is proposed to verify the algorithm with simulation, and we find that the slip ratio corresponds to the detection of the adhesion limit in real time. Finally, the proposed strategy is applied to traction control system (TCS). The results showed that the method can effectively identify the state of wheel and calculate the optimal slip ratio without wheel speed sensor; in the meantime, it can improve the accelerated stability of electric vehicle with traction control system (TCS).

Objective: To observe the effect of AdipoRon, an adiponin receptor agonist, on insulin sensitivity in mouse myoblast cell line (C2C12) and to explore its mechanism., Methods: C2C12 was induced to differentiate into myoblasts by using horse serum. Then the cells were divided into 6 groups (9 double wells):blank control group, high dose AdipoRon group, low dose AdipoRon group, insulin group and the low dose AdipoRon with PI3K inhibitor (phosphatidylinositol 3 kinase) group and the insulin with PI3K inhibitor group. After cultured for 12 h, the supernatant was collected and glucose consumption was measured. Cell proliferation was tested by using CCK8. In the 6-well plate, C2C12 was induced to differentiate into myoblasts. The drug was incubated for 12 h and the mRNA level of GLUT4 was detected by RT-PCR., Results: Compared with the blank control group, the levels of glucose consumption in high dose AdipoRon group, low dose AdipoRon group and insulin group was increased significantly ( P <0.05). After adding PI3K inhibitor, the levels of glucose consumption in the above mentioned three groups were not different from that in blank control group. high dose AdipoRon group, low dose AdipoRon group and insulin group had proliferation, but only the insulin group was statistically significant ( P <0.05). Compared with the control group, the levels of GLUT4 mRNA in AdipoRon high dose group, low dose AdipoRon group and insulin group were all higher than those in control group ( P <0.05). After adding PI3K inhibitor, GLUT4 mRNA level was not statistically significant compared with blank control group., Conclusions: AdipoRon can increase the consumption of glucose without affecting cell proliferation, which may play a role in improving insulin sensitivity, but the specific mechanism remains to be further studied.

L-methionine has attracted a great deal of attention for its nutritional, pharmaceutical, and clinical applications. In this study, Escherichia coli W3110 was engineered via deletion of a negative transcriptional regulator MetJ and over-expression of homoserine O-succinyltransferase MetA together with efflux transporter YjeH, resulting in L-methionine overproduction which is up to 413.16 mg/L. The partial inactivation of the L-methionine import system MetD via disruption of metI made the engineered E. coli ΔmetJ ΔmetI/pTrcA*H more tolerant to high L-ethionine concentration and accumulated L-methionine to a level 43.65% higher than that of E. coli W3110 ΔmetJ/pTrcA*H. Furthermore, deletion of lysA, which blocks the lysine biosynthesis pathway, led to a further 8.5-fold increase in L-methionine titer of E. coli ΔmetJ ΔmetI ΔlysA/pTrcA*H. Finally, addition of Na 2 S 2 O 3 to the media led to an increase of fermentation titer of 11.45%. After optimization, constructed E. coli ΔmetJ ΔmetI ΔlysA/pTrcA*H was able to produce 9.75 g/L L-methionine with productivity of 0.20 g/L/h in a 5 L bioreactor. This novel metabolically tailored strain of E. coli provides an efficient platform for microbial production of L-methionine. Biotechnol. Bioeng. 2017;114: 843-851. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Paralysis following spinal cord injury (SCI) is due to interruption of axons and their failure to regenerate. It has been suggested that the small GTPase RhoA may be an intracellular signaling convergence point for several types of growth-inhibiting extracellular molecules. Even if this is true in vitro, it is not clear from studies in mammalian SCI, whether the effects of RhoA manipulations on axon growth in vivo are due to a RhoA-mediated inhibition of true regeneration or only of collateral sprouting from spared axons, since work on SCI generally is performed with partial injury models. RhoA also has been implicated in local neuronal apoptosis after SCI, but whether this reflects an effect on axotomy-induced cell death or an effect on other pathological mechanisms is not known. In order to resolve these ambiguities, we studied the effects of RhoA knockdown in the sea lamprey central nervous system (CNS), where after complete spinal cord transection (TX), robust but incomplete regeneration of large axons belonging to individually identified reticulospinal (RS) neurons occurs, and where some RS neurons show unambiguous delayed retrograde apoptosis after axotomy. RhoA protein was detected in neurons and axons of the lamprey brain and spinal cord, and its expression was increased post-TX. Knockdown of RhoA in vivo by retrogradely-delivered morpholino antisense oligonucleotides (MOs) to the RS neurons significantly reduced retrograde apoptosis signaling in identified RS neurons post-SCI, as indicated by Fluorochrome Labeled Inhibitor of Caspases (FLICA) in brain wholemounts. In individual RS neurons, the reduction of caspase activation by RhoA knockdown began at 2weeks post-TX and was still seen at 8weeks. RhoA knockdown slowed axon retraction and possibly increased early axon regeneration in the proximal stump. The number of axons regenerating beyond the lesion more than 5mm at 10weeks post-TX also was increased. Thus RhoA knockdown both enhanced true axon regeneration and inhibited retrograde apoptosis signaling after SCI., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Polyribosomes, mRNA, and other elements of translational machinery have been reported in peripheral nerves and in elongating injured axons of sensory neurons in vitro, primarily in growth cones. Evidence for involvement of local protein synthesis in regenerating central nervous system (CNS) axons is less extensive. We monitored regeneration of back-labeled lamprey spinal axons after spinal cord transection and detected mRNA in axon tips by in situ hybridization and microaspiration of their axoplasm. Poly(A)+mRNA was present in the axon tips, and was more abundant in actively regenerating tips than in static or retracting ones. Target-specific polymerase chain reaction (PCR) and in situ hybridization revealed plentiful mRNA for the low molecular neurofilament subunit and β-tubulin, but very little for β-actin, consistent with the morphology of their tips, which lack filopodia and lamellipodia. Electron microscopy showed ribosomes/polyribosomes in the distal parts of axon tips and in association with vesicle-like membranes, primarily in the tip. In one instance, there were structures with the appearance of rough endoplasmic reticulum. Immunohistochemistry showed patches of ribosomal protein S6 positivity in a similar distribution. The results suggest that local protein synthesis might be involved in the mechanism of axon regeneration in the lamprey spinal cord. J. Comp. Neurol. 524:3614-3640, 2016. © 2016 Wiley Periodicals, Inc., Competing Interests: STATEMENT LQJ, CRP, MES are/were employees of Temple University. KSJ was an undergraduate student. WR is a graduate student. None of the authors have known or potential conflicts of interest, including any financial, personal, or other relationships with other people or organizations within 5 years of beginning the submitted work that could inappropriately influence, or be perceived to influence, this work., (© 2016 Wiley Periodicals, Inc.)