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Background The unnatural amino acid, L-2-aminobutyric acid (L-ABA) is an essential chiral building block for various pharmaceutical drugs, such as the antiepileptic drug levetiracetam and the antituberculosis drug ethambutol. The present study aims at obtaining variants of ω-transaminase from Ochrobactrum anthropi (OATA) with high catalytic activity to α-ketobutyric acid through protein engineering. Results Based on the docking model using α-ketobutyric acid as the ligand, 6 amino acid residues, consisting of Y20, L57, W58, G229, A230 and M419, were chosen for saturation mutagenesis. The results indicated that L57C, M419I, and A230S substitutions demonstrated the highest elevation of enzymatic activity among 114 variants. Subsequently, double substitutions combining L57C and M419I caused a further increase of the catalytic efficiency to 3.2-fold. This variant was applied for threonine deaminase/OATA coupled reaction in a 50-mL reaction system with 300 mM L-threonine as the substrate. The reaction was finished in 12 h and the conversion efficiency of L-threonine into L-ABA was 94%. The purity of L-ABA is 75%, > 99% ee. The yield of L-ABA was 1.15 g. Conclusion This study provides a basis for further engineering of ω-transaminase for producing chiral amines from keto acids substrates.

Torreya grandis is an important economic tree species in China. It provides nutritional value and is important to the health care industry. There are ongoing issues with product quality which are primarily related to improper management and early harvest. This study was carried out during the fruit ripening processes to evaluate the influence of harvesting date on T. grandis quality, and to determine the optimal harvest period. The effects of harvest time on the variation of quality and nutritional parameters of T. grandis nuts and its oil were evaluated, and the optimal harvest period was determined. The results showed that harvest timing had a strong effect on both oil yield and quality. Prolonged ripening could induce higher levels of kernel rate, fruit inclusions, oil and nutritional quality. When the sample harvested in the mid-September, the kernel rate and oil content were increased by 1.88±0.31% and 6.65±0.47%, respectively, compared to samples harvested in the beginning of late-August. Similarly, the mid-September harvest resulted in total unsaturated fatty acids content of the oil being increased by 5.3±0.34%, the FFA and peroxide value being decreased by 40.7±0.15% and 76±0.08%, respectively, and total tocopherols and free amino acids were increased 7.5±0.24% and 47.3±0.15%, respectively, compared to the samples harvested on Aug. 25. The results indicated that the optimal harvest time of T. grandis fruits was mid-September as it was beneficial for improving the quality of T. grandis nut and its oil. It was suggested that T. grandis fruit should be harvested later.

HIGHLIGHTS The antifungal effect of the active substance in camellia seed cake is evaluated on Aspergillus flavus. The n-butanol phase exhibited an apparent antagonistic effect on A. flavus. A reference and a guide for natural antifungal agents is provided. Knowledge of the utilization of the by-products of camellia seed is broadened.

Background Nattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion. In order to increase nattokinase yields, fermentation conditions, strains, cultivation media, and feeding strategies have been optimized. Nattokinase has been expressed using several heterologous expression systems. Pichia pastoris heterologous expression system was the alternative. Results This report aimed to express high levels of nattokinase from B. subtilis natto (NK-Bs) using a Pichia pastoris heterologous expression system and assess its fibrinolytic activity in vivo. Multicopy expression strains bearing 1–7 copies of the aprN gene were constructed. The expression level of the target protein reached a maximum at five copies of the target gene. However, multicopy expression strains were not stable in shake-flask or high-density fermentation, causing significant differences in the yield of the target protein among batches. Therefore, P. pastoris bearing a single copy of aprN was used in shake-flask and high-density fermentation. Target protein yield was 320 mg/L in shake-flask fermentation and approximately 9.5 g/L in high-density fermentation. The recombinant nattokinase showed high thermo- and pH-stability. The present study also demonstrated that recombinant NK-Bs had obvious thrombolytic activity. Conclusions This study suggests that the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of nattokinase.

Pseudomonas protegens SN15-2, a typical non-spore-forming rhizosphere bacterium, has excellent biocontrol capabilities; thus, it is necessary to explore the stress resistance of SN15-2. The choline-glycine betaine pathway is considered as an important mechanism by which bacteria adapt to stressful environments. In this work, we demonstrated that the expression of the betA and betB genes, which are involved in the choline- glycine betaine pathway in SN15-2, was highly increased by 12-fold and 26-fold, respectively, by hyperosmotic stress and choline treatment. The accumulation of betaine in SN15-2 (5.54 g/L) was significantly higher than that in the mutants Δ betA (3.44 g/L) and Δ betB (2.68 g/L) under hyperosmotic stress and choline treatment. Moreover, choline enhanced the growth of SN15-2 greatly, but it did not enhance the growth of Δ betB under hyperosmotic stress. Choline combined with hyperosmotic adaptation significantly increased the lethal stress resistance of SN15-2 while the resistance of Δ betA and Δ betB was significantly decreased. This research illuminated a strategy underlying the adaptation to osmotic stress in P. protegens and provided an effective method to improve the stress resistance of this species, thus provided a theoretical basis for the practical application of P. protegens SN15-2.

Objective High altitude pulmonary edema (HAPE) is a life threatening condition occurring in otherwise healthy individuals who rapidly ascend to high altitude. However, the molecular mechanisms of its pathophysiology are not well understood. The objective of this study is to evaluate differential gene expression in patients with HAPE during acute illness and subsequent recovery. Methods Twenty-one individuals who ascended to an altitude of 3780 m were studied, including 12 patients who developed HAPE and 9 matched controls without HAPE. Whole-blood samples were collected during acute illness and subsequent recovery for analysis of the expression of hypoxia-related genes, and physiologic and laboratory parameters, including mean pulmonary arterial pressure (mPAP), heart rate, blood pressure, and arterial oxygen saturation (SpO2), were also measured. Results Compared with control subjects, numerous hypoxia-related genes were up-regulated in patients with acute HAPE. Gene network analyses suggested that HIF-1α played a central role in acute HAPE by affecting a variety of hypoxia-related genes, including BNIP3L, VEGFA, ANGPTL4 and EGLN1. Transcriptomic profiling revealed the expression of most HAPE-induced genes was restored to a normal level during the recovery phase except some key hypoxia response factors, such asBNIP3L, EGR1, MMP9 and VEGF, which remained persistently elevated. Conclusions Differential expression analysis of hypoxia-related genes revealed distinct molecular signatures of HAPE during acute and recovery phases. This study may help us to better understand HAPE pathogenesis and putative targets for further investigation and therapeutic intervention.

To seek a feasible technique for processing waste printed circuit boards (PCBs), pretreatment of PCBs by table separation and further bioleached by moderate thermophiles in a stirred tank reactor were investigated. The shaking table separation, conducted after grinding and sieving of PCBs, produced two fractions: metal-rich parts (RPCBs), which is more suitable for pyrometallurgy process than untreated PCBs, and metal-poor parts (PPCBs) with only 8.83% metals was then bioleached by a mixed culture of moderate thermophiles effectively. After adaptation, the mixed culture could tolerate 80 g/L PPCBs. The bioleaching results showed that metals recovery was 85.23% Zn, 76.59% Cu and 70.16% Al in only 7 days. Trace Pb and Sn were detected in the leachate because of precipitating. The microorganism community structure was analyzed by amplified ribosomal DNA restriction analysis. Two moderately thermophilic bacteria species were identified as Leptospirillum ferriphilum and Acidithiobacillus caldus. Furthermore, uncultured Thermoplasmatales archaeon was also detected in the leaching system. It was also shown that moderate thermophiles revealed best bioleaching ability when compared with mesophiles and the mixture of mesophiles and moderate thermophiles. Finally, we designed a two-stage process model according to the present study to achieve semi-industrial waste PCBs recycling and economic feasibility analysis indicated that the process was profitable.

Investigation of macrozoobenthos was carried out in East Lake Dongting Reserves in September, 2001. All total, 51 taxa were identified from thirty-two sampling sites, of which, 48 oligchaetes, 15 mollusks, 14 insects and 4 other forms. Mollusks constituted the most dominant group and accounted for more than 70% of the total abundance. Among which, four gastropods, Alocinma longicornis, Bellamya aeraginosa, Parafossarulus sinensis, P. striatulus and one oligochaete Branchiura sowerbyi were the dominant species. The richness and three biodiversity index values ((Margalef, Simpson and Shannon) per site were 6.3, 1.38, 4.05 and 1.47, respectively. Assemblages were composed mainly of scrapers (66.7%) and collector-gatherers (19.9%). and to a lesser extent to other functional feeding groups.The investigation data were compiled and a GIS database of macrozoobenthos was established. A vector boundary map of the lake was digitized and georeferenced, then used to introduce sampling sites. Under the support of GIS software Areview 3.2 and the relative extension modules (Spatial Analyst Extension 2.0 and Kringing Interpolator Extension 3.22 SA), the GIS database was employed to simulate spacial distribution patterns of parameters of macrozoobenthos and Hilsenhoff biotic index (BI). The simulation process was based on the principle of Kringing interpolation technique. The simulated results were stored as Areview Grid format with a grid precision of 20 m. The higher distributional pattern of main groups and five dominated species were overlapped based on the 2-4 times as mean abundance, and the 16 physical-chemical parameters were collected in these higher distributional regions.The simulated patterns of macrozoobenthos showed that these patterns were relatively high spatial heterogeneity, and distributions of most parameters of macrozoobenthos had usually 2-4 high value distributed zones in the whole lake. The oligochaete pattern showed basically no overlapping with two other groups, and mainly distributed in deeper region. ANOVA detected water depth was the key factor influencing the distribution patterns of macrozoobenthos, and SO42- mainly controlling the pattern of oligochaete, and hardness the maim factor affecting mollusks. The average of BI (8.18) revealed the Reserves endured higher organic pollution.

The cDNA of growth hormone receptor (GHR) was cloned from the liver of 2-year common carp (Cyprinus carpio L.) by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE). Its open reading frame (ORF) of 1806 nucleotides is translated into a putative peptide of 602 amino acids, including an extracellular ligand-binding domain of 244 amino acids (aa), a single transmembrane domain of 24 aa and an intracellular signal-transduction domain of 334 aa. Sequence analysis indicated that common carp GHR is highly homologous to goldfish (Carassius auratus) GHR at both gene and protein levels. Using a pair of gene-specific primers, a GHR fragment was amplified from the cDNA of 2-year common carp, a 224 bp product was identified in liver and a 321 bp product in other tissues. The sequencing of the products and the partial genomic DNA indicated that the difference in product size was the result of a 97 bp intron that alternatively spliced. In addition, the 321 bp fragment could be amplified from all the tissues of 4-month common carp including liver, demonstrating the occurrence of the alternative splicing of this intron during the development of common carp. Moreover, a semi-quantitative RT-PCR was performed to analyze the expression level of GHR in tissues of 2-year common carp and 4-month common carp. The result revealed that in the tissues of gill, thymus and brain, the expression level of GHR in 2-year common carp was significantly tower than that of 4-month common carp.

Gonadotropin-releasing hormone (GnRH) is a conservative neurodecapeptide family, which plays a crucial role in regulating the gonad development and in controlling the final sexual maturation in vertebrate. Two differing cGnRH-II cDNAs of common carp, namely cGnRH-II cDNA1 and cDNA2, were firstly cloned from the brain by rapid amplification of cDNA end (RACE) and reverse transcription- polymerase chain reaction (RT-PCR). The length of cGnRH-II cDNA1 and cDNA2 was 622 and 578 base pairs (bp), respectively. The cGnRH-II precursors encoded by two cDNAs consisted of 86 amino acids, including a signal peptide, cGnRH-II decapeptide and a GnRH-associated peptide (GAP) linked by a Gly-Lys-Arg proteolytic site. The results of intron trapping and Southern blot showed that two differing cGnRH-II genes in common carp genome were further identified, and that two genes might exist as a single copy. The multi-gene coding of common carp cGnRH-II gene offered novel evidence for gene duplication hypothesis. Using semi-quantitative RT-PCR, expression and relative expression levels of cGnRH-II genes were detected in five dissected brain regions, pituitary and gonad of common carp. With the exception of no mRNA2 in ovary, two cGnRH-II genes could be expressed in all the detected tissues. However, expression levels showed an apparent difference in different brain regions, pituitary and gonad. According to the expression characterization of cGnRH-II genes in brain areas, it was presumed that cGnRH-II might mainly work as the neurotransmitter and neuromodulator and also operate in the regulation for the GnRH releasing. Then, the expression of cGnRH-II genes in pituitary and gonad suggested that cGnRH-II might act as the autocrine or paracrine regulator.

Objective Heat shock protein 90 (HSP90) is a chaperone for several client proteins involved in transcriptional regulation, signal transduction, and cell cycle control. HSP90 is abundantly expressed by a variety of tumour types and has been recently targeted for cancer therapy. The objective of this study is to determine the role of Hsp90 in regulating the migration of Mesenchymal stem cells and to determine the mechanism. We hypothesised that inhibition of Hsp90 impairs the MSCs migration via PI3K/Akt and ERK1/2 signalling pathways. Methods The MSCs were cultured from femoral and tibia. The ability for MSCs cells to migrate is to be determined by the wound healing assay and transwell assay. The activity of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) were estimated by gelatin –zymography. The mRNA levels of MMP-2, MMP-9, CXCR4 and VCAM-1 were detected by real-time PCR. The protein expression of MMP-2, MMP-9 and ERK1/2, phospho-ERK1/2, Akt and phospho-Akt were determined by Western-blot. Results Treatment with RhHsp90α significantly enhances MSCs migration from 9.83±2.48 to 48.65±2.81 cells. Treatment with sirhsp90α significantly decreased MSCs migration compared with treatment of hsp90α from 63.33±9.61 to 13.00±4.38 cells. Pretreat with 17-AAG, wortmannin, U0126, decreased MSCs migration to 13.33±1.29, 15.33±2.1, 16.5±3.3 cells, respectively. Treatment with RhHsp90α enhanced the MSCs secretion of MMP-2 and MMP-9, as well as significantly increasing the activity of MMP-9, and increasing the expression of CXCR4 and VCAM-1. PI3K/Akt and ERK signalling pathways mediate the promotion of MSCs migration by RhHsp90α. Conclusions Our data showed that Hsp90 promotes MSCs migration, elevate the expression of MMP-2, MMP-9, CXCR4 and VCAM-1. PI3K/Akt and ERK signalling pathways mediates these effects. Hsp90 is a candidate drug for enhancement of MSCs migration.