Daily dose-response from short-term monocular deprivation in adult humans.

Short-term monocular deprivation (MD) shifts sensory eye balance in favour of the previously deprived eye. The effect of MD on eye balance is significant but brief in adult humans. Recently, researchers and clinicians have attempted to implement MD in clinical settings for adults with impaired binocular vision. Although the effect of MD has been studied in detail in single-session protocols, what is not known is whether the effect of MD on eye balance deteriorates after repeated periods of MD (termed 'perceptual deterioration'). An answer to this question is relevant for two reasons. Firstly, the effect of MD (i.e., dose-response) should not decrease with repeated use if MD is to be used therapeutically (e.g., daily for weeks). Second, it bears upon the question of whether the neural basis of the effects of MD and contrast adaptation, a closely related phenomenon, is the same. The sensory change from contrast adaptation depends on recent experience. If the observer has recently experienced the same adaptation multiple times for consecutive days, then the adaptation effect will be smaller because contrast adaptation exhibits perceptual deterioration, so it is of interest to know if the effects of MD follow suit. This study measured the effect of 2-h MD for seven consecutive days on binocular balance of 15 normally sighted adults. We found that the shift in eye balance from MD stayed consistent, showing no signs of deterioration after subjects experienced multiple periods of MD. This finding shows no loss of effectiveness of repeated daily doses of MD if used therapeutically to rebalance binocular vision in otherwise normal individuals. Furthermore, ocular dominance plasticity, which is the basis of the effects of short-term MD, does not seem to share the property of 'perceptual deterioration' with contrast adaptation, suggesting different neural bases for these two related phenomena.

Direct RBS Engineering of the biosynthetic gene cluster for efficient productivity of violaceins in E. coli.

BACKGROUND: Violaceins have attracted much attention as potential targets used in medicines, food additives, insecticides, cosmetics and textiles, but low productivity was the key factor to limit their large-scale applications. This work put forward a direct RBS engineering strategy to engineer the violacein biosynthetic gene cluster cloned from Chromobacterium violaceum ATCC 12,472 to efficiently improve the fermentation titers. RESULTS: Through four-rounds of engineering of the native RBSs within the violaceins biosynthetic operon vioABCDE, this work apparently broke through the rate-limiting steps of intermediates conversion, resulting in 2.41-fold improvement of violaceins production compared to the titers of the starting strain Escherichia coli BL21(DE3) (Vio12472). Furthermore, by optimizing the batch-fermentation parameters including temperature, concentration of IPTG inducer and fermentation time, the maximum yield of violaceins from (BCDE)m (tnaA-) reached 3269.7 µM at 2 mM tryptophan in the medium. Interestingly, rather than previous reported low temperature (20 ℃), we for the first time found the RBS engineered Escherichia coli strain (BCDE)m worked better at higher temperature (30 ℃ and 37 ℃), leading to a higher-level production of violaceins. CONCLUSIONS: To our knowledge, this is the first time that a direct RBS engineering strategy is used for the biosynthesis of natural products, having the potential for a greater improvement of the product yields within tryptophan hyperproducers and simultaneously avoiding the costly low temperature cultivation for large-scale industrial production of violaciens. This direct RBS engineering strategy could also be easily and helpfully used in engineering the native RBSs of other larger and value-added natural product biosynthetic gene clusters by widely used site-specific mutagenesis methods represented by inverse PCR or CRISPR-Cas9 techniques to increase their fermentation titers in the future.

Aliiroseovarius marinus sp. nov., isolated from seawater.

A novel Gram-stain-negative, rod-shaped, non-spore-forming, non-flagellated, strictly aerobic strain, designated A6024T, was isolated from coastal seawater near Rizhao, PR China (119.61° E 35.47° N). The organism grew optimally at 28 °C, in pH 6.0-7.0 and in the presence of 3.0 % (w/v) NaCl. The strain required seawater or artificial seawater for growth and NaCl alone did not support growth. Strain A6024T contained ubiquinone 10 as the sole respiratory quinone and C18 : 1 ω7c (75.2 %) as the most abundant fatty acid. The predominant polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, one unidentified aminolipid and two unidentified lipids. The DNA G+C content of strain A6024T was 59.9 mol%. The results of phylogenetic analysis based on 16S rRNA gene sequences showed that the novel strain was related most closely to Aliiroseovarius halocynthiae MA1-10T, Aliiroseovarius pelagivivens GYSW-22T and Aliiroseovarius crassostreae CV919-312T with 98.3, 97.6 and 97.4 % sequence similarities, respectively. The calculated average nucleotide identity values and DNA-DNA hybridization values between strain A6024T and the phylogenetically related Aliiroseovarius species were in the range 76.0-85.6 % and 19.6-29.4 %, respectively. On the basis of the results of polyphasic analyses, strain A6024T represents a novel species of the genus Aliiroseovarius, for which the name Aliiroseovarius marinus sp. nov. is proposed. The type strain is A6024T (=KCTC 72114T=MCCC 1K03595T).

Leucothrix sargassi sp. nov., isolated from a marine alga [Sargassum natans (L.) Gaillon].

A novel bacterial strain, C3212T, was isolated from a marine alga collected from the sea shore of Yantai, China. The strain was Gram-stain-negative, rod-shaped, aerobic, non-motile, and oxidase- and catalase-positive. Growth was observed at 8-37 °C (optimum, 28 °C), at pH 6.0-9.0 (optimum, pH 7.0) and in the presence of 1.0-7.0 % (w/v) NaCl (optimum, 4.0 %). The major respiratory quinone was ubiquinone-8 (Q-8). The polar lipids of strain C3212T consisted of diphosphatidylglycerol (cardiolipin), phosphatidylglycerol, phosphatidylethanolamine, an unidentified aminophospholipid, an unidentified phospholipid and an unidentified polar lipid. The major fatty acids were C16 : 1ω6c and/or C16 : 1ω7c, and C18 : 1ω6c and/or C18 : 1ω7c. The DNA G+C content of strain C3212T was 44.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that the novel strain was related most closely to Leucothrix pacifica XH122T, Leucothrix arctica IMCC 9719T and Leucothrix mucor DSM 2157T with similarities of 98.0, 97.5 and 94.3 %, respectively. Estimated DNA-DNA hybridization values were 14.2, 20.7 and 13.9 % between strain C3212T and L. pacifica XH122T, L. arctica IMCC 9719T and L. mucor DSM 2157T, respectively. Phenotypic, phylogenetic and genomic analyses revealed that strain C3212T represents a novel species of the genus Leucothrix, for which the name Leucothrix sargassi sp. nov. is proposed. The type strain is C3212T (=MCCC 1K03600T=KCTC 72121T).

Thalassotalea atypica sp. nov., isolated from seawater, and emended description of Thalassotalea eurytherma.

A novel Gram-stain-negative, straight or slightly curved rod-shaped, non-spore-forming, non-flagellated, strictly aerobic strain, designated RZG4-3-1T, was isolated from coastal seawater of Rizhao, China (119.625° E 35.517° N). The organism grew optimally at 24-28 °C, at pH 7.0 and in the presence of 2.0 % (w/v) NaCl. The strain required seawater or artificial seawater for growth, and NaCl alone did not support growth. Strain RZG4-3-1T contained ubiquinone 8 (Q-8) as the major respiratory quinone and contained C16 : 1ω7c and/or C16 : 1ω6c and C16 : 0 as the dominant fatty acids. The polar lipids of strain RZG4-3-1T were phosphatidylethanolamine, phosphatidylglycerol and one unidentified aminophospholipid. The DNA G+C content of strain RZG4-3-1T was 40.1 mol%. Strain RZG4-3-1T exhibited the highest 16S rRNA gene sequence similarity value (96.0 %) to Thalassotalea eurytherma JCM 18482T. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain RZG4-3-1T belonged to the genus Thalassotalea. On the basis of polyphasic analyses, strain RZG4-3-1T represents a novel species of the genus Thalassotalea, for which the name Thalassotalea atypica sp. nov. is proposed. The type strain is RZG4-3-1T (=JCM 31894T=KCTC 52745T=MCCC 1K03276T). An emended description of Thalassotalea eurytherma is also provided.

Aquimarina aggregata sp. nov., isolated from seawater.

A novel Gram-stain-negative, agarolytic, rod-shaped, non-flagellated, strictly aerobic strain with gliding motility, designated RZW4-3-2T, was isolated from coastal seawater of the Yellow Sea in China (119.625° E 35.517° N). The organism grew optimally at 28 °C, at pH 7.0 and in the presence of 2.0-3.0 % (w/v) NaCl. The strain required seawater or artificial seawater for growth, and NaCl alone did not support growth. Strain RZW4-3-2T contained MK-6 as the major respiratory quinone and iso-C17 : 0 3-OH, iso-C15 : 0, iso-C15 : 1 G and iso-C15 : 0 3-OH as the dominant fatty acids. The polar lipids of strain RZW4-3-2T comprised phosphatidylethanolamine, five unknown phospholipids and one unknown lipid. The DNA G+C content of strain RZW4-3-2T was 32.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that the novel strain was related most closely to Aquimarina pacifica SW150T, Aquimarina macrocephali JCM 15542T and Aquimarina muelleri LMG 22569T with 98.1, 97.4 and 97.1 % sequence similarities, respectively. The estimated DNA-DNA hybridization values were 20.00±2.31 %, 20.70±2.33 % and 20.70±2.32 % between strain RZW4-3-2T and A. pacifica SW150T, A. macrocephali JCM 15542T and A. muelleri LMG 22569T, respectively. On the basis of results of polyphasic analyses, strain RZW4-3-2T represents a novel species of the genus Aquimarina, for which the name Aquimarinaaggregata sp. nov. is proposed. The type strain is RZW4-3-2T (=JCM 30824T=MCCC 1K00698T).

Shewanella gelidii sp. nov., isolated from the red algae Gelidium amansii, and emended description of Shewanella waksmanii.

A novel Gram-stain-negative, straight or slightly curved rod-shaped, non-spore-forming, facultatively anaerobic bacterium with a single polar flagellum, designated RZB5-4T, was isolated from a sample of the red algae Gelidium amansii collected from the coastal region of Rizhao, PR China (119.625° E 35.517° N). The organism grew optimally between 24 and 28 °C, at pH 7.0 and in the presence of 2-3 % (w/v) NaCl. The strain required seawater or artificial seawater for growth, and NaCl alone did not support growth. Strain RZB5-4T contained C16 : 1ω7c and/or C16 : 1ω6c, C16 : 0 and iso-C15 : 0 as the dominant fatty acids. The respiratory quinones detected in strain RZB5-4T were ubiquinone 7, ubiquinone 8, menaquinone 7 and methylmenaquinone 7. The polar lipids of strain RZB5-4T comprised phosphatidylethanolamine, phosphatidylglycerol, phosphatidylmonomethylethanolamine, one unidentified glycolipid, one unidentified phospholipid and one unknown lipid. The DNA G+C content of strain RZB5-4T was 47 mol %. Phylogenetic analysis based on 16S rRNA and gyrase B (gyrB) gene sequences showed that strain RZB5-4T belonged to the genus Shewanella, clustering with Shewanella waksmanii ATCC BAA-643T. Strain RZB5-4T exhibited the highest 16S rRNA gene sequence similarity value (96.6 %) and the highest gyrB gene sequence similarity value (80.7 %), respectively, to S. waksmanii ATCC BAA-643T. On the basis of polyphasic analyses, strain RZB5-4T represents a novel species of the genus Shewanella, for which the name Shewanella gelidii sp. nov. is proposed. The type strain is RZB5-4T (=JCM 30804T=KCTC 42663T=MCCC 1K00697T).

Pseudofulvibacter marinus sp. nov., isolated from seawater.

A novel Gram-stain-negative, rod-shaped, non-spore-forming, non-flagellated, strictly aerobic strain, designated RZW2-1T, was isolated from coastal seawater of the Yellow Sea in China (35.475° N 119.613° E). The organism grew optimally at 24 °C, at pH 7.0 and in the presence of 3.0 % (w/v) NaCl. The strain requires seawater or artificial seawater for growth and NaCl alone does not support growth. Strain RZW2-1T contained MK-6 as the only respiratory quinone and iso-C15 : 0, iso-C15 : 1 G and 10-methyl C16 : 0 and/or iso-C17 : 1ω9c as the dominant fatty acids. The polar lipids of strain RZW2-1T were four unidentified phospholipids (PL1-PL4), two unknown lipids (L1, L2) and one unidentified aminolipid (AL1). The DNA G+C content of strain RZW2-1T was 32 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that the novel strain was most closely related to the type strain of the only described species of genus Pseudofulvibacter, Pseudofulvibacter geojedonensis YCS-9T, with 95.1 % 16S rRNA gene sequence similarity. On the basis of polyphasic analyses, strain RZW2-1T represents a novel species of the genus Pseudofulvibacter, for which the name Pseudofulvibacter marinus sp. nov. is proposed. The type strain is RZW2-1T ( = JCM 30826T = MCCC 1K00695T).

A 10-Year Follow-up Survey of Treatment Abandonment of Children With Acute Myeloid Leukemia in Suzhou, China.

A survey of the clinical data on acute myeloid leukemia (AML) over the past 10 years in the treatment center of Children's Hospital was presented. The aim of the study was to identify the factors influencing the treatment abandonment rate (AR) of AML. Of the 474 AML cases examined, 264 were abandoned (55.7%). The most important factor affecting AR appeared to be the AML subtype-that is, the M3 versus non-M3 (42% vs. 60%). Patient age was observed to be closely related to AR-the older the patients, the lower the AR-and infants had the highest prevalence of abandonment (84.2%). The patient's residential location was markedly correlated to AR, which was almost inversely proportional to the size of the township where the patient came from. From large cities, intermediate and small towns to countryside villages, the AR increased linearly. So was the correlation with health insurance coverage, which decreased in the same way. Sex and karyotypes did not affect AR. In conclusion, the patients' financial burden and the perceived incurability of AML were the 2 leading factors dominating the decision for abandonment in parents and caregivers.

A Rapid Microwave-Assisted Thermolysis Route to Highly Crystalline Carbon Nitrides for Efficient Hydrogen Generation.

Highly crystalline graphitic carbon nitride (g-C3 N4 ) with decreased structural imperfections benefits from the suppression of electron-hole recombination, which enhances its hydrogen generation activity. However, producing such g-C3 N4 materials by conventional heating in an electric furnace has proven challenging. Herein, we report on the synthesis of high-quality g-C3 N4 with reduced structural defects by judiciously combining the implementation of melamine-cyanuric acid (MCA) supramolecular aggregates and microwave-assisted thermolysis. The g-C3 N4 material produced after optimizing the microwave reaction time can effectively generate H2 under visible-light irradiation. The highest H2 evolution rate achieved was 40.5 µmol h-1 , which is two times higher than that of a g-C3 N4 sample prepared by thermal polycondensation of the same supramolecular aggregates in an electric furnace. The microwave-assisted thermolysis strategy is simple, rapid, and robust, thereby providing a promising route for the synthesis of high-efficiency g-C3 N4 photocatalysts.

[Efficacy and safety of the WT-2009 chemotherapy protocol in treatment of Wilms' tumor in children].

OBJECTIVE: To evaluate the efficacy and safety of the WT-2009 chemotherapy protocol for Wilms' tumor (WT) in children. METHODS: The clinical data of 34 children with newly-diagnosed WT between July 2009 and December 2013 were retrospectively analyzed. Among the 34 children, 2 died before treatment, 6 children did not accept therapy and 26 accepted the chemotherapy based on the WT-2009 chemotherapy protocol. Kaplan-Meier method was used to estimate the 2-year survival rate. RESULTS: The pathological analysis revealed the favorable histology WT was common (88%, 30/34). The most common first manifestation was abdominal masses (56%, 19/34). Among the 26 patients who accepted the chemotherapy based on the WT-2009 protocol, complete remission was achieved in 24 cases (92%), partial remission was achieved in 1 case (4%), and disease relapse was found in 1 case (4%). Severe pulmonary infection occurred in 1 case in the course of treatment. The 2-year overall survival rate and event-free survival rate were 100% and 89.7% respectively. CONCLUSIONS: Favorable histology is most common pathological type in children with WT. The chemotherapy based on the WT-2009 protocol for WT can produce a favorable prognosis and a high tolerance.

A novel mutation in the CD40 ligand gene in a Chinese boy with X-linked hyper-IgM syndrome.

X-linked hyper-IgM Syndrome (XHIGM) is caused by a mutation of CD40 ligand (CD40L), which is normally expressed on activated CD4+ T cells and is responsible for immunoglobulin class switching. A 7-year-old boy with recurrent sino-pulmonary infections since the age of 3 months had normal CD3+, CD4+, CD8+T lymphocytes, and CD19+B lymphocytes and NK cells, but significantly elevated IgM and extremely decreased IgG and IgA. Sequencing of genomic DNA revealed that the patient had a 34 base deletion in intron 3 and exon 4 of CD40L(g.8172_8205del34bp), which lead to the entire deletion of exon 4 in cDNA (c.347_409del63bp, i.e.,exon 4 skipping) and an in-frame deletion of 21 amino acids in CD40L protein. Moreover, the patient had negligible CD40L expression on activated CD3+CD8-T lymphocytes. His mother and sister were carriers of the CD40L mutation. Our studies demonstrated a novel mutation in CD40L, which, to our knowledge, has not been reported previously.

Reduced Monocular Luminance Promotes Fusion But Not Mixed Perception in Amblyopia.

Purpose: The purpose of this study was to understand how monocular luminance reduction affects binocular balance and examine whether it differentially influences fusion and mixed perception in amblyopia. Methods: Twenty-three normally sighted observers and 12 adults with amblyopia participated in this study. A novel binocular rivalry task was used to measure the phase duration of four perceptual responses (right- and left-tilts, fusion, and mixed perception) before and after a neutral density (ND) filter was applied at various levels to the dominant eye (DE) of controls and the fellow eye (FE) of patients with amblyopia. Phase durations were analyzed to assess whether the duration of fusion or mixed perception shifted after monocular luminance reduction. Moreover, we quantified ocular dominance and adjusted monocular contrast and luminance separately to investigate the relationship between changes in ocular dominance induced by the two manipulations. Results: In line with previous studies, binocular balance shifted in favor of the brighter eye in both normal adults and patients with amblyopia. As a function of the ND filter's density, the duration of fusion and mixed perception decreased in normal controls, whereas that of fusion but not mixed perception increased significantly in patients with amblyopia. In addition, changes in binocular balance from luminance reduction were more significant in more balanced amblyopes or normal observers. Furthermore, shifts in binocular balance after contrast and luminance modulation were correlated in both normal and amblyopic observers. Conclusions: The duration of fusion but not mixed perception increased in amblyopia after monocular luminance reduction in the FE. Moreover, our findings demonstrate that changes in ocular dominance from contrast-modulation and luminance-modulation are correlated in both normal and amblyopic observers.

Improving the Catalytic Properties of Xylanase from Alteromones Macleadii H35 Through Sequence Analysis.

Endo-1,4-ß-xylanase is a key xylanolytic enzyme, and our study aimed to enhance the catalytic properties of Alteromones Macleadii xylanase (Xyn ZT-2) through sequence-guided design approach. Analysis of the amino acid sequence revealed highly conserved residues near the active site, with few differences. Introducing various mutations allowed us to modify the enzyme's catalytic performance. Particularly, the A152G mutation led to a 9.8-fold increase in activity and a 23.2-fold increase in catalytic efficiency. Moreover, A152G exhibited an optimal temperature of 65 °C, 20 °C higher than that of Xyn ZT-2, while the T287S mutant showed a 4.9-fold increase in half-life. These results underscore the role of amino acid evolution in shaping xylanase catalysis. Through targeted sequence analysis and a focused mutation library, we effectively improved catalytic performance, providing a straightforward approach for enhancing enzyme efficiency.

Analysis of kidney biopsy data from a single center in the midland rural area of china, 1996-2010.

OBJECTIVE: To survey the clinical epidemiology and correlations between pathology and clinical features of major groups of kidney diseases in a rural area of China. METHODS: From January 1996 to December 2010, histologic diagnosis of renal disease was made on samples collected from 919 patients from a single center in the midland rural area of China. Demographic data were obtained from all patients, and clinical profiles were analyzed in 917 patients. RESULTS: The mean age of the whole group was 33.13 (14.13) years (range 16-72 years). Men accounted for 55.28% (n = 508) and women made up 45.72% (n = 408). Patients aged 16 to 50 years comprised 83.75% of the sample (n = 770). Lupus nephritis was the predominant diagnosis in women; renal diseases were predominant in men. In patients with nephrotic syndrome, mesangial proliferative glomerulonephritis was the most frequent pathologic pattern (39.46%), followed by IgA nephropathy (18.39%), whereas in patients with nephritic syndrome, IgA nephropathy (39.64%) was the most frequent pathologic pattern, followed by mesangial proliferative glomerulonephritis (32.38%). The most common pathologic pattern in patients with secondary glomerulonephritis was Henoch-Schoˇnlein purpura nephritis, followed by lupus nephritis. CONCLUSIONS: Mesangial proliferative glomerulonephritis was the most common renal pathologic pattern. Male adolescents were predominant in this group of patients. The most common clinical syndrome was nephrotic syndrome.

Gut Bacterial Diversity of Insecticide-Susceptible and Insecticide-Resistant Megalurothrips usitatus (Thysanoptera: Thripidae) and Elucidation of Their Putative Functional Roles.

The gut bacterial microbiota of insects plays a crucial role in physiological, metabolic, and innate immune processes. In the current study, the gut bacterial communities of an insecticide-susceptible (IS), and a resistant (IR) population of a major legume pest, Megalurothrips usitatus (Bagnall), were evaluated. The 16S rDNA V3 + V4 regions of M. usitatus infected with Beauveria brongniartii along with the intestinal flora of both populations were sequenced based on a High-throughput sequencing platform. Toxicological bioassays revealed that the IR population exhibited resistance to acetamiprid and B. brongniartii isolate SB010 at levels of 138.0-fold and 55.6-fold higher, respectively, compared to the IS population. Through 16S High-throughput sequencing, the results indicate that both resistant populations, as well as B. brongniartii infestation, reduce the number of species of M. usitatus gut microbes. Using KEGG function prediction, it was found that most intestinal bacteria were involved in various metabolic activities, and the abundance of resistant populations was higher than that of sensitive populations. The bacteria in the gut of M. usitatus are mainly involved in various metabolic activities to achieve the degradation of B. brongniartii. This study provides valuable insights into the interaction between gut bacteria, insecticide resistance, and Beauveria. brongniartii infection in Megalurothrips usitatus, which can help inform future pest control strategies.

RNA interference in cytochrome P450 monooxygenase (CYP) gene results in reduced insecticide resistance in Megalurothrips usitatus Bagnall.

Genes of the cytochrome P450 (CYP450) superfamily are known to be involved in the evolution of insecticide resistance. In this study, the transcriptomes of two Megalurothrips usitatus Bagnall (Thysanoptera: Thripidae) strains (resistant and susceptible) were screened for detoxification genes. MusiDN2722 encodes a protein composed of 504 amino acid residues with a relative molecular mass of 57.3 kDa. Multiple sequence alignment and phylogenetic analysis showed that MusiDN2722 is a member of the CYP450 family and has characteristics of the conserved CYP6 domain shared by typical CYP450 family members. RT-qPCR (real-time quantitative polymerase chain reaction) analysis showed that MusiDN2722 was upregulated in the acetamiprid-resistant strain compared with the susceptible strain (p < 0.05), and the relative expression level was significantly higher at 48 h after exposure than at 24 h after exposure. The interference efficiency of the injection method was higher than that of the membrane-feeding method. Silencing of MusiDN2722 through RNA interference significantly increased the sensitivity of M. usitatus to acetamiprid. Overall, this study revealed that MusiDN2722 plays a crucial role in the resistance of M. usitatus to acetamiprid. The findings will not only advance our understanding of the role of P450s in insecticide resistance but also provide a potential target for the sustainable control of destructive pests such as thrips.

One-pot biosynthesis of gastrodin using UDP-glycosyltransferase itUGT2 with an in situ UDP-glucose recycling system.

Gastrodin, the major effective ingredient in Tianma (Gastrodia elata), is a p-hydroxybenzoic acid derivative with various activities. Gastrodin has been widely investigated for food and medical applications. The last biosynthetic step for gastrodin is UDP-glycosyltransferase (UGT)-mediated glycosylation with UDP-glucose (UDPG) as glycosyl donor. In this study, we performed a one-pot reaction both in vitro and in vivo to synthesize gastrodin from p-hydroxybenzyl alcohol (pHBA) by coupling UDP-glucosyltransferase from Indigofera tinctoria (itUGT2) to sucrose synthase from Glycine max (GmSuSy) for regeneration of UDPG. The in vitro results showed that itUGT2 transferred a glucosyl group to pHBA to generate gastrodin. After 37 UDPG regeneration cycles with 2.5% (molar ratio) UDP, the pHBA conversion reached 93% at 8 h. Furthermore, a recombinant strain with itUGT2 and GmSuSy genes was constructed. Through optimizing the incubation conditions, a 95% pHBA conversion rate (220 mg/L gastrodin titer) was achieved in vivo without addition of UDPG, which was 2.6-fold higher than that without GmSuSy. This in situ system for gastrodin biosynthesis provides a highly efficient strategy for both in vitro gastrodin synthesis and in vivo biosynthesis of gastrodin in E. coli with UDPG regeneration.

Cloning, Expression, and Characterization of an GHF 11 Xylanase from Aspergillus niger XZ-3S.

A xylanase gene (xynZF-2) from the Aspergillus niger XZ-3S was cloned and expressed in Escherichia coli. The coding region of the gene was separated by only one intron with the 68 bp in length. It encoded 225 amino acid residues of a protein with a calculated molecular weight of 24.04 kDa plus a signal peptide of 18 amino acids. The amino acid sequence of the xynZF-2 gene had a high similarity with those of family 11 of glycosyl hydrolases reported from other microorganisms. The mature peptide encoding cDNA was subcloned into pET-28a(+) expression vector. The resultant recombinant plasmid pET-28a-xynZF-2 was transformed into E. coli BL21(DE3), and finally the recombinant strain BL21/xynZF-2 was obtained. A maximum activity of 42.33 U/mg was gained from cellular of E. coli BL21/xynZF-2 induced by IPTG. The optimum temperature and pH for recombinant enzyme which has a good stability in alkaline conditions were 40 °C and 5.0, respectively. Fe(3+) had an active effect on the enzyme obviously.

[Effects of Monoammonium Glycyrrhizinate on Stem Cell-like Characteristics, Oxidative Stress and Mitochondrial Function of Acute Promyelocytic Leukemia NB4 Cells].

OBJECTIVE: To investigate the effect of monoammonium glycyrrhizinate on the stem cell-like characteristics, oxidative stress and mitochondrial function of acute promyelocytic leukemia cells NB4. METHODS: CCK-8 method was used to detect the viability of acute promyelocytic leukemia cells NB4, and the appropriate dose was screened; Cloning method was used to detect the proliferation rate of NB4 cell; Western blot was used to detect the expression of cell cycle-related protein; flow cytometry was used to detect cell apoptosis and sort NB4 stem cells positive (CD133+); Stem cell markers (Oct4, ABCG2, Dclk1) were detected by RT-PCR; ROS was detected by fluorescence; The kit was used to detect the level of oxidative stress markers (MDA); The flow cytometry was used to detect the change of mitochondrial membrane potential; Western blot was used to detect the expression of mitochondrial damage index-related proteins (Bax/BCL-2). RESULTS: Compared with the control group, if the concentration of MAG was less than 5 µmol/L, the cell NB4 viability showed no significant difference; if the concentration was higher than 5 µmol/L, the inhibitory effect on the growth of cell NB4 increased and showed significant difference (P<0.05), according to the results of CCK-8 experiment, four groups were set based on the concentration of MAG 0 µmol/L, MAG 5 µmol/L, MAG 10 µmol/L, and MAG 20 µmol/L; compared with the control group (MAG 0 µmol/L), the cells in MAG 5 µmol/L group showed no significant difference, while the proliferation rate, cyclin expression, mitochondrial membrane potential, stem cell CD133+ ratio, and marker mRNA level ( Oct4, ABCG2, Dclk1) of NB4 cell were significantly reduced (P<0.05); the apoptosis rate, reactive oxygen species, MDA content and Bax/BCL-2 expression of NB4 cell significantly increased (P<0.05). CONCLUSION: Monoammonium glycyrrhizinate has a significant inhibitory effect on acute promyelocytic leukemia cells NB4, which may be related to the regulation of stem cell-like characteristics, oxidative stress and mitochondrial function.