Unleashing the Power of Evolution in Xylanase Engineering: Investigating the Role of Distal Mutation Regulation.

The drive to enhance enzyme performance in industrial applications frequently clashes with the practical limitations of exhaustive experimental screening, underscoring the urgency for more refined and strategic methodologies in enzyme engineering. In this study, xylanase Xyl-1 was used as the model, coupling evolutionary insights with energy functions to obtain theoretical potential mutants, which were subsequently validated experimentally. We observed that mutations in the nonloop region primarily aimed at enhancing stability and also encountered selective pressure for activity. Notably, mutations in this region simultaneously boosted the Xyl-1 stability and activity, achieving a 65% success rate. Using a greedy strategy, mutant M4 was developed, achieving a 12 °C higher melting temperature and doubled activity. By integration of spectroscopy, crystallography, and quantum mechanics/molecular mechanics molecular dynamics, the mechanism behind the enhanced thermal stability of M4 was elucidated. It was determined that the activity differences between M4 and the wild type were primarily driven by dynamic factors influenced by distal mutations. In conclusion, the study emphasizes the pivotal role of evolution-based approaches in augmenting the stability and activity of the enzymes. It sheds light on the unique adaptive mechanisms employed by various structural regions of proteins and expands our understanding of the intricate relationship between distant mutations and enzyme dynamics.

Silencing of long non-coding RNA CRNDE promotes autophagy and alleviates neonatal hypoxic-ischemic brain damage in rats.

Hypoxic-ischemic (HI) brain damage (HIBD) leads to high neonatal mortality and severe neurologic morbidity. Autophagy is involved in the pathogenesis of HIBD. This study aims to investigate the effect of long non-coding RNA colorectal neoplasia differentially expressed (CRNDE) on HIBD and to validate whether autophagy is involved in this process. A HIBD model in rat pups and a HI model in rat primary cerebrocortical neurons were established. Autophagy was evaluated by western blot. The HIBD in rats was evaluated by hematoxylin and eosin staining, TUNEL staining, triphenyl tetrazolium chloride staining, and morris water maze test. The HI injury in vitro was evaluated by determining cell viability and apoptosis. The results showed that CRNDE expression was time-dependently increased in the brain after HIBD. Administration with CRNDE shRNA-expressing lentiviruses alleviated pathological injury and apoptosis in rat hippocampus, decreased infarct volume, and improved behavior performance of rats subjected to HIBD. Furthermore, CRNDE silencing promoted cell viability and inhibited cell apoptosis in neurons exposed to HI. Moreover, CRNDE silencing promoted autophagy and the autophagy inhibitor 3-methyladenine counteracted the neuroprotective effect of CRNDE silencing on HI-induced neuronal injury both in vivo and in vitro. Collectively, CRNDE silencing alleviates HIBD, at least partially, through promoting autophagy.

Long non-coding RNA CRNDE deteriorates intrauterine infection-induced neonatal brain injury.

BACKGROUND: This study aimed to test the hypothesis that long non-coding RNA (lncRNA) colorectal neoplasia differentially expressed (CRNDE) could exacerbate brain injury caused by intrauterine infection in neonatal rats. METHODS: Intrauterine infection was induced in pregnant rats by lipopolysaccharide (LPS). After delivery, newborn rats with brain injury caused by intrauterine infection were randomly divided into control, control shRNA, and CRNDE shRNA groups. CRNDE expression in serum and amniotic fluid of pregnant rats and neonatal brain tissues were determined by quantitative real-time PCR (qRT-PCR). Morris water maze (MWM) task was used to test the spatial learning and memory ability. Histological examination and apoptosis detection were performed by hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, respectively. Immunohistochemistry was conducted to evaluate the activation of astrocytes and microglia. RESULTS: LncRNA CRNDE was highly expressed in serum and amniotic fluid of maternal rats and in brain tissues of offspring rats. Furthermore, shRNA-mediated CRNDE downregulation could rescue the spatial learning and memory ability, improve brain histopathological changes and cell death, and inhibit the activation of astrocytes and microglia caused by LPS. CONCLUSION: CRNDE silencing possessed a cerebral protective effect in neonatal rats with brain injury caused by interauterine infection.

Mechanisms and effective control of physiological browning phenomena in plant cell cultures.

Browning phenomena are ubiquitous in plant cell cultures that severely hamper scientific research and widespread application of plant cell cultures. Up to now, this problem still has not been well controlled due to the unclear browning mechanisms in plant cell cultures. In this paper, the mechanisms were investigated using two typical materials with severe browning phenomena, Taxus chinensis and Glycyrrhiza inflata cells. Our results illustrated that the browning is attributed to a physiological enzymatic reaction, and phenolic biosynthesis regulated by sugar plays a decisive role in the browning. Furthermore, to confirm the specific compounds which participate in the enzymatic browning reaction, transcriptional profile and metabolites of T. chinensis cells, and UV scanning and high-performance liquid chromatography-mass spectrometry (HPLC-MS) profile of the browning compounds extracted from the brown-turned medium were analyzed, flavonoids derived from phenylpropanoid pathway were found to be the main compounds, and myricetin and quercetin were deduced to be the main substrates of the browning reaction. Inhibition of flavonoid biosynthesis can prevent the browning occurrence, and the browning is effectively controlled via blocking flavonoid biosynthesis by gibberellic acid (GA3 ) as an inhibitor, which further confirms that flavonoids mainly contribute to the browning. On the basis above, a model elucidating enzymatic browning mechanisms in plant cell cultures was put forward, and effective control approaches were presented.

Optimization of an HPLC Method for Determining the Genomic Methylation Levels of Taxus Cells.

An HPLC method for quantifying total DNA methylation in Taxus chinensis cells is described. Optimal conditions for the method were established as follows: DNA was hydrolyzed with DNA degradase at 37°C for 3 h. The mobile phase was a mixture of Solvent A [50 mM potassium dihydrogen phosphate/triethylamine (100:0.2, v/v)] and Solvent B (methanol); the gradient was 10% (v/v) solvent B. The calibration curves for deoxycytidine monophosphate (dCMP) and methylated dCMP were linear within 1.0-160.0 µg mL(-1), with correlation coefficients of 0.9996 and 0.9998. The limits of detection for dCMP and 5-mdCMP were 0.482 and 0.301 ng mL(-1), respectively, and the limits of quantification were 1.6 and 1.0 ng mL(-1), respectively. The method has been validated according to the current International Conference Harmonization guidelines. The method was able to quantify the content of dCMP and methylated dCMP specifically, accurately and precisely. The global DNA methylation level in different Taxus cells was measured using as little as 3 µg of DNA according to the optimized procedure. In addition, degradation of 5-methylcytosine was prevented.

[Clinical application of nasal intermittent positive pressure ventilation in initial treatment of neonatal respiratory distress syndrome].

OBJECTIVE: To study the clinical effectiveness and safety of nasal intermittent positive pressure ventilation (NIPPV) in the initial treatment of neonatal respiratory distress syndrome (NRDS) and the initial setting of NIPPV parameters. METHODS: One hundred neonates with NRDS were divided into NIPPV group (n=50) and nasal continuous positive airway pressure (NCPAP) group (n=50). A randomized controlled study was conducted to compare the effectiveness of NIPPV versus NCPAP in the initial treatment of NRDS from the following aspects: reducing CO2 retention, improving oxygenation, reducing second endotracheal intubation and second use of pulmonary surfactant (PS), reducing the duration of invasive respiratory support, reducing the duration of oxygen use, and reducing the incidence of air leak, abdominal distension and ventilator-associated pneumonia. RESULTS: After 1 and 6 hours of noninvasive respiratory support, the NIPPV group was superior to the NCPAP group with respect to the reduction in CO2 retention and improvement in oxygenation (P<0.05); in addition, compared with the NCPAP group, the NIPPV group had significantly lower rates of second endotracheal intubation and second PS use, significantly shorter duration of invasive respiratory support and time of FiO2 >0.21, and significantly lower incidence of apnea and ventilator-associated pneumonia (P<0.05); there were no significant differences in the incidence of air leak and abdominal distention between the two groups. CONCLUSIONS: NIPPV is effective and safe in the initial treatment of NRDS and holds promise for clinical application.

Correlation analysis of the taxane core functional group modification, enzyme expression, and metabolite accumulation profiles under methyl jasmonate treatment.

Metabolomic and transcriptomic profiling data were obtained and integrated to elucidate the crucial network controls on taxol and its precursor biosynthesis during the taxane core functionalization within methyl jasmonate (MJ)-induced Taxus chinensis cells. Twelve metabolites were identified using liquid chromatography-electrospray ionization-mass spectrometry. These metabolites contain taxol (paclitaxel), baccatin III (B-III) and its analogs, a group structurally bearing multiple free hydroxyls (TAX), and another group of multiple acyl taxanes (MAT), including taxuyunnanine C (TC) and its analogs. The metabolomic profile showed a higher increase in TAX than in MAT. Particularly, the ratio of B-III and taxol to the total taxane content increased more significantly in TAX than in MAT. The MAT proportion did not significantly change, although they are predominant components in cell cultures compared with TAX. Quantitative real-time polymerase chain reaction (qRT-RCR) was used to determine the transcription level of 20 genes, among which 11 were reported responsible for taxol biosynthesis and 9 were obtained from our previous transcriptomic data. The total expression levels of hydroxylase after 24 h and 6 days were higher than those of acylase. The principal component analysis (PCA) results validated the metabolomic analysis data, indicating that hydroxylation was more crucial than acylation for controlling the flux toward TAX biosynthesis. Furthermore, the PCA contribution comparison showed that two undefined genes of OHX1 and ACX3 might have good potential in TAX upregulation and MAT downregulation. To the best of our knowledge, this study provides the first experimental evidence on the contribution of total hydroxylation to taxane biosynthesis.

Hexa-substituted polyoxometalates made of trivacant Dawson {P2W15} fragments and {Ni6} clusters under hydrothermal conditions.

Three novel polyoxometalates (POMs) containing [Ni6(µ3-OH)3](9-) and [P2W15O56](12-) units were first made, showing the first hexa-nuclearity transition-metal substituted POMs (TMSPs) based on monomeric lacunary Dawson fragments, which further indicates that the hydrothermal technique can offer an effective way for making new TMSPs through lacunary POM fragments incorporated with high-nuclear TM clusters.

Negative correlation between serum syndecan-1 and apolipoprotein A1 in patients with type 2 diabetes mellitus.

Heparan sulfate proteoglycans (HSPGs) are involved in the regulation of cell growth, apoptosis and lipid metabolism in vitro. Syndecans are the primary form of HSPGs. Syndecan-1 is involved in the processes of cell growth, differentiation, adhesion, wound healing and inflammation. Additionally, as a sinusoidal transmembrane HSPG facing the plasma compartment, syndecan-1 is a promising target to be involved in lipoprotein physiology. We aimed to examine the possible correlation of syndecan-1 and lipid profile in type 2 diabetes mellitus. In this study, serum syndecan-1 was detected by ELISA, and potential correlations between syndecan-1 and triglyceride, cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, lipoprotein a, apolipoprotein (apo) B, apoA1 and apoB/apoA1 were analyzed. Forty-one patients with type 2 diabetes and 31 age-matched, non-diabetic healthy subjects (controls) were enrolled. Syndecan-1 in patients with diabetes (26.15 ± 2.42 ng/ml) was significantly higher than that of the controls (16.85 ± 1.98 ng/ml, t = -2.98, P = 0.005). Serum syndecan-1 level correlated negatively with apoA1 (r = -0.46, P = 0.003). Multiple regression analysis showed that apoA1 (b = -0.43, P = 0.003) was a predictor of serum syndecan-1 levels in subjects with type 2 diabetes.

Transcriptional profile of Taxus chinensis cells in response to methyl jasmonate.

BACKGROUND: Methyl jasmonate (MeJA) has been successfully used as an effective elicitor to enhance production of taxol and other taxanes in cultured Taxus cells. However the mechanism of MeJA-mediated taxane biosynthesis remains unclear. Genomic information for species in the genus Taxus is currently unavailable. Therefore, information about the transcriptome of Taxus cells and specifically, description of changes in gene expression in response to MeJA, is needed for the better exploration of the biological mechanisms of MeJA-mediated taxane biosynthesis. RESULTS: In this research, the transcriptome profiles of T. chinensis cells at 16 hours (T16) after MeJA treatment and of mock-treated cells (T0) were analyzed by "RNA-seq" to investigate the transcriptional alterations of Taxus cell in response to MeJA elicitation. More than 58 million reads (200 bp in length) of cDNA from both samples were generated, and 46,581 unigenes were found. There were 13,469 genes found to be expressed differentially between the two timepoints, including all of the known jasmonate (JA) biosynthesis/JA signaling pathway genes and taxol-related genes. The qRT-PCR results showed that the expression profiles of 12 randomly selected DEGs and 10 taxol biosynthesis genes were found to be consistent with the RNA-Seq data. MeJA appeared to stimulate a large number of genes involved in several relevant functional categories, such as plant hormone biosynthesis and phenylpropanoid biosynthesis. Additionally, many genes encoding transcription factors were shown to respond to MeJA elicitation. CONCLUSIONS: The results of a transcriptome analysis suggest that exogenous application of MeJA could induce JA biosynthesis/JA signaling pathway/defence responses, activate a series of transcription factors, as well as increase expression of genes in the terpenoid biosynthesis pathway responsible for taxol synthesis. This comprehensive description of gene expression information could greatly facilitate our understanding of the molecular mechanisms of MeJA-mediated taxane biosynthesis in Taxus cells.

Hexaaqua-magnesium(II) bis-{[2-(1-phenyl-1H-tetra-zol-5-yl)sulfan-yl]acetate}.

The asymmetric unit of the title compound, [Mg(H(2)O)(6)](C(9)H(7)N(4)O(2)S)(2), contains one-half of a [Mg(H(2)O)(6)](2+) cation ( symmetry) and one uncoordinated 2-[(1-phenyl-1H-tetra-zol-5-yl)sulfan-yl]acetate anion. The Mg(II) cation is coordinated by six water mol-ecules, exhibiting a slightly distorted octa-hedral coordination. A two-dimensional network parallel to (001) is formed via extensive hydrogen-bonding inter-actions involving the water mol-ecules as donors and the tetra-zole N and carboxyl-ate O atoms of the anion as acceptors. The shortest distance between two adjacent parallel benzene rings is 3.315 (2) Å. The dihedral angle between the benzene ring and the tetra-zole ring is 40.98 (2)°.

Efficient extraction of RNA and analysis of gene expression in a long-term Taxus cell culture using real-time RT-PCR.

A simple, quick and efficient method for isolating total RNA from heavy browning cells was developed by adding polyvinylpyrrolidone, mercaptoethanol and 3 M NaAc during the process of the Trizol (a kind of a widely used RNA extraction buffer) method. High-quality total RNA was isolated and synthesized to cDNA. Transcript levels of four paclitaxel biosynthetic pathway genes: dxr, hmgr, ggpps and dbat were assayed by real-time RT-PCR. The results demonstrated that the transcript levels of these genes experienced a coincident descent in the past three years as well as a decreasing paclitaxel productivity. According to these results, the possible reason for the descending paclitaxel productivity during long-term Taxus media cv. Hicksii cell culture maybe due to a decreasing transcripts level of mass genes in close with a gross secondary metabolite level. Gene manipulation emphasized only on key enzyme genes in the paclitaxel biosynthesis pathway may not hamper the somaclonal variation trend of Taxus media cv. Hicksii cell culture.

[Effect of oral carcinoma-associated fibroblasts on extracellular signal-regulated kinases pathway in the lingual carcinoma cell line].

OBJECTIVE: To investigate the effects of oral carcinoma-associated fibroblasts (CAFs) on extracellular signal-regulated kinases (ERK) pathway in a lingual carcinoma cell line. METHODS: The lingual carcinoma cell line, Tca8113 was stimulated by conditioned medium from oral CAFs, or cocultured with oral CAFs for definite time. Total ERK and pERK in Tca8113 lysate were detected by Western blotting, and the ratio between pERK and ERK were calculated. RESULTS: Both stimulation by conditioned medium and coculture induced prompt phosphorylation of ERK, and increased the ratio between pERK and ERK. CONCLUSION: Oral CAFs can activate ERK pathway of carcinoma cells.

[Genetic diversity of F-ATPase subunits gene uncEBF amplified from Streptococcus mutans clinical isolates].

OBJECTIVE: The purpose of this research was to study the genetic diversity of F-ATPase subunit gene uncEBF derived from Streptococcus mutans (S. mutans) clinical isolates, furthermore to investigate the relationship between the genetic diversity of F-ATPase and S. mutans aciduric ability. METHODS: 38 S. mutans strains included 18 high acid tolerance strains and 20 low acid tolerance strains. Gene uncEBF of these isolates were amplified with specific primers from S. mutans genomic DNA, and the PCR products were analyzed by RFLP and sequenced. SPSS 11.0 statistic software assayed the results. RESULTS: It was testified that two genotypes A and B of PCR-RFLP were revealed when digested with Alu I and Dde I digested fragments of uncEBF displayed two different patterns C and D. Fisher exact two-tail test showed that the distributions of A and B genotype strains with different acidurance were different (P < 0.05), and the proportion of A genotype strains from high acidurance group was higher than that from low acidurance one. Some of these amplified uncEBF genes from different genotype were sequenced and testified that there existed variation of Alu I and Dde I recognized sites. CONCLUSION: This study indicated that uncEBF gene of S. mutans F-ATPase obviously exhibited genetic diversity.

[Investigation of the degradation and the mechanisms of artificial basement membrane co-cultured with oral carcinoma-associated fibroblasts].

OBJECTIVE: To investigate the degradation of artificial basement membrane (matrigel) co-cultured with oral carcinoma-associated fibroblasts (CAFs) and its possible mechanism. METHODS: CAFs and normal fibroblasts (NFs) were incubated on matrigel for 24, 48, 72 h. Equivalent amounts of conditioned medium were collected and assayed for total protein, hydroxyproline and matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) activity by gelatin zymography. RESULTS: Oral CAFs were superior to oral NFs in total protein and hydroxyproline density, CAFs present more pro-MMP-2 and activated MMP-2. CONCLUSION: CAFs were superior to NFs in degradation of matrigel. CAFs might play a key role in the reconstitution of extracellular matrix and the progression of tumor.

[Genetic diversity of Citrus succosa Hort. Ex Tanaka from seven provinces as revealed by AFLP analysis].

Using 28 selected primer combinations, 12 accessions of Bendizao tangerine from seven provinces were investigated by AFLP analysis. A total of 882 genetic sites were detected, among which 192 were polymorphic. Using Huangyan Bendizao as contrast, the polymorphic sites of the other 11 accessions from seven provinces are not very high (3.5%-10.54%), and the number of different sites between accessions from other provinces is higher than that from Zhejiang province, which indicates that the ecological difference can partially contribute the formation of the genetic diversity. Genetic distance and dendrogram showed considerably close relatedness among the 12 accessions, and the max genetic distance was 0.229. The genetic analysis of the 12 accessions from seven provinces can provide useful information for the relationship between the formation of genetic variation and environmental difference and can facilitate the genetic improvement of this cultivar.

Immunotherapy of tumors with protein vaccine based on chicken homologous Tie-2.

PURPOSE: Tie-2 is an endothelium-specific receptor tyrosine kinase known to play a key role in tumor angiogenesis. The present study explores the feasibility of immunotherapy of tumors by using a protein vaccine based on chicken Tie-2 as a model antigen to break the immune tolerance against Tie-2 in a cross-reaction between the xenogeneic homologous and self-Tie-2. EXPERIMENTAL DESIGN AND RESULTS: In this study, a chicken homologous Tie-2 protein vaccine (chTie-2) and a corresponding mouse Tie-2 vaccine as a control were prepared and the antitumor effect of these vaccines was tested in two tumor models (murine B16F10 melanoma and murine H22 hepatoma). Immunotherapy with chTie-2 was found effective in two tumor models. Autoantibodies against mouse Tie-2 were detected in sera of mice immunized with chTie-2 through Western blot analysis and ELISA assay. Anti-Tie-2 antibody-producing B cells were detectable by ELISPOT. Histologic examination revealed that autoantibodies were deposited on the endothelial cells of tumor tissues. Purified immunoglobulins from chTie-2-immunized mice could induce the apoptosis of human umbilical vein endothelial cells in vitro. Importantly, adoptive transfer of purified immunoglobulins led to antitumor effect in vivo; apparently, angiogenesis was significantly inhibited in these tumors. Furthermore, the antitumor activity and production of autoantibodies could be abrogated by depletion of CD4+ T lymphocytes. CONCLUSIONS: Our findings may provide a vaccine strategy for cancer therapy and show the potential utilization of interference with Tie-2 pathway.

[Colonization relationship between Porphyromonas gingivalis and Bacteroides forsythus in the infected root canals with chronic apical periodontitis].

PURPOSE: To detect the distribution of P. gingivalis and B. forsythus in the infected root canals from Chinese chronic apical periodontitis, and investigate the colonization relationship between them in the root canals. METHODS: P. gingivalis and B. forsythus in the root canal samples of thirty-eight teeth with chronic apical periodontitis from 31 subjects who were referred to the Sichuan University West China Dental Hospital for dental treatment were studied with a 16S rDNA-directed polymerase chain reaction method. Fisher's exact tset was used to detect B. forsythus in the infected root canals with or without P. gingivalis. OR was used to analyse the relationship between them. RESULTS: The prevalence in 38 teeth was 39.5% for P. gingivalis, and 26.3% for B. forsythus, respectively. Significant positive relationship was shown in the combination of P. gingivalis and B. forsythus (OR=12, P<0.05). CONCLUSION: Both P. gingivalis and B. forsythus colonized in the root canals with chronic apical periodontitis and there was a positive relationship between P. gingivalis and B. forsythus in the infected root canal flora.

[Expression and secretion of human tumor necrosis factor gene transfected on human embryo myoblasts].

OBJECTIVE: To observe human tumor necrosis factor-alpha (hTNF-alpha) expression and secreting level of human embryo myoblasts transfected by hTNF-alpha gene. METHODS: Human embryo myoblasts were transfected with shuttle plasmid pSV23SHTNF containing hTNF-alpha gene by cationic liposomes DOSPER. The control group was only given equivalent liposomes except plasmid. After culturing for 24, 48, 72 and 96 hours, hTNF-alpha expression level of human embryo myoblasts was observed with immunocytochemistry staining, and hTNF-alpha secreting of human embryo myoblasts was analyzed by ELISA. RESULTS: After transfected by hTNF-alpha gene for 24, 48, 72 and 96 hours, the human embryo myoblasts displayed significant secretion of hTNF-alpha in the cultural supernatant (P < 0.05), and overexpression in cytoplasma and cell membrane. CONCLUSION: Transfection of hTNF-alpha gene to human myoblasts made myoblasts secrete high concentration of hTNF-alpha, implying it is feasible that transfecting muscle cells surrounding tongue carcinoma lesion with hTNF-alpha gene can prevent tongue carcinoma from intruding into deeper muscle tissue.