Paeoniflorin ameliorates neuropathic pain-induced depression-like behaviors in mice by inhibiting hippocampal neuroinflammation activated via TLR4/NF-κB pathway.

Neuropathic pain (NP) that contributes to the comorbidity between pain and depression is a clinical dilemma. Neuroinflammatory responses are known to have potentially important roles in the initiation of NP and depressive mood. In this study, we aimed to investigate the effects of paeoniflorin (PF) on NP-induced depression-like behaviors by targeting the hippocampal neuroinflammation through the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling pathway. We used a murine model of NP caused by unilateral sciatic nerve cuffing (Cuff ). PF was injected intraperitoneally once a day for a total of 14 days. Pain and depression-like behavior changes were evaluated via behavioral tests. Pathological changes in the hippocampus of mice were observed by H&E staining. The levels of proinflammatory cytokines in the hippocampus were detected using ELISA. Activated microglia were measured by immunohistochemical staining. The TLR4/NF-κB signaling pathwayassociated protein expression in the hippocampus was detected by western blotting. We found that the PF could significantly alleviate Cuff-induced hyperalgesia and depressive behaviors, lessen the pathological damage to the hippocampal cell, reduce proinflammatory cytokines levels, and inhibit microglial over-activation. Furthermore, PF downregulated the expression levels of TLR4/NF-κB signaling pathwayrelated proteins in the hippocampus. These results indicate that PF is an effective drug for improving the comorbidity between NP and depression.

Multiplexed analysis of fixed tissue RNA using Ligation in situ Hybridization.

Clinical tissues are prepared for histological analysis and long-term storage via formalin fixation and paraffin embedding (FFPE). The FFPE process results in fragmentation and chemical modification of RNA, rendering it less suitable for analysis by techniques that rely on reverse transcription (RT) such as RT-qPCR and RNA-Seq. Here we describe a broadly applicable technique called 'Ligation in situ Hybridization' ('LISH'), which is an alternative methodology for the analysis of FFPE RNA. LISH utilizes the T4 RNA Ligase 2 to efficiently join adjacent chimeric RNA-DNA probe pairs hybridized in situ on fixed RNA target sequences. Subsequent treatment with RNase H releases RNA-templated ligation products into solution for downstream analysis. We demonstrate several unique advantages of LISH-based assays using patient-derived FFPE tissue. These include >100-plex capability, compatibility with common histochemical stains and suitability for analysis of decade-old materials and exceedingly small microdissected tissue fragments. High-throughput DNA sequencing modalities, including single molecule sequencing, can be used to analyze ligation products from complex panels of LISH probes ('LISH-seq'), which can be amplified efficiently and with negligible bias. LISH analysis of FFPE RNA is a novel methodology with broad applications that range from multiplexed gene expression analysis to the sensitive detection of infectious organisms.

Prostate cancer risk locus at 8q24 as a regulatory hub by physical interactions with multiple genomic loci across the genome.

Chromosome 8q24 locus contains regulatory variants that modulate genetic risk to various cancers including prostate cancer (PC). However, the biological mechanism underlying this regulation is not well understood. Here, we developed a chromosome conformation capture (3C)-based multi-target sequencing technology and systematically examined three PC risk regions at the 8q24 locus and their potential regulatory targets across human genome in six cell lines. We observed frequent physical contacts of this risk locus with multiple genomic regions, in particular, inter-chromosomal interaction with CD96 at 3q13 and intra-chromosomal interaction with MYC at 8q24. We identified at least five interaction hot spots within the predicted functional regulatory elements at the 8q24 risk locus. We also found intra-chromosomal interaction genes PVT1, FAM84B and GSDMC and inter-chromosomal interaction gene CXorf36 in most of the six cell lines. Other gene regions appeared to be cell line-specific, such as RRP12 in LNCaP, USP14 in DU-145 and SMIN3 in lymphoblastoid cell line. We further found that the 8q24 functional domains more likely interacted with genomic regions containing genes enriched in critical pathways such as Wnt signaling and promoter motifs such as E2F1 and TCF3. This result suggests that the risk locus may function as a regulatory hub by physical interactions with multiple genes important for prostate carcinogenesis. Further understanding genetic effect and biological mechanism of these chromatin interactions will shed light on the newly discovered regulatory role of the risk locus in PC etiology and progression.

eRNA: a graphic user interface-based tool optimized for large data analysis from high-throughput RNA sequencing.

BACKGROUND: RNA sequencing (RNA-seq) is emerging as a critical approach in biological research. However, its high-throughput advantage is significantly limited by the capacity of bioinformatics tools. The research community urgently needs user-friendly tools to efficiently analyze the complicated data generated by high throughput sequencers. RESULTS: We developed a standalone tool with graphic user interface (GUI)-based analytic modules, known as eRNA. The capacity of performing parallel processing and sample management facilitates large data analyses by maximizing hardware usage and freeing users from tediously handling sequencing data. The module miRNA identification" includes GUIs for raw data reading, adapter removal, sequence alignment, and read counting. The module "mRNA identification" includes GUIs for reference sequences, genome mapping, transcript assembling, and differential expression. The module "Target screening" provides expression profiling analyses and graphic visualization. The module "Self-testing" offers the directory setups, sample management, and a check for third-party package dependency. Integration of other GUIs including Bowtie, miRDeep2, and miRspring extend the program's functionality. CONCLUSIONS: eRNA focuses on the common tools required for the mapping and quantification analysis of miRNA-seq and mRNA-seq data. The software package provides an additional choice for scientists who require a user-friendly computing environment and high-throughput capacity for large data analysis. eRNA is available for free download at https://sourceforge.net/projects/erna/?source=directory.

Characterization of human plasma-derived exosomal RNAs by deep sequencing.

BACKGROUND: Exosomes, endosome-derived membrane microvesicles, contain specific RNA transcripts that are thought to be involved in cell-cell communication. These RNA transcripts have great potential as disease biomarkers. To characterize exosomal RNA profiles systemically, we performed RNA sequencing analysis using three human plasma samples and evaluated the efficacies of small RNA library preparation protocols from three manufacturers. In all we evaluated 14 libraries (7 replicates). RESULTS: From the 14 size-selected sequencing libraries, we obtained a total of 101.8 million raw single-end reads, an average of about 7.27 million reads per library. Sequence analysis showed that there was a diverse collection of the exosomal RNA species among which microRNAs (miRNAs) were the most abundant, making up over 42.32% of all raw reads and 76.20% of all mappable reads. At the current read depth, 593 miRNAs were detectable. The five most common miRNAs (miR-99a-5p, miR-128, miR-124-3p, miR-22-3p, and miR-99b-5p) collectively accounted for 48.99% of all mappable miRNA sequences. MiRNA target gene enrichment analysis suggested that the highly abundant miRNAs may play an important role in biological functions such as protein phosphorylation, RNA splicing, chromosomal abnormality, and angiogenesis. From the unknown RNA sequences, we predicted 185 potential miRNA candidates. Furthermore, we detected significant fractions of other RNA species including ribosomal RNA (9.16% of all mappable counts), long non-coding RNA (3.36%), piwi-interacting RNA (1.31%), transfer RNA (1.24%), small nuclear RNA (0.18%), and small nucleolar RNA (0.01%); fragments of coding sequence (1.36%), 5' untranslated region (0.21%), and 3' untranslated region (0.54%) were also present. In addition to the RNA composition of the libraries, we found that the three tested commercial kits generated a sufficient number of DNA fragments for sequencing but each had significant bias toward capturing specific RNAs. CONCLUSIONS: This study demonstrated that a wide variety of RNA species are embedded in the circulating vesicles. To our knowledge, this is the first report that applied deep sequencing to discover and characterize profiles of plasma-derived exosomal RNAs. Further characterization of these extracellular RNAs in diverse human populations will provide reference profiles and open new doors for the development of blood-based biomarkers for human diseases.

Molecular and biochemical characterization of a new alkaline active multidomain xylanase from alkaline wastewater sludge.

A xylanase gene, xyn-b39, coding for a multidomain glycoside hydrolase (GH) family 10 protein was cloned from the genomic DNA of the alkaline wastewater sludge of a paper mill. Its deduced amino acid sequence of 1,481 residues included two carbohydrate-binding modules (CBM) of family CBM_4_9, one catalytic domain of GH 10, one family 9 CBM and three S-layer homology (SLH) domains. xyn-b39 was expressed heterologously in Escherichia coli, and the recombinant enzyme was purified and characterized. Xyn-b39 exhibited maximum activity at pH 7.0 and 60 °C, and remained highly active under alkaline conditions (more than 80 % activity at pH 9.0 and 40 % activity at pH 10.0). The enzyme was thermostable at 55 °C, retaining more than 90 % of the initial activity after 2 h pre-incubation. Xyn-b39 had wide substrate specificity and hydrolyzed soluble substrates (birchwood xylan, beechwood xylan, oat spelt xylan, wheat arabinoxylan) and insoluble substrates (oat spelt xylan and wheat arabinoxylan). Hydrolysis product analysis indicated that Xyn-b39 was an endo-type xylanase. The K (m) and V (max) values of Xyn-b39 for birchwood xylan were 1.01 mg/mL and 73.53 U/min/mg, respectively. At the charge of 10 U/g reed pulp for 1 h, Xyn-b39 significantly reduced the Kappa number (P < 0.05) with low consumption of chlorine dioxide alone.

RILPL2 is associated with the progression and prognosis of cervical squamous cell carcinoma and endocervical adenocarcinoma.

Cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) is one of the most common tumors among females worldwide. RILPL2 was recently reported to be a promising biomarker for the treatment of breast cancer. This study aimed to investigate the potential role of RILPL2 in CESC. Totally 302 CESC patients' data were downloaded from The Cancer Genome Atlas database. All patients were divided into high or low RILPL2 groups according to the median expression of RILPL2. Subsequently, survival analysis, multivariate Cox regression, and experimental validation were performed on all CESC patient data. The Ualcan database was used to analyze the expression level and prognostic value of RILPL2 in pan-cancer. The Gene Set Cancer Analysis database was used for drug sensitivity analysis. Functional KEGG pathways were analyzed using gene set enrichment analysis. RILPL2 was generally down-regulated in a variety of tumors, and a high level of RILPL2 was associated with a better prognosis in CESC patients. Immunohistochemistry, western blotting, and qRT-PCR results showed that RILPL2 was significantly down-regulated in CESC cells and tissues. Besides, along with the increase of TNM Stage, the RILPL2 expression tended to decrease gradually. Patients with high RILPL2 expression showed lower resistance to small molecule drugs used in CESC progressions, such as Methotrexate, AZD7762, and Vinblastine, and a higher response rate to immunotherapy. Additionally, we identified 267 co-expressing genes of RILPL2, all of which jointly affected CESC progression through 15 complex pathways. Low RILPL2 expression was closely associated with the onset, progression, and poor prognosis of CESC. RILPL2 might be a promising optional biomarker for CESC patients' diagnosis and prognosis.

Novel Prognostic Biomarkers for Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma (CESC) Patients via Analysis of Competing Endogenous RNA (ceRNA) Network.

Background: Cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) is a common malignant gynecological cancer. The ceRNA networks play important roles in many tumors, while RILPL2-related ceRNA network has been seldom studied in CESC. Methods: All CESC data was obtained from TCGA database. Differentially expressed RNAs and predicted target RNAs were cross analyzed to construct ceRNA network. RNA and clinicopathological characteristics' influence on overall survival (OS) were determined by univariate and multivariate Cox regression analyses. Lasso regression was used to construct the prediction model. Coexpression analysis was performed to explore the association of gene expression with CESC. This was followed by an experimental validation based on these results. Results: Between high and low RILPL2 expression CESC patients, totally 1227 DEmRNAs, 39 DEmiRNAs, and 1544 DElncRNAs were identified. After multiple cross analyses, 1 miRNA hsa-miR-1293, 20 mRNAs, and 43 lncRNAs were maintained to construct ceRNA network. CADM3-AS1, LINC00092, and ZNF667-AS1 in ceRNA network were significantly associated with the OS of CESC patients, and patients with low expression of these lncRNAs had worse prognosis. Significant lower expressions of these lncRNAs were also observed in CESC cell line compared with normal cell line. Conclusion: Low expressions of CADM3-AS1, LINC00092, and ZNF667-AS1 in ceRNA network were probably promising poor prognostic biomarkers for CESC patients. The genes show a prospective research area for CESC-targeted treatment in the future.

Glutamine inhibits inflammation, oxidative stress, and apoptosis and ameliorates hyperoxic lung injury.

Glutamine (Gln) is the most widely acting and abundant amino acid in the body and has anti-inflammatory properties, regulates body metabolism, and improves immune function. However, the mechanism of Gln's effect on hyperoxic lung injury in neonatal rats is unclear. Therefore, this work focused on examining Gln's function in lung injury of newborn rats mediated by hyperoxia and the underlying mechanism. We examined body mass and ratio of wet-to-dry lung tissue weights of neonatal rats. Hematoxylin and eosin (HE) staining was performed to examine histopathological alterations of lung tissues. In addition, enzyme-linked immunoassay (ELISA) was conducted to measure pro-inflammatory cytokine levels within bronchoalveolar lavage fluid (BALF). Apoptosis of lung tissues was observed using TUNEL assay. Western blotting was performed for detecting endoplasmic reticulum stress (ERS)-associated protein levels. The results showed that Gln promoted body weight gain, significantly reduced pathological damage and oxidative stress in lung tissue, and improved lung function in neonatal rats. Gln reduced pro-inflammatory cytokine release as well as inflammatory cell production in BALF and inhibited apoptosis in lung tissue cells. Furthermore, we found that Gln could downregulate ERS-associated protein levels (GRP78, Caspase-12, CHOP) and inhibit c-Jun N-terminal kinase (JNK) and inositol-requiring enzyme 1 alpha (IRE1α) phosphorylation. These results in an animal model of bronchopulmonary dysplasia (BPD) suggest that Gln may have a therapeutic effect on BPD by reducing lung inflammation, oxidative stress, and apoptosis and improving lung function; its mechanism of action may be related to the inhibition of the IRE1α/JNK pathway.

A novel thermoacidophilic and thermostable endo-ß-1,4-glucanase from Phialophora sp. G5: its thermostability influenced by a distinct ß-sheet and the carbohydrate-binding module.

An endo-ß-1,4-glucanase gene, egG5, was cloned from the fungus Phialophora sp. G5. The 1,290-bp open reading frame encodes a bimodular cellulase composed of an N-terminal family 1 carbohydrate-binding module (CBM) and a C-terminal family 5 glycoside hydrolase catalytic module. Recombinant EgG5 produced in Pichia pastoris exhibited maximal activity at pH 4.0-5.0 and 70 °C, retained 40% of the maximal activity at pH 2.0, and was stable at pH 2.0-10.0. When compared with its closest homolog in Trichoderma sp. C-4 (70.6% identity), EgG5 had better thermostability (51.6% activity at 65 °C for 12 h vs 10% activity at 60 °C for 20 min). Sequence-structure analysis indicated that the distinct ß-sheet in EgG5 in place of a linking loop in Trichoderma sp. C-4 endoglucanase might be the reason. To verify its function, two mutants, EgG5-Mut (disrupting the ß-sheet with four amino acid substitutions) and EgG5-CBM (removing the CBM), were constructed, expressed in P. pastoris, and characterized. Both mutants had similar pH optima (pH 4.0) and temperature optima (70 °C) but varied in pH stabilities (pH 2.0-10.0 and pH 2.0-7.0, respectively) and thermostabilities. The thermostability of EgG5-Mut (13.4% activity vs 52.5% of EgG5 at 65 °C for 12 h) confirmed the effect of ß-sheet on enzyme thermostability. EgG5-CBM was more thermostable (94.9% activity at 65 °C for 12 h and 15.5% activity at 80 °C for 30 min) and had higher specific activity (711.6 vs 60.3 U mg(-1) of EgG5). This study presents an excellent endoglucanase with potential use in the bioconversion of lignocellulosic materials and provides good ideas for the improvement of enzyme thermostability.

High-level expression of a novel Penicillium endo-1,3(4)-ß-D-glucanase with high specific activity in Pichia pastoris.

A novel endo-1,3(4)-ß-D-glucanase gene (bgl16C1) from Penicillium pinophilum C1 was cloned and sequenced. The 945-bp full-length gene encoded a 315-residue polypeptide consisting of a putative signal peptide of 18 residues and a catalytic domain belonging to glycosyl hydrolase family 16. The deduced amino acid sequence showed the highest identity (82%) with the putative endo-1,3(4)-ß-glucanase from Talaromyces stipitatus ATCC 10500 and 60% identity with the characterized ß-1,3(4)-glucanase from Paecilomyces sp. FLH30. The gene was successfully overexpressed in Pichia pastoris. Recombinant Bgl16C1 constituted 95% of total secreted proteins (2.61 g l⁻¹) with activity of 28,721 U ml⁻¹ in a 15-l fermentor. The purified recombinant Bgl16C1 had higher specific activity toward barley ß-glucan (12,622 U mg⁻¹) than all known glucanases and also showed activity against lichenan and laminarin. The enzyme was optimally active at pH 5.0 and 55°C and exhibited good stability over a broad acid and alkaline pH range (>85% activity at pH 3.0-7.0 and even 30% at pH 11.0). All these favorable enzymatic properties make it attractive for potential applications in various industries.

A new xylanase from Streptomyces megasporus DSM 41476 with high yield of xylobiose.

A new xylanase gene, xynBM4, was cloned from Streptomyces megasporus DSM 41476 and expressed in Pichia pastoris. The full-length gene consists of 1,443 bp and encodes 480 amino acids including a putative 49-residue signal peptide. The deduced amino acid sequence of xynBM4 shows the highest identity of 66.3% to the xylanase Xys1L from Streptomyces halstedii JM8. The purified recombinant XYNBM4 had a high specific activity of 350.7 U mg(-1) towards soluble wheat arabinoxylan, exhibited optimal activity at pH 6.0 and 57°C, showed broad pH adaptability (>75% of the maximum activity at pH 2.5-9.0), was resistant to neutral proteases and most chemicals, and produced simple products. The hydrolysis products of birchwood xylan and corncob xylan were predominantly xylobiose (76.9 and 90.8%, respectively) and no xylose. These characteristics suggest that XYNBM4 has potential in various applications, especially in the food industry.

Molecular and biochemical characterization of a new alkaline ß-propeller phytase from the insect symbiotic bacterium Janthinobacterium sp. TN115.

A phytase-encoding gene (phyA115) was cloned from Janthinobacterium sp. TN115, a symbiotic bacterial strain isolated from the gut contents of Batocera horsfieldi larvae (Coleoptera: Cerambycidae), and expressed in Escherichia coli. The 1,884-bp full-length gene encodes a 28-residue putative signal peptide and a 599-residue mature protein with a calculated mass of 64 kDa. The deduced PhyA115 shares low identity with known sequences (47% at most) and contains an N-terminal incomplete domain (residues 29-297; domain N) and a typical ß-propeller phytase domain at the C terminus (residues 298-627; domain C). Distinct from other ß-propeller phytases that have neutral pH optima (pH 6.0-7.5), purified recombinant PhyA115 exhibits maximal activity at pH 8.5 and 45°C in the presence of 1 mM Ca(2+) and is highly active over a wider pH range (pH 6.0-9.0). These results indicate that PhyA115 is a ß-propeller phytase that has application potential in aquaculture feed. To our knowledge, this is the first report of cloning of a phytase gene from the symbiotic microbes of an insect digestive tract and from the genus Janthinobacterium. The N-terminal incomplete domain is found to have no phytase activity but can influence the pH property of PhyA115.

A novel low-temperature-active ß-glucosidase from symbiotic Serratia sp. TN49 reveals four essential positions for substrate accommodation.

A 2,373-bp full-length gene (bglA49) encoding a 790-residue polypeptide (BglA49) with a calculated mass of 87.8 kDa was cloned from Serratia sp. TN49, a symbiotic bacterium isolated from the gut of longhorned beetle (Batocera horsfieldi) larvae. The deduced amino acid sequence of BglA49 showed the highest identities of 80.1% with a conceptually translated protein from Pantoea sp. At-9b (EEW02556), 38.3% with the identified glycoside hydrolase (GH) family 3 ß-glucosidase from Clostridium stercorarium NCBI 11754 (CAB08072), and <15.0% with the low-temperature-active GH 3 ß-glucosidases from Shewanella sp. G5 (ABL09836) and Paenibacillus sp. C7 (AAX35883). The recombinant enzyme (r-BglA49) was expressed in Escherichia coli and displayed the typical characteristics of low-temperature-active enzymes, such as low temperature optimum (showing apparent optimal activity at 35°C), activity at low temperatures (retaining approximately 60% of its maximum activity at 20°C and approximately 25% at 10°C). Compared with the thermophilic GH 3 ß-glucosidase, r-BglA49 had fewer hydrogen bonds and salt bridges and less proline residues. These features might relate to the increased structure flexibility and higher catalytic activity at low temperatures of r-BglA49. The molecular docking study of four GH 3 ß-glucosidases revealed five conserved positions contributing to substrate accommodation, among which four positions of r-BglA49 (R192, Y228, D260, and E449) were identified to be essential based on site-directed mutagenesis analysis.

An acid and highly thermostable xylanase from Phialophora sp. G5.

An endo-ß-1,4-xylanase gene, designated xyn10G5, was cloned from Phialophora sp. G5 and expressed in Pichia pastoris. The 1,197-bp full-length gene encodes a polypeptide of 399 amino acids consisting of a putative signal peptide at residues 1-20, a family 10 glycoside hydrolase domain, a short Gly/Thr-rich linker and a family 1 carbohydrate-binding module (CBM). The deduced amino acid sequence of XYN10G5 shares the highest identity (53.4%) with a putative xylanase precursor from Aspergillus terreus NIH2624. The purified recombinant XYN10G5 exhibited the optimal activity at pH 4.0 and 70 °C, remained stable at pH 3.0-9.0 (>70% of the maximal activity), and was highly thermostable at 70 °C (retaining ~90% of the initial activity for 1 h). Substrate specificity studies have shown that XYN10G5 had the highest activity on soluble wheat arabinoxylan (350.6 U mg(-1)), and moderate activity to various heteroxylans, and low activity on different types of cellulosic substrates. Under simulated gastric conditions, XYN10G5 was stable and released more reducing sugars from soluble wheat arabinoxylan; when combined with a glucanase (CelA4), the viscosity of barley-soybean feed was significantly reduced. These favorable enzymatic properties make XYN10G5 a good candidate for application in the animal feed industry.

RNA-Seq of the xylose-fermenting yeast Scheffersomyces stipitis cultivated in glucose or xylose.

Xylose is the second most abundant lignocellulosic component besides glucose, but it cannot be fermented by the widely used ethanol-producing yeast Saccharomyces cerevisiae. The yeast Scheffersomyces stipitis, however, is well known for its high native capacity to ferment xylose. Here, we applied next-generation sequencing technology for RNA (RNA-Seq) to generate two high-resolution transcriptional maps of the S. stipitis genome when this yeast was grown using glucose or xylose as the sole carbon source. RNA-Seq revealed that 5,176 of 5,816 annotated open reading frames had a uniform transcription and that 214 open reading frames were differentially transcribed. Differential expression analysis showed that, compared with other biological processes, carbohydrate metabolism and oxidation-reduction reactions were highly enhanced in yeast grown on xylose. Measurement of metabolic indicators of fermentation showed that, in yeast grown on xylose, the concentrations of cysteine and ornithine were twofold higher and the concentrations of unsaturated fatty acids were also increased. Analysis of metabolic profiles coincided with analysis of certain differentially expressed genes involved in metabolisms of amino acid and fatty acid. In addition, we predicted protein-protein interactions of S. stipitis through integration of gene orthology and gene expression. Further analysis of metabolic and protein-protein interactions networks through integration of transcriptional and metabolic profiles predicted correlations of genes involved in glycolysis, the tricarboxylic acid cycle, gluconeogenesis, sugar uptake, amino acid metabolism, and fatty acid ß-oxidation. Our study reveals potential target genes for xylose fermentation improvement and provides insights into the mechanisms underlying xylose fermentation in S. stipitis.

Catalytic efficiency of HAP phytases is determined by a key residue in close proximity to the active site.

The maximum activity of Yersinia enterocolitica phytase (YeAPPA) occurs at pH 5.0 and 45 °C, and notably, its specific activity (3.28 ± 0.24 U mg(-1)) is 800-fold less than that of its Yersinia kristeensenii homolog (YkAPPA; 88% amino acid sequence identity). Sequence alignment and molecular modeling show that the arginine at position 79 (Arg79) in YeAPPA corresponding to Gly in YkAPPA as well as other histidine acid phosphatase (HAP) phytases is the only non-conserved residue near the catalytic site. To characterize the effects of the corresponding residue on the specific activities of HAP phytases, Escherichia coli EcAPPA, a well-characterized phytase with a known crystal structure, was selected for mutagenesis-its Gly73 was replaced with Arg, Asp, Glu, Ser, Thr, Leu, or Tyr. The results show that the specific activities of all of the corresponding EcAPPA mutants (17-2,400 U mg(-1)) were less than that of the wild-type phytase (3,524 U mg(-1)), and the activity levels were approximately proportional to the molecular volumes of the substituted residues' side chains. Site-directed replacement of Arg79 in YeAPPA (corresponding to Gly73 of EcAPPA) with Ser, Leu, and Gly largely increased the specific activity, which further verified the key role of the residue at position 79 for determining phytase activity. Thus, a new determinant that influences the catalytic efficiency of HAP phytases has been identified.

Two types of phytases (histidine acid phytase and ß-propeller phytase) in Serratia sp. TN49 from the gut of Batocera horsfieldi (coleoptera) larvae.

Microbial phytases play a major role in the mineralization of organic phosphorous, especially in symbiotic plants and animals. In this study, we identified two types of phytases in Serratia sp. TN49 that was harbored in the gut of Batocera horsfieldi (Coleoptera) larvae. The two phytases, an acidic histidine acid phosphatase (PhyH49) and an alkaline ß-propeller phytase (PhyB49), shared low identities with known phytases (61% at most). PhyH49 and PhyB49 produced in Escherichia coli exhibited maximal activities at pH 5.0 (60°C) and pH 7.5-8.0 (45°C), respectively, and are complementary in phytate degradation over the pH range 2.0-9.0. Serratia sp. TN49 harboring both PhyH49 and PhyB49 might make it more adaptive to environment change, corresponding to the evolution trend of microorganism.

Genetic and biochemical characterization of a protease-resistant mesophilic ß-mannanase from Streptomyces sp. S27.

A ß-mannanase gene, designated as man5S27, was cloned from Streptomyces sp. S27 using the colony polymerase chain reaction (PCR) method and expressed in Escherichia coli BL21 (DE3). The open reading frame consisted of 1,161 bp and encoded a 386-amino-acid polypeptide (Man5S27) with calculated molecular mass of 37.2 kDa. The encoded protein comprised a putative 38-residue signal peptide, a family 5 glycoside hydrolase domain, and a family 10 carbohydrate-binding module. Purified recombinant Man5S27 had high specific activity of 2,107 U mg⁻¹ and showed optimal activity at pH 7.0 and 65 °C. The enzyme remained stable at pH 5.0-9.0 and had good thermostability at 50°C. The K (m) values for locust bean gum and konjac flour were 0.16 and 0.41 mg ml⁻¹, with V(max) values of 3,739 and 1,653 µmol min⁻¹ mg⁻¹, respectively. Divalent metal ions such as Mn²+, Zn²+, Ca²+, Pb²+, and Fe²+ enhanced the enzyme activity, but Ag+ and Hg²+ strongly inhibited the activity. Man5S27 also showed resistance to various neutral proteases (retaining >95% activity after proteolytic treatment for 2 h).

TGM4: an immunogenic prostate-restricted antigen.

BACKGROUND: Prostate cancer is the second leading cause of cancer-related death in men in the USA; death occurs when patients progress to metastatic castration-resistant prostate cancer (CRPC). Although immunotherapy with the Food and Drug Administration-approved vaccine sipuleucel-T, which targets prostatic acid phosphatase (PAP), extends survival for 2-4 months, the identification of new immunogenic tumor-associated antigens (TAAs) continues to be an unmet need. METHODS: We evaluated the differential expression profile of castration-resistant prostate epithelial cells that give rise to CRPC from mice following an androgen deprivation/repletion cycle. The expression levels of a set of androgen-responsive genes were further evaluated in prostate, brain, colon, liver, lung, skin, kidney, and salivary gland from murine and human databases. The expression of a novel prostate-restricted TAA was then validated by immunostaining of mouse tissues and analyzed in primary tumors across all human cancer types in The Cancer Genome Atlas. Finally, the immunogenicity of this TAA was evaluated in vitro and in vivo using autologous coculture assays with cells from healthy donors as well as by measuring antigen-specific antibodies in sera from patients with prostate cancer (PCa) from a neoadjuvant clinical trial. RESULTS: We identified a set of androgen-responsive genes that could serve as potential TAAs for PCa. In particular, we found transglutaminase 4 (Tgm4) to be highly expressed in prostate tumors that originate from luminal epithelial cells and only expressed at low levels in most extraprostatic tissues evaluated. Furthermore, elevated levels of TGM4 expression in primary PCa tumors correlated with unfavorable prognosis in patients. In vitro and in vivo assays confirmed the immunogenicity of TGM4. We found that activated proinflammatory effector memory CD8 and CD4 T cells were expanded by monocyte-derived dendritic cell (moDCs) pulsed with TGM4 to a greater extent than moDCs pulsed with either PAP or prostate-specific antigen (PSA), and T cells primed with TGM4-pulsed moDCs produce functional cytokines following a prime/boost regiment or in vitro stimulation. An IgG antibody response to TGM4 was detected in 30% of vaccinated patients, while fewer than 8% of vaccinated patients developed antibody responses to PSA or prostate-specific membrane antigen (PSMA). CONCLUSIONS: These results suggest that TGM4 is an immunogenic, prostate-restricted antigen with the potential for further development as an immunotherapy target.