Identification and structural analysis of a carbohydrate-binding module specific to alginate, a representative of a new family, CBM96.

Carbohydrate-binding modules (CBMs) are the noncatalytic modules that assist functions of the catalytic modules in carbohydrate-active enzymes, and they are usually discrete structural domains in larger multimodular enzymes. CBMs often occur in tandem in different alginate lyases belonging to the CBM families 13, 16, and 32. However, none of the currently known CBMs in alginate lyases specifically bind to an internal alginate chain. In our investigation of the multidomain alginate lyase Dp0100 carrying several ancillary domains, we identified an alginate-binding domain denoted TM6-N4 using protein truncation analysis. The structure of this CBM domain was determined at 1.35 Å resolution. TM6-N4 exhibited an overall ß-sandwich fold architecture with two antiparallel ß-sheets. We identified an extended binding groove in the CBM using site-directed mutagenesis, docking, and surface electrostatic potential analysis. Affinity analysis revealed that residues of Lys10, Lys22, Lys25, Lys27, Lys31, Arg36, and Tyr159 located on the bottom or the wall of the shallow groove are responsible for alginate binding, and isothermal titration calorimetry analyses indicated that the binding cleft consists of six subsites for sugar recognition. This substrate binding pattern is typical for type B CBM, and it represents the first CBM domain that specifically binds internal alginate chain. Phylogenetic analysis supports that TM6-N4 constitutes the founding member of a new CBM family denoted as CBM96. Our reported structure not only facilitates the investigation of the CBM-alginate ligand recognition mechanism but also inspires the utilization of the CBM domain in biotechnical applications.

The molecular basis of endolytic activity of a multidomain alginate lyase from Defluviitalea phaphyphila, a representative of a new lyase family, PL39.

Alginate is a polymer containing two uronic acid epimers, ß-d-mannuronate (M) and α-l-guluronate (G), and is a major component of brown seaweed that is depolymerized by alginate lyases. These enzymes have diverse specificity, cleaving the chain with endo- or exotype activity and with differential selectivity for the sequence of M or G at the cleavage site. Dp0100 is a 201-kDa multimodular, broad-specificity endotype alginate lyase from the marine thermophile Defluviitalea phaphyphila, which uses brown algae as a carbon source, converting it to ethanol, and bioinformatics analysis suggested that its catalytic domain represents a new polysaccharide lyase family, PL39. The structure of the Dp0100 catalytic domain, determined at 2.07 Å resolution, revealed that it comprises three regions strongly resembling those of the exotype lyase families PL15 and PL17. The conservation of key catalytic histidine and tyrosine residues belonging to the latter suggests these enzymes share mechanistic similarities. A complex of Dp0100 with a pentasaccharide, M5, showed that the oligosaccharide is located in subsites -2, -1, +1, +2, and +3 in a long, deep canyon open at both ends, explaining the endotype activity of this lyase. This contrasted with the hindered binding sites of the exotype enzymes, which are blocked such that only one sugar moiety can be accommodated at the -1 position in the catalytic site. The biochemical and structural analyses of Dp0100, the first for this new class of endotype alginate lyases, have furthered our understanding of the structure-function and evolutionary relationships within this important class of enzymes.

mTOR activation is critical for betulin treatment in renal cell carcinoma cells.

Betulin, a natural product isolated from the bark of the birch trees, exhibits multiple anticancer effects. Activation of mTOR signaling pathway has been found in numerous cancers, including renal cell carcinoma (RCC). Here, we attempted to study whether mTOR signaling was essential for betulin to treat RCC. Based on cell survival and colony formation assays, we found that mTOR hyperactive RCC cell line 786-O cells were more sensitive to betulin treatment compared with mTOR-inactive Caki-2 cells. Knockdown of TSC2 in Caki-2 cells had similar results to 786-O cells, and mTOR silencing in 786-O cells rescued the inhibitory effect of betulin, indicating that betulin inhibited RCC cell proliferation in an mTOR-dependent manner. Furthermore, betulin treatment decreases the levels of glucose consumption and lactate production in 786-O cells, while minimal effects were observed in Caki-2 cells. In addition, betulin significantly inhibited the expression of PKM2 and HK2 in 786-O cells. Finally, knockdown of PKM2 or HK2 in 786-O reversed the anti-proliferative effects of betulin, and overexpression of PKM2 or HK2 in Caki-2 cells enhanced the sensitivity to betulin treatment. Taken together, these findings demonstrated the critical role of mTOR activation in RCC cells to betulin treatment, suggesting that betulin might be valuable for targeted therapies in RCC patients with mTOR activation.

Defluviitalea phaphyphila sp. nov., a Novel Thermophilic Bacterium That Degrades Brown Algae.

Brown algae are one of the largest groups of oceanic primary producers for CO2 removal and carbon sinks for coastal regions. However, the mechanism for brown alga assimilation remains largely unknown in thermophilic microorganisms. In this work, a thermophilic alginolytic community was enriched from coastal sediment, from which an obligate anaerobic and thermophilic bacterial strain, designated Alg1, was isolated. Alg1 shared a 16S rRNA gene identity of 94.6% with Defluviitalea saccharophila LIND6LT2(T). Phenotypic, chemotaxonomic, and phylogenetic studies suggested strain Alg1 represented a novel species of the genus Defluviitalea, for which the name Defluviitalea phaphyphila sp. nov. is proposed. Alg1 exhibited an intriguing ability to convert carbohydrates of brown algae, including alginate, laminarin, and mannitol, to ethanol and acetic acid. Three gene clusters participating in this process were predicted to be in the genome, and candidate enzymes were successfully expressed, purified, and characterized. Six alginate lyases were demonstrated to synergistically deconstruct alginate into unsaturated monosaccharide, followed by one uronic acid reductase and two 2-keto-3-deoxy-d-gluconate (KDG) kinases to produce pyruvate. A nonclassical mannitol 1-phosphate dehydrogenase, catalyzing D-mannitol 1-phosphate to fructose 1-phosphate in the presence of NAD(+), and one laminarase also were disclosed. This work revealed that a thermophilic brown alga-decomposing system containing numerous novel thermophilic alginate lyases and a unique mannitol 1-phosphate dehydrogenase was adopted by the natural ethanologenic strain Alg1 during the process of evolution in hostile habitats.

Characterization of a thermostable endo-1,3(4)-ß-glucanase from Caldicellulosiruptor sp. strain F32 and its application for yeast lysis.

ß-1,3-Glucans, important structural components of cell wall or nutritional components of the endosperm, are extensively found in bacteria, fungi, yeast, algae, and plants. The structural complexity of ß-1,3-glucans implies that the enzymatic depolymerization of polysaccharides needs combined activities of distinct enzymes. In this study, Lam16A-GH, the catalytic module of a putative glycoside hydrolase (GH) family 16 laminarinase/lichenase from thermophilic bacterium Caldicellulosiruptor sp. F32, was purified and characterized through heterologous expression in Escherichia coli. Lam16A-GH can hydrolyze both ß-1,3-glucan (laminarin) and ß-1,3-1,4-glucan (barley ß-glucan) revealed by analysis of the products of polysaccharide degradation using thin-layer chromatography (TLC). The time required for the loss of 50 % of its activity is 45 h under the optimal condition of 75 °C and pH 6.5. Oligosaccharides degradation assay indicated that Lam16A-GH can catalyze endo-hydrolysis of the ß-1,4 glycosidic linkage adjacent to a 3-O-substituted glucosyl residue in the mixed linked ß-glucans, as well as the ß-1,3 linkage. The survival rate of Saccharomyces cerevisiae cells depends on the addition of Lam16A-GH, and the cytoplasm protein was released from the apparently deconstructed yeast cells. These results indicate that the bi-functional thermostable Lam16A-GH exhibits unique enzymatic properties and potential for yeast lysis.

Brassicibacter thermophilus sp. nov., a thermophilic bacterium isolated from coastal sediment.

A novel thermophilic, obligately anaerobic bacterium, strain Cel2f(T), was isolated from a cellulolytic community enriched from coastal marine sediment. Cells were Gram-stain-negative, non-motile, non-spore-forming and rod-shaped. Optimal growth temperature and pH of strain Cel2f(T) were 55 °C and pH 7.0, respectively. NaCl was essential for the growth of strain Cel2f(T) and the strain showed enhanced growth in the presence of sea salt; the optimum sea salt concentration for growth was 7% (w/v). Thiosulfate, sulfate and sulfite were potential electron acceptors. The major fatty acids of strain Cel2f(T) were iso-C15 : 0, C16 : 0, and C18 : 0. Polar lipid analysis indicated the presence of phosphatidylethanolamine and phosphatidylglycerol. Strain Cel2f(T) contained menaquinone MK-7 as the isoprenoid quinone, and the DNA G+C content was 31.3 mol%. Phylogenetic analysis revealed that the nearest relative of strain Cel2f(T) was Brassicibacter mesophilus BM(T) with 93.8% 16S rRNA gene sequence similarity. Based on phenotypic, chemotaxonomic and phylogenetic characteristics, strain Cel2f(T) represents a novel species of genus Brassicibacter, for which the name Brassicibacter thermophilus sp. nov. is proposed. The type strain is Cel2f(T) ( = JCM 30480(T) = CGMCC 1.5200(T)).

Dysgonomonas macrotermitis sp. nov., isolated from the hindgut of a fungus-growing termite.

A Gram-stain-negative, facultatively anaerobic, non-motile and coccoid- to short-rod-shaped bacterium, designated strain Dys-CH1(T), was isolated from the hindgut of a fungus-growing termite Macrotermes barneyi. The optimal pH and cultivation temperature of strain Dys-CH1(T) were pH 7.2-7.6 and 35-37 °C, respectively. Sequence analysis of 16S rRNA gene showed that Dys-CH1(T) shared 94.6 % and 90.9 % similarity with Dysgonomonas capnocytophagoides JCM 16697(T) and Dysgonomonas gadei CCUG 42882(T), respectively. Strain Dys-CH1(T) was found to be different from other species of the genus Dysgonomonas with validly published names with respect to taxonomically important traits, including habitat, biochemical tests, DNA G+C content, bile resistance, fatty-acid composition and susceptibility to antimicrobial agents. On the basis of these characteristics, strain Dys-CH1(T) represents a novel species of the genus Dysgonomonas for which the name Dysgonomonas macrotermitis sp. nov. is proposed. The type strain is Dys-CH1(T) ( = JCM 19375(T) = DSM 27370(T)).

Ferrimonas sediminum sp. nov., isolated from coastal sediment of an amphioxus breeding zone.

A Gram-stain-negative, facultatively anaerobic rod with a polar flagellum, designated strain JYr13(T), was isolated from coastal sediment of an amphioxus breeding zone located in Qingdao, China. The isolate utilized Fe(III) oxyhydroxide, Fe(III) citrate, SeO3(2-) and SeO4(2-) as electron acceptors. Strain JYr13(T) grew optimally at 25-28 °C, pH 7.0-8.0 and in presence of 3 % (w/v) NaCl. Strain JYr13(T) contained menaquinone-7 and ubiquinones Q-7 and Q-8 as the predominant isoprenoid quinones, and C18 : 1ω9c, iso-C15 : 0, C17 : 1ω8c and C16 : 0 as the major fatty acids. The DNA G+C content of strain JYr13(T) was 59 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that the closest phylogenetic neighbour of strain JYr13(T) was Ferrimonas kyonanensis DSM 18153(T) (96.0 % 16S rRNA gene sequence similarity). Based on the phenotypic, chemotaxonomic and phylogenetic distinctiveness, strain JYr13(T) is considered to represent a novel species of the genus Ferrimonas, for which the name Ferrimonas sediminum sp. nov. is proposed. The type strain is JYr13(T) ( = DSM 23317(T) = LMG 25564(T)).

Roseovarius marisflavi sp. nov., isolated from an amphioxus breeding zone in the coastal region of the Yellow Sea, China.

A novel Gram stain-negative, catalase- and oxidase-positive, strictly aerobic bacterium, designated strain H50(T), was isolated from an amphioxus breeding zone in the coastal region of the Yellow Sea, China. Cells were observed to be ovoid or short rods, lacked flagella and were found to contain bacteriochlorophyll a. Poly-beta-hydroxybutyrate was found to be accumulated. The temperature range for growth was determined to be 0-37 °C (optimum 28-37 °C). The halotolerance range for growth is 1-15 % NaCl (optimum 2-7 %). The pH range for growth is 6.0-8.0 (optimum 7.0). The major fatty acids were identified as C18:1ω7c and C16:0. The following polar lipids were found to be present: diphosphatidylglycerol, phosphatidylglycerol and a lipid. The predominant respiratory quinone was determined to be Q-10. DNA G+C content was determined to be 57.7 mol%. Strain H50(T) exhibited the highest 16S rRNA gene sequence similarity to Pelagicola litoralis DSM 18290(T) (96.1 %), Roseovarius mucosus DSM 17069(T) (95.8 %) and Roseovarius tolerans DSM 11457(T) (95.7 %). In the phylogenetic trees, strain H50(T) was clustered with the genus Roseovarius but not Pelagicola. On the basis of phenotypic, chemotaxonomic and genotypic data, strain H50(T) is considered to represent a novel species in the genus Roseovarius, for which the name Roseovarius marisflavi sp. nov. is proposed. The type strain is H50(T) (=CGMCC 1.10799(T)=JCM 17553(T)).

Litoribacillus peritrichatus gen. nov. sp. nov., isolated from coastal sediment of an amphioxus breeding zone in Qingdao, China.

A Gram-negative, strictly aerobic, motile, short rod-shaped bacterium with peritrichous flagella, designated JYr12(T), was isolated from sediment of an amphioxus breeding zone in the coastal region of Qingdao, China. Strain JYr12(T) was found to grow optimally at 28 °C, at pH 7.0 and in the presence of 2-3 % (w/v) NaCl. The organism was determined to contain C(16:0) (28.3 %) and C(16:1) ω7c and/or C(16:1) ω6c (34.8 %) as the major fatty acids and ubiquinone-8 (90 %) as the predominant isoprenoid ubiquinone. The polar lipids were found to consist of phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol and an unknown glycolipid. The G+C content of the genomic DNA was determined to be 42.3 mol %. Comparative 16S rRNA gene sequence analysis revealed that strain JYr12(T) was placed in the class Gammaproteobacteria and shared ~91.20, 91.18, 91.01, 90.95, 90.80 and 90.40 % sequence similarities with representatives of the genus Thalassolituus, Nitrincola, Oceanospirillum, Marinobacterium, Marinomonas and Microbulbifer, respectively. On the basis of phenotypic, chemotaxonomic and genotypic data, strain JYr12(T) is considered to represent a novel species in a new genus within the Gammaproteobacteria, for which the name Litoribacillus peritrichatus gen. nov., sp. nov. is proposed. The type strain of Litoribacillus peritrichatus is JYr12(T) (=CGMCC 1.10796(T) = JCM17551(T)).

Litorilituus sediminis gen. nov. sp. nov., isolated from coastal sediment of an amphioxus breeding zone in Qingdao, China.

A Gram-negative, non-spore-forming, catalase- and oxidase-positive, aerobic, curved-rod shaped bacterium with polar or subpolar flagellum, designated strain JYr2(T), was isolated from a sediment sample collected from an amphioxus breeding zone in the coastal region of Qingdao, China. The organism grew optimally at 37 °C, pH 8-9 and in the presence of 3 % (w/v) NaCl or 3-4 % sea salts (w/v; Sigma). Salt was required for growth. The strain contained isoprenoid quinone 8 (Q-8, 100 %) as the predominant isoprenoid quinone and C16:0 (24.1 %) and C16:1 ω7c and/or C16:1 ω6c (35.8 %) as major fatty acids. Phosphatidylethanolamine, phosphatidylglycerol, aminophospholipid (PN) and two aminolipid (AL1, AL2) were the major constituents of the phospholipids. A phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JYr2(T) formed a distinct evolutionary lineage within the family Colwelliaceae. It showed <95.0 % sequence similarities to all validly published species in the family Colwelliaceae, except 95.2 % to Thalassomonas viridans DSM 13754(T) and 95.0 % to Colwellia polaris JCM 537(T). The G+C content of the DNA was 42.7 mol%. On the basis of the polyphasic taxonomic study, strain JYr2(T) (= CGMCC 1.10794(T) = JCM 17549(T)) was considered to represent a novel genus and species in Gammaproteobacteria, for which the name Litorilituus sediminis gen. nov. sp. nov. was proposed.

Hyperthermia synergizes with tissue factor knockdown to suppress the growth and hepatic metastasis of colorectal cancer in orthotopic tumor model.

BACKGROUND: Tissue factor (TF) is a significant risk factor for tumor growth and hepatic metastasis in patients with colorectal cancer (CRC). This study aimed to investigate whether hyperthermia has synergistic anti-tumor effects with TF knockdown in suppressing CRC progression and metastasis in vitro and in vivo. METHODS: Human colorectal cancer LOVO cells were treated by hyperthermia at 44°C for 2 hr or/and TF siRNA. Then the cells were subjected to colony formation assay. Apoptosis was analyzed by flow cytometry, confocal microscopy, and transmission electron microscopy. The cell migration and invasion abilities were analyzed by wound healing and matrigel assay. In addition, orthotopic nude mice model of CRC was established. RESULTS: Hyperthermia synergized with TF knockdown to reduce colony formation ability, induce apoptosis, and suppress the migration and invasion of LOVO cells in vitro. Moreover, hyperthermia in combination with TF depletion inhibited the growth and hepatic metastasis of CRC in orthotopic nude mice model. Mechanistically, the synergistic effects were at least partly mediated by inducing JNK mediated apoptosis and suppressing matrix metalloproteinases (MMPs) mediated invasion. CONCLUSIONS: Hyperthermia in combination with TF-targeted therapy could be a potential approach for CRC treatment.

A-kinase interacting protein 1 regulates the cell proliferation, invasion, migration and angiogenesis of clear cell renal cell carcinoma cells and affects the ERK/c-Myc signaling pathway by binding to Rac1.

A-kinase interacting protein 1 (AKIP1) has previously been demonstrated to be overexpressed in clear cell renal cell carcinoma (ccRCC) tissues and is associated with patient prognosis. The aim of the present study was to explore whether AKIP1 can affect the proliferation, invasion, migration and angiogenesis of ccRCC cells via its interaction with Rac1. Furthermore, the influence of AKIP1 and therefore Rac1 on the expression of the downstream ERK/cellular (c)-Myc signaling pathway was explored. The interaction between AKIP1 and Rac1 was determined using co-immunoprecipitation. The mRNA and protein expression levels of AKIP1 and Rac1 in normal renal epithelial cell lines and ccRCC cell lines were detected using reverse transcription-quantitative PCR (RT-qPCR) and western blotting, respectively. The transfection efficiency of small interfering RNA-AKIP1 and the Rac1 overexpression vector were also confirmed using RT-qPCR and western blotting. The viability, proliferation, invasion and migration of ccRCC cells following transfection were analyzed using the Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine staining, Transwell and wound healing assays, respectively. The tube formation ability of HUVECs was assessed using the tube formation assay. The protein expression levels of proliferation, invasion, migration and tube-formation-associated proteins as well as proteins associated with the ERK/c-Myc signaling pathway, were detected via western blotting. The results demonstrated that AKIP1 expression levels were increased in ccRCC cell lines. AKIP1 knockdown inhibited the proliferation, invasion and migration of ccRCC cells and HUVEC tube-formation. In addition, AKIP1 was demonstrated to bind to Rac1 in ccRCC cells and AKIP1 downregulation inhibited Rac1 expression. Furthermore, Rac1 overexpression reversed the effects of AKIP1 knockdown on ccRCC cells. AKIP1 knockdown also suppressed the ERK/c-Myc signaling pathway, which was reversed by Rac1 overexpression. In conclusion, AKIP1 knockdown potentially suppressed the proliferation, invasion, migration and angiogenesis of ccRCC cells and inhibited the ERK/c-Myc signaling pathway by binding to Rac1.

LncRNA PTAR activates the progression of bladder cancer by modulating miR-299-3p/CD164 axis.

Bladder cancer (BC) occurs in the urinary system which has high incidence and mortality. During past decades, lots of long noncoding RNAs (lncRNAs) have been identified to function in cancer progression, including BC. In our research, we targeted at investigating the functions and mechanisms of lncRNA pro-transition associated RNA (PTAR) in BC. Functional assays were implemented to access the changes of BC cell phenotype. Mechanistic assays were applied for confirming the interaction between RNAs. Based on the collected data, PTAR expression was high in BC cells and silenced PTAR repressed BC cell proliferative, migratory and invasive abilities but improved cell apoptotic ability. In vivo study also verified PTAR depletion inhibited BC tumor growth. Furthermore, miR-299-3p was confirmed to bind with PTAR and its overexpression suppressed malignant behaviors of BC cells. Cluster of differentiation 164 (CD164) was proved to be miR-299-3p target. Rescue experiments implied overexpressed CD164 offset the inhibitory function of PTAR depletion on BC cell phenotype. Additionally, CD164 was uncovered to combine with C-X-C motif chemokine receptor 4 (CXCR4) to switch on PI3K/AKT pathway. To conclude, PTAR facilitates BC development via regulating miR-299-3p/CD164 axis and activating PI3K/AKT pathway.

[Clinical study of diethylstilbestrol in hormone refractory prostate cancer].

OBJECTIVE: To summarize the clinical outcome and adverse events of estrogen therapy for hormone refractory prostate cancer. METHODS: A total of 32 patients with hormone refractory prostate cancer received diethylstilbestrol (DES) 2 mg daily at our institute. The data of PSA (prostate-specific antigen) change, time to progression, overall survival rate, disease-specific survival rate and adverse events were collected and analyzed. RESULTS: The data of 29 patients were complete. The mean duration of DES dosing was 8.6 ± 0.9 months. Among them, 8 (27.5%) patients achieved a PSA response with a 50% decrement of PSA or more. Seven (24.1%) patients had a stable level of PSA (50% < PSA < 125%) while 14 of 29 (48.3%) maintained a PSA progression with a 25% increment of PSA or more. The overall median time to progression was 4 (1 - 12) months. And the median time to progression was 6 (5-12) months in the PSA response group. The overall survival rate was 48.3% and disease-specific survival rate 55.2%. The main adverse events were gynecomastia (10/29, 34.5%) and deep vein thrombosis (3/29, 10.3%). CONCLUSION: When used for the treatment of hormone refractory prostate cancer, diethylstilbestrol at a daily dose of 2 mg can achieve a PSA response in 27.5% patients and a PSA stability in 24.1% patients. And the median time to progression is 4 months. Estrogen is efficacious for the patients with hormone refractory prostate cancer.

MicroRNA-372 functions as a tumor suppressor in cell invasion, migration and epithelial-mesenchymal transition by targeting ATAD2 in renal cell carcinoma.

In recent years, renal cell carcinoma (RCC) has exhibited an increasing incidence and mortality rate worldwide. Accumulating evidence has identified that microRNAs (miRNAs) function as negative or positive regulators of many malignant tumors; however, the roles of miR-372 in RCC remain unclear. The focus of the present study was the functions of miR-372 in RCC metastasis and EMT. Data revealed that miR-372 expression levels were significantly downregulated in RCC tissue samples and cells. Moreover, the decreased expression levels were strongly associated with the poor survival rates and adverse clinical characteristics of RCC patients. Accordingly, miR-372 overexpression markedly inhibited RCC cell invasion, migration and EMT. In terms of the potential mechanisms, ATAD2, the expression of which was inversely correlated with miR-372 expression in RCC, was identified as a direct functional target of miR-372. Notably, ATAD2 silence exerted suppressive functions in RCC cells, being similar to the effects of miR-372 overexpression. In conclusion, findings of this study indicate that miR-372 repressed RCC EMT and metastasis via targeting ATAD2, suggesting that the miR-372/ATAD2 axis may be therapeutic biomarkers for RCC.

miR-145-5p inhibits the proliferation and migration of bladder cancer cells by targeting TAGLN2.

MicroRNA-145-5p (miR-145-5p) is found to be involved in tumor development and progression. However, there are few studies on the effects of miR-145-5p on bladder cancer (BC). The role of miR-145-5p in BC was predicted by analysis of cell proliferation and migration in this study. The miR-145-5p and transgelin-2 (TAGLN2) expressions were evaluated via reverse transcription-quantitative PCR (RT-qPCR) or western blot analysis. The MTT and Transwell assay assessed cell proliferation and migration. TAGLN2 targeted to miR-145-5p was determined using luciferase assays. The results showed that the miR-145-5p downregulation was found in BC. miR-145-5p overexpression inhibited cell proliferation and migration in BC. Moreover, miR-145-5p directly targeted TAGLN2, and TAGLN2 expression was increased in BC. In addition, the high expression of TAGLN2 promoted cell proliferation and migration in BC. miR-145-5p appeared to regulate TAGLN2 in BC, and it also inhibited the cell proliferation and migration. The novel miR-145-5p/TAGLN2 axis may provide new therapeutic implications for BC.

Resveratrol-mediated apoptosis in renal cell carcinoma via the p53/AMP­activated protein kinase/mammalian target of rapamycin autophagy signaling pathway.

Resveratrol, known as phytoalexin, is a natural compound. Clinical studies have revealed that resveratrol has a variety of effects including anti­inflammatory, antivirus and tumor suppressor activities. It has been reported that it may serve an important role in renal cell carcinoma (RCC) however, the molecular mechanism underlying resveratrol­induced apoptosis in RCC is still unclear. The aim of the present study was to determine whether resveratrol could suppress RCC progression. Analysis of apoptosis demonstrated that resveratrol may act as a RCC suppressor in a dose­ and time­dependent manner. In addition, the results of the MTT and cell migration experiments revealed that resveratrol significantly decreased cell viability and migration. In addition, the expression of the anti­apoptosis gene B­cell lymphoma 2 (Bcl­2) was downregulated by resveratrol, and the expression of pro­apoptosis gene Bcl­2­associated X was upregulated at the mRNA and protein levels. Resveratrol also promoted the expression of p53 and activated phospho­AMP­activated protein kinase (AMPK). The phosphorylation of mammalian target of rapamycin (mTOR) was inhibited and the autophagy­associated genes, light chain 3, autophagy related (ATG)5 and ATG7, were upregulated at the mRNA and protein levels. In conclusion, resveratrol suppressed RCC viability and migration, and promoted RCC apoptosis via the p53/AMPK/mTOR­induced autophagy signaling pathway.

Substitution of one calcium-binding amino acid strengthens substrate binding in a thermophilic alginate lyase.

Ligand binding is sensitive to temperatures since noncovalent bonds between the binding site and ligand could be broken by heat. How metal ion-binding amino acids in alginate lyase evolve to achieve tight substrate binding in a hostile environment remains unknown. An endolytic alginate lyase AlgAT0 specifically cleaved the M-G glycosidic bond and released disaccharides as the main end product. Four conserved calcium-binding sites were predicted and the supplement of Ca2+ led to enhanced substrate binding and protein stability. Among the four conserved calcium-binding sites, one substitution of aspartate for glutamate in AlgAT0 was proved to stimulate Ca2+ affinity. This study suggested that substrate affinity of polysaccharide lyases could be improved by tight binding to Ca2+ via one amino acid substitution.

Direct bioconversion of brown algae into ethanol by thermophilic bacterium Defluviitalea phaphyphila.

BACKGROUND: Brown algae are promising feedstocks for biofuel production with inherent advantages of no structural lignin, high growth rate, and no competition for land and fresh water. However, it is difficult for one microorganism to convert all components of brown algae with different oxidoreduction potentials to ethanol. Defluviitalea phaphyphila Alg1 is the first characterized thermophilic bacterium capable of direct utilization of brown algae. RESULTS: Defluviitalea phaphyphila Alg1 can simultaneously utilize mannitol, glucose, and alginate to produce ethanol, and high ethanol yields of 0.47 g/g-mannitol, 0.44 g/g-glucose, and 0.3 g/g-alginate were obtained. A rational redox balance system under obligate anaerobic condition in fermenting brown algae was revealed in D. phaphyphila Alg1 through genome and redox analysis. The excess reducing equivalents produced from mannitol metabolism were equilibrated by oxidizing forces from alginate assimilation. Furthermore, D. phaphyphila Alg1 can directly utilize unpretreated kelp powder, and 10 g/L of ethanol was accumulated within 72 h with an ethanol yield of 0.25 g/g-kelp. Microscopic observation further demonstrated the deconstruction process of brown algae cell by D. phaphyphila Alg1. CONCLUSIONS: The integrated biomass deconstruction system of D. phaphyphila Alg1, as well as its high ethanol yield, provided us an excellent alternative for brown algae bioconversion at elevated temperature.