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Biocathode application in microbial fuel cell has been developed as a sustainable technology for heavy metal reduction. However, most biocathodes require pretreatment with acclimatization inversion. Here, a biocathode MFC based on strain Corynebacterium vitaeruminis LZU47-1 without acclimatization inversion was constructed for hexavalent chromium reduction. The maximum power generation by the biocathode MFC with C. vitaeruminis LZU47-1 increased by 24.5% and 53.4% in inversion and abiotic cathode groups, respectively. Compared with the inversion (72.52%) and abiotic cathode groups (64.75%), the biocathode group achieved a Cr(VI) removal efficiency of 98.63%. Furthermore, electrochemical analysis such as SEM-EDS, XPS and CV test were conducted to elucidate the adsorption-reduction mechanism for Cr(VI) reduction. MiSeq sequencing revealed that Geobacter (51.28%) was enriched on the anode biofilm in the biocathode group than inversion (38.52%) and abiotic cathode groups (31.74%). Therefore, this study provides a convenient and highly effective method for enhancing power output and Cr(VI) reduction in biocathode MFCs.

During meiosis, chromosomes exhibit dramatic changes in morphology and intranuclear positioning. How these changes influence homolog pairing, alignment, and recombination remain elusive. Using Hi-C, we systematically mapped 3D genome architecture throughout all meiotic prophase substages during mouse spermatogenesis. Our data uncover two major chromosome organizational features varying along the chromosome axis during early meiotic prophase, when homolog alignment occurs. First, transcriptionally active and inactive genomic regions form alternating domains consisting of shorter and longer chromatin loops, respectively. Second, the force-transmitting LINC complex promotes the alignment of ends of different chromosomes over a range of up to 20% of chromosome length. Both features correlate with the pattern of homolog interactions and the distribution of recombination events. Collectively, our data reveal the influences of transcription and force on meiotic chromosome structure and suggest chromosome organization may provide an infrastructure for the modulation of meiotic recombination in higher eukaryotes., During meiosis, chromosomes undergo dramatic changes in morphology and intranuclear positioning. Here the authors mapped the 3D genome architecture throughout mouse spermatogenesis by Hi-C of sorted cells to reveal the contributions of transcriptional activity and mechanical force in modulating homolog alignment and recombination.

Stereocilia are actin-based cell protrusions on the apical surface of inner ear hair cells, playing a pivotal role in hearing and balancing sensation. The development and maintenance of stereocilia is tightly regulated and deficits in this process usually lead to hearing or balancing disorders. The Rho GTPase cell division cycle 42 (CDC42) is a key regulator of the actin cytoskeleton. It has been reported to localize in the hair cell stereocilia and play important roles in stereocilia maintenance. In the present work, we utilized hair cell-specific Cdc42 knockout mice and CDC42 inhibitor ML141 to explore the role of CDC42 in stereocilia development. Our data show that stereocilia height and width as well as stereocilia resorption are affected in Cdc42-deficient cochlear hair cells when examined at postnatal day 8 (P8). Moreover, ML141 treatment leads to planar cell polarity (PCP) deficits in neonatal hair cells. We also show that overexpression of a constitutively active mutant CDC42 in cochlear hair cells leads to enhanced stereocilia developmental deficits. In conclusion, the present data suggest that CDC42 plays a pivotal role in regulating hair cell stereocilia development.

Long-range communication between intestinal symbiotic bacteria and extra-intestinal organs can occur through circulating bacterial signal molecules, through neural circuits, or through cytokines or hormones from host cells. Here we report that Nod1 ligands derived from intestinal bacteria act as signal molecules and directly modulate insulin trafficking in pancreatic beta cells. The cytosolic peptidoglycan receptor Nod1 and its downstream adapter Rip2 are required for insulin trafficking in beta cells in a cell-autonomous manner. Mechanistically, upon recognizing cognate ligands, Nod1 and Rip2 localize to insulin vesicles, recruiting Rab1a to direct insulin trafficking through the cytoplasm. Importantly, intestinal lysozyme liberates Nod1 ligands into the circulation, thus enabling long-range communication between intestinal microbes and islets. The intestine-islet crosstalk bridged by Nod1 ligands modulates host glucose tolerance. Our study defines a new type of inter-organ communication based on circulating bacterial signal molecules, which has broad implications for understanding the mutualistic relationship between microbes and host.

Germinal centers (GCs), which are the site of antibody diversification and affinity maturation, are vitally important for humoral immunity. GC B cell proliferation is essentially for these processes by providing enough templates for somatic hypermutation (SHM) and serving as a critical mechanism of positive selection. In the current study, we found a significant reduction of GC response in the spleens of GC B cell specific PHF14 knockout (PHF14GCB KO) mice compared with the wild-type control (PHF14GCB WT) when the mice were challenged with SRBCs or lymphocytic choriomeningitis virus. We also demonstrated that PHF14 did not affect the cell survival of GC B cells, but regulated the proliferation of GC B cells. In addition, PHF14 suppressed the expression of Cdkn1a (p21) though regulating the level of H3K4me3 to control the proliferation of GC B cells. Collectively, our data suggest that PHF14 plays an important role in the process of germinal center formation by regulating GC B cell proliferation in spleen.

Background Angelica sinensis seedlings are grown in alpine uncultivated meadow soil with rainfed agroecosystems to ensure the quality of A. sinensis after seedling transplantation. The aim was to investigate the rhizosphere bacterial and fungal communities during the growth stages of A. sinensis seedlings. Methods The bacterial and fungal communities were investigated by HiSeq sequencing of 16S and 18S rDNA, respectively. Results Proteobacteria and Bacteroidetes were bacterial dominant phyla throughout growth stages. Fungal dominant phyla varied with growth stages, dominant phyla Ascomycota and Chytridiomycota in AM5, dominant phyla Basidiomycota, Ascomycota and Zygomycota in BM5, and dominant phyla Basidiomycota and Ascomycota in CM5. There was no significant variation in the alpha-diversity of the bacterial and fungal communities, but significant variation was in the beta-diversity. We found that the variation of microbial community composition was accompanied by the changes in community function. The relative abundance of fungal pathogens increased with plant growth. We also identified the core microbes, significant-changing microbes, stage-specific microbes, and host-specific microbes. Plant weight, root length, root diameter, soil pH, rainfall, and climate temperature were the key divers to microbial community composition. Conclusions Our findings reported the variation and environmental drivers of rhizosphere bacterial and fungal communities during the growth of A. sinensis seedlings, which enhance the understanding of the rhizosphere microbial community in this habitat.

Overexpression of the enzyme phosphatase (PhoN/PhoK) in the radiation-resistant bacterium Deinococcus radiodurans could be an efficient strategy for uranium remediation. However, the presence of other metals in nuclear wastes often interferes with uranium bioprecipitation. In our study, the uranium-precipitating ability of the PhoN-expressing D. radiodurans strain (Deino-phoN) significantly decreased by 45.4% in 13 h in the presence of chromium (VI); however, it was partially recovered after supplementation with chromium (III). Therefore, the reduction of chromium (VI) to chromium (III) was obtained by the co-expression of the YieF protein and PhoN in D. radiodurans (Deino-phoN-yieF). As a result, an increase in the chromium (VI) reduction (25.1%) rate was observed in 24 h. Furthermore, uranium precipitation also increased by 28.0%. For the decontamination of groundwater, we immobilized Deino-phoN-yieF cells using Polyvinyl alcohol (PVA)-sodium alginate (SA) beads, followed by incubation in a bioreactor. Approximately 99% of chromium (VI) and uranium (VI) was removed after 4 continuous cycles operated for a period of over 20 days at room temperature (25 °C). Therefore, Deino-phoN-yieF could be used as a potential biological agent for mixed radioactive nuclear waste remediation.

Cell division cycle 42 (CDC42) plays important roles in polarity establishment and maintenance as well as cell cycle progression and cell division. Although disruption of cell polarity is a prerequisite in epithelial tumor initiation, the roles of CDC42 in tumorigenesis are still poorly understood. Here we find that Cdc42 deficiency inhibits the Kras G12D -induced lung alveoli tumor formation, while conversely promotes bronchiole tumor formation in mice. Bronchial Cdc42 loss destroys contact inhibition potentially through cell polarity disruption, and results in increased tumor formation. In contrast, deletion of Cdc42 in alveoli cells prevents Kras G12D -induced cell proliferation, which leads to reduced tumor formation. Further analyses of clinical specimens uncover a significant positive correlation between CDC42 and type II alveolar epithelial cells marker SP-A, indicating the potential importance of CDC42 in this specific subset of lung cancer. Collectively, we identify the lineage-specific function of CDC42 in lung tumorigenesis potentially through the regulation of cell polarity integrity.

BACKGROUND We investigated the influence of Resolvin D1 (RvD1) on the inflammatory response in PC12 cells (a cell model of Parkinson disease, PD). MATERIAL AND METHODS 4 mmol/L 1-methyl-4-phenylpyridinium ion (Mpp+) was used in PC12 cells for an in vitro PD model. 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to explore PC12 cell viability. Western blot (WB) experiments were used to identify nuclear factor-κB (NF-κB), phosphorylated extracellular signal-regulated kinase (p-ERK)/p-Jun N-terminal kinase (JNK)/p-P38 mitogen-activated protein kinase (MAPK), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 protein levels. Transcription levels of inflammatory factors, for instance, TNF-α and IL-6, were explored by real-time quantitative polymerase chain reaction (RT-QPCR). Lactic dehydrogenase (LDH) level was detected by enzyme-linked immunosorbent (ELISA). Cell apoptosis was assessed by Annexin-V Fluorescein (FITC) kit. RESULTS RvD1 dose-dependently inhibited MPP+ induced upregulation of PC12 cell apoptosis/cellular damage/TNF-α and p-P38/p-ERK/NF-κB as well as downregulation of PC12 cell viability. CONCLUSIONS We can draw the conclusion that RvD1 attenuates PD via inhibiting Mpp+-induced inflammation in PC12 cells.

P-nitrophenol is one of the most common contaminants in chemical industrial wastewater, and in situ real-time monitoring of PNP cannot be achieved by conventional analytical techniques. Here, a two-chamber microbial fuel cell with an aerobic anode chamber was tested as a biosensor for in situ real-time monitoring of PNP. Pseudomonas monteilii LZU-3, which was used as the biological recognition element, can form a biofilm on the anode electrode using PNP as a sole substrate. The optimal operation parameters of the biosensor were as follows: external resistance 1000Ω, pH 7.8, temperature 30°C, and maximum PNP concentration 50mgL(-1). Under these conditions, the maximum voltages showed a linear relationship with PNP concentrations ranging from 15±5 to 44±4.5mgL(-1). Furthermore, we developed a novel portable device for in situ real-time monitoring of PNP. When the device was applied to measure PNP in wastewater containing various additional aromatic compounds and metal ions, the performance of the biosensor was not affected and the correlation between the maximum voltages and the PNP concentrations ranging from 9±4mgL(-1) to 36 ± 5mgL(-1) was conserved. The results demonstrated that the MFC biosensor provides a rapid and cost-efficient analytical method for real-time monitoring of toxic and recalcitrant pollutants in environmental samples.

Triazophos (TAP), methamidophos (MAP) and carbofuran (CF) pesticides are highly toxic, soluble and absorbable. Efficient co-degradation of multi-pesticides is rare reported. The objectives of this study were to investigate TAP, MAP and CF co-degradative ability of Enterobacter sp. Z1 and study the degradation mechanisms. Strain Z1 was shown to efficiently co-degrade TAP, MAP and CF when they were used as primary carbon sources. The degradation occurred over a wide range of temperatures, pH values and pesticide concentrations and followed first-order kinetics. Under the optimum conditions (37 °C, pH 7 and 100 mg/L of each pesticide), the degradation efficiencies were 100%, 100%, and 95.3% for TAP, MAP and CF, respectively. In addition, strain Z1 could simultaneously degrade TAP, MAP, CF and total nitrogen in wastewater in a batch bioreactor, with high removal efficiencies of 98.3%, 100%, 98.7% and 100%, respectively. Genomics, proteomics, qRT-PCR and gene overexpression analyses revealed that the degradation mechanisms involved the activities of multiple proteins, among which, organophosphorus hydrolase (Oph) and 3-hydroxyacyl-CoA dehydrogenase (PaaC) are primarily responsible for TAP and MAP degradation, while carbofuran hydrolase (Mcd) and amidohydrolase (RamA) primarily degrade CF. Among these enzymes, PaaC and RamA are newly identified pesticide-degrading enzymes. Toxicity assays of strain Z1 using reporter recombinase gene (recA) and zebrafish showed that there was no accumulation of toxic metabolites during the degradation process. Biosafety test using zebrafish showed that the strain was nontoxic toward zebrafish. Strain Z1 provides a good purification effect for pesticides-containing wastewater and novel microbial pesticide-degrading mechanisms were discovered.

Biodegradation of recalcitrant organic compounds in microbial fuel cell (MFC) is limited, due to its strong electron affinity and persisted in anaerobic condition. In this study, Pseudomonas monteilii LZU-3 degraded p-nitrophenol (PNP) and generated current at 100 mg L-1 of PNP in anode MFC with the addition of oxygen. The highest PNP degradation was 4, 37.75, and 99.89% in anaerobic, aerobic, and aerated anode of MFC respectively, at 7 h. The maximum voltage generation in aerated anode was 183 mV, which was comparatively higher than aerobic (150 mV) and anaerobic (68 mV). The qRT-PCR results confirmed that the oxygenase genes in strain LZU-3 were up-regulated from 17.51 to 39.39-fold at 1.6-4.5 mg L-1 of oxygen concentrations resulted in PNP degradation in anode MFC. This study demonstrated that supplementation of oxygen into the anode MFC might be a potential approach for biodegradation of recalcitrant compounds and electricity generation.

In this study, a sediment microbial fuel cell (SMFC) system for the simultaneous biodegradation of organic matter and detoxification of hexavalent chromium Cr (VI) was investigated. The total organic carbon (TOC) removal rate of the SMFC with Cr (VI) was 30.07%, which was significantly higher than that in a SMFC without Cr (VI) (13.74%). In the SMFC with Cr (VI), the maximum values of open-circuit voltage (OCV) and power density were 408 mV and 4.8 mW/m 2 , respectively. During the long-term operation of the SMFC with Cr (VI), 25 mg/L of Cr (VI) were completely reduced from all four consecutive batches over 48 days. MiSeq sequencing revealed that the biofilm microbial community of the anode comprised of Bacteroidetes (42.9%), Proteobacteria (33.6%), Chloroflexi (7.5%), and Euryarchaeota (7.5%) as the predominant phyla. Compared with that of the sediment, certain families were enriched; they included Pseudomonadaceae (46.88-fold), Flavobacteriaceae (5.05-fold), and Syntrophaceae (4.48-fold), which are organic matter-degrading bacteria. These results suggest that SMFCs are useful for TOC removal and detoxification of heavy metals in remediation of contaminated lakes.

The aim of this study was to investigate the chemically defined nutritional requirements for Lactobacillus plantarum in milk. Twenty amino acids, five nucleotide precursors, ten vitamins, and ten mineral salts were added to skim milk. The selective removal of individual nutrient groups led to the identification of amino acids and nucleotide precursors as essential nutrients for L. plantarum growth. Mineral salts were not essential but had a growth-stimulating effect on L. plantarum . Further single-omission growth experiments revealed six key amino acids (isoleucine, leucine, valine, tyrosine, methionine, and phenylalanine) and at least one purine required as minimal nutrient for L. plantarum growth in milk. L. plantarum grew and propagated well in the fortified milk supplementation with six amino acids and adenine. The pH values of the fortified milk fermented using L. plantarum ST-III, WCFS1 and LPHS were 3.93, 3.84 and 4.02 at 72 h, respectively. However, the pH values of the control-fermented milk using L. plantarum ST-III, WCFS1 and LPHS were only 5.73, 5.61 and 5.84 at 72 h. The identification of the chemically defined requirements for growth of L. plantarum in milk without complex and undefined substances contributes to the understanding of the physiological properties and metabolic pathways of bacteria.

Downregulation of E-cadherin by the transcriptional repressor Snail is associated with acquisition of metastatic potential. Although hepatitis C virus (HCV) core protein has been implicated in hepatocarcinogenesis, it is unclear whether Snail is involved in HCV core-induced dysregulation of E-cadherin. Herein, we investigated the mechanism by which HCV core induces E-cadherin repression and the role of Snail in HCV core-mediated invasiveness and metastasis. We found that HCV infection, especially HCV core expression, effectively induced the epithelial-mesenchymal transition (EMT) in hepatoma cells by repressing E-cadherin. HCV core interacted with Snail and enhanced its binding to the E-box in the promoter region of E-cadherin, leading to decreased E-cadherin promoter activity. We found that HCV core, Snail, and the histone deacetylases HDAC1/HDAC2 formed a co-repressor complex at the E-cadherin promoter. Moreover, HCV core was shown to stabilize Snail through activation of the PI3K/Akt/GSK3β pathway. Silencing Snail expression restored E-cadherin expression and inhibited HCV core-promoted tumor growth and distant lung metastasis in vivo. Collectively, these results demonstrated that HCV core induced EMT by interacting with the transcriptional repressor complex Snail/HDACs at the E-cadherin promoter, which led to E-cadherin repression and increased invasiveness of hepatoma cells. These findings increase understanding of factors regulating metastasis in hepatoma and may ultimately lead to the development of novel treatment strategies for HCV-associated hepatocellular carcinoma.

Lung cancer is the leading cause of cancer-related death in the United States, and metastatic behavior is largely responsible for this mortality. Mutations in multiple 'driver' oncogenes and tumor suppressors are known to contribute to the lung tumorigenesis and in some cases represent therapeutic targets. Leucine Zipper Transcription Factor-like 1 (LZTFL1) is located in the chromosome region 3p21.3 where allelic loss and genetic alterations occur early and frequently in lung cancers. Previously, we found that LZTFL1 is downregulated in epithelial tumors, including lung cancer, and functions as a tumor suppressor in gastric cancers. However, the functional role of LZTFL1 in lung oncogenesis is undefined. We show here that downregulation of LZTFL1 expression in non-small cell lung cancer is associated with recurrence and poor survival, whereas re-expression of LZTFL1 in lung tumor cells inhibited extravasation/colonization of circulating tumor cells to the lung and inhibited tumor growth in vivo. Mechanistically, we found that LZTFL1 is expressed in ciliated human bronchial epithelial cells (HBECs) and its expression correlates with HBEC differentiation. LZTFL1 inhibits transforming growth factor β-activated mitogen-activated protein kinase and hedgehog signaling. Alteration of intracellular levels of LZTFL1 resulted in changes of expression of genes associated with epithelial-to-mesenchymal transition (EMT). We conclude that LZTFL1 inhibits lung tumorigenesis, possibly by maintaining epithelial cell differentiation and/or inhibition of signalings that lead to EMT and suggest that reactivation of LZTFL1 expression in tumor cells may be a novel lung cancer therapeutic approach.

Streptococcus thermophilus phages present hazards for yogurt processing. In order to avoid adverse effects, different component yogurt starters were designed. For 2-component yogurt starters consisting of an S. thermophilus sensitive and a Lactobacillus delbrueckii ssp. bulgaricus strain, phage infection was destructive. Phages resulted in increased fermentation time, unbalanced cocci to rod ratios, decreased viscosity values, and varied sensory properties. For 3-component yogurt starters consisting of S. thermophilus sensitive, S. thermophilus insensitive, and L. bulgaricus strains, phage invasion did not result in the undesirable effects of slow acidification, decreased viscosity values, and unbalanced cocci to rod ratios. The 3-component yogurt starter stabilized fermentation and improved the practical fermentation performance in the presence of phages. Combinations of different S. thermophilus strains in the design and use of yogurt starters should be considered.

The influence of several Chinese teas on the ability of Lactobacillus casei to ferment milk was assessed. The three kinds of tea significantly stimulated the growth and acidification of L. casei. The viable cell counts in tea-supplemented fermented milk were higher than those of the control and were maintained during cold storage. Flavour component content increased significantly, and an additional eight components were detected in fermented milk supplemented with 5% green tea infusion (GTI5%) compared with control fermented milk. Moreover, the increase in total free amino acids after fermentation in GTI5% was 2.5-fold higher than in the control, suggesting that tea stimulated the growth and metabolism of L. casei during milk fermentation. Higher cell numbers, reduced fermentation time, abundant flavour components and free amino acids were achieved during the tea-supplemented fermentation of L. casei. Green tea effectively enhances the growth of L. casei, therefore could be considered for industrial purposes.

Hexavalent chromium [Cr(VI)] is one of the major environmental contaminants in the Lanzhou reaches of the Yellow River, and actinomycetes' Cr(VI) bioremediation within this area is still scarce. In the present study, twenty actinomycete strains were isolated from the Lanzhou reaches which could resist 1.2 mM Cr(VI) on Starch-Casein (SC) agar plates. Based on the 16S rRNA sequence data, sixteen strains belong to Streptomyces and four other strains belong to Gordonia, Nocardiopsis, Nocardia and Cellulosimicrobium. One strain named LZ-26-1 which is closely related to Streptomyces violaceoruber demonstrated a notable ability of Cr(VI) reduction. It could reduce 92.86% of the 0.6 mM Cr(VI) to trivalent chromium [Cr(III)] in SC liquid medium in 144 h. The optimum temperature and pH were 28 °C and 7.0 for Cr(VI) reduction. Transmission electron microscopy images showed that chromium was precipitated inside the cells. In resting cells and crude chromate reductase assays, strain LZ-26-1's Cr(VI) reduction was stimulated when NADPH was used as an electron donor and Cd2+ treatment inhibited Cr(VI) reduction. Furthermore, thioredoxin operon, which consists of three genes encoding a thioredoxin, thioredoxin reductase (NADPH) and a possible membrane protein, was upregulated by Cr(VI) treatment. These results suggested that LZ-26-1 might utilize a thioredoxin pathway to reduce Cr(VI).

The aim of this study was to investigate the diversity of phages from Chinese yoghurt plants. Restriction analysis of six isolated Streptococcus thermophilus (St) phages showed that Φ101, Φ102 and Φ108 with different origins but with the same host strain St1 were different while phages Φ901, Φ903 and Φ907 with host strain St9 from the same origin were homologous. DNA packaging mechanism analysis proved the isolated phages to be pac -type except Φ108. The sensitive strain St1 could be infected by two different packaging mechanisms. Probing and sequencing of the variable region (VR2) fragment revealed that VR2 in aggressive phages (Φ101, Φ102 and Φ108) infecting host strain St1 was deleted. Phage Φ901 has an 845 bp amplicon and the similarity coefficient of the sequence was lower than 96% compared with other reported phages, which shows phage Φ901 did not belong to any known grouping.

Background: Trypanosoma brucei, a causative agent of African Trypanosomiasis, is known to cross the blood brain barrier during the second stage of the disease. It was previously suggested that this parasite crosses the blood brain barrier in a manner similar to that of lymphocytes. This would imply that trypanosomes possess integrins that are required to interact with adhesion molecules located on the blood brain barrier microvascular endothelial cells, as a first step in traversal. To date, no T. brucei integrin has been described. However, one T. brucei putative FG-GAP repeat containing protein (typical of integrins) encoded by the Tb927.11.720 gene, was predicted to be involved in cell-cell/cell-matrix adhesion. Therefore, this study sought to characterize a putative FG-GAP repeat containing protein (FG-GAP RCP) and to determine its cellular localization as a basis for further exploration of its potential role in cell-cell or cell-matrix adhesion. Methods: In this study, we successfully cloned, characterized, expressed and localized this protein using antibodies we produced against its VCBS domain in T. brucei. Results: Contrary to what we initially suspected, our data showed that this protein is localized to the mitochondria but not the plasma membrane. Our data showed that it contains putative calcium binding motifs within the FG-GAP repeats suggesting it could be involved in calcium signaling/binding in the mitochondrion of T. brucei. Conclusion: Based on its localization we conclude that this protein is unlikely to be a trypanosomal integrin and thus that it may not be involved in traversal of the blood brain barrier. However, it could be involved in calcium signaling in the mitochondrion.

Gemini surfactant diglycol bis-N-tetradecyl nicotinate dibromide (designed as EQ14-2-14) has been synthesized. The interaction between EQ14-2-14 and bovine serum albumin (BSA) was studied by dual polarization interferometry (DPI), proton nuclear magnetic resonance spectroscopy ( 1 H NMR), Fourier transform infrared spectroscopy (FTIR) and molecular docking. Owing to the binding of EQ14-2-14, the thickness and mass of BSA increased; refractive index (RI) and density firstly raised and then tended to a plateau. In addition, a decrease of α-helix was observed from 54.01% to 31.56% with an increase in random structure from 7.86% to 21.76%. Due to BSA intertwining, the proton resonance signals of EQ14-2-14 shifted up-field and relaxation time decreased with increasing concentration of BSA. The study of molecular docking indicated that EQ14-2-14 embedded into subdomain II of BSA by π–π stacking between the electron-deficit pyridinium rings in EQ14-2-14 and the electron-abundant pyrrole ring in Trp residues of BSA, by hydrogen bonding and by hydrophobic interaction. Therefore the present work offers a whole view of the interaction of BSA with a new gemini surfactant.

Based on inhibition of acid production, 100 individual colonies of Streptococcus thermophilus isolated from commercial Direct Vat Set yoghurt starter DVS-M were typed into 2 groups of sensitive and insensitive to phage φ001, which was isolated from the lysis culture of the turbidity test. The ratio between the sensitive and insensitive isolates in DVS-M was calculated at 3:2. Whole-cell protein profile (WCPP) and randomly amplified polymorphic DNA (RAPD) analysis showed that both WCPP and RAPD patterns between sensitive and insensitive isolates were distinct, whereas isolates with the same sensitivity or insensitivity shared common WCPP and RAPD patterns. It suggested that all the sensitive isolates belong to one S. thermophilus strain and all the insensitive isolates belong to another one. This study revealed that DVS-M yoghurt starter consists of 2 S. thermophilus strains with different phage sensitivity.

The plant homeodomain (PHD) finger is identified in many chromatin-binding proteins, and functions as a ‘reader’ that recognizes specific epigenetic marks on histone tails, bridging transcription factors and their associated complexes to chromatin, and regulating gene expression. PHD finger-containing proteins perform many biological functions and are involved in many human diseases including cancer. PHF14 is predicted to code for a protein with multiple PHD fingers. However, its function is unidentified. The aim of this study is to characterize PHF14 and investigate its biological significance by employing multiple approaches including mouse gene-targeting knockout, and molecular cloning and characterization. Three transcripts of PHF14 in human cell lines were identified by reverse transcriptase polymerase chain reaction. Two isoforms of PHF14 (PHF14α and PHF14β) were cloned in this study. It was found that PHF14 was ubiquitously expressed in mouse tissues and human cell lines. PHF14α, the major isoform of PHF14, was localized in the nucleus and also bound to chromatin during cell division. Interestingly, co-immunoprecipitation results suggested that PHF14α bound to histones via its PHD fingers. Strikingly, gene-targeting knockout of PHF14 in mice resulted in a neonatal lethality due to respiratory failure. Pathological analysis revealed severe disorders of tissue and cell structures in multiple organs, particularly in the lungs. These results indicated that PHF14 might be an epigenetic regulator and play an important role in the development of multiple organs in mouse. Keywords plant homeodomain (PHD) finger; PHF14; epigenetic; knockout mice

PHF14 is a newly identified regulator of mesenchyme growth in embryonic tissues. Previous studies have shown that phf14-null mutants die just after birth due to interstitial tissue hyperplasia in major organs, including the kidneys. The aim of this study was to investigate PHF14 function in renal fibrosis. By studying the chronic kidney injury mouse model, we found that PHF14 was upregulated in fibrotic kidneys after renal insults induced by folic acid administration. Compared with wild-type mice, PHF14-null mice showed more severe renal fibrosis after pro-fibrotic stimuli. Moreover, PHF14 in rat renal fibroblasts was upregulated by transforming growth factor-β (TGF-β) stimulation; while this upregulation was inhibited when smad3 phosphorylation was blocked. A chromatin immunoprecipitation (ChIP) assay further indicated that phospho-smad3 (p-smad3) acted as a transcription factor to enhance PHF14 expression. A lack of PHF14 expression enhanced collagen I and α-smooth muscle actin (α-SMA) synthesis induced by TGF-β in vitro. PHF14 was involved in inhibition of platelet-derived growth factor (PDGF) signaling overactivation by selectively repressing PDGF receptor-α (PDGFR-α) transcription. In summary, PHF14 expression was upregulated in fibrotic models in vivo and in vitro, and the TGF-β/smad3/PHF14 pathway acted as a self-limiting mechanism in the TGF-β-dominated renal pro-fibrotic process by suppressing PDGFR-α expression.

The effects of cabbage waste (CW) addition on methane production in cow dung and corn straw co-fermentation systems were investigated. Four experimental groups, each containing 55 g of substrate, were set up as follows: 100% cow dung (C); 36% cabbage and 64% cow dung (CC); 36% straw and 64% cow dung (SC); and 18% cabbage, 18% straw, and 64% cow dung (CSC). After seven days of fermentation, the maximum methane yield was 134 mL in the CSC group, which was 2.81-fold, 1.78-fold, and 1340-fold higher than that obtained in the CC, SC, and C groups, respectively. CW treatment of the CSC group enhanced cellulase activity and enriched culturable cellulose-degrading bacterial strains. Miseq sequencing data revealed that the predominant phylum in the CSC group was Bacteroidetes, which contains most of the cellulose-degrading bacteria. Our results suggested that CW treatment elevated cellulose degradation and promoted methane production.

Partitioning-defective 3 (Par3), a key component of the evolutionarily conserved polarity PAR complex (Par3/Par6/aPKC), controls cell polarity and contributes to cell migration, proliferation and tumor development. Emerging evidence indicates that cell polarity proteins function as upstream modulators that regulate the Hippo pathway. However, little is known about Par3’s involvement in the Hippo pathway. Here, we find Par3 and YAP dynamically co-localize in different subcellular compartments; that is, the membrane, cytoplasm and nucleus, in a cell-density-dependent manner. Interestingly, Par3 knockdown promotes YAP phosphorylation, leading to a significant impairment of YAP nuclear translocation at low cell density, but not at high density, in MDCK cells. Furthermore, via its third PDZ domain, Par3 directly binds to the PDZ-binding motif of YAP. The interaction is required for regulating YAP phosphorylation and nuclear localization. Mechanistically, Par3, as a scaffold protein, associates with LATS1 and protein phosphatase 1, α subunit (PP1A) in the cytoplasm and nucleus. Par3 promotes the dephosphorylation of LATS1 and YAP, thus enhancing YAP activation and cell proliferation. Strikingly, we also find that under the condition of PP1A knockdown, Par3 expression promotes YAP hyperphosphorylation, leading to the suppression of YAP activity and its downstream targets. Par3 expression results in differential effects on YAP phosphorylation and activation in different tumor cell lines. These findings indicate that Par3 may have a dual role in regulating the activation of the Hippo pathway, in a manner possibly dependent on cellular context or cell type in response to cell–cell contact and cell polarity signals.

Stimulator of interferon genes (STING) is critical for cytosolic DNA-triggered innate immunity. STING is modified by several types of polyubiquitin chains. Here, we report that the deubiquitinase CYLD sustains STING signaling by stabilizing the STING protein. CYLD deficiency promoted the K48-linked polyubiquitination and degradation of STING, attenuating the induction of IRF3-responsive genes after HSV-1 infection or the transfection of DNA ligands. Additionally, CYLD knockout mice were more susceptible to HSV-1 infection than their wild-type (WT) littermates. Mechanistically, STING translocated from the ER to the Golgi upon HSV-1 stimulation; CYLD partially accumulated with STING and interacted selectively with K48-linked polyubiquitin chains on STING, specifically removing the K48-linked polyubiquitin chains from STING and ultimately boosting the innate antiviral response. Our study reveals that CYLD is a novel checkpoint in the cGAS-STING signaling pathway and sheds new light on the dynamic regulation of STING activity by ubiquitination., Author summary STING is critical for mediating the production of type I interferons and other proinflammatory cytokines. The appropriate activation of STING signaling is precisely modulated to maintain immune homeostasis. It is well established that covalent modification of STING by different types of polyubiquitin chains serves to fine-tune STING activity in response to extracellular and intracellular stresses. However, it remains poorly understood how these polyubiquitin chains on STING are dynamically removed in response to different stimuli. In this study, we characterized the deubiquitinase CYLD, which partially accumulates with STING upon HSV-1 infection and interacts selectively with the K48-linked polyubiquitin chains on STING. CYLD specifically removes K48-linked polyubiquitin chains from STING and thus promotes antiviral responses. Our study reveals a novel function of CYLD in the STING signaling pathway and indicates that CYLD is an important target for modulating the host response to infections caused by DNA pathogens.

The adaptive immune CRISPR/Cas and CRISPR/Cmr systems of the crenarchaeal thermoacidophile Sulfolobus were challenged by a variety of viral and plasmid genes, and protospacers preceded by different dinucleotide motifs. The genes and protospacers were constructed to carry sequences matching individual spacers of CRISPR loci, and a range of mismatches were introduced. Constructs were cloned into vectors carrying pyrE/pyrF genes and transformed into uracil auxotrophic hosts derived from Sulfolobus solfataricus P2 or Sulfolobus islandicus REY15A. Most constructs, including those carrying different protospacer mismatches, yielded few viable transformants. These were shown to carry either partial deletions of CRISPR loci, covering a broad spectrum of sizes and including the matching spacer, or deletions of whole CRISPR/Cas modules. The deletions occurred independently of whether genes or protospacers were transcribed. For family I CRISPR loci, the presence of the protospacer CC motif was shown to be important for the occurrence of deletions. The results are consistent with a low level of random dynamic recombination occurring spontaneously, either inter-genomically or intra-genomically, at the repeat regions of Sulfolobus CRISPR loci. Moreover, the relatively high incidence of single-spacer deletions observed for S. islandicus suggests that an additional more directed mechanism operates in this organism.

Microorganisms can utilize different sugars as energy and carbon sources and the genes involved in sugar metabolism often exhibit highly regulated expression. To study cis-acting elements controlling arabinose-responsive expression in archaea, the promoter of the Sulfolobus solfataricus araS gene encoding an arabinose binding protein was characterized using an Sulfolobus islandicus reporter gene system. The minimal active araS promoter (P(araS)) was found to be 59 nucleotides long and harboured four promoter elements: an ara-box, an upstream transcription factor B-responsive element (BRE), a TATA-box and a proximal promoter element, each of which contained important nucleotides that either greatly decreased or completely abolished promoter activity upon mutagenesis. The basal araS promoter was virtually inactive due to intrinsically weak BRE element, and the upstream activating sequence (UAS) ara-box activated the basal promoter by recruiting transcription factor B to its BRE. While this UAS ensured a general expression from an inactive or weak basal promoter in the presence of other tested carbon resources, it exhibited a strong arabinose-responsive transcriptional activation. To our knowledge, this represents the first example of an archaeal UAS that exhibits differential activation to the expression on the same promoter in the presence of different carbon sources.

C-terminal Src kinase (Csk)-binding protein (Cbp) is a transmembrane adaptor protein that localizes exclusively in lipid rafts, where it regulates Src family kinase (SFK) activities through recruitment of Csk. Although SFKs are well known for their involvement in cancer, the function of Cbp in carcinogenesis remains largely unknown. In this study, we reported overexpression of Cbp in more than 70% of renal cell carcinoma (RCC) specimens and in the majority of tested RCC cell lines. Depletion of Cbp in RCC cells by RNA interference led to remarkable inhibition of cell proliferation, migration, anchorage-independent growth as well as tumorigenicity in nude mice. Strikingly, silencing of Cbp negatively affected the sustaining of Erk1/2 activation but not c-Src activation induced by serum. Besides, the RhoA activity in RCC cells was remarkably impaired when Cbp was knocked down. Overexpression of wild-type Cbp, but not its mutant Cbp/DeltaCP lacking C-terminal PDZ-binding motif, significantly enhanced RhoA activation and cell migration of RCC cells. These results provided new insights into the function of Cbp in modulating RhoA activation, by which Cbp might contribute to renal cell carcinogenesis.

Sulfolobus islandicus is being used as a model for studying archaeal biology, geo-biology and evolution. However, no genetic system is available for this organism. To produce an S. islandicus mutant suitable for genetic analyses, we screened for colonies with a spontaneous pyrEF mutation. One mutant was obtained containing only 233 bp of the original pyrE sequence in the mutant allele and it was used as a host to delete the beta-glycosidase (lacS) gene. Two unmarked gene deletion methods were employed, namely plasmid integration and segregation, and marker replacement and looping out, and unmarked lacS mutants were obtained by each method. A new alternative recombination mechanism, i.e., marker circularization and integration, was shown to operate in the latter method, which did not yield the designed deletion mutation. Subsequently, Sulfolobus-E. coli plasmid shuttle vectors were constructed, which genetically complemented DeltapyrEFDeltalacS mutation after transformation. Thus, a complete set of genetic tools was established for S. islandicus with pyrEF and lacS as genetic markers.

Cell-cell contacts are crucial for the integrity of all tissues. Contrasting reports have been published about the role of Cdc42 in epithelial cell-cell contacts in vitro. In keratinocytes, it was suggested that Rac1 and not Cdc42 is crucial for the formation of mature epithelial junctions, based on dominant negative inhibition experiments. Deletion of the Cdc42 gene in keratinocytes in vivo slowly impaired the maintenance of cell-cell contacts by an increased degradation of beta-catenin. Whether Cdc42 is required for the formation of mature junctions was not tested. We show now that Cdc42-deficient immortalized and primary keratinocytes form only punctate primordial cell contacts in vitro, which cannot mature into belt-like junctions. This defect was independent of enhanced degradation of beta-catenin, but correlated to an impaired activation and localization of aPKCzeta in the Cdc42-null keratinocytes. Inhibition of aPKCzeta by the inhibitor Gö6983 reproduced the phenotype, suggesting that decreased activation of aPKCzeta was sufficient to explain the defective junctional maturation. In the absence of Cdc42, Rac1 activation was strongly decreased, indicating that Cdc42 is upstream of Rac1 activation. These data reveal that Cdc42 is crucial for the formation of mature epithelial cell junctions between keratinocytes by regulating activation of aPKCzeta.

Sulfolobus belongs to the hyperthermophilic archaea and it serves as a model organism to study archaeal molecular biology and evolution. In the last few years, we have focused on developing genetic systems for Sulfolobus islandicus using pyrEF as a selection marker and versatile genetic tools have been developed, including methods for constructing gene knockouts and for identifying essential genes. These genetic tools enable us to conduct genetic analysis on the functions of the genes involved in DNA replication and repair processes in S. islandicus and they should also facilitate in vivo analysis of functions of other genes in this model organism.

The evolutionarily conserved cell polarity protein Par3, a scaffold-like PDZ-containing protein, plays a critical role in the establishment and maintenance of epithelial cell polarity. Although the role of Par3 in establishing cell polarity in epithelial cells has been intensively explored, the function of Par3 in hematopoietic cells remains elusive. To address this issue, we generated GST-fusion proteins of Par3 PDZ domains. By combining the GST-pull-down approach with liq- uid chromatography-tandem mass spectrometry, we identi- fied 10 potential novel binding proteins of PDZ domains of Par3 in Jurkat cells (a T-cell line). The interaction of Par3 with three proteins--nuclear transport protein importin-α4 and proteasome activators PA28β and PA28γ--was con- firmed using in vitro binding assay, co-immunoprecipitation assay and immunofluorescence microscopy. Our results have the potential to uncover novel functions of the cell polarity protein Par3 in blood cells.

We previously reported that the β-1,4-Mannanase ( manB ) gene from Bacillus pumilus functions as a good reporter gene in Lactobacillus casei . Two vectors were constructed. One carries the signal peptide of secretion protein Usp45 (SPUsp45) from Lactococcus lactis (pELSH), and the other carries the full-length S-layer protein, SlpA, from L. acidophilus (pELWH). In this work, another vector, pELSPH, was constructed to include the signal peptide of protein SlpA (SPSlpA), and the capacity of all three vectors to drive expression of the manB gene in L. casei was evaluated. The results showed that SPUsp45 is functionally recognized and processed by the L. casei secretion machinery. The SPUsp45-mediated secretion efficiency was ∼87%, and SPSlpA drove the export of secreted ManB with ∼80% efficiency. SPSlpA secretion was highly efficient, and expressed SlpA was anchored to the cell wall by an unknown secretion mechanism. Full-length SlpA drove the cell wall-anchored expression of an SlpA-ManB fusion protein but at a much lower level than that of protein SlpA.

Two heme-dependent catalase genes were amplified from genomic DNA of Lactobacillus plantarum WCFS1 (KatE1) and Lactobacillus brevis ATCC 367 (KatE2), respectively, and a manganese-containing superoxide dismutase from Lactobacillus casei MCJΔ1 (MnSOD) were cloned into plasmid pELX1, yielding pELX1-KatE1, pELX1-KatE2 and pELX1-MnSOD, then the recombinant plasmids were transferred into L. casei MCJΔ1. The strains of L. casei MCJΔ1/pELX1-KatE1 and L. casei MCJΔ1/pELX1-KatE2 were tolerant at 2 mM H2O2. The survival rates of L. casei MCJΔ1/pELX1-KatE1 and L. casei MCJΔ1/pELX1-KatE2 were 270-fold and 300-fold higher than that of the control strain on a short-term H2O2 exposure, and in aerated condition, the survival cells counts were 146- and 190-fold higher than that of the control strain after 96 h of incubation. Furthermore, L. casei MCJΔ1/pELX1-MnSOD was the best in three recombinants which was superior in the living cell viability during storage when co-storage with Lactobacillus delbrueckii subsp. lactis LBCH-1.

Accurate chromosome segregation during mitosis requires the physical separation of sister chromatids which depends on correct position of mitotic spindle relative to membrane cortex. Although recent work has identified the role of PLK1 in spindle orientation, the mechanisms underlying PLK1 signaling in spindle positioning and orientation have not been fully illustrated. Here, we identified a conserved signaling axis in which NDR1 kinase activity is regulated by PLK1 in mitosis. PLK1 phosphorylates NDR1 at three putative threonine residues (T7, T183 and T407) at mitotic entry, which elicits PLK1-dependent suppression of NDR1 activity and ensures correct spindle orientation in mitosis. Importantly, persistent expression of non-phosphorylatable NDR1 mutant perturbs spindle orientation. Mechanistically, PLK1-mediated phosphorylation protects the binding of Mob1 to NDR1 and subsequent NDR1 activation. These findings define a conserved signaling axis that integrates dynamic kinetochore-microtubule interaction and spindle orientation control to genomic stability maintenance.

The conserved polarity complex, comprising the partitioning-defective (Par) proteins Par3 and Par6, and the atypical protein kinase C, functions in various cell-polarization events and asymmetric cell divisions. However, little is known about whether and how external stimuli-induced signals may regulate Par3 function in epithelial cell polarity. Here, we found that Par3 was tyrosine phosphorylated through phosphoproteomic profiling of pervanadate-induced phosphotyrosine proteins. We also demonstrated that the tyrosine phosphorylation event induced by multiple growth factors including epidermal growth factor (EGF) was dependent on activation of Src family kinase (SFK) members c-Src and c-Yes. The tyrosine residue 1127 (Y1127) of Par3 was identified as the major EGF-induced phosphorylation site. Moreover, we found that Y1127 phosphorylation reduced the association of Par3 with LIM kinase 2 (LIMK2), thus enabling LIMK2 to regulate cofilin phosphorylation dynamics. Substitution of Y1127 for phenylalanine impaired the EGF-induced Par3 and LIMK2 dissociation and delayed epithelial tight junction (TJ) assembly considerably. Collectively, these data suggest a novel, phosphotyrosine-dependent fine-tuning mechanism of Par3 in epithelial TJ assembly controlled by the EGF receptor-SFK signaling pathway.

The binding of insulin to its receptor triggers a signaling cascade regulated by protein complexes via tyrosine phosphorylation events on a multitude of associated proteins. To search novel phosphotyrosine proteins or associated proteins involved in insulin signaling pathway, we employed a method in which Rat1 cells stably expressing the human insulin receptor were stimulated with or without insulin and sub-fractionated prior to enrichment of phosphotyrosine proteins by immunoprecipitation and analysis by LC-MS/MS. Bioinformatic analysis and manual confirmation of peptide phosphorylation site assignments led to identification of 35 phosphotyrosine sites derived from 31 protein groups. Over 50% of these proteins were reported for the first time as tyrosine phosphorylated, including gigaxonin, XIAP and CDK10. In addition, we also found that calcium/calmodulin-dependent protein serine kinase (CASK), a key protein in protein-targeting and vesicle transport in neurons, forms a complex with two unidentified phosphotyrosine proteins pp100 and pp95 in response to insulin-stimulation, though CASK is not itself tyrosine phosphorylated. Furthermore, insulin was able to decrease CASK nuclear location, as well as down-regulate the expression of CASK targeted genes. Our results imply CASK as a novel joint knot connecting CASK-mediated pathways with the insulin signaling. Our data provide a wealth of information potentially paving the way to identify new components in the insulin signaling network.

The epididymis is a key structure of the male reproductive system; its function is to mature, transport, and store sperm. Most of the research examining the epididymis to date has been limited to the study of the secreted proteins involved in the maturation of spermatozoa. However, it is also very important to understand the protein components, regulation and function of the tissue itself since these are the basis for all of its physiological processes. We investigated the differential expression of proteins among the caput, corpus, and cauda regions of rat epididymis and considered the possible links between the localization of these proteins and the different functions of these epididymal regions. High-resolution 2-D gel electrophoresis followed by mass spectrometry (MS) revealed 28 distinct proteins whose expression levels varied from the caput to the cauda epididymis. Sixteen of them were reported for first time to be expressed in the epididymis. Expression patterns of some proteins were validated by Northern blot or Western blot. Immunohistochemistry revealed that inducible carbonyl reductase (iCR), an important enzyme in the anti-oxidative system, exhibits primary and cell-type specific distribution in the distal cauda region. Moreover, analysis of iCR transcription in castrated animals showed that its expression is androgen-dependent. Together with its known functions, iCR may also be involved in androgen metabolism and maintaining a steady microenvironment in the duct of epididymis.

Murine IL-4 is a pleiotropic cytokine with undefined core functional region for eliciting downstream signaling. We used molecular modeling to predict the binding sites recognized by an anti-IL-4-neutralizing mAb (11B.11) and peptide phage display to delineate their makeup. The results of these approaches were confirmed by site-directed mutagenesis analysis. The results suggest that the amino acid residues spanning from 79 to 86 (QRLFRAFR) on IL-4 are of the major binding site for 11B.11. Furthermore, the functional experiments demonstrate that the residues R80, R83 and R86, which are located in the helix C of murine IL-4, play a crucial role in binding to the IL-4R alpha-chain. Taken together, a new core functional region of murine IL-4 is identified, which provides new insight into the interaction between IL-4 and IL-4Ralpha. In addition, the results demonstrate that 11B.11 binds to a core functional region of murine IL-4, which prevents this cytokine from interacting with its cognate receptor.

Slit is a secreted protein known to function through the Roundabout (Robo) receptor as a chemorepellent in axon guidance and neuronal migration, and as an inhibitor in leukocyte chemotaxis. Here we show Slit2 expression in a large number of solid tumors and Robo1 expression in vascular endothelial cells. Recombinant Slit2 protein attracted endothelial cells and promoted tube formation in a Robo1- and phosphatidylinositol kinase-dependent manner. Neutralization of Robo1 reduced the microvessel density and the tumor mass of human malignant melanoma A375 cells in vivo. These findings demonstrate the angiogenic function of Slit-Robo signaling, reveal a mechanism in mediating the crosstalk between cancer cells and endothelial cells, and indicate the effectiveness of blocking this signaling pathway in treating cancers.

Hexavalent chromium (Cr(VI)) is a well-known toxic heavy metal in industrial wastewater, but in situ and real time monitoring cannot be achieved by current methods used during industrial wastewater treatment processes. In this study, a Sediment Microbial Fuel Cell (SMFC) was used as a biosensor for in situ real-time monitoring of Cr(VI), which was the organic substrate is oxidized in the anode and Cr(VI) is reduced at the cathode simultaneously. The pH 6.4 and temperature 25 °C were optimal conditions for the operation. Under the optimal conditions, linearity (R2 = 0.9935) of the generated voltage was observed in the Cr(VI) concentration range from 0.2 to 0.7 mg/L. The system showed high specificity for Cr(VI), as other co-existing ions such as Cu2+, Zn2+, and Pb2+ did not interfere with Cr(VI) detection. In addition, when the sediment MFC-based biosensor was applied for measuring Cr(VI) in actual wastewater samples, a low deviation (

Reporter gene systems are useful for studying bacterial molecular biology, including the regulation of gene expression and the histochemical analysis of protein products. Here, two genes, β-1,4-mannanase (manB) from Bacillus pumilus and β-glucuronidase (gusA) from Escherichia coli K12, were cloned into the expression vector pELX1. The expression patterns of these reporter genes in Lactobacillus casei were investigated by measuring their enzymatic activities and estimating their recombinant protein yields using western blot analysis. Whereas mannanase activity was positively correlated with the accumulation of ManB during growth, GusA activity was not; western blot analysis indicated that while the amount of GusA protein increased during later growth stages, GusA activity gradually decreased, indicating that the enzyme was inactive during cell growth. A similar trend was observed in E. coli JM109. We chose to use the more stable mannanase gene as the reporter to test secretion expression in L. casei. Two pELX1-based secretion vectors were constructed: one carried the signal peptide of the unknown secretion protein Usp45 from Lactococcus lactis (pELSH), and the other contained the full-length SlpA protein from the S-layer of L. acidophilus (pELWH). The secretion of ManB was detected in the supernatant of the pELSH-ManB transformants and in the S-layer of the cell surface of the pELWH-ManB transformants. This is the first report demonstrating that the B. pumilus manB gene is a useful reporter gene in L. casei and E.coli.

To gain new insight into the molecular mechanism underlying the pathogenesis of human primary hepatocellular carcinoma (HCC), we searched for HCC-specific molecules through screening genes that are differentially expressed between cancerous and noncancerous counterparts of liver and identified a novel HCC-associated gene, HCCA1 encoding a approximately 80 kDa cytoplasmic protein that contains several proline-rich motifs likely for SH3-binding. HCCA1 transcript, albeit present in some adult tissues, is up-regulated selectively in HCC but not in other tumor cells. High expression of HCCA1 occurs as a late event frequently (89.2%) in HCCs and correlated significantly with the degree of tumor progression. When treated with antisense oligonucleotides to HCCA1, HCCA1 expression in HCC cells (HuH-7) was effectively suppressed and cell growth was down-regulated in a time- and dose-dependent manner. Furthermore, HuH-7 cells harboring the HCCA1 antisense expression clone displayed a remarkably reduced efficiency in colony formation. Together, these data strongly suggest that HCCA1 is a positive effector in cell proliferation and contributes to HCC carcinogenesis and progression. We believe that this protein will serve as a novel useful marker for HCC and is a potential target for pharmaceutical intervention of this malignant disease.

Lactobacillus casei, a probiotic, and Streptococcus thermophilus, a fast acidifying lactic acid bacterial strain, are both used in the food industry. The aim of this study was to investigate the interaction between L. casei and S. thermophilus in the presence or absence of S. thermophilus-specific bacteriophage during milk fermentation. The acidification capability of L. casei co-cultured with S. thermophilus was significantly higher than that observed for L. casei or S. thermophilus cultured alone. However, the probiotic content (i.e., L. casei cell viability) was low. The fastest acidification and the highest viable L. casei cell count were observed in co-cultures of L. casei and S. thermophilus with S. thermophilus phage. In these co-cultures, S. thermophilus compensated for the slow acid production of L. casei in the early exponential growth phase. Thereafter, phage-induced lysis of the S. thermophilus cells eliminated the competition for nutrients, allowing L. casei to grow well. Additionally, the ruptured S. thermophilus cells released intracellular factors, which further promoted the growth and function of the probiotic bacteria. Crude cellular extract isolated from S. thermophilus also significantly accelerated the growth and propagation of L. casei, supporting the stimulatory role of the phage on this micro-ecosystem.

Several growth factors and cytokines, including EGF, are known to induce tyrosine phosphorylation of Signal Regulatory Proteins (SIRPs). Consistent with the idea that increased phosphorylation activates SIRP function, we overexpressed human SIRPalpha1 in U87MG glioblastoma cells in order to examine how SIRPalpha1 modulates EGFR signaling pathways. Endogenous EGFR proteins are overexpressed in U87MG cells and these cells exhibit survival and motility phenotypes that are influenced by EGFR kinase activity. Overexpression of the SIRPalpha1 cDNA diminished EGF-induced phosphoinositide-3-OH kinase (PI3-K) activation in U87MG cells. Reduced EGF-stimulated activation of PI3-K was mediated by interactions between carboxyl terminus of SIRPalpha1 and the Src homology-2 (SH2)-containing phosphotyrosine phosphatase, SHP2. SIRPalpha1 overexpression also reduced the EGF-induced association between SHP2 and the p85 regulatory subunit of PI3-K. Inhibition of transformation and enhanced apoptosis following gamma-irradiation were observed in SIRPalpha1-overexpressing U87MG cells, and enhanced apoptosis was associated with reduced levels of bcl-xL protein. Furthermore, SIRPalpha1-overexpressing U87MG cells displayed reduced cell migration and cell spreading that was mediated by association between SIRPalpha1 and SHP2. However, SIRPalpha1-overexpressing U87MG clonal derivatives exhibited no differences in cell growth or levels of mitogen-activated protein kinase (MAPK) activation. These data reveal a pathway that negatively regulates EGFR-induced PI3-K activation in glioblastoma cells and involves interactions between SHP2 and tyrosine phosphorylated SIRPalpha1. These results also suggest that negative regulation of PI3-K pathway activation by the SIRP family of transmembrane receptors may diminish EGFR-mediated motility and survival phenotypes that contribute to transformation of glioblastoma cells. Oncogene (2000) 19, 3999 - 4010.

Signal-regulatory proteins (SIRPs) comprise a novel transmembrane glycoprotein family involved in the negative regulation of receptor tyrosine kinase-coupled signaling pathways. To analyze the expression and function of SIRPs, we prepared soluble recombinant fusion proteins of the extracellular regions of SIRP1 and SIRP2, as well as a variety of monoclonal antibodies (MoAbs) against these domains. The antibodies reacted predominantly with monocytes, granulocytes, dendritic cells, and their precursors, as well as with bone marrow CD34+, AC133+, CD90+hematopoietic stem/progenitor cells. In contrast, SIRP expression was absent or significantly reduced on the majority of myeloid blasts from patients with acute myeloid leukemia (AML) or chronic myeloid leukemia (CML). Functional studies showed that the extracellular domains of SIRP1 and SIRP2 support adhesion of a number of primary hematopoietic cells and cell lines. This interaction could be blocked by 4 of 7 SIRP1-reactive MoAbs. In addition, SIRP1 and SIRP2 competed for the same cell binding site, suggesting a common widely expressed SIRP ligand. In an approach to identify this molecule, MoAbs were generated against the SIRP-binding cell line CCRF-CEM, and MoAb CC2C6 was selected because of its capacity to inhibit cell binding to SIRP1. Further analysis showed that this antibody recognized CD47, a ubiquitously expressed plasma membrane protein previously implicated in integrin function, host defense action, and neutrophil migration. In this study, we identify CD47 as the extracellular ligand for human SIRP and show that these two counterreceptors are involved in cellular adhesion.