Construction of amperometric uric acid biosensor based on uricase immobilized on PBNPs/cMWCNT/PANI/Au composite

A chitosan-glutaraldehyde crosslinked uricase was immobilized onto Prussian blue nanoparticles (PBNPs) absorbed onto carboxylated multiwalled carbon nanotube (c-MWCNT) and polyaniline (PANI) layer, electrochemically deposited on the surface of Au electrode. The nanohybrid-uricase electrode was characterized by scanning electron microscopic (SEM), Fourier transform infrared spectroscopy (FTIR) and cyclic voltammetry. An amperometric uric acid biosensor was fabricated using uricase/c-MWCNT/PBNPs/Au electrode as working electrode, Ag/AgCl as standard and Pt wire as auxiliary electrode connected through a potentiostat. The biosensor showed optimum response within 4 s at pH 7.5 and 40 °C, when operated at 0.4 V vs. Ag/AgCl. The linear working range for uric acid was 0.005-0.8 mM, with a detection limit of 5 μM. The sensor was evaluated with 96% recovery of added uric acid in sera and 4.6 and 5.4% within and between batch of coefficient of variation respectively and a good correlation (r = 0.99) with standard enzymic colorimetric method. This sensor measured uric acid in real serum samples. The sensor lost only 37% of its initial activity after its 400 uses over a period of 7 months, when stored at 4 °C.     

Kinetic and equilibrium studies on the biosorption of reactive black 5 dye by Aspergillus foetidus

An isolated fungus, Aspergillus foetidus had the ability to decolourize growth unsupportive medium containing 100 mg L(-1) of reactive black 5 (RB5) dye with >99% efficiency at acidic pH (2-3). Pre-treatment of fungal biomass by autoclaving or exposure to 0.1M sodium hydroxide facilitated more efficient uptake of dye as compared to untreated fungal biomass. Pre-equilibrium biosorption of RB5 dye onto fungus under different temperatures followed pseudo-second-order kinetic model with high degree of correlation coefficients (R(2)>0.99). Biosorption isotherm data fitted better into Freundlich model for lower concentrations of dye probably suggesting the heterogeneous nature of sorption process. Based on the Langmuir isotherm plots the maximum biosorption capacity (Q(0)) value was calculated to be 106 mg g(-1) at 50 degrees C for fungal biomass pre-treated with 0.1M NaOH. Thermodynamic studies revealed that the biosorption process was favourable, spontaneous and endothermic in nature. Recovery of both adsorbate (dye) and adsorbent (fungal biomass) was possible using sodium hydroxide. Recovered fungal biomass could be recycled number of times following desorption of dye using 0.1M NaOH. Fungal biomass pre-treated with NaOH was efficient in decolourizing solution containing mixture of dyes as well as composite raw industrial effluent generated from leather, pharmaceutical and dye manufacturing company.     

Synergistic repression of anaerobic genes by Mot3 and Rox1 in Saccharomyces cerevisiae

Two groups of anaerobic genes (genes induced in anaerobic cells and repressed in aerobic cells) are negatively regulated by heme, a metabolite present only in aerobic cells. Members of both groups, the hypoxic genes and the DAN/TIR/ERG genes, are jointly repressed under aerobic conditions by two factors. One is Rox1, an HMG protein, and the second, originally designated Rox7, is shown here to be Mot3, a global C2H2 zinc finger regulator. Repression of anaerobic genes results from co-induction of Mot3 and Rox1 in aerobic cells. Repressor synthesis is triggered by heme, which de-represses a mechanism controlling expression of both MOT3 and ROX1 in anaerobic cells; it includes Hap1, Tup1, Ssn6 and a fourth unidentified factor. The constitutive expression of various anaerobic genes in aerobic rox1Δ or mot3Δ cells directly implies that neither factor can repress by itself at endogenous levels and that stringent aerobic repression results from the concerted action of both. Mot3 and Rox1 are not essential components of a single complex, since each can repress independently in the absence of the other, when artificially induced at high levels. Moreover, the two repression mechanisms appear to be distinct: as shown here repression of ANB1 by Rox1 alone requires Tup1–Ssn6, whereas repression by Mot3 does not. Though artificially high levels of either factor can repress well, the absolute efficiency observed in normal cells when both are present—at much lower levels—demonstrates a novel inhibitory synergy. Evidently, expression levels for the two mutually dependent repressors are calibrated to permit a range of variation in basal aerobic expression at different promoters with differing operator site combinations.     

Genomic sequence of an otitis media isolate of nontypeable Haemophilus influenzae: comparative study with H. influenzae serotype d, strain KW20

In 1995, the Institute for Genomic Research completed the genome sequence of a rough derivative of Haemophilus influenzae serotype d, strain KW20. Although extremely useful in understanding the basic biology of H. influenzae, these data have not provided significant insight into disease caused by nontypeable H. influenzae, as serotype d strains are not pathogens. In contrast, strains of nontypeable H. influenzae are the primary pathogens of chronic and recurrent otitis media in children. In addition, these organisms have an important role in acute otitis media in children as well as other respiratory diseases. Such strains must therefore contain a gene repertoire that differs from that of strain Rd. Elucidation of the differences between these genomes will thus provide insight into the pathogenic mechanisms of nontypeable H. influenzae. The genome of a representative nontypeable H. influenzae strain, 86-028NP, isolated from a patient with chronic otitis media was therefore sequenced and annotated. Despite large regions of synteny with the strain Rd genome, there are large rearrangements in strain 86-028NP's genome architecture relative to the strain Rd genome. A genomic island similar to an island originally identified in H. influenzae type b is present in the strain 86-028NP genome, while the mu-like phage present in the strain Rd genome is absent from the strain 86-028NP genome. Two hundred eighty open reading frames were identified in the strain 86-028NP genome that were absent from the strain Rd genome. These data provide new insight that complements and extends the ongoing analysis of nontypeable H. influenzae virulence determinants.     

Protein as Chemical Cue: Non-Nutritional Growth Enhancement by Exogenous Protein in Pseudomonas putida KT2440

Research pertaining to microbe-microbe and microbe-plant interactions has been largely limited to small molecules like quorum sensing chemicals. However, a few recent reports have indicated the role of complex molecules like proteins and polysaccharides in microbial communication. Here we demonstrate that exogenous proteins present in culture media can considerably accelerate the growth of Pseudomonas putida KT2440, even when such proteins are not internalized by the cells. The growth enhancement is observed when the exogenous protein is not used as a source of carbon or nitrogen. The data show non-specific nature of the protein inducing growth; growth enhancement was observed irrespective of the protein type. It is shown that growth enhancement is mediated via increased siderophore secretion in response to the exogenous protein, leading to better iron uptake. We highlight the ecological significance of the observation and hypothesize that exogenous proteins serve as chemical cues in the case of P.putida and are perceived as indicator of the presence of competitors in the environment. It is argued that enhanced siderophore secretion in response to exogenous protein helps P.putida establish numerical superiority over competitors by way of enhanced iron assimilation and quicker utilization of aromatic substrates.     

Human vocal cord infection with the Microsporidium Anncaliia algerae

We describe a biopsy proven case of microsporidial infection of the false vocal cords in a 69-yr-old male with a history of chronic lymphocytic leukemia. The patient had hoarseness for several weeks before his admission to the hospital for shortness of breath. He had received chemotherapy with fludarabine 6 wk before this hospital admission. A biopsy of vocal cord nodules demonstrated an organism that was identified as Anncaliia algerae by electron microscopy. Molecular analysis of the small subunit RNA gene amplified by polymerase chain reaction further confirmed the identification of this organism as A. algerae. This case illustrates the ability of this insect pathogen to cause disease in immune-compromised mammalian hosts.     

Spatial and seasonal changes of net plankton and zoobenthos in Lake Tonle Sap, Cambodia

To clarify spatial and seasonal differences in net plankton and zoobenthos in Lake Tonle Sap, Cambodia, quantitative surveys were carried out at 14 stations in the north and south basins in high- and low-water seasons during 2003–2005. In the phytoplankton communities, a diatom Aulacoseira granulata dominated throughout the lake in the high-water seasons, while blue-green algae, mostly composed of Microcystis, surpassed other algae in the low-water season when the lake water was very turbid and the Secchi disk readings were only a few centimeters. In the low-water seasons, a bloom of floating blue-green algae occurred everywhere, especially prominent in the coastal areas. Protozoans and rotifers dominated the zooplankton communities. In the open-water stations, diversity was higher in high-water seasons in phytoplankton, while it was not significantly different between seasons in zooplankton. Composition of plankton communities in Lake Tonle Sap appears to have changed little since the 1950s, at least in phytoplankton, while the phytoplankton density appears to be higher in the present study. Among the macrozoobenthos, mollusks, oligochaetes and chironomids dominated in density, and mollusks exceeded others in biomass in both basins and seasons. The total densities of macrozobenthos were not high, being fewer than 1,300 m⁻² throughout the stations and seasons. Possible reasons for the low zoobenthos abundance in the lake may include high predation pressures by benthivorous fish or unfavorable unstable and flocculant substrates.     

Molecular architectures of trimeric SIV and HIV-1 envelope glycoproteins on intact viruses: strain-dependent variation in quaternary structure

The initial step in target cell infection by human, and the closely related simian immunodeficiency viruses (HIV and SIV, respectively) occurs with the binding of trimeric envelope glycoproteins (Env), composed of heterodimers of the viral transmembrane glycoprotein (gp41) and surface glycoprotein (gp120) to target T-cells. Knowledge of the molecular structure of trimeric Env on intact viruses is important both for understanding the molecular mechanisms underlying virus-cell interactions and for the design of effective immunogen-based vaccines to combat HIV/AIDS. Previous analyses of intact HIV-1 BaL virions have already resulted in structures of trimeric Env in unliganded and CD4-liganded states at ∼20 Å resolution. Here, we show that the molecular architectures of trimeric Env from SIVmneE11S, SIVmac239 and HIV-1 R3A strains are closely comparable to that previously determined for HIV-1 BaL, with the V1 and V2 variable loops located at the apex of the spike, close to the contact zone between virus and cell. The location of the V1/V2 loops in trimeric Env was definitively confirmed by structural analysis of HIV-1 R3A virions engineered to express Env with deletion of these loops. Strikingly, in SIV CP-MAC, a CD4-independent strain, trimeric Env is in a constitutively “open” conformation with gp120 trimers splayed out in a conformation similar to that seen for HIV-1 BaL Env when it is complexed with sCD4 and the CD4i antibody 17b. Our findings suggest a structural explanation for the molecular mechanism of CD4-independent viral entry and further establish that cryo-electron tomography can be used to discover distinct, functionally relevant quaternary structures of Env displayed on intact viruses.     

Balance of bacterial pro- and anti-inflammatory mediators dictates net effect of enteropathogenic Escherichia coli on intestinal epithelial cells

Enteropathogenic Escherichia coli (EPEC) virulence requires a type III secretion system (TTSS) to deliver effector molecules in host cells. Although the TTSS is crucial to EPEC pathogenesis, its function in EPEC-induced inflammation is not known. The aim of this study was to investigate the role of the TTSS in EPEC-induced inflammation. HT-29 intestinal epithelial cells were infected with wild-type (WT) EPEC or select mutant strains or exposed to corresponding filter-sterilized supernatants (SN), and interleukin-8 (IL-8) secretion was determined by ELISA. EPEC SN stimulated significantly greater IL-8 production than EPEC organisms. Flagellin, as well as a TTSS-independent >50-kDa nonflagellin protein, was found to significantly contribute to this response. Dose-response studies showed that increasing concentrations of WT SN proportionally increased IL-8, whereas increasing multiplicity of infection of EPEC inversely correlated with IL-8 secretion, suggesting that EPEC dampens this host response. Infection with DeltaescN (nonfunctional TTSS) markedly increased IL-8 compared with WT, indicating that a functional TTSS is required for this anti-inflammatory property; complementation of escN restored the attenuated response. Mutation of espB also enhanced the IL-8 response, and complementation returned IL-8 to near WT levels, suggesting involvement of this effector. The anti-inflammatory effect extends to both bacterial and host-derived proinflammatory stimuli, since prior infection with EPEC suppressed the IL-8 response to tumor necrosis factor-alpha, IL-1beta, and enterohemorrhagic E. coli flagellin. These findings indicate that EPEC-induced inflammation is a balance between pro- and anti-inflammatory proteins; extracellular factors, including flagellin and an unidentified TTSS-independent, >50-kDa protein, trigger inflammation while intracellular TTSS-dependent factors, including EspB, attenuate this response.     

Ecto-5'-nucleotidase (CD73) decreases mortality and organ injury in sepsis

The extracellular concentrations of adenosine are increased during sepsis, and adenosine receptors regulate the host's response to sepsis. In this study, we investigated the role of the adenosine-generating ectoenzyme, ecto-5'-nucleotidase (CD73), in regulating immune and organ function during sepsis. Polymicrobial sepsis was induced by subjecting CD73 knockout (KO) and wild type (WT) mice to cecal ligation and puncture. CD73 KO mice showed increased mortality in comparison with WT mice, which was associated with increased bacterial counts and elevated inflammatory cytokine and chemokine concentrations in the blood and peritoneum. CD73 deficiency promoted lung injury, as indicated by increased myeloperoxidase activity and neutrophil infiltration, and elevated pulmonary cytokine levels. CD73 KO mice had increased apoptosis in the thymus, as evidenced by increased cleavage of caspase-3 and poly(ADP-ribose) polymerase and increased activation of NF-κB. Septic CD73 KO mice had higher blood urea nitrogen levels and increased cytokine levels in the kidney, indicating increased renal dysfunction. The increased kidney injury of CD73 KO mice was associated with augmented activation of p38 MAPK and decreased phosphorylation of Akt. Pharmacological inactivation of CD73 in WT mice using α, β-methylene ADP augmented cytokine levels in the blood and peritoneal lavage fluid. These findings suggest that CD73-derived adenosine may be beneficial in sepsis.