Community structure of microbial biofilms associated with membrane-based water purification processes as revealed using a polyphasic approach

The microbial communities of membrane biofilms occurring in two full-scale water purification processes employing microfiltration (MF) and reverse osmosis (RO) membranes were characterized using a polyphasic approach that employed bacterial cultivation, 16S rDNA clone library and fluorescence in situ hybridization techniques. All methods showed that the alpha-Proteobacteria was the largest microbial fraction in the samples, followed by the gamma-Proteobacteria. This suggested that members of these two groups could be responsible for the biofouling on the membranes studied. Furthermore, the microbial community structures between the MF and RO samples were considerably different in composition of the most predominant 16S rDNA clones and bacterial isolates from the alpha-Proteobacteria and only shared two common groups ( Bradyrhizobium, Bosea) out of more than 17 different bacterial groups observed. The MF and RO samples further contained Planctomycetes and Fibroacter/ Acidobacteria as the second predominant bacterial clones, respectively, and differed in minor bacterial clones and isolates. The community structure differences were mainly attributed to differences in feed water, process configurations and operating environments, such as the pressure and hydrodynamic conditions present in the water purification systems.     

Systematic analysis of snake neurotoxins' functional classification using a data warehousing approach

MOTIVATION: Sequence annotations, functional and structural data on snake venom neurotoxins (svNTXs) are scattered across multiple databases and literature sources. Sequence annotations and structural data are available in the public molecular databases, while functional data are almost exclusively available in the published articles. There is a need for a specialized svNTXs database that contains NTX entries, which are organized, well annotated and classified in a systematic manner. RESULTS: We have systematically analyzed svNTXs and classified them using structure-function groups based on their structural, functional and phylogenetic properties. Using conserved motifs in each phylogenetic group, we built an intelligent module for the prediction of structural and functional properties of unknown NTXs. We also developed an annotation tool to aid the functional prediction of newly identified NTXs as an additional resource for the venom research community. AVAILABILITY: We created a searchable online database of NTX proteins sequences (http://research.i2r.a-star.edu.sg/Templar/DB/snake_neurotoxin). This database can also be found under Swiss-Prot Toxin Annotation Project website (http://www.expasy.org/sprot/).     

Purification and biochemical characterization of a pyruvate-specific class II aldolase, HpaI

HpaI, a class II pyruvate-specific aldolase involved in the catabolic pathway of hydroxyphenylacetate, is overexpressed and purified. A previous suggestion that phosphate is involved in proton transfer of pyruvate, based on the crystal structure of the homologous 2-dehydro-3-deoxygalactarate aldolase, is not substantiated from biochemical studies with HpaI. Thus, specific activities of the enzyme for the substrate 4-hydroxy-2-ketopentanoate in sodium HEPES and Tris-acetate buffers are higher than in sodium phosphate buffer. The enzyme also catalyzed the partial reaction of pyruvate proton exchange with an initial rate of 0.77 mmol min(-)(1) mg(-)(1) in phosphate-free buffer, as monitored by nuclear magnetic resonance. Steady-state kinetic analysis shows that the enzyme is also able to catalyze the aldol cleavage of 4-hydroxy-2-ketohexanoate and 3-deoxy-d-manno-oct-2-ulosonic acid (KDO). The enzyme exhibits significant oxaloacetate decarboxylase activity, with a k(cat) value 2.4-fold higher than the corresponding value for the aldol cleavage of 4-hydroxy-2-ketopentanoate. Sodium oxalate, an analogue of the enolate intermediate of the enzyme-catalyzed reaction, is a competitive inhibitor of the enzyme, with a K(i) value of 5.5 microM. Replacement of an active site arginine residue (R70) with alanine by site-specific mutagenesis resulted in an enzyme that lacks both aldolase and decarboxylase activities. The mutant enzyme is also unable to catalyze pyruvate proton exchange. The dissociation constant for pyruvate in the R70A mutant, determined by fluorescence titration, is similar to that of the wild-type enzyme, indicating that pyruvate binding is not affected by this mutation. Together, the results show that R70 influences catalysis in HpaI, particularly at the pyruvate proton exchange step.     

White spot syndrome virus (WSSV) infects specific hemocytes of the shrimp Penaeus merguiensis

White spot syndrome virus (WSSV) was specifically detected by PCR in Penaeus merguiensis hemocytes, hemolymph and plasma. This suggested a close association between the shrimp hemolymph and the virus. Three types of hemocyte from shrimp were isolated using flow cytometry. Dynamic changes of the hemocyte subpopulations in P. merguiensis at different times after infection were observed, indicating that the WSSV infection selectively affected specific subpopulations. Immunofluorescence assay (IFA) and a Wright-Giemsa double staining study of hemocyte types further confirmed the cellular localization of the virus in the infected hemocytes. Electron microscopy revealed virus particles in both vacuoles and the nucleus of the semigranular cells (SGC), as well as in the vacuoles of the granular cells (GC). However, no virus could be detected in the hyaline cells (HC). Our results suggest that the virus infects 2 types of shrimp hemocytes--GCs and SGCs. The SGC type contains higher virus loads and exhibits faster infection rates, and is apparently more susceptible to WSSV infection.     

Length–weight relationships of seven fish species from a fish landing port at Sungai Udang,Penang, Malaysia

The length–weight relationships (LWRs) are described for seven fish species from four families (Ariidae, Clupeidae, Leiognathidae and Sciaenidae) inhabiting Penang waters. A total of 471 specimens were sampled and measured from 19 to 20 January 2015, using a bottom trawl net with a 38 mm mesh size of cod‐end. No information regarding LWRs was reported previously in FishBase for Cryptarius truncatus, Escualosa thoracata, Johnius carouna, Nuchequula blochii, Plicofollis argyropleuron, Secutor hanedai or Secutor megalolepis, and the study also provides a new FishBase record of maximum total length for Escualosa thoracata (10.6 cm), Secutor hanedai (9.1 cm) and Secutor megalolepis (7.5 cm).     

Characterization of a phage specific to hemorrhagicEscherichia coli O157:H7 and disclosure of variations in host outer membrane protein OmpC

Phage AR1, previously known to infectEscherichia coli O157:H7 with high specificity, was further characterized for its genetic properties. The phage DNA sequences including capsid genes and a putative -glucosyltransferase gene(-gt) have been deduced. These sequences are conservative but not identical to those of T4 phage. However, a nonessential gene,SegD, organized within the capsid gene cluster of T4 is missing in the corresponding region of AR1 genome, and this characteristic has not been observed among T-even related phages. The difference between AR1 and T4 was further exemplified by their distinct host ranges. Strains ofE. coli O157:H7 collected from different sources were permissive to AR1 but resistant to T4 that normally infects K-12 strains ofE. coli through contact with the outer membrane protein OmpC. Thus, the O157:H7 strains must have a varied OmpC. Indeed, the OmpC sequence of O157:H7 strains was proved to differ from that of K-12 strains by a total of 15 amino acid substitutions and two gaps (a five-residue deletion and a four-residue insertion). The OmpC molecules are relatively conserved across the gram-negative bacteria, and this is the first time OmpC divergence has been found within the sameE. coli species. Since OmpC is located in the outer membrane and its expression is regulated by environmental conditions, alteration of the structure in pathogenic O157:H7 strains may have biological significance.     

Mebendazole in the treatment of helminthiasis.

Mebendazole, a new broad-spectrum anthelmintic, was used to treat patients with nematode infections--ascariasis, trichuriasis and hookworm. The dosage for adults was 100 mg twice daily for 3 days and for children, 50 mg twice daily for 3 days. Pretreatment and post-treatment egg counts on stool specimens showed that after mebendazole there was a reduction of over 99% in egg count per gram of stool in all three types of infection. The overall cure rates for the infections were as follows: Ascaris lumbricoides, 86.8% (59/68); Trichuris trichiura, 86.0% (37/43); and hookworm, 85.7% (24/28). The drug was equally effective in light and heavy infections. No important side effect was noted with this drug. It is suggested that mebendazole is the drug of choice for trichuriasis and mixed nematode infection.     

The tautomeric half-reaction of BphD, a C-C bond hydrolase. Kinetic and structural evidence supporting a key role for histidine 265 of the catalytic triad

BphD of Burkholderia xenovorans LB400 catalyzes an unusual C-C bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to afford benzoic acid and 2-hydroxy-2,4-pentadienoic acid (HPD). An enol-keto tautomerization has been proposed to precede hydrolysis via a gem-diol intermediate. The role of the canonical catalytic triad (Ser-112, His-265, Asp-237) in mediating these two half-reactions remains unclear. We previously reported that the BphD-catalyzed hydrolysis of HOPDA (lambda(max) is 434 nm for the free enolate) proceeds via an unidentified intermediate with a red-shifted absorption spectrum (lambda(max) is 492 nm) (Horsman, G. P., Ke, J., Dai, S., Seah, S. Y. K., Bolin, J. T., and Eltis, L. D. (2006) Biochemistry 45, 11071-11086). Here we demonstrate that the S112A variant generates and traps a similar intermediate (lambda(max) is 506 nm) with a similar rate, 1/tau approximately 500 s(-1). The crystal structure of the S112A:HOPDA complex at 1.8-A resolution identified this intermediate as the keto tautomer, (E)-2,6-dioxo-6-phenyl-hex-3-enoate. This keto tautomer did not accumulate in either the H265A or the S112A/H265A double variants, indicating that His-265 catalyzes tautomerization. Consistent with this role, the wild type and S112A enzymes catalyzed tautomerization of the product HPD, whereas H265A variants did not. This study thus identifies a keto intermediate, and demonstrates that the catalytic triad histidine catalyzes the tautomerization half-reaction, expanding the role of this residue from its purely hydrolytic function in other serine hydrolases. Finally, the S112A:HOPDA crystal structure is more consistent with hydrolysis occurring via an acyl-enzyme intermediate than a gem-diol intermediate as solvent molecules have poor access to C6, and the closest ordered water is 7 A away.     

Cyclophosphamide in Treatment of Systemic Lupus Erythematosus: 7 Years' Experience

This paper describes our experience with cyclophosphamide in the treatment of systemic lupus erythematosus. Since 1965 42 such patients have been treated either singly with cyclophosphamide or in combination with steroid. Serious complications have been rare except for amenorrhoea, which occurred in 14 out of 32 patients within the reproductive period. Our experience suggests that cyclophosphamide has an important, though not primary, part to play in the therapy of this disease.