The effects of metabolite molecules produced by drinking water-isolated bacteria on their single and multispecies biofilms

The elucidation of the mechanisms by which diverse species survive and interact in drinking water (DW) biofilm communities may allow the identification of new biofilm control strategies. The purpose of the present study was to investigate the effects of metabolite molecules produced by bacteria isolated from DW on biofilm formation. Six opportunistic bacteria, viz. Acinetobacter calcoaceticus, Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata and Staphylococcus sp. isolated from a drinking water distribution systems (DWDS) were used to form single and multispecies biofilms in the presence and absence of crude cell-free supernatants produced by the partner bacteria. Biofilms were characterized in terms of mass and metabolic activity. Additionally, several physiological aspects regulating interspecies interactions (sessile growth rates, antimicrobial activity of cell-free supernatants, and production of iron chelators) were studied to identify bacterial species with biocontrol potential in DWDS. Biofilms of Methylobacterium sp. had the highest growth rate and M. mucogenicum biofilms the lowest. Only B. cepacia was able to produce extracellular iron-chelating molecules. A. calcoaceticus, B. cepacia, Methylobacterium sp. and M. mucogenicum biofilms were strongly inhibited by crude cell-free supernatants from the other bacteria. The crude cell-free supernatants of M. mucogenicum and S. capsulata demonstrated a high potential for inhibiting the growth of counterpart biofilms. Multispecies biofilm formation was strongly inhibited in the absence of A. calcoaceticus. Only crude cell-free supernatants produced by B. cepacia and A. calcoaceticus had no inhibitory effects on multispecies biofilm formation, while metabolite molecules of M. mucogenicum showed the most significant biocontrol potential.     

Biofouling control using microparticles carrying a biocide

This study presents a new technological approach to minimize the use of antimicrobial (AMB) agents and their deleterious effects, based on the principle of drug-delivery systems whereby the AMB chemicals are transported on microparticles. The efficacy of microparticles carrying the quaternary ammonium compound (QAC), benzyldimethyldodecyl ammonium chloride (BDMDAC), was assessed against Pseudomonas fluorescens in both the planktonic and the biofilm state. The microparticles were prepared using a layer-by-layer (LBL) self-assembly technique. Oppositely charged molecules of polyethyleneimine (PEI), sodium polystyrene sulfonate (PSS), and BDMDAC were assembled on polystyrene (PS) cores. BDMDAC-coated particles were observed by CryoSEM and their composition analyzed by X-ray microanalysis. Zeta potential measurements indicated that changes in surface charge were compatible with a BDMDAC/particle interaction. This biocidal carrier structure had significant stability, verified by the release of only 15% of the BDMDAC when immersed in water for 18 months. Biocidal carrier activity was evaluated by determining the survival ratio of P. fluorescens planktonic and biofilm cells after different exposure periods to BDMDAC-coated particles. Tests with biofilm cells were also performed with the free QAC. An efficient AMB effect (minimum bactericidal concentration) against suspended cells was found for a concentration of 9.2 mg l^?1 of BDMDAC on coated particles after incubation for 30 min and 6.5 mg l^?1 of BDMDAC on coated particles after 60 min. Exposure of biofilms to PS-PEI/PSS/BDMDAC (0.87 mg l^?1) resulted in a decrease in viability of 60.5% and 66.5% of the total biofilm population for 30 and 60 min exposure times, respectively. Exposure for 60 min to 6.33 mg l^?1 and 11.75 mg l^?1 of BDMDAC in PS-PEI/PSS/BDMDAC particles promoted inactivation of 80.6% and 87.2% of the total population, respectively. The AMB effects obtained with the application of free BDMDAC were statistically similar to those promoted by the application of BDMDAC coated particles. The overall results indicate that this novel AMB strategy has potential for the control of microbial growth of planktonic cells and biofouling. Moreover, the technique allows the reuse of AMB molecules and consequently reduces the environmental risks associated with excessive use of AMB agents, thereby providing real benefits to public health.     

Ultrastructure of enamel and dentine in extant dolphins (Cetacea: Delphinoidea and Inioidea)

Longitudinal and cross sections of teeth from 17 species of the Recent dolphins (Delphinoidea and Inioidea) were examined under scanning electron microscope to study the arrangement and ultrastructure of dental tissues with reference to phylogenetic and functional constraints. For most species, enamel had a simple bi-layered structure of radial enamel and an outer layer of prismless enamel. The outer prismless layer varied from 5 to 30 % of enamel thickness. The enamel of Burmeister’s porpoise (Phocoena spinipinnis) was entirely prismless. The prisms had an open sheath; tubules and tuft-like structures were common at the enamel-dentine junction. Cetacean dentine was characterized by irregularly distributed dentinal tubules in a relatively homogenous dentinal matrix. Radial enamel was observed in all Delphinoidea and in the franciscana (Pontoporia blainvillei), whereas the Amazon river dolphin (Inia geoffrensis) had prisms organized in Hunter–Schreger bands. HSB in enamel are regarded as a device for resisting propagation of cracks. These may occur due to increased functional demands, possibly related to the hardness of the species diet. Simplification in tooth shape and reduced biomechanical demands plausibly explain the primitive radial organization among delphinoids and Pontoporia. The HSB structure in the Amazon river dolphin, similar to those of extinct archaeocetes, seems to have secondary functional implications. However, the distribution of HSB in more-basal odontocetes is too poorly known to judge whether the HSB of Inia are a retained plesiomorphic feature or convergence.     

Cryptosporidium hominis Infection Diagnosed by Real-Time PCR-RFLP

There are approximately 20 known species of the genus Cryptosporidium, and among these, 8 infect immunocompetent or immunocompromised humans. C. hominis and C. parvum most commonly infect humans. Differentiating between them is important for evaluating potential sources of infection. We report here the development of a simple and accurate real-time PCR-based restriction fragment length polymorphism (RFLP) method to distinguish between C. parvum and C. hominis. Using the CP2 gene as the target, we found that both Cryptosporidium species yielded 224 bp products. In the subsequent RFLP method using TaqI, 2 bands (99 and 125 bp) specific to C. hominis were detected. Using this method, we detected C. hominis infection in 1 of 21 patients with diarrhea, suggesting that this method could facilitate the detection of C. hominis infections.     

Validation of a specific prolylcarboxypeptidase activity assay and its suitability for plasma and serum measurements

Prolylcarboxypeptidase (PRCP, EC 3.4.16.2), a lysosomal carboxypeptidase, was discovered 45 years ago. However, research has been hampered by a lack of well-validated assays that are needed to measure low activities in biological samples. Two reversed-phase high-performance liquid chromatography (RP-HPLC) methods for quantifying PRCP activity in crude homogenates and plasma samples were optimized and validated. PRCP activity was determined by measuring the hydrolysis of N-benzyloxycarbonyl-l-proline (Z-Pro)-Phe. The enzymatically formed Z-Pro and Phe were measured independently under different HPLC conditions. The in-house methods showed good precision, linearity, accuracy, and specificity. Based on Michaelis–Menten constants, Z-Pro-Phe was chosen over Z-Pro-Ala as the substrate of preference. Cross-reactivity studies with dipeptidyl peptidases (DPPs) 2, 4, and 9 and prolyl oligopeptidase (PREP) confirmed the specificity of the PRCP activity assay. The average PRCP activity in plasma and serum of 32 healthy individuals was found to be 0.65 ± 0.02 and 0.72 ± 0.03 U/L, respectively. Both methods can be used to measure PRCP activity specifically in different biological samples and are well suited to evaluate PRCP inhibitors. These well-validated methods are valuable tools for studying PRCP’s role in cardiovascular diseases, stroke, inflammation, and metabolic syndrome.     

Deep Whole-Genome Sequencing of 100 Southeast Asian Malays

Whole-genome sequencing across multiple samples in a population provides an unprecedented opportunity for comprehensively characterizing the polymorphic variants in the population. Although the 1000 Genomes Project (1KGP) has offered brief insights into the value of population-level sequencing, the low coverage has compromised the ability to confidently detect rare and low-frequency variants. In addition, the composition of populations in the 1KGP is not complete, despite the fact that the study design has been extended to more than 2,500 samples from more than 20 population groups. The Malays are one of the Austronesian groups predominantly present in Southeast Asia and Oceania, and the Singapore Sequencing Malay Project (SSMP) aims to perform deep whole-genome sequencing of 100 healthy Malays. By sequencing at a minimum of 30× coverage, we have illustrated the higher sensitivity at detecting low-frequency and rare variants and the ability to investigate the presence of hotspots of functional mutations. Compared to the low-pass sequencing in the 1KGP, the deeper coverage allows more functional variants to be identified for each person. A comparison of the fidelity of genotype imputation of Malays indicated that a population-specific reference panel, such as the SSMP, outperforms a cosmopolitan panel with larger number of individuals for common SNPs. For lower-frequency (<5%) markers, a larger number of individuals might have to be whole-genome sequenced so that the accuracy currently afforded by the 1KGP can be achieved. The SSMP data are expected to be the benchmark for evaluating the value of deep population-level sequencing versus low-pass sequencing, especially in populations that are poorly represented in population-genetics studies.     

Synthesis and biological evaluation of novel 3,4-diaryl lactam derivatives as triple reuptake inhibitors

A series of 3,4-diarylpyrrolidin-2-one was designed, prepared and evaluated as triple reuptake inhibitors for antidepressant. Most compounds exhibited comparable in vitro efficacy as norepinephrine and dopamine transporter reuptake inhibitors. Especially, 2i showed better potency than GBR-12909 (IC₅₀ = 14 nM) which was used as reference compound for dopamine transporter. In addition, 2a and 2b showed inhibition (5.17 μM–85.6 nM) for three transporters.