Physiological methods to study biofilm disinfection

This report reviews the development of a rapidin situ approach to study the physiological responses of bacteria within biofilms to disinfectants. One method utilized direct viable counts (DVC) to assess the disinfection efficacy when thin biofilms were exposed to chlorine or monochloramine. Results obtained using the DVC method were one log higher than plate count (PC) estimates of the surviving population after disinfection. Other methods incorporated the use of fluorogenic stains, a cryotomy technique to yield thin (5-m) sections of biofilm communities and examination by fluorescence microscopy. The fluorogenic stains used in this approach included 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which indicates cellular electron transport activity and Rhodamine 123, which responds specifically to proton motive force. The use of these stains allowed the microscopic discrimination of physiologically active bacteria as well as heterogeneities of active cells within thicker biofilms. The results of experiments using these techniques with pure culture and binary population biofilms on stainless steel coupons indicated biocidal activity of chlorine-based disinfectants occurred initially at the bulk-fluid interface of the communities and progressed toward the substratum. This approach provided a unique opportunity to describe the spatial response of bacteria within biofilms to antimicrobial agents and address mechanisms explaining their comparative resistance to disinfection in a way that has not been possible using traditional approaches. Results obtained using this alternative approach were also consistently higher than PC data following disinfection. These observations suggest that traditional methods involving biofilm removal and bacterial enumeration by colony formation overestimate biocide efficacy. Hence the alternative approach described here more accurately indicates the ability of bacteria surviving disinfection to recover and grow as well as demonstrate spatial heterogeneities in cellular physiological activities within biofilms.     

Micronuclei as a marker for medical screening of subjects continuously occupationally exposed to low doses of ionizing radiation

Context: Genotoxicity assays are widely employed in human biomonitoring studies to assess genetic damage inflicted by genotoxic agents.

Objective: Evaluation of micronuclei (MN) as a screening marker of occupational ionizing radiation (IR) exposure.

Materials and methods: Using micronucleus test, peripheral blood lymphocytes (PBL) of 402 control and exposed subjects were screened for genetic damage.

Results: The mean frequencies of micronucleus test parameters were significantly higher in exposed persons. Increase of micronucleus yield with duration of exposure (DOE) by 0.303MN/year was revealed.

Discussion and conclusion: The obtained data encourage us to consider MN as valuable markers for preventive medical screening of occupationally exposed groups.     

Arsenic and Heavy Metal Concentrations in Drinking Water in Pakistan and Risk Assessment: A Case Study

The present study was performed to assess drinking water quality and potential health risk in the Nowshera District, Khyber Pakhtunkhwa, Pakistan. For this purpose drinking water samples were collected from local available sources and analyzed for physico-chemical characteristics, arsenic (As) and heavy metals. Results revealed high levels of toxic heavy metals such as chromium (Cr), nickel (Ni), lead (Pb), cadmium (Cd), and As contaminations in the drinking water. Results were evaluated for chronic risk including average daily intake (ADI) and hazard quotient (HQ). Among heavy metals the HQ values were highest for Cd (5.80) and As (2.00). Therefore, populations in the study area may be at a low level of chronic toxicity and carcinogenic risk. Statistical analyses showed that contribution of different drinking water sources to the mean contaminant levels in the study area was insignificant (p =.53). Correlation analysis further revealed that anthropogenic activities were the main sources of contamination, rather than geogenic. This study strongly recommends the treatment of urban and industrial wastewater in the vicinity of the study area and provision of safe drinking water.     

EhMAPK, the mitogen-activated protein kinase from Entamoeba histolytica is associated with cell survival

Mitogen Activated Protein Kinases (MAPKs) are a class of serine/threonine kinases that regulate a number of different cellular activities including cell proliferation, differentiation, survival and even death. The pathogen Entamoeba histolytica possess a single homologue of a typical MAPK gene (EhMAPK) whose identification was previously reported by us but its functional implications remained unexplored. EhMAPK, the only mitogen-activated protein kinase from the parasitic protist Entamoeba histolytica with Threonine-X-Tyrosine (TXY) phosphorylation motif was cloned, expressed in E. coli and functionally characterized under different stress conditions. The expression profile of EhMAPK at the protein and mRNA level remained similar among untreated, heat shocked and hydrogen peroxide-treated samples in all cases of dose and time. But a significant difference was obtained in the phosphorylation status of the protein in response to different stresses. Heat shock at 43°C or 0.5 mM H(2)O(2) treatment enhanced the phosphorylation status of EhMAPK and augmented the kinase activity of the protein whereas 2.0 mM H(2)O(2) treatment induced dephosphorylation of EhMAPK and loss of kinase activity. 2.0 mM H(2)O(2) treatment reduced parasite viability significantly but heat shock and 0.5 mM H(2)O(2) treatment failed to adversely affect E. histolytica viability. Therefore, a distinct possibility that activation of EhMAPK is associated with stress survival in E. histolytica is seen. Our study also gives a glimpse of the regulatory mechanism of the protein under in vivo conditions. Since the parasite genome lacks any typical homologue of mammalian MEK, the dual specificity kinases which are the upstream activators of MAPK, indications of the existence of some alternate regulatory mechanisms of the EhMAPK activity is perceived. These may include the autophosphorylation activity of the protein itself in combination with some upstream phosphatases which are not yet identified.     

Multivariate analysis for various quantitative traits in wheat advanced lines

Genetic diversity play key role in the germplasm improvement which is directly correlated with the crop production. Various statistical techniques have been used to study diversity among different genotypes. Among these techniques multivariate is most frequently used one for the genetic association of genotypes. In the present study a total of 64 advance lines included one check cultivar were evaluated under the field conditions of Cereal Crop Research Institute, Pirsabaq Nowshera, Pakistan during September 2017. Data were recorded for nine different parameters. Multivariate analysis divided the total 64 genotypes into four groups. The first five PCs with Eigen values > 1 contributed 86.95% of the variability amongst genotypes. Characters with maximum values in PC1 were Spikelets spike^-1 (SPPS) (0.732), spike length (SPL) (0.722) and biological yield (BY) (0.607), PC2 comprised of 100-grain weight (TGW) (0.605), grain yield (GY) (0.482) while days to heading (DH) (0.393), for PC3 major contributors were BY (0.550) and number of tillers meter square^-1 (NTPS) (0.289), the contribution of PC4 were flag leaf area (FLA) (0.716) and SPL (0.298) and the maximum values for various traits in PC5 were SPPS (0.732), SPL (0.722) and BY (0.607). From the findings of present study best performing lines can be directly recommended for general cultivation or to be used in future breeding programs.     

An asymmetric enlargement of the monolayer surfaces mechanism of membrane fusion

A mathematical model of one of the mechanisms of membrane fusion is described. From the model, it follows that when the outer leaflet of a membrane formed of bilayer stabilizing phospholipids is enlarged over the inner leaflet, convexities are formed on the membrane surface. This asymmetric enlargement of the outer layer over the inner layer occurs when fusogenic peptides, such as cobra venom cytotoxin and bee venom melittin, interact with the outer membrane monolayer. This phenomenon facilitates not only membrane fusion, but it might also play an important role in physiological processes, such as inter- and intracellular communications and cellular motility.     

Ligand-based and structure-based approaches in identifying ideal pharmacophore against c-Jun N-terminal kinase-3

Structure and ligand based pharmacophore modeling and docking studies carried out using diversified set of c-Jun N-terminal kinase-3 (JNK3) inhibitors are presented in this paper. Ligand based pharmacophore model (LBPM) was developed for 106 inhibitors of JNK3 using a training set of 21 compounds to reveal structural and chemical features necessary for these molecules to inhibit JNK3. Hypo1 consisted of two hydrogen bond acceptors (HBA), one hydrogen bond donor (HBD), and a hydrophobic (HY) feature with a correlation coefficient (r²) of 0.950. This pharmacophore model was validated using test set containing 85 inhibitors and had a good r² of 0.846. All the molecules were docked using Glide software and interestingly, all the docked conformations showed hydrogen bond interactions with important hinge region amino acids (Gln155 and Met149) and these interactions were compared with Hypo1 features. The results of ligand based pharmacophore model (LBPM) and docking studies are validated each other. The structure based pharmacophore model (SBPM) studies have identified additional features, two hydrogen bond donors and one hydrogen bond acceptor. The combination of these methodologies is useful in designing ideal pharmacophore which provides a powerful tool for the discovery of novel and selective JNK3 inhibitors.     

Progranulin directly binds to the CRD2 and CRD3 of TNFR extracellular domains

We previously reported that PGRN directly bound to TNF receptors (TNFR) in vitro and in chondrocytes (Tang, et al., Science, 2011). Here we report that PGRN also associated with TNFR in splenocytes, and inhibited the binding of TNFα to immune cells. Proper folding of PGRN is essential for its binding to TNFR, as DTT treatment abolished its binding to TNFR. In contrast, the binding of PGRN to Sortilin was enhanced by DTT. Protein interaction assays with mutants of the TNFR extracellular domain demonstrated that CRD2 and CRD3 of TNFR are important for the interaction with PGRN, similar to the binding to TNFα. Taken together, these findings provide the molecular basis underlying PGRN/TNFR interaction and PGRN-mediated anti-inflammatory activity in various autoimmune diseases and conditions.     

Evaluating surface drying and re-drying for wheat seed priming with polyamines: effects on emergence,early seedling growth and starch metabolism

This study was conducted to investigate the benefits associated with re-drying after seed priming with polyamines. Wheat (cv. AS-2002) seeds were soaked in 10 and 20 mg L⁻¹ aerated solutions of spermidine (Spd), putrescine (Put) and spermine (Spm), and distilled water (CK2) for 12 h at 28 ± 2°C. Untreated seeds (CK1) and priming in distilled water (CK2) were taken as control treatments. Seeds were primed in two sets: In one set, after each treatment, seeds were given three surface washings with distilled water and dried closer to original moisture; in the other, seeds were only surface dried and used immediately. Use of surface-dried seeds after priming was more effective since it reduced emergence time and synchronized the emergence. Moreover, final emergence, shoot and root length, seedling fresh and dry weight were also improved. Improved starch metabolism was considered possible reason of seed invigoration. All the seed treatments resulted in a lower electrical conductivity of seed leachates compared with control; however, there was more decrease in seeds re-dried after priming than the seeds surface dried after priming. Although the effect of all the polyamines was stimulatory, Spd was the more effective for most of the attributes studied. Nonetheless, Put was more effective for seedling fresh and dry weights. All the polyamines were more effective at lower concentrations except Spm, which improved the coefficient of uniformity of emergence at high concentration. To conclude, if immediate sowing is possible, use of surface-dried seeds after priming may be more effective; seed priming with 10 mg L⁻¹ Spd was the most effective technique when surface dried.