The beta-glucoside genes of Klebsiella aerogenes: conservation and divergence in relation to the cryptic bgl genes of Escherichia coli

The ability to metabolize aromatic beta-glucosides such as salicin and arbutin varies among members of the Enterobacteriaceae. The ability of Escherichia coli to degrade salicin and arbutin appears to be cryptic, subject to activation of the bgl genes, whereas many members of the Klebsiella genus can metabolize these sugars. We have examined the genetic basis for beta-glucoside utilization in Klebsiella aerogenes. The Klebsiella equivalents of bglG, bglB and bglR have been cloned using the genome sequence database of Klebsiella pneumoniae. Nucleotide sequencing shows that the K. aerogenes bgl genes show substantial similarities to the E. coli counterparts. The K. aerogenes bgl genes in multiple copies can also complement E. coli mutants deficient in bglG encoding the antiterminator and bglB encoding the phospho-beta-glucosidase, suggesting that they are functional homologues. The regulatory region bglR of K aerogenes shows a high degree of similarity of the sequences involved in BglG-mediated regulation. Interestingly, the regions corresponding to the negative elements present in the E. coli regulatory region show substantial divergence in K aerogenes. The possible evolutionary implications of the results are discussed. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.v. All rights reserved.

Multicomponent solids of lamotrigine with some selected coformers and their characterization by thermoanalytical, spectroscopic and X-ray diffraction methods

The present study investigates the structural and pharmaceutical properties of different multicomponent crystalline forms of lamotrigine (LTG) with some pharmaceutically acceptable coformers viz. nicotinamide (1), acetamide (2), acetic acid (3), 4-hydroxy-benzoic acid (4) and saccharin (5). The structurally homogeneous phases were characterized in the solid state by DSC/TGA, FT-IR and XRD (powder and single crystal structure analysis) as well as in the solution phase. Forms 1 and 2 were found to be cocrystal hydrate and cocrystal, respectively, while in forms 3, 4 and 5, proton transfer was observed from coformer to drug. The enthalpy of formation of multicomponent crystals from their components was determined from the enthalpy of solution of the cocrystals and the components separately. Higher exothermic values of the enthalpy of formation for molecular complexes 3, 4 and 5 suggest these to be more stable than 1 and 2. The solubility was measured in water as well as in phosphate buffers of varying pH. The salt solvate 3 exhibited the highest solubility of the drug in water as well as in buffers over the pH range 7-3 while the cocrystal hydrate 1 showed the maximum solubility in a buffer of pH 2. A significant lowering of the dosage profile of LTG was observed for 1, 3 and 5 in the animal activity studies on mice.

Catalytic activity of Peptidase N is required for adaptation of Escherichia coli to nutritional downshift and high temperature stress

Peptidase N (PepN), the sole M1 family member in Escherichia coli, displays broad substrate specificity and modulates stress responses: it lowers resistance to sodium salicylate (NaSal)-induced stress but is required during nutritional downshift and high temperature (NDHT) stress. The expression of PepN does not significantly change during different growth phases in LB or NaSal-induced stress; however, PepN amounts are lower during NDHT stress. To gain mechanistic insights on the roles of catalytic activity of PepN in modulating these two stress responses, alanine mutants of PepN replacing E264 (GAMEN motif) and E298 (HEXXH motif) were generated. There are no major structural changes between purified wild type (WT) and mutant proteins, which are catalytically inactive. Importantly, growth profiles of Delta pepN upon expression of WT or mutant proteins demonstrated the importance of catalytic activity during NDHT but not NaSal-induced stress. Further fluorescamine reactivity studies demonstrated that the catalytic activity of PepN is required to generate higher intracellular amounts of free N-terminal amino acids; consequently, the lower growth of Delta pepN during NDHT stress increases with high amounts of casamino acids. Together, this study sheds insights on the expression and functional roles of the catalytic activity of PepN during adaptation to NDHT stress. (C) 2012 Elsevier GmbH. All rights reserved.

Heat Shock Protein 90 Inhibitors as Broad Spectrum Anti-Infectives

Combating stress is one of the prime requirements for any organism. For parasitic microbes, stress levels are highest during the growth inside the host. Their survival depends on their ability to acclimatize and adapt to new environmental conditions. Robust cellular machinery for stress response is, therefore, both critical and essential especially for pathogenic microorganisms. Microbes have cleverly exploited stress proteins as virulence factors for pathogenesis in their hosts. Owing to its ability to sense and respond to the stress conditions, Heat shock protein 90 (Hsp90) is one of the key stress proteins utilized by parasitic microbes. There are growing evidences for the critical role played by Hsp90 in the growth of pathogenic organisms like Candida, Giardia, Plasmodium, Trypanosoma, and others. This review, therefore, explores potential of exploiting Hsp90 as a target for the treatment of infectious diseases. This molecular chaperone has already gained attention as an effective anti-cancer drug target. As a result, a lot of research has been done at laboratory, preclinical and clinical levels for several Hsp90 inhibitors as potential anti-cancer drugs. In addition, lot of data pertaining to toxicity studies, pharmacokinetics and pharmacodynamics studies, dosage regime, drug related toxicities, dose limiting toxicities as well as adverse drug reactions are available for Hsp90 inhibitors. Therefore, repurposing/repositioning strategies are also being explored for these compounds which have gone through advanced stage clinical trials. This review presents a comprehensive summary of current status of development of Hsp90 as a drug target and its inhibitors as candidate anti-infectives. A particular emphasis is laid on the possibility of repositioning strategies coupled with pharmaceutical solutions required for fulfilling needs for ever growing pharmaceutical infectious disease market.

Chitosan-dextran sulphate nanocapsule drug delivery system as an effective therapeutic against intraphagosomal pathogen Salmonella

Objectives: The ability to target conventional drugs efficiently inside cells to kill intraphagosomal bacteria has been a major hurdle in treatment of infective diseases. We aimed to develop an efficient drug delivery system for combating infection caused by Salmonella, a well-known intracellular and intraphagosomal pathogen. Chitosan dextran sulphate (CD) nanocapsules were assessed for their efficiency in delivering drugs against Salmonella. Methods: The CD nanocapsules were prepared using the layer-by-layer method and loaded with ciprofloxacin or ceftriaxone. Antibiotic-loaded nanocapsules were analysed in vitro for their ability to enter epithelial and macrophage cells to kill Salmonella. In vivo pharmacokinetics and organ distribution studies were performed to check the efficiency of the delivery system. The in vivo antibacterial activity of free antibiotic and antibiotic loaded into nanocapsules was tested in a murine salmonellosis model. Results: In vitro and in vivo experiments showed that this delivery system can be used effectively to clear Salmonella infection, CD nanocapsules were successfully employed for efficient targeting and killing of the intracellular pathogen at a dosage significantly lower than that of the free antibiotic. The increased retention time of ciprofloxacin in the blood and organs when it was delivered by CD nanocapsules compared with the conventional routes of administration may be the reason underlying the requirement for a reduced dosage and frequency of antibiotic administration. Conclusions: CD nanocapsules can be used as an efficient drug delivery system to treat intraphagosomal pathogens, especially Salmonella infection, This delivery system might be used effectively for other vacuolar pathogens including Mycobacteria, Brucella and Legionella.

Pre-clinical toxicity & immunobiological evaluation of DNA rabies vaccine & combination rabies vaccine in rhesus monkeys (Macaca mulatta)

Background & objectives: Pre-clinical toxicology evaluation of biotechnology products is a challenge to the toxicologist. The present investigation is an attempt to evaluate the safety profile of the first indigenously developed recombinant DNA anti-rabies vaccine DRV (100 mu g)] and combination rabies vaccine CRV (100 mu g DRV and 1.25 IU of cell culture-derived inactivated rabies virus vaccine)], which are intended for clinical use by intramuscular route in Rhesus monkeys. Methods: As per the regulatory requirements, the study was designed for acute (single dose - 14 days), sub-chronic (repeat dose - 28 days) and chronic (intended clinical dose - 120 days) toxicity tests using three dose levels, viz. therapeutic, average (2x therapeutic dose) and highest dose (10 x therapeutic dose) exposure in monkeys. The selection of the model i.e. monkey was based on affinity and rapid higher antibody response during the efficacy studies. An attempt was made to evaluate all parameters which included physical, physiological, clinical, haematological and histopathological profiles of all target organs, as well as Tiers I, II, III immunotoxicity parameters. Results: In acute toxicity there was no mortality in spite of exposing the monkeys to 10XDRV. In sub chronic and chronic toxicity studies there were no abnormalities in physical, physiological, neurological, clinical parameters, after administration of test compound in intended and 10 times of clinical dosage schedule of DRV and CRV under the experimental conditions. Clinical chemistry, haematology, organ weights and histopathology studies were essentially unremarkable except the presence of residual DNA in femtogram level at site of injection in animal which received 10X DRV in chronic toxicity study. No Observational Adverse Effects Level (NOAEL) of DRV is 1000 ug/dose (10 times of therapeutic dose) if administered on 0, 4, 7, 14, 28th day. Interpretation & conclusions: The information generated by this study not only draws attention to the need for national and international regulatory agencies in formulating guidelines for pre-clinical safety evaluation of biotech products but also facilitates the development of biopharmaceuticals as safe potential therapeutic agents.

Note on high aspect-ratio SU-8 micromechanical structures using mask-less direct laser writing

We report high aspect-ratio micromechanical structures made of SU-8 polymer, which is a negative photoresist. Mask-less direct writing with 405 nm laser is used to pattern spin-cast SU-8 films of thickness of more than 600 um. As compared with X-ray lithography, which helps pattern material to give aspect ratios of 1:50 or higher, laser writing is a less expensive and more accessible alternative. In this work, aspect ratios up to 1:30 were obtained on narrow pillars and cantilever structures. Deep vertical patterning was achieved in multiple exposures of the surface with varying dosages given at periodic intervals of sufficient duration. It was found that a time lag between successive exposures at the same location helps the material recover from the transient changes that occur during exposure to the laser. This gives vertical sidewalls to the resulting structures. The time-lags and dosages were determined by conducting several trials. The micromechanical structures obtained with laser writing are compared with those obtained with traditional UV lithography as well as e-beam lithography. Laser writing gives not only high aspect ratios but also narrow gaps whereas e-beam can only give narrow gaps over very small depths. Unlike traditional UV lithography, laser writing does not need a mask. Furthermore, there is no adjustment for varying the dosage in traditional UV lithography. A drawback of this method compared to UV lithography is that the writing time increases. Some test structures as well as a compliant microgripper are fabricated.

Biofouling and microbially influenced corrosion of stainless steels

Stainless steels are among the most investigated materials on biofouling and microbially-influenced corrosion (MIC). Although, generally corrosion-resistant owing to tenacious and passive surface film due to chromium, stainless steels are susceptible to extensive biofouling in subsoil, fresh water and sea water and chemical process environments. Biofilms influence their corrosion behavior due to corrosion potential ennoblement and sub-surface pitting. Both aerobic and anaerobic microorganisms catalyse microbial corrosion of stainless steels through biotic and abiotic mechanisms. MIC of stainless steels is common adjacent to welds at the heat-affected zone. Both austenite and delta ferrite phases may be susceptible. Even super stainless steels are found to be amenable to biofouling and MIC. Microbiological, electrochemical as well as physicochemical aspects of MIC pertaining to stainless steels in different environments are analyzed.

Nanostructure Patterning on Flexible Substrates Using Electron Beam Lithography

Patterning nanostructures on flexible substrates plays a key role in the emerging flexible electronics technology. The flexible electronic devices are inexpensive and can be conformed to any shape. The potential applications for such devices are sensors, displays, solar cells, RFID, high-density biochips, optoelectronics etc. E-beam lithography is established as a powerful tool for nanoscale fabrication, but its applicability on insulating flexible substrates is often limited because of surface charging effects. This paper presents the fabrication of nanostructures on insulating flexible substrates using low energy E-beam lithography along with metallic layers for charge dissipation. Nano Structures are patterned on different substrates of materials such as acetate and PET foils. The fabrication process parameters such as the proximity gap of exposure, the exposure dosage and developing conditions have been optimized for each substrate.

Multimodal therapy for complete regression of malignant melanoma using constrained nonlinear optimal dynamic inversion

Using a realistic nonlinear mathematical model for melanoma dynamics and the technique of optimal dynamic inversion (exact feedback linearization with static optimization), a multimodal automatic drug dosage strategy is proposed in this paper for complete regression of melanoma cancer in humans. The proposed strategy computes different drug dosages and gives a nonlinear state feedback solution for driving the number of cancer cells to zero. However, it is observed that when tumor is regressed to certain value, then there is no need of external drug dosages as immune system and other therapeutic states are able to regress tumor at a sufficiently fast rate which is more than exponential rate. As model has three different drug dosages, after applying dynamic inversion philosophy, drug dosages can be selected in optimized manner without crossing their toxicity limits. The combination of drug dosages is decided by appropriately selecting the control design parameter values based on physical constraints. The process is automated for all possible combinations of the chemotherapy and immunotherapy drug dosages with preferential emphasis of having maximum possible variety of drug inputs at any given point of time. Simulation study with a standard patient model shows that tumor cells are regressed from 2 x 107 to order of 105 cells because of external drug dosages in 36.93 days. After this no external drug dosages are required as immune system and other therapeutic states are able to regress tumor at greater than exponential rate and hence, tumor goes to zero (less than 0.01) in 48.77 days and healthy immune system of the patient is restored. Study with different chemotherapy drug resistance value is also carried out. (C) 2014 Elsevier Ltd. All rights reserved.

Fabrication and characterization of sub 100 nm period polymer gratings for photonics applications

We report on the fabrication of polymethylmethacrylate (PMMA) nanogratings on silicon (Si) and glass substrates using electron beam lithography technique. Various aspects of proximity corrections using Monte Carlo simulation have been discussed. The fabrication process parameters such as proximity gap of exposure, exposure dosage and developing conditions have been optimized for high-density PMMA nanogratings structure on Si and glass substrates. Electron beam exposure is adjusted in such a way that PMMA acts as a negative tone resist and at the same time resolution loss due to proximity effect is minimum. Both reflection and transmission-type, nanometre period gratings have been fabricated and their diffraction characteristics are evaluated.

Why is the influence of soil macrofauna on soil structure only considered by soil ecologists?

These last twenty years have seen the development of an abundant literature on the influence of soil macrofauna on soil structure. Amongst these organisms, earthworms, termites and ants are considered to play a key role in regulating the physical, chemical and microbiological properties of soils. Due to these influential impacts, soil ecologists consider these soil macro-invertebrates as `soil engineers' and their diversity and abundance are nowadays considered as relevant bioindicators of soil quality by many scientists and policy makers. Despite this abundant literature, the soil engineering concept remains a `preach to the choir' and bioturbation only perceived as important for soil ecologists. We discussed in this article the main mechanisms by which soil engineers impact soil structure and proposed to classify soil engineers with respect to their capacity to produce biostructures and modify them. We underlined the lack of studies considering biostructure dynamics and presented recent techniques in this purpose. We discussed why soil engineering concept is mainly considered by soil ecologists and call for a better collaboration between soil ecologists and soil physicists. Finally, we summarized main challenges and questions that need to be answered to integrate soil engineers activities in soil structure studies. (C) 2014 Elsevier B.V. All rights reserved.

Production and characterization of bioflocculants for mineral processing applications

A comparative study of two bacterial strains namely, Bacillus licheniformis and Bacillus firmus in the production of bioflocculants was made. The highest bioflocculant yield of 16.55 g/L was obtained from B. licheniformis (L) and 10 g/L from B. firmus (F). The bioflocculants obtained from the bacterial species were water soluble and insoluble in organic solvents. FTIR spectral analysis revealed the presence of hydroxyl, carboxyl and sugar derivatives in the bioflocculants. Thermal characterization by differential scanning calorimetry (DSC) showed the crystalline transition and the melting point (T-m) at 90-100 degrees C. Effects of bioflocculant dosage and pH on the flocculation of clay fines were evaluated. Highest bioflocculation efficiency on kaolin clay suspensions was observed at an optimum bioflocculant dosage of 5 g/L. The optimum pH range for the maximum bioflocculation was at pH 7-9. Bioflocculants exhibited high efficiency in dye decolorization. The maximum Cr (VI) removal was found to be 85 % for L (bioflocculant dosage at 2 g/L). This study demonstrates that microbial bioflocculants find potential applications in mineral processing such as selective flocculation of mineral fines, decolorization of dye solutions and in the remediation of toxic metal solutions. (C) 2015 Elsevier B.V. All rights reserved.

Cytotoxicity of Ultrasmall Gold Nanoparticles on Planktonic and Biofilm Encapsulated Gram-Positive Staphylococci

The emergence of multidrug resistant bacteria, especially biofilm-associated Staphylococci, urgently requires novel antimicrobial agents. The antibacterial activity of ultrasmall gold nanoparticles (AuNPs) is tested against two gram positive: S. aureus and S. epidermidis and two gram negative: Escherichia coli and Pseudomonas aeruginosa strains. Ultrasmall AuNPs with core diameters of 0.8 and 1.4 nm and a triphenylphosphine-monosulfonate shell (Au0.8MS and Au1.4MS) both have minimum inhibitory concentration (MIC) and minimum bactericidal concentration of 25 x 10(-6)m Au]. Disc agar diffusion test demonstrates greater bactericidal activity of the Au0.8MS nanoparticles over Au1.4MS. In contrast, thiol-stabilized AuNPs with a diameter of 1.9 nm (AuroVist) cause no significant toxicity in any of the bacterial strains. Ultrasmall AuNPs cause a near 5 log bacterial growth reduction in the first 5 h of exposure, and incomplete recovery after 21 h. Bacteria show marked membrane blebbing and lysis in biofilm-associated bacteria treated with ultrasmall AuNP. Importantly, a twofold MIC dosage of Au0.8MS and Au1.4MS each cause around 80%-90% reduction in the viability of Staphylococci enveloped in biofilms. Altogether, this study demonstrates potential therapeutic activity of ultrasmall AuNPs as an effective treatment option against staphylococcal infections.

Chitosan-dextran sulphate nanocapsule drug delivery system as an effective therapeutic against intraphagosomal pathogen Salmonella

Objectives: The ability to target conventional drugs efficiently inside cells to kill intraphagosomal bacteria has been a major hurdle in treatment of infective diseases. We aimed to develop an efficient drug delivery system for combating infection caused by Salmonella, a well-known intracellular and intraphagosomal pathogen. Chitosan dextran sulphate (CD) nanocapsules were assessed for their efficiency in delivering drugs against Salmonella. Methods: The CD nanocapsules were prepared using the layer-by-layer method and loaded with ciprofloxacin or ceftriaxone. Antibiotic-loaded nanocapsules were analysed in vitro for their ability to enter epithelial and macrophage cells to kill Salmonella. In vivo pharmacokinetics and organ distribution studies were performed to check the efficiency of the delivery system. The in vivo antibacterial activity of free antibiotic and antibiotic loaded into nanocapsules was tested in a murine salmonellosis model. Results: In vitro and in vivo experiments showed that this delivery system can be used effectively to clear Salmonella infection, CD nanocapsules were successfully employed for efficient targeting and killing of the intracellular pathogen at a dosage significantly lower than that of the free antibiotic. The increased retention time of ciprofloxacin in the blood and organs when it was delivered by CD nanocapsules compared with the conventional routes of administration may be the reason underlying the requirement for a reduced dosage and frequency of antibiotic administration. Conclusions: CD nanocapsules can be used as an efficient drug delivery system to treat intraphagosomal pathogens, especially Salmonella infection, This delivery system might be used effectively for other vacuolar pathogens including Mycobacteria, Brucella and Legionella.