Antibacterial and cytotoxic assessment of poly (methyl methacrylate) based hybrid nanocomposites.

Poly (methyl methacrylate) (PMMA) is an extensively used implant material in biomedical devices. Biofilm formation creates issues in PMMA-based biomedical implants, while emergence of drug resistant pathogens poses an additional complication. Hence development of surfaces that resist bacterial colonisation is extremely desirable. In this context, nanomaterials are among the potential choices. In the present work, nanocomposites (NCs) were developed by incorporation of chemically synthesized nanoparticles of CuO, cetyl trimethyl ammonium bromide (CTAB) capped CuO and ZnO (singly and in combination) in PMMA. The efficacy of these NCs was assessed against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) bacteria which are prevalent in many implant-associated infections. Results revealed species-specific response of the bacteria towards nanomaterials. CuO NC (0.1% (w/v)) was more effective against E. coli, while CTAB capped CuO NC and ZnO NC were very effective against S. aureus. Furthermore, combination of nanoparticles improved efficacy of nanocomposites against both the bacterial species. In vitro cytotoxicity assay using L6 myoblast cell line showed that all NCs at 0.1% (w/v) were biocompatible, showing >85% cell viability. The present study suggests that combination of NPs is a promising option to combat implant infection by multiple organisms.

A combined experimental and theoretical approach towards mechanophenotyping of biological cells using a constricted microchannel.

We report a combined experimental and theoretical technique that enables the characterization of various mechanical properties of biological cells. The cells were infused into a microfluidic device that comprises multiple parallel micro-constrictions to eliminate device clogging and facilitate characterization of cells of different sizes and types on a single device. The extension ratio λ and transit velocity Uc of the cells were measured using high-speed and high-resolution imaging which were then used in a theoretical model to predict the Young's modulus Ec = f(λ, Uc) of the cells. The predicted Young's modulus Ec values for three different cell lines (182 ± 34.74 Pa for MDA MB 231, 360 ± 75 Pa for MCF 10A and, 763 ± 93 Pa for HeLa) compare well with those reported in the literature from micropipette measurements and atomic force microscopy measurement within 10% and 15%, respectively. Also, the Young's modulus of MDA-MB-231 cells treated with 50 µM 4-hyrdroxyacetophenone (for localization of myosin II) for 30 min was found out to be 260 ± 52 Pa. The entry time te of cells into the micro-constrictions was predicted using the model and validated using experimentally measured data. The entry and transit behaviors of cells in the micro-constriction including cell deformation (extension ratio λ) and velocity Uc were experimentally measured and used to predict various cell properties such as the Young's modulus, cytoplasmic viscosity and induced hydrodynamic resistance of different types of cells. The proposed combined experimental and theoretical approach leads to a new paradigm for mechanophenotyping of biological cells.

Transcriptional regulation of microsomal prostaglandin E synthase 1 by the proto-oncogene, c-myc, in the pathogenesis of inflammation and cancer.

Pro-inflammatory molecules play a key role in the progression of various types of cancers highlighting the importance of studying the pathways that regulate the inflammatory cytokine production. To this end, prostaglandins have been reported to correlate with exacerbated cancer phenotypes that may be prevented by using anti-inflammatory drugs in humans. To understand how the prostaglandin E synthase 1 (mPGES1) may be regulated we analyzed its promoter sequence and identified myc-binding sites. Functional validation was performed by mutating the sites that led to attenuated promoter activation of mPGES1. The known c-myc inhibitor (10058-F4) also blocked PGE2 activity, indicating the importance of c-Myc in PGE2 synthesis. Isocoumarin analogs were able to reduce the expressions of both c-myc as well as mPGES1 and also inhibit the production of PGE2. Based on these data and the well-established role of c-myc in oncogenesis, we have demonstrated an additional role of c-myc in exacerbating cancers via PGE2 production, which may provide a therapeutic opportunity to treat these diseases.

A microfluidic device with focusing and spacing control for resistance-based sorting of droplets and cells.

This paper reports a novel hydrodynamic technique for sorting of droplets and cells based on size and deformability. The device comprises two modules: a focusing and spacing control module and a sorting module. The focusing and spacing control module enables focusing of objects present in a sample onto one of the side walls of a channel with controlled spacing between them using a sheath fluid. A 3D analytical model is developed to predict the sheath-to-sample flow rate ratio required to facilitate single-file focusing and maintain the required spacing between a pair of adjacent objects. Experiments are performed to demonstrate focusing and spacing control of droplets (size 5-40 µm) and cells (HL60, size 10-25 µm). The model predictions compare well with experimental data in terms of focusing and spacing control within 9%. In the sorting module, the main channel splits into two branch channels (straight and side branches) with the flow into these two channels separated by a "dividing streamline". A sensing channel and a bypass channel control the shifting of the dividing streamline depending on the object size and deformability. While resistance offered by individual droplets of different sizes has been studied in our previous work (P. Sajeesh, M. Doble and A. K. Sen, Biomicrofluidics, 2014, 8, 1-23), here we present resistance of individual cells (HL60) as a function of size. A theoretical model is developed and used for the design of the sorter. Experiments are performed for size-based sorting of droplets (sizes 25 and 40 µm, 10 and 15 µm) and HL60 cells (sizes 11 µm and 19 µm) and deformability-based sorting of droplets (size 10 ± 1.0 µm) and polystyrene microbeads (size 10 ± 0.2 µm). The performance of the device for size- and deformability-based sorting is characterized in terms of sorting efficiency. The proposed device could be potentially used as a diagnostic tool for sorting of larger tumour cells from smaller leukocytes.

Hydrodynamic resistance and mobility of deformable objects in microfluidic channels.

This work reports experimental and theoretical studies of hydrodynamic behaviour of deformable objects such as droplets and cells in a microchannel. Effects of mechanical properties including size and viscosity of these objects on their deformability, mobility, and induced hydrodynamic resistance are investigated. The experimental results revealed that the deformability of droplets, which is quantified in terms of deformability index (D.I.), depends on the droplet-to-channel size ratio [Formula: see text] and droplet-to-medium viscosity ratio [Formula: see text]. Using a large set of experimental data, for the first time, we provide a mathematical formula that correlates induced hydrodynamic resistance of a single droplet [Formula: see text] with the droplet size [Formula: see text] and viscosity [Formula: see text]. A simple theoretical model is developed to obtain closed form expressions for droplet mobility [Formula: see text] and [Formula: see text]. The predictions of the theoretical model successfully confront the experimental results in terms of the droplet mobility [Formula: see text] and induced hydrodynamic resistance [Formula: see text]. Numerical simulations are carried out using volume-of-fluid model to predict droplet generation and deformation of droplets of different size ratio [Formula: see text] and viscosity ratio [Formula: see text], which compare well with that obtained from the experiments. In a novel effort, we performed experiments to measure the bulk induced hydrodynamic resistance [Formula: see text] of different biological cells (yeast, L6, and HEK 293). The results reveal that the bulk induced hydrodynamic resistance [Formula: see text] is related to the cell concentration and apparent viscosity of the cells.

Influence of surface characteristics on biofouling formed on polymers exposed to coastal sea waters of India.

Biofouling on six different (silicone rubber, polydimethylsiloxane, polypropylene, high density polyethylene, polyvinylchloride, and polycarbonate) substrata with varying surface energy (18-40 mN/m) and surface roughness (R(a) 45-175 µm) was studied in the Eastern coastal waters of India over a short period of time (3 days). The results showed that the substrata surface energy (SE) followed by the surface roughness (R(a)) had profound effect on attachment of fouling organisms. After one day of immersion, viable count of bacteria in the biofilm was positively correlated with surface energy (r=0.69, p<0.05) and not with surface roughness (r=-0.02) of the substratum. Whereas, Pseudomonas count was inversely correlated with surface energy (r=-0.66, p<0.05) and surface roughness (r=-0.52, p<0.05). The attachment of macrofouler and the surface characteristics were also well correlated with SE 0.48 and with roughness 0.62, p<0.05. A positive correlation was observed amongst the various biofouling constituents such as bacteria, ATP, carbohydrates and organic matter on almost all the substrata. However after the first day, the surface characteristics of the substratum became less important and the conditioning film that was formed on the substrata appeared to directly influence further fouling on the surfaces, as evidenced by poor correlation between surface energy and macrofouler attachment (r=-0.11). The observation of high numbers of Hydroides elegans on PVC could be solely due to the influence of surface roughness (r=0.62). Though there is no marked difference in the 'primary film', and the composition of the biofilm, the amount of attached macrofouler is minimal on silicone rubber and polydimethylsiloxane on subsequent days of immersion, which reveals the foul release quality of these substrata probably due to their flexible nature.

Barnacle cement: an etchant for stainless steel 316L?

Localized corrosion of stainless steel beneath the barnacle-base is an unsolved issue for the marine industry. In this work, we clearly bring out for the first time the role of the barnacle cement in acting as an etchant, preferentially etching the grain boundaries, and initiating the corrosion process in stainless steel 316L. The investigations include structural characterization of the cement and corroded region, and also chemical characterization of the corrosion products generated beneath the barnacle-base. Structural characterization studies using scanning electron microscopy (SEM) reveals the morphological changes in the cement structure across the interface of the base-plate and the substrate, modification of the steel surface by the cement and the corrosion pattern beneath the barnacle-base. Fourier transform infrared spectroscopy (FTIR) of the corrosion products show that they are composed of mainly oxides of iron thereby implying that the corrosion is aerobic in nature. A model for the etching and corrosion mechanism is proposed based on our observations.

Barnacle cement: An etchant for stainless steel 316L?

Localized corrosion of stainless steel beneath the barnacle-base is an unsolved issue for the marine industry. In this work, we clearly bring out for the first time the role of the barnacle cement in acting as an etchant, preferentially etching the grain boundaries, and initiating the corrosion process in stainless steel 316L. The investigations include structural characterization of the cement and corroded region, and also chemical characterization of the corrosion products generated beneath the barnacle-base. Structural characterization studies using scanning electron microscopy (SEM) reveals the morphological changes in the cement structure across the interface of the base-plate and the substrate, modification of the steel surface by the cement and the corrosion pattern beneath the barnacle-base. Fourier transform infrared spectroscopy (FTIR) of the corrosion products show that they are composed of mainly oxides of iron thereby implying that the corrosion is aerobic in nature. A model for the etching and corrosion mechanism is proposed based on our observations.

Synthesis, antibacterial activity evaluation and QSAR studies of novel dispiropyrrolidines.

A series of novel dispiropyrrolidines have been synthesized through 1,3-dipolar cycloaddition of an azomethine ylide generated from sarcosine and isatin with the dipolarophile 3-benzylidene-1-methyl-pyrrolidine-2,5-dione. Their antibacterial activity was evaluated against Bacillus subtilis NCIM 2718, Staphylococcus aureus NCIM5021, Salmonella typhi NCIM2501, Pseudomonas aeruginosa NCIM 5029 and Proteus vulgaris NCIM2813 by two fold dilution method. Compound 6e exhibits reasonably good activity and compound 6c exhibits poor activity against all the organisms. The QSAR's were developed for all antibacterial activities. The models had either one or two descriptors (r(2) = 0.81-0.97, r(adj)(2)=0.75-0.96, q(2) = 0.57-0.92, F-ratio = 12.73-162.76). Topology, shape, charge distribution and hydrophobic nature of the molecules had pronounced effect on their antibacterial activity.

Antibacterial activity and QSAR of chalcones against biofilm-producing bacteria isolated from marine waters.

Biofouling in the marine environment is a major problem. In this study, three marine organisms, namely Bacillus flexus (LD1), Pseudomonas fluorescens (MD3) and Vibrio natriegens (MD6), were isolated from biofilms formed on polymer and metal surfaces immersed in ocean water. Phylogenetic analysis of these three organisms indicated that they were good model systems for studying marine biofouling. The in vitro antifouling activity of 47 synthesized chalcone derivatives was investigated by estimating the minimum inhibitory concentration against these organisms using a twofold dilution technique. Compounds C-5, C-16, C-24, C-33, C-34 and C-37 were found to be the most active. In the majority of the cases it was found that these active compounds had hydroxyl substitutions. A quantitative structure-activity relationship (QSAR) was developed after dividing the total data into training and test sets. The statistical measures r(2), [image omitted] (>0.6) q(2) (>0.5) and the F-ratio were found to be satisfactory. Spatial, structural and electronic descriptors were found to be predominantly affecting the antibiofouling activity of these compounds. Among the spatial descriptors, Jurs descriptors showed their contribution in all the three antibacterial QSARs.

Synthesis, antibacterial activity evaluation and QSAR studies of novel dispiropyrrolidines.

A series of novel dispiropyrrolidines have been synthesized through 1,3-dipolar cycloaddition of an azomethine ylide generated from sarcosine and isatin with the dipolarophile 3-benzylidene-1-methyl-pyrrolidine-2,5-dione. Their antibacterial activity was evaluated against Bacillus subtilis NCIM 2718, Staphylococcus aureus NCIM5021, Salmonella typhi NCIM2501, Pseudomonas aeruginosa NCIM 5029 and Proteus vulgaris NCIM2813 by two fold dilution method. Compound 6e exhibits reasonably good activity and compound 6c exhibits poor activity against all the organisms. The QSAR's were developed for all antibacterial activities. The models had either one or two descriptors (r2 = 0.81-0.97, , q2 = 0.57-0.92, F-ratio = 12.73-162.76). Topology, shape, charge distribution and hydrophobic nature of the molecules had pronounced effect on their antibacterial activity.

Characterization of glycolipid biosurfactant from Pseudomonas aeruginosa CPCL isolated from petroleum-contaminated soil.

AIMS: To isolate and characterize the biosurfactant-producing micro-organism from petroleum-contaminated soil as well as to determine the biochemical properties of the biosurfactant. METHODS AND RESULTS: A novel rhamnolipid-producing Pseudomonas aeruginosa (GenBank accession number GQ241355) strain was isolated from a petroleum-contaminated soil. Surface active compound was separated by solvent extraction of the acidified culture supernatant. The extract was able to reduce the surface tension of water from 72 to 44 mN m(-1) at a critical micelle concentration of 11.27 +/- 1.85 mg l(-1). It showed better activity (based on microdilution method) against Gram-positive (or= 125 mg ml(-1)) with mild toxicity (HC(50)- 38 +/- 8.22 microg ml(-1)) to red blood cells. Fourier transform infrared spectroscopy revealed the presence of aliphatic chain, hydroxyl groups, ester and glycosidic bonds. Presence of nineteen rhamnolipid homologues with variation in chain length and saturation was revealed from liquid chromatography coupled to mass spectrometry with electrospray ionization. CONCLUSION: The results indicate that the isolated biosurfactant has a novel combination of rhamnolipid congeners with unique properties. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a biosurfactant, which can be used as a biocontrol agent against phytopathogens (Fusarium proliferatum NCIM 1105 and Aspergillus niger NCIM 596) and exploited for biomedical applications.

Synergistic interaction of eugenol with antibiotics against Gram negative bacteria.

Eugenol, the principal chemical component of clove oil from Eugenia aromatica has been long known for its analgesic, local anesthetic, anti-inflammatory, and antibacterial effects. The interaction of the eugenol with ten different hydrophobic and hydrophilic antibiotics was studied against five different Gram negative bacteria. The MIC of the combination was found to decrease by a factor of 5-1000 with respect to their individual MIC. This synergy is because of the membrane damaging nature of eugenol, where 1mM of its concentration is able to damage nearly 50% of the bacterial membrane. Eugenol was also able to enhance the activities of lysozyme, Triton X-100 and SDS in damaging the bacterial cell membrane. The hydrophilic antibiotics such as vancomycin and beta-lactam antibiotics which have a marginal activity on these gram negative bacteria exhibit an enhanced antibacterial activity when pretreated with eugenol. Reduced usage of antibiotics could be employed as a treatment strategy to slow down the onset of antibiotic resistance as well as decrease its toxicity. Experiments performed with human blood cells indicated that the concentration of eugenol used for the combination studies were below its cytotoxic values. Pharmacodynamic studies of the combinations need to be performed to decide on the effective dosage.

Quantitative structure-activity relationships for commercially available inhibitors of COX-2.

Quantitative structure activity relationship (QSAR) studies of selective COX-2 inhibitors of commercial interest (drugs in market and on clinical trials) were performed. The COX-2 inhibitory activity (pIC(50)=-logIC(50)) of these twelve compounds was correlated with nineteen descriptors including steric, electronic and constitutional parameters. pIC(50) activity showed high positive correlation with both volume and HOMO (Highest occupied molecular orbital). A Biparametric model was developed that included both these descriptors. The predictive capability (q(2)= 0.66) of this equation was satisfactory. So it can be used to design newer templates or modify existing templates. Volume is an important parameter for the selective COX-2 inhibitory activity, because the secondary pocket in the active site of this enzyme is bigger than the active site of COX-1 enzyme (by 17%). HOMO is a measure of the nucleophilicity of the molecule and a molecule with high HOMO energy is ready to donate its electrons and thus is more reactive than molecule with low values. Binding studies were performed between the COX-2 enzyme and these molecules. The inhibitory activity increased with decrease in binding energy (or interaction energy) between the compounds with the COX-2 enzyme (with a correlation coefficient = -0.65). Calculated Log BBB (Blood Brain barrier), Log P (octonol water partition) and HBD (hydrogen bond donor) values were in the acceptable range (i.e., BBB = -1 to 0.3; LogP= 0 to 5; HBD < 5).

Luminal osmolarity downregulates gene expression of Na+/H+ exchanger (NHE3) in rat colon mucosa.

Water-coupled Na&sup+ absorption in the colon is mediated principally by Na+/H+ exchange (isoforms NHE2 and NHE3). To determine whether luminal ion composition or osmolarity influences NHE expression in colon mucosa, two groups (n = 6 in each) of adult male Sprague-Dawley rats underwent sham laparotomy or loop ileostomy. In these studies, diversion did not markedly alter mRNA levels for NHE2, NHE3, or Na+/K+, at 8 or 21 days, indicating that loss of luminal volume does not alter NHE gene expression. To evaluate the effects of specific luminal components, we infused equal volumes of half-normal (154 mOsm) or iso-osmolar (308 mOsm) solutions of saline and mannitol into the diverted colon. All solutions elicited significant (45% to 60%; P <0.05) decreases in mRNA levels for NHE3, with iso-osmolar mannitol eliciting the greatest changes. Decreases in NHE2 and Na+/K+ mRNA levels were observed following these infusions but were not as marked as the changes for NHE3. These findings suggest that (1) loss of luminal Na+ is not, in itself, a signal that regulates NHE expression and (2) infusion of any solute, including Na+ itself, provides a signal to downregulate expression of NHE3 in colon mucosa.

Intercellular communication mediated by the extracellular calcium-sensing receptor.

Agonist-evoked, intracellular Ca2+-signalling events are associated with active extrusion of Ca2+ across the plasma membrane, implying a local increase in Ca2+ concentration ([Ca2+]) at the extracellular face of the cell. The possibility that these external [Ca2+] changes may have specific physiological functions has received little consideration in the past. Here we show that, at physiological ambient [Ca2+], Ca2+ mobilization in one cell produces an extracellular signal that can be detected in nearby cells expressing the extracellular Ca2+-sensing receptor (CaR), a cell-surface receptor for divalent cations with a widespread tissue distribution. The CaR may therefore mediate a universal form of intercellular communication that allows cells to be informed of the Ca2+-signalling status of their neighbours.

Selective decreases in levels of mRNA encoding a water channel (AQP3) in ileal mucosa after ileostomy in the rat.

Water channels (aquaporins) provide pathways for water permeation in a variety of epithelia. Aquaporin-3 (AQP3) has been localized to the basolateral membranes of epithelial cells in the small intestine, but mechanisms that regulate its expression and function have not been explored. To determine whether luminal content may influence intestinal AQP3 gene expression, adult Sprague-Dawley rats underwent sham laparotomy (N = 11) or loop ileostomy (N = 9) and were killed 8 days after procedures. Northern blot analysis was used to measure messenger RNA (mRNA) levels for AQP3 and the Na(+)/K(+) ATPase, a housekeeping transporter that regulates cellular levels of Na(+) and K(+). At sacrifice, histologic examination revealed only minimal changes in mucosal morphology. In sham animals, Na/K mRNA levels increased moderately in distal regions of the small intestine. Ileostomy did not alter these levels in any region. In contrast, in sham animals, AQP3 mRNA levels increased along the length of the intestine and were markedly higher in the distal ileum. Diversion of luminal contents decreased AQP3 mRNA levels in the postileostomy region by 30% to 50%. These findings indicate regional variations in expression of the AQP3 water channel in mucosa of the small intestine. In addition, they suggest that AQP3 gene expression may depend on the presence of luminal contents.

The influence of the obstetrician in the relationship between epidural analgesia and cesarean section for dystocia.

BACKGROUND: The association between epidural analgesia for labor and the risk of cesarean section for dystocia remains controversial The authors hypothesized that if epidural analgesia were an important factor in determining cesarean section rates, then obstetricians with higher rates of utilization of epidural analgesia for labor would have higher rates of cesarean section for dystocia. METHODS: The frequency of use of epidural analgesia and frequency of occurrence of various patient risk factors for cesarean section were calculated for 110 obstetricians caring for > or = 50 low-risk parturients. These frequencies were compared by linear regression to obstetricians' rates of cesarean section for dystocia. Stepwise regression was used to attempt to predict obstetricians' cesarean rates from the incidence of various patient and provider risk factors. RESULTS: There was no relationship between frequency of epidural analgesia and rate of cesarean section for dystocia across practitioners (R2 = 0.019; P = 0.156). Weighting each obstetrician's data for the number of patients cared for during the study period did not change this result. Stepwise linear regression only modestly predicted obstetricians' cesarean section rates for dystocia, yielding a model containing 12 variables not including epidural analgesia (gestational age, induction of labor, maternal age, provider volume, nulliparity, and seven interactions; adjusted R2 = 0.312; P < 0.0001). CONCLUSIONS: The frequency of use of epidural analgesia does not predict obstetricians' rates of cesarean section for dystocia. After accounting for a number of known patient risk factors, obstetrical practice style appears to be a major determinant of rates of cesarean section.