To compare central corneal thickness measurements obtained by Pentacam with those obtained by IOLMaster 700, Cirrus anterior segment optical coherence tomography and Tomey specular microscopy in normal healthy eyes.

Purpose: To compare central corneal thickness measurements obtained by Pentacam with those obtained by IOL Master 700, Cirrus Anterior segment optical coherence tomography and Tomey Specular microscopy in normal healthy eyes. Methods: Two hundred and six eyes of healthy subjects were included in the study. Each subject was assessed by four different methods of measuring central corneal thickness using Pentacam, IOL Master 700, Cirrus AS-OCT and Tomey Specular microscopy by a single examiner. Results: The mean CCT [± standard deviation (SD)] for Pentacam, IOL Master 700, Cirrus AS-OCT and Tomey Specular microscopy were Pentacam (Oculus), AS-OCT (Cirrus), IOL Master 700 and Specular microscopy (Tomey) were 523.75 (±27.75), 525.29 (±28.81),517.13 (±28.43) and 512.82 (±27.60) µm, respectively. All the means were significantly different from one another (P < 0.000). The differences between pairs of mean central corneal thickness (CCT) for Pentacam and IOL Master, Pentacam and anterior segment- optical coherence tomography (AS-OCT), and Pentacam and Specular microscopy are statistically significant. Bland-Altman plots showed that pentacam and IOL Master 700 have the closest agreement, followed by AS-OCT. Specular microscopy was found to have the poorest agreement with Pentacam. Conclusion: We found that CCT measurements of Pentacam did not correlate with measurements of IOL Master, or AS-OCT or Specular microscopy. In clinical practice, the devices analyzed should not be used interchangeably due to low agreement regarding CCT values.

Development of an electroactive aerobic biocathode for microbial fuel cell applications.

Microbial biocathodes are gaining interest due to their low cost, environmental friendliness and sustainable nature. In this study, a microbial consortium was enriched from activated sludge obtained from a common textile effluent treatment plant in the absence of organic carbon source to produce an electroactive biofilm. Chronoamperometry method of enrichment was carried out for over 70 days to select for electroactive bacteria that could be used as a cathode catalyst in microbial fuel cells (MFC). The resultant biofilm produced an average peak current of -0.7 mA during the enrichment and produced a maximum power density of 64.6 ± 3.5 mW m-2 compared to platinum (72.7 ± 1.2 mW m-2 ) in a Shewanella-based MFC. Microbial community analysis of the initial sludge sample and enriched samples, based on 16S rRNA gene sequencing, revealed the selection of chemolithotrophs with the most dominant phylum being Bacteroidetes, Proteobacteria, Firmicutes, Actinobacteria and Acidobacteria in the enriched samples. A variety of CO2 fixing and nitrate-reducing bacteria was present in the resultant biofilm on the cathode. This study suggests that microbial consortia are capable of replacing expensive platinum as a cathode catalyst in MFCs.

Metabolic engineering of Ashbya gossypii for enhanced FAD production through promoter replacement of FMN1 gene.

Riboflavin (vitamin B2), Flavin Mononucleotide (FMN), Flavin Adenine Dinucleotide (FAD) are essential biomolecules for carrying out various metabolic activities of oxidoreductases and other enzymes. Riboflavin is mainly used as food and feed supplement while the more expensive FAD has pharmacological importance. Although Ashbya gossypii has been metabolically engineered for industrial production of riboflavin, there are no reports on FAD production. In the present study, a transcriptional analysis of the time course of flavin genes expression, indicated that riboflavin to FMN conversion by riboflavin kinase enzyme encoded by FMN1 gene could be the major rate limiting step in FAD synthesis. Overexpression of FMN1 gene was attempted by placing the ORF of FMN1 under control of the stronger constitutively expressed GPD (Glyceraldehyde-3-phosphate dehydrogenase) promoter replacing its native promoter. A 2.25Kb promoter replacement cassette (PRC) for FMN1 gene was synthesized from cloned pUG6-GPDp vector and used for transformation of Ashbya gossypii. Resultant recombinant strain CSAgFMN1 had 35.67-fold increase in riboflavin kinase enzyme activity. A 14.02-fold increase in FAD production up to 86.56 ±â€¯3.88 mg L-1 at 120 h incubation was obtained compared to wild type. While there was a marginal increase in riboflavin synthesis by the clone, FMN accumulation was not detected and could be attributed to other metabolic fluxes channeling FMN. This is the first report on development of FAD overproducing strain of A. gossypii.

Kluyvera georgiana MCC 3673: A Novel Electrogen Enriched in Microbial Fuel Cell Fed with Oilseed Cake.

A novel electrogenic bacterial species, Kluyvera georgiana MCC 3673, was isolated by enrichment in microbial fuel cells (MFC) using oilseed cake as a growth substrate. CHNS analyses showed that oilseed cakes are rich in carbon and nitrogen content. Utilization of these compounds by bacteria was evident from 50% reduction in chemical oxygen demand. The maximum power density of 379 ± 8 mW/m2 was produced from sesame seed cake media with mixed consortia inoculum from lake sediment. Enrichment was carried out for over 15 cycles by renewing the media periodically on drop of the voltage. A pure culture of enriched electrogen was isolated by dilution plate technique. Physiological and biochemical studies were performed on the isolate as per standard methods. Genetic analysis by 16S rDNA sequencing revealed that this organism is closely related to Kluyvera georgiana. When inoculated in MFC as pure culture, the maximum power density of 158 ± 11 mW/m2 and 172 ± 13 mW/m2 was produced with sesame and groundnut oilseed cake media, respectively. The performance increased in LB media producing maximum power density of 394 ± 6 mW/m2. This bacterium has also scope for application in wide range of MFC as it can produce electricity even in suspended culture. To our knowledge, this is the first report on bio-electricity generation using oilseed cake as substrate in MFC.

Effect of neutral red incorporation on Al-doped ZnO thin films and its bio-electrochemical interaction with NAD+/NADP+ dependent enzymes.

A new approach to deposition of electroactive ZnO thin films have been carried out, by one-pot chemical bath deposition with Al dopant and incorporation of neutral red as organic mediator. The morphological, structural and functional characterization of the neutral red incorporated, Al-doped ZnO (NR-AZO) film was carried out using electron microscopy, FTIR, XRD and EIS respectively. The incorporated neutral red was found to induce strain in the crystal of AZO proportional to the concentration used in depositing solution which further affected the charge transfer resistance of the films in solution. One mM neutral red was found to be the optimum concentration for both conductivity and response to NADH/NADPH. The response of the films was further validated by immobilizing NAD+ dependent alcohol dehydrogenase (ADH) and NADP+ dependent glucose dehydrogenase (GDH) independently. The ADH/NR-AZO showed a sensitivity of 3.2 µA cm-2 mM-1 with a LoD of 1.7 µM of ethanol in the range 5.6 µM-7 mM, whereas GDH/NR-AZO showed a sensitivity of 4.33 µA cm-2 mM-1 with a LoD of 27 µM of glucose in the range 90 µM-4 mM. This method serves as a simple alternative to immobilize the organic redox dyes into the inorganic thin films in a single step making it electroactive towards specific biomolecules.

New approach to biosensing of co-enzyme nicotinamide adenine dinucleotide (NADH) by incorporation of neutral red in aluminum doped nanostructured ZnO thin films.

Biosensing of NADH on bare electrodes has drawbacks such as high over-potential and poisoning during the oxidation reaction. To overcome this challenge a different approach has been undertaken by incorporating neutral red (NR) in Al doped ZnO (AZO) thin films using one-pot chemical bath deposition (CBD). The surface morphology of the films was hexagonal nanorods along the c-axis, perpendicular to the substrate. The thickness of the thin films were ranging from 400 to 3000nm varying dependent on time of deposition (30 to 150min). The average diameter of the nanorods was larger in the presence of neutral red (NR-AZO) with ~300nm in contrast to its absence (AZO) with ~200nm. The density of the packing of nanorods was dependent on the citrate concentration used during deposition. Control over the dopant concentration in the films was achieved by varying the area of Al foil used in the deposition solution. The selected area diffraction (SAED) and X-ray diffraction (XRD) indicated 002 plane of orientation in the nanorods. FTIR and FT-Raman analysis revealed conserved structure of NR and AZO. Chronoamperometric (CA) analysis showed a sensitivity of 0.45µAcm-2mM-1 and LoD of 22µM within the range 0.075-4mM of NADH. The biological sensing of NADH was validated by physical adsorption of NAD+ dependent-lactate dehydrogenase (LDH) on NR-AZO. CA showed sensitivity of 0.56µAcm-2mM-1 and LoD for lactate was 27µM in the range of 0.1-1mM of lactate. Further validation with real-time serum sample shows that LDH/NR-AZO correlates with the clinical values. The distinction in this study is that the organic mediator like neutral red has been incorporated into the grain structure of the ZnO thin film whereas other study with the mediators have only attempted surface functionalization. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.

Mediator-free interaction of glucose oxidase, as model enzyme for immobilization, with Al-doped and undoped ZnO thin films laser-deposited on polycarbonate supports.

Al doped and undoped ZnO thin films were deposited by pulsed-laser deposition on polycarbonate sheets. The films were characterized by optical transmission, Hall effect measurement, XRD and SEM. Optical transmission and surface reflectometry studies showed good transparency with thicknesses ∼100nm and surface roughness of 10nm. Hall effect measurements showed that the sheet carrier concentration was -1.44×1015cm-2 for AZO and -6×1014cm-2 for ZnO. The films were then modified by drop-casting glucose oxidase (GOx) without the use of any mediators. Higher protein concentration was observed on ZnO as compared to AZO with higher specific activity for ZnO (0.042Umg-1) compared to AZO (0.032Umg-1), and was in agreement with cyclic voltemmetry (CV). X-ray photoelectron spectroscopy (XPS) suggested that the protein was bound by dipole interactions between AZO lattice oxygen and the amino group of the enzyme. Chronoamperometry showed sensitivity of 5.5µAmM-1cm-2 towards glucose for GOx/AZO and 2.2µAmM-1cm-2 for GOx/ZnO. The limit of detection (LoD) was 167µM of glucose for GOx/AZO, as compared to 360µM for GOx/ZnO. The linearity was 0.28-28mM for GOx/AZO whereas it was 0.6-28mM for GOx/ZnO with a response time of 10s. Possibly due to higher enzyme loading, the decrease of impedance in presence of glucose was larger for GOx/ZnO as compared to GOx/AZO in electrochemical impedance spectroscopy (EIS). Analyses with clinical blood serum samples showed that the systems had good reproducibility and accuracy. The characteristics of novel ZnO and AZO thin films with GOx as a model enzyme, should prove useful for the future fabrication of inexpensive, highly sensitive, disposable electrochemical biosensors for high throughput diagnostics.

Live cell fluorescence imaging for early expression and localization of RIB1 and RIB3 genes in Ashbya gossypii.

Ashbya gossypii is a riboflavin overproducing filamentous fungus. RIB1 and RIB3 genes encode GTP-cyclohydrolase II (GCH II) and DHBP synthase, respectively, the two rate limiting enzymes of the riboflavin biosynthetic pathway. The genes encoding yeast enhanced green fluorescent protein (yEGFP) and mCherry were fused with RIB1 and RIB3 genes, under their native promoters by PCR-based gene tagging method for their early in vivo expression and cellular localization in A. gossypii. In the integrative transformants, the fusion proteins were expressed as cytoplasmic proteins from the germ bubble stage, in all the cells throughout the hypha. This was evident from the observation that mCherry fusion proteins were seen outside the vacuoles in the cytoplasm. The older matured cells of 14 h hyphae developed large vacuoles which showed green autofluorescence due to riboflavin. It is concluded that RIB1 and RIB3 genes are constitutively expressed in all the cells of this multicellular multinucleate fungus.