IndiGenomes: a comprehensive resource of genetic variants from over 1000 Indian genomes.

With the advent of next-generation sequencing, large-scale initiatives for mining whole genomes and exomes have been employed to better understand global or population-level genetic architecture. India encompasses more than 17% of the world population with extensive genetic diversity, but is under-represented in the global sequencing datasets. This gave us the impetus to perform and analyze the whole genome sequencing of 1029 healthy Indian individuals under the pilot phase of the 'IndiGen' program. We generated a compendium of 55,898,122 single allelic genetic variants from geographically distinct Indian genomes and calculated the allele frequency, allele count, allele number, along with the number of heterozygous or homozygous individuals. In the present study, these variants were systematically annotated using publicly available population databases and can be accessed through a browsable online database named as 'IndiGenomes' http://clingen.igib.res.in/indigen/. The IndiGenomes database will help clinicians and researchers in exploring the genetic component underlying medical conditions. Till date, this is the most comprehensive genetic variant resource for the Indian population and is made freely available for academic utility. The resource has also been accessed extensively by the worldwide community since it's launch.

Tec1 and Ste12 transcription factors play a role in adaptation to low pH stress and biofilm formation in the human opportunistic fungal pathogen Candida glabrata.

Eukaryotic cells respond to environmental cues through mitogen activated protein kinase (MAPK) signaling pathways. Each MAPK cascade is specific to particular stimuli and mediates specialized responses through activation of transcription factors. In the budding yeast, Saccharomyces cerevisiae, the pheromone-induced mating pathway and the starvation-responsive invasive growth/filamentation pathway generate their distinct outputs through the transcription factors Ste12 and Tec1, respectively. In this study, we report the functional characterization of these transcription factors in the closely related human opportunistic pathogenic yeast Candida glabrata. Two homologues each for S. cerevisiae TEC1 and STE12 were identified in C. glabrata. Both C. glabrata Tec1 proteins contain the N-terminal TEA DNA-binding domain characteristic of the TEA/ATTS transcription factor family. Similarly, the DNA-binding homeodomain shared by members of the highly conserved fungal Ste12 transcription factor family is present in N-terminus of both C. glabrata Ste12 transcription factors. We show that both C. glabrata STE12 genes are at least partial functional orthologues of S. cerevisiae STE12 as they can rescue the mating defect of haploid S. cerevisiae ste12 null mutant. Knockout of one of the STE12 genes (ORF CAGL0H02145g) leads to decreased biofilm development; a stronger biofilm-impaired phenotype results from loss of both CgSTE12 genes in the double deletion mutant (Cgste12ΔΔ). The transcript levels of one of the TEC1 genes (ORF CAGL0M01716g) were found to be upregulated upon exposure to low pH; its deletion causes slightly increased sensitivity to higher concentrations of acetic acid. Heat shock leads to increase in mRNA levels of one of the STE12 genes (ORF CAGL0M01254g). These findings suggest a role of Tec1 and Ste12 transcription factors in the regulation of some traits (biofilm formation, response to low pH stress and elevated temperature) that contribute to C. glabrata's ability to colonize various host niches and to occasionally cause disease.

Cell adhesion and twitching motility influence strong biofilm formation in Pseudomonas aeruginosa.

In the present study, biofilm formation was quantified in UTI isolates of Pseudomonas aeruginosa (n = 22) using the crystal violet assay and was categorized into; strong (n = 16), weak (n = 4), and moderate (n = 2) biofilm producers. Further experiments were done using strong (n = 4) and weak (n = 4) biofilm producers. Biofilm formation was greater in Luria broth followed by natural urine and artificial urine on silicone and silicone-coated latex. Cell adhesion and twitching motility were greater in strong biofilm producers. The presence of thick biofilm with an increased number of dead and total number of cells of strong biofilm producers was observed using CLSM. The concentrations of exopolymeric substances (eDNA, protein, and pel polysaccharide) were high in strong biofilm producers. FEG-SEM visualization of biofilm produced by strong biofilm producers showed more cells encased in thick biofilm matrix than weak ones. Overall results provide evidence for increased cell adhesion and twitching motility in strong biofilm producers.

DOPA and DHN pathway orchestrate melanin synthesis in Aspergillus species.

Melanins are high molecular weight hydrophobic pigments that have been studied for their role in the virulence of fungal pathogens. We investigated the amount and type of melanin in 20 isolates of Aspergillus spp.; A. niger (n = 3), A. flavus (n = 5), A. tamarii (n = 3), A. terreus (n = 3), A. tubingensis (n = 3), A. sydowii (n = 3). Aspergillus spp. were identified by sequencing the internal transcribed spacer (ITS) region. Extraction of melanin from culture filtrate and fungal biomass was done and followed by qualitative and quantitative analysis of melanin pigment. Ultraviolet (UV), Fourier transformed infrared (FT-IR), and electron paramagnetic resonance (EPR) spectra analyses confirmed the presence of melanin. The melanin pathway was studied by analyzing the effects of inhibitors; kojic acid, tropolone, phthalide, and tricyclazole. The results indicate that in A. niger and A. tubingensis melanin was found in both culture filtrate and fungal biomass. For A. tamarii and A. flavus melanin was extracted from biomass only, whereas melanin was found only in culture filtrate for A. terreus. A negligible amount of melanin was found in A. sydowii. The maximum amount of melanin from culture filtrate and fungal biomass was found in A. niger and A. tamarrii, respectively. The DOPA (3,4-dihydroxyphenylalanine) pathway produces melanin in A. niger, A. tamarii and A. flavus, whereas the DHN (1,8-dihydroxynaphthalene) pathway produces melanin in A. tubingensis and A. terreus. It can be concluded that the amount and type of melanin in aspergilli largely differ from species to species.

Qualitative and Quantitative Analysis of Siderophore Production from Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is known for its production of a diverse range of virulence factors to establish infections in the host. One such mechanism is the scavenging of iron through siderophore production. P. aeruginosa produces two different siderophores: pyochelin, which has lower iron-chelating affinity, and pyoverdine, which has higher iron-chelating affinity. This report demonstrates that pyoverdine can be directly quantified from bacterial supernatants, while pyochelin needs to be extracted from supernatants before quantification. The primary method for qualitatively analyzing siderophore production is the Chrome Azurol Sulfonate (CAS) agar plate assay. In this assay, the release of CAS dye from the Fe3+-Dye complex leads to a color change from blue to orange, indicating siderophore production. For the quantification of total siderophores, bacterial supernatants were mixed in equal proportions with CAS dye in a microtiter plate, followed by spectrophotometric analysis at 630 nm. Pyoverdine was directly quantified from the bacterial supernatant by mixing it in equal proportions with 50 mM Tris-HCl, followed by spectrophotometric analysis. A peak at 380 nm confirmed the presence of pyoverdine. As for Pyochelin, direct quantification from the bacterial supernatant was not possible, so it had to be extracted first. Subsequent spectrophotometric analysis revealed the presence of pyochelin, with a peak at 313 nm.

Estrogen mediated protection of cytoskeleton against oxidative stress.

BACKGROUND & OBJECTIVES: Cytoskeletal proteins are deregulated during oxidative stress and cataract formation. However, estrogen which protects against cataract formation and harmful effects of oxidative stress has not been tested on the cytoskeleton of lens epithelial cells (LECs). The current study was undertaken to assess if the protection rendered to LECs by estrogen was mediated by preserving the cytoskeletal proteins. METHODS: Oxidative stress was induced by 50 µM of H 2 O 2 in cultured goat LECs (gLECs) and effect of 1 µM 17ß-estradiol (E 2 ) was tested. After treatment, morphological analysis of cells was carried out using haematoxylin-eosin staining and cell density was also quantified. Cell viability was determined using Hoechst (Ho), YO-Pro (YP) and propidium iodide (PI). F-actin and vimentin were localized using phalloidin and anti-vimentin antibody, respectively, and viewed under fluorescence microscopy. Vimentin was further analysed at protein level by Western blotting. RESULTS: H 2 O 2 led to increased condensation of nucleus, cell death and apoptosis but these were prevented with pre- and co-treatment of E 2 with increase in cell viability (P<0.001). E 2 also prevented H 2 O 2 mediated depolymerization of cytoskeleton but was not able to reverse the changes when given after induction of oxidative stress. INTERPRETATION & CONCLUSIONS: Our findings showed that E 2 helped in preventing deteriorating effect of H 2 O 2 , inhibited cell death, apoptosis and depolymerisation of cytoskeletal proteins in LECs. However, the exact mechanism by which estrogen renders this protection to cytoskeleton of lens epithelial cells remains to be determined.

Diversity and Distribution of ß-Lactamase Genes Circulating in Indian Isolates of Multidrug-Resistant Klebsiella pneumoniae.

Klebsiella pneumoniae (Kp) has gained prominence in the last two decades due to its global spread as a multidrug-resistant (MDR) pathogen. Further, carbapenem-resistant Kp are emerging at an alarming rate. The objective of this study was (1) to evaluate the prevalence of ß-lactamases, especially carbapenemases, in Kp isolates from India, and (2) determine the most prevalent sequence type (ST) and plasmids, and their association with ß-lactamases. Clinical samples of K. pneumoniae (n = 65) were collected from various pathology labs, and drug susceptibility and minimum inhibitory concentrations (MIC) were detected. Whole genome sequencing (WGS) was performed for n = 22 resistant isolates, including multidrug-resistant (MDR) (n = 4), extensively drug-resistant (XDR) (n = 15), and pandrug-resistant (PDR) (n = 3) categories, and genomic analysis was performed using various bioinformatics tools. Additional Indian MDRKp genomes (n = 187) were retrieved using the Pathosystems Resource Integration Center (PATRIC) database. Detection of ß-lactamase genes, location (on chromosome or plasmid), plasmid replicons, and ST of genomes was carried out using CARD, mlplasmids, PlasmidFinder, and PubMLST, respectively. All data were analyzed and summarized using the iTOL tool. ST231 was highest, followed by ST147, ST2096, and ST14, among Indian isolates. blaampH was detected as the most prevalent gene, followed by blaCTX-M-15 and blaTEM-1. Among carbapenemase genes, blaOXA-232 was prevalent and associated with ST231, ST2096, and ST14, which was followed by blaNDM-5, which was observed to be prevalent in ST147, ST395, and ST437. ST231 genomes were most commonly found to carry Col440I and ColKP3 plasmids. ST16 carried mainly ColKP3, and Col(BS512) was abundantly present in ST147 genomes. One Kp isolate with a novel MLST profile was identified, which carried blaCTX-M-15, blaOXA-1, and blaTEM-1. ST16 and ST14 are mostly dual-producers of carbapenem and ESBL genes and could be emerging high-risk clones in India.

Matrix metalloproteinase-2 and -9 activities in the human lens epithelial cells and serum of steroid induced posterior subcapsular cataracts.

PURPOSE: To evaluate the level of matrix metalloproteinase (MMP)-2 and MMP-9 activities in patients with steroid induced posterior subcapsular cataract (PSC). METHODS: This prospective, observational study comprised of 156 patients having either steroid induced PSC (n=50) or non-steroidal PSC (n=106) were performed to evaluate the level of MMP-2 and MMP-9 activities in the lens epithelial cells (LECs) and the serum. Anterior lens capsules harboring LECs were obtained during phacoemulsification and peripheral blood was collected from patients before administration of anesthesia. Serum was separated by centrifugation at 10,000× g for 15 min at 4 °C. The LECs and serum samples were processed to analyze MMP-2 and MMP-9 activities using succinylated gelatin assay. Quantitative real time-PCR (qRT-PCR) was performed to determine the mRNA levels of MMP-2 and MMP-9 in LECs. The mRNA levels were expressed as a ratio, using the delta-delta method for comparing the relative expression results between cases with steroid induced PSC and cases with non-steroidal PSC. MMP-2 and MMP-9 levels were also compared in the two groups using immunolocalization. RESULTS: The level of MMP-2 and MMP-9 activity was found to be high in LECs and serum of cases with steroid induced PSC. Further in all steroid induced cases, a 1.4 fold increase was observed in MMP-2 activity in LECs and a 1.4 fold increase in MMP-9 activity in the serum. Both qRT-PCR and immunolocalization showed increased expression of MMP-2 and MMP-9 activity. CONCLUSIONS: MMP-2 and MMP-9 activity in both LECs and serum was significantly higher in cases with steroid induced PSC. The possible use of MMP-9 as a non-invasive biomarker in ascertaining the presence of steroid induced PSC should be evaluated using a larger sample size.

Pseudomonas aeruginosa persister cell formation upon antibiotic exposure in planktonic and biofilm state.

Persister cell (PC) is dormant, tolerant to antibiotics, and a transient reversible phenotype. These phenotypes are observed in P. aeruginosa and cause bacterial chronic infection as well as recurrence of biofilm-mediated infection. PC formation requires stringent response and toxin-antitoxin (TA) modules. This study shows the P. aeruginosa PC formation in planktonic and biofilm stages on ceftazidime, gentamicin, and ciprofloxacin treatments. The PC formation was studied using persister assay, flow cytometry using Redox Sensor Green, fluorescence as well as Confocal Laser Scanning Microscopy, and gene expression of stringent response and TA genes. In the planktonic stage, ceftazidime showed a high survival fraction, high redox activity, and elongation of cells was observed followed by ciprofloxacin and gentamicin treatment having redox activity and rod-shaped cells. The gene expression of stringent response and TA genes were upregulated on gentamicin followed by ceftazidime treatment and varied among the isolates. In the biofilm stage, gentamicin and ciprofloxacin showed the biphasic killing pattern, redox activity, gene expression level of stringent response and TA varied across the isolates. Ceftazidime treatment showed higher persister cells in planktonic growth while all three antibiotics were able to induce persister cell formation in the biofilm stage.

Genomic evolution of the globally disseminated multidrug-resistant Klebsiella pneumoniae clonal group 147.

The rapid emergence of multidrug-resistant Klebsiella pneumoniae is being driven largely by the spread of specific clonal groups (CGs). Of these, CG147 includes 7-gene multilocus sequence typing (MLST) sequence types (STs) ST147, ST273 and ST392. CG147 has caused nosocomial outbreaks across the world, but its global population dynamics remain unknown. Here, we report a pandrug-resistant ST147 clinical isolate from India (strain DJ) and define the evolution and global emergence of CG147. Antimicrobial-susceptibility testing following European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines and genome sequencing (Illumina and Oxford Nanopore Technologies, Unicycler assembly) were performed on strain DJ. Additionally, we collated 217 publicly available CG147 genomes [National Center for Biotechnology Information (NCBI), May 2019]. CG147 evolution was inferred within a temporal phylogenetic framework (beast) based on a recombination-free sequence alignment (Roary/Gubbins). Comparative genomic analyses focused on resistance and virulence genes and other genetic elements (BIGSdb, Kleborate, PlasmidFinder, phaster, ICEfinder and CRISPRCasFinder). Strain DJ had a pandrug-resistance phenotype. Its genome comprised the chromosome, seven plasmids and one linear phage-plasmid. Four carbapenemase genes were detected: blaNDM-5 and two copies of blaOXA-181 in the chromosome, and a second copy of blaNDM-5 on an 84 kb IncFII plasmid. CG147 genomes carried a mean of 13 acquired resistance genes or mutations; 63 % carried a carbapenemase gene and 83 % harboured blaCTX-M. All CG147 genomes presented GyrA and ParC mutations and a common subtype I-E CRISPR-Cas system. ST392 and ST273 emerged in 2005 and 1995, respectively. ST147, the most represented phylogenetic branch, was itself divided into two main clades with distinct capsular loci: KL64 (74 %, DJ included, emerged in 1994 and disseminated worldwide, with carbapenemases varying among world regions) and KL10 (20 %, emerged in 2002, predominantly found in Asian countries, associated with carbapenemases NDM and OXA-48-like). Furthermore, subclades within ST147-KL64 differed at the yersiniabactin locus, OmpK35/K36 mutations, plasmid replicons and prophages. The absence of IncF plasmids in some subclades was associated with a possible activity of a CRISPR-Cas system. K. pneumoniae CG147 comprises pandrug-resistant or extensively resistant isolates, and carries multiple and diverse resistance genes and mobile genetic elements, including chromosomal blaNDM-5. Its emergence is being driven by the spread of several phylogenetic clades marked by their own genomic features and specific temporo-spatial dynamics. These findings highlight the need for precision surveillance strategies to limit the spread of particularly concerning CG147 subsets.

Fungal scleral keratitis caused by Phomopsis phoenicicola.

We report a case of scleral keratitis caused by Phomopsis phoenicicola. Pterygium surgery was a predisposing factor, and the patient was treated with natamycin and fluconazole eye drops and oral fluconazole. The fungus was identified by sequencing of the internal transcribed spacer (ITS) region of the fungal ribosomal DNA (rDNA) locus and confirmed on the basis of its typical pycnidia and conidia.

Pharmacogenomic landscape of COVID-19 therapies from Indian population genomes.

Aim: Numerous drugs are being widely prescribed for COVID-19 treatment without any direct evidence for the drug safety/efficacy in patients across diverse ethnic populations. Materials & methods: We analyzed whole genomes of 1029 Indian individuals (IndiGen) to understand the extent of drug-gene (pharmacogenetic), drug-drug and drug-drug-gene interactions associated with COVID-19 therapy in the Indian population. Results: We identified 30 clinically significant pharmacogenetic variants and 73 predicted deleterious pharmacogenetic variants. COVID-19-associated pharmacogenes were substantially overlapped with those of metabolic disorder therapeutics. CYP3A4, ABCB1 and ALB are the most shared pharmacogenes. Fifteen COVID-19 therapeutics were predicted as likely drug-drug interaction candidates when used with four CYP inhibitor drugs. Conclusion: Our findings provide actionable insights for future validation studies and improved clinical decisions for COVID-19 therapy in Indians.

Genetic epidemiology of autoinflammatory disease variants in Indian population from 1029 whole genomes.

BACKGROUND: Autoinflammatory disorders are the group of inherited inflammatory disorders caused due to the genetic defect in the genes that regulates innate immune systems. These have been clinically characterized based on the duration and occurrence of unprovoked fever, skin rash, and patient's ancestry. There are several autoinflammatory disorders that are found to be prevalent in a specific population and whose disease genetic epidemiology within the population has been well understood. However, India has a limited number of genetic studies reported for autoinflammatory disorders till date. The whole genome sequencing and analysis of 1029 Indian individuals performed under the IndiGen project persuaded us to perform the genetic epidemiology of the autoinflammatory disorders in India. RESULTS: We have systematically annotated the genetic variants of 56 genes implicated in autoinflammatory disorder. These genetic variants were reclassified into five categories (i.e., pathogenic, likely pathogenic, benign, likely benign, and variant of uncertain significance (VUS)) according to the American College of Medical Genetics and Association of Molecular pathology (ACMG-AMP) guidelines. Our analysis revealed 20 pathogenic and likely pathogenic variants with significant differences in the allele frequency compared with the global population. We also found six causal founder variants in the IndiGen dataset belonging to different ancestry. We have performed haplotype prediction analysis for founder mutations haplotype that reveals the admixture of the South Asian population with other populations. The cumulative carrier frequency of the autoinflammatory disorder in India was found to be 3.5% which is much higher than reported. CONCLUSION: With such frequency in the Indian population, there is a great need for awareness among clinicians as well as the general public regarding the autoinflammatory disorder. To the best of our knowledge, this is the first and most comprehensive population scale genetic epidemiological study being reported from India.

Quantification of dolichol in the human lens with different types of cataracts.

PURPOSE: To quantify and characterize dolichol species in cataractous and clear human lenses. METHODS: Whole lenses were collected from cadaver eyeballs from the C.H. Nagri Eye Bank and Red Cross Society Eye Bank (Ahmedabad, India). Cataractous nuclei were collected after extracapsular cataract extraction (ECCE). Wet weight for all the lenses was taken and were stored at -50 degrees C until used. Dolichol was extracted using a standard protocol and then analyzed using High Performance Liquid Chromatography (HPLC) on a 4.6 mmx60 mm Hypersil-Octadecylsilane (ODS; 3 microm) reversed phase column using a Waters dual pump apparatus, a Waters gradient programmer, and an ultraviolet (UV) detector set at 210 nm. Dolichol 13 was used as an internal standard, and dolichol mixture from the liver was used as an external qualitative standard. RESULTS: The highest dolichol concentration was found in nuclear cataract (2.54+/-0.6 microg) followed by posterior subcapsular cataract (1.4+/-0.35 microg), and the lowest levels were observed in cortical cataract (0.37+/-0.06 microg). The level of dolichol concentration in cataractous lenses was statistically significantly higher than the levels in clear lenses (1.0+/-04.3 microg; p<0.01). CONCLUSIONS: The dolichol concentration was significantly higher in lenses with nuclear cataract. A significant difference in dolichol concentration was observed between the different types of cataract. It suggests that dolichol and other isoprenoids may be associated with cataractogenesis.

Analysis of single nucleotide polymorphisms of CRYGA and CRYGB genes in control population of western Indian origin.

AIM: Polymorphisms in gamma-crystallins ( CRYG ) can serve as markers for lens differentiation and eye disorders leading to cataract. Several investigators have reported the presence of sequence variations within crystallin genes, with or without apparent effects on the function of the proteins both in mice and humans. Delineation of these polymorphic sites may explain the differences observed in the susceptibility to cataract observed among various ethnic groups. An easier Restriction Fragment Length Polymorphism (RFLP)-based method has been used to detect the frequency of four single nucleotide polymorphisms (SNPs) in CRYGA / CRYGB genes in control subjects of western Indian origin. MATERIALS AND METHODS: A total of 137 healthy volunteers from western India were studied. Examination was performed to exclude volunteers with any ocular defects. Polymerase chain reaction (PCR)-RFLP based method was developed for genotyping of G198A (Intron A), T196C (Exon 3) of CRYGA and T47C (Promoter), G449T (Exon 2) of CRYGB genes. RESULTS: The exonic SNPs in CRYGA and CRYGB were found to have an allele frequency 0.03 and 1.00 for ancestral allele respectively, while frequency of non-coding SNP in CRYGA was 0.72. Allele frequency of T90C of CRYGB varied significantly ( P = 0.02) among different age groups. An in-silico analysis reveals that this sequence variation in CRYGB promoter impacts the binding of two transcription factors, ACE2 (Member of CLB2 cluster) and Progesterone Receptor (PR) which may impact the expression of CRYGB gene. CONCLUSIONS: This study establishes baseline frequency data for four SNPs in CRYGA and CRYGB genes for future case control studies on the role of these SNPs in the genetic basis of cataract.

Effect of vitamin C and E activity on surgically removed cataractous human lens epithelium cells.

Oxidative stress has been proposed as a common underlying mechanism of cataractogenesis. Experimental and observational data suggest that micronutrients like vitamin C and vitamin E with antioxidant capabilities may retard the development of age-related cataract. Effect of these factors on lens epithelium cells, center of lens metabolic activities, is not completely elucidated. The aim of present study was to examine the effect of vitamin C and E on surgically removed lens epithelium cells of patients with cataract. Capsulorhexis samples were collected from 170 patients, admitted for cataract surgery. Catalase specific activity was estimated in lens epithelium cells with and without vitamin (C or E) treatment at different concentration for different time duration. Student's t-test was employed for data analysis. We observed that in ex-vivo condition, a) both vitamin C and E bring about a decrease in catalase activity in lens epithelial cells. b) vitamin C showed toxic effect at high concentration. c) 100µM was the optimum concentration at which both vitamins showed maximum antioxidant activity. It was concluded that both vitamin C and E has direct effect on lens epithelium cells. At optimum concentration, they can reduce oxidative stress in these cells thus can support to prevent or delay cataract development.

High Adhesion and Increased Cell Death Contribute to Strong Biofilm Formation in Klebsiella pneumoniae.

Klebsiella pneumoniae (Kp), is a frequent cause of hospital and community-acquired infections and WHO had declared it as a "priority pathogen". Biofilm is a major virulence factor of Kp and yet the mechanism of strong biofilm formation in Kp is unclear. A key objective of the present study is to investigate the differences between strong and weak biofilms formed by clinical isolates of Kp on various catheters and in different media conditions and to identify constituents contributing to strong biofilm formation. Quantification of matrix components (extracellular DNA (eDNA), protein, exopolysaccharides (EPS), and bacterial cells), confocal laser scanning microscopy (CLSM), field emission gun scanning electron microscopy (FEG-SEM) and flow-cytometry analysis were performed to compare strong and weak biofilm matrix. Our results suggest increased biofilm formation on latex catheters compared to silicone and silicone-coated latex catheters. Higher amounts of eDNA, protein, EPS, and dead cells were observed in the strong biofilm of Kp. High adhesion capacity and cell death seem to play a major role in formation of strong Kp biofilms. The enhanced eDNA, EPS, and protein in the biofilm matrix appear as a consequence of increased cell death.

Evaluation of collagen derived antiangiogenic factors and matrix metalloproteinases in anterior lens epithelial cells of pediatric eyes with persistent fetal vasculature.

Purpose: To measure levels of collagen-derived antiangiogenic factors (arresten, canstatin, tumstatin, endostatin) and matrix metalloproteinases (MMP-2 and MMP-9) in anterior lens epithelial cells (LECs) and anterior capsules of children with cataract and persistent fetal vasculature (PFV) as cases and cataract without PFV as controls. Methods: Anterior capsules harboring LECs were collected from pediatric cataract patients with (n = 13) and without PFV (n = 13) during surgery. Samples were immediately subjected to RNA extraction and cDNA preparation. Quantitative real time PCR was performed to determine the mRNA levels of antiangiogenic factors and matrix metalloproteinases. GAPDH (Glyceraldehyde 3-Phosphate Dehydrogenase) and ß Actin were used as the housekeeping control. The mRNA levels were expressed as a ratio, using the delta-delta method for comparing the relative expression results between controls and cases. The non-parametric Mann-Whitney U test was applied for statistical evaluation. P values < 0.05 were statistically significant. Results: The relative mRNA levels of arresten, canstatin, tumstatin, endostatin, MMP-2 and MMP-9 in cases were 6.20E-03 ± 0.003, 1.49E-01 ± 0.02, 1.70E-01 ± 0.007, 3.20E-03 ± 0.003, 1.11E-03 ± 0.0009 and 3.72E-04 ± 0.0001. The mRNA levels of arresten was 1.6 times lower (P = 0.01) while mRNA levels of MMP-2, tumstatin and canstatin were 4, 2.5, and 2.3 times higher in cases than in controls. No change was observed in mRNA levels of MMP-9 and endostatin (P = 0.82). Conclusion: A significant difference in the levels of arresten, canstatin, tumstatin, and MMP-2 was found in LECs with PFV.

Comparison of 2 moxifloxacin regimens for preoperative prophylaxis: prospective randomized triple-masked trial. Part 1: aqueous concentration of moxifloxacin.

PURPOSE: To evaluate the aqueous concentration of moxifloxacin following 2 dosing regimens of topically administered moxifloxacin hydrochloride ophthalmic solution 0.5% (Vigamox). SETTING: Iladevi Cataract & IOL Research Centre, Ahmedabad, India. METHODS: In this prospective randomized triple-masked clinical trial, 156 patients having cataract surgery were randomly assigned to 1 of 2 regimens of preoperative prophylaxis. In Group A (n = 76), Vigamox was instilled 4 times a day 1 day before surgery plus 1 drop 2 hours before surgery (total of 5 drops). In Group B (n = 76), Vigamox was first instilled 2 hours before surgery and then every 15 minutes for 1 hour (total of 5 drops). In both groups, aqueous samples (0.1 mL) were collected within 2 hours of the first instillation on the day of surgery and stored at -80 degrees C. The antibiotic concentration in all aqueous aspirates was determined using high-performance liquid chromatography. Data were analyzed using the Kolmogorov-Smirnov t test; 95% confidence intervals (CIs) were calculated. RESULTS: The mean aqueous humor concentration of moxifloxacin was 1.58 microg/mL +/- 0.80 (SD) in Group A and 2.05 +/- 0.72 microg/mL in Group B (P<.0001; 95% CI, -0.72 to -0.22). CONCLUSIONS: Both dosing regimens produced substantially higher aqueous concentrations than the known minimum inhibitory concentration for Staphylococcus epidermidis. Topical moxifloxacin administered 2 hours before surgery achieved significantly higher aqueous concentrations than topical moxifloxacin administered 1 day before surgery with 1 drop given on the day of surgery.