Showing papers by "Manipal University published in 2012"

Diverse effects of dimethyl sulfoxide (DMSO) on the differentiation potential of human embryonic stem cells

Abstract: In vitro disease modeling using pluripotent stem cells can be a fast track screening tool for toxicological testing of candidate drug molecules. Dimethyl sulfoxide (DMSO) is one of the most commonly used solvents in drug screening. In the present investigation, we exposed 14- to 21-day-old embryoid bodies (EBs) to three different concentrations of DMSO [0.01% (low dose), 0.1% (medium dose) and 1.0% (high dose)] to identify the safest dose that could effectively be used as solvent. We found that DMSO treatment substantially altered the morphology and attachment of cells in concurrence with a significant reduction in cell viability in a dose-dependent manner. Gene expression studies revealed a selective downregulation of key markers associated with stemness (Oct-4, Sox-2, Nanog and Rex-1); ectoderm (Nestin, TuJ1, NEFH and Keratin-15); mesoderm (HAND-1, MEF-2C, GATA-4 and cardiac-actin); and endoderm (SOX-17, HNF-3β, GATA-6 and albumin), indicating an aberrant and untimely differentiation trajectory. Furthermore, immunocytochemistry, flow cytometry and histological analyses demonstrated substantial decrease in the levels of albumin and CK-18 proteins coupled with a massive reduction in the number of cells positive for PAS staining, implicating reduced deposits of glycogen. Our study advocates for the first time that DMSO exposure not only affects the phenotypic characteristics but also induces significant alteration in gene expression, protein content and functionality of the differentiated hepatic cells. Overall, our experiments warrant that hESC-based assays can provide timely alerts about the outcome of widespread applications of DMSO as drug solvent, cryoprotectant and differentiating agent.

Self-medication patterns among medical students in South India

Abstract: Background Self‐medication results in wastage of resources, increases resistance of pathogens and generally causes serious health hazards such as adverse drug reactions, prolonged suffering and drug dependence. This study was undertaken to determine the reasons for self‐medication and the pattern of self‐medication among medical students. Method This cross‐sectional descriptive study was conducted at the K.S. Hegde Medical Academy, Mangalore. The participants were medical students from first to final year. Medical students were selected through convenience sampling. The data was collected using a pre‐tested semi‐structured questionnaire. The data was analysed using SPSS version 16 and the results expressed as proportions. Results A total of 200 students, 121 (60.5%) female and 79 (39.5%) male, were included in the study. Of the medical students surveyed, self‐medication was reported among 92%. The respondents who used self‐medication found it to be timesaving in providing relief from minor ailments. The most common ailments for which self‐medication were used were: the common cold (69%), fever (63%) and headache (60%). The students consulted their textbooks (39%) and seniors or classmates (38%) for the medications. Antipyretics (71%), analgesics (65%), antihistamines (37%) and antibiotics (34%) were the most common selfmedicated drugs. Of the respondents, 33% were unaware of the adverse effects of the medication and 5% had experienced adverse reactions. The majority (64%) of students advised medications to others, more often to family and friends. Conclusion The prevalence of self‐medication among medical students is high, facilitated by the easy availability of drugs and information from textbooks or seniors. A significant number of students are unaware of the adverse effects of the medication that they themselves take and suggest to others. Therefore, potential problems of self‐medication should be emphasised to the students.

Poor sperm quality and advancing age are associated with increased sperm DNA damage in infertile men.

Abstract: With increasing evidence for faulty paternal contribution to reproduction, there has been a steady increase in studies highlighting an association between sperm DNA damage, failed/delayed fertilisation and aberrant embryo development. Owing to prevailing ambiguity, the aims of the study were to analyse the genetic integrity of the male gamete and then to understand its association with age, standard semen parameters, lifestyle and occupational factors. The study included 504 subjects, attending university infertility clinic for fertility evaluation and treatment. Semen characteristics were analysed by standard criteria; terminal deoxynucelotidyl transferase-mediated nick end-labelling assay was employed for DNA damage assessment. The average incidence of sperm DNA damage in patients with normozoospermic semen parameters was <10%. Patients with oligozoospermia, severe oligozoospermia, oligoasthenoteratospermia, asthenoteratozoospermia and necrozoospermia had significantly higher level of sperm DNA damage (P < 0.001). Patients above 40 years of age had significantly high levels of DNA damage (P < 0.001) compared with their counterparts. Patients with varicocele and a history of alcohol consumption had higher incidence of spermatozoa with DNA damage (P < 0.01). Poor sperm characteristics in the ejaculate are associated with increased sperm DNA damage. Age-related increase in sperm DNA damage and association of the same with varicocele and alcohol consumption are also demonstrated.

APOL1 Risk Variants Predict Histopathology and Progression to ESRD in HIV-Related Kidney Disease

Abstract: With earlier institution of antiretroviral therapy, kidney diseases other than HIV-associated nephropathy (HIVAN) predominate in HIV-infected persons. Outcomes for these diseases are typically worse among those infected with HIV, but the reasons for this are not clear. Here, we examined the role of APOL1 risk variants in predicting renal histopathology and progression to ESRD in 98 HIV-infected African Americans with non-HIVAN kidney disease on biopsy. We used survival analysis to determine time to ESRD associated with APOL1 genotype. Among the 29 patients with two APOL1 risk alleles, the majority (76%) had FSGS and 10% had hypertensive nephrosclerosis. In contrast, among the 54 patients with one APOL1 risk allele, 47% had immune-complex GN as the predominant lesion and only 23% had FSGS. Among the 25 patients with no APOL1 risk allele, 40% had immune-complex GN and 12% had FSGS. In 310 person-years of observation, 29 patients progressed to ESRD. In adjusted analyses, individuals with two APOL1 risk alleles had a nearly three-fold higher risk for ESRD compared with those with one or zero risk alleles (P=0.03). In summary, these data demonstrate an association between APOL1 variants and renal outcomes in non-HIVAN kidney disease, suggesting a possible use for APOL1 genotyping to help guide the care of HIV-infected patients.

Design of RF energy harvesting system for energizing low power devices

Abstract: Electromagnetic energy harvesting holds a promising future for energizing low power electronic devices in wireless communication circuits. This article presents an RF energy harvesting system that can harvest energy from the ambient surroundings at the downlink radio frequency range of GSM-900 band. The harvesting system is aimed to provide an alternative source of energy for energizing low power devices. The system design consists of three modules: a single wideband 377› E-shaped patch antenna, a pi matching network and a 7-stage voltage doubler circuit. These three modules were fabricated on a single printed circuit board. The antenna and Pi matching network have been optimized through electromagnetic simulation software, Agilent ADS 2009 environment. The uniqueness of the system lies in the partial ground plane and the alignment of induced electric fleld for maximum current ∞ow in the antenna that maximizes the captured RF energy. The design and simulation of the voltage doubler circuit were performed using Multisim software. All the three modules were integrated and fabricated on a double sided FR 4 printed circuit board. The DC voltage obtained from the harvester system in the fleld test at an approximate distance of 50m from GSM cell tower was 2.9V. This voltage was enough to power the STLM20 temperature sensor.

Image Analysis Tools for Evaluation of Microscopic Views of Immunohistochemically Stained Specimen in Medical Research---a Review

Abstract: The aim of this study is to review the methods being used for image analysis of microscopic views of immunohistochemically stained specimen in medical research. The solutions available range from general purpose software to commercial packages. Many studies have developed their own custom written programs based on some general purpose software available. Many groups have reported development of computer aided image analysis programs aiming at obtaining faster, simpler and cheaper solutions. Image analysis tools namely Aperio, Lucia, Metaview, Metamorph, ImageJ, Scion, Adobe Photoshop, Image Pro Plus are also used for evaluation of expressions using immunohistochemical staining. An overview of such methods used for image analysis is provided in this paper. This study concludes that there is good scope for development of freely available software for staining intensity quantification, which a medical researcher could easily use without requiring high level computer skills.

Prevalence of depression and its associated factors using Beck Depression Inventory among students of a medical college in Karnataka.

Abstract: Background and Objective: Depression among medical students is an area of increasing concern worldwide. This study aimed to assess the prevalence of depression and its associated factors among medical students. Materials and Methods: A stratified random sample of 400 students was assessed using Beck Depression Inventory by investigators. Associations between depression and class of studying, social factors like alcohol use, drug addiction, family problems, family history of depression, and staying away from home were analyzed by univariate analysis. Results: The overall prevalence of depression was found to be 71.25%. Among those with depression, a majority (80%) had mild and moderate degree of depression. The study showed that 46.3% (132) of the depressed were females and 53.7% (153) were males. According to cut-off scores, 115 students (29.8%) scored as normal (0-9), 111 (27.8%) as mild (10-18), 117 (29.3%) as moderate (19-29), 30 (7.5%) as severe (30-40), and 27 (6.7%) as very severe (>40) depression. The prevalence of depression was comparatively less among 1 st and 2 nd year medical students (57% and 50%, respectively) and the difference between the grade of depression and year of studying was found to be significant (χ2 =122, P Conclusion: Depression is highly prevalent among medical students in this area. Our findings point to the importance of broad screening and psychiatric counseling of this vulnerable population.

Awareness and Attitude towards Human Papillomavirus (HPV) Vaccine among Medical Students in a Premier Medical School in India

Abstract: Background: As preventing cancer with the help of a vaccine is a comparatively new concept, awareness and education about it will have important implication in the implementation of this strategy. Materials and Methods: Present explorative questionnaire based survey included 618 MBBS students for final analysis. Results: Majority of participants (89.6%) were well aware of the preventable nature of cervical cancer. Most of them (89.2%) knew that necessary factor responsible for cervical cancer is infection with high risk HPV. Awareness regarding the availability of vaccine against cervical cancer was 75.6%. Females had a better awareness regarding availability of vaccine, target population for vaccination and about the catch up program. Overall acceptance of HPV vaccine among the population studied was 67.8%. Medical teaching had a definitive impact on the understanding of this important public health issue. Females seemed to be more ready to accept the vaccine and recommend it to others. For our study population the most common source of information was medical school teaching. Majority of participants agreed that the most important obstacle in implementation of HPV vaccination program in our country is inadequate information and 86.2% wanted to be educated by experts in this regard. Conclusion: HPV vaccine for primary prevention of cervical cancer is a relatively new concept. Health professional will be able to play a pivotal role in popularizing this strategy.

Polycomb Repressor Complex-2 Is a Novel Target for Mesothelioma Therapy

Abstract: Purpose: Polycomb group (PcG) proteins are critical epigenetic mediators of stem cell pluripotency, which have been implicated in the pathogenesis of human cancers. This study was undertaken to examine the frequency and clinical relevance of PcG protein expression in malignant pleural mesotheliomas (MPM). Experimental Design: Microarray, quantitative reverse transcriptase PCR (qRT-PCR), immunoblot, and immunohistochemistry techniques were used to examine PcG protein expression in cultured MPM, mesothelioma specimens, and normal mesothelial cells. Lentiviral short hairpin RNA techniques were used to inhibit EZH2 and EED expression in MPM cells. Proliferation, migration, clonogenicity, and tumorigenicity of MPM cells either exhibiting knockdown of EZH2 or EED , or exposed to 3-deazaneplanocin A (DZNep), and respective controls were assessed by cell count, scratch and soft agar assays, and murine xenograft experiments. Microarray and qRT-PCR techniques were used to examine gene expression profiles mediated by knockdown of EZH2 or EED , or DZNep. Results: EZH2 and EED , which encode components of polycomb repressor complex-2 (PRC-2), were overexpressed in MPM lines relative to normal mesothelial cells. EZH2 was overexpressed in approximately 85% of MPMs compared with normal pleura, correlating with diminished patient survival. Overexpression of EZH2 coincided with decreased levels of miR-101 and miR-26a. Knockdown of EZH2 or EED , or DZNep treatment, decreased global H3K27Me3 levels, and significantly inhibited proliferation, migration, clonogenicity, and tumorigenicity of MPM cells. Common as well as differential gene expression profiles were observed following knockdown of PRC-2 members or DZNep treatment. Conclusions: Pharmacologic inhibition of PRC-2 expression/activity is a novel strategy for mesothelioma therapy. Clin Cancer Res; 18(1); 77–90. ©2011 AACR .

Study of Nurses' Knowledge about Palliative Care: A Quantitative Cross-sectional Survey.

Abstract: Context: Studies have documented that nurses and other health care professionals are inadequately prepared to care for patients in palliative care. Several reasons have been identified including inadequacies in nursing education, absence of curriculum content related to pain management, and knowledge related to pain and palliative care. Aims: The objective of this paper was to assess the knowledge about palliative care amongst nursing professionals using the palliative care knowledge test (PCKT). Settings and Design: Cross-sectional survey of 363 nurses in a multispecialty hospital. Materials and Methods: The study utilized a self-report questionnaire- PCKT developed by Nakazawa et al., which had 20 items (statements about palliative care) for each of which the person had to indicate 'correct', 'incorrect', or 'unsure.' The PCKT had 5 subscales (philosophy- 2 items, pain- 6 items, dyspnea- 4 items, psychiatric problems- 4 items, and gastro-intestinal problems- 4 items). Statistical Analysis Used: Comparison across individual and professional variables for both dimensions were done using one-way ANOVA, and correlations were done using Karl-Pearson's co-efficient using SPSS version 16.0 for Windows. Results: The overall total score of PCKT was 7.16 ± 2.69 (35.8%). The philosophy score was 73 ± .65 (36.5%), pain score was 2.09 ± 1.19 (34.83%), dyspnea score was 1.13 ± .95 (28.25%), psychiatric problems score was 1.83 ± 1.02 (45.75%), and gastro-intestinal problems score was 1.36 ± .97 (34%). (P = .00). The female nurses scored higher than their male counterparts, but the difference was not significant (P > .05). Conclusions: Overall level of knowledge about palliative care was poor, and nurses had a greater knowledge about psychiatric problems and philosophy than the other aspects indicated in PCKT.

Mithramycin Represses Basal and Cigarette Smoke–Induced Expression of ABCG2 and Inhibits Stem Cell Signaling in Lung and Esophageal Cancer Cells

Abstract: Cigarette smoking at diagnosis or during therapy correlates with poor outcome in patients with lung and esophageal cancers, yet the underlying mechanisms remain unknown. In this study, we observed that exposure of esophageal cancer cells to cigarette smoke condensate (CSC) led to upregulation of the xenobiotic pump ABCG2, which is expressed in cancer stem cells and confers treatment resistance in lung and esophageal carcinomas. Furthermore, CSC increased the side population of lung cancer cells containing cancer stem cells. Upregulation of ABCG2 coincided with increased occupancy of aryl hydrocarbon receptor, Sp1, and Nrf2 within the ABCG2 promoter, and deletion of xenobiotic response elements and/or Sp1 sites markedly attenuated ABCG2 induction. Under conditions potentially achievable in clinical settings, mithramycin diminished basal as well as CSC-mediated increases in AhR, Sp1, and Nrf2 levels within the ABCG2 promoter, markedly downregulated ABCG2, and inhibited proliferation and tumorigenicity of lung and esophageal cancer cells. Microarray analyses revealed that mithramycin targeted multiple stem cell-related pathways in vitro and in vivo. Collectively, our findings provide a potential mechanistic link between smoking status and outcome of patients with lung and esophageal cancers, and support clinical use of mithramycin for repressing ABCG2 and inhibiting stem cell signaling in thoracic malignancies.

Comparative cellular and molecular analyses of pooled bone marrow multipotent mesenchymal stromal cells during continuous passaging and after successive cryopreservation.

Abstract: The clinical application of human bone marrow derived multipotent mesenchymal stromal cells (MSC) requires expansion, cryopreservation, and transportation from the laboratory to the site of cell implantation The cryopreservation and thawing process of MSCs may have important effects on the viability, growth characteristics and functionality of these cells both in vitro and in vivo More importantly, MSCs after two rounds of cryopreservation have not been as well characterized as fresh MSCs from the transplantation perspective The objective of this study was to determine if the effect of successive cryopreservation of pooled MSCs during the exponential growth phase could impair their morphology, phenotype, gene expression, and differentiation capabilities MSCs cryopreserved at passage 3 (cell bank) were thawed and expanded up to passage 4 and cryopreserved for the second time These cells (passive) were then thawed and cultured up to passage 6, and, at each passage MSCs were characterized As control, pooled passage 3 cells (active) after one round of cryopreservation were taken all the way to passage 6 without cryopreservation We determined the growth rate of MSCs for both culture conditions in terms of population doubling number (PDN) and population doubling time (PDT) Gene expression profiles for pluripotency markers and tissue specific markers corresponding to neuroectoderm, mesoderm and endoderm lineages were also analyzed for active and passive cultures of MSC The results show that in both culture conditions, MSCs exhibited similar growth properties, phenotypes and gene expression patterns as well as similar differentiation potential to osteo-, chondro-, and adipo-lineages in vitro To conclude, it appears that successive or multiple rounds of cryopreservation of MSCs did not alter the fundamental characteristics of these cells and may be used for clinical therapy

PLGA 50:50 nanoparticles of paclitaxel: Development, in vitro anti-tumor activity in BT-549 cells and in vivo evaluation

Abstract: Clinical administration of paclitaxel is hindered due to its poor solubility, which necessitates the formulation of novel drug delivery systems to deliver such extreme hydrophobic drug. To formulate nanoparticles which makes suitable to deliver hydrophobic drugs effectively (intravenous) with desired pharmacokinetic profile for breast cancer treatment; in this context in vitro cytotoxic activity was evaluated using BT-549 cell line. PLGA nanoparticles were prepared by emulsion solvent evaporation technique and evaluated for physicochemical parameters, in vitro anti-tumor activity and in vivo pharmacokinetic studies in rats. Particle size obtained in optimized formulation was <200 nm. Encapsulation efficiency was higher at polymer-to-drug ratio of 20:1. In vitro drug release exhibited biphasic pattern with initial burst release followed by slow and continuous release (15 days). In vitro anti-tumor activity of optimized formulation inhibited cell growth for a period of 168 h against BT-549 cells. AUC(0−∞) and t1/2 were found to be higher for nanoparticles with low clearance rate.

In Vivo Effects Of Traditional Ayurvedic Formulations in Drosophila melanogaster Model Relate with Therapeutic Applications

Abstract: Background Ayurveda represents the traditional medicine system of India. Since mechanistic details of therapy in terms of current biology are not available in Ayurvedic literature, modern scientific studies are necessary to understand its major concepts and procedures. It is necessary to examine effects of the whole Ayurvedic formulations rather than their “active” components as is done in most current studies.

DNA double-strand break analysis by γ-H2AX foci: a useful method for determining the overreactors to radiation-induced acute reactions among head-and-neck cancer patients.

Abstract: Purpose Interindividual variability in normal tissue toxicity during radiation therapy is a limiting factor for successful treatment. Predicting the risk of developing acute reactions before initiation of radiation therapy may have the benefit of opting for altered radiation therapy regimens to achieve minimal adverse effects with improved tumor cure. Methods and Materials DNA double-strand break (DSB) induction and its repair kinetics in lymphocytes of head-and-neck cancer patients undergoing chemoradiation therapy was analyzed by counting γ-H2AX foci, neutral comet assay, and a modified version of neutral filter elution assay. Acute normal tissue reactions were assessed by Radiation Therapy Oncology Group criteria. Results The correlation between residual DSBs and the severity of acute reactions demonstrated that residual γ-H2AX foci in head-and-neck cancer patients increased with the severity of oral mucositis and skin reaction. Conclusions Our results suggest that γ-H2AX analysis may have predictive implications for identifying the overreactors to mucositis and skin reactions among head-and-neck cancer patients prior to initiation of radiation therapy.

(-)-Epigallocatechin-3-gallate induces apoptosis and inhibits invasion and migration of human cervical cancer cells.

Abstract: Invasion and metastasis are the major causes of cancer-related death. Pharmacological or therapeutic interventions such as chemoprevention of the progression stages of neoplastic development could result in substantial reduction in the incidence of cancer mortality. (-)-Epigallocatechin-3-gallate (EGCG), a promising chemopreventive agent, has attracted extensive interest for cancer therapy utilizing its antioxidant, antiproliferative and inhibitory effects on angiogenesis and tumor cell invasion. In this study, we assessed the influence of EGCG on the proliferative potential of HeLa cells by cell viability assay and authenticated the results by nuclear morphological examination, DNA laddering assay and cell cycle analysis. Further we analyzed the anti-invasive properties of EGCG by wound migration assay and gene expression of MMP-9 and TIMP-1 in HeLa cells. Our results indicated that EGCG induced growth inhibition of HeLa cells in a dose- and timedependent manner. It was observed that cell death mediated by EGCG was through apoptosis. Interestingly, EGCG effectively inhibited invasion and migration of HeLa cells and modulated the expression of related genes (MMP-9 and TIMP-1) . These results indicate that EGCG may effectively suppress promotion and progression stages of cervical cancer development.

The effect of smoking on the ocular surface and the precorneal tear film

Abstract: Background:Smoking, both active and passive, creates a plethora of health‐related problems, which primarily affect the cardiovascular and respiratory systems. There is very little evidence on the effects of tobacco smoke on the eye, especially regarding anterior ocular surface related pathology. This study was undertaken to determine the effects of smoking on the ocular surface and the tear film in smokers. Methods:A total of 51 (102 eyes) smokers and 50 (100 eyes) age‐ andgender‐matched healthy non‐smokers were included in thisstudy. The ocular surface was evaluated by measuring tear film break‐up time, surface staining with fluorescein, and corneal and conjunctival sensitivities, and by completing the Schirmer’s II test. Data was analysed using Statistical Package for Social Sciences (SPSS) version 11.5. A p value less than 0.05 was considered statistically significant. Results: The smoker group had significantly lower tear film break‐up time, and corneal and conjunctival sensitivity than the nonsmoker group. Punctate staining was significantly higher in the smoker group than the non‐smoker group. There was no statistically significant difference in Schirmer’s II test results between the smoker and non‐smoker group. Conclusion: Smoking caused adverse effects on the precorneal tear film and there was strong association between smoking and tear film instability. Although a causative relationship could not be determined, there is a need for further longitudinal studies.

Simultaneous detection of periodontal pathogens in subgingival plaque and placenta of women with hypertension in pregnancy

Abstract: There are many studies documenting increased prevalence of periodontal infection in women with preeclampsia. But, very few studies have attempted to establish causal relationship between the two. To find out causal circumstantial evidence by isolating specific periodontal pathogens in oral and placental samples. Antenatal periodontal screening and subgingival plaque collection was carried out in ten women with hypertension in pregnancy and ten normotensive controls on their hospital admission at term for cesarean delivery. Placental biopsy was obtained after aseptic placental collection at the time of elective cesarean delivery. Subgingival plaque and placental biopsy were studied for Porphyromonas gingivalis, Fusobacterium nucleatum, Treponema denticola, Prevotella intermedia and Aggregatibacter actinomycetemcomitans using quantitative polymerase chain reaction technique. Periodontist and laboratory personnel were unaware of case or control status. Periodontal status was not informed to the obstetrician recruiting the cases and laboratory. Microbiology report was not revealed till end of the study. Periodontal pathogens were found to be high in the group with hypertension than the controls. P gingivalis was found in all the samples from subgingival plaque and placenta, irrespective of the periodontal disease status. In cases with hypertension, periodontal pathogens are present in higher proportion in subgingival plaque and placenta.

Role of Actin Dependent Nuclear Deformation in Regulating Early Gene Expression

Abstract: The nucleus of a living cell is constantly undergoing changes in shape and size as a result of various mechanical forces in physiology. These changes correlate with alterations in gene expression, however it is unclear whether nuclear deformation alone is sufficient to elicit these alterations. We used T-cell activation as a model system to test the coupling between nuclear deformation (elongation) and gene expression. Naive T-cell activation with surrogate antigens resulted in actin dependent nuclear elongation. This was accompanied with Erk and NF-κB signaling to the nucleus to induce CD69 expression. Importantly, inhibiting actin polymerization abolished both nuclear elongation and CD69 expression, while inhibiting Erk, NF-κB or microtubule depolymerization only abolished expression but not elongation. Immobilization of antigen-coated beads, under conditions where actin polymerization was inhibited, rescued both nuclear elongation and CD69 expression. In addition, fibroblast cells plated on fibronectin micropatterns of different sizes showed correlation between nuclear shape index and tenascin C expression. Upon inhibiting the signaling intermediate Erk, tenascin C expression was down regulated although the nuclear shape index remained unaltered. Our results highlight the importance of specific signaling intermediates accompanied with nuclear deformation in the modulation of cellular genomic programs.

Calibration-free laser-induced breakdown spectroscopy for quantitative elemental analysis of materials

Abstract: The application of calibration-free laser-induced breakdown spectroscopy (CF-LIBS) for quantitative analysis of materials, illustrated by CF-LIBS applied to a brass sample of known composition, is presented in this paper The LIBS plasma is produced by a 355 nm pulsed Nd:YAG laser with a pulse duration of 6 ns focussed onto a brass sample in air at atmospheric pressure The time-resolved atomic and ionic emission lines of Cu and Zn from the LIBS spectra recorded by an Echelle spectrograph coupled with a gated intensified charge coupled detector are used for the plasma characterization and the quantitative analysis of the sample The time delay where the plasma is optically thin and is also in local thermodynamic equilibrium (LTE), necessary for the elemental analysis of samples from the LIBS spectra, is deduced An algorithm relating the experimentally measured spectral intensity values with the basic physics of the plasma is developed Using the algorithm, the Zn and Cu concentrations in the brass sample are determined The analytical results obtained from the CF-LIBS technique agree well with the certified values of the elements in the sample, with an accuracy error <1%