Protease production and molecular characterization of a protease dipeptidyl-aminopeptidase gene from different strains of Sordaria fimicola / Produção de protease e caracterização molecular de um gene de protease - DPAP (dipeptidil-aminopeptidase) de diferentes cepas de Sordaria fimicola

The current research was designed to reach extracellular protease production potential in different strains of Sordaria fimicola which were previously obtained from Dr. Lamb (Imperial College, London) from North Facing Slope and South Facing Slope of Evolution Canyon. After initial and secondary screening, two hyper-producers strains S2 and N6 were selected for submerged fermentation and cultural conditions including temperature, pH, incubation period, inoculum size, substrate concentration, and different carbon and nitrogen sources were optimized for enzyme production. S2 strain showed maximum protease production of 3.291 U/mL after 14 days of incubation at 30 °C with 7 pH, 1% substrate concentration and 1 mL inoculum, While N6 strain showed maximum protease production of 1.929 U/mL under fermentation optimized conditions. Another aim of the present research was to underpin the biodiversity of genetics and post-translational modifications (PTMs) of protease DPAP (peptidyl-aminopeptidase) in Sordaria fimicola. Five polymorphic sites were observed in amino acid sequence of S. fimicola strains with reference to Neurospora crassa. PTMs prediction from bioinformatics tools predicted 38 phosphorylation sites on serine residues for protease peptidyl-aminopeptidase in S1 strain of S. fimicola while 45 phosphorylation sites on serine in N7 strain and 47 serine phosphorylation modifications were predicted in N. crassa. Current research gave an insight that change in genetic makeup effected PTMs which ultimately affected the production of protease enzyme in different strains of same organism (S. fimicola). The production and molecular data of the research revealed that environmental stress has strong effects on the specific genes through mutations which may cause genetic diversity. S. fimicola is non- pathogenic fungus and has a short life cycle. This fungus can be chosen to produce protease enzyme on a commercial scale.

A pesquisa atual foi projetada para alcançar o potencial de produção de protease extracelular em diferentes cepas de Sordaria fimicola que foram previamente obtidas do Dr. Lamb (Imperial College, Londres) de North Facing Slope e South Facing Slope de Evolution Canyon. Após a triagem inicial e secundária, duas cepas hiperprodutoras S2 e N6 foram selecionadas para fermentação submersa e condições culturais, incluindo temperatura, pH, período de incubação, tamanho do inóculo, concentração de substrato, e diferentes fontes de carbono e nitrogênio foram otimizadas para produção de enzima. A cepa S2 apresentou produção máxima de protease de 3,291 U/mL após 14 dias de incubação a 30 °C com pH 7, concentração de substrato de 1% e inóculo de 1 mL, enquanto a cepa N6 apresentou produção máxima de protease de 1,929 U/mL em condições otimizadas de fermentação. Outro objetivo da presente pesquisa foi sustentar a biodiversidade da genética e modificações pós-tradicionais (PTMs) da protease DPAP (peptidil-aminopeptidase) em Sordaria fimicola. Cinco sítios polimórficos foram observados na sequência de aminoácidos de cepas de S. fimicola com referência a Neurospora crassa. A previsão de PTMs a partir de ferramentas de bioinformática previu 38 locais de fosforilação em resíduos de serina para protease peptidil-aminopeptidase na cepa S1 de S. fimicola, enquanto 45 locais de fosforilação em serina na cepa N7 e 47 modificações de fosforilação de serina foram previstas em N. crassa. A pesquisa atual deu uma ideia de que a mudança na composição genética afetou os PTMs que, em última análise, afetaram a produção da enzima protease em diferentes cepas do mesmo organismo (S. fimicola). A produção e os dados moleculares da pesquisa revelaram que o estresse ambiental tem fortes efeitos sobre genes específicos por meio de mutações que podem causar diversidade genética. S. fimicola é um fungo não patogênico e tem um ciclo de vida curto. Esse fungo pode ser escolhido para produzir enzima protease em escala comercial.

Evaluation of the persistent organic pollutants association with type 2 diabetes: A prospective study from Karachi, Pakistan / Avaliação da associação de poluentes orgânicos persistentes com diabetes tipo 2: um estudo prospectivo de Karachi, Paquistão

The aim of this study is to determine the association between environmental organic pollutants with type 2 diabetes. This prospective study was conducted in Federal Urdu University of Arts, Science and Technology (FUUAST) Gulshan-e-Iqbal Campus Karachi in duration from January 2016 to June 2017. This study was ethically approved from the Institutional Review Board of FUUAST. The study included 50 male and female convenient subjects with type 2 diabetes. Subject with other type of diabetes was excluded. Consent was obtained by each individual. Self-structured questionnaire was used for data collection. The comparative results suggest that the maximum level of summation polychlorinated biphenyls (PCBs) mean value was found in age group 27-33 as 0.695 mg/kg in 73% having total individual eleven. Median (interquartile range) of pesticides levels among subjects with normal weight, over weight and obesity were 0.49 (0.26-2.13), 1.53 (0.60-2.65), and 1.60 (1.23-2.05) respectively. It was observed that Organochlorine pesticides (OCS) levels of subjects with overweight and obesity were almost similar (P-value > 0.05) but significantly higher as compared to subjects with normal weight (P-value < 0.05). No significant differences were observed between PCB levels of subjects in terms of body mass index (BMI). In present study we trace the important elements involve in the deposition of persistent organic pollutants and established an association between pollutants with etiology of diabetes and associated disorders such as obesity.

O objetivo deste estudo é determinar a associação entre poluentes orgânicos ambientais com diabetes tipo 2. Este estudo prospectivo foi conduzido na Universidade Federal Urdu de Artes, Ciência e Tecnologia (FUUAST), Gulshan-e-Iqbal, Campus Karachi, com duração de janeiro de 2016 a junho de 2017. Este estudo foi eticamente aprovado pelo Comitê de Revisão Institucional da FUUAST. O estudo incluiu 50 indivíduos convenientes do sexo masculino e feminino com diabetes tipo 2. Indivíduos com outro tipo de diabetes foram excluídos. O consentimento foi obtido por cada indivíduo. Um questionário autoestruturado foi utilizado para a coleta de dados. Os resultados comparativos sugerem que o nível máximo de soma de bifenilas policloradas (PCBs) valor médio foi encontrado na faixa etária 27-33 como 0,695 mg / kg em 73%, tendo total de 11 indivíduos. A mediana (intervalo interquartil) dos níveis de pesticidas entre indivíduos com peso normal, sobrepeso e obesidade foi de 0.49 (0.26-2.13), 1.53 (0.60-2.65) e 1.60 (1.23-2.05), respectivamente. Observou-se que os níveis de pesticidas organoclorados (OCS) de indivíduos com sobrepeso e obesidade eram quase semelhantes (valor P > 0.05), mas significativamente maiores em comparação com indivíduos com peso normal (valor P < 0.05). Não foram observadas diferenças significativas entre os níveis de PCB dos indivíduos em termos de índice de massa corporal (IMC). No presente estudo, rastreamos os elementos importantes envolvidos na deposição de poluentes orgânicos persistentes e estabelecemos uma associação entre os poluentes com a etiologia do diabetes e doenças associadas, como a obesidade.

Molecular analysis of Panton-Valentine Leucocidin (pvl) gene among MRSA and MSSA isolates.

The present study was conducted in order to determine the frequency of pvl gene among the pathogenic and healthy population isolates of Methicillin Resistant Staphylococcus aureus (MRSA) and Methicillin Sensitive Staphylococcus aureus (MSSA). For this purpose, nasal swab samples were collected from the healthy individuals (to be used as controls, all the samples were collected irrespective of the sex and age factors), the pathogenic samples were collected from different patients suffering from skin &soft tissue infections caused by S. aureus (to be used as test samples).Both of these population samples were analyzed for the presence of pvl gene. S.aureus were identified through conventional microbiological identification procedures. In the case of normal samples, 70 nasal swabs were collected and only 33 (47%) proved to be S. aureus while 20 pathogenic samples were collected and all (100%) were cleared as S. aureus. For further distribution of samples into MRSA and MSSA, antibiotic susceptibility pattern was checked by using the standard protocols of Kirby-Bauer disc diffusion method. Two antibiotic discs Oxacillin (OX: 1ug) and cefoxitin (FOX: 30ug) were used. Among healthy population, MRSA was found to be 79% (n=26) and MSSA were present as 21% (n= 7). Among pathogenic strains 100% MRSA was detected where n= 20. Detection of pvl gene among the MRSA and MSSA isolates was done by using the uniplex PCR followed by gel electrophoresis. MRSA and MSSA of normal healthy population carried 49% and 7% pvl gene respectively. While, pathogenic MRSA samples carried 46% pvl gene among them. Potentially alarming percentage of pvl gene is present among the normal healthy individuals which indicates a future threat and a major health concern.

Molecular analysis of Panton-Valentine Leucocidin (pvl) gene among MRSA and MSSA isolates / Análise molecular do gene Panton-Valentine Leucocidin (pvl) entre isolados de MRSA e MSSA

The present study was conducted in order to determine the frequency of pvl gene among the pathogenic and healthy population isolates of Methicillin Resistant Staphylococcus aureus (MRSA) and Methicillin Sensitive Staphylococcus aureus (MSSA). For this purpose, nasal swab samples were collected from the healthy individuals (to be used as controls, all the samples were collected irrespective of the sex and age factors), the pathogenic samples were collected from different patients suffering from skin &soft tissue infections caused by S. aureus (to be used as test samples).Both of these population samples were analyzed for the presence of pvl gene. S.aureus were identified through conventional microbiological identification procedures. In the case of normal samples, 70 nasal swabs were collected and only 33 (47%) proved to be S. aureus while 20 pathogenic samples were collected and all (100%) were cleared as S. aureus. For further distribution of samples into MRSA and MSSA, antibiotic susceptibility pattern was checked by using the standard protocols of Kirby-Bauer disc diffusion method. Two antibiotic discs Oxacillin (OX: 1ug) and cefoxitin (FOX: 30ug) were used. Among healthy population, MRSA was found to be 79% (n=26) and MSSA were present as 21% (n= 7). Among pathogenic strains 100% MRSA was detected where n= 20. Detection of pvl gene among the MRSA and MSSA isolates was done by using the uniplex PCR followed by gel electrophoresis. MRSA and MSSA of normal healthy population carried 49% and 7% pvl gene respectively. While, pathogenic MRSA samples carried 46% pvl gene among them. Potentially alarming percentage of pvl gene is present among the normal healthy individuals which indicates a future threat and a major health concern.

O presente estudo almejou determinar a frequência do gene PVL entre os isolados patogênicos e saudáveis da população de Staphylococcus aureus resistente à meticilina (MRSA) e Staphylococcus aureus sensível à meticilina (MSSA). Para este propósito, amostras de swab nasal foram coletadas de indivíduos saudáveis (utilizadas como controle, todas as amostras foram coletadas independentemente do sexo e idade), e de diferentes pacientes com infecções de pele e tecidos moles causadas por S. aureus (utilizadas como amostras de teste). Ambas as amostras populacionais foram analisadas quanto à presença do gene PVL. S.aureus foram identificados através de procedimentos convencionais de identificação microbiológica. No caso de amostras normais, 70 swabs nasais foram coletados e apenas 33 (47%) provaram ser S. aureus, enquanto 20 amostras patogênicas foram coletadas e todas (100%) foram eliminadas como S. aureus. Para distribuição posterior de amostras em MRSA e MSSA, o padrão de suscetibilidade a antibióticos foi verificado a partir dos protocolos padrão do método de difusão de disco de Kirby-Bauer. Foram utilizados dois discos de antibióticos Oxacilina (OX: 1ug) e cefoxitina (FOX: 30ug). Entre a população saudável, MRSA foi encontrado em 79% (n = 26) e MSSA estava presente em 21% (n = 7). Entre as cepas patogênicas, 100% de MRSA foi detectado onde n = 20. A detecção do gene PVL entre os isolados de MRSA e MSSA foi feita usando a PCR uniplex seguida de eletroforese em gel. MRSA e MSSA da população saudável normal carregavam 49% e 7% do gene PVL, respectivamente. Enquanto as amostras patogênicas de MRSA carregavam 46% do gene PVL entre elas. Uma porcentagem potencialmente alarmante do gene PVL foi detectada entre os indivíduos saudáveis normais, o que indica uma ameaça futura e um grande problema de saúde.

Persistent organic pollutants distribution in plasma lipoprotein fractions / Distribuição persistente de poluentes orgânicos em frações de lipoproteínas plasmáticas

This study determines the associations among serum lipid profiles, risk of cardiovascular disease, and persistent organic pollutants. Using Gas chromatography technique, the intensity of toxic pollutant residues in serum samples of Hypertensive patients were measured. Based on statistical analysis, the effects of different covariates namely pesticides, age, systolic blood pressure, diastolic blood pressure, and lipid profile duration was checked using the logistic regression model. Statistical computation was performed on SPSS 22.0. The P-values of F-Statistic for each lipid profile class are greater than 0.01 (1%), therefore we cannot reject the null hypothesis for all cases. The estimated coefficients, their standard errors, Wald Statistic, and odds ratio of the binary logistic regression model for different lipid profile parameters indicate if pesticides increase then the logit value of different lipid profile parameters changes from -0.46 to -0.246 except LDL which increases by 0.135. The study reports a significantly increased threat of cardiovascular disease with increased concentrations of toxic pollutants.(AU)

Este estudo determina as associações entre o perfil lipídico sérico, o risco de doença cardiovascular e os poluentes orgânicos persistentes. Por meio da técnica de cromatografia gasosa, mediu-se a intensidade dos resíduos de poluentes tóxicos em amostras de soro de pacientes hipertensos. Com base na análise estatística, os efeitos de diferentes covariáveis ou seja, pesticidas, idade, pressão arterial sistólica, pressão arterial diastólica e duração do perfil lipídico foram verificados usando o modelo de regressão logística. O cálculo estatístico foi realizado no SPSS 22.0. Os valores P da estatística F para cada classe de perfil lipídico são maiores que 0,01 (1%), portanto não podemos rejeitar a hipótese nula para todos os casos. Os coeficientes estimados, seus erros padrão, estatística de Wald e odds ratio do modelo de regressão logística binária para diferentes parâmetros do perfil lipídico indicam se os pesticidas aumentam, então o valor logit de diferentes parâmetros do perfil lipídico muda de -0,46 para -0,246, exceto LDL, que aumenta em 0,135. O estudo relata um aumento significativo da ameaça de doença cardiovascular com aumento das concentrações de poluentes tóxicos.(AU)

Persistent organic pollutants distribution in plasma lipoprotein fractions / Distribuição persistente de poluentes orgânicos em frações de lipoproteínas plasmáticas

Abstract This study determines the associations among serum lipid profiles, risk of cardiovascular disease, and persistent organic pollutants. Using Gas chromatography technique, the intensity of toxic pollutant residues in serum samples of Hypertensive patients were measured. Based on statistical analysis, the effects of different covariates namely pesticides, age, systolic blood pressure, diastolic blood pressure, and lipid profile duration was checked using the logistic regression model. Statistical computation was performed on SPSS 22.0. The P-values of F-Statistic for each lipid profile class are greater than 0.01 (1%), therefore we cannot reject the null hypothesis for all cases. The estimated coefficients, their standard errors, Wald Statistic, and odds ratio of the binary logistic regression model for different lipid profile parameters indicate if pesticides increase then the logit value of different lipid profile parameters changes from -0.46 to -0.246 except LDL which increases by 0.135. The study reports a significantly increased threat of cardiovascular disease with increased concentrations of toxic pollutants.

Resumo Este estudo determina as associações entre o perfil lipídico sérico, o risco de doença cardiovascular e os poluentes orgânicos persistentes. Por meio da técnica de cromatografia gasosa, mediu-se a intensidade dos resíduos de poluentes tóxicos em amostras de soro de pacientes hipertensos. Com base na análise estatística, os efeitos de diferentes covariáveis ​​- ou seja, pesticidas, idade, pressão arterial sistólica, pressão arterial diastólica e duração do perfil lipídico - foram verificados usando o modelo de regressão logística. O cálculo estatístico foi realizado no SPSS 22.0. Os valores P da estatística F para cada classe de perfil lipídico são maiores que 0,01 (1%), portanto não podemos rejeitar a hipótese nula para todos os casos. Os coeficientes estimados, seus erros padrão, estatística de Wald e odds ratio do modelo de regressão logística binária para diferentes parâmetros do perfil lipídico indicam se os pesticidas aumentam, então o valor logit de diferentes parâmetros do perfil lipídico muda de -0,46 para -0,246, exceto LDL, que aumenta em 0,135. O estudo relata um aumento significativo da ameaça de doença cardiovascular com aumento das concentrações de poluentes tóxicos.

Persistent organic pollutants distribution in plasma lipoprotein fractions / Distribuição persistente de poluentes orgânicos em frações de lipoproteínas plasmáticas

This study determines the associations among serum lipid profiles, risk of cardiovascular disease, and persistent organic pollutants. Using Gas chromatography technique, the intensity of toxic pollutant residues in serum samples of Hypertensive patients were measured. Based on statistical analysis, the effects of different covariates namely pesticides, age, systolic blood pressure, diastolic blood pressure, and lipid profile duration was checked using the logistic regression model. Statistical computation was performed on SPSS 22.0. The P-values of F-Statistic for each lipid profile class are greater than 0.01 (1%), therefore we cannot reject the null hypothesis for all cases. The estimated coefficients, their standard errors, Wald Statistic, and odds ratio of the binary logistic regression model for different lipid profile parameters indicate if pesticides increase then the logit value of different lipid profile parameters changes from -0.46 to -0.246 except LDL which increases by 0.135. The study reports a significantly increased threat of cardiovascular disease with increased concentrations of toxic pollutants.

Este estudo determina as associações entre o perfil lipídico sérico, o risco de doença cardiovascular e os poluentes orgânicos persistentes. Por meio da técnica de cromatografia gasosa, mediu-se a intensidade dos resíduos de poluentes tóxicos em amostras de soro de pacientes hipertensos. Com base na análise estatística, os efeitos de diferentes covariáveis – ou seja, pesticidas, idade, pressão arterial sistólica, pressão arterial diastólica e duração do perfil lipídico – foram verificados usando o modelo de regressão logística. O cálculo estatístico foi realizado no SPSS 22.0. Os valores P da estatística F para cada classe de perfil lipídico são maiores que 0,01 (1%), portanto não podemos rejeitar a hipótese nula para todos os casos. Os coeficientes estimados, seus erros padrão, estatística de Wald e odds ratio do modelo de regressão logística binária para diferentes parâmetros do perfil lipídico indicam se os pesticidas aumentam, então o valor logit de diferentes parâmetros do perfil lipídico muda de -0,46 para -0,246, exceto LDL, que aumenta em 0,135. O estudo relata um aumento significativo da ameaça de doença cardiovascular com aumento das concentrações de poluentes tóxicos.

Persistent organic pollutants distribution in plasma lipoprotein fractions

Abstract This study determines the associations among serum lipid profiles, risk of cardiovascular disease, and persistent organic pollutants. Using Gas chromatography technique, the intensity of toxic pollutant residues in serum samples of Hypertensive patients were measured. Based on statistical analysis, the effects of different covariates namely pesticides, age, systolic blood pressure, diastolic blood pressure, and lipid profile duration was checked using the logistic regression model. Statistical computation was performed on SPSS 22.0. The P-values of F-Statistic for each lipid profile class are greater than 0.01 (1%), therefore we cannot reject the null hypothesis for all cases. The estimated coefficients, their standard errors, Wald Statistic, and odds ratio of the binary logistic regression model for different lipid profile parameters indicate if pesticides increase then the logit value of different lipid profile parameters changes from -0.46 to -0.246 except LDL which increases by 0.135. The study reports a significantly increased threat of cardiovascular disease with increased concentrations of toxic pollutants.

Resumo Este estudo determina as associações entre o perfil lipídico sérico, o risco de doença cardiovascular e os poluentes orgânicos persistentes. Por meio da técnica de cromatografia gasosa, mediu-se a intensidade dos resíduos de poluentes tóxicos em amostras de soro de pacientes hipertensos. Com base na análise estatística, os efeitos de diferentes covariáveis ou seja, pesticidas, idade, pressão arterial sistólica, pressão arterial diastólica e duração do perfil lipídico foram verificados usando o modelo de regressão logística. O cálculo estatístico foi realizado no SPSS 22.0. Os valores P da estatística F para cada classe de perfil lipídico são maiores que 0,01 (1%), portanto não podemos rejeitar a hipótese nula para todos os casos. Os coeficientes estimados, seus erros padrão, estatística de Wald e odds ratio do modelo de regressão logística binária para diferentes parâmetros do perfil lipídico indicam se os pesticidas aumentam, então o valor logit de diferentes parâmetros do perfil lipídico muda de -0,46 para -0,246, exceto LDL, que aumenta em 0,135. O estudo relata um aumento significativo da ameaça de doença cardiovascular com aumento das concentrações de poluentes tóxicos.

Evaluation of the persistent organic pollutants association with type 2 diabetes: A prospective study from Karachi, Pakistan.

The aim of this study is to determine the association between environmental organic pollutants with type 2 diabetes. This prospective study was conducted in Federal Urdu University of Arts, Science and Technology (FUUAST) Gulshan-e-Iqbal Campus Karachi in duration from January 2016 to June 2017. This study was ethically approved from the Institutional Review Board of FUUAST. The study included 50 male and female convenient subjects with type 2 diabetes. Subject with other type of diabetes was excluded. Consent was obtained by each individual. Self-structured questionnaire was used for data collection. The comparative results suggest that the maximum level of summation polychlorinated biphenyls (PCBs) mean value was found in age group 27-33 as 0.695 mg/kg in 73% having total individual eleven. Median (interquartile range) of pesticides levels among subjects with normal weight, over weight and obesity were 0.49 (0.26-2.13), 1.53 (0.60-2.65), and 1.60 (1.23-2.05) respectively. It was observed that Organochlorine pesticides (OCS) levels of subjects with overweight and obesity were almost similar (P-value > 0.05) but significantly higher as compared to subjects with normal weight (P-value < 0.05). No significant differences were observed between PCB levels of subjects in terms of body mass index (BMI). In present study we trace the important elements involve in the deposition of persistent organic pollutants and established an association between pollutants with etiology of diabetes and associated disorders such as obesity.

Protease production and molecular characterization of a protease dipeptidyl-aminopeptidase gene from different strains of Sordaria fimicola.

The current research was designed to reach extracellular protease production potential in different strains of Sordaria fimicola which were previously obtained from Dr. Lamb (Imperial College, London) from North Facing Slope and South Facing Slope of Evolution Canyon. After initial and secondary screening, two hyper-producers strains S2 and N6 were selected for submerged fermentation and cultural conditions including temperature, pH, incubation period, inoculum size, substrate concentration, and different carbon and nitrogen sources were optimized for enzyme production. S2 strain showed maximum protease production of 3.291 U/mL after 14 days of incubation at 30 °C with 7 pH, 1% substrate concentration and 1 mL inoculum, While N6 strain showed maximum protease production of 1.929 U/mL under fermentation optimized conditions. Another aim of the present research was to underpin the biodiversity of genetics and post-translational modifications (PTMs) of protease DPAP (peptidyl-aminopeptidase) in Sordaria fimicola. Five polymorphic sites were observed in amino acid sequence of S. fimicola strains with reference to Neurospora crassa. PTMs prediction from bioinformatics tools predicted 38 phosphorylation sites on serine residues for protease peptidyl-aminopeptidase in S1 strain of S. fimicola while 45 phosphorylation sites on serine in N7 strain and 47 serine phosphorylation modifications were predicted in N. crassa. Current research gave an insight that change in genetic makeup effected PTMs which ultimately affected the production of protease enzyme in different strains of same organism (S. fimicola). The production and molecular data of the research revealed that environmental stress has strong effects on the specific genes through mutations which may cause genetic diversity. S. fimicola is non- pathogenic fungus and has a short life cycle. This fungus can be chosen to produce protease enzyme on a commercial scale.

Persistent organic pollutants distribution in plasma lipoprotein fractions.

This study determines the associations among serum lipid profiles, risk of cardiovascular disease, and persistent organic pollutants. Using Gas chromatography technique, the intensity of toxic pollutant residues in serum samples of Hypertensive patients were measured. Based on statistical analysis, the effects of different covariates namely pesticides, age, systolic blood pressure, diastolic blood pressure, and lipid profile duration was checked using the logistic regression model. Statistical computation was performed on SPSS 22.0. The P-values of F-Statistic for each lipid profile class are greater than 0.01 (1%), therefore we cannot reject the null hypothesis for all cases. The estimated coefficients, their standard errors, Wald Statistic, and odds ratio of the binary logistic regression model for different lipid profile parameters indicate if pesticides increase then the logit value of different lipid profile parameters changes from -0.46 to -0.246 except LDL which increases by 0.135. The study reports a significantly increased threat of cardiovascular disease with increased concentrations of toxic pollutants.