Molecular detection of Mycoplasma gallisepticum in different poultry breeds of Abbottabad and Rawalpindi, Pakistan / Detecção molecular de Mycoplasma gallisepticum em diferentes raças de aves de Abbottabad e Rawalpindi, Paquistão

The poultry sector in Pakistan is contributing mainly in bridging gap between demand and supply for protein. Mycoplasma gallisepticum is an emerging bacterium causing serious problems in poultry industry of Pakistan. A cross-sectional study was conducted to evaluate the M. gallisepticum load in poultry populated regions of Pakistan. Total 600 serum and 600 swab samples were collected, 200 from each broiler, layers and breeders poultry in Rawalpindi and Abbottabad districts. Serum samples were analyzed through ELISA for seroprevalence. Swabs were cultured on Frey’s medium followed by PCR and partial mgc2 gene sequencing. Results of seroprevalence of M. gallisepticum showed that layers (75%, n=150) are more positive as compared to breeders (70%, n=140) and broilers (50%, n=100). Typical colonies of the M. gallisepticum were observed in breeder (26.5%), followed by layer (21%) and broilers (9%). A total of 37.1% (n=42) samples were identified positive through PCR out of total 113 cultured based positive samples. A total of six M. gallisepticum isolates of current study showed 98-99 percent similarity with previously reported isolates on the basis of mgc2 gene partial sequencing. The M. gallisepticum was found highly prevalent in different poultry breads. Results of this study would add into basic data and provide a direction for livestock sector to strengthen a control strategy for mycoplasmosis in poultry farms.

O setor avícola do Paquistão está contribuindo principalmente para preencher a lacuna entre a demanda e a oferta de proteína. Mycoplasma gallisepticum é uma bactéria emergente que causa sérios problemas na indústria avícola do Paquistão. Um estudo transversal foi conduzido para avaliar a carga de M. gallisepticum em regiões de avicultura do Paquistão. Um total de 600 amostras de soro e 600 amostras de esfregaço foi coletado, 200 de cada frango de corte, poedeiras e aves reprodutoras nos distritos de Rawalpindi e Abbottabad. Amostras de soro foram analisadas por ELISA para soroprevalência. As zaragatoas foram cultivadas em meio Frey, seguido de PCR e sequenciação parcial do gene mgc2. Os resultados da soroprevalência de M. gallisepticum mostraram que as poedeiras (75%, n = 150) são mais positivas em comparação com matrizes (70%, n = 140) e frangos de corte (50%, n = 100). Colônias típicas de M. gallisepticum foram observadas em reprodutoras (26,5%), seguidas de poedeiras (21%) e frangos de corte (9%). Um total de 37,1% (n = 42) das amostras foi identificado como positivas por PCR de um total de 113 amostras positivas baseadas em cultura. Um total de seis isolados de M. gallisepticum do estudo atual mostrou 98-99% de similaridade com isolados relatados anteriormente com base no sequenciamento parcial do gene mgc2. O M. gallisepticum foi encontrado com alta prevalência em diferentes pães de aves. Os resultados deste estudo acrescentariam dados básicos e forneceriam orientação para o setor pecuário fortalecer uma estratégia de controle da micoplasmose em granjas avícolas.

Molecular detection of Mycoplasma gallisepticum in different poultry breeds of Abbottabad and Rawalpindi, Pakistan

Abstract The poultry sector in Pakistan is contributing mainly in bridging gap between demand and supply for protein. Mycoplasma gallisepticum is an emerging bacterium causing serious problems in poultry industry of Pakistan. A cross-sectional study was conducted to evaluate the M. gallisepticum load in poultry populated regions of Pakistan. Total 600 serum and 600 swab samples were collected, 200 from each broiler, layers and breeders poultry in Rawalpindi and Abbottabad districts. Serum samples were analyzed through ELISA for seroprevalence. Swabs were cultured on Freys medium followed by PCR and partial mgc2 gene sequencing. Results of seroprevalence of M. gallisepticum showed that layers (75%, n=150) are more positive as compared to breeders (70%, n=140) and broilers (50%, n=100). Typical colonies of the M. gallisepticum were observed in breeder (26.5%), followed by layer (21%) and broilers (9%). A total of 37.1% (n=42) samples were identified positive through PCR out of total 113 cultured based positive samples. A total of six M. gallisepticum isolates of current study showed 98-99 percent similarity with previously reported isolates on the basis of mgc2 gene partial sequencing. The M. gallisepticum was found highly prevalent in different poultry breads. Results of this study would add into basic data and provide a direction for livestock sector to strengthen a control strategy for mycoplasmosis in poultry farms.

Resumo O setor avícola do Paquistão está contribuindo principalmente para preencher a lacuna entre a demanda e a oferta de proteína. Mycoplasma gallisepticum é uma bactéria emergente que causa sérios problemas na indústria avícola do Paquistão. Um estudo transversal foi conduzido para avaliar a carga de M. gallisepticum em regiões de avicultura do Paquistão. Um total de 600 amostras de soro e 600 amostras de esfregaço foi coletado, 200 de cada frango de corte, poedeiras e aves reprodutoras nos distritos de Rawalpindi e Abbottabad. Amostras de soro foram analisadas por ELISA para soroprevalência. As zaragatoas foram cultivadas em meio Frey, seguido de PCR e sequenciação parcial do gene mgc2. Os resultados da soroprevalência de M. gallisepticum mostraram que as poedeiras (75%, n = 150) são mais positivas em comparação com matrizes (70%, n = 140) e frangos de corte (50%, n = 100). Colônias típicas de M. gallisepticum foram observadas em reprodutoras (26,5%), seguidas de poedeiras (21%) e frangos de corte (9%). Um total de 37,1% (n = 42) das amostras foi identificado como positivas por PCR de um total de 113 amostras positivas baseadas em cultura. Um total de seis isolados de M. gallisepticum do estudo atual mostrou 98-99% de similaridade com isolados relatados anteriormente com base no sequenciamento parcial do gene mgc2. O M. gallisepticum foi encontrado com alta prevalência em diferentes pães de aves. Os resultados deste estudo acrescentariam dados básicos e forneceriam orientação para o setor pecuário fortalecer uma estratégia de controle da micoplasmose em granjas avícolas.

Molecular detection of Mycoplasma gallisepticum in different poultry breeds of Abbottabad and Rawalpindi, Pakistan / Detecção molecular de Mycoplasma gallisepticum em diferentes raças de aves de Abbottabad e Rawalpindi, Paquistão

Abstract The poultry sector in Pakistan is contributing mainly in bridging gap between demand and supply for protein. Mycoplasma gallisepticum is an emerging bacterium causing serious problems in poultry industry of Pakistan. A cross-sectional study was conducted to evaluate the M. gallisepticum load in poultry populated regions of Pakistan. Total 600 serum and 600 swab samples were collected, 200 from each broiler, layers and breeders poultry in Rawalpindi and Abbottabad districts. Serum samples were analyzed through ELISA for seroprevalence. Swabs were cultured on Frey's medium followed by PCR and partial mgc2 gene sequencing. Results of seroprevalence of M. gallisepticum showed that layers (75%, n=150) are more positive as compared to breeders (70%, n=140) and broilers (50%, n=100). Typical colonies of the M. gallisepticum were observed in breeder (26.5%), followed by layer (21%) and broilers (9%). A total of 37.1% (n=42) samples were identified positive through PCR out of total 113 cultured based positive samples. A total of six M. gallisepticum isolates of current study showed 98-99 percent similarity with previously reported isolates on the basis of mgc2 gene partial sequencing. The M. gallisepticum was found highly prevalent in different poultry breads. Results of this study would add into basic data and provide a direction for livestock sector to strengthen a control strategy for mycoplasmosis in poultry farms.

Resumo O setor avícola do Paquistão está contribuindo principalmente para preencher a lacuna entre a demanda e a oferta de proteína. Mycoplasma gallisepticum é uma bactéria emergente que causa sérios problemas na indústria avícola do Paquistão. Um estudo transversal foi conduzido para avaliar a carga de M. gallisepticum em regiões de avicultura do Paquistão. Um total de 600 amostras de soro e 600 amostras de esfregaço foi coletado, 200 de cada frango de corte, poedeiras e aves reprodutoras nos distritos de Rawalpindi e Abbottabad. Amostras de soro foram analisadas por ELISA para soroprevalência. As zaragatoas foram cultivadas em meio Frey, seguido de PCR e sequenciação parcial do gene mgc2. Os resultados da soroprevalência de M. gallisepticum mostraram que as poedeiras (75%, n = 150) são mais positivas em comparação com matrizes (70%, n = 140) e frangos de corte (50%, n = 100). Colônias típicas de M. gallisepticum foram observadas em reprodutoras (26,5%), seguidas de poedeiras (21%) e frangos de corte (9%). Um total de 37,1% (n = 42) das amostras foi identificado como positivas por PCR de um total de 113 amostras positivas baseadas em cultura. Um total de seis isolados de M. gallisepticum do estudo atual mostrou 98-99% de similaridade com isolados relatados anteriormente com base no sequenciamento parcial do gene mgc2. O M. gallisepticum foi encontrado com alta prevalência em diferentes pães de aves. Os resultados deste estudo acrescentariam dados básicos e forneceriam orientação para o setor pecuário fortalecer uma estratégia de controle da micoplasmose em granjas avícolas.

Correlation of eGFR By MDRD and CKD-EPI Formula with Creatinine Clearance Estimation in CKD Patients and Healthy Subjects.

This cross sectional comparative study was conducted in the Nephrology and Medicine outdoor and in-patients department of Mymensingh Medical College Hospital, Bangladesh from April 2014 to March 2015. A total of 100 patients with CKD and 100 healthy subjects were included in the study. Data were collected by interview of the patients, clinical examination and laboratory investigations using a semi-structured case record form. Among all subjects, 50.0% had no CKD and 50.0% patients had CKD: Stage 3 CKD were 8.5%, CKD Stage 4 CKD were 21.0%, CKD Stage 5 CKD were 20.5%. Serum creatinine was 4.32±3.08mg/dl in patients with CKD and 1.00±0.22mg/dl was in healthy subjects. Mean±SD of CCR/ml/min was found 17.67±11.63ml/min in patients with CKD and 79.31±13.31ml/min was found in healthy subjects. On the other hand, Mean±SD CCCR/ml/m/1.73m² was found 19.79±12.85 ml/m/1.73m² in patient with CKD and healthy subjects had 83.83±13.33 ml/m/1.73m². Urinary creatinine was 45.59±15.63 & 57.66±11.45mg/dl respectively. CKD-EPI eGFR was 22.10±15.02 & 90.61±23.27ml/m/1.73m²; MDRD eGFR was 22.15±14.18 & 89.35±26.19 ml/m/1.73m² respectively. Difference between all the variables between CKD group and healthy group was found statistically significant (p<0.001). CKD-EPIeGFR and MDRDeGFR were increased both in CKD patients and healthy subjects in respect to CCR and CCCR. There was a strong positive correlation between CCCR (ml/m/1.73m2) and CKD-EPI (ml/m/1.73m²) among all patients (r=0.934 and p<0.001) and also a positive correlation of CCCR with MDRD among all patients (r=0.913 and p<0.001). A positive correlation of CCCR was found with CKD-EPIeGFR among CKD patients (r=0.848 and p<0.001). A positive correlation of CCCR was also found with MDRDeGFR among CKD patients (r=0.841, p<0.001). There are positive correlations between CCCR and CKD/EPI among healthy subjects (r=0.616 and p<0.05) and between CCCR with MDRD among healthy subjects (r=0.568 and p<0.05). Various formulae were used to calculate GFR on the basis of serum creatinine levels. The Overall correlation of population (healthy and CKD patients) between CCCR and CKD EPI and MDRD formula was (r=0.93 and 0.91) respectively, among CKD patients it was (r=0.848 and r=0.841) in healthy subjects it was (r=0.616 and r=0.568) respectively. CKD EPI eGFR and MDRD eGFR formula had fairly good correlation with conventional 24 hours creatinine clearance in both CKD patient and healthy subjects, there was even more strong correlation especially in CKD patients. The performance of CKD-EPI equation is better than MDRD equation to estimate the eGFR in both CKD patients and healthy subjects.

Trypanosoma brucei RAP1 Has Essential Functional Domains That Are Required for Different Protein Interactions.

RAP1 is a telomere protein that is well conserved from protozoa to mammals. It plays important roles in chromosome end protection, telomere length control, and gene expression/silencing at both telomeric and nontelomeric loci. Interaction with different partners is an important mechanism by which RAP1 executes its different functions in yeast. The RAP1 ortholog in Trypanosoma brucei is essential for variant surface glycoprotein (VSG) monoallelic expression, an important aspect of antigenic variation, where T. brucei regularly switches its major surface antigen, VSG, to evade the host immune response. Like other RAP1 orthologs, T. brucei RAP1 (TbRAP1) has conserved functional domains, including BRCA1 C terminus (BRCT), Myb, MybLike, and RAP1 C terminus (RCT). To study functions of various TbRAP1 domains, we established a strain in which one endogenous allele of TbRAP1 is flanked by loxP repeats, enabling its conditional deletion by Cre-mediated recombination. We replaced the other TbRAP1 allele with various mutant alleles lacking individual functional domains and examined their nuclear localization and protein interaction abilities. The N terminus, BRCT, and RCT of TbRAP1 are required for normal protein levels, while the Myb and MybLike domains are essential for normal cell growth. Additionally, the Myb domain of TbRAP1 is required for its interaction with T. brucei TTAGGG repeat-binding factor (TbTRF), while the BRCT domain is required for its self-interaction. Furthermore, the TbRAP1 MybLike domain contains a bipartite nuclear localization signal that is required for its interaction with importin α and its nuclear localization. Interestingly, RAP1's self-interaction and the interaction between RAP1 and TRF are conserved from kinetoplastids to mammals. However, details of the interaction interfaces have changed throughout evolution.IMPORTANCETrypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, to evade the host immune response. VSGs are expressed from subtelomeres in a monoallelic fashion. TbRAP1, a telomere protein, is essential for cell viability and VSG monoallelic expression and suppresses VSG switching. Although TbRAP1 has conserved functional domains in common with its orthologs from yeasts to mammals, the domain functions are unknown. RAP1 orthologs have pleiotropic functions, and interaction with different partners is an important means by which RAP1 executes its different roles. We have established a Cre-loxP-mediated conditional knockout system for TbRAP1 and examined the roles of various functional domains in protein expression, nuclear localization, and protein-protein interactions. This system enables further studies of TbRAP1 point mutation phenotypes. We have also determined functional domains of TbRAP1 that are required for several different protein interactions, shedding light on the underlying mechanisms of TbRAP1-mediated VSG silencing.

Seroprevalence of Bluetongue Virus in small ruminants in Balochistan province, Pakistan.

Bluetongue (BT), caused by bluetongue virus (BTV), is a vector-borne disease of small ruminants that has the potential to spread across international borders. Despite large populations of susceptible animals and borders with BTV endemic countries, little is known of the disease burden and prevalent serotypes in the province of Balochistan in Pakistan. We conducted a cross-sectional study to determine seroconversion and prevalent serotypes in selected districts of the province using a competitive enzyme-linked immunosorbent assay (cELISA) and real-time polymerase chain reaction (RT-PCR). Sera (n = 876) were collected from clinically healthy sheep and goats originating from the districts of Quetta (n = 300), Mastung (n = 201), Killa Saifullah (n = 75) and Kech (n = 300). None of the study herds (n = 97) were seronegative for BTV, and at the individual level, the overall prevalence of BTV seroconversion was 47.26% (n = 414/876, 95% CI = 43.92%-50.63%). A higher percentage of goats (50.87%, 95% CI = 45.99%-55.73%) were seropositive for anti-VP7 immunoglobulins (IgG) than sheep (44.21%, 95% CI = 39.81%-48.70%). Odds ratios of seroconversion for goats were associated with breed type (χ2  = 16.84, p = .01), parity (χ2  = 23.66, p = .00) and presence of vector (χ2  = 2.63, p = .10), whereas for sheep, it was associated with breed type (χ2  = 13.80, p = .01) and parity (χ2  = 53.40, p = .00). Serotype 8 was the most prevalent (26.82%, 95% CI = 14.75%-43.21%) followed by an equal prevalence of serotypes 2 and 9 (7.31%, 95% CI = 1.91%-21.01%). To the best of our knowledge, this is the first study conducted in Balochistan province and the results indicate that there is a necessity to initiate intervention strategies to control BT disease burden not only in this region of Pakistan but also in adjacent areas of the neighbouring countries, Iran and Afghanistan.

5-Hydroxytryptophan, a major product of tryptophan degradation, is essential for optimal replication of human parainfluenza virus.

Interferon (IFN) exerts its antiviral effect by inducing a large family of cellular genes, named interferon (IFN)-stimulated genes (ISGs). An intriguing member of this family is indoleamine 2,3-dioxygenase (IDO), which catalyzes the first and rate-limiting step of the main branch of tryptophan (Trp) degradation, the kynurenine pathway. We recently showed that IDO strongly inhibits human parainfluenza virus type 3 (PIV3), a significant respiratory pathogen. Here, we show that 5-hydoxytryptophan (5-HTP), the first product of an alternative branch of Trp degradation and a serotonin precursor, is essential to protect virus growth against IDO in cell culture. We also show that the apparent antiviral effect of IDO on PIV3 is not due to the generation of the kynurenine pathway metabolites, but rather due to the depletion of intracellular Trp by IDO, as a result of which this rare amino acid becomes unavailable for the alternative, proviral 5-HTP pathway.

Identification of Interferon-Stimulated Gene Proteins That Inhibit Human Parainfluenza Virus Type 3.

A major arm of cellular innate immunity is type I interferon (IFN), represented by IFN-α and IFN-ß. Type I IFN transcriptionally induces a large number of cellular genes, collectively known as IFN-stimulated gene (ISG) proteins, which act as antivirals. The IFIT (interferon-induced proteins with tetratricopeptide repeats) family proteins constitute a major subclass of ISG proteins and are characterized by multiple tetratricopeptide repeats (TPRs). In this study, we have interrogated IFIT proteins for the ability to inhibit the growth of human parainfluenza virus type 3 (PIV3), a nonsegmented negative-strand RNA virus of the Paramyxoviridae family and a major cause of respiratory disease in children. We found that IFIT1 significantly inhibited PIV3, whereas IFIT2, IFIT3, and IFIT5 were less effective or not at all. In further screening a set of ISG proteins we discovered that several other such proteins also inhibited PIV3, including IFITM1, IDO (indoleamine 2,3-dioxygenase), PKR (protein kinase, RNA activated), and viperin (virus inhibitory protein, endoplasmic reticulum associated, interferon inducible)/Cig5. The antiviral effect of IDO, the enzyme that catalyzes the first step of tryptophan degradation, could be counteracted by tryptophan. These results advance our knowledge of diverse ISG proteins functioning as antivirals and may provide novel approaches against PIV3. IMPORTANCE: The innate immunity of the host, typified by interferon (IFN), is a major antiviral defense. IFN inhibits virus growth by inducing a large number of IFN-stimulated gene (ISG) proteins, several of which have been shown to have specific antiviral functions. Parainfluenza virus type 3 (PIV3) is major pathogen of children, and no reliable vaccine or specific antiviral against it currently exists. In this article, we report several ISG proteins that strongly inhibit PIV3 growth, the use of which may allow a better antiviral regimen targeting PIV3.

Effects of disulfiram on apoptosis in PANC-1 human pancreatic cancer cell line.

Pancreatic carcinoma is currently considered as a rapidly progressive and fatal disease, and is typically diagnosed late in its natural course. It is characterized by a poor diagnosis and lack of response to conventional therapy. Recent studies have suggested that disulfiram (DSF), a member of the dithiocarbamate family, may have antitumor activity. This study aimed to evaluate the in vitro effect of DSF on apoptosis in human pancreatic cancerous cell line (PANC-1). PANC-1 cells were cultured and treated with DSF at doses of 5, 10, 13 µM for 24 h and apoptosis was measured. Methylation specific PCR (MS-PCR) and real-time quantitative PCR were carried out to detect the methylation pattern and to estimate the mRNA expression levels of RASSF1A, p21 and Bax. MS-PCR analysis demonstrated that no unmethylated band was apeared in PANC-1 cell line after DSF treatments. The real-time quantitative PCR results showed no significant mRNA expression for RASSF1A (p>0.05); whereas p21 and Bax expression were significantly (p<0.01) enhanced after treatment with DSF. The results of the current study indicated that DSF can induce appoptosis in PANC-1 through p21 and Bax pathway but not through RASSF1A.

Prevalence of nonalcoholic fatty liver disease (NAFLD) in patients of cardiovascular diseases and its association with hs-CRP and TNF-α.

BACKGROUND: There is increasing recognition of association of nonalcoholic fatty liver disease (NAFLD) with cardiovascular disease (CVD). Metabolic syndrome is common in both NAFLD and cardiovascular diseases. Our study is designed to investigate the association of NAFLD with cardiovascular disease. METHODS: It's a cross-sectional study which included 104 patients of coronary artery disease and hypertensive heart disease. Those patients having secondary causes of steatosis were excluded. Complete cardiovascular evaluation which included assessment of metabolic syndrome, routine biochemistries, viral markers, Ultrasonography (USG) abdomen, hs-CRP and TNF-α levels were obtained for all patients. RESULTS: Of all patients with cardiovascular disease, 19.2% (20/104) had essential hypertension with hypertensive heart disease the remaining 80.8% (84/104) patients had ischemic heart disease (IHD). On USG 69.2% (72/104) had NAFLD, these 50% (36/72) had grade 1 NAFLD and the rest grade 2 NAFLD. The hs-CRP levels and TNF-α were significantly higher in patients with NAFLD (p-value <0.001) and within patients with NAFLD the levels were higher in patients with grade 2 NAFLD. Also, binary logistic regression showed that high body-mass index (BMI), raised serum triglyceride levels, increased waist circumference and hypertension were significantly associated with the presence of NAFLD. CONCLUSION: Our data indicates that NALD is highly prevalent in patients of cardiovascular disease (69.2%) and is significantly associated with metabolic syndrome and its individual components. The levels of hs-CRP and TNF-α were significantly higher in patients with NAFLD and showed an increasing trend with the severity of fatty liver.

The apoptotic effects of sirtuin1 inhibitor on the MCF-7 and MRC-5 cell lines.

Sirtuin1 (SIRT1) is an enzyme that deacetylates histones and several nonhistone proteins including p53 during stress and plays an important role in the survival of tumor cells. Hereby, this study describes the potency of salermide as a SIRT1 inhibitor to induce apoptosis in the MCF-7 and MRC-5 cell lines. MCF7 and MRC-5 cell lines were cultured in RPMI-1640 and treated with or without salermide at concentration of 80.56 µmol/L, based on the half-maximal inhibitory concentration (IC50) index at different times (24, 48 and72 h). The IC50 value was established for the salermide in MCF-7. The percentage of apoptotic cells was measured by flow cytometry. Real-time quantitative RT-PCR was performed to estimate the mRNA expression of sirtuin1 in MCF-7 and MRC-5 with salermide at different times. ELISA and Bradford protein techniques were used to detect endogenous levels of total and acetylated p53 protein generated in MCF-7 and MRC-5 cells. Our findings indicated that salermide can induce apoptosis in MCF-7 significantly more effective than MRC-5 cells. We showed that the expression of SIRT1 was dramatically down-regulated by increasing the time of salermide treatment in MCF-7 but not MRC-5 and that the acetylated and total p53 protein levels were increased more in MCF-7 than MRC-5. Salermide, by decreasing the expression of sirtuin1 gene, can induce acetylation of P53 protein and consequently induce significant cell death in MCF-7 that was well tolerated in MRC-5.

Quantitative evaluation of DNMT3B promoter methylation in breast cancer patients using differential high resolution melting analysis.

DNA methylation plays an important role in carcinogenesis through epigenetic silencing of tumor suppressor genes. Aberrant methylation usually results from changes in the activity of DNA methyltransferases (DNMTs). Some studies show that the overexpression of the DNMTs may lead to aberrant methylation of tumor suppressor genes. Also the overexpression of DNMTs may be related to methylation status of their genes. Due to limited number of studies on DNMT3B promoter methylation, this study was performed to quantitatively measure the methylation level of DNMT3B gene in archival formalin fixed paraffin embedded (FFPE) tissues from breast cancer patients. Using differential high resolution melting analysis (D-HRMA) technology, the methylation level of DNMT3B gene promoter was quantified in 98 breast cancer FFPE tissues and also 10 fresh frozen normal tissue samples. Statistical analyses used for analyzing the correlation between the methylation and clinical variables. All the normal samples were found to be methylated at the DNMT3B promoter (the average methylation level 3.34%). Patients were identified as hypo-methylated (mean methylation level 0.8%), methylated (mean methylation level 2.48%) and hyper-methylated (mean methylation level 10.5%). Statistical analysis showed a significant correlation between the methylation status and the sample type, cancer type and tumor size. Also the methylation level was significantly associated with histologic grade. It is concluded that quantification of DNMT3B promoter methylation might be used as a reliable and sensitive diagnostic and prognostic tool in breast cancer. Also D-HRMA is demonstrated as a rapid and cost effective method for quantitative evaluation of promoter methylation.

Isolation of genomic DNA from defatted oil seed residue of rapeseed (Brassica napus).

A simple protocol for obtaining pure, restrictable and amplifiable megabase genomic DNA from oil-free seed residue of Brassica napus, an important oil seed plant, has been developed. Oil from the dry seeds was completely recovered in an organic solvent and quantified gravimetrically followed by processing of the residual biomass (defatted seed residue) for genomic DNA isolation. The isolated DNA can be cut by a range of restriction enzymes. The method enables simultaneous isolation and recovery of lipids and genomic DNA from the same test sample, thus allowing two independent analyses from a single sample. Multiple micro-scale oil extraction from the commercial seeds gave approximately 39% oil, which is close to the usual oil recovery from standard oil seed. Most of the amplified fragments were scored in the range of 2.5 to 0.5 kb, best suited for scoring as molecular diagnostics.

The role of N53Q mutation on the rat mu-opioid receptor function.

Glycosylation of the mu-opioid receptor may play an important role on its function. Using nested PCR, N53Q mutation was prepared in the N-terminal region of the rat mu-opioid receptor cDNA and cloned into the pcDNA3 vector. The plasmids containing the wild-type and mutated receptor cDNA were transfected into the COS-7 cells. Intracellular cAMP was measured in the morphine-treated and untreated transfected cells using an ELISA kit. Plasmid expression was evaluated using X-gal staining. Intracellular concentration of cAMP in the N53Q-mutated cells was not significantly different from the wild-type. The expression of the transfected plasmids was confirmed. Therefore, based on these results, it seems that glycosylation at the N53 site of the rat mu-opioid receptor does not influence the function of this receptor significantly.

Genetic diversity associated with agronomic traits using microsatellite markers in Pakistani rice landraces

Genetic diversity underlies the improvement of crops by plant breeding. Land races of rice (Oryza sativa L.) can contain some valuable alleles not common in modern germplasm. The aim here was to measure genetic diversity and its effect on agronomic traits among rice land-race genotypes grown in Pakistan. Diversity was measured using thirty-five microsatellite markers and seventy-five genotypes. Among the markers used a total of 142 alleles were detected at 32 polymorphic SSR loci, while three loci were monomorphic in Pakistani rice landraces. The number of alleles identified by each marker ranged from 2 to 13 with a mean of 4.4. Size differences between the smallest and largest alleles varied from 11bp to 71bp. Polymorphism information content ranged from 0.124 to 0.836, with an average of 0.569. At nine microsatellite loci, basmati-type landraces amplified more different alleles than those in the coarse-type. DNA markers RM70 and RM72 divided the rice landraces on the basis of days to flowering. A dendrogram based on total microsatellite polymorphism grouped 75 genotypes into four major clusters at 0.40 similarity coefficient, differentiating tall, late maturing and slender aromatic types from the short, early and bold non-aromatic ones. It inferred that Pakistani landraces have diverse genetic bases and can be utilized in future breeding programs. The DNA markers developed will assist in genotype identification, purity testing and plant variety protection.

The role of strain variation in BAX and BCL-2 expression in murine bleomycin-induced pulmonary fibrosis.

This study hypothesized that the expression of apoptosis-regulatory genes, such as BCL-2 and BAX may be affected by genetic variation in bleomycin-induced pulmonary fibrosis in C57BL/6 and NMRI mice. Pulmonary fibrosis induced by single intratracheal dose of bleomycin (3 U kg(-1)). After 2 weeks, lung samples were analyzed for collagen deposition, pathological changes and expression of BCL-2 and BAX. The fibrotic lung changes were similar in both strains. The immunohistochemical assay using a biotin-streptavidin technique showed no significant difference in immunoreactivity for BCL-2 protein between the controls and bleomycin-treated C57BL/6 mice. However, in NMRI mice, the expression of BCL-2 was significantly (p<0.05) upregulated in myofibroblasts and neutrophils. The expression of BAX protein was significantly (p<0.05) upregulated in alveolar epithelial cells of both strains and downregulated in myofibroblasts and lymphocytes of the lung tissues of C57BL/6 mice and also in lymphocytes of NMRI mice at 2 weeks after bleomycin instillation. These results confirm the role of BCL-2 and BAX proteins in the pathogenesis of pulmonary fibrosis and suggest that the expression of apoptotic regulatory genes may be specific in different cell types in various strains.

Efficacy of dried colostrum powders in the prevention of diarrhea in neonatal Holstein calves.

One hundred and twenty healthy Holstein calves were assigned randomly at birth to one of four groups; each group contained 30 calves and the calves were received as follows: group A, 85 ± 5 g of lyophilized colostrum powder dissolved in 3 kg of whole milk; group B, 85 ± 5 g of spray-dried colostrum powder dissolved in 3 kg of whole milk; group C, 750 ml frozen and thawed colostrum in 3 kg of whole milk, and group D, only 3 kg of whole milk without colostrum supplement. Each group was subdivided into 3 sub-groups of 10 calves as follows: calves fed colostrum supplement at 24 or 48 or 72 h after birth. All calves fed fresh colostrum within 6 h after birth at the amount of 5% of BW by bottle. Calves which were fed lyophilized colostrum supplement had a significant lower incidence of diarrhea in comparison to control calves. In addition, calves were received lyophilized colostrum supplement at 24 h after birth showed less incidence of diarrhea than calves fed the supplement at 48 and 72 h. According to the results of present study providing lyophilized colostrum supplement at 24 h after birth might have been preventive effect on calf diarrhea. The spray-dried colostrum supplement had not the same effect.

Radiocontrast nephropathy: is it dose related or not?

OBJECTIVE: To assess the safety of high dose non-ionic contrast media during a single radiological procedure in patients with pre-existing renal impairment. METHODS: One hundred eighteen patients, with serum Creatinine greater than 1.3 mg/dl who were undergoing coronary angiography or percutaneous transluminal coronary angiography (PTCA) were included in the study. All patients received the nonionic dye ULTRAVIST (lopromide). Serum creatinine were measured before, 48 hours and 1 week after the administration of contrast agent. An acute contrast induced reduction in renal function was defined as an increase in Serum Creatinine concentration of >=0.5 mg/dl, 48 hours after the administration of contrast agent. All patients with end stage renal disease or patients undergoing coronary bypass surgery within a week after coronary angiography or had any concomitant factors that could cause acute renal failure e.g., sepsis, hypotention, etc., were excluded. Patients receiving a dose of upto 100 ml of contrast agent (low dose group) were separated from those who received greater than 100 ml of contrast agent (high dose group). Patients in both groups had similar characteristics in terms of sex, age, weight and underlying disease. Student's t-test was used for statistical analysis. RESULTS: The mean age of our patients was 62.3+/-8.83 (range 40-84 years). There were 93 (78.8%) males and 25 (21.2%) females. The mean pre-contrast creatinine in the low contrast group was 1.97+/-0.92 and high dose group was 2.16+/-1.90 (p=0.48). The post-contrast Creatinine at 48 hours was 2.11+/-1.11 and 2.06+/-1.39 in the groups receiving low and high dose contrast agents respectively (p=0.830), while at 7 days post-contrast it was 2.17+/-1.28 and 1.95+/-1.43 respectively in the two groups (p=0.391). The contrast-induced reduction in renal function (rise in serum Cr >=0.5 mg/dl above base line) occurred in 14% (n=8) of patients in low dose and in 11% (n=7) in high dose contrast group (p=0.830, insignificant). CONCLUSION: The results of our study confirm that high dose non-ionic contrast is not associated with increased risk of contrast-mediated nephrotoxicity in patients with pre-existing renal insufficiency undergoing cardiac angiography (p=0.830, insignificant).

Maximum tolerable dose of cyclophosphamide and azathioprine in Pakistani patients with primary renal disease.

OBJECTIVE: The immunosuppressive regimens, at present, mainly rely on western guidelines that were derived from studies conducted in western populations. No such study exists for South Asian population, which is home to almost two billion people different in both genetics and environment from west. Locally derived thresholds for side effects markedly different from western figures may warrant re-adjustment of current local immunosuppressive regimens that are at present based largely on western guidelines. In order to define optimum dose for Cyclophosphamide (CYC) and Azathioprine (AZA) based immunosuppressive therapy, we conducted this study to find out maximum tolerable doses of azathioprine (AZA) and cyclophosphamide (CYC) beyond which neutropenia and thrombocytepenia are most likely to occur in patients with primary renal pathology. METHOD: Patients with systemic vasculitis and idiopathic glomerulonephritis who were on CYC and AZA were identified through review of medical records at a tertiary care hospital in Pakistan (The Aga Khan University Hospital, Karachi). Patients were categorized under three principal diagnosis i.e. systemic lupus erythematosus (SLE), primary (idiopathic) glomerulonephritis (GN) and Wegener's granulomatosis (WG). The Receiver Operating Curve (ROC) was used to calculate the maximum tolerable dose for both CYC and AZA. RESULTS: We identified 94 patients aged 6-82 years (median 44.5 years) with primary renal disease (Wegener's granulomatosis n=13, Systemic lupus erythematosis n=62 and idiopathic glomerulonephritis n=19) who received CYC or AZA. Of these 94 patients, 36.2% (n=34) received CYC and 63.8% (n=60) received AZA. The mean dose of CYC was 1.54 +/- 0.50 mg/kg of body weight (range: 0.77-2.93). The mean dose of AZA was 1.64 +/- 0.59 mg/kg of body weight (range: 0.47-2.97). The maximum tolerable doses calculated for CYC and AZA were 1.25 mg/kg and 1.30 mg/kg of body weight respectively. The maximum tolerable dose for CYC and AZA among males could not be calculated, because of insufficient number of patients who developed neutropenia and thrombocytopenia. The maximum tolerable doses for CYC and AZA among females were 1.34 mg/kg and 1.03 mg/kg of body weight respectively. Also we found out that AZA was relatively more likely to cause neutropenia and thrombocytopenia (p=0.07). CONCLUSION: We thereby recommend that CYC should be initiated at a dose no more than 1 mg/kg of body weight and AZA at an initial dose of 0.75-1.0 mg/kg of body weight. The dose may be adjusted later on the basis of clinical response and laboratory reports.

Direct labelling of octreotide with 99mTc: effect of different concentration of reducing agents and amount of sodium pertechnetate on radiolabelling efficiency.

Octreotide, a synthetic analog of natural hormone somatostatin, was labelled with 99mTc. Labelling was accomplished by reduction of the cysteine bridge, which provided sulfhydryl groups for chelating with 99mTc. Sodium ascorbate and sodium dithionite in different concentrations were used as reducing agents. Different amounts of sodium pertechnetate were used for labelling of peptide. When the mass ratio of peptide and sodium ascorbate was 1:100 and the final concentration of dithionite in the labelling vial was 0.2-0.4 microg/microl with 0.18-1.48 GBq sodium pertechnetate more than 80% radiolabelling efficiency was confirmed by RP-HPLC, ITLC-SG and C18 Cartridge analysis. The stability of the 99mTc-peptide bond was evaluated by human serum challenge and that showed the stability was 90% after 4h.