Abstract: Water plays a major role in maintaining life on earth, but it can also serve as a matrix for pathogenic organisms, posing substantial health threats to humans. Although, outbreaks of diseases attributable to drinking water may not be common in industrialized countries, they still occur and can lead to serious acute, chronic, or sometimes fatal health consequences. The analysis of drinking water samples from different regions of Kuwait was performed in this study for bacterial and viral contaminations. Drinking tap water samples were collected from 15 different locations of the six Kuwait governorates. All samples were analyzed by confocal microscopy for the presence of bacteria. The samples were cultured in vitro to detect cultivable organisms. DNA was isolated from the cultured organisms and the identity of the bacteria was determined by sequencing the bacterial 16S rRNA genes, followed by BLAST analysis in the database of NCBI, USA. RNA was extracted from water samples and analyzed by real-time PCR for the detection of viruses with potential health risks, i.e. Astrovirus, Enterovirus, Norovirus, Rotavirus, and Hepatitis A. Confocal microscopy showed the presence of bacteria in some water samples. The 16S rRNA gene sequencing of culture grown organisms, followed by BLAST analysis, identified the presence of several non-pathogenic bacterial species. However, one sample had Acinetobacter baumannii, which often causes opportunistic infections in immunocompromised people, but none of the studied viruses could be detected in the drinking water samples analyzed. The results indicate that drinking water samples analyzed from various locations in Kuwait are relatively safe for drinking and do not contain many harmful pathogens.
Abstract: Betong chicken is a slow growing and a lean strain of chicken, while the rapid growth of broiler is accompanied by increased fat. We investigated the growth performance, fat accumulations, lipid serum biochemical levels and lipoprotein lipase (LPL) gene expression of female Betong (KU line) at the age of 4 and 6 weeks. A total of 80 female Betong chickens (KU line) and 80 female broiler chickens were reared under open system (each group had 4 replicates of 20 chicks per pen). The results showed that feed intake and average daily gain (ADG) of broiler chicken were significantly higher than Betong (KU line) (P < 0.01), while feed conversion ratio (FCR) of Betong (KU line) at week 6 were significantly lower than broiler chicken (P < 0.01) at 6 weeks. At 4 and 6 weeks, two birds per replicate were randomly selected and slaughtered. Carcass weight did not significantly differ between treatments; the percentage of abdominal fat and subcutaneous fat yield was higher in the broiler (P < 0.01) at 4 and 6 week. Total cholesterol and LDL level of broiler were higher than Betong (KU line) at 4 and 6 weeks (P < 0.05). Abdominal fat samples were collected for total RNA extraction. The cDNA was amplified using primers specific for LPL gene expression and analysed using real-time PCR. The results showed that the expression of LPL gene was not different when compared between Betong (KU line) and broiler chickens at the age of 4 and 6 weeks (P > 0.05). Our results indicated that broiler chickens had high growth rate and fat accumulation when compared with Betong (KU line) chickens, whereas LPL gene expression did not differ between breeds.
Abstract: Halal authentication and verification in supplement capsules are highly required as the gelatine available in the market can be from halal or non-halal sources. It is an obligation for Muslim to consume and use the halal consumer goods. At present, real-time polymerase chain reaction (RT-PCR) is the most common technique being used for the detection of porcine and bovine DNA in gelatine due to high sensitivity of the technique and higher stability of DNA compared to protein. In this study, twenty samples of supplements capsules from different products with different Halal logos were analyzed for porcine and bovine DNA using RT-PCR. Standard bovine and porcine gelatine from eurofins at a range of concentration from 10-1 to 10-5 ng/µl were used to determine the linearity range, limit of detection and specificity on RT-PCR (SYBR Green method). RT-PCR detected porcine (two samples), bovine (four samples) and mixture of porcine and bovine (six samples). The samples were also tested using FT-IR technique where normalized peak of IR spectra were pre-processed using Savitsky Golay method before Principal Components Analysis (PCA) was performed on the database. Scores plot of PCA shows three clusters of samples; bovine, porcine and mixture (bovine and porcine). The RT-PCR and FT-IR with chemometrics technique were found to give same results for porcine gelatine samples which can be used for Halal authentication.
Abstract: This study was conducted to evaluate the effect of Echinacea purpurea on the expression of cyclooxygenase-2 (COX-2), interleukin-17F (IL-17F) in seven-day-old broiler chickens. Four groups were fed with concentration of 0 g/kg, 5 g/kg, 10 g/kg and 20 g/kg from the root of E. purpurea in the basal diet and two other groups were only fed with the basal diet for 21 days. At the 28th day, lipopolysaccharide (LPS, 2 mg/kg diet) was injected in four groups and the basal diet group was injected by saline as control. The chickens’ spleen RNA expression was measured for the COX-2 and IL-17F genes by Real-Time PCR. The results have shown that chickens which were fed E. purpurea had a lower COX-2 and IL-17F mRNA expression. The chickens who have received LPS only, lymphocyte was lower than other treatments. Vital organ weights were not significantly different, but body weight loss was recovered by dietary herbs inclusion. The results of this study have shown the positive effect of an anti-inflammatory herb to prevent the undesirable effect of inflammation.
Abstract: Agriculture is the backbone of economy of Pakistan
and cotton is the major agricultural export and supreme source of raw
fiber for our textile industry. To combat severe problems of insect
and weed, combination of three genes namely Cry1Ac, Cry2A and
EPSPS genes was transferred in locally cultivated cotton variety
MNH-786 with the use of Agrobacterium mediated genetic
transformation. The present study focused on the molecular screening
of transgenic cotton plants at T3 generation in order to confirm
integration and expression of all three genes (Cry1Ac, Cry2A and
EPSP synthase) into the cotton genome. Initially, glyphosate spray
assay was used for screening of transgenic cotton plants containing
EPSP synthase gene at T3 generation. Transgenic cotton plants which
were healthy and showed no damage on leaves were selected after 07
days of spray. For molecular analysis of transgenic cotton plants in
the laboratory, the genomic DNA of these transgenic cotton plants
were isolated and subjected to amplification of the three genes. Thus,
seventeen out of twenty (Cry1Ac gene), ten out of twenty (Cry2A
gene) and all twenty (EPSP synthase gene) were produced positive
amplification. On the base of PCR amplification, ten transgenic plant
samples were subjected to protein expression analysis through
ELISA. The results showed that eight out of ten plants were actively
expressing the three transgenes. Real-time PCR was also done to
quantify the mRNA expression levels of Cry1Ac and EPSP synthase
gene. Finally, eight plants were confirmed for the presence and active
expression of all three genes at T3 generation.
Abstract: Persea declinata (Bl.) Kosterm is a member of the
Lauraceae family, widely distributed in Southeast Asia. It is from the
same genus with avocado (Persea americana Mill), which is widely
consumed as food and for medicinal purposes. In the present study,
we examined the anticancer properties of Persea declinata (Bl.)
Kosterm bark methanolic crude extract (PDM). PDM exhibited a
potent antiproliferative effect in MCF-7 human breast cancer cells,
with an IC50 value of 16.68 .g/mL after 48h of treatment. We
observed that PDM caused cell cycle arrest and subsequent apoptosis
in MCF-7 cells, as exhibited by increased population at G0/G1 phase,
higher lactate dehydrogenase (LDH) release, and DNA
fragmentation. Mechanistic studies showed that PDM caused
significant elevation in ROS production, leading to perturbation of
mitochondrial membrane potential, cell permeability, and activation
of caspases-3/7. On the other hand, real-time PCR and Western blot
analysis showed that PDM treatment increased the expression of the
proapoptotic molecule, Bax, but decreased the expression of
prosurvival proteins, Bcl-2 and Bcl-xL, in a dose-dependent manner.
These findings imply that PDM could inhibit proliferation in MCF-7
cells via cell cycle arrest and apoptosis induction, indicating its
potential as a therapeutic agent worthy of further development.
Abstract: Osteoporosis is a common multifactorial disease with
a strong genetic component characterized by reduced bone mass and
increased risk of fractures. Genetic factors play an important role in
the pathogenesis of osteoporosis. The aim of our study was to
identify the genotype and allele distribution of T245G polymorphism
in OPG gene in Slovak postmenopausal women. A total of 200
unrelated Slovak postmenopausal women with diagnosed
osteoporosis and 200 normal controls were genotyped for T245G
(rs3134069) polymorphism of OPG gene. Genotyping was performed
using the Custom Taqman®SNP Genotyping assays. Genotypes and
alleles frequencies showed no significant differences (p=0.5551;
p=0.6022). The results of the present study confirm the importance of
T245G polymorphism in OPG gene in the pathogenesis of
osteoporosis.
Abstract: Osteoporosis is a complex health disease characterized by low bone mineral density, which is determined by an interaction of genetics with metabolic and environmental factors. Current research in genetics of osteoporosis is focused on identification of responsible genes and polymorphisms. TNFRSF11B gene plays a key role in bone remodeling. The aim of this study was to investigate the genotype and allele distribution of A163G (rs3102735) osteoprotegerin gene promoter and G1181C (rs2073618) osteoprotegerin first exon polymorphisms in the group of 180 unrelated postmenopausal women with diagnosed osteoporosis and 180 normal controls. Genomic DNA was isolated from peripheral blood leukocytes using standard methodology. Genotyping for presence of different polymorphisms was performed using the Custom Taqman®SNP Genotyping assays. Hardy-Weinberg equilibrium was tested for each SNP in the groups of participants using the chi-square (χ2) test. The distribution of investigated genotypes in the group of patients with osteoporosis were as follows: AA (66.7%), AG (32.2%), GG (1.1%) for A163G polymorphism; GG (19.4%), CG (44.4%), CC (36.1%) for G1181C polymorphism. The distribution of genotypes in normal controls were follows: AA (71.1%), AG (26.1%), GG (2.8%) for A163G polymorphism; GG (22.2%), CG (48.9%), CC (28.9%) for G1181C polymorphism. In A163G polymorphism the variant G allele was more common among patients with osteoporosis: 17.2% versus 15.8% in normal controls. Also, in G1181C polymorphism the phenomenon of more frequent occurrence of C allele in the group of patients with osteoporosis was observed (58.3% versus 53.3%). Genotype and allele distributions showed no significant differences (A163G: χ2=0.270, p=0.605; χ2=0.250, p=0.616; G1181C: χ2= 1.730, p=0.188; χ2=1.820, p=0.177). Our results represents an initial study, further studies of more numerous file and associations studies will be carried out. Knowing the distribution of genotypes is important for assessing the impact of these polymorphisms on various parameters associated with osteoporosis. Screening for identification of “at-risk” women likely to develop osteoporosis and initiating subsequent early intervention appears to be most effective strategy to substantially reduce the risks of osteoporosis.
Abstract: Infectious bronchitis virus (IBV) is a very dynamic and evolving virus, causing major economic losses to the global poultry industry. Recently, the Libyan poultry industry faced severe outbreak of respiratory distress associated with high mortality and dramatic drop in egg production. Tracheal and cloacal swabs were analyzed for several poultry viruses. IBV was detected using SYBR Green I real-time PCR detection based on the nucleocapsid (N) gene. Sequence analysis of the partial N gene indicated high similarity (~ 94%) to IBV strain 3382/06 that was isolated from Taiwan. Even though the IBV strain 3382/06 is more similar to that of the Mass type H120, the isolate has been implicated associated with intertypic recombinant of 3 putative parental IBV strains namely H120, Taiwan strain 1171/92 and China strain CK/CH/LDL/97I. Complete sequencing and antigenicity studies of the Libya IBV strains are currently underway to determine the evolution of the virus and its importance in vaccine induced immunity. In this paper we documented for the first time the presence of possibly variant IBV strain from Libya which required dramatic change in vaccination program.
Abstract: Cytogenetic analysis still remains the gold standard method for prenatal diagnosis of trisomy 21 (Down syndrome, DS). Nevertheless, the conventional cytogenetic analysis needs live cultured cells and is too time-consuming for clinical application. In contrast, molecular methods such as FISH, QF-PCR, MLPA and quantitative Real-time PCR are rapid assays with results available in 24h. In the present study, we have successfully used a novel MGB TaqMan probe-based real time PCR assay for rapid diagnosis of trisomy 21 status in Down syndrome samples. We have also compared the results of this molecular method with corresponding results obtained by the cytogenetic analysis. Blood samples obtained from DS patients (n=25) and normal controls (n=20) were tested by quantitative Real-time PCR in parallel to standard G-banding analysis. Genomic DNA was extracted from peripheral blood lymphocytes. A high precision TaqMan probe quantitative Real-time PCR assay was developed to determine the gene dosage of DSCAM (target gene on 21q22.2) relative to PMP22 (reference gene on 17p11.2). The DSCAM/PMP22 ratio was calculated according to the formula; ratio=2 -ΔΔCT. The quantitative Real-time PCR was able to distinguish between trisomy 21 samples and normal controls with the gene ratios of 1.49±0.13 and 1.03±0.04 respectively (p value
Abstract: Atherosclerosis was identified as a chronic inflammatory process resulting from interactions between plasma lipoproteins, cellular components (monocyte, macrophages, T lymphocytes, endothelial cells and smooth muscle cells) and the extracellular matrix of the arterial wall. Several types of genes were known to express during formation of atherosclerosis. This study is carried out to identify unknown differentially expressed gene (DEG) in atherogenesis. Rabbit’s aorta tissues were stained by H&E for histomorphology. GeneFishing™ PCR analysis was performed from total RNA extracted from the aorta tissues. The DNA fragment from DEG was cloned, sequenced and validated by Real-time PCR. Histomorphology showed intimal thickening in the aorta. DEG detected from ACP-41 was identified as cathepsin B gene and showed upregulation at week-8 and week-12 of atherogenesis. Therefore, ACP-based GeneFishing™ PCR facilitated identification of cathepsin B gene which was differentially expressed during development of atherosclerosis.
Abstract: Attachment of the circulating monocytes to the
endothelium is the earliest detectable events during formation of
atherosclerosis. The adhesion molecules, chemokines and matrix
proteases genes were identified to be expressed in atherogenesis.
Expressions of these genes may influence structural integrity of the
luminal endothelium. The aim of this study is to relate changes in the
ultrastructural morphology of the aortic luminal surface and gene
expressions of the endothelial surface, chemokine and MMP-12 in
normal and hypercholesterolemic rabbits. Luminal endothelial
surface from rabbit aortic tissue was examined by scanning electron
microscopy (SEM) using low vacuum mode to ascertain
ultrastructural changes in development of atherosclerotic lesion. Gene
expression of adhesion molecules, MCP-1 and MMP-12 were studied
by Real-time PCR. Ultrastructural observations of the aortic luminal
surface exhibited changes from normal regular smooth intact
endothelium to irregular luminal surface including marked globular
appearance and ruptures of the membrane layer. Real-time PCR
demonstrated differentially expressed of studied genes in
atherosclerotic tissues. The appearance of ultrastructural changes in
aortic tissue of hypercholesterolemic rabbits is suggested to have
relation with underlying changes of endothelial surface molecules,
chemokine and MMP-12 gene expressions.