Abstract: The most demanded organic foods worldwide are those that are consumed fresh, such as fruits and vegetables. However, there is a knowledge gap about some aspects of organic food microbiological quality and safety. Organic fruits and vegetables are more exposed to pathogenic microorganisms due to surface contact with natural fertilizers such as animal manure, wastes and vermicompost used during farming. Therefore, the objective of this work was to study the contamination of organic fresh green leafy vegetables by two emergent pathogens, Arcobacter spp. and Helicobacter pylori. For this purpose, a total of 24 vegetable samples, 13 lettuce and 11 spinach were acquired from 10 different ecological supermarkets and greengroceries and analyzed by culture and PCR. Arcobacter spp. was detected in five samples (20%) by PCR, four spinach and one lettuce. One spinach sample was found to be also positive by culture. For H. pylori, the H. pylori VacA gene-specific band was detected in 12 vegetable samples (50%), 10 lettuces and two spinach. Isolation in the selective medium did not yield any positive result, possibly because of low contamination levels together with the presence of the organism in its viable but non-culturable form. Results showed significant levels of H. pylori and Arcobacter contamination in organic vegetables that are generally consumed raw, which seems to confirm that these foods can act as transmission vehicles to humans.
Abstract: E10A is a kind of replication-defective adenovirus which carries the human endostatin gene to inhibit the growth of tumors. Kringle 5(K5) has almost the same function as angiostatin to also inhibit the growth of tumors since they are all the byproduct of the proteolytic cleavage of plasminogen. Tumor size increasing can be suppressed because both of the endostatin and K5 can restrain the angiogenesis process. Therefore, in order to improve the treatment effect on tumor, 2A peptide is used to construct a fusion gene carrying both E10A and K5. Using 2A peptide is an ideal strategy when a fusion gene is expressed because it can avoid many problems during the expression of more than one kind of protein. The overlap extension PCR is also used to connect 2A peptide with E10A and K5. The final construction of fusion gene E10A-2A-K5 can provide a possible new method of the anti-angiogenesis treatment with a better expression performance.
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: 5-FU is a chemotherapeutic agent that has been used in colorectal cancer (CRC) treatment. However, it is usually associated with the acquired resistance, which decreases the therapeutic effects of 5-FU. miR-200c is involved in chemotherapeutic drug resistance, but its mechanism is not fully understood. In this study, the effect of inhibition of miR-200c in sensitivity of HCT-116 CRC cells to 5-FU was evaluated. HCT-116 cells were transfected with LNA-anti- miR-200c for 48 h. mRNA expression of miR-200c was evaluated using quantitative real- time PCR. The protein expression of phosphatase and tensin homolog (PTEN) and E-cadherin were analyzed by western blotting. Annexin V and propidium iodide staining assay were applied for apoptosis detection. The caspase-3 activation was evaluated by an enzymatic assay. The results showed LNA-anti-miR-200c inhibited the expression of PTEN and E-cadherin protein, apoptosis and activation of caspase 3 compared with control cells. In conclusion, these results suggest that miR-200c as a prognostic marker can overcome to 5-FU chemoresistance in CRC.
Abstract: Microsporidia comprises various pathogenic species can infect humans by means of water. Moreover, chlorine disinfection of drinking-water has limitations against this protozoan pathogen. A total of 48 water samples were collected from two drinking water treatment plants having two different filtration systems (slow sand filter and rapid sand filter) during one year period. Samples were collected from inlet and outlet of each plant. Samples were separately filtrated through nitrocellulose membrane (142 mm, 0.45 µm), then eluted and centrifuged. The obtained pellet from each sample was subjected to DNA extraction, then, amplification using genus-specific primer for microsporidia. Each microsporidia-PCR positive sample was performed by two species specific primers for Enterocytozoon bieneusi and Encephalitozoon intestinalis. The results of the present study showed that the percentage of removal for microsporidia through different treatment processes reached its highest rate in the station using slow sand filters (100%), while the removal by rapid sand filter system was 81.8%. Statistically, the two different drinking water treatment plants (slow and rapid) had significant effect for removal of microsporidia. Molecular identification of microsporidia-PCR positive samples using two different primers for Enterocytozoon bieneusi and Encephalitozoon intestinalis showed the presence of the two pervious species in the inlet water of the two stations, while Encephalitozoon intestinalis was detected in the outlet water only. In conclusion, the appearance of virulent microsporidia in treated drinking water may cause potential health threat.
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: Gastric Cancer (GC) has high morbidity and fatality
rate in various countries. It is still one of the most frequent and
deadly diseases. Gastrokine1 (GKN1) and gastrokine2 (GKN2) genes
are highly expressed in the normal stomach epithelium and play
important roles in maintaining the integrity and homeostasis of
stomach mucosal epithelial cells. In this study, 47 paired samples that
were grouped according to the types of gastric cancer and the clinical
characteristics of the patients, including gender and average of age.
They were investigated with gene expression analysis and mutation
screening by monitoring RT-PCR, SSCP and nucleotide sequencing
techniques. Both GKN1 and GKN2 genes were observed significantly
reduced found by (Wilcoxon signed rank test; p
Abstract: Nitrification is essential to biological processes
designed to remove ammonia and/or total nitrogen. It removes excess
nitrogenous compound in wastewater which could be very toxic to
the aquatic fauna or cause serious imbalance of such aquatic
ecosystem. Efficient nitrification is linked to an in-depth knowledge
of the structure and dynamics of the nitrifying community structure
within the wastewater treatment systems. In this study, molecular
technique was employed for characterizing the microbial structure of
activated sludge [ammonia oxidizing bacteria (AOB) and nitrite
oxidizing bacteria (NOB)] in a municipal wastewater treatment with
intention of linking it to the plant efficiency. PCR based phylogenetic
analysis was also carried out. The average operating and
environmental parameters as well as specific nitrification rate of plant
was investigated during the study. During the investigation the average temperature was 23±1.5oC.
Other operational parameters such as mixed liquor suspended solids
and chemical oxygen demand inversely correlated with ammonia
removal. The dissolved oxygen level in the plant was constantly
lower than the optimum (between 0.24 and 1.267 mg/l) during this
study. The plant was treating wastewater with influent ammonia
concentration of 31.69 and 24.47 mg/L. The influent flow rates
(ML/Day) was 96.81 during period. The dominant nitrifiers include:
Nitrosomonas spp. Nitrobacter spp. and Nitrospira spp. The AOB
had correlation with nitrification efficiency and temperature. This
study shows that the specific ammonia oxidizing rate and the specific
nitrate formation rates can serve as good indicator of the plant overall
nitrification performance.
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: A simple, rapid and non-invasive electromagnetic sensor (C-FAST device) was- patented; for diagnosis of HCV RNA. Aim: To test the validity of the device compared to standard HCV PCR. Subjects and Methods: The first phase was done as pilot in Egypt on 79 participants; the second phase was done in five centers: one center from Egypt, two centers from Pakistan and two centers from India (800, 92 and 113 subjects respectively). The third phase was done nationally as multicenter study on (1600) participants for ensuring its representativeness. Results: When compared to PCR technique, C-FAST device revealed sensitivity 95% to 100%, specificity 95.5% to 100%, PPV 89.5% to 100%, NPV 95% to 100% and positive likelihood ratios 21.8% to 38.5%. Conclusion: It is practical evidence that HCV nucleotides emit electromagnetic signals that can be used for its identification. As compared to PCR, C-FAST is an accurate, valid and non-invasive device.
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: Legionella pneumophila is involved in more than 95%
cases of severe atypical pneumonia. Infection is mainly by
inhalation the indoor aerosols through the water-coolant systems.
Because some Legionella strains may be viable but not culturable,
therefore, Taq polymerase, DNA amplification and semi-nested-PCR
were carried out to detect Legionella-specific 16S-rDNA sequence.
For this purpose, 1.5 litter of water samples from 77 water-coolant
system were collected from four different hospitals, two nursing
homes and one student hostel in Kerman city of Iran, each in a brand
new plastic bottle during summer season of 2006 (from April to
August). The samples were filtered in the sterile condition through
the Millipore Membrane Filter. DNA was extracted from membrane
and used for PCR to detect Legionella spp. The PCR product was
then subjected to semi-nested PCR for detection of L. pneumophila.
Out of 77 water samples that were tested by PCR, 30 (39%) were
positive for most species of Legionella. However, L. pneumophila
was detected from 14 (18.2%) water samples by semi-nested PCR.
From the above results it can be concluded that water coolant
systems of different hospitals and nursing homes in Kerman city of
Iran are highly contaminated with L. pneumophila spp. and pose
serious concern. So, we recommend avoiding such type of coolant
system in the hospitals and nursing homes.
Abstract: The full length mitochondrial small subunit ribosomal
(mt-rns) gene has been characterized for Ophiostoma novo-ulmi
subspecies americana. The gene was also characterized for
Ophiostoma ulmi and a group II intron was noted in the mt-rns gene
of O. ulmi. The insertion in the mt-rns gene is at position S952 and it
is a group IIB1 intron that encodes a double motif LAGLIDADG
homing endonuclease from an open reading frame located within a
loop of domain III. Secondary structure models for the mt-rns RNA
of O. novo-ulmi subsp. americana and O. ulmi were generated to
place the intron within the context of the ribosomal RNA. The in vivo
splicing of the O.ul-mS952 group II intron was confirmed with
reverse transcription-PCR. A survey of 182 strains of Dutch Elm
Diseases causing agents showed that the mS952 intron was absent in
what is considered to be the more aggressive species O. novo-ulmi
but present in strains of the less aggressive O. ulmi. This observation
suggests that the O.ul-mS952 intron can be used as a PCR-based
molecular marker to discriminate between O. ulmi and O. novo-ulmi
subsp. americana.
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.
Abstract: Brucellosis is a zoonotic disease; its symptoms and appearances are not exclusive in human and its traditional diagnosis is based on culture, serological methods and conventional PCR. For more sensitive, specific detection and differentiation of Brucella spp., the real time PCR method is recommended. This research has performed to determine the presence and prevalence of Brucella spp. and differentiation of Brucella abortus and Brucella melitensis in house mouse (Mus musculus) in west of Iran. A TaqMan analysis and single-step PCR was carried out in total 326 DNA of Mouse's spleen samples. From the total number of 326 samples, 128 (39.27%) gave positive results for Brucella spp. by conventional PCR, also 65 and 32 out of the 128 specimens were positive for B. melitensis, B. abortus, respectively. These results indicate a high presence of this pathogen in this area and that real time PCR is considerably faster than current standard methods for identification and differentiation of Brucella species. To our knowledge, this study is the first prevalence report of direct identification and differentiation of B. abortus and B. melitensis by real time PCR in mouse tissue samples in Iran.
Abstract: Many high-risk pathogens that cause disease in
humans are transmitted through various food items. Food-borne
disease constitutes a major public health problem. Assessment of the
quality and safety of foods is important in human health. Rapid and
easy detection of pathogenic organisms will facilitate precautionary
measures to maintain healthy food. The Polymerase Chain Reaction
(PCR) is a handy tool for rapid detection of low numbers of bacteria.
We have designed gene specific primers for most common food
borne pathogens such as Staphylococci, Salmonella and E.coli.
Bacteria were isolated from food samples of various food outlets and
identified using gene specific PCRs. We identified Staphylococci,
Salmonella and E.coli O157 using gene specific primers by rapid and
direct PCR technique in various food samples. This study helps us in
getting a complete picture of the various pathogens that threaten to
cause and spread food borne diseases and it would also enable
establishment of a routine procedure and methodology for rapid
identification of food borne bacteria using the rapid technique of
direct PCR. This study will also enable us to judge the efficiency of
present food safety steps taken by food manufacturers and exporters.