The ability to isolate and generate a DNA profile from human DNA recovered from tropical bed bugs (Cimex hemipterus) for identifying individuals can be useful for public health, forensic, and medical entomology. In this study, genomic DNA was recovered from both male and female bed bugs at every time interval tested (0, 1, 3, 5, 7, 14, 30, and 45 days post blood meal). The total DNA concentrations recovered from male bed bugs ranged from 12.93 to 65.97 ng/µL, while the total DNA concentrations from female bed bugs ranged from 8.93 to 44.53 ng/µL. However, based on the results from the BLAST search and PCR products, human DNA could be detected from female bed bugs at 0, 3, 5, 14, and 30 days post blood meal using the D18S51 marker. Concentrations of PCR products of the D18S51 locus from male bed bugs ranged from 4.20 to 35.50 ng/µL, whereas, for female bed bugs, concentrations ranged from 4.31 to 22.47 ng/µL. These were generally higher compared to the PCR products of the first hypervariable part (HVR1) marker. The results indicate the HVR1 locus was less sensitive than the D18S51 locus.
A test for assessing pork adulteration in meatballs, using TaqMan probe real-time polymerase chain reaction, was developed. The assay combined porcine-specific primers and TaqMan probe for the detection of a 109 bp fragment of porcine cytochrome b gene. Specificity test with 10 ng DNA of eleven different species yielded a threshold cycle (Ct) of 15.5 ± 0.20 for the pork and negative results for the others. Analysis of beef meatballs with spiked pork showed the assay can determine 100-0.01% contaminated pork with 102% PCR efficiency, high linear regression (r(2) = 0.994) and ≤ 6% relative errors. Residuals analysis revealed a high precision in all determinations. Random analysis of commercial meatballs from pork, beef, chicken, mutton and goat, yielded a Ct between 15.89 ± 0.16 and 16.37 ± 0.22 from pork meatballs and negative results from the others, showing the suitability of the assay to determine pork in commercial meatballs with a high accuracy and precision.
This study was carried out to determine the antibody responses and protective capacity of an inactivated recombinant vaccine expressing the fimbrial protein of Pasteurella multocida B:2 following intranasal vaccination against hemorrhagic septicemia in goats. Goats were vaccinated intranasal with 10(6) CFU/mL of the recombinant vaccine (vaccinated group) and 10(6) CFU/mL of pET32/LIC vector without fimbrial protein (control group). All three groups were kept separated before all goats in the three groups were challenged with 10(9) CFU/mL of live pathogenic P. multocida B:2. During the course of study, both serum and lung lavage fluid were collected to evaluate the antibody levels via enzyme-linked immunosorbent assay. It was found that goats immunized with the inactivated recombinant vaccine developed a strong and significantly (p
The present study was conducted to determine the prevalence and antibiotic resistance of Salmonella sp. isolated from
African catfish (Clarias gariepinus). A total of 30 catfish were harvested from four different farms and four different
wet markets. A total of 60 samples (30 catfish skins and 30 catfish intestines) were used for Salmonella sp. isolation
(pellet-method), its biochemical and serological test. Confirmation of Salmonella sp. were determined by polyvalent
O antisera and polymerase chain reaction (PCR) using genus specific primers for invA genes (DNA amplification
showed one distinct band with molecular weight of 389 bp) and the species of isolated Salmonella sp. were identified
by serotyping. The result showed 6/30 (20%) of fish or 6/60 (10%) of organ samples were positive for Salmonella sp.
Among those positive for Salmonella sp., 4/6 were from intestine samples and 2/6 were from skin samples. No significant
difference was found in the prevalence of Salmonella sp. isolates between fish harvested from farms and wet markets
(p-value= 0.406). The Salmonella serovars identified were Salmonella corvallis (n=3), Salmonella mbandaka (n=2)
and Salmonella typhmurium (n=1). Salmonella sp. isolates were resistance to Penicillin (P 10, 100%), Clindamycin
(DA 2, 100%), Tetracycline (TE 30, 100%) and Rifampicin (RD 5, 100%) and all of the isolates were susceptible or
intermediate resistance to Ceftazidime (CAZ 30) and Trimethopin (W 5). Multiple antibiotic resistance (MAR) index of
all Salmonella sp. isolates in current study was 0.67 indicating that fish sampled in the present study was under high
risk of been exposed to the tested antibiotics.
Vancomycin-resistant enterococci (VRE) have been reported to be present in humans, chickens, and pigs in Malaysia. In the present study, representative samples of VRE isolated from these populations were examined for similarities and differences by using the multilocus sequence typing (MLST) method. Housekeeping genes of Enterococcus faecium (n = 14) and Enterococcus faecalis (n = 11) isolates were sequenced and analyzed using the MLST databases eBURST and goeBURST. We found five sequence types (STs) of E. faecium and six STs of E. faecalis existing in Malaysia. Enterococcus faecium isolates belonging to ST203, ST17, ST55, ST79, and ST29 were identified, and E. faecium ST203 was the most common among humans. The MLST profiles of E. faecium from humans in this study were similar to the globally reported nosocomial-related strain lineage belonging to clonal complex 17 (CC17). Isolates from chickens and pigs have few similarities to those from humans, except for one isolate from a chicken, which was identified as ST203. E. faecalis isolates were more diverse and were identified as ST4, ST6, ST87, ST108, ST274, and ST244, which were grouped as specific to the three hosts. E. faecalis, belonging to the high-risk CC2 and CC87, were detected among isolates from humans. In conclusion, even though one isolate from a chicken was found clonal to that of humans, the MLST analysis of E. faecium and E. faecalis supports the findings of others who suggest VRE to be predominantly host specific and that clinically important strains are found mainly among humans. The infrequent detection of a human VRE clone in a chicken may in fact suggest a reverse transmission of VRE from humans to animals.
Resistance to activated protein C (APC-R) is the commonest inherited cause of thrombosis among Caucasians. Few studies have been carried out on its prevalence in Asians. We conducted a prospective study on 60 patients with thromboembolism to determine its prevalence in our local population. The Factor V Leiden (VaQ506) mutation associated with this condition was detected by amplification of the Factor V gene by polymerase chain reaction (PCR) and digestion of the fragment with Mnl I. Three patients were found to be heterozygous for this mutation. None of the 3 patients had other concomitant hypercoagulable states. In addition, we studied the prevalence of this condition in Malays which was found to be 0.5%. Our study suggests that the incidence of APC-R is much lower here compared to the West.
A prospective study was carried out to evaluate the sensitivity of dengue NS1 antigen-capture ELISA in comparison with dengue virus isolation, conventional RT-PCR and real-time RT-PCR for laboratory confirmation of acute dengue based on single-acute serum samples. Four primary healthcare centres were involved to recruit patients with clinical diagnosis of dengue illness. Patient's demographic, epidemiological and clinical information were collected on a standardized data entry form and 5 ml of venous blood was collected upon consent. In the laboratory, six types of laboratory tests were performed on each of the collected acute serum sample. Of the 558 acute serum samples collected from 558 patients with clinical diagnosis of dengue from mid-August 2006 to March 2009, 174 serum samples were tested positive by the dengue NS1 antigen-capture ELISA, 77 by virus isolation, 92 by RT-PCR and 112 by real-time RT-PCR. A total of 190 serum samples were tested positive by either one or a combination of the four methods whereas, only 59 serum samples were tested positive by all four methods. Thus, based on single-acute serum samples, 190 of the 558 patients (34.1%) were laboratory-confirmed acute dengue. The overall test sensitivity was 91.6%, 40.5%, 48.4% and 58.9% for dengue NS1 antigen-capture ELISA, virus isolation, conventional RT-PCR and real-time RT-PCR respectively. Statistically, dengue NS1 antigen-capture ELISA was the most sensitive and virus isolation was the least sensitive test for the laboratory confirmation of acute dengue based on single-acute serum specimens. Real-time RT-PCR was significantly more sensitive than the conventional RT-PCR.
Dental pulp tissue contains dental pulp stem cells (DPSCs). Dental pulp cells (also known as dental pulp-derived mesenchymal stem cells) are capable of differentiating into multilineage cells including neuron-like cells. The aim of this study was to examine the capability of DPSCs to differentiate into neuron-like cells without using any reagents or growth factors. DPSCs were isolated from teeth extracted from 6- to 8-week-old mice and maintained in complete medium. The cells from the fourth passage were induced to differentiate by culturing in medium without serum or growth factors. RT-PCR molecular analysis showed characteristics of Cd146(+) , Cd166(+) , and Cd31(-) in DPSCs, indicating that these cells are mesenchymal stem cells rather than hematopoietic stem cells. After 5 days of neuronal differentiation, the cells showed neuron-like morphological changes and expressed MAP2 protein. The activation of Nestin was observed at low level prior to differentiation and increased after 5 days of culture in differentiation medium, whereas Tub3 was activated only after 5 days of neuronal differentiation. The proliferation of the differentiated cells decreased in comparison to that of the control cells. Dental pulp stem cells are induced to differentiate into neuron-like cells when cultured in serum- and growth factor-free medium.
A major challenge in the application of mesenchymal stem cells in cartilage reconstruction is that whether the cells are able to differentiate into fully mature chondrocytes before grafting. The aim of this study was to isolate mouse dental pulp stem cells (DPSC) and differentiate them into chondrocytes. For this investigation, morphological, molecular, and biochemical analyses for differentiated cells were used. To induce the chondrocyte differentiation, DPSC were cultured in chondrogenic medium (Zen-Bio, Inc.). Based on morphological analyses using toluidine blue staining, proteoglycan products appear in DPSC after 21 days of chondrocyte induction. Biochemical analyses in differentiated group showed that alkaline phosphatase activity was significantly increased at day 14 as compared to control (P < 0.05). Cell viability analyses during the differentiation to chondrocytes also showed that these cells were viable during differentiation. However, after the 14th day of differentiation, there was a significant decrease (P < 0.05) in the viability proportion among differentiated cells as compared to the control cells. In RT-PCR molecular analyses, mouse DPSC expressed Cd146 and Cd166 which indicated that these cells belong to mesenchymal stem cells. Coll I and Coll II markers showed high expression after 14 and 21 days, respectively. In conclusion, this study showed that DPSC successfully differentiated into chondrocytes.
This study was conducted to investigate the sensitivity and detection of porcine DNA in raw materials, ingredients and finished bakery products by polymerase chain reaction (PCR) - southern hybridization on chip analysis. A total of 20 samples (n=20* 3) with three replicates for each samples were obtained from a bakery factory located in Bangi, Selangor from January to December 2012. The sensitivity level of PCR-southern hybridization on chip was 0.001 ng. The species-specific oligonucleotide primers used in PCR-southern hybridization were targeted on the mitochondria DNA (mtDNA) of cytochrome b (cyt b) gene sequence, namely cty b biotin-labeled oligonucleotide primers. The amplicon from PCR amplification was 276 bp in size. None of the raw materials, ingredients and finished bakery product samples was positive towards porcine DNA, except for the positive control. The results in the present study demonstrated that the PCR- southern-hybridization technique on the gene chip (OliproTM Porcine gene chip) is a sensitive tool for monitoring the porcine component in highly processed ingredients and finished bakery products.
A real-time quantitative polymerase chain reaction (qPCR) was developed for detection and discrimination of Mycobacterium tuberculosis (H37Rv and H37Ra) and M. bovis bacillus Calmette-Guérin (BCG) of the Mycobacterium tuberculosis complex (MTBC) from mycobacterial other than tuberculosis (MOTT). It was based on the melting curve (Tm) analysis of the gyrB gene using SYBR(®) Green I detection dye and the LightCycler 1.5 system. The optimal conditions for the assay were 0.25 μmol/L of primers with 3.1 mmol/L of MgCl(2) and 45 cycles of amplification. For M. tuberculosis (H37Rv and H37Ra) and M. bovis BCG of the MTBC, we detected the crossing points (Cp) at cycles of 16.96 ± 0.07, 18.02 ± 0.14, and 18.62 ± 0.09, respectively, while the Tm values were 90.19 ± 0.06 °C, 90.27 ± 0.09 °C, and 89.81 ± 0.04 °C, respectively. The assay was sensitive and rapid with a detection limit of 10 pg of the DNA template within 35 min. In this study, the Tm analysis of the qPCR assay was applied for the detection and discrimination of MTBC from MOTT.
Magnoflorine, a major bioactive metabolite isolated from Tinospora crispa, has been reported for its diverse biochemical and pharmacological properties. However, there is little report on its underlying mechanisms of action on immune responses, particularly on macrophage activation. In this study, we aimed to investigate the effects of magnoflorine, isolated from T. crispa on the pro-inflammatory mediators generation induced by LPS and the concomitant NF-κB, MAPKs, and PI3K-Akt signaling pathways in U937 macrophages. Differentiated U937 macrophages were treated with magnoflorine and the release of pro-inflammatory mediators was evaluated through ELISA, while the relative mRNA expression of the respective mediators was quantified through qRT-PCR. Correspondingly, western blotting was executed to observe the modulatory effects of magnoflorine on the expression of various markers related to NF-κB, MAPK and PI3K-Akt signaling activation in LPS-primed U937 macrophages. Magnoflorine significantly enhanced the upregulation of TNF-α, IL-1β, and PGE2 production as well as COX-2 protein expression. Successively, magnoflorine prompted the mRNA transcription level of these pro-inflammatory mediators. Magnoflorine enhanced the NF-κB activation by prompting p65, IκBα, and IKKα/β phosphorylation as well as IκBα degradation. Besides, magnoflorine treatments concentration-dependently augmented the phosphorylation of JNK, ERK, and p38 MAPKs as well as Akt. The immunoaugmenting effects were further confirmed by investigating the effects of magnoflorine on specific inhibitors, where the treatment with specific inhibitors of NF-κB, MAPKs, and PI3K-Akt proficiently blocked the magnoflorine-triggered TNF-α release and COX-2 expression. Magnoflorine furthermore enhanced the MyD88 and TLR4 upregulation. The results suggest that magnoflorine has high potential on augmenting immune responses.
The EphA4 receptor tyrosine kinase is involved in numerous cell-signalling activities during embryonic development. EphA4 has the ability to bind to both types of ephrin ligands, the ephrinAs and ephrinBs. The C57BL/6J-Epha4rb-2J/GrsrJ strain, denoted Epha4(rb-2J/rb-2J), is a spontaneous mouse mutant that arose at The Jackson Laboratory. These mutants exhibited a synchronous hind limb locomotion defect or "hopping gait" phenotype, which is also characteristic of EphA4 null mice. Genetic complementation experiments suggested that Epha4(rb-2J) corresponds to an allele of EphA4, but details of the genomic defect in this mouse mutant are currently unavailable. We found a single base-pair deletion in exon 9 resulting in a frame shift mutation that subsequently resulted in a premature stop codon. Analysis of the predicted structure of the truncated protein suggests that both the kinase and sterile α motif (SAM) domains are absent. Definitive determination of genotype is needed for experimental studies of mice carrying the Epha4(rb-2J) allele, and we have also developed a method to ease detection of the mutation through RFLP. Eph-ephrin family members are reportedly expressed as numerous isoforms. Hence, delineation of the specific mutation in EphA4 in this strain is important for further functional studies, such as protein-protein interactions, immunostaining and gene compensatory studies, investigating the mechanism underlying the effects of altered function of Eph family of receptor tyrosine kinases on phenotype.
Neurodegenerative diseases (NDDs) are pathological conditions characterised by progressive damage of neuronal cells leading to eventual loss of structure and function of the cells. Due to implication of multi-systemic complexities of signalling pathways in NDDs, the causes and preventive mechanisms are not clearly delineated. The study was designed to investigate the potential signalling pathways involved in neuroprotective activities of purely isolated glucomoringin isothiocyanate (GMG-ITC) against H2O2-induced cytotoxicity in neuroblastoma (SH-SY5Y) cells. GMG-ITC was isolated from Moringa oleifera seeds, and confirmed with NMR and LC-MS based methods. Gene expression analysis of phase II detoxifying markers revealed significant increase in the expression of all the genes involved, due to GMG-ITC pre-treatment. GMG-ITC also caused significant decreased in the expression of NF-kB, BACE1, APP and increased the expressions of IkB and MAPT tau genes in the differentiated cells as confirmed by multiplex genetic system analysis. The effect was reflected on the expressed proteins in the differentiated cells, where GMG-ITC caused increased in expression level of Nrf2, SOD-1, NQO1, p52 and c-Rel of nuclear factor erythroid factor 2 (Nrf2) and nuclear factor kappa-B (NF-kB) pathways respectively. The findings revealed the potential of GMG-ITC to abrogate oxidative stress-induced neurodegeneration through Nrf2 and NF-kB signalling pathways.
This paper reports total nematode anthelmintic resistance towards albendazole, fenbendazole, levamisole and ivermectin in a commercial sheep farm located in Terengganu, Malaysia. Faecal Egg Count Reduction Test (FECRT) was conducted on 25 sheep, where five sheep in each group were treated with the respective four anthelmintics based on live bodyweight. The balance of five sheep placed in the control group were not treated with any anthelmintics. At day 13 post-treatment, faecal egg count was conducted and nematode worm egg count reduction percentage was calculated to determine the resistance status towards the respective anthelmintics tested. Results showed that nematodes were resistant to all the anthelmintics tested, namely albendazole, fenbendazole, levamisole and ivermectin with reduction percentage of 87%, 46%, 94% and 68%, respectively. Subsequently, the third stage larvae of Haemonchus contortus and Trichostrongylus colubriformis recovered from post-treatment faecal cultures were subjected to allele-specific polymerase chain reaction (AS-PCR) assay to determine the presence of the benzimidazole resistance gene. This study reports the occurrence of the classical F200Y mutation in the isotype 1 βtubulin gene, for the first time in Malaysia.
Basal stem rot (BSR) disease caused by the fungus Ganoderma boninense is the most serious disease affecting the oil palm; this is because the disease escapes the early disease detection. The biocontrol agent Trichoderma harzianum can protect the disease only at the early stage of the disease. In the present study, the expression levels of three oil palm (Elaeis guineensis Jacq.) chitinases encoding EgCHI1, EgCHI2, and EgCHI3 at 2, 5, and 8 weeks inoculation were measured in oil palm leaves from plants treated with G. boninense or T. harzianum alone or both.
Background. Quercus infectoria (QI) is a plant used in traditional medicines in Asia. The plant was reported to contain various active phytochemical compounds that have potential to stimulate bone formation. However, the precise mechanism of the stimulation effect of QI on osteoblast has not been elucidated. The present study was carried out to isolate QI semipurified fractions from aqueous QI extract and to delineate the molecular mechanism of QI semipurified fraction that enhanced bone formation by using hFOB1.19 human fetal osteoblast cell model. Methods. Isolation of QI semipurified fractions was established by means of column chromatography and thin layer chromatography. Established QI semipurified fractions were identified using Liquid Chromatography-Mass Spectrometry (LC-MS). Cells were treated with derived QI semipurified fractions and investigated for mineralization deposition and protein expression level of BMP-2, Runx2, and OPN by ELISA followed gene expression analysis of BMP-2 and Runx2 by RT-PCR. Results. Column chromatography isolation and purification yield Fractions A, B, and C. LC-MS analysis reveals the presence of polyphenols in each fraction. Results show that QI semipurified fractions increased the activity and upregulated the gene expression of BMP-2 and Runx2 at day 1, day 3, and day 7. OPN activity increased in cells treated with QI semipurified fractions at day 1 and day 3. Meanwhile, at day 7, expression of OPN decreased in activity. Furthermore, the study showed that combination of Fractions A, B, and C with osteoporotic drug (pamidronate) further increased the activity and upregulated the gene expression of BMP-2 and Runx2. Conclusions. These findings demonstrated that polyphenols from semipurified fractions of QI enhanced bone formation through expression of the investigated bone-related marker that is its potential role when combined with readily available osteoporotic drug.
We describe a rare tumor site in a 46 year old man who presented with a two week history of headache. Physical examination revealed bilateral papilloedema with no other localizing signs. Computed Tomographic Scan as well as Magnetic Resonance Imaging of the brain revealed a lesion with a dura tail located adjacent to the falx cerebri of the right frontal lobe. This lesion was not invading the inner table of the skull base. A tumor blush was seen on angiogram. There were no abnormalities on CT scan of the abdomen and fundoscopy was normal. Intraoperatively a vascular tumor not attached to the dura was noted and removed totally. Histopathological examination was typical of a hemangioblastoma. Analysis revealed no mutations of the VHL gene in 5 regions, exon 5-8 of the p53 gene, exon 1-2 of the p16 gene and exon 5,6 and 8 of the PTEN gene. This is the first case report of a supratentorial hemangioblastoma in a non-Von Hippel Lindau patient with genetic evidence.
Frequent loss of heterozygosity (LOH) and mutations of the tumor suppressor gene PTEN (phosphatase and tensin homologue deleted from chromosome 10) have been found in sporadic gliomas. The most documented regions of allelic losses include 9p21, 10q23-25 and 17p1 3 whereas PTEN aberrations are preferentially found in glioblastoma multiformes. This research aimed to detect the incidence of allelic losses on chromosomes 10q, 9p, 17p and 13q and mutations on exons 5, 6 and 8 of PTEN in malignant gliomas. Malignant glioma specimens obtained were classified histopathologically according to the WHO criteria. Each tumor was then subjected to polymerase chain reaction (PCR)-LOH analysis using microsatellite markers and single-stranded conformational polymorphism (SSCP) analysis. Twelve of 23 (52%) malignant glioma cases showed allelic losses whereas 7 of 23 (30%) samples showed aberrant band patterns and mutations of PTEN. Four of these cases showed LOH in 10q23 and mutations of PTEN. The data on LOH indicated the involvement of different genes in the genesis of glioma whereas mutations of PTEN indicated the role of PTEN tumor suppressor gene in the progression of glioma in Malay population.
Background: Brain tumors, constituting one of the most deadly forms of cancer worldwide, result from the accumulation of multiple genetic and epigenetic alterations in genes and signaling pathways. Isocitrate dehydrogenase enzyme isoform 1 (IDH1) mutations are frequently identified in primary brain tumors and acute myeloid leukemia. Studies on IDH1 gene mutations have been extensively performed in various populations worldwide but not in Malaysia. This work was conducted to study the prevalence of IDH1 c.395G>A (R132H) hotspot mutations in a group of Malaysian patients with brain tumors in order to gain local data for the IDH1 mutation profile in our population. Methods: Mutation analysis of c.395G>A (R132H) of IDH1 was performed in 40 brain tumor specimens by the polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) and then verified by direct sequencing. Associations between the IDH1 c.395G>A (R132H) mutation and clinicopathologic characteristics were also analyzed. Results: The IDH1 c.395G>A (R132H) mutation was detected in 14/40 patients (35%). A significant association was found with histological tumor types, but not with age, gender and race. Conclusions: IDH1 is frequently mutated and associated with histological subtypes in Malay brain tumors.