Avian Influenza viruses belonging to the Orthomyxoviridae family are enveloped viruses with segmented negative sense RNA genome surrounded by a helical symmetry capsid. Influenza viruses, especially the highly pathogenic avian influenza virus (HPAI) such as H5 or H7 subtype are the most important pathogens for the poultry industry in recent times. The haemagglutinin protein and neuraminidase, serves as the target for the immune response of the host. Due to recurrent genetic reassortments between avian and human influenza viruses, global pandemics may emerge and the naive human immunity could not withstand pressure by the novel hybrid virus. The emergence of genetic engineering technology provided the industry with new methods of manufacturing diagnostics tools and vaccines. After extraction of RNA from the cell culture of strain influenza A/Chicken/Malaysia/2004(H5N1) of AIV, the viral RNA was converted to cDNA by a specific primer. The cDNA was amplified by the polymerase chain reaction (PCR) and analyzed
by agarose gel electrophoresis. The intact PCR product of full length haemagglutinin gene was cloned in TO POTM TA Cloning vector. The full-length HA-encoding gene of H5N1 AIV was subcloned into a pPICZA vector. After successful ligation, the constructed plasmid was transformed into E.coli.Top10, Plasmid DNA from transformed bacteria was extracted in white colony and positive clones were confirmed by restriction digestion with Sacl and Not1 restriction enzymes, colony PCR screening and nucleotide sequencing. Construction of a recombinant pPICZA/H5HA plasmid containing the full length haemagglutinin gene was achieved as a first step
towards the expression in Pichia pastoris.
Gelatin is a highly purified animal protein of pig, cow, and fish origins and is extensively used in food, pharmaceuticals, and personal care products. However, the acceptability of gelatin products greatly depends on the animal sources of the gelatin. Porcine and bovine gelatins have attractive features but limited acceptance because of religious prohibitions and potential zoonotic threats, whereas fish gelatin is welcomed in all religions and cultures. Thus, source authentication is a must for gelatin products but it is greatly challenging due to the breakdown of both protein and DNA biomarkers in processed gelatins. Therefore, several methods have been proposed for gelatin identification, but a comprehensive and systematic document that includes all of the techniques does not exist. This up-to-date review addresses this research gap and presents, in an accessible format, the major gelatin source authentication techniques, which are primarily nucleic acid and protein based. Instead of presenting these methods in paragraph form which needs much attention in reading, the major methods are schematically depicted, and their comparative features are tabulated. Future technologies are forecasted, and challenges are outlined. Overall, this review paper has the merit to serve as a reference guide for the production and application of gelatin in academia and industry and will act as a platform for the development of improved methods for gelatin authentication.
Staphylococcus aureus aregram positive cocci which colonizethe skin and mucous membranes particularly the anterior nares. Prevalence of nosocomial infections associated with methicillin resistant S. aureus have been reported in hospitals (HA-MRSA) for over five decades. Recently,community-acquired MRSA (CA-MRSA) has emerged as a cause of skin and soft tissue infections in healthy individuals. These strains are sensitive to antimicrobials, carry genes for Panton-Valentine leukocidin (PVL) toxin and belong to the staphylococcal cassette chromosome (SCC) mec type IV or V. The suspected mode of transmission involves close contact with carriers leading to skin or nasal colonization that resultin subsequent active infection. Molecular typing is used to determine the mode of transmission of CA-MRSA in the community.General typing methods such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and specific methods for Staphylococci such as SCCmec typing and spa typing have the capability to characterize bacterial chromosomes and mobile genetic elements. Combination of these molecular typing methods is necessary as each method has its own advantages with respect to discriminatory power, rapidity, cost effectiveness, reproducibility, and ease of performance.
Edible bird's nest (EBN) is an expensive animal bioproduct due to its reputation as a food and delicacy with diverse medicinal properties. One kilogram of EBN costs ~$6000 in China. EBN and its products are consumed in mostly Asian countries such as China, Hong Kong, Taiwan, Singapore, Malaysia, Indonesia, Vietnam and Thailand, making up almost 1/3 of world population. The rapid growth in EBN consumption has led to a big rise in the trade scale of its global market. Presently, various fake materials such as tremella fungus, pork skin, karaya gum, fish swimming bladder, jelly, agar, monosodium glutamate and egg white are used to adulterate EBNs for earning extra profits. Adulterated or fake EBN may be hazardous to the consumers. Thus, it is necessary to identify of the adulterants. Several sophisticated techniques based on genetics, immunochemistry, spectroscopy, chromatography and gel electrophoresis have been used for the detection of various types of adulterants in EBN. This article describes the recent advances in the authentication methods for EBN. Different genetic, immunochemical, spectroscopic and analytical methods such as genetics (DNA) based techniques, enzyme-linked immunosorbent assays, Fourier transform infrared and Raman spectroscopic techniques, and chromatographic and gel electrophoretic methods have been discussed. Besides, significance of the reported methods that might pertain them to applications in EBN industry has been described. Finally, efforts have been made to discuss the challenges and future perspectives of the authentication methods for EBN.
Little is known about the evolution, adaptation and pathogenesis of Burkholderia pseudomallei within host during acute melioidosis infection. Melioidosis is a potential life threatening disease contracted through inhalation, ingestion, inoculation or direct entry of the organism into the blood stream via wounds or skin abrasions from contaminated soil and water. Environmental B. pseudomallei strain (Bp MARAN ), isolated during a melioidosis outbreak in Pahang, Malaysia was injected intra-peritoneally into a mouse and passaged strain was recovered from spleen (Bpmouse-adapted). A gel-based comparative proteomics profiling approach was used, to map and identify differentially expressed proteins (fold-change ≥ 2; p-value ≤ 0.05) between the strains. A total of 730 and 685 spots were visualised in the Bp MARAN and Bpmouse-adapted strains, respectively. Of the 730 spots (Bp MARAN as reference gel), 87 spots were differentially regulated (44 up- and 43 down-regulated). The identified proteins were classified as proteins related to metabolism, stress response, virulence, signal transduction, or adhesion. In comparison, it was found that those proteins related to adhesins, virulence factors and stress- response were up-regulated and could possibly explain the adaptation of the bacteria in the host. Investigating the differentially expressed proteins may provide better perspective of bacterial factors which aid survivability of B. pseudomallei in host.
Genetic polymorphism of the cytochrome P450 (CYP) genes particularly affects CYP2D6 and CYP2C19 to a functionally relevant extent, and it is therefore crucial to elucidate the enzyme kinetic and molecular basis for altered catalytic activity of these allelic variants. This study explored the expression and function of the reported alleles CYP2D6*2, CYP2D6*10, CYP2D6*17, CYP2C19*23, CYP2C19*24, and CYP2C19*25 with respect to gene polymorphisms. Site-directed mutagenesis (SDM) was carried out to generate these six alleles. After DNA sequencing, the CYP2D6 and CYP2C19 wild types alongside with their alleles were each independently co-expressed with NADPH-CYP oxidoreductase (OxR) in Escherichia coli. The expressed proteins were analyzed using Western blotting, reduced carbon monoxide (CO) difference spectral scanning, and cytochrome c reductase assay. Results from Western blot revealed the presence of all CYP wild-type and allelic proteins in E. coli membrane fractions. The reduced CO difference spectra scanning presented the distinct peak of absorbance at 450 nm, and the cytochrome c reductase assay has confirmed that spectrally active OxR was expressed in each protein preparation. As a conclusion, the results obtained from this study have proven the CYP variants to be immunoreactive and spectrally active and are suitable for use to examine biotransformation and interaction mechanism of the enzymes.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
A. niger produced alpha-glucosidase, alpha-amylase and two forms of glucoamylase when grown in a liquid medium containing raw tapioca starch as the carbon source. The glucoamylases, which formed the dominant components of amylolytic activity manifested by the organism, were purified to homogeneity by ammonium sulfate precipitation, ion-exchange and two cycles of gel filtration chromatography. The purified enzymes, designated GA1 and GA2, a raw starch digesting glucoamylase, were found to have molar masses of 74 and 96 kDa and isoelectric points of 3.8 and 3.95, respectively. The enzymes were found to have pH optimum of 4.2 and 4.5 for GA1 and GA2, respectively, and were both stable in a pH range of 3.5-9.0. Both enzymes were thermophilic in nature with temperature optimum of 60 and 65 degrees C, respectively, and were stable for 1 h at temperatures of up to 60 degrees C. The kinetic parameters Km and V showed that with both enzymes the branched substrates, starch and amylopectin, were more efficiently hydrolyzed compared to amylose. GA2, the more active of the two glucoamylases produced, was approximately six to thirteen times more active towards raw starches compared to GA1.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
The serological responses to Cryptococcus neoformans proteins of blood donors and HIV patients with active cryptococcosis from a tropical region were investigated in this study. Exposure to C. neoformans, an organism ubiquitous in the environment, contributes to the antibody responses observed in the blood donors. IgG responses to cryptococcal proteins were stronger than IgM responses in most sera tested in this study. A 53-kDa cryptococcal protein fragment was identified as the most immunoreactive protein on the IgM immunoblots of both blood donors and patients. Overall, there was no obvious difference in IgG responses of patients when compared with blood donors. Some immunogenic protein fragments (27.5, 76, 78 and 91.5 kDa) were detected at least two times more frequently on IgM immunoblots of patients compared with those of blood donors. It is yet to be investigated whether the proteins identified in this study may have any potential to be used as biomarker for cryptococcosis.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Leptospira species were studied in water and soils from selected urban sites in Malaysia. A total of 151 water (n=121) and soil (n=30) samples were collected from 12 recreational lakes and wet markets. All samples were filtered and inoculated into semi-solid Ellinghausen and McCullough modified by Johnson and Harris (EMJH) media supplemented with additional 5-fluorouracil. The cultures were then incubated at 30°C and observed under a dark field microscope with intervals of 10 days. A PCR assay targeting the rrs gene was used to confirm the genus Leptospira among the isolates. Subsequently, the pathogenic status of the isolates was determined using primer sets G1/G2 and Sapro1/Sapro2, which target the secY and rrs genes, respectively. The isolates were identified at serogroup level using the microscopic agglutination test (MAT) while their genetic diversity was assessed by pulsed field gel electrophoresis (PFGE). Based on dark field microscopy, 23.1% (28/121) water and 23.3% (7/30) soil cultures were positive for Leptospira spp. Of the 35 positive cultures, only 8 were pure and confirmed as Leptospira genus by PCR assay. Two out of 8 isolates were confirmed as pathogenic, 5 were saprophytic and one was intermediate. These 8 isolates were negative for the 25 reference hyperimmune rabbit sera tested in the MAT. PFGE showed that all 8 of these environmental Leptospira spp. were genetically diverse. In conclusion, the presence of pathogenic Leptospira spp. in the urban Malaysian environment may indicate and highlight the importance of water screening, especially in recreational lakes, in order to minimize any chance of Leptospira infection.
A novel cationic polymer, dextran-spermine (D-SPM), has been found to mediate gene expression in a wide variety of cell lines and in vivo through systemic delivery. Here, we extended the observations by determining the optimal conditions for gene expression of D-SPM/plasmid DNA (D-SPM/pDNA) in cell lines and in the lungs of BALB/c mice via instillation delivery. In vitro studies showed that D-SPM could partially protect pDNA from degradation by nuclease and exhibited optimal gene transfer efficiency at D-SPM to pDNA weight-mixing ratio of 12. In the lungs of mice, the levels of gene expression generated by D-SPM/pDNA are highly dependent on the weight-mixing ratio of D-SPM to pDNA, amount of pDNA in the complex, and the assay time postdelivery. Readministration of the complex at day 1 following the first dosing showed no significant effect on the retention and duration of gene expression. The study also showed that there was a clear trend of increasing size of the complexes as the amount of pDNA was increased, where the sizes of the D-SPM/pDNA complexes were within the nanometer range.
Ninety-two strains of lactic acid bacteria (LAB) were isolated from a Malaysian food ingredient, chili bo, stored for up to 25 days at 28 degreesC with no benzoic acid (product A) or with 7,000 mg of benzoic acid kg-1 (product B). The strains were divided into eight groups by traditional phenotypic tests. A total of 43 strains were selected for comparison of their sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) whole-cell protein patterns with a SDS-PAGE database of LAB. Isolates from product A were identified as Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus farciminis, Pediococcus acidilactici, Enterococcus faecalis, and Weissella confusa. Five strains belonging to clusters which could not be allocated to existing species by SDS-PAGE were further identified by 16S rRNA sequence comparison. One strain was distantly related to the Lactobacillus casei/Pediococcus group. Two strains were related to Weissella at the genus or species level. Two other strains did not belong to any previously described 16S rRNA group of LAB and occupied an intermediate position between the L. casei/Pediococcus group and the Weissella group and species of Carnobacterium. The latter two strains belong to the cluster of LAB that predominated in product B. The incidence of new species and subspecies of LAB in chili bo indicate the high probability of isolation of new LAB from certain Southeast Asian foods. None of the isolates exhibited bacteriocin activity against L. plantarum ATCC 14917 and LMG 17682.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Protein profiling has revealed the presence of glacontryphan-M, a peptide toxin identified only in the sea snail genus Conus, in the wings of Hebomoia glaucippe (HG). The wings and body of HG were homogenized and the proteins were extracted and analyzed by 2D gel electrophoresis with subsequent in-gel digestion. Posttranslational protein modifications were detected and analyzed by nano-LC-MS/MS. An antibody was generated against glacontryphan-M, and protein extracts from the wings of HG samples from Malaysia, Indonesia, and the Philippines were tested by immunoblotting. Glacontryphan-M was unambiguously identified in the wings of HG containing the following posttranslational protein modifications: monoglutamylation at E55, methylation at E53, quinone modification at W61, cyanylation at C56, and amidation of the C terminus at G63. Immunoblotting revealed the presence of the toxin in the wings of HG from all origins, showing a single band for glacontryphan-M in HG samples from Malaysia and Philippines and a double band in HG samples from Indonesia. Intriguingly, sequence analysis indicated that the Conus glacontryphan is identical to that of HG. The toxin may function as a defense against diverse predators, including ants, mantes, spiders, lizards, green frogs, and birds.
The nucleocapsid (N) protein of Nipah virus (NiV) is a major constituent of the viral proteins which play a role in encapsidation, regulating the transcription and replication of the viral genome. To investigate the use of a fusion system to aid the purification of the recombinant N protein for structural studies and potential use as a diagnostic reagent, the NiV N gene was cloned into the pFastBacHT vector and the His-tagged fusion protein was expressed in Sf9 insect cells by recombinant baculovirus. Western blot analysis of the recombinant fusion protein with anti-NiV antibodies produced a band of approximately 62 kDa. A time course study showed that the highest level of expression was achieved after 3 days of incubation. Electron microscopic analysis of the NiV recombinant N fusion protein purified on a nickel-nitrilotriacetic acid resin column revealed different types of structures, including spherical, ring-like, and herringbone-like particles. The light-scattering measurements of the recombinant N protein also confirmed the polydispersity of the sample with hyrdrodynamic radii of small and large types. The optical density spectra of the purified recombinant fusion protein revealed a high A(260)/A(280) ratio, indicating the presence of nucleic acids. Western blotting and enzyme-linked immunosorbent assay results showed that the recombinant N protein exhibited the antigenic sites and conformation necessary for specific antigen-antibody recognition.
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel
Pulsed-field gel electrophoresis (PFGE) of XbaI-digested chromosomal DNA was performed on 133 strains of Salmonella enterica serovar Typhi obtained from Papua New Guinea, with the objective of assessing the temporal variation of these strains. Fifty-two strains that were isolated in 1992 and 1994 were of one phage type, D2, and only two predominant PFGE profiles, X1 and X2, were present. Another 81 strains isolated between 1997 and 1999 have shown divergence, with four new phage types, UVS I (n = 63), UVS (n = 5), VNS (n = 4), and D1 (n = 9), and more genetic variability as evidenced by the multiple and new PFGE XbaI profiles (21 profiles; Dice coefficient, F = 0.71 to 0.97). The two profiles X1 and X2 have remained the stable, dominant subtypes since 1992. Cluster analysis based on the unweighted pair group method using arithmetic averages algorithm identifies two main clusters (at 87% similarity), indicating that the divergence of the PFGE subtypes was probably derived from some genomic mutations of the X1 and X2 subtypes. The majority of isolates were from patients with mild and moderate typhoid fever and had various XbaI profiles. A single isolate from a patient with fatal typhoid fever had a unique X11 profile, while four of six isolates from patients with severe typhoid fever had the X1 pattern. In addition, 12 paired serovar Typhi isolates recovered from the blood and fecal swabs of individual patients exhibited similar PFGE patterns, while in another 11 individuals paired isolates exhibited different PFGE patterns. Three pairs of isolates recovered from three individuals had different phage types and PFGE patterns, indicating infection with multiple strains. The study reiterates the usefulness of PFGE in assessing the genetic diversity of S. enterica serovar Typhi for both long-term epidemiology and in vivo stability and instability within an individual patient.
The incidence of food-borne salmonellosis due to Salmonella enterica serotype Weltevreden is reported to be on the increase in Malaysia. The pulsed-field gel electrophoresis (PFGE) subtyping method was used to assess the extent of genetic diversity and clonality of Salmonella serotype Weltevreden strains from humans and the environment. PFGE of XbaI-digested chromosomal DNA from 95 strains of Salmonella serotype Weltevreden gave 39 distinct profiles with a wide range of Dice coefficients (0.27 to 1.00), indicating that PFGE is very discriminative and that multiple clones of Salmonella serotype Weltevreden exist among clinical and environmental isolates. Strains of one dominant pulsotype (pulsotype X1/X2) appeared to be endemic in this region, as they were consistently recovered from humans with salmonellosis between 1996 and 2001 and from raw vegetables. In addition, the sharing of similar PFGE profiles among isolates from humans, vegetables, and beef provides indirect evidence of the possible transmission of salmonellosis from contaminated raw vegetables and meat to humans. Furthermore, the recurrence of PFGE profile X21 among isolates found in samples of vegetables from one wet market indicated the persistence of this clone. The environment in the wet markets may represent a major source of cross-contamination of vegetables with Salmonella serotype Weltevreden. Antibiotic sensitivity tests showed that the clinical isolates of Salmonella serotype Weltevreden remained drug sensitive but that the vegetable isolates were resistant to at least two antibiotics. To the best of our knowledge, this is the first study to compare clinical and environmental isolates of Salmonella serotype Weltevreden in Malaysia.
Molecular characterization of a total of 52 human isolates of Salmonella typhi from Papua New Guinea was performed by using pulsed-field gel electrophoresis (PFGE) after digestion of chromosomal DNA with three restriction endonucleases, XbaI (5'-TCTAGA-3'), AvrII (5'-CCTAGG-3'), and SpeI (5'-ACTAGT-3'). Of the 52 isolates tested, 11 were obtained from patients with fatal typhoid fever and 41 were obtained from patients with nonfatal disease. The 52 isolates showed limited genetic diversity as evidenced by only three different PFGE patterns detected following digestion with XbaI (patterns X1 to X3; F [coefficient of similarity] = 0.86 to 1.0), four patterns detected following digestion with AvrII (patterns A1 to A4; F =0.78 to 1.0), and two patterns detected following digestion with SpeI (patterns S1 and S2; F = 0.97 to 1.0). Of the 52 isolates, 37 were phage typed, and all belonged to phage type D2. All 11 isolates obtained from patients with fatal typhoid fever were identical (F = 1.0) and possessed the PFGE pattern combination X1S1A1, whereas the 41 isolates from patients with nonfatal typhoid fever had various PFGE pattern combinations, the most common being X2S1A2 (39%), X1S1A1 (24%), and X1S1A2 (15%). Thus, all the isolates from patients with the fatal disease had the X1 and A1 patterns, whereas the majority of the isolates from patients with nonfatal typhoid fever possessed the X2 and A2 patterns. The data suggest that there is an association among strains of S. typhi between genotype, as assessed by PFGE patterns, and the capability to cause fatal illness. Analysis of blood and fecal isolates of S. typhi from the same patient also indicated that some genetic changes occur in vivo during the course of infection.
Pulsed-field gel electrophoresis (PFGE) was used to compare and analyze 158 isolates of Salmonella typhi from five well-defined outbreaks of typhoid fever in Malaysia and also isolates involved in sporadic cases of typhoid fever occurring during the same period. Digestion of chromosomal DNAs from these S. typhi isolates with the restriction endonucleases XbaI (5'-TCTAGA-3'), SpeI (5'-ACTAGT-3'), and AvrII (5'-CCTAGG-3') and then PFGE produced restriction endonuclease analysis (REA) patterns consisting of 11 to 24 DNA fragments ranging in size from 20 to 630 kbp. Analysis of the REA patterns generated by PFGE after digestion with XbaI and SpeI indicated that the S. typhi isolates obtained from sporadic cases of infection were much more heterogeneous (at least 13 different REA patterns were detected; Dice coefficient, between 0.73 and 1.0) than those obtained during outbreaks of typhoid fever. The clonal nature and the close genetic identities of isolates from outbreaks in Alor Setar, Penang, Kota Kinabalu, Johor Bahru, and Kota Bahru were suggested by the fact that only a limited number of REA patterns, which mostly differed by only a single band, were detected (one to four patterns; Dice coefficient, between 0.82 and 1.0), although a different pattern was associated with each of these outbreaks. Comparison of REA patterns with ribotyping for 18 S. typhi isolates involved in sporadic cases of infection showed a good correlation, in that 72% of the isolates were in the same group. There was no clear correlation of phage types with a specific REA pattern. We conclude that PFGE of s. typhi chromosomal DNA digested with infrequently cutting restriction endonucleases is a useful method for comparing and differentiating S. typhi isolates for epidemiological purposes.
Bacteriocin release proteins (BRPs) can be used for the release of heterologous proteins from the Escherichia coli cytoplasm into the culture medium. The gene for a highly thermostable alkaline protease was cloned from Bacillus stearothermophilus F1 by the polymerase chain reaction. The recombinant F1 protease was efficiently excreted into the culture medium using E. coli XL1-Blue harboring two vectors: pTrcHis bearing the protease gene and pJL3 containing the BRPs. Both vectors contain the E. coli lac promoter-operator system. In the presence of 40 microM IPTG, the recombinant F1 protease and the BRP were expressed and mature F1 protease was released into the culture medium. This opens the way for the large-scale production of this protease in E. coli. The recombinant enzyme was purified through a one-step heat treatment at 70 degrees C for 3h and this method purified the protease to near homogeneity. The purified enzyme showed a pH optimum of 9.0, temperature optimum of 80 degrees C, and was stable at 70 degrees C for 24h in the pH range from 8.0 to 10.0. The enzyme exhibited a high degree of thermostability with a half-life of 4 h at 85 degrees C, 25 min at 90 degrees C, and was inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF).
Matched MeSH terms: Electrophoresis, Polyacrylamide Gel