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Fulltext An overview of atypical bacterial in congenital pneumonia

Zurina Zainuddin, Zainab Jumai Kassim, Siti Norbaya Masri, Putri Yubbu, Norlijah Othman, Zainab Jumai Kassim

Malaysian Journal of Paediatrics and Child Health, 2018;24(2):1-13.
MyJurnal

Congenital pneumonia is one of the common causes of respiratory distress at birth with significant morbidity and mortality in infants. Estimates show that neonatal pneumonia including congenital pneumonia contributes to between 750 000 and 1.2 million neonatal deaths every year which accounts for 10% global child mortality. Etiological agents are many and vary but atypical bacterial causes are few. The commonest cause for atypical bacteria is Ureaplasma urealyticum. Congenital pneumonia is often clinically difficult to diagnose owing to poor specificity of clinical signs, with similarities in radiologic presentation with other respiratory conditions of the newborn. Isolation of causative organism (s) by culture from nasopharyngeal aspirates or tracheal aspirates obtained within 8 hours of life is the gold standard of its diagnosis. However, this technique is elaborate and time consuming in identifying atypical bacteria. Development of a more sensitive modality such as polymerase chain reaction (PCR) has dramatically altered the microbiological diagnosis of congenital pneumonia.

Matched MeSH terms: Polymerase Chain Reaction
Fulltext Molecular characterization and epidemiology of rotavirus isolates obtained from children with diarrhoea in Malaysia

Zuridah H, Kirkwood CD, Bishop RF, Bogdanovic-Sakran N, Yap KL

Med J Malaysia, 2009 Sep;64(3):193-6.
PMID: 20527266 MyJurnal

This retrospective study examined the G/P type of rotavirus in RNA samples that have previously been e-typed by RNA-PAGE in 1996. The results were then compared to 2007 samples to ascertain the extent of changes that may have occurred in this 11-years time interval. The G and P genotypes were determined by hemi-nested PCR and further analysed by phylogenetic study. In 1996, the G/P combination G1P[8], G(UT)P[8] and G1P(UT) prevalence rate were 81%, 9% and 7%, respectively. As expected, the G9 genotype which has already emerged worldwide was identified in 42% of the 2007 samples with the remaining 33% G1P[8] and 25% G1P(UT) Analysis of the RNA pattern showed that majority of the isolates were long e-type in both series, nevertheless minor differences within electropherotypes were observed. Genetic diversity in some strains of the human group A rotaviruses was analysed by phylogenetic methods. These findings will help in the decision to introduce rotavirus vaccines within the next decade.

Matched MeSH terms: Polymerase Chain Reaction
First Report of Ralstonia solanacearum Race 2 Biovar 1 Causing Moko Disease of Banana in Malaysia

Zulperi D, Sijam K

Plant Dis, 2014 Feb;98(2):275.
PMID: 30708756 DOI: 10.1094/PDIS-03-13-0321-PDN

During March 2011 to June 2012, 50 banana plants of cultivar Musa × paradisiaca 'Horn' with Moko disease symptoms were randomly sampled in 12 different locations of 5 outbreak states in Peninsular Malaysia comprising Kedah, Selangor, Pahang, Negeri Sembilan, and Johor, with disease incidence exceeding 90% in some severely affected plantations. The disease symptoms observed in the infected plants included yellowing and wilting of the oldest leaves, which became necrotic, and eventually led to their dieback or collapse. The pulp of banana fruits also became discolored and exuded bacterial ooze. Vascular tissues in pseudostems were discolored. Fragments from symptomatic plant samples were excised and cultured on Kelman's-tetrazolium salt (TZC) medium. Twenty positive samples produced fluidal colonies that were either entirely white or white with pink centers after incubation for 24 to 48 h at 28°C on Kelman's-TZC medium and appeared as gram-negative rods after Gram staining. They were also positive for potassium hydroxide (KOH), Kovacs oxidase, and catalase tests, but negative for utilization of disaccharides and hexose alcohols, which are characteristics of biovar 1 Ralstonia solanacearum. For the pathogenicity test, 30 μl of 108 CFU/ml bacterial suspension of three selected virulent strains were injected into banana (Musa × paradisiaca 'Horn') leaves explants grown in plastic pots of 1,440 cm3 volume in a greenhouse, with temperature range from 26 to 35°C. Leaves that were infiltrated with sterile distilled water served as a negative control. Inoculations with all isolates were performed in three replications, as well as the uninoculated control leaves explants. The inoculated plants produced the same symptoms as observed on naturally diseased samples, whereas control plants remained asymptomatic. Strain cultures were re-isolated and possessed the morphological and biochemical characteristics as previously described. PCR amplification using race 2 R. solanacearum primers ISRso19-F (5'-TGGGAGAGGATGGCGGCTTT-3') and ISRso19-R (5'-TGACCCGCCTTTCGGTGTTT-3') (3) produced a 1,900-bp product from DNA of all bacterial strains. BLAST searches resulted that the sequences were 95 to 98% identical to published R. solanacearum strain race 2 insertion sequence ISRso19 (GenBank Accession No. AF450275). These genes were later deposited in GenBank (KC812051, KC812052, and KC812053). Phylotype-specific multiplex PCR (Pmx-PCR) and Musa-specific multiplex PCR (Mmx-PCR) were performed to identify the phylotype and sequevar of all isolates (4). Pmx-PCR showed that all isolates belonged to phylotype II, whereas Mmx-PCR showed that they belonged to phylotype II sequevar 4 displaying 351-bp amplicon. Although there were previously extensive studies on R. solanacearum associated with bacterial wilt disease of banana crops in Malaysia, none related to Moko disease has been reported (1,2). The result has a great importance to better understand and document R. solanacearum race 2 biovar 1, since banana has been identified as the second most important commercial fruit crop with a high economic value in Malaysia. References: (1) R. Khakvar et al. Plant Pathol. J. 7:162, 2008. (2) R. Khakvar et al. Am. J. Agri. Biol. Sci. 3:490, 2008. (3) Y. A. Lee and C. N. Khor. Plant Pathol. Bull. 12:57, 2003. (4) P. Prior et al. Pages 405-414 in: Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex. The American Phytopathological Society, St. Paul, MN, 2005.

Matched MeSH terms: Multiplex Polymerase Chain Reaction
Identification of Vibrio parahaemolyticus isolates by PCR targeted to the toxR gene and detection of virulence genes

Zulkifli, Y., Alitheen, N.B., Son, R., Yeap, S.K., Lesley, M.B., Raha, A.R.

International Food Research Journal, 2009;16(3):289-296.
MyJurnal

Vibrio parahaemolyticus is a gram negative bacterium and causes gastrointestinal illness in humans. In this study, twenty five out of fifty cockle samples from Padang, Indonesia produced purple colonies when they were grown on selective medium, CHROMagarTM Vibrio. Specific–PCR for toxR gene detection gave positive results in which a band with 368 base pairs size appeared on the gel for all the isolates that confirmed the presence of V. parahaemolyticus. In the virulence properties test, all the isolates showed negative results for tdh and trh genes detection. The results indicate that the isolates under this study do not contain virulence properties that correlate to the ability of infection and diseases, which means that they are nonpathogenic.

Matched MeSH terms: Polymerase Chain Reaction
Fulltext Random amplified polymorphic DNA-PCR and ERIC PCR analysis on Vibrio parahaemolyticus isolated from cockles in Padang, Indonesia

Zulkifli, Y., Alitheen, N.B., Son, R., Raha, A.R., Samuel, L., Yeap, S.K., et al.

International Food Research Journal, 2009;16(2):141-150.
MyJurnal

In this study, RAPD-PCR and ERIC-PCR were used to study the epidemiology of V. parahaemolyticus isolated from cockles in Padang, Indonesia. The Gold Oligo OPAR3 primer produced bands ranged from 1-8 with sizes from 0.2 – 5.0 kb and the Gold Oligo OPAR8 primer produced 1-7 bands with sizes 0.7 – 1.5 kb. Both primers produced twenty five RAPD patterns with a few isolates failed to produce any products. Based on phylogenetic dendrogram, all the isolates can be divided into 6 major clusters with similarity between 0 to 52%. For the ERIC primer, it produced bands ranged from 3-15 with sizes from 0.1 – 5.0 kb and twenty seven different ERIC patterns. Construction of the phylogenetic dendogram showed the isolates can be divided into 4 major clusters with similarity between 56 to 86%. The high diversity of both processes may be due to the multiple contamination sources of V. parahaemolyticus.

Matched MeSH terms: Polymerase Chain Reaction
Fulltext Predominance of G to A codon 12 mutation K-ras gene in Dukes' B colorectal cancer

Zulhabri O, Rahman J, Ismail S, Isa MR, Wan Zurinah WN

Singapore Med J, 2012 Jan;53(1):26-31.
PMID: 22252179

K-ras gene mutations in codons 12 and 13 are one of the earliest events in colon carcinogenesis.

Matched MeSH terms: Polymerase Chain Reaction/methods
Selective inhibition of genes in methicillin resistant Staphylococcus aureus (MRSA) treated with Melastoma malabathricum methanol extract

Zulaikah Mohamed, Nazlina Ibrahim, Ismail Ahmad

Sains Malaysiana, 2008;37(1):107-113.

Methanol extract of Melastoma malabathricum leaves inhibited the growth of Staphylococcus aureus and six clinical isolates of Methicilin Resistant Stapyhlococcus aureus (MRSA 1-6). The minimum inhibitory concentration (MIC) of test substance was 1.565mg/ml and the minimum bacteriocidal concentration (MBC) was 3.125 mg/ml. The methanol extract suppressed RNA synthesis at 10 mg/ml as shown by RNA profile which was devoid of three bands compared to the control. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis using seven primer pairs was only successful in amplifying four cDNA amplicons. The failure to amplify three cDNA amplicons for three primer pairs corresponding to gyrA, femA and nuc genes, implied the possibility of suppression of the corresponding mRNA. Electrophoretic separation of endogenous and exogenuos bacterial proteins showed that three and five protein, respectively were not expressed. One endogenous and three exogenous proteins were over-expressed in treated MRSA compared with untreated control. The results of the molecular and proteomic analyses are in agreement, and based on primers being used, methanol extract of M. malabathricum leaves possibly inhibits MRSA growth through inhibition of DNA synthesis, peptidoglycan production, and nuclease production.
Keywords: Methicillin resistant Staphylococcus aureus; Melastoma malabathricum; gene expression; protein production

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Development of a human salivary gland organoid model

Zulaiha A. Rahman, Colin D. Bingle, Lynne Bingle

Malaysian Journal of Medicine and Health Sciences, 2019;15(107):1-1.
MyJurnal

Introduction: Currently, organoid technology provides a useful tool for modelling human organ development and pathologies in vitro. Salivary gland (SG) organoids developed from mice SG cells display self-organizing properties closely mimic the native organ. Thus, this study would like to investigate the potential of this organoid system to de-velop a human salivary gland in vitro. Methods: Organoids were developed from biopsy samples of normal human sublingual gland tissue. Cells were isolated and cultured in Matrigel at an Air Liquid Interface (ALI) for up to 14 days in an enriched media supplementing with Wnt-3A, R-spondin1, EGF, and FGF2. Specific differentiation factors like TGFβ, BMP, and LIMK inhibitors were added to enriched media for further differentiation studies. Haematoxylin and eosin-stained sections of the cultures were used to visualise growth. RT-PCR, immunohistochemistry and immunoflu-orescence were used to determine the differential expression of cell-specific markers. Results: Human SG organoids developed when the cells were grown in Matrigel at ALI in a defined culture system. The addition of TGFβ inhibitor and all the inhibitors (TGFβ, BMP and LIMK) to the culture media affected SG organoids development by displaying distinct characteristics that closely resemble native glands and expressed specific cell-type markers; BPIFA2, AQP5, CK5 and E-cadherin. The inhibition of BMP signalling demonstrated SG organoids growth more into ductal-like struc-tures and expressed ductal cell marker, CK7. While LIM kinase inhibition signalling showed significantly higher of amylase activity assay. Conclusion: This study certainly offers valuable insight into determining the optimal culture conditions for developing human SG organoids.

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
Fulltext Detection of Strongyloides stercoralis infection among cancer patients in a major hospital in Kelantan, Malaysia

Zueter AM, Mohamed Z, Abdullah AD, Mohamad N, Arifin N, Othman N, et al.

Singapore Med J, 2014 Jul;55(7):367-71.
PMID: 25091885

INTRODUCTION: Strongyloidiasis is one of the most commonly neglected but clinically important parasitic infections worldwide, especially among immunocompromised patients. Evidence of infection among immunocompromised patients in Malaysia is, however, lacking. In this study, microscopy, real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISAs) were used to detect Strongyloides stercoralis (S. stercoralis) infection among cancer patients in a Malaysian hospital.

METHODS: A total of 192 stool and serum samples were collected from cancer patients who were receiving chemotherapy with or without steroid treatment at a hospital in northeastern Malaysia. Stool samples were examined for S. stercoralis using parasitological methods and real-time PCR. Serology by ELISA was performed to detect parasite-specific immunoglobulin G (IgG), IgG4 and immunoglobulin E (IgE) antibodies. For comparison, IgG4- and IgG-ELISAs were also performed on the sera of 150 healthy individuals from the same area.

RESULTS: Of the 192 samples examined, 1 (0.5%) sample was positive for S. stercoralis by microscopy, 3 (1.6%) by real-time PCR, 8 (4.2%) by IgG-ELISA, 6 (3.1%) by IgG4-ELISA, and none was positive by IgE-ELISA. In comparison, healthy blood donors had significantly lower prevalence of parasite-specific IgG (2.67%, p < 0.05) and IgG4 (2.67%, p < 0.05) responses.

CONCLUSION: This study showed that laboratory testing may be considered as a diagnostic investigation for S. stercoralis among immunocompromised cancer patients.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Administration of Bacillus subtilis strains in the rearing water enhances the water quality, growth performance, immune response, and resistance against Vibrio harveyi infection in juvenile white shrimp, Litopenaeus vannamei

Zokaeifar H, Babaei N, Saad CR, Kamarudin MS, Sijam K, Balcazar JL

Fish Shellfish Immunol, 2014 Jan;36(1):68-74.
PMID: 24161773 DOI: 10.1016/j.fsi.2013.10.007

In this study, vegetative cell suspensions of two Bacillus subtilis strains, L10 and G1 in equal proportions, was administered at two different doses 10(5) (BM5) and 10(8) (BM8) CFU ml(-1) in the rearing water of shrimp (Litopenaeus vannamei) for eight weeks. Both probiotic groups showed a significant reduction of ammonia, nitrite and nitrate ions under in vitro and in vivo conditions. In comparison to untreated control group, final weight, weight gain, specific growth rate (SGR), food conversion ratio (FCR) and digestive enzymatic activity were significantly greater in the BM5 and BM8 groups. Significant differences for survival were recorded in the BM8 group as compared to the control. Eight weeks after the start of experiment, shrimp were challenged with Vibrio harveyi. Statistical analysis revealed significant differences in shrimp survival between probiotic and control groups. Cumulative mortality of the control group was 80%, whereas cumulative mortality of the shrimp that had been given probiotics was 36.7% with MB8 and 50% with MB5. Subsequently, real-time RT-PCR was employed to determine the mRNA levels of prophenoloxidase (proPO), peroxinectin (PE), lipopolysaccharide- and β-1,3-glucan- binding protein (LGBP) and serine protein (SP). The expression of all immune-related genes studied was only significantly up-regulated in the BM5 group compared to the BM8 and control groups. These results suggest that administration of B. subtilis strains in the rearing water confers beneficial effects for shrimp aquaculture, considering water quality, growth performance, digestive enzymatic activity, immune response and disease resistance.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext Effects of Bacillus subtilis on the growth performance, digestive enzymes, immune gene expression and disease resistance of white shrimp, Litopenaeus vannamei

Zokaeifar H, Balcázar JL, Saad CR, Kamarudin MS, Sijam K, Arshad A, et al.

Fish Shellfish Immunol, 2012 Oct;33(4):683-9.
PMID: 22659618 DOI: 10.1016/j.fsi.2012.05.027

We studied the effect of two probiotic Bacillus subtilis strains on the growth performance, digestive enzyme activity, immune gene expression and disease resistance of juvenile white shrimp (Litopenaeus vannamei). A mixture of two probiotic strains, L10 and G1 in equal proportions, was administered at two different doses 10(5) (BM5) and 10(8) (BM8) CFU g(-1) feed to shrimp for eight weeks. In comparison to untreated control group, final weight, weight gain and digestive enzyme activity were significantly greater in shrimp fed BM5 and BM8 diets. Significant differences for specific growth rate (SGR) and survival were recorded in shrimp fed BM8 diet as compared with the control; however, no significant differences were recorded for food conversion ratio (FCR) among all the experimental groups. Eight weeks after the start of the feeding period, shrimp were challenged with Vibrio harveyi. Statistical analysis revealed significant differences in shrimp survival between probiotic and control groups. Cumulative mortality of the control group was 63.3%, whereas cumulative mortality of the shrimp that had been given probiotics was 20.0% with BM8 and 33.3% with BM5. Subsequently, real-time PCR was employed to determine the mRNA levels of prophenoloxidase (proPO), peroxinectin (PE), lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) and serine protein (SP). The expression of all immune-related genes studied was significantly up-regulated (P

Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction/veterinary; Real-Time Polymerase Chain Reaction/veterinary
Fulltext Selection and identification of non-pathogenic bacteria isolated from fermented pickles with antagonistic properties against two shrimp pathogens

Zokaeifar H, Balcázar JL, Kamarudin MS, Sijam K, Arshad A, Saad CR

J Antibiot (Tokyo), 2012 Jun;65(6):289-94.
PMID: 22491136 DOI: 10.1038/ja.2012.17

In this study, potential probiotic strains were isolated from fermented pickles based on antagonistic activity against two shrimp pathogens (Vibrio harveyi and Vibrio parahaemolyticus). Two strains L10 and G1 were identified by biochemical tests, followed by16S ribosomal RNA gene sequence analysis as Bacillus subtilis, and characterized by PCR amplification of repetitive bacterial DNA elements (Rep-PCR). Subsequently, B. subtilis L10 and G1 strains were tested for antibacterial activity under different physical conditions, including culture medium, salinity, pH and temperature using the agar well diffusion assay. Among the different culture media, LB broth was the most suitable medium for antibacterial production. Both strains showed the highest level of antibacterial activity against two pathogens at 30 °C and 1.0% NaCl. Under the pH conditions, strain G1 showed the greatest activity against V. harveyi at pH 7.3-8.0 and against V. parahaemolyticus at pH 6.0-8.0, whereas strain L10 showed the greatest activity against two pathogens at pH 7.3. The cell-free supernatants of both strains were treated with four different enzymes in order to characterize the antibacterial substances against V. harveyi. The result showed considerable reduction of antibacterial activity for both strains, indicating the proteinaceous nature of the antibacterial substances. A wide range of tolerance to NaCl, pH and temperature was also recorded for both strains. In addition, both strains showed no virulence effect in juvenile shrimp Litopenaeus vannamei. On the basis of these results and safety of strains to L. vannamei, they may be considered for future challenge experiments in shrimp as a very promising alternative to the use of antibiotics.

Matched MeSH terms: Polymerase Chain Reaction
An improved rapid genotyping method for the study of beta-2 adrenergic receptor gene polymorphisms using single tube allele specific multiplex PCR

Zilfalil BA, Hoh BP, Nizam MZ, Liza-Sharmini AT, Teh LK, Ismail R

J Clin Pharm Ther, 2006 Dec;31(6):637-40.
PMID: 17176369

Seventeen single nucleotide polymorphisms (SNPs) have been identified so far, within the beta-2 receptor (beta(2) AR) gene. The presence of so many SNPs within the beta(2) AR gene causes a problem, for those studying beta(2) AR pharmacogenetics, in relation to which SNPs to choose. Most of the work has focused on the three common SNPs within the coding block (alleles 16, 27 and 164) and the techniques developed have been for these three functionally important alleles.

Matched MeSH terms: Polymerase Chain Reaction/methods*
Current analytical methods for porcine identification in meat and meat products

Zia Q, Alawami M, Mokhtar NFK, Nhari RMHR, Hanish I

Food Chem, 2020 Sep 15;324:126664.
PMID: 32380410 DOI: 10.1016/j.foodchem.2020.126664

Authentication of meat products is critical in the food industry. Meat adulteration may lead to religious apprehensions, financial gain and food-toxicities such as meat allergies. Thus, empirical validation of the quality and constituents of meat is paramount. Various analytical methods often based on protein or DNA measurements are utilized to identify meat species. Protein-based methods, including electrophoretic and immunological techniques, are at times unsuitable for discriminating closely related species. Most of these methods have been replaced by more accurate and sensitive detection methods, such as DNA-based techniques. Emerging technologies like DNA barcoding and mass spectrometry are still in their infancy when it comes to their utilization in meat detection. Gold nanobiosensors have shown some promise in this regard. However, its applicability in small scale industries is distant. This article comprehensively reviews the recent developments in the field of analytical methods used for porcine identification.

Matched MeSH terms: Polymerase Chain Reaction
Molecular characterization of a Toxocara variant from cats in Kuala Lumpur, Malaysia

Zhu XQ, Jacobs DE, Chilton NB, Sani RA, Cheng NA, Gasser RB

Parasitology, 1998 Aug;117 ( Pt 2):155-64.
PMID: 9778638

The ascaridoid nematode of cats from Kuala Lumpur, Malaysia, previously identified morphologically as Toxocara canis, was characterized using a molecular approach. The nuclear ribosomal DNA (rDNA) region spanning the first internal transcribed spacer (ITS-1), the 5.8S gene and the second internal transcribed spacer (ITS-2) was amplified and sequenced. The sequences for the parasite from Malaysian cats were compared with those for T. canis and T. cati. The sequence data showed that this taxon was genetically more similar to T. cati than to T. canis in the ITS-1, 5.8S and ITS-2. Differences in the ITS-1 and ITS-2 sequences between the taxa (9.4-26.1%) were markedly higher than variation between samples within T. canis and T. cati (0-2.9%). The sequence data demonstrate that the parasite from Malaysian cats is neither T. canis nor T. cati and indicate that it is a distinct species. Based on these data, PCR-linked restriction fragment length polymorphism (RFLP) and single-strand conformation polymorphism (SSCP) methods were employed for the unequivocal differentiation of the Toxocara variant from T. canis and T. cati. These methods should provide valuable tools for studying the life-cycle, transmission pattern(s) and zoonotic potential of this parasite.

Matched MeSH terms: Polymerase Chain Reaction/veterinary
EGFR Intron 1 polymorphism in Asian Populations and its correlation with EGFR gene expression and amplification in breast tumor tissues

Zhou Q, Cheung YB, Jada SR, Lim WT, Kuo WL, Gray JW, et al.

Cancer Biol Ther, 2006 Nov;5(11):1445-9.
PMID: 17102595

AIM: The purpose of this study was to test the hypothesis if longer CA dinucleotide repeats are more common in the Asian population and also to gain insights into the interplay between the CA dinucleotide repeats and the frequencies of EGFR gene expression and amplifications as this might have therapeutic implications with regards to treatment with tyrosine kinase inhibitors.
MATERIALS AND METHODS: The EGFR intron 1 polymorphism was analysed in three distinct healthy Asian subjects, namely, Chinese (N = 96), Malays (N = 98) and Indians (N = 100). Comparative genomic hybridisation was performed to investigate for changes in DNA copy number in relation to the polymorphic CA dinucleotide repeats in breast tumor tissues (N = 22).
RESULTS: The frequency of short alleles with 14 and 15 CA repeats were most common in the Asian populations and significantly higher than those reported for Caucasians. The frequency of 20 CA repeats was 5%, almost 13-fold lower than previous reports. EGFR amplifications were detected in 23% and 11% of breast tumor tissues harboring short and long CA repeats, respectively.
CONCLUSION: Our results show that the frequency of alleles encoding for short CA dinucleotide repeats is common in Asian populations. EGFR expression and amplification levels were also higher in Asian breast tumor tissues with short CA dinucleotide repeats. These findings suggest that the EGFR intron 1 polymorphism may influence response to treatment with tyrosine kinase inhibitors in breast cancer patients and further studies are warranted.

Matched MeSH terms: Polymerase Chain Reaction
Fulltext DACH1, a zona glomerulosa selective gene in the human adrenal, activates transforming growth factor-β signaling and suppresses aldosterone secretion

Zhou J, Shaikh LH, Neogi SG, McFarlane I, Zhao W, Figg N, et al.

Hypertension, 2015 May;65(5):1103-10.
PMID: 25776071 DOI: 10.1161/HYP.0000000000000025

Common somatic mutations in CACNAID and ATP1A1 may define a subgroup of smaller, zona glomerulosa (ZG)-like aldosterone-producing adenomas. We have therefore sought signature ZG genes, which may provide insight into the frequency and pathogenesis of ZG-like aldosterone-producing adenomas. Twenty-one pairs of zona fasciculata and ZG and 14 paired aldosterone-producing adenomas from 14 patients with Conn's syndrome and 7 patients with pheochromocytoma were assayed by the Affymetrix Human Genome U133 Plus 2.0 Array. Validation by quantitative real-time polymerase chain reaction was performed on genes >10-fold upregulated in ZG (compared with zona fasciculata) and >10-fold upregulated in aldosterone-producing adenomas (compared with ZG). DACH1, a gene associated with tumor progression, was further analyzed. The role of DACH1 on steroidogenesis, transforming growth factor-β, and Wnt signaling activity was assessed in the human adrenocortical cell line, H295R. Immunohistochemistry confirmed selective expression of DACH1 in human ZG. Silencing of DACH1 in H295R cells increased CYP11B2 mRNA levels and aldosterone production, whereas overexpression of DACH1 decreased aldosterone production. Overexpression of DACH1 in H295R cells activated the transforming growth factor-β and canonical Wnt signaling pathways but inhibited the noncanonical Wnt signaling pathway. Stimulation of primary human adrenal cells with angiotensin II decreased DACH1 mRNA expression. Interestingly, there was little overlap between our top ZG genes and those in rodent ZG. In conclusion, (1) the transcriptome profile of human ZG differs from rodent ZG, (2) DACH1 inhibits aldosterone secretion in human adrenals, and (3) transforming growth factor-β signaling pathway is activated in DACH1 overexpressed cells and may mediate inhibition of aldosterone secretion in human adrenals.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext Detection of circulating tumor DNA in plasma of patients with primary CNS lymphoma by digital droplet PCR

Zhong Y, Tan GW, Bult J, Veltmaat N, Plattel W, Kluiver J, et al.

BMC Cancer, 2024 Apr 02;24(1):407.
PMID: 38566053 DOI: 10.1186/s12885-024-12191-z

BACKGROUND: Primary central nervous system lymphoma (PCNSL) are rare mature B-cell lymphoproliferative diseases characterized by a high incidence of MYD88 L265P and CD79B Y196 hotspot mutations. Diagnosis of PCNSL can be challenging. The aim of the study was to analyze the detection rate of the MYD88 L265P and CD79B Y196 mutation in cell free DNA (cfDNA) in plasma of patients with PCNSL.
METHODS: We analyzed by digital droplet PCR (ddPCR) to determine presence of the MYD88 L265P and CD79B Y196 hotspot mutations in cfDNA isolated from plasma of 24 PCNSL patients with active disease. Corresponding tumor samples were available for 14 cases. Based on the false positive rate observed in 8 healthy control samples, a stringent cut-off for the MYD88 L265P and CD79B Y196 mutation were set at 0.3% and 0.5%, respectively.
RESULTS: MYD88 L265P and CD79B Y196 mutations were detected in 9/14 (64%) and 2/13 (15%) tumor biopsies, respectively. In cfDNA samples, the MYD88 L265P mutation was detected in 3/24 (12.5%), while the CD79B Y196 mutation was not detected in any of the 23 tested cfDNA samples. Overall, MYD88 L265P and/or CD79B Y196 were detected in cfDNA in 3/24 cases (12.5%). The detection rate of the combined analysis did not improve the single detection rate for either MYD88 L265P or CD79B Y196.
CONCLUSION: The low detection rate of MYD88 L265P and CD79B Y196 mutations in cfDNA in the plasma of PCNSL patients argues against its use in routine diagnostics. However, detection of MYD88 L265P by ddPCR in cfDNA in the plasma could be considered in challenging cases.

Matched MeSH terms: Polymerase Chain Reaction
Optimization and Implementation of the Virus Extraction Method for Hepatitis E Virus Detection from Raw Pork Liver

Zhao MY, Li D

Food Environ Virol, 2021 03;13(1):74-83.
PMID: 33449335 DOI: 10.1007/s12560-020-09452-y

Hepatitis E virus (HEV) has been frequently detected from pork liver and liver products, which can usually cause self-limiting diseases in healthy adults, yet may result in fatality in immunosuppressed groups. Nevertheless, there is so far no standardized method for HEV detection available from pork liver and/or liver products. The present study aimed to optimize the virus extraction method of HEV from raw pork liver, which is often consumed in Asia undercooked to avoid a grainy texture. By comparing different sample preparation protocols and by applying the selected protocol to 60 samples collected from Singapore retail markets, we demonstrated that homogenization of 0.25 g raw pork liver with FastPrep™ Lysing Matrix Y containing yttria-stabilized zircondium oxide beads in 2 ml tubes and with harsh mechanical force at 6 ms-1, 40 s/cycle, for 5 cycles with 300 s pause time after each cycle is promising in both releasing the potentially intracellular viruses and resulting in satisfactory virus recovery rates (> 1%). A high prevalence (52%) of HEV genome was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) from the 60 samples collected from Singapore retail markets imported from Indonesia, Australia and Malaysia. However, RNase treatment decreased the HEV prevalence to 33.3%, and all of the 20 positive samples were with high RT-qPCR Ct values above 35, suggesting that the positive RT-qPCR signals maybe largely due to the inactive viruses and/or exposed HEV RNA traces in raw pork liver products. Therefore, conscious care should be taken when interpreting molecular detection results of viruses from food samples to be correlated with public health risks.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Detection of mutations and polymorphism of N-acetyltransferase 1 gene in Indian, Malay and Chinese populations

Zhao B, Lee EJ, Yeoh PN, Gong NH

Pharmacogenetics, 1998 Aug;8(4):299-304.
PMID: 9731716

The xenobiotic metabolizing enzymes N-acetyltransferases (NATs) are important for the biotransformation and/or bioactivation of drugs and carcinogens. NATs are coded for in humans by two distinct genes, designated NAT1 and NAT2. NAT1, which was originally thought to be monomorphic, was recently reported to exhibit variation in human populations. Recent studies suggested that a genetic polymorphism of NAT1 may be associated with colorectal cancer risk. The distributions of NAT1 allele and genotype frequencies in unrelated individuals among Indian (n = 140), Malay (n = 122) and Chinese (n = 181) populations in Singapore were characterized by polymerase chain reaction-restriction fragment length polymorphism and allele-specific-polymerase chain reaction. The allelic frequencies of NAT1*3, NAT1*4, NAT1*10 and NAT1*11 among Indians were 0.3, 0.51, 0.17 and 0.02, respectively. The corresponding NAT1 allelic frequencies in Malays were 0.29, 0.30, 0.39 and 0.02, respectively, and were similar to those in Chinese in the region. The allelic frequencies of NAT1*3, NAT1*4, NAT1*10 and NAT1*11 among Chinese were 0.33, 0.35, 0.30 and 0.02, respectively. These findings are of importance for the determination of cancer risk in these populations. In addition, nucleotide changes at positions 350-351 (GG to CC) and 497-499 (GGG to CCC) of the NAT1 gene were not found in the alleles of the populations studied.

Matched MeSH terms: Polymerase Chain Reaction

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