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Double gene targeting PCR assay for the detection of Crocodylus porosus in commercial products

Ahmad Nizar NN, Ali ME, Hossain MAM, Sultana S, Ahamad MNU

Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2018 Jun;35(6):1038-1051.
PMID: 29447579 DOI: 10.1080/19440049.2018.1440644

The demand for crocodile meat is quickly growing because of its exotic and organoleptic appeal and also the low content of cholesterol and lipids. Moreover, crocodile oil and blood have been used in alternative medicines for treating asthma and several other ailments since ancient times. Furthermore, crocodile hides have great demand in leather industries. All of these have collectively contributed to the extensive hunting, illegal trading and consequent decline of crocodiles in most parts of the world. To keep space with the growing demands, some crocodile species such as Crocodylus porosus have been raised in farms and its commercial trades have been legalised. However, demand for wild crocodiles in foods and medicines has continued in high gear. Recently, several DNA-based methods have been proposed for crocodile detection, but those assays are based on single gene and longer-sized amplicon targets that break down during extensive processing. To address this gap, here we developed and validated a highly stable double gene targeted multiplex PCR assay for the identification of C. porosus materials in commercial products. The assay involved two short sites from C. porosus atp6 (77 bp) and cytb (127 bp) genes and a universal internal control (99 bp) for eukaryotes. The PCR primers were cross-tested against 18 species and validated under pure and mixed matrices under extensive boiling, autoclaving and microwave cooking conditions. Finally, it was used to identify five crocodile-based commercial products. The lower limits of detection for atp6 and cytb genes were 0.001 ng and 0.01 ng DNA, respectively, in pure meat and 1% under mixed matrices. Some inherent features, such as 77-127 bp amplicon sizes, exceptional stability and superior sensitivity, suggested the assay could be used for the identification of C. porosus in any forensic specimen.

Matched MeSH terms: Mitochondrial Proton-Translocating ATPases/metabolism
Model-aided atpE gene knockout strategy in Escherichia coli for enhanced succinic acid production from glycerol

Mienda BS, Shamsir MS, Md Illias R

J Biomol Struct Dyn, 2016 Aug;34(8):1705-16.
PMID: 26513379 DOI: 10.1080/07391102.2015.1090341

Succinic acid is an important platform chemical with a variety of applications. Model-guided metabolic engineering strategies in Escherichia coli for strain improvement to increase succinic acid production using glucose and glycerol remain largely unexplored. Herein, we report what are, to our knowledge, the first metabolic knockout of the atpE gene to have increased succinic acid production using both glucose and alternative glycerol carbon sources in E. coli. Guided by a genome-scale metabolic model, we engineered the E. coli host to enhance anaerobic production of succinic acid by deleting the atpE gene, thereby generating additional reducing equivalents by blocking H(+) conduction across the mutant cell membrane. This strategy produced 1.58 and .49 g l(-1) of succinic acid from glycerol and glucose substrate, respectively. This work further elucidates a model-guided and/or system-based metabolic engineering, involving only a single-gene deletion strategy for enhanced succinic acid production in E. coli.

Matched MeSH terms: Bacterial Proton-Translocating ATPases/metabolism*
Vacuolar-ATPase (V-ATPase) Mediates Progesterone-Induced Uterine Fluid Acidification in Rats

Karim K, Giribabu N, Muniandy S, Salleh N

J. Membr. Biol., 2016 04;249(1-2):65-76.
PMID: 26403527 DOI: 10.1007/s00232-015-9848-z

We hypothesized that progesterone-induced decrease in uterine fluid pH involves V-ATPase. In this study, expression and functional activity of V-ATPase in uterus were investigated under progesterone influence. Ovariectomized adult female rats received subcutaneous injection of estradiol-17β (1 µg/kg/day) or progesterone (20 mg/kg/day) for 3 days or 3 days estradiol-17β followed by 3 days vehicle, progesterone, or estradiol-17β plus progesterone. Mifepristone, a progesterone receptor blocker, was concomitantly given to the rats which received progesterone. A day after last injection, rate of uterine fluid secretion, its HCO3 (-) concentration, and pH were determined via in vivo uterine perfusion in rats under anesthesia. V-ATPase inhibitor, bafilomycin, was introduced into the perfusion buffer, and changes in these parameters were observed. Expression of V-ATPase A1 and B1/2 proteins and mRNAs in uterus were quantified by Western blotting and real-time PCR, respectively. Distribution of these proteins was observed by immunohistochemistry. Our findings showed that under progesterone influence, uterine fluid secretion rate, HCO3 (-) concentration, and pH were significantly reduced. Administration of bafilomycin did not cause significant changes in fluid secretion rate; however, HCO3 (-) concentration and pH were significantly elevated. In parallel with these changes, expression of V-ATPase A1 and B1/2 proteins and mRNAs were significantly increased with these proteins highly distributed in uterine luminal and glandular epithelia. In conclusion, increased expression and functional activity of V-ATPase were most likely responsible for the decreased in uterine fluid pH observed under progesterone influence.

Matched MeSH terms: Vacuolar Proton-Translocating ATPases/metabolism*
Fulltext Variations in DREB1A and VP1.1 Genes Show Association with Salt Tolerance Traits in Wild Tomato (Solanum pimpinellifolium)

Rao ES, Kadirvel P, Symonds RC, Geethanjali S, Thontadarya RN, Ebert AW

PLoS One, 2015;10(7):e0132535.
PMID: 26161546 DOI: 10.1371/journal.pone.0132535

Association analysis was conducted in a core collection of 94 genotypes of Solanum pimpinellifolium to identify variations linked to salt tolerance traits (physiological and yield traits under salt stress) in four candidate genes viz., DREB1A, VP1.1, NHX1, and TIP. The candidate gene analysis covered a concatenated length of 4594 bp per individual and identified five SNP/Indels in DREB1A and VP1.1 genes explaining 17.0% to 25.8% phenotypic variation for various salt tolerance traits. Out of these five alleles, one at 297 bp in DREB1A had in-frame deletion of 6 bp (CTGCAT) or 12 bp (CTGCATCTGCAT), resulting in two alleles, viz., SpDREB1A_297_6 and SpDREB1A_297_12. These alleles individually or as haplotypes accounted for maximum phenotypic variance of about 25% for various salt tolerance traits. Design of markers for selection of the favorable alleles/haplotypes will hasten marker-assisted introgression of salt tolerance from S. pimpinellifolium into cultivated tomato.

Matched MeSH terms: Vacuolar Proton-Translocating ATPases/metabolism
pink1, atp13a2 and uchl1 expressions are affected by inflammation in the brain

Moriya S, Tan VP, Yee AK, Parhar IS

Neurosci Lett, 2019 08 24;708:134330.
PMID: 31201839 DOI: 10.1016/j.neulet.2019.134330

In Parkinson's disease (PD), several genes have been identified as the PD-related genes, however, the regulatory mechanisms of these gene expressions have not been fully identified. In this study, we investigated the effect of inflammation, one of the major risk factors in PD on expressions of the PD-related genes. Lipopolysaccharide (LPS) was intraperitoneally administered to mature male zebrafish and gene expressions in the brains were examined by real-time PCR. In the inflammation-related genes, expressions of tnfb, il1b and il6 were increased at 2 days post administration in the 10 μg group, and tnfb expression was also increased at 4 days post administration in the 1 μg and 10 μg group. In the PD-related genes, pink1 expression was significantly decreased at 4 days, atp13a2 expression was significantly increased at 7 days, and uchl1 expression was significantly decreased at 7 days. This suggests that pink1, atp13a2 and uchl1 expressions are regulated by inflammation, and this regulatory mechanism might be involved in the progress of PD.

Matched MeSH terms: Proton-Translocating ATPases/metabolism*
Multiplex PCR assay discriminates rabbit, rat and squirrel meat in food chain

Ahamad MNU, Ali ME, Hossain MAM, Asing A, Sultana S, Jahurul MHA

Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2017 Dec;34(12):2043-2057.
PMID: 28748739 DOI: 10.1080/19440049.2017.1359752

Rabbit meat is receiving increasing attention because it contains a high level of proteins with relatively little fat. On the other hand, squirrel meat is served in upper-class meals in certain countries, so is sold at higher prices. The other side of the coin is rat meat, which has family ties with rabbit and squirrel but poses substantial threats to public health because it is a potential carrier of several zoonotic organisms. Recently, rat meat was mislabelled and sold as lamb after chemical modification. Thus, the chances of rabbit and squirrel meat substitution by rat meat cannot be ruled out. For the first time, a multiplex PCR assay was developed in Malaysia for the discriminatory identification of rat, rabbit and squirrel in the food chain. Rabbit (123 bp), rat (108 bp) and squirrel (243 bp) targets were amplified from ATP6 and cytb genes, along with a eukaryotic internal control (141bp). The products were sequenced and cross-tested against 22 species. A total of 81 reference samples and 72 meatball specimens were screened to validate the assay. Analyte stability was evaluated through boiling, autoclaving and micro-oven cooking. The tested lower limits of detection were 0.01 ng DNA for pure meat and 0.1% for meatballs.

Matched MeSH terms: Mitochondrial Proton-Translocating ATPases/metabolism
Fulltext Identification of proteins of altered abundance in oil palm infected with Ganoderma boninense

Al-Obaidi JR, Mohd-Yusuf Y, Razali N, Jayapalan JJ, Tey CC, Md-Noh N, et al.

Int J Mol Sci, 2014;15(3):5175-92.
PMID: 24663087 DOI: 10.3390/ijms15035175

Basal stem rot is a common disease that affects oil palm, causing loss of yield and finally killing the trees. The disease, caused by fungus Ganoderma boninense, devastates thousands of hectares of oil palm plantings in Southeast Asia every year. In the present study, root proteins of healthy oil palm seedlings, and those infected with G. boninense, were analyzed by 2-dimensional gel electrophoresis (2-DE). When the 2-DE profiles were analyzed for proteins, which exhibit consistent significant change of abundance upon infection with G. boninense, 21 passed our screening criteria. Subsequent analyses by mass spectrometry and database search identified caffeoyl-CoA O-methyltransferase, caffeic acid O-methyltransferase, enolase, fructokinase, cysteine synthase, malate dehydrogenase, and ATP synthase as among proteins of which abundances were markedly altered.

Matched MeSH terms: Proton-Translocating ATPases/metabolism
Kidney activity increases in copper exposed goldfish (Carassius auratus auratus)

Moyson S, Liew HJ, Fazio A, Van Dooren N, Delcroix A, Faggio C, et al.

Comp Biochem Physiol C Toxicol Pharmacol, 2016 Dec;190:32-37.
PMID: 27521798 DOI: 10.1016/j.cbpc.2016.08.003

In the present study, the effect of copper was examined in the common goldfish (Carassius auratus auratus). Fish were fasted and exposed to either a high (0.84μM), a low (0.34μM) or a control copper concentration (0.05μM) for 1 and 7days. Swimming performance was not affected by either fasting or copper exposure. Food deprivation alone had no effect on ionoregulation, but low plasma osmolality levels and plasma Na(+) were noticed in fasted fish exposed to Cu for 7days. Both gill Na(+)/K(+)-ATPase and H(+)-ATPase activities were undisturbed, while both kidney ATPase activities were up-regulated when challenged with the high Cu levels. Up-regulated kidney ATPase activities likely acted as compensatory strategy to enhance Na(+) reabsorption. However, this up-regulation was not sufficient to restore Na(+) to control levels in the highest exposure group.

Matched MeSH terms: Proton-Translocating ATPases/metabolism*
Effects of Marantodes pumilum (Kacip Fatimah) on vaginal pH and expression of vacoular ATPase and carbonic anhydrase in the vagina of sex-steroid deficient female rats

Giribabu N, Karim K, Salleh N

Phytomedicine, 2018 Oct 01;49:95-105.
PMID: 30217266 DOI: 10.1016/j.phymed.2018.05.018

BACKGROUND: In sex-steroid deficiency, increased in the pH of vaginal fluid is due to low estrogen levels.
HYPOTHESIS: Consumption of Marantodes pumilum leaves helps to ameliorate increased in vaginal fluid pH in sex-steroid deficient condition.
PURPOSE: To investigate changes in vaginal fluid pH and expression of proteins that participate in pH changes i.e vacoular (V)-ATPases and carbonic anhydrases (CA) in the vagina following M. pumilum leaves consumption.
METHODS: Ovariectomized adult female rats were treated orally with M. pumilum leaves extract (MPE) at 100, 250 and 500 mg/kg.b.w and estradiol at 0.2 µg/kg/b.w for 28 days. At the end of the treatment, vaginal fluid pH was measured in anesthetised rats by using micropH probe. Following sacrificed, levels of V-ATPase and CA proteins and mRNAs in the vagina were identified by Western blotting and real-time PCR, respectively. Protein distribution was visualized by immunohistochemistry.
RESULTS: Administration of MPE causes the pH of vaginal fluid to decrease and expression and distribution of vaginal V-ATPase A & B and CA II, III, IX, XII and XIII to increase.
CONCLUSIONS: The decrease in vaginal fluid pH following MPE treatment suggested that this herb has potential to be used to ameliorate vaginal fluid pH changes in sex-steroid deficient condition.

Matched MeSH terms: Vacuolar Proton-Translocating ATPases/metabolism*