SNP Analysis of TLR4 Promoter and Its Transcriptional Factor Binding Profile in Relevance to Bovine Subclinical Mastitis.

Bovine mastitis is a complex infectious disease that develops in the mammary gland, predominantly caused by a bacterial infection of mammary tissue. Genetic variability of mastitis is well established and depends upon different quantitative trait loci (QTL) related to mastitis resistance or susceptibility. The susceptibility is often attributed to single-nucleotide polymorphisms (SNPs) in the variable cow breed genomes. Several global investigative attempts have resulted in studies mapping mastitis to the variations in the relevant genes. Reports have been attributed to dramatic genetic expression changes in Toll-Like Receptor 4 (TLR4) genes in mastitis-positive cows. However, the mechanism behind this variable genetic expression of TLR4 genes has been studied poorly. The present study aims to investigate SCM through various screening tests like somatic cell count (SCC), electric conductivity (EC), pH, and California mastitis test (CMT) in milk samples. This study also aims to investigate possible mechanisms behind this variable expression of TLR4 by comparative SNP evaluation and transcriptional factor profile mining. So that the important genetic mutations and effects thereof can be exploited in selecting specific breeds with higher mastitis resistance and milk yield. Seventy Holstein Frisian (HF) crossbred dairy cows were selected in the present study. The animals were screened based on various diagnostic tests (SCC, pH, EC, and CMT). Blood samples (5 mL) were collected for extraction of DNA followed by amplification of PPR1 and PPR2 of the promoter region and 5'UTR of the bovine TLR4 gene using specific primers. Sanger's enzymatic DNA sequencing technique sequenced the amplified PCR products. Further, the identification of SNPs was done through various bioinformatic tools used in this study. The findings of the present study revealed that CMT, EC, pH, and SCC could be used for the early detection of subclinical mastitis. In the present study, a significant increase in the EC, pH, and SCC in milk samples of animals affected with SCM was found in comparison to the healthy animals. The present study also revealed 16 SNPs falling in TLR4 promoter and 5' untranslated region (5'UTR) sequences in mastitis-positive genotypes compared to reference genomes. The study also investigates the potential transcriptional factor program deployed in response to variable mastitis development resistance. In the present study, the allelic and genotype frequencies of all SNP variants in the three regions viz., PPR1, PPR2, and 5'UTR, were the same indicating the absence of heterozygous condition at the respective loci. The present study has wide applicability for researchers developing mastitis-resistant breeding programs and the data generated may aid in the selection of better genetic breeds. The transcription factor binding profiles can serve as concrete leads about the studies on bovine mastitis at the molecular level and may also aid global research groups working on transcription factor (TF)-based molecular pathology of mastitis.

Biochemical and Computational Assessment of Acute Phase Proteins in Dairy Cows Affected with Subclinical Mastitis.

Subclinical mastitis (SCM) is a predominant form of mastitis wherein major visible signs of disease are absent. The present study aimed to determine acute phase proteins (APPs) like ferritin, C-reactive protein (CRP), and microalbumin (Malb) in 135 composite milk and serum samples of healthy (n = 25) and SCM (n = 110) cows. As bovine mastitis is an inflammatory disease, the present study also aimed at finding novel anti-inflammatory compounds from natural sources by repurposing approach using computational studies. The findings of the present study revealed substantial elevation (p < 0.001) in milk SCC and an increase in ferritin, CRP, and Malb (p < 0.001) in milk and sera of the SCM group as compared to healthy animals. Receiver operating characteristics of milk SCC, milk, and serum APPs unraveled statistically substantial alteration (p < 0.001). Further, SCC was correlated with milk APPs ferritin (r = 0.26 **, p < 0.002), CRP (r = 0.19 *, p < 0.02), and Malb (r = 0.21 *, p < 0.01). Additionally, milk SCC was correlated with serum ferritin (r = 0.28 **, p < 0.001), CRP (r = 0.16, p > 0.05), and Malb (r = 0.16, p > 0.05). The findings of molecular docking revealed that Chaetoglobosin U was the most effective molecule that showed the highest binding affinity (kcal/mol) of -10.1 and -8.5 against ferritin and albumin. The present study concluded that the estimation of cow-side tests, SCC, and APPs in milk/serum is suitable to detect SCM and screening herd community. Furthermore, Chaetoglobosin U could be developed as a promising anti-inflammatory inhibitor; however, further studies are required to validate these findings.

Milk-Compositional Study of Metabolites and Pathogens in the Milk of Bovine Animals Affected with Subclinical Mastitis.

Bovine milk is an important food component in the human diet due to its nutrient-rich metabolites. However, bovine subclinical mastitis alters the composition and quality of milk. In present study, California mastitis testing, somatic cell count, pH, and electrical conductivity were used as confirmatory tests to detect subclinical mastitis. The primary goal was to study metabolome and identify major pathogens in cows with subclinical mastitis. In this study, 29 metabolites were detected in milk using gas chromatography−mass spectrometry. Volatile acidic compounds, such as hexanoic acid, hexadecanoic acid, lauric acid, octanoic acid, n-decanoic acid, tricosanoic acid, tetradecanoic acid, and hypogeic acid were found in milk samples, and these impart good flavor to the milk. Metaboanalyst tool was used for metabolic pathway analysis and principal component estimation. In this study, EC and pH values in milk were significantly increased (p < 0.0001), whereas fat (p < 0.04) and protein (p < 0.0002) significantly decreased in animals with subclinical mastitis in comparison to healthy animals. Staphylococcus aureus was the predominant pathogen found (n = 54), followed by Escherichia coli (n = 30). Furthermore, antibiotic sensitivity revealed that Staphylococcus aureus was more sensitive to gentamicin (79.6%), whereas Escherichia coli showed more sensitivity to doxycycline hydrochloride (80%).

In Silico Tools for Analysis of Single-Nucleotide Polymorphisms in the Bovine Transferrin Gene.

Dairy cattle with a high milk yield are susceptible to many infectious diseases, such as mastitis. Subclinical mastitis (SCM) is the most prevalent form of mastitis that predominantly affects animal health, and causes adverse effects on the quality and quantity of milk. In dairy animals, subclinical mastitis often remains undetected, as no gross changes in udder characteristics are visible. In the present study, 135 Holstein Friesian dairy animals were selected and screened as healthy (n = 25) and mastitic (n = 110) based on diagnostic tests such as the California mastitis test, pH, electrical conductivity, and somatic cell count. In this study, the somatic cell count was used as a gold-standard test in differentiating subclinical mastitis animals from healthy ones. The present study was carried out to study polymorphisms in the bovine transferrin gene in cows (with subclinical mastitis and healthy). For the early detection of resistant/or susceptible animals, a useful marker could be provided by the detection of single-nucleotide polymorphisms (SNPs) in the transferrin gene, which are often associated with mammary innate immune response. The sequencing results revealed three nucleotide substitutions: two transversions (230 A > C, 231 C > A) and one transition (294 A > G) in susceptible cows as compared to disease-free subjects. The nucleotide variations at position 230 (GAC > GCA) and 231 (GAC > GCA) were nonsynonymous, and corresponded to an amino acid change from aspartic acid to alanine; whereas at position 294 (GAA > GAG), the mutation was synonymous. In the present study, many in silico tools were taken into consideration to determine the effect of SNPs on protein structure and function. The PROVEAN tool found the amino acid substitution to be neutral and deleterious. PolyPhen-2 revealed the amino acid variations at positions 320 and 321 to most likely be damaging; and at the 341 position, the variations were benign. The I-Mutant and MUpro tools found that the protein stability decreased for nonsynonymous variations. The SIFT tool revealed the protein function was likely to be affected in nonsynonymous variations, with no change in the case of synonymous ones. Phylogenetic analysis of the bovine transferrin gene revealed a close relation of the CA allele with the Bos taurus transferrin, while the G allele was closely related to a cross of Bos indicus × Bos taurus serotransferrins, followed by the Bison bison transferrin. The least relation was shown by both alleles to Capra hircus, Ovis aries, and Bubalus bubalis.

Zingerone (4-(four-hydroxy-3-methylphenyl) butane-two-1) modulates adjuvant-induced rheumatoid arthritis by regulating inflammatory cytokines and antioxidants.

OBJECTIVE: Ginger (Zingiber officinale Roscoe) is considered to be one of the most commonly consumed dietary condiments of the world. The present study was designed to explicate the protective role of zingerone; an active ingredient of ginger in complete Freund's adjuvant (FCA)-immunized arthritic rats. METHODS: 24 Wistar rats were divided into 4 groups with 6 rats each. Group I as control followed by group II, III and IV were treated with single intradermal injection of FCA (0.1 ml = 100 µg) to induce rheumatoid arthritis. Group III and IV were also administered with zingerone orally at 25 mg/kg b.w for 3 weeks at two different time points. RESULTS: Adjuvant-treated rats exhibited a significant increase in lipid peroxidation and a reduction in the enzymatic antioxidants such as SOD, catalase and GPx, in the liver and joint tissues. Moreover, FCA inoculation resulted in the increase in levels of NF-κB, TGF-ß, TNF-α, IL-1ß, IL-6 and Hs-CRP and a decrease in IL-10 levels. Zingerone significantly reduced the levels of NF-κB, TGF-ß, TNF-α, IL-1ß, IL-6 and Hs-CRP and markedly increased IL-10 levels. Levels of antioxidant enzymes were also restored by zingerone treatment. DISCUSSION: Oral administration of zingerone ameliorated inflammatory outburst and decreased oxidative stress, suggesting its role in the prevention of rheumatoid arthritis. Further mechanistic insights are necessary to study the exact mechanism involved.

Zingerone prevents lead-induced toxicity in liver and kidney tissues by regulating the oxidative damage in Wistar rats.

Among the heavy metal poisonings, lead is considered as a major toxic metal causing hematological, neurological, immunological, hepatic, and renal dysfunctions. Lead causes inhibition of ALAD leading to the ALA accumulation inside the cells. Lead also leads to disruption of the anti-oxidative enzyme system, organ function, and lipid membranes of the cell causing oxidative stress. Zingerone, a phenolic alkanone, is an active edible ingredient present in the ginger that possess varied pharmacological properties. The aim of our study was to evaluate the protective effect of zingerone in lead-induced toxicity in wistar rats. ALAD concentration was improved in kidney and liver tissues treated with zingerone. Protective effect of zingerone was observed in terms of significant improvement in kidney and liver histology, anti-oxidant enzyme activity (CAT, SOD, GPx, and GR), organ function parameters, lipid profile, and decreased level of LPO. Therefore, zingerone pretreatment can be a promising agent for alleviation of lead-induced oxidative damage in cells. PRACTICAL APPLICATIONS: Published reports have revealed that consumption of certain bioactive nutrients for example, flavonoids, mineral elements, and vitamins can offer defense from the environmental lead contamination. Zingerone is a strong anti-oxidant, with very less side effects and has exceptional property of scavenging free radicals, hence reducing the oxidative stresses. This fundamental property of zingerone can alone help in countering the heavy metal toxicity. Different groups have published reported numerous properties of zingerone but as per our understanding till date no study about alleviation of lead toxicity by zingerone in animal model has been undertaken. Hence, we conducted this research to explore the preventive effect of zingerone in lead induced kidney and liver toxicity. The outcome of our study shows potent anti-oxidant effect and ALAD modulatory property of zingerone which makes it suitable edible candidate for use in countering lead toxicity.

Zingerone (4-(4-hydroxy-3-methylphenyl) butan-2-one) protects against alloxan-induced diabetes via alleviation of oxidative stress and inflammation: Probable role of NF-kB activation.

Diabetes is considered as the most common metabolic disease affecting millions of people all around the world. Use of natural herbal medicines can be effective in treating diabetes. Zingerone (4-(4-hydroxy-3-methylphenyl) butan-2-one) a polyphenolic alkanone extracted from ginger has a broad spectrum of pharmacological properties and thus can be used as a promising candidate against various ailments. In the current study we aimed at demonstrating the protective effect of zingerone against diabetes mellitus and elucidating its possible mechanism. Five groups of animals (I-V) were made with ten animals each. Group I (control) was given normal saline orally. Group II (diabetic positive control) was given alloxan at the dose rate of 100 mg/kg bwt once. Group III and IV was given alloxan once at the dose rate of 100 mg/kg bwt. and received oral treatment of zingerone at a dose rate of 50 and 100 mg/kg bwt respectively daily for 21 days. Group V was given alloxan at the dose of 100 mg/kg bwt. and was treated with standard drug glibenclamide at the dose rate of 4.5 mg/kg bwt. daily for 21 days. According to our findings we confirmed that zingerone restrained the alloxan induced oxidative stress by increasing the activity of reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and reducing the peroxidative damage. We also confirmed that zingerone suppressed the level of redox sensitive transcription factor NFκB and downregulated other downstream inflammatory cytokines like interleukins (IL1-ß IL-2, IL-6) and tumor necrosis factor alpha (TNF-α). Moreover, the experimental findings suggested that zingerone improved the insulin levels. Taken together our results indicated that zingerone effectively ameliorated the diabetes induced complications which provide a strong theoretical basis for zingerone to be used clinically for treatment of diabetes.

Diagnostic utility of glycosyltransferase mRNA expression in gastric cancer.

OBJECTIVE/BACKGROUND: Posttranslational modification of proteins, including glycosylation, is known to differ between normal and tumor cells. Altered glycosyltransferase levels have been observed in tumor tissues and their role in tumor metastasis and invasion has been implicated. In this study the role of altered glycosyltransferase messenger RNA (mRNA) levels in serum of gastric cancer patients as early markers of gastric cancer was evaluated. METHODS: In this case control study the expression profile of ppGalNAc-T6, GlcNAcT-V, ST3Gal I, ST3 Gal IV, and ST6GalNAc-I in normal healthy control and gastric cancer patients was compared. Serum was isolated from blood samples of gastric cancer patients (n = 200) and controls (n = 200). Following RNA extraction, reverse transcription was carried out and transcript levels of glycosyltransferases were determined using real-time quantitative polymerase chain reaction and normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. The amount of target gene, normalized to an endogenous reference gene relative to calibrator was calculated by using ΔΔCT method. Transcript levels in the serum samples of gastric cancer patients were compared with those of controls; also the same was correlated within sex and different stages of disease. RESULTS: The mRNA expression of ppGalNAc-T6 and ST6GalNAc-I was significantly higher in serum samples of gastric cancer patients on comparison with controls (p = .008), however, there was no significant difference in mRNA expression of GlcNAcT-V, ST3Gal I, and ST3 Gal IV in serum samples of gastric cancer patients and controls (p = .097). In addition, no significant association of mRNA expression of these glycosyltransferases was found within sex and stages in this study. CONCLUSION: This study revealed the potential of ppGalNAc-T6 and ST6GalNAc-I mRNA transcript levels in serum as markers of gastric cancer. Further studies on the wider range of glycosyltransferases in various cancers are needed to establish signature mRNA batteries as minimally invasive markers of gastric cancer.

Antifibrotic effects of D-limonene (5(1-methyl-4-[1-methylethenyl]) cyclohexane) in CCl4 induced liver toxicity in Wistar rats.

This study was designed to assess the potential antifibrotic effect of D-Limonene-a component of volatile oils extracted from citrus plants. D-limonene is reported to have numerous therapeutic properties. CCl4 -intduced model of liver fibrosis in Wistar rats is most widely used model to study chemopreventive studies. CCl4 -intoxication significantly increased serum aminotransferases and total cholesterol these effects were prevented by cotreatment with D-Limonene. Also, CCl4 -intoxication caused depletion of glutathione and other antioxidant enzymes while D-Limonene preserved them within normal values. Hydroxyproline and malondialdehyde content was increased markedly by CCl4 treatment while D-Limonene prevented these alterations. Levels of TNF-α, TGF-ß, and α-SMA were also assessed; CCl4 increased the expression of α-SMA, NF-κB and other downstream inflammatory cascade while D-Limonene co-treatment inhibited them. Collectively these findings indicate that D-Limonene possesses potent antifibrotic effect which may be attributed to its antioxidant and anti-inflammatory properties.

Effect of Naringenin (A naturally occurring flavanone) Against Pilocarpine-induced Status Epilepticus and Oxidative Stress in Mice.

BACKGROUND: Epilepsy is a disorder of the central nervous system characterized by recurrent seizures. It is a very common disease in which approximately 30% of patients do not respond favourably to treatment with anticonvulsants. Oxidative stress is associated with neuronal damage arising from epileptic seizures. The present study investigated the effects of naringenin in pilocarpine-induced epilepsy in mice. Naringenin, one of the most frequently occurring flavanone in citrus fruits, was evaluated for its shielding effect against the pilocarpine induced behavioural, oxidative and histopathological alterations in rodent model of epilepsy. METHODOLOGY: Epilepsy was induced by giving pilocarpine (300mg/kg) and sodium valproate (300mg/kg) was given as standard anti-epileptic drug Pilocarpine was administered (300 mg /kg body weight) intraperitoneally to the mice on 15th day while naringenin was administered orally (20 and 40 mg/kg body weight) for 15 days prior to administration of pilocarpine. RESULTS: The intraperitoneal administration of pilocarpine enhanced lipid peroxidation, caused reduction in antioxidant enzymes, viz., catalase, superoxide dismutase and glutathione reductase. Treatment of mice orally with naringenin (20 mg/kg body weight and 40 mg/kg body weight) resulted in a significant decrease in lipid peroxidation. There was significant recovery of glutathione content and all the antioxidant enzymes studied. Also in case of behavioural parameters studied, naringenin showed decrease in seizure severity. All these changes were supported by histological observations, which revealed excellent improvement in neuronal damage. CONCLUSION: The higher dose of naringenin was more potent in our study and was comparable to the standard drug (sodium valproate) in effectiveness. SUMMARY: Naringenin ameliorated the development of ROS formation in hippocamus.Naringenin helped in recovery of antioxidant enzymes.Naringenin decreased seizure severity.Naringenin treatment reduced lipid peroxidation. Abbreviations used: 6-OHDA: 6-hydroxydopamine, AED: Anti epileptic drugs, AIDS: Acquired immune deficiency syndrome, ANOVA: Analysis of variance, ATP: Adenosine triphosphate, CA: Cornu ammonis, CAT: Catalase, DG: Dentate gyrus, EDTA: Ethylenediamine tetra acetic acid, GR: Glutathione reductase, GSH: Glutathione reduced, HCl: Hydrochloric acid, IL-1ß: Interleukin 1 beta, LPO: Lipid peroxidation, MDA: Malondialdehyde, NADPH: Nicotinamide adenine dinucleotide phosphate, PMS: post mitochondrial supernatant, SE: Status epilepticus, SEM: Standard error of the mean, SOD Superoxide dismutase, TBA: Thiobarbituric acid, TBARS: Thiobarbituric acid reactive substance, TLE: Temporal lobe epilepsy, TNF-α: Tumor necrosis factor alpha.

A Review on Pharmacological Properties of Zingerone (4-(4-Hydroxy-3-methoxyphenyl)-2-butanone).

Humans have been using natural products for medicinal use for ages. Natural products of therapeutic importance are compounds derived from plants, animals, or any microorganism. Ginger is also one of the most commonly used condiments and a natural drug in vogue. It is a traditional medicine, having some active ingredients used for the treatment of numerous diseases. During recent research on ginger, various ingredients like zingerone, shogaol, and paradol have been obtained from it. Zingerone (4-(4-hydroxy-3-methoxyphenyl)-2-butanone) is a nontoxic and inexpensive compound with varied pharmacological activities. It is the least pungent component of Zingiber officinale. Zingerone is absent in fresh ginger but cooking or heating transforms gingerol to zingerone. Zingerone closely related to vanillin from vanilla and eugenol from clove. Zingerone has potent anti-inflammatory, antidiabetic, antilipolytic, antidiarrhoeic, antispasmodic, and so forth properties. Besides, it displays the property of enhancing growth and immune stimulation. It behaves as appetite stimulant, anxiolytic, antithrombotic, radiation protective, and antimicrobial. Also, it inhibits the reactive nitrogen species which are important in causing Alzheimer's disease and many other disorders. This review is written to shed light on the various pharmacological properties of zingerone and its role in alleviating numerous human and animal diseases.