Mango Seed Kernel Fat as a Cocoa Butter Substitute Suitable for the Tropics.

Cocoa butter is a key ingredient in many chocolate products but its partial substitution with mango (Mangifera indica L.) seed kernel fat (MSKF) has the potential to reduce chocolate production costs and improve shelf-life. Here, MSKF was extracted from three cultivars of mango grown in Pakistan: Lal Badshah, Anwar Retual, and Chaunsa. Physicochemical and antioxidant properties of the MSKF samples were studied at 0, 30, and 60 days of storage at 30 °C, a temperature reflecting typical storage conditions in the tropics. Overall, the Lal Badshah MSKF had the most favorable physicochemical properties, including the highest DPPH antioxidant activity among the three cultivars. Thus, Lal Badshah MSKF was used to formulate cocoa butter substitute chocolate (CBSC), substituting the cocoa butter at 20 to 80 g/100 g. CBSC had a lower value for hardness (3.80 N) compared with the control chocolate (4.42 N). Color values L* , a* , and b* were not significantly affected by the different rates of substitution or by length of storage. Oxidative stability and antioxidant potential of CBSC increased with both higher substitution levels of MSKF and length of storage. The results suggest that MSKF can be utilized as a cocoa butter substitute at levels up to 60 g/100 g. This potential for substitution is particularly valuable for tropical regions where refrigerated storage may not be available or financially viable. PRACTICAL APPLICATION: Mango seed kernel fat (MSKF) has potential to be used as a cocoa butter substitute in confectionery products, particularly chocolate. The mango industry could utilize fat extraction from mango seeds, which are normally a waste product, for value adding.

Mequindox induces apoptosis, DNA damage, and carcinogenicity in Wistar rats.

Mequindox (MEQ) is a synthetic antibacterial agent. Recent studies showed that MEQ and its primary metabolites exhibit strong genotoxicity to mammalian cells, and MEQ induced carcinogenicity in mice. These findings suggest that chronic exposure to MEQ could lead to an increased risk of cancer later in life. In the present study, four groups of Wistar rats (55 rats/sex/group) were fed with diets containing MEQ (0, 25, 55, and 110 mg/kg) for 2 years. The results showed that the hematological system, liver, kidneys, and adrenal glands, as well as the developmental and reproductive systems, were the main targets for MEQ. Liver toxicity mediated by MEQ was associated with apoptosis and the nuclear factor κB (NF-κB) signaling pathway. In addition, MEQ increased the incidence of tumors in rats. Phosphorylated histone H2AX (γ-H2AX) is identified as a biomarker of cellular response to DNA double-strand breaks (DSB). Our data demonstrated that γ-H2AX expression was significantly increased in tumors. Thus, high levels of DSB might be responsible for carcinogenesis in rats, and further investigation is absolutely required to clarify the exact molecular mechanisms for carcinogenicity caused by MEQ in vivo.

The Involvement of the Cas9 Gene in Virulence of Campylobacter jejuni.

Campylobacter jejuni is considered as the leading cause of gastroenteritis all over the world. This bacterium has the CRISPR-cas9 system, which is used as a gene editing technique in different organisms. However, its role in bacterial virulence has just been discovered; that discovery, however, is just the tip of the iceberg. The purpose of this study is to find out the relationship between cas9 and virulence both phenotypically and genotypically in C. jejuni NCTC11168. Understanding both aspects of this relationship allows for a much deeper understanding of the mechanism of bacterial pathogenesis. The present study determined virulence in wild and mutant strains by observing biofilm formation, motility, adhesion and invasion, intracellular survivability, and cytotoxin production, followed by the transcriptomic analysis of both strains. The comparative gene expression profile of wild and mutant strains was determined on the basis of De-Seq transcriptomic analysis, which showed that the cas9 gene is involved in enhancing virulence. Differential gene expression analysis revealed that multiple pathways were involved in virulence, regulated by the CRISPR-cas9 system. Our findings help in understanding the potential role of cas9 in regulating the other virulence associated genes in C. jejuni NCTC11168. The findings of this study provide critical information about cas9's potential involvement in enhancing the virulence of C. jejuni, which is a major public health threat.

The Reproductive Toxicity of Mequindox in a Two-Generation Study in Wistar Rats.

Mequindox (MEQ), belonging to quinoxaline-di-N-oxides (QdNOs), has been extensively used as a synthetic antibacterial agent. To evaluate the reproductive toxicity of MEQ, different concentrations of MEQ were administered to Wistar rats by feeding diets containing 0, 25, 55, 110, and 275 mg/kg, respectively. Each group consisting of 25 males and 25 females (F0) was treated with different concentrations of MEQ for 12-week period time, prior to mating and during mating, gestation, parturition and lactation. At weaning, 25 males and 25 females of F1 generation weanlings per group were randomly selected as parents for the F2 generation. Selected F1 weanlings were exposed to the same diet and treatment as their parents. The number of live litter and indexes of mating and fertility were significantly decreased in the F1 and F2 generation at 110 and 275 mg/kg groups. Significant decrease in pup vitality during lactation was observed in F1 litter at 275 mg/kg group, in F2 litter at 55, 110, and 275 mg/kg groups. A downward trend in the body weights was observed in F1 pups at 55, 110, and 275 mg/kg MEQ groups, and in F2 pups at 110 and 275 mg/kg MEQ groups. The changed levels of ALT, AST, CREA, BUN, UA, Na, and K were noted in the serum of rats. The histopathologic examination showed that MEQ induced toxicity in the liver, kidney, adrenal, uterus and testis. The no-observed-adverse-effect level (NOAEL) for reproduction toxicity of MEQ was 25 mg/kg diet. The malformations and severe maternal toxicity of MEQ caused adverse effects on the conceptus and embryo, which result in fetal malformations and fetal deaths. In summary, the present study showed that MEQ induced maternal, embryo and reproductive toxicities as well as teratogenicity in rats.

Mequindox-Induced Kidney Toxicity Is Associated With Oxidative Stress and Apoptosis in the Mouse.

Mequindox (MEQ), belonging to quinoxaline-di-N-oxides (QdNOs), is a synthetic antimicrobial agent widely used in China. Previous studies found that the kidney was one of the main toxic target organs of the QdNOs. However, the mechanisms underlying the kidney toxicity caused by QdNOs in vivo still remains unclear. The present study aimed to explore the molecular mechanism of kidney toxicity in mice after chronic exposure to MEQ. MEQ led to the oxidative stress, apoptosis, and mitochondrial damage in the kidney of mice. Meanwhile, MEQ upregulated Bax/Bcl-2 ratio, disrupted mitochondrial permeability transition pores, caused cytochrome c release, and a cascade activation of caspase, eventually induced apoptosis. The oxidative stress mediated by MEQ might led to mitochondria damage and apoptosis in a mitochondrial-dependent apoptotic pathway. Furthermore, upregulation of the Nrf2-Keap1 signaling pathway was also observed. Our findings revealed that the oxidative stress, mitochondrial dysfunction, and the Nrf2-Keap1 signaling pathway were associated with the kidney apoptosis induced by MEQ in vivo.

Corrigendum: The Reproductive Toxicity of Mequindox in a Two-Generation Study in Wistar Rats.

[This corrects the article DOI: 10.3389/fphar.2018.00870.].

Microbial Shifts in the Intestinal Microbiota of Salmonella Infected Chickens in Response to Enrofloxacin.

Fluoroquinolones (FQs) are important antibiotics used for treatment of Salmonella infection in poultry in many countries. However, oral administration of fluoroquinolones may affect the composition and abundance of a number of bacterial taxa in the chicken intestine. Using 16S rRNA gene sequencing, the microbial shifts in the gut of Salmonella infected chickens in response to enrofloxacin treatments at different dosages (0, 0.1, 4, and 100 mg/kg b.w.) were quantitatively evaluated. The results showed that the shedding levels of Salmonella were significantly reduced in the high dosage group as demonstrated by both the culturing method and 16S rRNA sequencing method. The average values of diversity indices were higher in the control group than in the three medicated groups. Non-metric multidimensional scaling (NMDS) analysis results showed that the microbial community of high dosage group was clearly separated from the other three groups. In total, 25 genera were significantly enriched (including 6 abundant genera: Lactococcus, Bacillus, Burkholderia, Pseudomonas, Rhizobium, and Acinetobacter) and 23 genera were significantly reduced in the medicated groups than in the control group for the treatment period, but these bacterial taxa recovered to normal levels after therapy withdrawal. Additionally, 5 genera were significantly reduced in both treatment and withdrawal periods (e.g., Blautia and Anaerotruncus) and 23 genera (e.g., Enterobacter and Clostridium) were significantly decreased only in the withdrawal period, indicating that these genera might be the potential targets for the fluoroquinolones antimicrobial effects. Specially, Enterococcus was significantly reduced under high dosage of enrofloxacin treatment, while significantly enriched in the withdrawal period, which was presumably due to the resistance selection. Predicted microbial functions associated with genetic information processing were significantly decreased in the high dosage group. Overall, enrofloxacin at a dosage of 100 mg/kg b.w. significantly altered the microbial community membership and structure, and microbial functions in the chicken intestine during the medication. This study fully investigates the chicken intestinal microbiota in response to enrofloxacin treatment and identifies potential targets against which the fluoroquinolones may have potent antimicrobial effects. These results provide insights into the effects of the usage of enrofloxacin on chicken and will aid in the prudent and rational use of antibiotics in poultry industry.

Bacteria vs. Bacteriophages: Parallel Evolution of Immune Arsenals.

Bacteriophages are the most common entities on earth and represent a constant challenge to bacterial populations. To fend off bacteriophage infection, bacteria evolved immune systems to avert phage adsorption and block invader DNA entry. They developed restriction-modification systems and mechanisms to abort infection and interfere with virion assembly, as well as newly recognized clustered regularly interspaced short palindromic repeats (CRISPR). In response to bacterial immune systems, bacteriophages synchronously evolved resistance mechanisms, such as the anti-CRISPR systems to counterattack bacterial CRISPR-cas systems, in a continuing evolutionary arms race between virus and host. In turn, it is fundamental to the survival of the bacterial cell to evolve a system to combat bacteriophage immune strategies.

Pharmacokinetics and Metabolism of Cyadox and Its Main Metabolites in Beagle Dogs Following Oral, Intramuscular, and Intravenous Administration.

Cyadox (Cyx) is an antibacterial drug of the quinoxaline group that exerts markedly lower toxicity in animals, compared to its congeners. Here, the pharmacokinetics and metabolism of Cyx after oral (PO), intramuscular (IM), and intravenous (IV) routes of administration were studied to establish safety criteria for the clinical use of Cyx in animals. Six beagle dogs (3 males, 3 females) were administered Cyx through PO (40 mg kg(-1) b.w.), IM (10 mg kg(-1) b.w.), and IV (10 mg kg(-1) b.w.) routes with a washout period of 2 weeks in a crossover design. Highly sensitive high-performance liquid chromatography with ultraviolet detection (HPLC-UV) was employed for determination of Cyx and its main metabolites, 1, 4-bisdesoxycyadox (Cy1), cyadox-1-monoxide (Cy2), N-(quinoxaline-2-methyl)-cyanide acetyl hydrazine (Cy4), and quinoxaline-2-carboxylic acid (Cy6) in plasma, urine and feces of dogs. The oral bioavailability of Cyx was 4.75%, suggesting first-pass effect in dogs. The concentration vs. time profile in plasma after PO administration indicates that Cyx is rapidly dissociated into its metabolites and eliminated from plasma earlier, compared to its metabolites. The areas under the curve (AUC) of Cyx after PO, IM and IV administration were 1.22 h × µg mL(-1), 6.3 h × µg mL(-1), and 6.66 h × µg mL(-1), while mean resident times (MRT) were 7.32, 3.58 and 0.556 h, respectively. Total recovery of Cyx and its metabolites was >60% with each administration route. In feces, 48.83% drug was recovered after PO administration, while 18.15% and 17.11% after IM and IV injections, respectively, suggesting renal clearance as the major route of excretion with IM and IV administration and feces as the major route with PO delivery. Our comprehensive evaluation of Cyx has uncovered detailed information that should facilitate its judicious use in animals by improving understanding of its pharmacology.

Prevalence and distribution of soil-borne zoonotic pathogens in Lahore district of Pakistan.

A multidisciplinary, collaborative project was conducted to determine the prevalence and distribution of soil-borne zoonotic pathogens in Lahore district of Pakistan and ascertain its Public Health Significance. Using a grid-based sampling strategy, soil samples (n = 145) were collected from villages (n = 29, 5 samples/village) and examined for Bacillus anthracis, Burkholderia mallei/pseudomallei, Coxiella burnetii, Francisella tularensis, and Yersinia pestis using real time PCR assays. Chemical analysis of soil samples was also performed on these samples. The relationship between soil composition and absence or presence of the pathogen, and seven risk factors was evaluated. DNA of B. anthracis (CapB), B. mallei/pseudomallei (chromosomal gene), C. burnetii (IS1111, transposase gene), and F. tularensis (lipoprotein/outer membrane protein) was detected in 9.6, 1.4, 4.8, and 13.1% of soil samples, respectively. None of the samples were positive for protective antigen plasmid (PA) of B. anthracis and Y. pestis (plasminogen activating factor, pPla gene). The prevalence of B. anthracis (CapB) was found to be associated with organic matter, magnesium (Mg), copper (Cu), chromium (Cr), manganese (Mn), cobalt (Co), cadmium (Cd), sodium (Na), ferrous (Fe), calcium (Ca), and potassium (K). Phosphorous (P) was found to be associated with prevalence of F. tularensis while it were Mg, Co, Na, Fe, Ca, and K for C. burnetii. The odds of detecting DNA of F. tularensis were 2.7, 4.1, and 2.7 higher when soil sample sites were >1 km from animal markets, >500 m from vehicular traffic roads and animal density of < 1000 animals, respectively. While the odds of detecting DNA of C. burnetii was 32, 11.8, and 5.9 higher when soil sample sites were >500 m from vehicular traffic roads, presence of ground cover and animal density of < 1000 animals, respectively. In conclusion, the distribution pattern of the soil-borne pathogens in and around the areas of Lahore district puts both human and animal populations at a high risk of exposure. Further studies are needed to explore the genetic nature and molecular diversity of prevailing pathogens together with their seroconversion in animals and humans.

Complete genome sequence of a velogenic neurotropic avian paramyxovirus 1 isolated from peacocks (Pavo cristatus) in a wildlife park in Pakistan.

Avian paramyxovirus serotype 1 (APMV-1) was isolated from an acute and highly contagious outbreak in peacocks (Pavo cristatus) in a wildlife park in Pakistan. A velogenic neurotropic form of APMV-1 caused a 100% case fatality rate and killed 190 peacocks within a week. Biological and serological characterizations showed features of a velogenic strain of APMV-1, and these results were further confirmed by sequence analysis of the cleavage site in the fusion protein. The complete genome of one of the isolates was sequenced, and phylogenetic analysis was conducted. The analysis showed that this isolate belonged to genotype VII, specifically, to subgenotype VIIa, and clustered closely with isolates characterized from Indonesia in the 1990s. Interestingly, the isolate showed significant differences from previously characterized APMV-1 isolates from commercial and rural chickens in Pakistan. The work presented here is the first complete genome sequence of any APMV-1 isolate from wild birds in the region and therefore highlights the need for increased awareness and surveillance in such bird species.