Allicin extracted from Allium sativum shows potent anti-cancer and antioxidant properties in zebrafish.

Garlic (Allium sativum) is an important flavouring component in Indian cuisine. Allicin, a sulphur containing compound, is the most abundant component of garlic and has been widely studied for its antimicrobial and antioxidant properties. It is also known to play a role in the regulation of blood pressure and cholesterol levels. Despite the known health benefits associated with allicin, systematic studies on its anti-cancer properties using animal models are very limited. This study aimed to develop a simple method for the extraction of allicin from fresh garlic, study the stability of the extracted compound at various temperatures, and evaluate the antioxidant, anti-proliferative, pro-apoptotic and anti-angiogenic properties in zebrafish. A five-month stability study indicated that allicin remains significantly stable at temperatures 4 °C and below but shows extensive degradation if stored at room temperature. The in vivo studies in zebrafish using a combination of mutants and transgenic lines demonstrated the antioxidant, anti-proliferative, apoptotic and anti-angiogenic properties of allicin. The study highlights the importance of natural bioactive compounds as potential anti-cancer agents that can be studied further.

The current state of research and potential applications of insects for resource recovery and aquaculture feed.

Concerns about fishmeal use and its ecological footprints must be addressed for the aquaculture industry to move on as a sustainable food production sector. Through recent research outcomes, the insect-based meals in fish diets have promise and harnessed promises for commercial applications. In this midst, the efficiency of the selected insects in valorizing biological waste, as well as the nutritional profile of the harvested insects for use in fish diets, will be the driving forces behind such an approach. More extensive research has been published on the suitability of the waste substrate, the nutritional profiling of the meals, the level of substitution, the effects on growth, the immune physiology, and the flesh quality of the animals. Previously, there are only a few reviews available in insect protein applications in aqua feed that focused particularly on the nutritional quality and substitution levels. Considering the dearth of available work, the goal of this review is to provide a more comprehensive account of the resource recovery potential of insects and its derivatives, with a special emphasis on quality as determined by substrate used and processing techniques. Suggestions and policy implications for a sustainable approach to achieving a circular bio-economy of insect farming and its application in aquaculture are discussed for progression and advancement of the existing state of the art.

Screening for microplastics in drinking water and its toxicity profiling in zebrafish.

Microplastics (MPs) have emerged as a major environmental problem in freshwater and marine environments. The effects of these polymers on aquatic life are well studied; however, there is limited knowledge of MP-associated health hazards in humans. We estimated the presence of MPs in different brands of bottled water available in India using the Nile red (NR) staining method. The FTIR examination revealed the presence of polystyrene (PS), polyethylene (PE), and polyamide (PA) in the bottled water samples with PE being the most prevalent one. Zebrafish embryos exposed to different concentrations of fluorescent-tagged polyethylene microplastics (PE-MPs) (10-150 µm) showed accumulation patterns at different time points in various organs. The exposure to PE MPs induced a concentration-dependent ROS activity. The expression of first-line antioxidative defense marker genes were significantly downregulated in embryos exposed to varying concentrations of PE-MPs, suggesting concentration and time-dependent effects on zebrafish. The results of this study suggest that the potential negative consequences on human health could be due to the oxidative stress and time-dependent toxicity of MPs.

Investigation of anti-proliferative and anti-angiogenic properties of Parkia javanica bark and fruit extracts in zebrafish.

The use of herbal products as traditional medicines has been a practice in India for centuries. Due to high ethnic diversity, the pool of herbal medicines is enormous, and they are often preferred over modern medicines in certain parts of the country. Cancer is one of the major non-communicable diseases affecting people worldwide. Despite considerable research, cancer is a disease that is still not understood completely, and there have been constant efforts towards the identification of novel drugs or approaches in cancer management. Parkia javanica, an important medicinal plant and a rich source of flavonoids and terpenoids, is widely studied for its antioxidant and anti-inflammatory activities. Traditionally, the fruit and bark extracts of P. javanica find use as home remedy for dysentery and piles in NE India. Moreover, the fruits are consumed by the people of North-East (NE) India as vegetables, either in steamed or cooked form. In this study, crude extracts of P. javanica fruit and bark were obtained, the sub-lethal dose was determined and were then analyzed for anti-proliferative and anti-angiogenic properties using a battery of assays in zebrafish embryos. The sub-lethal concentration 50 (LC50) was found to be 28.66 mg/L and 346.66 mg/L for bark and fruit extract respectively, indicating a decreased toxicity of the fruit extract compared to that of the bark. The anti-proliferative and anti-angiogenic properties were more pronounced for the fruit extract compared to the bark extract. Although preliminary, the results of the study suggest that P. javanica fruits possess potent anti-angiogenic and anti-proliferative properties, which can be further studied for the isolation of active phytochemicals for use as therapeutic agents.

Wild-type and cancer-prone zebrafish exhibit distinct gut microbial diversity and differential anti-inflammatory response upon infection.

The study investigated the gut microbial diversity and the role of gut-associated microorganisms in modulating the immune responses in normal (wild-type) and TP53M214K (cancer-prone) zebrafish. Biochemical tests, genus/species-specific PCR, and 16S rDNA sequencing were performed to characterize the bacteria isolated from the gut of wild-type (WT) and cancer-prone zebrafish. Gut microbiome analysis revealed greater diversity but reduced bacterial load in wild-type zebrafish compared with cancer-prone zebrafish, which had lesser diversity but higher bacterial load. Interestingly, the gut in cancer-prone fish showed selective colonization by opportunistic pathogens. The bacterial isolates showed resistance to antibiotics such as tetracycline, nalidixic acid, and ciprofloxacin. Gnotobiotic zebrafish embryos were established, and mono-colonization with the isolated bacteria was done to examine the expression of anti-inflammatory genes using real-time PCR. Variable expression of IL10 and IL4 was observed in germ-free (GF) wild-type embryos when mono-colonized with Staphylococcus sciuri and Vibrio cholerae. In contrast, germ-free TP53 mutant embryos showed a consistent downregulation of both the anti-inflammatory genes. Thus, a better immune response in WT embryos against S. sciuri or V. cholerae infection than in cancer-prone fish was observed, suggesting that genetic predisposition could contribute to disabling the immune system against infection.

Loop-mediated isothermal amplification (LAMP): a sensitive molecular tool for detection of Staphylococcus aureus in meat and dairy product.

Staphylococcus aureus-mediated food poisoning is a primary concern worldwide. The presence of the organism in food is an indicative of poor sanitation during production, and it is essential to have efficient methods for detecting this pathogen. A novel molecular diagnostic technique called loop-mediated isothermal amplification (LAMP) serves as a rapid and sensitive detection method, which amplifies nucleic acids at isothermal conditions. In this study, a LAMP-based diagnostic assay was developed to detect Staphylococcus aureus (S. aureus) using two target genes femA and arcC. The optimum reaction temperature was found to be 65 °C and at 60 °C for femA and arcC genes, respectively. The developed assay specifically amplified DNA from S. aureus, not from other related bacterial species and compared to PCR, and a 100-fold higher sensitivity was observed. Furthermore, the LAMP assay could detect the pathogen from food samples mainly meat and dairy samples when analyzed in both intact and enriched conditions. Thirteen samples were found positive for S. aureus with LAMP showing a greater number of positive samples in comparison to PCR. This study established a highly sensitive and a rapid diagnostic procedure for the detection and surveillance of this major foodborne pathogen.

Phenotypic and Molecular Heterogeneity in Mandibulofacial Dysostoses: A Case Series From India.

OBJECTIVE: Facial dysostosis is a group of rare craniofacial congenital disabilities requiring multidisciplinary long-term care. This report presents the phenotypic and genotypic information from South India. DESIGN: The study is a case series. SETTING: This was an international collaborative study involving a tertiary craniofacial clinic and medical genetics unit. PATIENTS, PARTICIPANTS: The participants were 9 families with 17 affected individuals of facial dysostosis. INTERVENTION: Exome analysis focused on known genes associated with acrofacial and mandibulofacial syndromes. MAIN OUTCOME MEASURE: The outcome measure was to report phenotyptic and genetic heterogeneity in affected individuals. RESULTS: A Tessier cleft was seen in 7 (41%), lower eyelid coloboma in 12 (65%), ear anomalies in 10 (59%), uniolateral or bilateral aural atresia in 4 (24%), and deafness in 6 (35%). The facial gestalt of Treacher Collins syndrome (TCS) showed extensive phenotypic variations. Pathogenic variants in TCOF1 (Treacher Collins syndrome) were seen in six families, POLR1A (acrofacial dysostosis, Cincinnati type) and EFTUD2 (mandibulofacial dysostosis with microcephaly) in one each. One family (11.1%) had no detectable variation. Five out of six probands with Treacher Collins syndrome had other affected family members (83.3%), including a non-penetrant mother, identified after sequencing. CONCLUSION: Our report illustrates the molecular heterogeneity of mandibulofacial dysostosis in India.

TP53 codon 72 polymorphism and type 2 diabetes: a case-control study in South Indian population.

TP53 functions primarily as a tumor suppressor, controlling a myriad of signalling pathways that prevent a cell from undergoing malignant transformation. This tumor suppressive function requires an activation and stabilization of TP53 in response to cell stressors. However, besides its cancer-preventive functions, TP53 is also known to be involved in diverse cellular processes including metabolism, reproduction, stem cell renewal and development. Indeed, several lines of evidence strongly suggest that TP53 plays crucial role in diabetes. A number of studies have evaluated the association of genetic alterations (single nucleotide variations) in TP53 gene with the development of diabetes. However, the results have not been consistent. The aim of this study was to evaluate whether the C/G polymorphism at codon 72 (Pro72/Arg72), located in exon 4 of TP53, is associated with type 2 diabetes in South Indian population. A total of 74 type 2 diabetic patients and 54 non-diabetic subjects were screened. None of the three genotypes, namely C/C (Pro/Pro), C/G (Pro/Arg), and G/G (Arg/Arg) was found to be significantly associated with type 2 diabetes in our study group. The findings of this study indicate that TP53 codon 72 polymorphism is not associated with increased risk of type 2 diabetes in South Indian population. Further studies with a large cohort size would be necessary to corroborate the observations of this study. Nevertheless, this study represents the first genetic analysis of TP53 variants in South Indian type 2 diabetic patients.

Development of a Rapid and Low-Cost Method for the Extraction of Dermatophyte DNA.

Background: Polymerase chain reaction (PCR) is the most optimized method for the rapid detection and analysis of any environmental or clinically significant organism. While PCR amplification directly from samples has been shown effective for several bacteria and viruses, for filamentous fungus and yeast, extraction of genomic DNA is a must. The extraction of DNA from fungal cultures is often reported using user-friendly commercially available kits, which are designed to decrease the time, extensive manual work in extraction procedures but are often expensive. Dermatophytes pose an added drawback to efficient DNA extraction due to their poor recovery on culture media and slow growth rate. Aims and Objectives: In the present study, we developed and validated a method for effective genomic DNA extraction from dermatophytes. Materials and Methods: DNA yield from standard dermatophytes extracted from spore suspensions and mycelia mat by commercially available kits was compared. A modified method using lyticase buffer and phenol-chloroform extraction was developed. The yield obtained was compared with the existing methods (kit-based method and cetyl trimethyl ammonium bromide method). The yield and quality of the total genomic DNA were estimated spectrophotometrically and by successful PCR amplification of the ITS region. The results were validated using 21 clinical isolates from recalcitrant dermatophytosis. Results: Minimal fungal DNA was obtained from the spores compared to that obtained from mycelial mat. Commercially available kits yielded lower amounts of DNA compared to the CATB method. The modified method developed in this study yielded better quality and quantity of DNA. Conclusion: Of the three extraction methods evaluated, the developed method gave significantly higher total genomic DNA yield and better purity than the reference methods. In addition, the turnaround time for DNA extraction was reduced to half based on modifications in culture conditions.

Non-invasive detection of EGFR mutations by cell-free loop-mediated isothermal amplification (CF-LAMP).

Targeting epidermal growth factor receptor (EGFR) through tyrosine kinase inhibitors (TKI) is a successful therapeutic strategy in non-small cell lung cancer. However, the response to TKI therapy depends on specific activating and acquired mutations in the tyrosine kinase domain of the EGFR gene. Therefore, confirming the EGFR status of patients is crucial, not only for determining the eligibility, but also for monitoring the emergence of mutations in patients under TKI therapy. In this study, our aim was to develop a cost effective, yet sensitive, technique that allows the detection of therapeutically-relevant EGFR hotspot mutations at isothermal conditions in a non-invasive manner. Previously, we developed an allele-specific loop-mediated isothermal amplification (AS-LAMP) assay for screening germline and somatic de novo T790M EGFR mutation in lung cancer patients. In this study, we used cell free DNA as a template in AS-LAMP assay (CF-LAMP) for non-invasive detection of two hotspot EGFR mutations (T790M, and L858R) and compared its efficiency with ultrasensitive droplet digital PCR (ddPCR) assay. The results of CF-LAMP assay were consistent with those obtained in ddPCR assay, indicating the robustness of the method. CF-LAMP may serve as a valuable and cost-effective alternative for liquid biopsy techniques used in molecular diagnosis of non-small cell lung cancer.

Detection of epidermal growth factor receptor T790M mutation by allele-specific loop mediated isothermal amplification.

INTRODUCTION: Targeted therapy using specific inhibitors against tyrosine kinases (TKs) is a paradigm in non-small-cell lung cancer management. However, the success of TK inhibitor (TKI) therapy depends on certain activating or acquired mutations, which render sensitivity or resistance to TKIs in the patients. The acquisition of epidermal growth factor receptor (EGFR) T790M point mutation is the most common mechanism of resistance to TKI in non-small cell lung cancer. A number of molecular strategies are now available for molecular testing of non-small cell lung cancers. However, almost all of them are cost-intensive and laborious and require high-end advanced equipment. Thus, assays that are rapid, simple, and cost-effective, yet sensitive, are most ideal in clinical settings for screening such therapeutically relevant mutations. MATERIALS AND METHODS: Allele-specific loop-mediated isothermal amplification assay (AS-LAMP), which is a variant of the original LAMP assay, is a promising diagnostic technique for screening single-nucleotide polymorphisms. Using commercially available plasmid constructs as template DNA, AS-LAMP assay for EGFR T790M mutation was optimized with six different sets of reaction mixture containing varying concentrations of buffer and primers. The results of AS-LAMP assay were further validated by ultrasensitive droplet digital polymerase chain reaction. RESULTS: Only one of the six sets of reaction mixture could accurately distinguish between wild type and mutated DNA, indicating that the primers and buffer are the two most critical components that determine the accuracy of AS-LAMP. The optimized AS-LAMP assay was further used to screen germ line and somatic T790M mutations in non-small cell lung cancer using blood and tissue samples collected from patients. CONCLUSION: Development of an accurate and rapid diagnostic assay that can detect resistant mutations without the need for sequencing is highly useful for clinicians in deciding on the eligibility of patients for TKI therapy. Considering its several inherent advantages, AS-LAMP assay could become an effective molecular tool for screening baseline or acquired EGFR T790M mutations in non-small cell lung cancer patients.

Loop Mediated Isothermal Amplification: A Promising Tool for Screening Genetic Mutations.

Mutation screening is elemental for clinical diagnosis and in determining therapeutic strategies. Nucleic acid-based techniques are considered to be the most accurate tools in genetic diagnosis. One such technique is loop-mediated isothermal amplification (LAMP) assay, which has seen tremendous applications in recent years. The advantages of the assay lie in its rapidity, efficiency, sensitivity, and cost. It works in isothermal conditions and amplifies the target gene using DNA polymerases that have strand displacement activity. To date, the assay has been widely used in different fields of research, including pathogen detection, crop development, and disease diagnosis. However, despite the potential, its application in mutation screening has been minimal. This review highlights the LAMP assay and its variants that have been developed for screening single-nucleotide polymorphisms and gene translocations in cancer.

Mismatch amplification mutation assay-polymerase chain reaction: A method of detecting fluoroquinolone resistance mechanism in bacterial pathogens.

The mismatch amplification assay is a modified version of polymerase chain reaction (PCR) that permits specific amplification of gene sequences with single base pair change. The basis of the technique relies on primer designing. The single nucleotide mismatch at the 3' proximity of the reverse oligonucleotide primer makes Taq DNA polymerase unable to carry out extension process. Thus, the primers produce a PCR fragment in the wild type, whereas it is not possible to yield a product with a mutation at the site covered by the mismatch positions on the mismatch amplification mutation assay (MAMA) primer from any gene. The technique offers several advantages over other molecular methods, such as PCR-restriction fragment length polymorphism (RFLP) and oligonucleotide hybridization, which is routinely used in the detection of known point mutations. Since multiple point mutations in the quinolone resistance determining region play a major role in high-level fluoroquinolone resistance in Gram-negative bacteria, the MAMA-PCR technique is preferred for detecting these mutations over PCR-RFLP and sequencing technology.

Comparison of Mismatch Amplification Mutation Assay PCR and PCR-Restriction Fragment Length Polymorphism for Detection of Major Mutations in gyrA and parC of Escherichia coli Associated with Fluoroquinolone Resistance.

Fluoroquinolones are the drug of choice for most of the infections caused by Escherichia coli, and their indiscriminate use has resulted in increased selective pressure for antibiotic resistance. At present, sequencing is the only reliable and direct technique to detect mutations in the quinolone resistance determining region (QRDR). In this study, a rapid and reliable mismatch amplification mutation assay (MAMA) PCR to detect mutations in the QRDR was evaluated and compared to PCR-restriction fragment length polymorphism (PCR-RFLP). One hundred one clinical isolates of E. coli were subjected to MAMA-PCR and PCR-RFLP to detect QRDR mutations. Overall, 92 (91.08%) resistant isolates harbored a point mutation of S83L in gyrA. Double mutations in gyrA were also detected in 45 (44.55%) isolates. Similarly, 41 (40.59%) isolates possessed a point mutation at parC 80, and 25 (24.75%) isolates possessed a point mutation at parC 84. Additionally, MAMA-PCR-the first of its kind-was also standardized to detect mutations in regions gyrB 447 and parE 416, although no mutations were detected in these regions. The rapid and sensitive MAMA-PCR method evaluated in this study would be helpful in exploring the underlying mechanism of fluoroquinolone resistance to enhance control strategies.

Identification of a bacteria-like ferrochelatase in Strongyloides venezuelensis, an animal parasitic nematode.

Heme is an essential molecule for vast majority of organisms serving as a prosthetic group for various hemoproteins. Although most organisms synthesize heme from 5-aminolevulinic acid through a conserved heme biosynthetic pathway composed of seven consecutive enzymatic reactions, nematodes are known to be natural heme auxotrophs. The completely sequenced Caenorhabditis elegans genome, for example, lacks all seven genes for heme biosynthesis. However, genome/transcriptome sequencing of Strongyloides venezuelensis, an important model nematode species for studying human strongyloidiasis, indicated the presence of a gene for ferrochelatase (FeCH), which catalyzes the terminal step of heme biosynthesis, whereas the other six heme biosynthesis genes are apparently missing. Phylogenetic analyses indicated that nematode FeCH genes, including that of S. venezuelensis (SvFeCH) have a fundamentally different evolutionally origin from the FeCH genes of non-nematode metazoa. Although all non-nematode metazoan FeCH genes appear to be inherited vertically from an ancestral opisthokont, nematode FeCH may have been acquired from an alpha-proteobacterium, horizontally. The identified SvFeCH sequence was found to function as FeCH as expected based on both in vitro chelatase assays using recombinant SvFeCH and in vivo complementation experiments using an FeCH-deficient strain of Escherichia coli. Messenger RNA expression levels during the S. venezuelensis lifecycle were examined by real-time RT-PCR. SvFeCH mRNA was expressed at all the stages examined with a marked reduction at the infective third-stage larvae. Our study demonstrates the presence of a bacteria-like FeCH gene in the S. venezuelensis genome. It appeared that S. venezuelensis and some other animal parasitic nematodes reacquired the once-lost FeCH gene. Although the underlying evolutionary pressures that necessitated this reacquisition remain to be investigated, it is interesting that the presence of FeCH genes in the absence of other heme biosynthesis genes has been reported only for animal pathogens, and this finding may be related to nutritional availability in animal hosts.

Transcriptomic analysis of four developmental stages of Strongyloides venezuelensis.

Strongyloides venezuelensis is one of some 50 species of genus Strongyloides, obligate gastrointestinal parasites of vertebrates, responsible for strongyloidiasis in humans and other domestic/companion animals. Although S. venezuelensis has been widely used as a model species for studying human/animal strongyloidiasis, the sequence information for this species has been quite limited. To create a more comprehensive catalogue of expressed genes for identification of genes potentially involved in animal parasitism, we conducted a de novo sequencing analysis of the transcriptomes from four developmental stages of S. venezuelensis, using a Roche 454 GS FLX Titanium pyrosequencing platform. A total of 14,573 contigs were produced after de novo assemblies of over 2 million sequencing reads and formed a dataset "Vene454". BLAST homology search of Vene454 against proteome and transcriptome data from other animal-parasitic and non-animal-parasitic nematode species revealed several interesting genes, which may be potentially related to animal parasitism, including nicotinamide phosphoribosyltransferase and ferrochelatase. The Vene454 dataset analysis also enabled us to identify transcripts that are specifically enriched in each developmental stage. This work represents the first large-scale transcriptome analysis of S. venezuelensis and the first study to examine the transcriptome of the lung L3 developmental stage of any Strongyloides species. The results not only will serve as valuable resources for future functional genomics analyses to understand the molecular aspects of animal parasitism, but also will provide essential information for ongoing whole genome sequencing efforts in this species.

Immunostimulatory effects of natural human interferon-alpha (huIFN-alpha) on carps Cyprinus carpio L.

Human interferon-alpha (huIFN-alpha) is an important immunomodulatory substance used in the treatment and prevention of numerous infectious and immune-related diseases in animals. However, the immunostimulatory effects of huIFN-alpha in fish remain to be investigated. In the current study, the immune responses of the carp species Cyprinus carpio L. to treatment with huIFN-alpha were analyzed via measurement of superoxide anion production, phagocytic activity and the expression of cytokine genes including interleukin-1beta, tumor necrosis factor-alpha and interleukin 10. Low doses of huIFN-alpha were administered orally once a day for 3 days, and sampling was carried out at 1, 3 and 5 days post-treatment. Our results indicate that a low dose of huIFN-alpha significantly increased phagocytic activity and superoxide anion production in the carp kidney. The huIFN-alpha-treated fish also displayed a significant upregulation in cytokine gene expression. The current study demonstrates the stimulatory effects of huIFN-alpha on the carp immune system and highlights the immunomodulatory role of huIFN-alpha in fish.

Establishment of loop-mediated isothermal amplification method (LAMP) for the detection of Vibrio nigripulchritudo in shrimp.

LAMP is a novel method that amplifies DNA with high specificity and rapidity under isothermal conditions. In this study, using the LAMP method, a diagnostic protocol was developed for the detection of Vibrio nigripulchritudo in shrimps. Vibrio nigripulchritudo is associated with distinct shrimp diseases (vibriosis) and is considered one of the threatening pathogens in shrimp industry. After initial cloning and sequencing of the intergenic spacer region (ITS) between 16S and 23S rRNA genes of V. nigripulchritudo, a set of four primers - two inner and two outer - were designed for use in the LAMP reaction. Reaction time and temperature were optimized for 60 min at 63 degrees C, respectively. The detection limit of V. nigripulchritudo by LAMP was 10(2) CFU mL(-1) but PCR could detect up to 10(3) CFU mL(-1). The LAMP method could detect the presence of V. nigripulchritudo from heart, lymphoid organ, and muscle of experimentally infected shrimps with V. nigripulchritudo. This study established a highly sensitive and a rapid diagnostic procedure for detection of V. nigripulchritudo in shrimps. The method developed in this study could be very useful for routine shrimp disease diagnostics.