Alessandra Mezzelani

Development of a Universal Microarray Based on the Ligation Detection Reaction and 16S rRNA Gene Polymorphism To Target Diversity of Cyanobacteria

ABSTRACT The cyanobacteria are photosynthetic prokaryotes of significant ecological and biotechnological interest, since they strongly contribute to primary production and are a rich source of bioactive compounds. In eutrophic fresh and brackish waters, their mass occurrences (water blooms) are often toxic and constitute a high potential risk for human health. Therefore, rapid and reliable identification of cyanobacterial species in complex environmental samples is important. Here we describe the development and validation of a microarray for the identification of cyanobacteria in aquatic environments. Our approach is based on the use of a ligation detection reaction coupled to a universal array. Probes were designed for detecting 19 cyanobacterial groups including Anabaena/Aphanizomenon, Calothrix, Cylindrospermopsis, Cylindrospermum, Gloeothece, halotolerants, Leptolyngbya, Palau Lyngbya, Microcystis, Nodularia, Nostoc, Planktothrix, Antarctic Phormidium, Prochlorococcus, Spirulina, Synechococcus, Synechocystis, Trichodesmium, and Woronichinia. These groups were identified based on an alignment of over 300 cyanobacterial 16S rRNA sequences. For validation of the microarrays, 95 samples (24 axenic strains from culture collections, 27 isolated strains, and 44 cloned fragments recovered from environmental samples) were tested. The results demonstrated a high discriminative power and sensitivity to 1 fmol of the PCR-amplified 16S rRNA gene. Accurate identification of target strains was also achieved with unbalanced mixes of PCR amplicons from different cyanobacteria and an environmental sample. Our universal array method shows great potential for rapid and reliable identification of cyanobacteria. It can be easily adapted to future development and could thus be applied both in research and environmental monitoring.

Environment, dysbiosis, immunity and sex-specific susceptibility: A translational hypothesis for regressive autism pathogenesis

Abstract Background Autism is an increasing neurodevelopmental disease that appears by 3 years of age, has genetic and/or environmental etiology, and often shows comorbid situations, such as gastrointestinal (GI) disorders. Autism has also a striking sex-bias, not fully genetically explainable. Objective Our goal was to explain how and in which predisposing conditions some compounds can impair neurodevelopment, why this occurs in the first years of age, and, primarily, why more in males than females. Methods We reviewed articles regarding the genetic and environmental etiology of autism and toxins effects on animal models selected from PubMed and databases about autism and toxicology. Discussion Our hypothesis proposes that in the first year of life, the decreasing of maternal immune protection and child immune-system immaturity create an immune vulnerability to infection diseases that, especially if treated with antibiotics, could facilitate dysbiosis and GI disorders. This condition triggers a vicious circle between immune system impairment and increasing dysbiosis that leads to leaky gut and neurochemical compounds and/or neurotoxic xenobiotics production and absorption. This alteration affects the ‘gut-brain axis’ communication that connects gut with central nervous system via immune system. Thus, metabolic pathways impaired in autistic children can be affected by genetic alterations or by environment–xenobiotics interference. In addition, in animal models many xenobiotics exert their neurotoxicity in a sex-dependent manner. Conclusions We integrate fragmented and multi-disciplinary information in a unique hypothesis and first disclose a possible environmental origin for the imbalance of male:female distribution of autism, reinforcing the idea that exogenous factors are related to the recent rise of this disease.

Osteogenic Differentiation of MSC through Calcium Signaling Activation: Transcriptomics and Functional Analysis.

The culture of progenitor mesenchymal stem cells (MSC) onto osteoconductive materials to induce a proper osteogenic differentiation and mineralized matrix regeneration represents a promising and widely diffused experimental approach for tissue-engineering (TE) applications in orthopaedics. Among modern biomaterials, calcium phosphates represent the best bone substitutes, due to their chemical features emulating the mineral phase of bone tissue. Although many studies on stem cells differentiation mechanisms have been performed involving calcium-based scaffolds, results often focus on highlighting production of in vitro bone matrix markers and in vivo tissue ingrowth, while information related to the biomolecular mechanisms involved in the early cellular calcium-mediated differentiation is not well elucidated yet. Genetic programs for osteogenesis have been just partially deciphered, and the description of the different molecules and pathways operative in these differentiations is far from complete, as well as the activity of calcium in this process. The present work aims to shed light on the involvement of extracellular calcium in MSC differentiation: a better understanding of the early stage osteogenic differentiation program of MSC seeded on calcium-based biomaterials is required in order to develop optimal strategies to promote osteogenesis through the use of new generation osteoconductive scaffolds. A wide spectrum of analysis has been performed on time-dependent series: gene expression profiles are obtained from samples (MSC seeded on calcium-based scaffolds), together with related microRNAs expression and in vivo functional validation. On this basis, and relying on literature knowledge, hypotheses are made on the biomolecular players activated by the biomaterial calcium-phosphate component. Interestingly, a key role of miR-138 was highlighted, whose inhibition markedly increases osteogenic differentiation in vitro and enhance ectopic bone formation in vivo. Moreover, there is evidence that Ca-P substrate triggers osteogenic differentiation through genes (SMAD and RAS family) that are typically regulated during dexamethasone (DEX) induced differentiation.

Role of mycotoxins in the pathobiology of autism: A first evidence

Objectives: Gene–environment interaction is an emerging hypothesis to expound not only the autism pathogenesis but also the increased incidence of neurodevelopmental disorders (such as autistic spectrum disorder, attention-deficit, hyperactivity disorder). Among xenobiotics, mycotoxins are worldwide contaminants of food that provoke toxicological effects, crucially resembling several symptoms associated with autism such as oxidative stress, intestinal permeability, and inflammation. Here, we focused on a group of mycotoxins to test their role in the manifestation of autism, try to explain their mechanism of action, and discuss possible preventive and therapeutic interventions. Methods: Autistic children (n = 52) and healthy children [n = 58 (31 siblings and 27 unrelated subjects)] were recruited and body fluids and clinical data collected. The diagnosis of autism was made according to DSM V criteria, then with GMDS 0-2, WPPSI, and ADOS. Ochratoxin A (OTA), gliotoxin, zearalenone, and sphingosine/sphinganine ratio were determined by LC analysis in sera and urines. Statistical analysis was performed by the Wilcoxon Rank Sum (Mann–Whitney) test and Spearman test. Results: By comparing the results of autistic patients with those of unrelated controls, a significant association was found for OTA levels in urines (P = 0.0002) and sera (P = 0.0017), and also comparing patients with siblings and unrelated controls together (P = 0.0081). Discussion: Our results are the first describing a possible role of OTA in the pathobiology of autism. Recalling the male prevalence of ASD (male/female = 4–5/1), it is noted that, in animal models, OTA exerts its neurotoxicity especially in males. Moreover, in vitro, OTA increases microRNA-132 that is dysregulated in autistic patients and involved in reciprocal regulation of the autism-related genes MeCP2 and PTEN. A personalized diet coupled with probiotic administration, especially OTA adsorbing Lactobacillus, could ameliorate autistic symptoms in OTA-positive patients.

Association Analysis of Noncoding Variants in Neuroligins 3 and 4X Genes with Autism Spectrum Disorder in an Italian Cohort

Since involved in synaptic transmission and located on X-chromosome, neuroligins 3 and 4X have been studied as good positional and functional candidate genes for autism spectrum disorder pathogenesis, although contradictory results have been reported. Here, we performed a case-control study to assess the association between noncoding genetic variants in NLGN3 and NLGN4X genes and autism, in an Italian cohort of 202 autistic children analyzed by high-resolution melting. The results were first compared with data from 379 European healthy controls (1000 Genomes Project) and then with those from 1061 Italian controls genotyped by Illumina single nucleotide polymorphism (SNP) array 1M-duo. Statistical evaluations were performed using Plink v1.07, with the Omnibus multiple loci approach. According to both the European and the Italian control groups, a 6-marker haplotype on NLGN4X (rs6638575(G), rs3810688(T), rs3810687(G), rs3810686(C), rs5916269(G), rs1882260(T)) was associated with autism (odd ratio = 3.58, p-value = 2.58 × 10−6 for the European controls; odds ratio = 2.42, p-value = 6.33 × 10−3 for the Italian controls). Furthermore, several haplotype blocks at 5-, 4-, 3-, and 2-, including the first 5, 4, 3, and 2 SNPs, respectively, showed a similar association with autism. We provide evidence that noncoding polymorphisms on NLGN4X may be associated to autism, suggesting the key role of NLGN4X in autism pathophysiology and in its male prevalence.

Study on the association among mycotoxins and other variables in children with autism

Environmental factors and genetic susceptibility are implicated in the increased risk of autism spectrum disorder (ASD). Mycotoxins are agricultural contaminants of fungal origin that represent real risk factors for human health and especially for children. Thus, the main hypothesis of this work is that the deterioration of the clinical manifestation of autism in children may result from the exposure to mycotoxins through the consumption of contaminated food. Within a cross-sectional study, a group of autistic children (n = 172) and a group of controls (n = 61) (siblings and non-parental) were recruited in North and South Italy. All children had blood and urine samples taken, for testing some mycotoxins by a LC–MS/MS validated method. Blood samples were also tested for assessing specific IgG against food and fungal antigens and cytokines. The analyses outputs highlighted statistically significant differences comparing mycotoxins levels between (i) children groups both in urine (deoxynivalenol and de-epoxydeoxynivalenol, p = 0.0141 and p = 0.0259, respectively) and serum (aflatoxin M1, ochratoxin A and fumonisin B1, p = 0.0072, p = 0.0141 and p = 0.0061, respectively); (ii) a group of selected fungal IgGs, and IgGs against wheat and gluten and (iii) cytokines. These results suggest the need for a deeper examination of the role that mycotoxins may have on the etiology of ASD.

Apolipoprotein E and Transferrin Genotyping by Ligation Detection Reaction and Universal Array

Genetic studies in Alzheimer disease (AD) have indicated that its etiology is multifactorial. The apolipoprotein E locus ( APOE ) is a known major susceptibility factor, and additional genetic loci have been associated with disease development (1)(2). Among many others, the transferrin gene ( TF ) has been suggested (3) as a candidate locus for AD because it is the major transport protein for iron, which itself is an important factor in free-radical generation. Oxidative stress and free-radical damage occur in AD, which justifies the interest in this protein. Previous studies have shown contrasting results regarding the influence of combinations of TF and APOE alleles (4)(5). We therefore designed a large-scale study of AD patients and controls to ascertain the relevance of TF as a risk factor for AD in conjunction with APOE . Here, we present the method that we have established for the simultaneous typing of the APOE and TF genes based on the ligation detection reaction (LDR)/universal array approach proposed by Gerry et al. (6). This method (Supplemental Fig. 1⇓ , A and B, accompanying the online version of this Technical Brief at http://www.clinchem.org/content/vol49/issue9/) is based on the PCR amplification of the regions including the polymorphic loci for TF and APOE genes. The resulting products are subjected to a multiplexed cycled ligation reaction that uses oligonucleotides designed to differentiate all possible alleles and that includes positive controls useful for normalizing the signals. This approach requires the design of a common LDR probe and two differentiating oligonucleotides for each polymorphic site. The common probe is phosphorylated on the 5′ end and contains a zip-code complement on its 3′-terminal …

Sex-specific neurotoxicity as possible cause of regressive autism

In India there is no present government policy to survey and evaluate adverse drug events (ADEs) / Pharmacovigilance programme in veterinary medicine. Therefore, essential information such as frequency, severity of treated animal ADRs and reliable data about frequent ADR-producing drugs remains unknown. The objective of the study is to assess and communicate risks and benefits in the market. Ultimately to educate the veterinarians and the stake holders on the safety and efficacy of veterinar y drugs and biologicals. To this purpose, a 12 month pilot study based on WHO r ecommendations was conducted to monitor ADRs in the livestock treated by field veterinarian in the state of T amil Nadu in India for frequently used drugs and common labeled signs. The present study warrants for the need for sustained veterinary pharmacovigilance programmers in livestock for timely ADR presenting drug reactions and drug safety improvement.O of the ways adopted by person for suicide is drowning. Criminals also hide their crime by throwing person in water after murder. To identify antemortem drowning or postmortem drowning, many postmortem findings are considered along with diatom test from bone marrow but many fallacies are there and false results are obtained. To overcome all these difficulties, the new method is proposed which is easy, less cumbersome, economical and confirmatory test; drowning by diatom test from tracheal fluid obtained by distilled water washed tracheal fluid.H susceptibility to xenobiotics, such as pharmaceutical drugs and genotoxicants, is highly variable. Much variability is conferred by polymorphisms in P450 genes and in housekeeping genes involved in basic DNA repair and metabolism. Since humans express multiple P450 genes, it is often difficult to discern which variant P450 gene confers xenobiotic susceptibility. In addition, epidemiological studies are often limited due to small patient populations. As a model xenobiotic, the potent liver carcinogen aflatoxin B1 (AFB1) was used, which is metabolically activated by CYP1A2 and CYP3A4. Since (budding yeast) do not express P450 genes that can metabolically activate AFB1, it was possible to phenotype CYP1A2 variants and 2) profile the yeast genome for resistance to AFB1. AFB1 activation based on DNA adducts, Rad51 polymorphisms, cell survival, and genome instability was characterized. Considering that 31% of (Saccharomyces cerevisiae) budding yeast genes are very similar to human genes, it was hypothesized that genes that confer AFB1 resistance in budding yeast will also confer resistance in humans. CYP1A2 was introduced into the diploid yeast collection containing ~5000 single gene deletions. Using state-of-the-art next generation DNA sequencing, ~500 genes were identified that confer AFB1 resistance, including genes that function in DNA repair, checkpoint response and adaptation, DNA damage tolerance, and oxidative stress. Future studies will be focused on identifying whether down-regulation of these human orthologs also confer AFB1 sensitivity. The author and his co-workers are now profiling resistance to additional P450-activated xenobiotics and phenotyping CYP1A1 polymorphisms.C kidney disease (CKD) is associated with a decreased expression and activity of several cytochrome P450 enzymes. This may result in drug-associated toxicity in CKD patients taking drugs that are metabolized by affected isozymes. The objective of this study was to determine the mechanism of hepatic drug metabolizing enzyme down-regulation in CKD. Hepatic CYP3A1, CYP3A2 and CYP2C11 mRNA expression were determined in rats with surgically induced CKD. Chromatin Immunoprecipitation (ChIP) was performed to determine nuclear receptor and epigenetic mediated differences in the promoter region of these enzymes. Hepatic CYP3A and CYP2C11 mRNA expression was significantly decreased in CKD rats compared to controls (P<0.05). RNA polymerase II binding to the CYP3A and CYP2C11 promoter regions was decreased in CKD rats (P<0.05). ChIP also revealed a decreased PXR binding to the CYP3A2 promoter in CKD rats (P<0.05). HNF4α binding to the CYP3A and CYP2C11 promoter regions was also decreased compared to controls (P<0.05). The decrease in PXR and HNF4α binding was concurrent with diminished histone 4 acetylation in the CYP3A2 promoter locus for nuclear receptor activation. The uremic toxin indoxyl sulfate also mediates a decrease in CYP3A expression. A novel mechanism of drug metabolizing enzyme regulation in CKD was demonstrated. The results show that decreased CYP3A and CYP2C11 mRNA expression is secondary to decreased PXR and HNF4α binding as a result of histone modulation in CKD. These data may partially explain why patients with CKD have a higher incidence of adverse medication events than patients with normal kidney function.oxic Epidermal Necrolysis (TEN) is a rare, life threatening skin reaction that is usually drug-induced. Anti-convulsants such as phenytoin, carbamazepine and phenobarbital and some antibiotics such as co-trimoxazole, quinolones and cephalosporins have been identified as common causes of drug-induced TEN. TEN has many serious complications including dehydration, increased energy expenditure and local or systemic infections. Many studies and case reports were published in the literature supporting the association between phenytoin and the development of TEN. Also many other factors can put the patient at higher risk for developing TEN while on phenytoin those include advanced age, malignancy and radiation exposure. More than one mechanism has been proposed to explain TEN pathophysiology. Hypersensitivity due to toxic metabolites of involved drugs is one theory. Genetic basis for drug-induced TEN has been proposed where there is inherited or acquired deficiency in phase 2 detoxification enzymes. Few studies have also indicated an association between HLA*1502 and phenytoin induced TEN. Family history of hypersensitivity reactions to medications should be documented and discussed with the patients. TEN’s treatment requires multidisciplinary approach to identify and withdraw the causative agent, controlling fluid and temperature homeostasis, preventing multi-organ damage, and treating systemic complications. Supportive therapy is the main strategy of treatment. Phenytoin-induced TEN carries a high mortality and morbidity rate, so accurate diagnosis and rapid treatment is essential to treat and prevent complications. It’s vital to make sure that the “right” patient is taking the “right” dose of the “right” medication. Medications history documentation with special drug allergy card indicating any history of drug reaction is recommended.T indiscriminate discharge of pollutants, such as heavy metals, surfactants and drugs, generated by anthropogenic activities is causing a tremendous hazard to both aquatic and terrestrial habitats and to human health. Therefore in recent years, the interest towards the toxic effects of these molecules on living organisms has increased. Plants have to cope with the detrimental effects of pollutants, e.g., typical symptoms of their toxicity are decrease of growth rate and chlorophylls, root detachment and leaf chlorosis and necrosis. The ability of plants to survive depends on the metabolic responsiveness of detoxification mechanisms. In fact, consequence to the toxicity is the elicitation of stress response that involved changes in the activity of enzymes, such as peroxidases, as well as enzymes of phenylpropanoid pathway, which is responsible for the synthesis of a diverse array of phenolic metabolites. These compounds are often induced by stress and serve specific roles in plant protection as well as structural components of the cell wall. The ability of plants to withstand the toxicity and accumulate pollutants is the base of environmental phytotechnologies, since numerous species can be an interesting tool for remediation of contaminated soils and waters. The mechanisms of plant defence against pollutant toxicity have been studied in floating macrophyte-based model systems. The results obtained will be discussed together with the future perspective of phytoremediation of polluted waters.Results: The total of 609 battery manufacturing male workers were consisting of 285(46.8%) workers with blood lead level (BLL) <40 μg/dl and 324 (53.2%) workers with BLL ≥40 μg/dl. High frequency (3, 4, 6, 8 Khz) hearing loss at hearing threshold above 25 dB in either ear was significantly more prevalent in workers with blood lead level ≥40 μg/dl (Adjusted Odds ratio=2.66, 95%CI: 1.86 3.80, P<0.001 and Adjusted Odds ratio=1.60, 95%CI: 1.13-2.27, P<0.008 for age and work duration respectively). Mean noise exposure level was 84.0 dBALeq.