Diverse microtubule-targeted anticancer agents kill cells by inducing chromosome missegregation on multipolar spindles.

Microtubule-targeted agents are commonly used for cancer treatment, though many patients do not benefit. Microtubule-targeted drugs were assumed to elicit anticancer activity via mitotic arrest because they cause cell death following mitotic arrest in cell culture. However, we recently demonstrated that intratumoral paclitaxel concentrations are insufficient to induce mitotic arrest and rather induce chromosomal instability (CIN) via multipolar mitotic spindles. Here, we show in metastatic breast cancer and relevant human cellular models that this mechanism is conserved among clinically useful microtubule poisons. While multipolar divisions typically produce inviable progeny, multipolar spindles can be focused into near-normal bipolar spindles at any stage of mitosis. Using a novel method to quantify the rate of CIN, we demonstrate that cell death positively correlates with net loss of DNA. Spindle focusing decreases CIN and causes resistance to diverse microtubule poisons, which can be counteracted by addition of a drug that increases CIN without affecting spindle polarity. These results demonstrate conserved mechanisms of action and resistance for diverse microtubule-targeted agents. Trial registration: clinicaltrials.gov, NCT03393741.

Greek Fire, Poison Arrows, and Scorpion Bombs: How Tumor Cells Defend Against the Siege Weapons of Cytotoxic T Lymphocytes.

CD8+ cytotoxic T lymphocytes (CTLs) are the main cellular effectors of the adaptive immune response against cancer cells, which in turn have evolved sophisticated cellular defense mechanisms to withstand CTL attack. Herein we provide a critical review of the pertinent literature on early and late attack/defense events taking place at the CTL/target cell lytic synapse. We examine the earliest steps of CTL-mediated cytotoxicity ("the poison arrows") elicited within seconds of CTL/target cell encounter, which face commensurately rapid synaptic repair mechanisms on the tumor cell side, providing the first formidable barrier to CTL attack. We examine how breach of this first defensive barrier unleashes the inextinguishable "Greek fire" in the form of granzymes whose broad cytotoxic potential is linked to activation of cell death executioners, injury of vital organelles, and destruction of intracellular homeostasis. Herein tumor cells deploy slower but no less sophisticated defensive mechanisms in the form of enhanced autophagy, increased reparative capacity, and dysregulation of cell death pathways. We discuss how the newly discovered supra-molecular attack particles (SMAPs, the "scorpion bombs"), seek to overcome the robust defensive mechanisms that confer tumor cell resistance. Finally, we discuss the implications of the aforementioned attack/defense mechanisms on the induction of regulated cell death (RCD), and how different contemporary RCD modalities (including apoptosis, pyroptosis, and ferroptosis) may have profound implications for immunotherapy. Thus, we propose that understanding and targeting multiple steps of the attack/defense process will be instrumental to enhance the efficacy of CTL anti-tumor activity and meet the outstanding challenges in clinical immunotherapy.

The mitochondrial poison carbonyl cyanide 3-chlorophenyl hydrazone (CCCP) induces aneugenic effects in primary human fibroblasts: a possible link between mitochondrial dysfunction and chromosomal loss.

An association between proper chromosome segregation and intact mitochondria has been extensively reported. This could be related to the effects on the progression of cell division of altered energy production, increased oxidative stress, and deregulated calcium homeostasis. However, evidence for a direct relationship is still lacking. The present study was aimed at investigating the possible effect of mitochondrial dysfunction on chromosomal instability as detected in primary human cells treated with the mitochondrial poison carbonyl cyanide 3-chlorophenyl hydrazone (CCCP). Chromosome instability was analyzed in anaphase and interphase cells to follow the fate of chromosome damage during the progression of mitosis and the subsequent cell cycle. Through the combination of cytogenetic approaches and molecular analyses, i.e. morphological cell analysis, formation and characterization of micronucleus content, Comet assay, and gene expression, it was demonstrated that the prevalent DNA damage associated with CCCP treatment was the induction of chromosome loss, while primary DNA damage was not detected. No alterations in the shape of anaphase cells were observed nor induction of multipolar spindles. The proper activation of mitotic checkpoint was maintained. A linear dose-response curve characterizing the CCCP effects suggested that multiple cellular targets could be affected by the CCCP-induced mitochondrial dysfunctions triggering aneuploidy. Conversely, a steep increase was induced by the positive control vinblastine, known to have tubulin as a unique target. In addition, the effect of CCCP on mitochondrial function was demonstrated by changes in mitochondrial DNA copy number and in the expression of genes involved in mitochondrial maintenance. Overall, these results indicate that the mitochondrial poison CCCP may induce aneugenic effects.

Fungal mycotoxin penisuloxazin A, a novel C-terminal Hsp90 inhibitor and characteristics of its analogues on Hsp90 function related to binding sites.

Hsp90 is a promising drug target for cancer therapy. However, toxicity and moderate effect are limitations of current inhibitors owing to broad protein degradation. The fungal mycotoxin penisuloxazin A (PNSA) belongs to a new epipolythiodiketopiperazines (ETPs) possessing a rare 3H-spiro[benzofuran-2,2'-piperazine] ring system. PNSA bound to cysteine residues C572/C598 of CT-Hsp90 with disulfide bonds and inhibits Hsp90 activity, resulting in apoptosis and growth inhibition of HCT116 cells in vitro and in vivo. We identified that analogues PEN-A and HDN-1 bound to C572/C597 and C572 of CT-Hsp90α respectively, with binding pattern very similar to PNSA. These ETPs exhibited different effects on ATPase activity, dimerization formation and selectivity on client protein of Hsp90, indicating client recognition of Hsp90 can be exactly regulated by different sites of Hsp90. Our findings not only offer new chemotypes for anticancer drug development, but also help to better understand biological function of Hsp90 for exploring inhibitor with some client protein bias.

Fitness Cost of Aflatoxin Production in Aspergillus flavus When Competing with Soil Microbes Could Maintain Balancing Selection.

Selective forces that maintain the polymorphism for aflatoxigenic and nonaflatoxigenic individuals of Aspergillus flavus are largely unknown. As soils are widely considered the natural habitat of A. flavus, we hypothesized that aflatoxin production would confer a fitness advantage in the soil environment. To test this hypothesis, we used A. flavus DNA quantified by quantitative PCR (qPCR) as a proxy for fitness of aflatoxigenic and nonaflatoxigenic field isolates grown in soil microcosms. Contrary to predictions, aflatoxigenic isolates had significantly lower fitness than did nonaflatoxigenic isolates in natural soils across three temperatures (25, 37, and 42°C). The addition of aflatoxin to soils (500 ng/g) had no effect on the growth of A. flavus Amplicon sequencing showed that neither the aflatoxin-producing ability of the fungus nor the addition of aflatoxin had a significant effect on the composition of fungal or bacterial communities in soil. We argue that the fitness disadvantage of aflatoxigenic isolates is most likely explained by the metabolic cost of producing aflatoxin. Coupled with a previous report of a selective advantage of aflatoxin production in the presence of some insects, our findings give an ecological explanation for balancing selection resulting in persistent polymorphisms in aflatoxin production.IMPORTANCE Aflatoxin, produced by the fungus Aspergillus flavus, is an extremely potent hepatotoxin that causes acute toxicosis and cancer, and it incurs hundreds of millions of dollars annually in agricultural losses. Despite the importance of this toxin to humans, it has remained unclear what the fungus gains by producing aflatoxin. In fact, not all strains of A. flavus produce aflatoxin. Previous work has shown an advantage to producing aflatoxin in the presence of some insects. Our current work demonstrates the first evidence of a disadvantage to A. flavus in producing aflatoxin when competing with soil microbes. Together, these opposing evolutionary forces could explain the persistence of both aflatoxigenic and nonaflatoxigenic strains through evolutionary time.

Aflatoxin B1 Induces Reactive Oxygen Species-Mediated Autophagy and Extracellular Trap Formation in Macrophages.

Aflatoxins are a group of highly toxic mycotoxins with high carcinogenicity that are commonly found in foods. Aflatoxin B1 (AFB1) is the most toxic member of the aflatoxin family. A recent study reported that AFB1 can induce autophagy, but whether AFB1 can induce extracellular traps (ETs) and the relationships among innate immune responses, reactive oxygen species (ROS), and autophagy and the ETs induced by AFB1 remain unknown. Here, we demonstrated that AFB1 induced a complete autophagic process in macrophages (MΦ) (THP-1 cells and RAW264.7 cells). In addition, AFB1 induced the generation of MΦ ETs (METs) in a dose-dependent manner. In particular, the formation of METs significantly reduced the AFB1 content. Further analysis using specific inhibitors showed that the inhibition of either autophagy or ROS prevented MET formation caused by AFB1, indicating that autophagy and ROS were required for AFB1-induced MET formation. The inhibition of ROS prevented autophagy, indicating that ROS generation occurred upstream of AFB1-induced autophagy. Taken together, these data suggest that AFB1 induces ROS-mediated autophagy and ETs formation and an M1 phenotype in MΦ.

In vitro activation of dibromoacetonitrile to cyanide by myeloperoxidase.

Dibromoacetonitrile (DBAN) is a disinfection by-product classified as a potential human and animal carcinogen. This study aimed at investigating the ability of myeloperoxidase (MPO) to oxidize DBAN to cyanide (CN-) in vitro Detection of CN- served as a marker for the possible generation of free radical intermediates implicated in DBAN-induced toxicity. Optimum conditions for the oxidation of DBAN to CN- were characterized with respect to pH, temperature, and time of incubation as well as DBAN, MPO, potassium chloride, and hydrogen peroxide (H2O2) concentrations in incubation mixtures. Maximum reaction velocity and Michaelis-Menten constant were assessed. Addition of sodium hypochlorite to the reaction mixtures significantly enhanced the rate of the reaction. Addition of the MPO inhibitors, sodium azide, 4-amino benzoic acid hydrazine, or indomethacin to the reaction mixtures significantly decreased the rate of DBAN oxidation. Inclusion of the antioxidant enzyme superoxide dismutase in the incubation mixtures significantly decreased the rate of reaction. Inclusion of the sulfhydryl compounds as reduced glutathione, N-acetylcysteine, d-penicillamine, or l-cysteine enhanced the rate of DBAN oxidation. These results demonstrate the ability of MPO/H2O2/chloride ion system to oxidize DBAN to CN- and provide insight for the elucidation of DBAN chronic toxicity.

Continued implication of the banned pesticides carbofuran and aldicarb in the poisoning of domestic and wild animals of the Canary Islands (Spain).

Although nowadays the intentional poisoning of domestic and wild animals is a crime in EU, in the past the poison was used in rural areas of a number of European countries to kill animals that were considered harmful for human activities. In Spain evidences indicate that intentional poisonings continue to occur throughout the entire country nowadays. This situation seems to be of particular concern in the Canary Islands (Spain), where this study was performed. Our results confirmed that 225 animals were poisoned by pesticides over the study period (32 months; 2010-2013). The intentionality of the poisoning was confirmed in 117 cases. It has to be highlighted that the other 108 animals also died by pesticide poisoning, although the intentionality was only suspected. This incidence is currently the highest reported in any region from European Union. The pesticides carbofuran, bromadiolone, brodifacoum and aldicarb were the most frequently detected involved. Among the affected species, it has to be highlighted that endangered species are frequently affected in poisoning incidents. Notably, chemicals banned in the EU (carbofuran and aldicarb) were identified in approximately 75% of cases, and in almost 100% of baits, which suggests that these pesticides are still available to the population. Several circumstances may explain these results. Firstly, little control over the sale and possession of pesticide products, and the potential existence of an illegal market of pesticides banned in the European Union in the neighbouring African continent. In addition, the limited awareness of the population about the dangerousness of these compounds, for the environment, animals, or even people, make the situation very worrying in these islands. Stronger regulations, control of legal and illegal pesticide use, development of educational programs and legal action in poisoning incidents are needed to decrease the impact of pesticide misuse on wildlife and domestic animals.

Histological description of the skin glands of Phyllobates bicolor (Anura: Dendrobatidae) using three staining techniques / Descripción histológica de las glándulas cutáneas de Phyllobates bicolor (Anura: Dendrobatidae) usando tres técnicas de tinción

The mechanisms to obtain and store skin toxins in frogs in of the family Dendrobatidae are not completely understood. In order to contribute to understand how toxins are stored, we provide a histological description of the cutaneous glands of the species Phyllobates bicolor. The skin of two adult frogs was examined through three histological staining techniques (hematoxilin-eosin, PAS and Masson Trichrome) using conventional optic microscopy. The skin of Phyllobates bicolor contains two types of exocrine glands: mucous and serous, which empty their products to the epidermal surface through an intra-epithelial duct that leads to a stoma. The mucous and serous glands and the intercalated ducts are surrounded by a discontinuous sheath of myoepithelial cells, which colapse the lumen of the acinus and the lumen of ducts and facilitate the secretion and release of their content. The serous glands have a polarized syncytium of tall cuboidal or columnar epithelial cells. Both glands have a mixed secretion, thus, the contents of mucous glands tend to be neutral and basophilic, while the contents of the serous glands are basophilic and acidophilic.

A la fecha no existe mayor información con respecto a los mecanismos para obtener y almacenar las toxinas cutáneas de ranas de la familia Dendrobatidae. Con el fin de contribuir y entender cómo son almacenadas estas toxinas, realizamos una descripción histológica de las glándulas cutáneas de la especie Phyllobates bicolor. La piel de dos ranas adultas se examinó mediante tres técnicas de tinción histológica (hematoxilina-eosina, PAS y tricrómico de Masson) mediante microscopía óptica convencional. La piel de P. bicolor contiene dos tipos de glándulas exocrinas: mucosas y serosas, que vierten sus productos a la superficie epidérmica a través de un conducto intra-epitelial que conduce a un estoma. Las glándulas mucosas, serosas y los conductos intercalados están rodeados por una funda discontinua de células mioepiteliales, las que colapsan el lumen de los acinos y conductos, facilitando la secreción y liberación de su contenido. Las glándulas serosas tienen un sincitio polarizado de células epiteliales columnares cúbicas. Ambas glándulas tienen una secreción mixta, por lo tanto, los contenidos de las glándulas mucosas tienden a ser neutral y basófilas, mientras que los contenidos de las glándulas serosas son basófilas y acidófilas.

XRCC1 Arg194Trp and Arg399Gln polymorphisms and arsenic methylation capacity are associated with urothelial carcinoma.

The association between DNA repair gene polymorphisms and bladder cancer has been widely studied. However, few studies have examined the correlation between urothelial carcinoma (UC) and arsenic or its metabolites. The aim of this study was to examine the association between polymorphisms of the DNA repair genes, XRCC1 Arg194Trp, XRCC1 Arg399Gln, XRCC3 Thr241Met, and XPD Lys751Gln, with urinary arsenic profiles and UC. To this end, we conducted a hospital-based case-control study with 324 UC patients and 647 age- and gender-matched non-cancer controls. Genomic DNA was used to examine the genotype of XRCC1 Arg194Trp, XRCC1 Arg399Gln, XRCC3 Thr241Met, and XPD Lys751Gln by PCR-restriction fragment length polymorphism analysis (PCR-RFLP). Urinary arsenic profiles were measured by high performance liquid chromatography (HPLC) linked with hydride generator and atomic absorption spectrometry. The XRCC1 399 Gln/Gln and 194 Arg/Trp and Trp/Trp genotypes were significantly related to UC, and the odds ratio (OR) and 95% confidence interval (95%CI) were 1.68 (1.03-2.75) and 0.66 (0.48-0.90), respectively. Participants with higher total urinary arsenic levels, a higher percentage of inorganic arsenic (InAs%) and a lower percentage of dimethylarsinic acid (DMA%) had a higher OR of UC. Participants carrying XRCC1 risk diplotypes G-C/G-C, A-C/A-C, and A-T/G-T, and who had higher total arsenic levels, higher InAs%, or lower DMA% compared to those with other XRCC1 diplotypes had a higher OR of UC. Our results suggest that the XRCC1 399 Gln/Gln and 194 Arg/Arg DNA repair genes play an important role in poor arsenic methylation capacity, thereby increasing the risk of UC in non-obvious arsenic exposure areas.

Occupational exposure to Aspergillus and aflatoxins among food-grain workers in India.

BACKGROUND: Aflatoxins are a metabolite of Aspergillus molds and are widespread in the natural environment. Workers who handle food grains are at increased risk of exposure to aflatoxins and subsequently certain respiratory conditions. In India, more than half of the employed population is engaged in some type of agricultural work, yet little known about the respiratory problems as a result of exposure to aflatoxins among workers who handle food grains in India. OBJECTIVES: The aim of this study was to determine the risk of occupational exposure to aflatoxins in food-grain workers compared to workers who are not occupationally exposed to food grains. METHODS: Bronchoalveolar lavage (BAL) and serum samples from 46 food-grain workers and 44 non-food-grain workers were analyzed for the presence of aflatoxins. Microscopy and culture of BAL samples were performed to detect Aspergillus species. RESULTS: Aflatoxins were detected in 32·6% of the food-grain workers and 9·1% of non food grain workers (P<0·01). A significant difference was also found in BAL culture for Aspergillus (P<0·01) between the two groups. About 47·8% of the food-grain workers and 11·4% of non-food-grain workers had chronic respiratory symptoms. CONCLUSION: Occupational exposure to aflatoxins in food-grain workers was found to be associated with the increased presence of respiratory symptoms.

Context-dependent antagonism between Akt inhibitors and topoisomerase poisons.

Signaling through the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, which is aberrantly activated in >50% of carcinomas, inhibits apoptosis and contributes to drug resistance. Accordingly, several Akt inhibitors are currently undergoing preclinical or early clinical testing. To examine the effect of Akt inhibition on the activity of multiple widely used classes of antineoplastic agents, human cancer cell lines were treated with the Akt inhibitor A-443654 [(2S)-1-(1H-indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxypropan-2-amine; ATP-competitive] or MK-2206 (8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3-one;dihydrochloride; allosteric inhibitor) or with small interfering RNA (siRNA) targeting phosphoinositide-dependent kinase 1 (PDK1) along with cisplatin, melphalan, camptothecin, or etoposide and assayed for colony formation. Surprisingly different results were observed when Akt inhibitors were combined with different drugs. Synergistic effects were observed in multiple cell lines independent of PI3K pathway status when A-443654 or MK-2206 was combined with the DNA cross-linking agents cisplatin or melphalan. In contrast, effects of the Akt inhibitors in combination with camptothecin or etoposide were more complicated. In HCT116 and DLD1 cells, which harbor activating PI3KCA mutations, A-443654 over a broad concentration range enhanced the effects of camptothecin or etoposide. In contrast, in cell lines lacking activating PI3KCA mutations, partial inhibition of Akt signaling synergized with camptothecin or etoposide, but higher A-443654 or MK-2206 concentrations (>80% inhibition of Akt signaling) or PDK1 siRNA antagonized the topoisomerase poisons by diminishing DNA synthesis, a process that contributes to effective DNA damage and killing by these agents. These results indicate that the effects of combining inhibitors of the PI3K/Akt pathway with certain classes of chemotherapeutic agents might be more complicated than previously recognized.

Seasonal and gestation stage associated differences in aflatoxin exposure in pregnant Gambian women.

OBJECTIVE: Aflatoxin is known to cross the placental barrier and exposures in utero could influence genomic programming, foetal growth and development, resulting in long-term health effects. We aimed to determine aflatoxin exposure in Gambian women at two stages of pregnancy and during the rainy and dry seasons. METHODS: We examined aflatoxin exposure in pregnant Gambian women at early (<16 weeks) and later (16 weeks onward) stages of pregnancy and at different times of the year, during the rainy (June to October 2009) or dry (November to May 2010) season, using aflatoxin-albumin adducts (AF-alb). RESULTS: Mean AF-alb was higher during the dry season than in the rainy season, in both early and later pregnancy although the difference was strongest in later pregnancy. There was a modest increase in AF-alb in later than early pregnancy (geometric mean 41.8 vs. 34.5 pg/mg, P < 0.05), but this was restricted to the dry season when exposures were generally higher. CONCLUSIONS: The study confirmed that Gambian pregnant women were exposed to aflatoxin throughout the pregnancy, with higher levels in the dry season. There was some evidence in the dry season that women in later pregnancy had higher AF-alb levels than those in earlier pregnancy. Further research on the effects of exposure to this potent mutagen and carcinogen throughout pregnancy, including the epigenetic modification of foetal gene expression and impact on pre- and post-natal growth and development, are merited.

A new headspace gas chromatographic method for the determination of methanol content in paper materials used for food and drink packaging.

This study reports on a method for determination of methanol in paper products by headspace gas chromatography (HS-GC). The method is based on the hydrolysis of the pulp or paper matrix, using a phosphoric acid solution (42.5%) as the medium at 120 °C in 5 h (excluding air contact) in order to release matrix-entrapped methanol, which is then determined by HS-GC. Data show that, under the given conditions of hydrolysis, no methanol was formed from the methoxyl groups in the material. Reproducibility tests of the method generated a relative standard deviation of <3.5%, with recovery in the range of 93.4-102%. The present method is reliable, accurate, and suitable for use in batch testing of the methanol content in paper-related materials. The method can play an important role in addressing food safety concerns that may be raised regarding the use of paper materials in food and beverage packaging.

Spatial distribution of ciguateric fish in the Republic of Kiribati.

Ciguatera is food poisoning caused by human consumption of reef fish contaminated with ciguatoxins (CTXs). The expanding international trade of tropical fish species from ciguatera-endemic regions has resulted in increased global incidence of ciguatera, and more than 50000 people are estimated to suffer from ciguatera each year worldwide. The Republic of Kiribati is located in the Pacific Ocean; two of its islands, Marakei and Tarawa, have been suggested as high-risk areas for ciguatera. The toxicities of coral reef fish collected from these islands, including herbivorous, omnivorous and carnivorous fish (24% [n=41], 8% [n=13] and 68% [n=117], respectively), were analyzed using the mouse neuroblastoma assay (MNA) after CTX extraction. The MNA results indicated that 156 fish specimens, or 91% of the fish samples, were ciguatoxic (CTX levels >0.01 ng g(-1)). Groupers and moray eels were generally more toxic by an order of magnitude than other fish species. All of the collected individuals of eight species (n=3-19) were toxic. Toxicity varied within species and among locations by up to 10000-fold. Cephalapholis argus and Gymnothorax spp. collected from Tarawa Island were significantly less toxic than those from Marakei Island, although all individuals were toxic based on the 0.01 ng g(-1) threshold. CTX concentrations in the livers of individuals of two moray eel species (Gymnothorax spp., n=6) were nine times greater than those in muscle, and toxicity in liver and muscle showed a strong positive correlation with body weight. The present study provides quantitative information on the ciguatoxicity and distribution of toxicity in fish for use in fisheries management and public health.

Aflatoxin B1--a potential endocrine disruptor--up-regulates CYP19A1 in JEG-3 cells.

Previous studies have indicated that aromatase (CYP19A1) is involved in the metabolism of aflatoxin B1 (AFB1). We hypothesized that exposure to AFB1 contaminated food during pregnancy could disrupt the normal production of steroid hormones in placenta. We examined the capability of AFB1 exposure to disrupt CYP19A1 expression as a putative endocrine disrupter, and to investigate the metabolism of AFB1 by CYP19A1. JEG-3 cells, as model for placental cells, were exposed alone and in combination to AFB1 and estrogen receptor ligands for 24-96 h. AFB1 (0.3-1.0 µM) induced the expression of CYP19A1 by 163%-339% compared to control at the 96 h time point, although no induction was observed at 24 h. AFB1 concentrations higher than 1 µM were cytotoxic to JEG-3 cells, and the cytotoxicity was inhibited by the aromatase inhibitor, finrozole. AFB1 was metabolized to aflatoxicol (AFL) by JEG-3 cells and CYP19A1 recombinant protein. AFL formation was partially inhibited by addition of tamoxifen and finrozole to the JEG-3 cells. AFB1 had no effect on the expression of CYP1A2 and CYP3A4 in JEG-3 cells. These results reveal that AFB1 can affect the expression of aromatase enzyme, indicating that chronic exposure to AFB1 may cause endocrine disruption in the foetoplacental unit.

Differential localization of vesicular glutamate transporters and peptides in corneal afferents to trigeminal nucleus caudalis.

Trigeminal afferents convey nociceptive information from the corneal surface of the eye to the trigeminal subnucleus caudalis (Vc). Trigeminal afferents, like other nociceptors, are thought to use glutamate and neuropeptides as neurotransmitters. The current studies examined whether corneal afferents contain both neuropeptides and vesicular glutamate transporters. Corneal afferents to the Vc were identified by using cholera toxin B (CTb). Corneal afferents project in two clusters to the rostral and caudal borders of the Vc, regions that contain functionally distinct nociceptive neurons. Thus, corneal afferents projecting to these two regions were examined separately. Dual immunocytochemical studies combined CTb with either calcitonin gene-related peptide (CGRP), substance P (SP), vesicular glutamate transporter 1 (VGluT1), or VGluT2. Corneal afferents were more likely to contain CGRP than SP, and corneal afferents projecting to the rostral region were more likely to contain CGRP than afferents projecting caudally. Overall, corneal afferents were equally likely to contain VGluT1 or VGluT2. Together, 61% of corneal afferents contained either VGluT1 or VGluT2, suggesting that some afferents lack a VGluT. Caudal corneal afferents were more likely to contain VGluT2 than VGluT1, whereas rostral corneal afferents were more likely to contain VGluT1 than VGluT2. Triple-labeling studies combining CTb, CGRP, and VGluT2 showed that very few corneal afferents contain both CGRP and VGluT2, caudally (1%) and rostrally (2%). These results suggest that most corneal afferents contain a peptide or a VGluT, but rarely both. Our results are consistent with a growing literature suggesting that glutamatergic and peptidergic sensory afferents may be distinct populations.

Conopeptide characterization and classifications: an analysis using ConoServer.

Cone snails are carnivorous marine gastropods that have evolved potent venoms to capture their prey. These venoms comprise a rich and diverse cocktail of peptide toxins, or conopeptides, whose high diversity has arisen from an efficient hypermutation mechanism, combined with a high frequency of post-translational modifications. Conopeptides bind with high specificity to distinct membrane receptors, ion channels, and transporters of the central and muscular nervous system. As well as serving their natural function in prey capture, conopeptides have been utilized as versatile tools in neuroscience and have proven valuable as drug leads that target the nervous system in humans. This paper examines current knowledge on conopeptide sequences based on an analysis of gene and peptide sequences in ConoServer (http://www.conoserver.org), a specialized database of conopeptide sequences and three-dimensional structures. We describe updates to the content and organization of ConoServer and discuss correlations between gene superfamilies, cysteine frameworks, pharmacological families targeted by conopeptides, and the phylogeny, habitat, and diet of cone snails. The study identifies gaps in current knowledge of conopeptides and points to potential directions for future research.

Aflatoxin B1 misregulates the activity of serine proteases: possible implications in the toxicity of some mycotoxin.

Aflatoxins are highly hazardous contaminants of common food and feed. Aflatoxin B1 in particular, the most predominant among aflatoxins, was thoroughly demonstrated to be highly toxic, mutagenic, teratogenic and carcinogenic in many animal species. Besides its established targets and effects, this work investigates on the possible direct interaction between aflatoxin B1 and three major serine proteases, namely elastase, thrombin and trypsin. These proteases belongs to a class of structurally and functionally related proteins pivotal in both direct and indirect regulation of a number of cellular events. Additionally, several pathological processes, including cancer, inflammatory processes and thrombosis, rely upon the subtle equilibrium between these enzymes and their potential modulators: in fact, their misregulation, caused by foreign molecules, could facilitate (or be the cause for) the occurrence of these pathologies. Our results provide the evidence for a reversible binding between AFB1 and these enzymes, likely to have profound implications in the manifestation of aflatoxicosis. Precisely, the toxin behaved as a moderate competitive inhibitor toward the enzymatic activity of the serine proteases in the low micromolar range.

Urinary biomarkers of aflatoxin exposure in young children from Egypt and Guinea.

Aflatoxins are a major risk factor for hepatocellular carcinoma (HCC), and thus understanding the pattern of aflatoxin exposure in different regions is important in order to develop targeted intervention strategies. Given the early onset of HCC in many countries early life exposures may be important. This study investigated aflatoxin exposure in Egyptian children (n=50, aged 1-2.5 years) by assessing urinary aflatoxin metabolite (AFM(1), AFB(1), AFB(2), AFG(1), AFG(2)) levels. Samples from Guinean children (n=50, aged 2-4 years) were analyzed in parallel providing a comparison to a region of established frequent aflatoxin exposure. Aflatoxins were isolated from urine using C18-cartridges followed by immunoaffinity clean-up, and quantified by HPLC with fluorescence detection. Overall aflatoxins were less frequently present in Egyptian (38%) than Guinean urine samples (86%) (p<0.001), which was particularly related to differences in detection rates of AFM(1) (8% compared to 64%, respectively, (p<0.001)). For AFM(1) the geometric mean level in Guinea (16.3 pg/ml; 95% CI: 10.1, 26.6 pg/ml) was 6-fold higher (p<0.001) than in Egypt (2.7 pg/ml; 95% CI: 2.5, 2.8 pg/ml). Urinary aflatoxins from healthy children in these two regions have not previously been reported, and exposure appears modest in Egypt compared to Guinea. These data suggest that measures to reduce aflatoxin exposure in both regions are important, though particularly in Guinea.