A consensus recombinant elapid long-chain α-neurotoxin and how protein folding matters for antibody recognition and neutralization of elapid venoms.

Antivenoms are essential in the treatment of the neurotoxicity caused by elapid snakebites. However, there are elapid neurotoxins, e.g., long-chain α-neurotoxins (also known as long-chain three-finger toxins) that are barely neutralized by commercial elapid antivenoms; so, recombinant elapid neurotoxins could be an alternative or complements for improving antibody production against the lethal long-chain α-neurotoxins from elapid venoms. This work communicates the expression of a recombinant long-chain α-neurotoxin, named HisrLcNTx or rLcNTx, which based on the most lethal long-chain α-neurotoxins reported, was constructed de novo. The gene of rLcNTx was synthesized and introduced into the expression vector pQE30, which contains a proteolytic cleavage region for exscinding the mature protein, and His residues in tandem for affinity purification. The cloned pQE30/rLcNTx was transfected into Escherichia coli Origami cells to express rLcNTx. After expression, it was found in inclusion bodies, and folded in multiple Cys-Cys structural isoforms. To observe the capability of those isoforms to generate antibodies against native long-chain α-neurotoxins, groups of rabbits were immunized with different cocktails of Cys-Cys rLcNTx isoforms. In vitro, and in vivo analyses revealed that rabbit antibodies raised against different rLcNTx Cys-Cys isoforms were able to recognize pure native long-chain α-neurotoxins and their elapid venoms, but they were unable to neutralize bungarotoxin, a classical long-chain α-neurotoxin, and other elapid venoms. The rLcNTx Cys-Cys isoform 2 was the immunogen that produced the best neutralizing antibodies in rabbits. Yet to neutralize the elapid venoms from the black mamba Dendroaspis polylepis, and the coral shield cobra Aspidelaps lubricus, it was required to use two types of antibodies, the ones produced using rLcNTx Cys-Cys isoform 2 and antibodies produced using short-chain α-neurotoxins. Expression of recombinant elapid neurotoxins as immunogens could be an alternative to improve elapid antivenoms; nevertheless, recombinant elapid neurotoxins must be well-folded to be used as immunogens for obtaining neutralizing antibodies.

In Vitro and In Vivo Antibiotic Capacity of Two Host Defense Peptides.

Two nonamidated host defense peptides named Pin2[G] and FA1 were evaluated against three types of pathogenic bacteria: two (Staphylococcus aureus UPD13 and Pseudomonas aeruginosa UPD3) isolated from diabetic foot ulcer patients, and another (Salmonella enterica serovar Typhimurium [ATCC 14028]) from a commercial collection. In vitro experiments showed that the antimicrobial performance of the synthetic peptides Pin2[G] and FA1 was modest, although FA1 was more effective than Pin2[G]. In contrast, Pin2[G] had superior in vivo anti-infective activity to FA1 in rabbit wound infections by the diabetic foot ulcer pathogens S. aureus UPD13 and P. aeruginosa UPD3. Indeed, Pin2[G] reduced bacterial colony counts of both S. aureus UPD13 and P. aeruginosa UPD3 by >100,000-fold after 48 to 72 h on skin wounds of infected rabbits, while in similar infected wounds, FA1 had no major effects at 72 to 96 h of treatment. Ceftriaxone was equally effective versus Pseudomonas but less effective versus S. aureus infections. Additionally, the two peptides were evaluated in mice against intragastrically inoculated S. enterica serovar Typhimurium (ATCC 14028). Only Pin2[G] at 0.56 mg/kg was effective in reducing systemic (liver) infection by >67-fold, equivalent to the effect of treatment with levofloxacin. Pin2[G] showed superior immunomodulatory activity in increasing chemokine production by a human bronchial cell line and suppressing polyinosinic-polycytidylic acid (poly[I:C])-induced proinflammatory IL-6 production. These data showed that the in vitro antimicrobial activity of these peptides was not correlated with their in vivo anti-infective activity and suggest that other factors such as immunomodulatory activity were more important.

Key amino acid residues involved in mammalian and insecticidal activities of Magi4 and Hv1b, cysteine-rich spider peptides from the δ-atracotoxin family.

δ-Atracotoxins, also known as δ-hexatoxins, are spider neurotoxic peptides, lethal to both vertebrates and insects. Their mechanism of action involves the binding to of the S3/S4 loop of the domain IV of the voltage-gated sodium channels (Nav). Because of the chemical difficulties of synthesizing folded synthetic δ-atracotoxins correctly, here we explore an expression system that is designed to produce biologically active recombinant δ-atracotoxins, and a number of variants, in order to establish certain amino acids implicated in the pharmacophore of this lethal neurotoxin. In order to elucidate and verify which amino acid residues play a key role that is toxic to vertebrates and insects, amino acid substitutes were produced by aligning the primary structures of several lethal δ-atracotoxins with those of δ-atracotoxins-Hv1b; a member of the δ-atracotoxin family that has low impact on vertebrates and is not toxic to insects. Our findings corroborate that the substitutions of the amino acid residue Y22 from δ-atracotoxin-Mg1a (Magi4) to K22 in δ-atracotoxin-Hv1b reduces its mammalian activity. Moreover, the substitutions of the amino acid residues Y22 and N26 from δ-atracotoxin-Mg1a (Magi4) to K22 and N26 in δ-atracotoxin-Hv1b reduces its insecticidal activity. Also, the basic residues K4 and R5 are important for keeping such insecticidal activity. Structural models suggest that such residues are clustered onto two bioactive surfaces, which share similar areas, previously reported as bioactive surfaces for scorpion α-toxins. Furthermore, these bioactive surfaces were also found to be similar to those found in related spider and anemone toxins, which affect the same Nav receptor, indicating that these motifs are important not only for scorpion but may be also for animal toxins that affect the S3/S4 loop of the domain IV of the Nav.

Myocardial longitudinal strain, fitness, and heart failure risk factors in young adults.

AIMS: To investigate the relationship between fitness, heart failure (HF) risk factors (age, blood pressure, and obesity), and global/regional myocardial longitudinal strain in young adults undergoing stress testing. METHODS: Individuals 25-55 years old without any significant medical history, not taking medications, and with a normal maximal stress echocardiogram were eligible. Global and regional longitudinal strain (LS) was evaluated by 2D speckle tracking echocardiography. RESULTS: One hundred and seventy patients were included, of which 60% were males. The mean age was 43 years old, 49% had optimal blood pressure, and 30% were obese. On average, patients achieved 10.5 (3) METS, and the global LS was -19.9 (3.1) %. Reduced fitness was associated with decreased global longitudinal strain (GLS). Those in the top GLS quartile walked on average 1 minute and 21 seconds longer compared with the lowest quartile (P < .001). The effect of fitness on LS was preferential to the mid and apex, such that there was an apex-to-base gradient. Obesity was also independently associated with reduced GLS. However, the reduction in LS in obese individuals was more prominent at the base and mid-walls with relative sparing of the apex. Similar to fitness, aging was also associated with an increase in the apex-to-base gradient of LS. Furthermore, diastolic filling parameters correlated distinctively with regional LS. CONCLUSIONS: In young adults without cardiovascular disease, low fitness and obesity are independently associated with reduced left ventricular longitudinal strain. There is a differential effect of HF risk factors on regional longitudinal function.

cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom.

A mRNA transcript that codes for a phospholipase (PLA2) was isolated from a single venom gland of the Bothrops ammodytoides viper. The PLA2 transcript was cloned onto a pCR®2.1-TOPO vector and subsequently expressed heterologously in the E. coli strain M15, using the pQE30 vector. The recombinant phospholipase was named rBamPLA2_1, and is composed of an N-terminal fusion protein of 16 residues, along with 122 residues from the mature protein that includes 14 cysteines that form 7 disulfide bonds. Following bacterial expression, rBamPLA2_1 was obtained from inclusion bodies and extracted using a chaotropic agent. rBamPLA2_1 had an experimental molecular mass of 15,692.5 Da that concurred with its theoretical molecular mass. rBamPLA2_1 was refolded in in vitro conditions and after refolding, three main protein fractions with similar molecular masses, were identified. Although, the three fractions were considered to represent different oxidized cystine isoforms, their secondary structures were comparable. All three recombinant isoforms were active on egg-yolk phospholipid and recognized similar cell membrane phospholipids to be native PLA2s, isolated from B. ammodytoides venom. A mixture of the three rBamPLA2_1 cystine isoforms was used to immunize a horse in order to produce serum antibodies (anti-rBamPLA2_1), which partially inhibited the indirect hemolytic activity of B. ammodytoides venom. Although, anti-rBamPLA2_1 antibodies were not able to recognize crotoxin, a PLA2 from the venom of a related but different viper genus, Crotalus durissus terrificus, they recognized PLA2s in other venoms from regional species of Bothrops.

Echocardiographic and clinical markers of left ventricular ejection fraction and moderate or greater systolic dysfunction in left ventricular noncompaction cardiomyopathy.

BACKGROUND: Left ventricular noncompaction (LVNC) is associated with progressive LV systolic dysfunction and dilated cardiomyopathy. We aimed to investigate the echocardiographic and clinical characteristics associated with LV ejection fraction (LVEF) and moderate or greater systolic dysfunction in patients with LVNC. METHODS: Our institutional echocardiography database was retrospectively reviewed between 2008 and 2014, and 62 patients with LVNC were identified. Forty-three (69%) had moderate or greater LV systolic dysfunction (LVEF ≤ 40%) and were compared with 19 (31%) patients with preserved or mildly reduced LVEF (>40%). Linear regression analyses were utilized to identify markers associated with LVEF. RESULTS: The mean age was 63 ± 17 years and noncompacted-to-compacted ratio was 2.3 ± 0.5, and was larger in patients with LVEF ≤ 40% (2.4 vs 2.1; P = .02). Patients with LVEF ≤ 40% were older, had more congestive heart failure, significant QRS interval prolongation, and greater LV remodeling and worse mean global longitudinal strain (GLS). Multivariate regression analysis revealed increased age (standardized regression coefficient (ß) = -0.17; P = .04) and QRS duration (ß = -0.13; P = .08), congestive heart failure (ß = -0.18; P = .04), and worsened GLS (ß = -0.40; P = .001) were independently associated with decreased LVEF in the cohort (overall model fit R2  = 0.71; P < .0001). Increased age (ß = -0.49; P = .01) and QRS duration (ß = -0.50; P = .002), and worsened GLS (ß = -0.33; P = .04), were also associated with a lower LVEF in patients with LVEF > 40%. CONCLUSIONS: The independent markers associated with LVEF and moderate or greater LV systolic dysfunction in patients with LVNC, in particular GLS and QRS duration, may detect high-risk candidates for more aggressive clinical surveillance and medical therapy.

Enhanced vasculotoxic metal excretion in post-myocardial infarction patients following a single edetate disodium-based infusion.

Toxic metals have been associated with cardiovascular mortality and morbidity. We have hypothesized that enhanced excretion of vasculotoxic metals might explain the positive results of the Trial to Assess Chelation Therapy (TACT). The purpose of this study was to determine whether a single infusion of the edetate disodium- based infusion used in TACT led to enhanced excretion of toxic metals known to be associated with cardiovascular events. METHODS: Twenty six patients (post-MI, age > 50 years, serum creatinine ≤ 2.0mg/dL) were enrolled in this open-label study. Urinary levels of 20 toxic metals normalized to urinary creatinine concentrations were measured at baseline in overnight urine collections, for 6h following a placebo infusion of 500mL normal saline and 1.2% dextrose, and for 6h following a 3g edetate disodium-based infusion. Self-reported metal exposure, smoking status, food frequency, occupational history, drinking water source, housing and hobbies were collected at baseline by a metal exposure questionnaire. RESULTS: The mean age was 65 years (range 51-81 years). All patients were male. 50% had diabetes mellitus and 58% were former smokers. Mean (SD) serum creatinine was 0.95 (0.31) mg/dL. Toxic metals were detected in the baseline urine of >80% of patients. After placebo infusion there were no significant changes in total urinary metal levels. After edetate infusion, total urinary metal level increased by 71% compared to baseline (1500 vs. 2580µg/g creatinine; P<0.0001). The effect of edetate was particularly large for lead (3835% increase) and cadmium (633% increase). CONCLUSIONS: Edetate disodium-based infusions markedly enhanced the urinary excretion of lead and cadmium, toxic metals with established epidemiologic evidence and mechanisms linking them to coronary and vascular events.

Antimicrobial Activity and Stability of Short and Long Based Arachnid Synthetic Peptides in the Presence of Commercial Antibiotics.

Four antimicrobial peptides (AMPs) named Pin2[G], Pin2[14], P18K and FA1 were chemically synthesized and purified. The four peptides were evaluated in the presence of eight commercial antibiotics against four microorganisms of medical importance: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae. The commercial antibiotics used were amoxicillin, azithromycin, ceftriaxone, gentamicin, levofloxacin, sulfamethoxazole, trimethoprim and vancomycin. The best AMP against P. aeruginosa was the peptide FA1, and the best AMP against S. aureus was Pin2[G]. Both FA1 and Pin2[G] were efficient against E. coli, but they were not effective against K. pneumoniae. As K. pneumoniae was resistant to most of the commercial antibiotics, combinations of the AMPs FA1 and Pin2[G] were prepared with these antibiotics. According to the fractional inhibitory concentration (FIC) index, the best antimicrobial combinations were obtained with concomitant applications of mixtures of FA1 with levofloxacin and sulfamethoxazole. However, combinations of FA1 or Pin2[G] with other antibiotics showed that total inhibitory effect of the combinations were greater than the sum of the individual effects of either the antimicrobial peptide or the antibiotic. We also evaluated the stability of the AMPs. The AMP Pin2[G] manifested the best performance in saline buffer, in supernatants of bacterial growth and in human blood plasma. Nevertheless, all AMPs were cleaved using endoproteolytic enzymes. These data show advantages and disadvantages of AMPs for potential clinical treatments of bacterial infections, using them in conjunction with commercial antibiotics.

Antimicrobial, toxicological, and antigenic characteristics of three scorpion venoms from Colombia: Centruroides margaritatus, Tityus pachyurus and Tityus n. sp. aff. metuendus.

The venom fractions of three buthid scorpion species from Colombia, C. margaritatus, T. pachyurus and T. n. sp. aff. metuendus, were examined for antimicrobial and toxicity toward mice and insects. The three venoms were separated into individual fractions using RP-HPLC, resulting in 85 fractions from C. margaritatus, 106 from T. pachyurus, and 70 from T. n. sp. aff. metuendus. The major fractions from the three scorpion venoms, which were eluted between 35 and 50 min, were tested for antimicrobial activity and toxicity. It was confirmed that the venom of the three species contains fractions with antimicrobial peptides that were evaluated against two bacterial strains of public health importance, Pseudomonas aeruginosa and Staphylococcus aureus. The venom of C. margaritatus had two antimicrobial fractions that showed activity against the named tested strains. The venom of T. pachyurus had three fractions that showed activity against S. aureus and two against both bacterial strains. Finally, the venom of T. n. sp. aff. metuendus had one fraction that showed activity against S. aureus, and five fractions showed activity against both bacterial strains. Also, some peptide fractions from the three venoms were toxic to mice. Last, the venoms of C. margaritatus and T. pachyurus were used as immunogens to obtain neutralizing antibodies against its respective venoms and to observe antibody recognition to related and unrelated scorpion venoms. A total of 15 mg of lyophilized antibodies were able to neutralize 1.5⋅LD50 of the venoms from T. n. sp. aff. metuendus, T. pachyurus and C. margaritatus, respectively. This information provides valuable insights into the diversity of each species' venom and their potential role in antimicrobial and venom toxicity.

An overview of some enzymes from buthid scorpion venoms from Colombia: Centruroides margaritatus, Tityus pachyurus, and Tityus n. sp. aff. metuendus.

Background: In Colombia, several species of Buthidae scorpions belonging to the genera Centruroides and Tityus coexist, and their stings are considered life-threatening to humans because of their venom neurotoxins. Despite previous studies focusing on neurotoxins from these scorpion genera, little is known about the enzymes present in their venoms and their relationship with whole venom toxicity. Methods: Here, using proteomic and biochemical protocols the enzymatic activities of the venoms of three Colombian scorpion species, C. margaritatus, T. pachyurus, and T. n. sp. aff. metuendus, were compared to establish the presence and absence of enzymes such as phospholipases, hyaluronidases, and proteases that could be related to venom toxicity. Results: C. margaritatus was positive for hyaluronidases, T. n. sp. aff. metuendus for proteases, and T. pachyurus exhibited activity for all three mentioned enzymes. Conclusion: This information provides valuable insights into the specific enzyme diversity of each species' venom and their potential role in venom toxicity, which could contribute to the development of better treatments and prevention strategies for scorpion envenomation.

Cupiennius spiders (Trechaleidae) from southern Mexico: DNA barcoding, venomics, and biological effect.

Background: Members of the genus Cupiennius Simon, 1891 are categorized as wandering spiders and are part of the family Trechaleidae. The genomics and proteomics of Cupiennius spiders from North America remain uncharacterized. The present study explores for the first time molecular data from the endemic species Cupiennius chiapanensis Medina, 2006, and also presents new data for Cupiennius salei (Keyserling, 1878), both collected in southern Mexico. Methods: In total, 88 Cupiennius specimens were collected from southern Mexico and morphologically identified. DNA was extracted and the mitochondrial COI fragment was amplified. COI sequences were analyzed, and a phylogenetic tree was inferred for species from the Americas. Genetic diversity was analyzed using haplotype networks and gene distances. Venom was obtained from C. chiapanensis and C. salei by electrostimulation. The venom was separated by HPLC, visualized using SDS-PAGE, and quantified for use in toxicity bioassays in mice and insects. Results: Analysis of COI sequences from C. chiapanensis showed 94% identity with C. salei, while C. salei exhibited 94-97% identity with sequences from Central and South American conspecifics. The venom from C. chiapanensis exhibited toxic activity against crickets. Venoms from C. chiapanensis and C. salei caused death in Anastrepha obliqua flies. Analysis of venom fractions from C. salei and C. chiapanensis revealed molecular masses of a similar size as some previously reported toxins and neurotoxic components. We determined the amino acid sequences of ChiaTx1 and ChiaTx2, toxins that are reported here for the first time and which showed toxicity against mice and insects. Conclusion: Our work is the first to report COI-based DNA barcoding sequences from southern Mexican Cupiennius spiders. Compounds with toxic activity were identified in venom from both species.

Cardiac geometry, function and mechanics in left ventricular non-compaction cardiomyopathy with preserved ejection fraction.

BACKGROUND: Left ventricular non-compaction (LVNC) cardiomyopathy in adults has primarily been studied with a phenotypic expression of low ejection fraction (EF) and dilated cardiomyopathy; however, data on LVNC with preserved EF is scarce. The present study aimed to evaluate cardiac geometry and mechanics in LVNC patients with preserved EF. METHODS: A retrospective cohort study of patients diagnosed with LVNC and a preserved EF between 2008 and 2019 was performed. LVNC was defined according to the presence of established transthoracic 2D echocardiographic (TTE) criteria as follows: (1) prominent LV trabeculations with deep recesses; (2) bi-layered myocardial appearance; and, (3) systolic non-compacted:compacted ratio≥ 2. Subjects were matched 1:1 to controls without LVNC referred for routine TTE. Geometric, functional and mechanics parameters were analyzed in the two cohorts using 2D and speckle-tracking TTE. RESULTS: Seventeen patients with LVNC and preserved EF were identified. Compared with controls, patients with LVNC had similar LV systolic function and chamber dimensions, but a larger mass and relative wall thickness, and more abnormal LV geometry (76% vs. 18%, p = 0.002), LA remodeling, and pulmonary hypertension. Global longitudinal strain was significantly decreased (-15.4 ± 3.2 vs. -18.9 ± 2.8%, p = < 0.01) and the prevalence of rigid body rotation was significantly increased (57% vs. 14%, p = 0.05) in the LVNC population. The peak twist values were comparable in both cohorts. CONCLUSIONS: Impaired LV geometry and longitudinal mechanics, as well as increased myocardial stiffness as expressed by rigid body rotation, characterize LVNC with preserved EF when compared with controls.

Recombinant expression and antigenicity of two peptide families of neurotoxins from Androctonus sp.

Background: Scorpion neurotoxins such as those that modify the mammalian voltage-gated sodium ion channels (Nav) are the main responsible for scorpion envenomation. Their neutralization is crucial in the production of antivenoms against scorpion stings. Methods: In the present study, two in silico designed genes - one that codes for a native neurotoxin from the venom of the Anatolian scorpion Androctonus crassicauda, named Acra 4 - and another non-native toxin - named consensus scorpion toxin (SccTx) obtained from the alignment of the primary structures of the most toxic neurotoxins from the Middle Eastern and North African scorpions - were recombinantly expressed in E. coli Origami. Results: Following bacterial expression, the two expressed neurotoxins, hereafter named HisrAcra4 and HisrSccTx, were obtained from inclusion bodies. Both recombinant neurotoxins were obtained in multiple Cys-Cys isoforms. After refolding, the active protein fractions were identified with molecular masses of 8,947.6 and 9,989.1 Da for HisrAcra4 and HisrSccTx, respectively, which agreed with their expected theoretical masses. HisrAcra4 and HisrSccTx were used as antigens to immunize two groups of rabbits, to produce either anti-HisrAcra4 or anti-HisrSccTx serum antibodies, which in turn could recognize and neutralize neurotoxins from venoms of scorpion species from the Middle East and North Africa. The antibodies obtained from rabbits neutralized the 3LD50 of Androctonus australis, Leiurus quinquestriatus hebraeus and Buthus occitanus venoms, but they did not neutralize A. crassicauda and A. mauritanicus venoms. In addition, the anti-HisrAcra4 antibodies did not neutralize any of the five scorpion venoms tested. However, an antibody blend of anti-HisrAcra4 and anti-HisrSccTx was able to neutralize A. crassicauda and A. mauritanicus venoms. Conclusions: Two recombinant Nav neurotoxins, from different peptide families, were used as antigens to generate IgGs for neutralizing scorpion venoms of species from the Middle East and North Africa.

Role of alkaline phosphatase from Manduca sexta in the mechanism of action of Bacillus thuringiensis Cry1Ab toxin.

Cry toxins produced by Bacillus thuringiensis have been recognized as pore-forming toxins whose primary action is to lyse midgut epithelial cells in their target insect. In the case of the Cry1A toxins, a prepore oligomeric intermediate is formed after interaction with cadherin receptor. The Cry1A oligomer then interacts with glycosylphosphatidylinositol-anchored receptors. Two Manduca sexta glycosylphosphatidylinositol-anchored proteins, aminopeptidase (APN) and alkaline phosphatase (ALP), have been shown to bind Cry1Ab, although their role in toxicity remains to be determined. Detection of Cry1Ab binding proteins by ligand blot assay revealed that ALP is preferentially expressed earlier during insect development, because it was found in the first larval instars, whereas APN is induced later after the third larval instar. The binding of Cry1Ab oligomer to pure preparations of APN and ALP showed that this toxin structure interacts with both receptors with high affinity (apparent K(d) = 0.6 nM), whereas the monomer showed weaker binding (apparent K(d) = 101.6 and 267.3 nM for APN and ALP, respectively). Several Cry1Ab nontoxic mutants located in the exposed loop 2 of domain II or in beta-16 of domain III were affected in binding to APN and ALP, depending on their oligomeric state. In particular monomers of the nontoxic domain III, the L511A mutant did not bind ALP but retained APN binding, suggesting that initial interaction with ALP is critical for toxicity. Our data suggest that APN and ALP fulfill two roles. First APN and ALP are initial receptors promoting the localization of toxin monomers in the midgut microvilli before interaction with cadherin. Then APN and ALP function as secondary receptors mediating oligomer insertion into the membrane. However, the expression pattern of these receptors and the phenotype of L511A mutant suggest that ALP may have a predominant role in toxin action because Cry toxins are highly effective against the neonate larvae that is the target for pest control programs.

Low-Level Cadmium Exposure and Atherosclerosis.

PURPOSE OF REVIEW: Cadmium has been recognized as a potential risk factor for cardiovascular disease (CVD). We present a review of cadmium toxicity, its effect on cellular activities, and a summary of reported association between environmental cadmium exposure and CVD. We also discuss the possible therapeutic benefit of cadmium chelation. RECENT FINDINGS: Experimental data suggest that cadmium affects several signaling pathways which may lead to endothelial dysfunction and vascular tissue damage, promoting atherosclerosis. This is further supported by epidemiological studies that have shown an association of even low-level cadmium exposure with an increased risk of clinical cardiovascular events. The Trial to Assess Chelation Therapy (TACT) provided inferential evidence for the cardiovascular benefit of treating toxic metal burden. However, at the present time, there is no direct evidence, but suggestive findings from clinical trials indicating that removal of cadmium from body stores may be associated with improved cardiovascular outcomes. An evolving body of evidence supports environmental cadmium exposure as a pro-atherosclerosis risk factor in CVD; however, the mechanisms for the proatherogenic effect of cadmium are still not completely understood. Further studies in translational toxicology are needed to fill the knowledge gaps regarding the molecular mechanisms of cadmium toxicity and the promotion of atherosclerosis.

Identification of a crocodylian ß-defensin variant from Alligator mississippiensis with antimicrobial and antibiofilm activity.

ß-defensin host defense peptides are important components of the innate immune system of vertebrates. Although evidence of their broad antimicrobial, antibiofilm and immunomodulatory activities in mammals have been presented, ß-defensins from other vertebrate species, like crocodylians, remain largely unexplored. In this study, five new crocodylian ß-defensin variants from Alligator mississippiensis and Crocodylus porosus were selected for synthesis and characterization based on their charge and hydrophobicity values. Linear peptides were synthesized, folded, purified and then evaluated for their antimicrobial and antibiofilm activities against the bacterial pathogens, Salmonella enterica serovar Typhimurium, Staphylococcus aureus, Enterobacter cloacae and Acinetobacter baumannii. The Am23SK variant (SCRFSGGYCIWNWERCRSGHFLVALCPFRKRCCK) from A. mississippiensis displayed promising activity against both planktonic cells and bacterial biofilms, outperforming the human ß-defensin 3 under the experimental conditions. Moreover, Am23SK exhibited no cytotoxicity towards mammalian cells and exerted immunomodulatory effects in vitro, moderately suppressing the production of proinflammatory mediators from stimulated human bronchial epithelial cells. Overall, our results have expanded the activity landscape of crocodylian and reptilian ß-defensin in general.

Cellular senescence evaluated by P16INK4a immunohistochemistry is a prevalent phenomenon in advanced calcific aortic valve disease.

BACKGROUND: Fibrosis, calcification, and ossification are histopathologic hallmarks of calcific aortic valve disease (CAVD), a leading cause of morbidity and mortality in the aging population. Cellular senescence contributes to a functional decay in chronic diseases by intensifying tissue remodeling and impairing tissue regeneration. We evaluated the expression of P16INK4A and P53 as surrogate markers of senescence in CAVD. METHODS: Aortic valves from 27 individuals with severe CAVD requiring aortic valve replacement were selected for routine histologic processing. Immunohistochemical expression of P16INK4A and P53 was quantified using computerized image analysis on fields matching compartments with varying degrees of tissue remodeling. RESULTS: All aortic valves demonstrated P16INK4A and P53-positive cells. The percentage of P16INK4A -positive cells, but not of P53, was higher in areas of calcification and/or ossification (57.21%±26.31, n=40) and severe fibrosis (54.79%±27.19, n=25) than in areas with minimal to mild tissue remodeling (13.69% ± 11.88, n=16, P<.0001). P16INK4A expression was observed in interstitial valve cells within all compartments proportional to the degree of fibrosis and did not correlate with age, severity of aortic stenosis, or P53 expression. Multiple linear regression analysis by backward elimination revealed P16INK4A expression was lower among statin users (P<.01). CONCLUSIONS: P16INK4A- expression is ubiquitous in calcified aortic valves and correlates with severity of tissue remodeling, suggesting a role of cellular senescence in the progression of CAVD. Further research is needed to identify possible treatment modalities as disease modifying agents for CAVD.

Domain II loop 3 of Bacillus thuringiensis Cry1Ab toxin is involved in a "ping pong" binding mechanism with Manduca sexta aminopeptidase-N and cadherin receptors.

Bacillus thuringiensis Cry toxins are used worldwide as insecticides in agriculture, in forestry, and in the control of disease transmission vectors. In the lepidopteran Manduca sexta, cadherin (Bt-R(1)) and aminopeptidase-N (APN) function as Cry1A toxin receptors. The interaction with Bt-R(1) promotes cleavage of the amino-terminal end, including helix alpha-1 and formation of prepore oligomer that binds to APN, leading to membrane insertion and pore formation. Loops of domain II of Cry1Ab toxin are involved in receptor interaction. Here we show that Cry1Ab mutants located in domain II loop 3 are affected in binding to both receptors and toxicity against Manduca sexta larvae. Interaction with both receptors depends on the oligomeric state of the toxin. Monomers of loop 3 mutants were affected in binding to APN and to a cadherin fragment corresponding to cadherin repeat 12 but not with a fragment comprising cadherin repeats 7-12. In contrast, the oligomers of loop 3 mutants were affected in binding to both Bt-R(1) fragments but not to APN. Toxicity assays showed that either monomeric or oligomeric structures of Cry1Ab loop 3 mutations were severely affected in insecticidal activity. These data suggest that loop 3 is differentially involved in the binding with both receptor molecules, depending on the oligomeric state of the toxin and also that possibly a "ping pong" binding mechanism with both receptors is involved in toxin action.

Characterization of the mechanism of action of the genetically modified Cry1AbMod toxin that is active against Cry1Ab-resistant insects.

Bacillus thuringiensis Cry toxins are used in the control of insect pests. They are pore-forming toxins with a complex mechanism that involves the sequential interaction with receptors. They are produced as protoxins, which are activated by midgut proteases. Activated toxin binds to cadherin receptor, inducing an extra cleavage including helix alpha-1, facilitating the formation of a pre-pore oligomer. The toxin oligomer binds to secondary receptors such as aminopeptidase and inserts into lipid rafts forming pores and causing larval death. The primary threat to efficacy of Bt-toxins is the evolution of insect resistance. Engineered Cry1AMod toxins, devoid of helix alpha-1, could be used for the control of resistance in lepidopterans by bypassing the altered cadherin receptor, killing resistant insects affected in this receptor. Here we analyzed the mechanism of action of Cry1AbMod. We found that alkaline pH and the presence of membrane lipids facilitates the oligomerization of Cry1AbMod. In addition, tryptophan fluorescence emission spectra, ELISA binding to pure aminopeptidase receptor, calcein release assay and analysis of ionic-conductance in planar lipid bilayers, indicated that the secondary steps in mode of action that take place after interaction with cadherin receptor such as oligomerization, receptor binding and pore formation are similar in the Cry1AbMod and in the wild type Cry1Ab. Finally, the membrane-associated structure of Cry1AbMod oligomer was analyzed by electron crystallography showing that it forms a complex with a trimeric organization.