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.

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.

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.

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.

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.

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.

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.

Transthoracic versus intra-operative transesophageal echocardiography in right heart assessment.

METHODS: Fifty-four patients who had combined mitral and tricuspid valve surgery were included. Right heart measurements were performed in the TTE apical 4-chamber (A4C) and RV inflow views, and TEE mid-esophageal 4-chamber (ME4C) and transgastric RV inflow views at end-diastole. Spearman correlation coefficients (r) were applied to test for associations between the imaging modalities. RESULTS: The mean age was 65 years and 39% were male. All patients had ≥ moderate tricuspid regurgitation (TR), and a secondary/functional etiology was present in 89%. The median TAd and RV basal (RVd) diameters in the TTE-A4C view measured 37 mm [interquartile range (IQR), 34-44] and 43 mm (IQR, 40-51), respectively. The TTE-A4C TAd strongly correlated with the TEE-ME4C measurement (r=0.72), with an overestimation of 1 mm (IQR, -2 to 4) by TEE (P<0.01). For RVd, the TTE-A4C measurement correlated moderately with the TEE-ME4C view (r=0.61), underestimating the RVd by -1 mm (IQR, -4 to 3.3) (P<0.01). No correlation was observed between TAPSE measured by TTE and TEE (r=0.22, P=0.13). CONCLUSIONS: Intra-operative TEE may reliably quantitate TA and RV size and geometry. The current findings are best interpreted as hypothesis-generating for future validative studies.

Tricuspid regurgitation and in-hospital outcomes after transcatheter aortic valve replacement in high-risk patients.

BACKGROUND: The prognostic impact of tricuspid regurgitation (TR) following transcatheter aortic valve replacement (TAVR) is uncertain, and the management of patients with severe aortic stenosis and significant TR undergoing TAVR is unclear. METHODS: Retrospective study investigating the role of TR severity on hospital outcomes in high risk patients with severe aortic stenosis undergoing TAVR. RESULTS: A total of 174 participants were included in the present study. The median age was 84 years and 48% were women. The median (IR) STS score was 7.3 (4.7-13.6). The pre-procedural mean (SD) aortic valve area (AVA) was 0.69 (0.2) cm2 and the average (SD) peak and mean gradients were 71 [23]/42 [15] mmHg. Pre TAVR, 28.7% of patients had significant (moderate or severe) TR. Significant TR pre-TAVR increased the risk of in-hospital cardiovascular (CV) and all-cause and mortality [adjusted relative risk (RR) (95% CI): 14.67 (1.35-159.51) and 5.09 (1.14-22.72), respectively], and those with severe TR post-TAVR had longer hospital stay [median (IR): 9.9 (2.9-17.0) days]. No improvement or worsened TR (greater than mild) post-TAVR was associated with higher CV and all-cause mortality [adjusted RR (95% CI): 21.5 (1.81-255.96) and 8.19 (1.67-40.29), respectively]. Right ventricular systolic pressure (RVSP) was independently associated with TR severity pre and post TAVR. CONCLUSIONS: Significant TR was common among patients undergoing high risk TAVR, and is associated with increased in hospital mortality and longer hospital stay. Patients with elevated RVSP and persistent moderate or severe TR after TAVR are at higher risk of in hospital death.

Urinary Metal Levels after Repeated Edetate Disodium Infusions: Preliminary Findings.

Environmentally acquired lead and cadmium are associated with increased cardiovascular disease risk. In the Trial to Assess Chelation Therapy, up to 40 infusions with edetate disodium over an approximately one-year period lowered the cardiovascular disease risk in patients with a prior myocardial infarction. We assessed whether a reduction in surrogate measures of total body lead and cadmium, post-edetate disodium urine lead and pre-edetate urine cadmium, could be detected after repeated edetate disodium-based infusions compared to the baseline. Fourteen patients with coronary artery disease received multiple open-label edetate disodium infusions. The urine metals pre- and post-edetate infusion, normalized for urine creatinine, were compared to urine levels pre and post final infusion by a paired t-test. Compared with the pre-edetate values, post-edetate urine lead and cadmium increased by 3581% and 802%, respectively, after the first infusion. Compared to baseline, post-edetate lead decreased by 36% (p = 0.0004). A reduction in post-edetate urine lead was observed in 84% of the patients after the final infusion. Pre-edetate lead decreased by 60% (p = 0.003). Pre-edetate lead excretion became undetectable in nearly 40% of patients. This study suggests that edetate disodium-based infusions may decrease the total body burden of lead. However, our data suggest no significant reduction in the body burden of cadmium.

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.

Left Atrial "Pinball" Thrombus Causing Recurrent Syncope.

A 75-year-old female was admitted due to a syncopal episode. Transesophageal echocardiography demonstrated severe mitral stenosis with a 5 cm, free-floating, left atrial thrombus intermittently obstructing the mitral valve orifice; she underwent thrombus removal and mitral valve replacement.

A Novel Insecticidal Spider Peptide that Affects the Mammalian Voltage-Gated Ion Channel hKv1.5.

Spider venoms include various peptide toxins that modify the ion currents, mainly of excitable insect cells. Consequently, scientific research on spider venoms has revealed a broad range of peptide toxins with different pharmacological properties, even for mammal species. In this work, thirty animal venoms were screened against hKv1.5, a potential target for atrial fibrillation therapy. The whole venom of the spider Oculicosa supermirabilis, which is also insecticidal to house crickets, caused voltage-gated potassium ion channel modulation in hKv1.5. Therefore, a peptide from the spider O. supermirabilis venom, named Osu1, was identified through HPLC reverse-phase fractionation. Osu1 displayed similar biological properties as the whole venom; so, the primary sequence of Osu1 was elucidated by both of N-terminal degradation and endoproteolytic cleavage. Based on its primary structure, a gene that codifies for Osu1 was constructed de novo from protein to DNA by reverse translation. A recombinant Osu1 was expressed using a pQE30 vector inside the E. coli SHuffle expression system. recombinant Osu1 had voltage-gated potassium ion channel modulation of human hKv1.5, and it was also as insecticidal as the native toxin. Due to its novel primary structure, and hypothesized disulfide pairing motif, Osu1 may represent a new family of spider toxins.

The effect of EDTA-based chelation on patients with diabetes and peripheral artery disease in the Trial to Assess Chelation Therapy (TACT).

OBJECTIVE: Approximately 1 in 7 US adults have diabetes; and over 60% of deaths in patients with diabetes have cardiac disease as a principal or contributing cause. Both coronary and peripheral artery disease (PAD) identify high-risk cohorts among patients with diabetes. We have previously demonstrated improved cardiovascular outcomes with edetate disodium-based chelation in post-MI patients with diabetes, enrolled in the Trial to Assess Chelation Therapy (TACT). In these analyses we further studied the effect size of patients with diabetes and severe disease in 2 vascular beds; coronaries, and lower extremity arteries. We questioned whether greater atherosclerotic burden would attenuate the observed beneficial effect of edetate disodium infusions. RESEARCH DESIGN AND METHODS: The multicenter TACT used a double blind, placebo controlled, 2 × 2 factorial design with 1708 participants, randomly assigned to receive edetate disodium-based chelation, or placebo and high dose oral vitamins or placebo. There were 162 (9.5% of 1708) post-MI patients with a diagnosis of diabetes mellitus and PAD for this post hoc analysis. Patients received up to 40 double-blind intravenous infusions of edetate disodium-based chelation, or placebo. The composite primary endpoint of TACT consisted of death from any cause, myocardial infarction, stroke, coronary revascularization and hospitalization for angina. RESULTS: The median age was 66 years, 15% female, 5% non-Caucasian, and BMI was 31. Insulin was used by 32% of patients. Active infusions significantly reduced the primary endpoint compared with placebo infusions (HR, 0.52; 95% CI, 0.30-0.92; P = 0.0069), with a 30% absolute risk reduction in the primary endpoint. There was a marked reduction in total mortality from 24% to 11%, although of borderline significance (P = 0.052). CONCLUSION: Atherosclerotic disease in multiple vascular beds did not attenuate the beneficial effect of edetate disodium infusions in post MI patients with diabetes. Studies now in progress will prospectively test this post hoc finding.