Application of behavior data to predictive exploratory models of metritis self-cure and treatment failure in dairy cows.

The objective was to evaluate the performance of exploratory models containing routinely available on-farm data, behavior data, and the combination of both to predict metritis self-cure (SC) and treatment failure (TF). Holstein cows (n = 1,061) were fitted with a collar-mounted automated- health monitoring device (AHMD) from -21 ± 3 to 60 ± 3 d relative to calving to monitor rumination and activity. Cows were examined for diagnosis of metritis at 4 ± 1, 7 ± 1, and 9 ± 1 DIM. Cows diagnosed with metritis (n = 132), characterized by watery, fetid, reddish/brownish vaginal discharge (VD) were randomly allocated to one of 2 treatments: Control (CON; n = 62) - no treatment at the time of metritis diagnosis (d 0); Ceftiofur (CEF; n = 70) - subcutaneous injection of 6.6 mg/kg of ceftiofur crystalline-free acid on d 0 and 3 relative to diagnosis. Cure was determined 12 d after diagnosis and was considered when VD became mucoid and not fetid. Cows in CON were used to determine SC and cows in CEF were used to determine TF. Univariable analyses were performed using farm-collected data (parity, calving season, calving-related disorders, body condition score, rectal temperature, and days in milk at metritis diagnosis) and behavior data (i.e., daily averages of rumination, activity generated by AHMD, and derived variables) to assess their association with metritis SC or TF. Variables with a P ≤ 0.20 were included in the multivariable logistic regression exploratory models. To predict SC, the area under the curve (AUC) for the exploratory model containing only data routinely available on-farm was 0.75. The final exploratory model to predict SC combining routinely available on-farm data and behavior data increased the AUC to 0.87, sensitivity (Se) 87% and specificity (Sp) 71%. To predict TF, the AUC for the exploratory model containing only data routinely available on-farm was 0.90. The final exploratory model combining routinely available on-farm data and behavior data increased the AUC to 0.93, Se of 93% and Sp of 82%. Cross-validation analysis revealed that generalizability of the exploratory models was poor, which indicates that the findings are applicable to the conditions of the present exploratory study. In summary, the addition of behavior data contributed to increasing the prediction of SC and TF. Developing and validating accurate prediction models for SC could lead to a reduction in antimicrobial use, whereas accurate prediction of cows that would have TF may allow for better management decisions.

Changes in Uterine Metabolome Associated with Metritis Development and Cure in Lactating Holstein Cows.

The objective of this study was to identify alterations in the vaginal discharge (VD) metabolome and potential biomarkers to predict metritis development and a cure in dairy cows. This prospective cohort study was conducted on two dairies located in CA and TX. Vaginal discharge was evaluated and collected using the Metricheck® device. Cows were examined for metritis at 4, 7, and 9 days in milk (DIM). Cows with a fetid, watery, and reddish-brown uterine discharge were classified as having metritis and randomized to receive ceftiofur (n = 10) or remain untreated (n = 7). A cure was defined as the absence of a fetid, watery, reddish-brown uterine discharge at 14 d after enrollment. Vaginal discharge samples were collected from 86 cows within 6 h after parturition, at 4 and 7 DIM, at metritis diagnosis, and at 4 and 7 days after metritis diagnosis. Cows with metritis (MET; n = 17) were paired with counterparts without metritis (HTH) of a similar DIM and parity (n = 34). The uterine metabolome was evaluated using untargeted gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). Metabolomic data were analyzed using the MetaboAnalyst 5.0. Data were log-transformed and auto-scaled for normalization. Univariate analyses, including the fold-change, were performed to identify the metabolites linked to metritis development and its cure and principal component analysis and partial least squares discriminant analysis were performed to explain metabolite variance between animals developing or not developing metritis and being cured or not being cured of metritis. Comparing HTH with MET cows at calving, 12 metabolites were upregulated, and one was downregulated. At four and seven DIM, 51 and 74 metabolites, respectively, were altered between MET and HTH cows. After metritis development, three and five metabolites were upregulated in cows that were cured and in cows that received treatment and were cured, respectively. In all scenarios, the metabolites lignoceric, malic, and maleic acids, ornithine, and hypotaurine, which are associated with arginine/aminoacyl-tRNA biosynthesis and taurine/purine metabolism, were upregulated in HTH cows. Metritis was associated with changes in the uterine metabolome. Cows not being cured of metritis had changes in the uterus metabolome independent of receiving ceftiofur or remaining untreated. Metabolome analysis may be an important tool to understand the vaginal discharge changes during postpartum and the dynamics of metritis development and cures and help to identify biomarkers to predict metritis being cured.

SARS-CoV-2 uses CD4 to infect T helper lymphocytes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent of a major global outbreak of respiratory tract disease known as Coronavirus Disease 2019 (COVID-19). SARS-CoV-2 infects mainly lungs and may cause several immune-related complications, such as lymphocytopenia and cytokine storm, which are associated with the severity of the disease and predict mortality. The mechanism by which SARS-CoV-2 infection may result in immune system dysfunction is still not fully understood. Here, we show that SARS-CoV-2 infects human CD4+ T helper cells, but not CD8+ T cells, and is present in blood and bronchoalveolar lavage T helper cells of severe COVID-19 patients. We demonstrated that SARS-CoV-2 spike glycoprotein (S) directly binds to the CD4 molecule, which in turn mediates the entry of SARS- CoV-2 in T helper cells. This leads to impaired CD4 T cell function and may cause cell death. SARS-CoV-2-infected T helper cells express higher levels of IL-10, which is associated with viral persistence and disease severity. Thus, CD4-mediated SARS-CoV-2 infection of T helper cells may contribute to a poor immune response in COVID-19 patients.

Interactive euthanasia training program for the dairy industry: Implementation and perceived respondent knowledge change.

This study described the development of an interactive euthanasia training program and its potential to improve dairy workers' perceived euthanasia decision-making skills and awareness of timely euthanasia by using a survey instrument before and after the program. Training material encompassed euthanasia information over 2 production stages (calves and cows or heifers) and material was delivered on-farm in a case-scenario format (14 cases). During a 3-mo period, 30 different dairy farms were visited and 81 participants were enrolled in this study. Each participant was required to complete a survey pretraining, to complete the case studies from the production stage in which their job responsibility was more closely aligned with (estimated completion time of 1 h), and to complete a survey post-training. Surveys contained 8 statements regarding participants' perceived knowledge of euthanasia practices. The questions were answered on a 5-point scale: (1) strongly disagree, (2) disagree, (3) neither agree nor disagree, (4) agree, or (5) strongly agree. Multivariable mixed-effects logistic regression models were created for each question to investigate the effect of age, sex, dairy experience, farm size, role at the farm, race, previous experience with euthanasia, veterinarian degree, and production stage in the score change, defined by the presence or absence of an increase in the 5-point scale score. Upon completion of the training, respondents were more confident in identifying compromised animals (score change = 0.35), determining when an animal should be euthanized (score change = 0.64), and understanding the importance of timely euthanasia (score change = 0.26). Age and euthanasia experience were significantly associated with the respondents' perceived knowledge; suggesting that younger, less-experienced caretakers on-farm should be prioritized to receive training. The proposed interactive case-based euthanasia training program has proven to be valuable to dairy participants and veterinarians as it provides a means to improve dairy welfare.

Dairy workers' attitudes toward dairy cattle euthanasia.

Implementing timely and humane euthanasia in dairy farms remains a critical concern. One of the possible barriers for the implementation of timely euthanasia on-farm is dairy workers' attitudes toward the act. The objectives of this study were to investigate dairy workers' attitudes toward dairy cattle euthanasia and their association to individuals' demographic characteristics. A total of 81 workers from 30 dairy farms (ranging in size from less than 500 to more than 3,000 cows) participated in the survey and most participants were caretakers (n = 45; 55.6%) or farm managers (n = 16; 19.8%), with an average work experience of 14.8 years. Dairy workers' attitudes toward dairy cattle (empathy affect, empathy attribution, and negative attitudes about cattle), working environment (relying on others, perceived time constraints) and euthanasia decision-making (feeling comfortable with euthanasia, feeling confident, seeking knowledge, using different sources to obtain advice, having negative attitudes about euthanasia, having insufficient knowledge, having trouble deciding when to euthanize and avoiding if possible) were identified and used for cluster analyses. Cluster analyses identified 3 distinct clusters: (1) confident but uncomfortable with euthanasia (n = 40); (2) confident and comfortable with euthanasia (n = 32); and (3) unconfident, lacking knowledge and detached from cattle (n = 9). Dairy workers' demographic characteristics (age, sex, race and ethnicity, dairy experience, role on-farm, farm size, and previous euthanasia experience) were used as predictors for the risk factor analyses. The risk analysis demonstrated that there were no predictors for cluster 1, but White workers (P = 0.04) and caretakers that had previous euthanasia experience tended to be more likely to be members of cluster 2 (P = 0.07) whereas respondents that worked in farms with 501-1,000 cows were more likely to be grouped in cluster 3. This study provides vital information about variability in dairy workers' attitudes toward dairy euthanasia as well as its association with race and ethnicity, farm size, and previous euthanasia experience. This information can be used to implement appropriate training and euthanasia protocols to increase both human and dairy cattle welfare on-farm.

Behavioral changes of metritic primiparous cows treated with chitosan microparticles or ceftiofur.

The main objective was to characterize behavioral changes in metritic primiparous cows treated with chitosan microparticles (CM) or ceftiofur (CEF). A secondary objective was to compare behavioral patterns of metritic cows with nonmetritic (NMET) cows. Nulliparous Holstein cows (n = 311) were fitted with a neck-mounted automated health-monitoring device (AHMD) from -21 to 60 d relative to calving. Cows diagnosed with metritis (d 0), characterized by watery, fetid, red-brownish uterine discharge within 21 d in milk were assigned randomly to CM (n = 45), intrauterine infusion of 24 g of CM dissolved in 40 mL of sterile distilled water on d 0, 2, and 4; CEF (n = 47), subcutaneous injection of 6.6 mg/kg ceftiofur crystalline-free acid on d 0 and 3; and control (CON; n = 39), no treatment. For comparison, NMET cows (n = 180) were matched with metritic cows according to age at calving and calving date. Postdiagnosis, there was an effect of treatment and an interaction between treatment and time on rumination and activity. The interaction showed that CM had lesser rumination than CEF from d 1 to 11, d 18, and d 20; CM had lesser rumination than CON from d 2 to 8; and CEF was not different from CON. The interaction showed that CM had lesser activity than CON on d 2, from d 6 to 11, and d 13 to 14; CM was not different from CEF; and CEF had lesser activity than CON on d 8, 9, 13, and 14. Prediagnosis, cows in CM, CEF, and CON had lesser rumination and activity than cows in NMET. Postdiagnosis, cows in CM, CEF, and CON had lesser rumination than NMET from d 0 to 2 and had lesser activity than NMET from d 0 to 5. In summary, CM decreased rumination and activity compared with CON, which indicates a negative systemic effect of CM. This may be associated with exacerbated inflammation in the uterus. Additionally, metritic cows had decreased rumination and activity prediagnosis, which may allow for the use of AHMD for metritis diagnosis.

Dairy Cattle Euthanasia-Focus Groups Exploring the Perspectives of Brazilians Working in the Dairy Cattle Industry.

The objective of this study was to explore perspectives and attitudes about euthanasia specific to the Brazilian dairy cattle industry. Twenty-five Brazilian citizens (13 veterinarians, 4 animal scientists, 3 professors, 3 researchers, 1 dairy owner, and 1 caretaker) participated in one of three focus groups conducted and recorded online (10, 8, and 7 participants per group). Questions regarding euthanasia were posed by a moderator, and the focus group discussions were then transcribed verbatim for analysis. After the initial data analysis, themes were evaluated and collapsed into three major categories: Euthanasia Training and Farm and Human Components. A complex interconnection between the three main themes and multiple subthemes specific to dairy cattle euthanasia was also revealed. The lack of nationally recognized euthanasia guidelines for dairy cattle paired with ineffective and inaccessible euthanasia tools makes it difficult for dairy veterinarians to implement humane protocols for on-farm euthanasia. In addition, logistical factors, particularly, the financial cost of euthanasia and the human-animal bond, play a role in the failure to perform euthanasia when warranted. Future studies should focus on the development of science-based standards and producer training to improve the consistency of on-farm euthanasia in Brazilian dairy operations.

Homogenised and pasteurised human milk: lipid profile and effect as a supplement in the enteral diet of Wistar rats.

The retention of human milk (HM) fat in nasogastric probes of infusion pumps can be observed during the feed of infants unable to suck at the mother's breast. The lack of homogenisation of HM could contribute to the fat holding. Therefore, the present study evaluated (i) the influence of homogenisation on milk fat retaining in infant feeding probes and (ii) the in vivo effect of the homogenisation on lipid absorption by Wistar rats. The animals were fed with HM treated following two processing conditions, that is, pasteurised and homogenised-pasteurised. The animals were randomly subdivided into four experimental groups: water-fed (control), pasteurised milk, homogenised-pasteurised milk and pasteurised-skimmed milk. The results of food consumption, mass body gain, corporate metrics and plasma blood levels of total cholesterol did not show any difference (P < 0·05) among the three types of HM used in the experiments. The liver, intestine and intra-abdominal adipose tissue of the four groups of animals presented normal and healthy histology. The composition of fatty acids in the brain tissue of animals fed with homogenised HM increased when compared with the groups fed with non-homogenised HM. These values were 11·08 % higher for arachidonic acids, 6·59 % for DAH and 47·92 % for nervous acids. The ingestion of homogenised HM promoted higher absorption of milk nutrients. Therefore, the addition of the homogenisation stage in HM processing could be an alternative to reduce fat retention in probes and to improve the lipids' absorption in the body.

Angiotensin II Type 1 Receptor Tachyphylaxis Is Defined by Agonist Residence Time.

Several GPCRs (G-protein-coupled receptors) have been reported to exhibit tachyphylaxis, which is an acute loss of functional receptor response after repeated stimuli with an agonist. GPCRs are important clinical targets for a wide range of disorders. Therefore, elucidation of the ligand features that contribute to receptor tachyphylaxis and signaling events underlying this phenomenon is important for drug discovery and development. In this study, we examined the role of ligand-binding kinetics in the tachyphylaxis of AT1R (angiotensin II type 1 receptor) using bioluminescence resonance energy transfer assays to monitor signaling events under both kinetic and equilibrium conditions. We investigated AT1R signal transduction and translocation promoted by the endogenous tachyphylactic agonist Ang II (angiotensin II) and its analogs, described previously for inducing reduced receptor tachyphylaxis. Estimation of binding kinetic parameters of the ligands revealed that the residence time of Ang II was higher than that of the analogs, resulting in more sustained Gq protein activation and recruitment of ß-arrestin than that promoted by the analogs. Furthermore, we observed that Ang II led to more sustained internalization of the receptor, thereby retarding its recycling to the plasma membrane and preventing further receptor responses. These results show that the apparent lack of tachyphylaxis in the studied analogs resulted from their short residence time at the AT1R. In addition, our data highlight the relevance of complete characterization of novel GPCR drug candidates, taking into account their receptor binding kinetics as well.

Effect of Chitosan Microparticles on the Uterine Microbiome of Dairy Cows with Metritis.

The objective of this study was to evaluate the effect of chitosan microparticles on the uterine microbiome of cows with metritis. Dairy cows with metritis (n = 89) were assigned to 1 of 3 treatments: chitosan microparticles (n = 21), in which the cows received an intrauterine infusion of chitosan microparticles at metritis diagnosis (day 0), day 2, and day 4; ceftiofur (n = 25), in which the cows received a subcutaneous injection of ceftiofur on day 0 and day 3; and no intrauterine or subcutaneous treatment (n = 23). Nonmetritic cows (n = 20) were healthy cows matched with cows with metritis by the number of days postpartum at metritis diagnosis. Uterine swab samples collected on days 0, 3, 6, 9, and 12 were used for 16S rRNA gene sequencing and 16S RNA gene copy number quantification by quantitative PCR. Principal-coordinate analysis showed that the microbiome of the ceftiofur-treated and metritic untreated groups progressed toward that of the nonmetritic group by day 3, whereas that of the chitosan microparticle-treated group remained unchanged. The differences on day 3 were mainly due to a greater relative abundance of Fusobacteria, particularly Fusobacterium, in the chitosan microparticle-treated group than in the ceftiofur-treated and metritic untreated groups. Furthermore, the microbiome of the ceftiofur-treated group became similar to that of the nonmetritic group by day 9, whereas the microbiome of the chitosan microparticle-treated and metritic untreated groups became similar to that of the nonmetritic group only by day 12. The total bacterial 16S rRNA gene counts in the chitosan microparticle-treated group were greater than those in the metritic untreated controls on days 6 and 9, whereas the ceftiofur treatment group was the only group in which the total bacterial 16S rRNA gene count became similar to that in the nonmetritic group by day 12. In summary, chitosan microparticles slowed the progression of the uterine microbiome toward a healthy state, whereas ceftiofur hastened the progression toward a healthy state.IMPORTANCE Third-generation cephalosporins, such as ceftiofur, are commonly used to treat metritis in dairy cows. Chitosan microparticles has been shown to have a broad spectrum of activity in vitro and to be effective against uterine pathogens in vivo; therefore, they have been hailed as a possible alternative to traditional antibiotics. Nonetheless, in the present study, we saw that chitosan microparticle treatment slowed the progression of the uterine microbiome of cows with metritis toward a healthy state, whereas ceftiofur treatment hastened the progression toward a healthy state. Given the lack of an effective alternative to traditional antibiotics and an increased concern about antimicrobial resistance, a greater effort should be devoted to the prevention of metritis in dairy cows.

In vivo effects of the association of the psychoactive phenotiazine thioridazine on antitumor activity and hind limb paralysis induced by the native polypeptide crotamine.

Crotamine is a cationic polypeptide composed by 42 amino acid residues with several pharmacological and biological properties, including the selective ability to enter and kill actively proliferating tumour cells, which led us to propose its use as a theranostic agent for cancer therapy. At the moment, the improvement of crotamine antitumoral efficacy by association with chemotherapeutic adjuvants is envisioned. In the present work, we evaluated the association of crotamine with the antitumoral adjuvant phenotiazine thioridazine (THD). In spite of the clear efficacy of these both compounds as anticancer agents in long-term in vivo treatment of animal model bearing implanted xenograph melanoma tumor, the expected mutual potentiation of the antitumor effects was not observed here. Moreover, this association revealed for the first time the influence of THD on crotamine ability to trigger the hind limb paralysis in mice, and this discovery may represent the first report suggesting the potential involvement of the CNS in the action of this snake polypeptide on the skeletal muscle paralysis, which was classically believed to be essentially limited to a direct action in peripheral tissues as the skeletal muscle. This is also supported by the observed ability of crotamine to potentiate the sedative effects of THD which action was consistently demonstrated to be based on its central action. The better characterization of crotamine properties in CNS may certainly bring important insights for the knowledge needed to pave the way toward the use of this molecule as a theranostic compound in human diseases as cancer.

Crotamine Cell-Penetrating Nanocarriers: Cancer-Targeting and Potential Biotechnological and/or Medical Applications.

Crotamine is a basic, 42-residue polypeptide from snake venom that has been shown to possess cell-penetrating properties. Here we describe the preparation, purification, biochemical and biophysical analysis of venom-derived, recombinant, chemically synthesized, and fluorescent-labeled crotamine. We also describe the formation and characterization of crotamine-DNA and crotamine-RNA nanoparticles; and the delivery of these nanoparticles into cells and animals. Crotamine forms nanoparticles with a variety of DNA and RNA molecules, and crotamine-plasmid DNA nanoparticles are selectively delivered into actively proliferating cells in culture or in living organisms such as mice, Plasmodium, and worms. As such, these nanoparticles could form the basis for a nucleic acid drug-delivery system. We also describe here the design and characterization of crotamine-functionalized gold nanoparticles, and the delivery of these nanoparticles into cells. We also evaluated the viability of using the combination of crotamine with silica nanoparticles in animal models, aiming to provide slow delivery, and to decrease the crotamine doses needed for the biological effects. In addition, the efficacy of administering crotamine orally was also demonstrated.

Effects of the Natural Peptide Crotamine from a South American Rattlesnake on Candida auris, an Emergent Multidrug Antifungal Resistant Human Pathogen.

Invasive Candida infections are an important growing medical concern and treatment options are limited to a few antifungal drug classes, with limited efficacies depending on the infecting organism. In this scenario, invasive infections caused by multiresistant Candida auris are emerging in several places around the world as important healthcare-associated infections. As antimicrobial peptides (AMPs) exert their activities primarily through mechanisms involving membrane disruption, they have a lower chance of inducing drug resistance than general chemical antimicrobials. Interestingly, we previously described the potent candicidal effect of a rattlesnake AMP, crotamine, against standard and treatment-resistant clinical isolates, with no hemolytic activity. We evaluated the antifungal susceptibility of several Candida spp. strains cultured from different patients by using the Clinical and Laboratory Standards Institute (CLSI) microdilution assay, and the antifungal activity of native crotamine was evaluated by a microbial growth inhibition microdilution assay. Although all Candida isolates evaluated here showed resistance to amphotericin B and fluconazole, crotamine (40-80 µM) exhibited in vitro activity against most isolates tested. We suggest that this native polypeptide from the South American rattlesnake Crotalus durissus terrificus has potential as a structural model for the generation of a new class of antimicrobial compounds with the power to fight against multiresistant Candida spp.

Camu-camu (Myrciaria dubia) from commercial cultivation has higher levels of bioactive compounds than native cultivation (Amazon Forest) and presents antimutagenic effects in vivo.

BACKGROUND: Camu-camu (Myrciaria dubia) is a typical Amazonian fruit and has high antioxidant capacity due to its high levels of vitamin C and phenolic compounds. This study aimed to determine the phytochemicals, antioxidant capacity and antimutagenic effects of camu-camu fruits with different maturity stages grown in dry (commercial cultivation) or flooded environments (native cultivation, Amazon). RESULTS: Total polyphenols, ascorbic acid and in vitro antioxidant capacity levels were higher in ripe fruits grown in a commercial cultivation. The extracts from ripe camu-camu grown in a commercial cultivation exerted antioxidant effects and high percentage of protection against doxorubicin and 1,2-dimethylhydrazine in all tested systems (liver, bone marrow and gut), for three camu-camu extract concentrations (17, 85 and 170 mg kg-1 body weight), as follows: bone marrow minocronucleus (37.91%, 41.75%, 43.95%); micronucleus gut test (61.01%, 64.40%, 50.28%); apoptosis index (60.26%, 62.44%, 58.22%); comet assay through the tail moment (71.64%, 72.31%, 70.70%), percent DNA in the tail (64.54%, 68.75%, 76.79%) and tail intensity (76.43%, 81.02%, 68.33%). CONCLUSION: The results of this study contribute to increasing the production of camu-camu fruits grown in dry environments and their use as a health-promoting food. © 2018 Society of Chemical Industry.

Effects of the natural peptide crotamine from a south american rattlesnake on Candida auris, an emergent multidrug antifungal resistant human pathogen

Invasive Candida infections are an important growing medical concern and treatment options are limited to a few antifungal drug classes, with limited efficacies depending on the infecting organism. In this scenario, invasive infections caused by multiresistant Candida auris are emerging in several places around the world as important healthcare-associated infections. As antimicrobial peptides (AMPs) exert their activities primarily through mechanisms involving membrane disruption, they have a lower chance of inducing drug resistance than general chemical antimicrobials. Interestingly, we previously described the potent candicidal effect of a rattlesnake AMP, crotamine, against standard and treatment-resistant clinical isolates, with no hemolytic activity. We evaluated the antifungal susceptibility of several Candida spp. strains cultured from different patients by using the Clinical and Laboratory Standards Institute (CLSI) microdilution assay, and the antifungal activity of native crotamine was evaluated by a microbial growth inhibition microdilution assay. Although all Candida isolates evaluated here showed resistance to amphotericin B and fluconazole, crotamine (40–80 µM) exhibited in vitro activity against most isolates tested. We suggest that this native polypeptide from the South American rattlesnake Crotalus durissus terrificus has potential as a structural model for the generation of a new class of antimicrobial compounds with the power to fight against multiresistant Candida spp.

Effects of the natural peptide crotamine from a south american rattlesnake on Candida auris, an emergent multidrug antifungal resistant human pathogen

Invasive Candida infections are an important growing medical concern and treatment options are limited to a few antifungal drug classes, with limited efficacies depending on the infecting organism. In this scenario, invasive infections caused by multiresistant Candida auris are emerging in several places around the world as important healthcare-associated infections. As antimicrobial peptides (AMPs) exert their activities primarily through mechanisms involving membrane disruption, they have a lower chance of inducing drug resistance than general chemical antimicrobials. Interestingly, we previously described the potent candicidal effect of a rattlesnake AMP, crotamine, against standard and treatment-resistant clinical isolates, with no hemolytic activity. We evaluated the antifungal susceptibility of several Candida spp. strains cultured from different patients by using the Clinical and Laboratory Standards Institute (CLSI) microdilution assay, and the antifungal activity of native crotamine was evaluated by a microbial growth inhibition microdilution assay. Although all Candida isolates evaluated here showed resistance to amphotericin B and fluconazole, crotamine (40–80 µM) exhibited in vitro activity against most isolates tested. We suggest that this native polypeptide from the South American rattlesnake Crotalus durissus terrificus has potential as a structural model for the generation of a new class of antimicrobial compounds with the power to fight against multiresistant Candida spp.

Crotamine induces browning of adipose tissue and increases energy expenditure in mice.

Crotamine, originally isolated from rattlesnake venom, has been extensively studied due to its pleiotropic biological properties, and special attention has been paid to its antitumor activity. However, long-term treatment with crotamine was accompanied by a reduction in animal body weight gain and by increases in glucose tolerance. As cancer is commonly associated with cachexia, to preclude the possible cancer cachexia-like effect of crotamine, herein this polypeptide was administered in healthy wild-type C57/BL6 mice by the oral route daily, for 21 days. Reduced body weight gain, in addition to decreased white adipose tissue (WAT) and increased brown adipose tissue (BAT) mass were observed in healthy animals in the absence of tumor. In addition, we observed improved glucose tolerance and increased insulin sensitivity, accompanied by a reduction of plasma lipid levels and decreased levels of biomarkers of liver damage and kidney disfunctions. Importantly, long-term treatment with crotamine increased the basal metabolic rate in vivo, which was consistent with the increased expression of thermogenic markers in BAT and WAT. Interestingly, cultured brown adipocyte cells induced to differentiation in the presence of crotamine also showed increases in some of these markers and in lipid droplets number and size, indicating increased brown adipocyte maturation.

Oral treatment with a rattlesnake native polypeptide crotamine efficiently inhibits the tumor growth with no potential toxicity for the host animal and with suggestive positive effects on animal metabolic profile.

The efficacy of crotamine as antitumoral was first demonstrated by daily intraperitoneal (IP) injections of low doses of this toxin in an animal model bearing melanoma tumors. Significant inhibition of tumor growth and increased lifespan of mice bearing tumor was also noticed after 21 consecutive days of this daily IP administration of crotamine. However, due to the limited acceptance of treatments by IP route in clinical conditions, herein, we evaluated the antitumor effect of this native polypeptide employing the oral route. The efficacy of crotamine in inhibiting the melanoma growth in vivo, even after passing through the gastrointestinal tract of the animal, was confirmed here. In addition, biochemical biomarkers and also histopathological analysis showed both the absence of any potential toxic effects in tissues or organs of the animal in which the highest accumulation of crotamine is expected. Interestingly, a reduction of weight gain was observed mainly in animals with tumor treated with crotamine by IP route, but not by oral administration. Albeit, oral administered crotamine was able to significantly decrease the body weight gain of healthy animals without tumor. Taking advantage of this same experimental animal models receiving crotamine by oral route, it was possible to show metabolic changes as the increased capacity of glucose clearance, which was accompanied by a reduction of the total cholesterol, and by increased high-density lipoprotein levels, both observed mainly in the absence of tumor. Triglycerides and low-density lipoprotein were also significantly decreased, but only in the absence of tumor. Taken together, these data suggest a clear trend for metabolic positive effects and mischaracterize unhealthy condition of animals, with or without tumors, treated with crotamine for 21 days. In addition, this study confirmed the efficacy of crotamine administered by oral route as antitumor agent, which besides the additional advantage of administration convenience and decreased risk of toxic effects, allowed the serendipitous observation of several positive metabolic effects on treated animals.

Design and characterization of crotamine-functionalized gold nanoparticles.

This paper describes the development of a facile and environmentally friendly strategy for supporting crotamine on gold nanoparticles (GNPs). Our approach was based on the covalent binding interaction between the cell penetrating peptide crotamine, which is a snake venom polypeptide with preference to penetrate dividing cells, and a polyethylene glycol (PEG) ligand, which is a nontoxic, water-soluble and easily obtainable commercial polymer. Crotamine was derivatized with ortho-pyridyldisulfide-polyethyleneglycol-N-hydroxysuccinimide (OPSS-PEG-SVA) cross-linker to produce OPSS-PEG-crotamine as the surface modifier of GNP. OPSS-PEG-SVA can serve not only as a surface modifier, but also as a stabilizing agent for GNPs. The successful PEGylation of the nanoparticles was demonstrated using different physicochemical techniques, while the grafting densities of the PEG ligands and crotamine on the surface of the nanoparticles were estimated using a combination of electron microscopy and mass spectrometry analysis. In vitro assays confirmed the internalization of these GNPs, into living HeLa cells. The results described herein suggest that our approach may serve as a simple platform for the synthesis of GNPs decorated with crotamine with well-defined morphologies and uniform dispersion, opening new roads for crotamine biomedical applications.

Development and Validation of Prediction Equations for Spinal Curve Angles Based on Skin Surface Measurements.

OBJECTIVE: The purpose of this study was to develop, assess the reliability of, and validate prediction equations that estimate the sagittal curves of the spine from the skin surface. METHODS: Forty digital panoramic radiographs were used to develop the prediction equation, and 59 radiographs were used to assess reliability and validate the equations. For evaluation of the thoracic and lumbar curves, anatomical reference points were marked on the vertebral body, spinous process, and skin surface at the C6, C7, T2, T4, T6, T8, T10, T12, L2, L4, and S2 vertebrae. Three third-degree polynomials were obtained, estimated with the least squares method: inner curves from the centroid of the vertebral bodies and from the apex of the spinous processes and external curve from the skin surface. The magnitude of the curves of each region was estimated based on the angle between tangent lines at several vertebral levels. Prediction equations were obtained (simple linear regression) for the vertebral levels that had the best correlation between the inner and surface curves. The validation of the prediction equations was confirmed using Pearson's correlation (r), Student t test, and root mean square error. The reliability of the method was confirmed using the intraclass correlation coefficient, standard error of measurement, and minimal detectable change (α = 0.05). RESULTS: The best correlations were obtained between the T4-T12 (thoracic) and T10-S2 (lumbar) levels (r > 0.85). For the intrarater and interrater reliability, the correlation was higher than 0.965 and higher than 0.896, respectively. There was a significant and strong correlation between estimated and actual values for the thoracic and lumbar curves, which was confirmed by the t-test results and by the root mean square error inferior to 1°. CONCLUSION: Prediction equations can precisely and accurately estimate the angles of the internal sagittal curves of the spine from the skin surface.