•BjussuLAAO-II is a snake venom toxin with cytotoxic effects towards Caco-2 cells.•The toxin induces necrosis, apoptosis, oxidative stress, and DNA damage.•The toxin reduces cell viability by acting ...on multiple intracellular targets.•The toxin upregulates the inflammatory cytokine genes TNF and IL6 expression.•The toxin downregulates the apoptotic-related genes BAX, BCL2 and RELA expression.
Colorectal carcinoma is one of the most common cancers in adults. As chemotherapy, the first-choice treatment for colorectal carcinoma, is often infeasible due to acquired tumor resistance and several adverse effects, it is important to discover and explore new molecules with better therapeutic action. Snake venom toxins have shown promising results with high cytotoxicity against tumor cells, but their mechanisms of action remain unclear. Here we examined how BjussuLAAO-II, an L-amino acid oxidase isolated from Bothrops jararacussu snake venom, exerts cytotoxicity towards colorectal adenocarcinoma human cells (Caco-2) and human umbilical vein endothelial cell line (HUVEC). A 24-h treatment with BjussuLAAO-II at 0.25 − 5.00 μg/mL diminished cell viability by decreasing (i) mitochondrial activity, assessed by reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and resazurin; (ii) the activity of acid phosphatases; and (iii) lysosomal function, assessed by neutral red uptake. BjussuLAAO-II also increased intracellular levels of reactive oxygen species and DNA damage, as assessed by fluorescence and the comet assay, respectively. BjussuLAAO-II altered the expression of cell proliferation-related genes, as determined by RT-qPCR: it elevated the expression of the inflammatory cytokine genes TNF and IL6, and lowered the expression of the apoptotic-related genes BAX, BCL2, and RELA. Therefore, BjussuLAAO-II induces Caco-2 cells death by acting on multiple intracellular targets, providing important data for further studies to assess whether these effects are seen in both tumor and normal cells, with the aim of selecting this drug for possible therapeutic purposes in the future.
The objectives were to study the diversity of Enterococcus spp. isolated from mastitis cases, milking equipment and the environment of dairy cows; and to determine in vitro resistance of isolates to ...antimicrobials that are relevant to human and animal health. Ten dairy farms were visited to collect samples from mastitis cases, faeces, bedding, aisles, water and milking equipment. Identification of Enterococcus at the species level and antimicrobial resistance testing was performed by MALDI‐TOF and the disk‐diffusion method, respectively. Of 365 isolates, Enterococcus hirae was the most prevalent, being more likely to be isolated from faeces than from milk (odds ratio (OR) = 39·2), liners (OR = 5·4) or bedding (OR = 2·2). Enterococcus saccharolyticus was the most prevalent in milk samples. The chances of isolating Enterococcus faecalis from milk were higher than from aisles (OR = 12·5), faeces (OR = 5·3), bedding (OR = 3·6) or liners (OR = 3·0). The odds of isolating Enterococcus faecium from faeces were higher than from liners (OR = 7·3), bedding (OR = 2·5) or aisles (OR = 2·4). Of 360 tested isolates, 1·9, 0·3 and 0·6% were resistant to penicillin, vancomycin and teicoplanin, respectively. Our results suggest that Enterococcus species can occupy specific ecological niches on dairy farms and pose a risk to public and animal health.
Significance and Impact of the Study: Enterococcus spp. can cause diseases in both humans and animals and are widely found in the environment. Results of this study show that the distribution of Enterococcus species was not similar between farm sources, such as mastitis cases, milking equipment and the environment of the cows, suggesting that species can occupy specific ecological niches. We also observed isolates that were resistant to antimicrobials that are relevant to animal and human health. Thus, this study highlights the risk of Enterococcus for animal and public health and the need for epidemiological monitoring of such pathogens on dairy farms.
We investigated the genes kpsMTII, iucD, sfaDE, afaBC, papA and papC, (proposed to be involved in extra‐intestinal pathogenic Escherichia coli—ExPEC), phylogroup classification and the in vitro ...swimming and swarming motility in 50 E. coli isolated from bovine mastitis with different clinical severity scores (mild, moderate and severe). The aforementioned genes were detected in 12 (n = 12/50; 24·0%) isolates. kpsMTII and iucD were the most frequent genes identified in six (n = 6/50; 12·0%) and four (n = 4/50; 8·0%) of the isolates, respectively. In only one (n = 1/50; 2·0%) isolate, more than one gene was simultaneously identified: iucD and kpsMTll were detected whereas sfaDE and afaBC were not detected. Mild, moderate and severe clinical signs were observed in 40·0% (n = 20/50), 28·0% (n = 14/50) and 32·0% (n = 16/50) of the cases. Commensal phylogroups B1 (n = 19/50; 38·0%) and A (n = 19/50; 38·0%) were prevalent; whereas pathogenic phylogroups B2 and D were observed in only 10·0% (n = 5/50). Swarming and swimming motilities were observed in 90·0% (n = 45/50) and 68·0% (n = 34/50) of the isolates, respectively; and there was a significant association (P = 0·0036) between swarming motility and severe clinical cases (score 3). To the best of our knowledge, this is the first study where clinical severity of bovine mastitis cases and the genes proposed to classify ExPEC were assessed in relation to swarming and swimming motility.
Significance and Impact of the Study
Escherichia coli is classified as extra‐intestinal (ExPEC) when strains contain at least two of the genes kpsMTII, iucD, sfaDE, afaBC and papA and/or papC. We investigated in vitro motility and the presence of these genes in 50 E. coli isolated from bovine mastitis with different clinical scores (mild, moderate and severe). Clinical severity was not associated with the genes studied. Swarming motility was associated with severe cases (score 3) of clinical mastitis. Results of this study contribute to a better understanding of the factors that determine the severity of clinical mastitis.
Significance and Impact of the Study: Escherichia coli is classified as extra‐intestinal (ExPEC) when strains contain at least two of the genes kpsMTII, iucD, sfaDE, afaBC and papA and/or papC. We investigated in vitro motility and the presence of these genes in 50 E. coli isolated from bovine mastitis with different clinical scores (mild, moderate and severe). Clinical severity was not associated with the genes studied. Swarming motility was associated with severe cases (score 3) of clinical mastitis. Results of this study contribute to a better understanding of the factors that determine the severity of clinical mastitis.
Although more complex than usually described, the anticancer action mechanism of cisplatin is based on binding to DNA. Following this line of reasoning, most the metal-based compounds discovered soon ...after cisplatin were designed to acting as DNA-binding agents and their pharmacological properties were thought to be correlated with this mechanism. Apart from the DNA structure, a significant number of proteins and biochemical pathways have been described as drug targets for metal-based compounds. This paper is therefore aimed at discussing the most recent findings on the medicinal chemistry of metal-based drugs. It starts illustrating the design concept behind the bioinorganic chemistry of anticancer complexes. Anticancer metallic compounds that inhibit the protein kinases are concisely discussed as a case study. The accuracy and limitations of molecular docking programs currently available to predict the binding mode of metallic complexes in molecular targets are further discussed. Finally, the advantages and disadvantages of different in vitro screenings are briefly commented.
Carbohydrate biopolymers of fungal-origin are an important natural resource in the search for new bioagents with therapeutic and nutraceutical potential. In this study the mutagenic, genotoxic, ...antigenotoxic and antioxidant properties of the fungal exopolysaccharide botryosphaeran, a (1→3)(1→6)-β-D-glucan, from Botryosphaeria rhodina MAMB-05, was evaluated. The mutagenicity was assessed at five concentrations in Salmonella typhimurium by the Ames test. Normal and tumor (Jurkat cells) human T lymphocyte cultures were used to evaluate the genotoxicity and antigenotoxicity (Comet assay) of botryosphaeran alone and in combination with the mutagen methyl methanesulfonate (MMS). The ability of botryosphaeran to reduce the production of reactive oxygen and nitrogen species (RONS) generated by hydrogen peroxide was assessed using the CM-H2DCFDA probe in lymphocyte cultures under different treatment times. None of the evaluated botryosphaeran concentrations were mutagenic in bacteria, nor induced genotoxicity in normal and tumor lymphocytes. Botryosphaeran protected lymphocyte DNA against damage caused by MMS under simultaneous treatment and post-treatment conditions. However, botryosphaeran was not able to reduce the RONS generated by H2O2. Besides the absence of genotoxicity, botryosphaeran exerted a protective effect on human lymphocytes against genotoxic damage caused by MMS. These results are important in the validation of botryosphaeran as a therapeutic agent targeting health promotion.
Field walking tests are commonly employed to evaluate exercise capacity, assess prognosis and evaluate treatment response in chronic respiratory diseases. In recent years, there has been a wealth of ...new literature pertinent to the conduct of the 6-min walk test (6MWT), and a growing evidence base describing the incremental and endurance shuttle walk tests (ISWT and ESWT, respectively). The aim of this document is to describe the standard operating procedures for the 6MWT, ISWT and ESWT, which can be consistently employed by clinicians and researchers. The Technical Standard was developed by a multidisciplinary and international group of clinicians and researchers with expertise in the application of field walking tests. The procedures are underpinned by a concurrent systematic review of literature relevant to measurement properties and test conduct in adults with chronic respiratory disease. Current data confirm that the 6MWT, ISWT and ESWT are valid, reliable and responsive to change with some interventions. However, results are sensitive to small changes in methodology. It is important that two tests are conducted for the 6MWT and ISWT. This Technical Standard for field walking tests reflects current evidence regarding procedures that should be used to achieve robust results.
This systematic review examined the measurement properties of the 6-min walk test (6MWT), incremental shuttle walk test (ISWT) and endurance shuttle walk test (ESWT) in adults with chronic ...respiratory disease. Studies that report the evaluation or use of the 6MWT, ISWT or ESWT were included. We searched electronic databases for studies published between January 2000 and September 2013. The 6-min walking distance (6MWD) is a reliable measure (intra-class correlation coefficients ranged from 0.82 to 0.99 in seven studies). There is a learning effect, with greater distance walked on the second test (pooled mean improvement of 26 m in 13 studies). Reliability was similar for ISWT and ESWT, with a learning effect also evident for ISWT (pooled mean improvement of 20 m in six studies). The 6MWD correlates more strongly with peak work capacity (r=0.59-0.93) and physical activity (r=0.40-0.85) than with respiratory function (r=0.10-0.59). Methodological factors affecting 6MWD include track length, encouragement, supplemental oxygen and walking aids. Supplemental oxygen also affects ISWT and ESWT performance. Responsiveness was moderate to high for all tests, with greater responsiveness to interventions that included exercise training. The findings of this review demonstrate that the 6MWT, ISWT and ESWT are robust tests of functional exercise capacity in adults with chronic respiratory disease.
The aims of this study were to evaluate the intra- and interobserver reproducibility of manual segmentation of bone sarcomas in magnetic resonance imaging (MRI) studies and to compare manual and ...semiautomatic segmentation methods. This retrospective study included twelve osteosarcoma and eight Ewing sarcoma MRI studies performed prior to any therapeutic intervention. All cases were histopathologically confirmed. Three radiologists used 3D-Slicer software to perform manual segmentation of bone sarcomas in a blinded and independent manner. One radiologist segmented manually and also performed semiautomatic segmentation with the GrowCut tool. Segmentation exercises were timed for comparison. The dice similarity coefficient (DSC) and Hausdorff distance (HD) were used to evaluate similarity between the segmentation results and further statistical analyses were performed to compare DSC, HD, and volumetric results. Manual segmentation was reproducible with intraobserver DSC varying from 0.83 to 0.97 and HD from 3.37 to 28.73 mm. Interobserver DSC of manual segmentation showed variation from 0.73 to 0.97 and HD from 3.93 to 33.40 mm. Semiautomatic segmentation compared to manual segmentation resulted in DSCs of 0.71-0.96 and HDs of 5.38-31.54 mm. Semiautomatic segmentation required significantly less time compared to manual segmentation (P value ≤0.05). Among all situations compared, tumor volumetry did not show significant statistical differences (P value >0.05). We found excellent intra- and interobserver agreement for manual segmentation of osteosarcoma and Ewing sarcoma. There was high similarity between manual and semiautomatic segmentation, with a significant reduction of segmentation time using the semiautomatic method.
The down-conversion process in Tb3+–Yb3+ co-doped Calibo glasses was studied. The emission, excitation and time-resolved measurements indicated the existence of an energy conversion through the ...excitation of Tb3+ ions to near-infrared emission by Yb3+ ions. The emission intensity dependence on excitation power confirms that the one-photon process is responsible for the Yb3+ emission. An enhanced Yb3+ emission was observed with Yb3+ doping and an optimal energy transfer efficiency of 32% was obtained before reaching near-infrared emission quenching. The mechanism of the non-resonant energy transfer from Tb3+ to Yb3+ is discussed in terms of the Tb3+–Yb3+ cross-relaxation and multiphonon decay processes.
► The down-conversion process was observed for the first time in Tb3+–Yb3+ co-doped Calibo glasses. ► Excitation power measurements allowed identification of the one-photon process in the energy transfer from Tb3+ to Yb3+. ► An enhanced near-infrared emission was observed from Yb3+ with almost 32% for the energy transfer efficiency. ► Tb3+–Yb3+ cross-relaxation and multiphonon decay processes explain all these results. ► This system has potential application as a down-converter material for the near-infrared region.
To characterize the chemical interactions and analyze the interface of adhesive systems containing 10-methacryloyloxydecyl dihydrogen phosphate (10-MDP) and N-methacryloyl glycine (methacrylamide) ...functional monomers with the dentin in noncarious cervical lesions (NCCLs) compared with artificial defects (ADs).
Twenty human teeth with natural NCCLs on the buccal surface were used. Class V cavities, similar to NCCLs, were created on the lingual surface to serve as controls. Teeth were randomly allocated to two groups according to the functional monomer in the adhesive (N=10): G1, 10-MDP; and G2, methacrylamide. NCCLs and ADs were characterized by their mineral composition (MC) and degree of demineralization (DD) using micro-Raman spectroscopy, adhesive/dentin chemical interactions (CIs) were assessed with infrared photoacoustic spectroscopy, and interface morphology was evaluated with scanning electron and light microscopy. MC, CI, and DD data were submitted to Shapiro-Wilk and Student t-tests ( p<0.05).
Compared with ADs, dentin in NCCLs was hypermineralized ( p<0.05). In G1, CI, and DD in the first 2 μm, and adhesive projections in NCCLs and ADs interfaces were similar. Additionally, a thin layer of dentin collagen was observed in ADs, while it was hardly present in NCCLs. In G2, although CI could not be identified, changes in the mineral components were observed. The DD in the ADs and NCCLs were statistically similar, while SEM showed a lack of adhesion at NCCLs interface. DD and collagen exposure in the ADs and NCCLs were more pronounced than in G1.
Results suggest that the G1 adhesive could be applied directly on the superficial sclerotic layer in NCCLs. In contrast, previous cavity preparation should be conducted to improve the micromechanical interaction of G2 with the dentin.
PDF
