A biofloc system avoids the adverse effects of diets with suboptimal protein levels on zootechnical performance, intestinal histomorphometry, and protein metabolism of Nile tilapia juvenile fed Spirulina biomass (Arthrospira platensis) as an alternative protein source.

This study aimed to evaluate the effect of the biofloc technology (BFT) system and the replacement of fish meal with Spirulina biomass on productive performance, intestinal histomorphometry, plasma biochemistry, and oxidative stress of Nile tilapia juveniles (Oreochromis niloticus) fed suboptimal levels of protein. Two factors were evaluated: production systems (clear water × BFT) and replacement of fish meal with Spirulina (0, 33, 66 e 100%). The design was in a 2 × 4 randomized factorial scheme with four replications, and the fish were evaluated for 48 days. Four isoproteic (28% crude protein) diets were formulated with gross energy values close to 4300 kcal kg-1. Nile tilapia juveniles (0.23 ± 0.01 g) were distributed in 16 circular tanks (70 L) at seven fish/tank. The diets were formulated with protein levels approximately 20% below that required for the species and life stage. No interaction was observed between the factors evaluated (production systems × Spirulina inclusion). Rearing the fish in the BFT system avoided the adverse effects of diets with suboptimal protein levels on performance, intestinal histomorphometry, and protein metabolism. Lower values lower lipid peroxidation and higher antioxidant capacity were observed in fish reared in the BFT system, showing evidence of improvements in antioxidant responses and lower levels of physiological oxidative stress. Spirulina completely replaced fish meal in the diets of Nile tilapia juveniles without adverse effects on intestinal morphometry, protein metabolism, and antioxidant response. Replacing 66% of fish meal with Spirulina improved the productive performance, regardless of the rearing system.

Dot blot platform as a novel diagnostic kit: rapid, accurate, and on-site detection of Schistosoma mansoni in urine samples of hard to detect individuals.

Rapid diagnostics provide actionable information for patient care at the time and site of an encounter with the health care system. The mainstay of infectious diseases care is early detection (case finding) and treatment completion, but for many, it is hard to identify positive individuals, as is the case of infection with low burden in schistosomiasis, a parasitic disease common in the tropics and subtropics. We developed a new, accurate, and fast Dot blot methodology (iDot) to indirectly detect Schistosoma mansoni in individuals with very low parasite burden using urine samples. Accuracy of 0.74 was obtained with a significant difference between negative and positive patients and a substantial agreement was found when iDot was compared with five available methods. Our analysis also revealed the superiority of iDot in detecting negative individuals from non-endemic sites, thus, presenting the lowest rate of false positives. This new method called iDot is convenient and suitable for qualitative and quantitative detection of schistosomiasis in individuals with low parasite burden.

L-Methionine inhibits growth of human pancreatic cancer cells.

We have previously shown that L-methionine inhibits proliferation of breast, prostate, and colon cancer cells. This study extends these findings to BXPC-3 (mutated p53) and HPAC (wild-type p53) pancreatic cancer cells and explores the reversibility of these effects. Cells were exposed to L-methionine (5 mg/ml) for 7 days or for 3 days, followed by 4 days of culture without L-methionine (recovery). Cell proliferation, apoptosis, and cell cycle effects were assessed by flow cytometry after staining for Ki-67 or annexin V/propidium iodide. Cell proliferation was reduced by 31-35% after 7 days of methionine exposure; the effect persisted in BXPC-3 and HPAC cells after 4 days of recovery. Methionine increased apoptosis by 40-75% in HPAC cells, but not in BXPC-3 cells. Continuous exposure to methionine caused accumulation of BXPC-3 cells in the S phase and HPAC cells in both the G0/G1 and S phases; however, after 4 days of recovery, these effects disappeared. In conclusion, L-methionine inhibits proliferation and interferes with the cell cycle of BXPC-3 and HPAC pancreatic cancer cells; the effects on apoptosis remarkably persisted after methionine withdrawal. Apoptosis was induced only in BXPC-3 cells. Some of the differences in the effects of methionine between cell lines may be related to disparate p53 status. These findings warrant further studies on the potential therapeutic benefit of L-methionine against pancreatic cancer.

A further pocket or conformational plasticity by mapping COX-1 catalytic site through modified-mofezolac structure-inhibitory activity relationships and their antiplatelet behavior.

Cyclooxygenase enzymes have distinct roles in cardiovascular, neurological, and neurodegenerative disease. They are differently expressed in different type of cancers. Specific and selective COXs inhibitors are needed to be used alone or in combo-therapies. Fully understand the differences at the catalytic site of the two cyclooxygenase (COX) isoforms is still opened to investigation. Thus, two series of novel compounds were designed and synthesized in fair to good yields using the highly selective COX-1 inhibitor mofezolac as the lead compound to explore a COX-1 zone formed by the polar residues Q192, S353, H90 and Y355, as well as hydrophobic amino acids I523, F518 and L352. According to the structure of the COX-1:mofezolac complex, hydrophobic amino acids appear to have free volume eventually accessible to the more sterically hindering groups than the methoxy linked to the phenyl groups of mofezolac, in particular the methoxyphenyl at C4-mofezolac isoxazole. Mofezolac bears two methoxyphenyl groups linked to C3 and C4 of the isoxazole core ring. Thus, in the novel compounds, one or both methoxy groups were replaced by the higher homologous ethoxy, normal and isopropyl, normal and tertiary butyl, and phenyl and benzyl. Furthermore, a major difference between the two sets of compounds is the presence of either a methyl or acetic moiety at the C5 of the isoxazole. Among the C5-methyl series, 12 (direct precursor of mofezolac) (COX-1 IC50 = 0.076 µM and COX-2 IC50 = 0.35 µM) and 15a (ethoxy replacing the two methoxy groups in 12; COX-1 IC50 = 0.23 µM and COX-2 IC50 > 50 µM) were still active and with a Selectivity Index (SI = COX-2 IC50/COX-1 IC50) = 5 and 217, respectively. The other symmetrically substituted alkoxyphenyl moietis were inactive at 50 µM final concentration. Among the asymmetrically substituted, only the 16a (methoxyphenyl on C3-isoxazole and ethoxyphenyl on C4-isoxazole) and 16b (methoxyphenyl on C3-isoxazole and n-propoxyphenyl on C4-isoxazole) were active with SI = 1087 and 38, respectively. Among the set of compounds with the acetic moiety, structurally more similar to mofezolac (SI = 6329), SI ranged between 1.4 and 943. It is noteworthy that 17b (n-propoxyphenyl on both C3- and C4-isoxazole) were found to be a COX-2 slightly selective inhibitor with SI = 0.072 (COX-1 IC50 > 50 µM and COX-2 IC50 = 3.6 µM). Platelet aggregation induced by arachidonic acid (AA) can be in vitro suppressed by the synthesized compounds, without affecting of the secondary hemostasia, confirming the biological effect provided by the selective inhibition of COX-1. A positive profile of hemocompatibility in relation to erythrocyte and platelet toxicity was observed. Additionally, these compounds exhibited a positive profile of hemocompatibility and reduced cytotoxicity. Quantitative structure activity relationship (QSAR) models and molecular modelling (Ligand and Structure based virtual screening procedures) provide key information on the physicochemical and pharmacokinetic properties of the COX-1 inhibitors as well as new insights into the mechanisms of inhibition that will be used to guide the development of more effective and selective compounds. X-ray analysis was used to confirm the chemical structure of 14 (MSA17).

N, N'-disubstituted Ureas as Novel Antiplatelet Agents: Synthesis, Pharmacological Evaluation and In Silico Studies.

INTRODUCTION: Thrombotic disorders are among the leading causes of morbidity and mortality worldwide. Drugs used in the prevention and treatment of atherothrombosis have pharmacokinetic limitations and adverse effects such as hemorrhagic conditions, highlighting the importance of developing more effective antiplatelet agents. ethod: In this work, we synthesized N,N'-disubstituted ureas 3a-3j and evaluated their antiplatelet profiles through in vitro, ex vivo, and in silico studies. The synthesized derivatives exhibited a selective inhibitory profile against platelet aggregation induced by arachidonic acid (AA) in vitro, without significantly affecting other aspects of primary hemostasis and blood coagulation. The compounds that showed inhibition greater than 85% were submitted to the analysis of their potency by calculating the concentration required to inhibit 50% of platelet aggregation induced by AA (IC50). Urea derivative 3a was the most potent with IC50 of 1.45 µM. Interestingly, this derivative inhibited more than 90% of platelet aggregation induced by AA ex vivo, with a similar effect to acetylsalicylic acid. In the hemolysis assay, most of the urea derivatives presented values below 10% suggesting good hemocompatibility. Additionally, the compounds tested at 100 µM also showed no cytotoxic effects in HepG2 and Vero cells. RESULT: The in silico results suggested that compound 3a may bind to the key residue of COX-1 similar to AA and known COX-1 inhibitors, and the results are also in agreement with our SAR, which suggests that the inhibition of this enzyme is the most likely mechanism of antiplatelet activity. CONCLUSION: Therefore, these results demonstrated that N,N'-disubstituted ureas are promising candidates for the development of novel antiplatelet agents.

In vivo evaluation of time-dependent antithrombotic effect of rivaroxaban-loaded poly(lactic-co-glycolic acid)/sodium lauryl sulfate or didodecyl dimethylammonium bromide nanoparticles in Wistar rats.

Rivaroxaban (RVX), an oral direct factor Xa inhibitor, is being explored as an alternative to traditional anticoagulans. However, RVX still faces pharmacokinetic limitations and adverse effects, highlighting the need for more effective formulations. In this regard, pharmaceutical nanotechnology, particularly the use of polymeric nanoparticles (PNPs), offers a promising approach for optimizing RVX delivery. This study aimed to develop and physicochemically characterize RVX-loaded poly(lactic-co-glycolic acid) (PLGA)/sodium lauryl sulfate (SLS) or didodecyl dimethylammonium bromide (DMAB) nanoparticles, and also evaluate their pharmacological and toxicological profiles as a potential therapeutic strategy. The PNPs exhibited sizes below 300 nm and spherical morphology, with both negative and positive surface charges, according to surfactant used. They demonstrated high encapsulation efficiency and suitable yields, as well as rapid initial liberation followed by sustained release in different pH environments. Importantly, in vivo evaluations revealed a time-dependent antithrombotic effect surpassing the free form of RVX when administered orally in SLS or DMAB PNP. No hemolytic or cytotoxic effects were observed at various concentrations of the PNPs. Interestingly, the PNPs did not induce hemorrhagic events or cause liver enzyme alterations in vivo. These findings suggest that RVX-loaded SLS or DMAB PNPs are promising innovative therapeutic alternatives for the treatment of thromboembolic diseases.

Enzymatic synthesis of ascorbyl oleate and evaluation of biological activities.

Compounds that reduce or neutralize free radicals have been evaluated for use as nutraceutical or antioxidant additives in processed foods. This study aimed to enzymatically produce ascorbyl oleate and assess its biological properties. The synthesis was performed under previously maximized conditions (L-ascorbic acid/oleic acid 1:9 molar ratio, 70 °C, 1 h reaction). Immobilized commercial lipase from Candida antarctica (NS 88011) was used as biocatalyst. The reaction product was isolated, and its structure was confirmed by High-Performance Liquid Chromatography and Nuclear Magnetic Resonance. Ascorbyl oleate showed antioxidant and antimicrobial activity, besides no toxicity, did not influencing blood coagulation and also not presenting hemolytic profile. Better storage stability was achieved under refrigerated conditions, and the oxidative stability demonstrated free radicals fighting efficiency, increasing olive oil's shelf life. In vitro gastrointestinal simulation showed that ascorbyl oleate maintained antioxidant potential up to the duodenum stage during the digestive process. Therefore, the synthesized natural compound presented a high potential to be applied in the food and pharmaceutical industries.

Glycemic Variability and Insulin Needs in Patients with Type 1 Diabetes Mellitus Supplemented with Vitamin D: A Pilot Study Using Continuous Glucose Monitoring System.

BACKGROUND: Recent studies suggest that glycemic variability could influence the risk of complications in Type 1 Diabetes Mellitus (T1DM). There are no data about the action of Vitamin D (VD) on glycemic variability. Our pilot study aims to evaluate glycemic variability and insulin needs in patients with T1DM supplemented with VD. METHODS: 22 Patients received doses of 4000 and 10000 IU/day of cholecalciferol for 12 weeks, according to the patient's baseline VD levels and underwent continuous glucose monitoring system. RESULTS: Correlations were found between percentage variation (Δ) of glycemia standard deviation (ΔSDG), calculated using continuous glucose monitoring, with Δ of basal (r = 0.6; p <0.01) and total insulin dose (r = 0.6; p <0.01). Correlations between VD status after supplementation and Δ of prandial (r = 0.5; p <0.05) and total insulin dose (r = 0.4; p <0.05) were found, suggesting that the dose of insulin needed by patients is lower when VD status is better. We divided patients in two subgroups: SDG improved (subgroup 1; N = 12 (55%)) and SDG worsened (subgroup 2; N = 10 (45%)). Group 1, compared to subgroup 2, required a lower insulin dose (Δbasal insulin dose = -8.0 vs. 6.3%; p <0.05) and had a lower frequency of hypoglycemia (27% vs. 64%, hypoglycemias/days evaluated; p <0.01). CONCLUSION: Our study suggests a relation between VD supplementation, improved glycemic variability, lower insulin needs and lower frequency of hypoglycemia in patients with T1DM.

Albuminuria Reduction after High Dose of Vitamin D in Patients with Type 1 Diabetes Mellitus: A Pilot Study.

BACKGROUND: Some studies suggest an association between diabetic kidney disease (DKD) and vitamin D (VD), but there is no data about the effect of high dose of VD on DKD in type 1 diabetes mellitus (T1DM). Our pilot study aims to evaluate albuminuria reduction in patients with T1DM supplemented with high dose of VD. METHODS: 22 patients received doses of 4,000 and 10,000 IU/day of cholecalciferol for 12 weeks according to patient's previous VD levels. They were submitted to continuous glucose monitoring system, 24 hours ambulatory blood pressure monitoring and urine albumin-to-creatinine ratio before and after VD supplementation. RESULTS: There was a reduction of DKD prevalence at the end of the study (68 vs 32%; p = 0.05), with no changes on insulin doses, glycated hemoglobin, glycemic variability and blood pressure values. A correlation between percentage variation of VD levels (ΔVD) and albuminuria at the end of the study was presented (r = -0.5; p < 0.05). Among T1DM patients with DKD at the beginning of the study, 8/13 (62%) had their DKD stage improved, while the other five ones (38%) showed no changes (p < 0.05). CONCLUSION: Our pilot study suggests an association between VD high dose supplementation, lower prevalence and improvement in stages of DKD in T1DM.

Present Insights on Cardiomyopathy in Diabetic Patients.

The pathogenesis of diabetic cardiomyopathy (DCM) is partially understood and is likely to be multifactorial, involving metabolic disturbances, hypertension and cardiovascular autonomic neuropathy (CAN). Therefore, an important need remains to further delineate the basic mechanisms of diabetic cardiomyopathy and to apply them to daily clinical practice. We attempt to detail some of these underlying mechanisms, focusing in the clinical features and management. The novelty of this review is the role of CAN and reduction of blood pressure descent during sleep in the development of DCM. Evidence has suggested that CAN might precede left ventricular hypertrophy and diastolic dysfunction in normotensive patients with type 2 diabetes, serving as an early marker for the evaluation of preclinical cardiac abnormalities. Additionally, a prospective study demonstrated that an elevation of nocturnal systolic blood pressure and a loss of nocturnal blood pressure fall might precede the onset of abnormal albuminuria and cardiovascular events in hypertensive normoalbuminuric patients with type 2 diabetes. Therefore, existing microalbuminuria could imply the presence of myocardium abnormalities. Considering that DCM could be asymptomatic for a long period and progress to irreversible cardiac damage, early recognition and treatment of the preclinical cardiac abnormalities are essential to avoid severe cardiovascular outcomes. In this sense, we recommend that all type 2 diabetic patients, especially those with microalbuminuria, should be regularly submitted to CAN tests, Ambulatory Blood Pressure Monitoring and echocardiography, and treated for any abnormalities in these tests in the attempt of reducing cardiovascular morbidity and mortality.

Vitamin D on Early Stages of Diabetic Kidney Disease: A Cross-sectional Study in Patients with Type 1 Diabetes Mellitus.

CONTEXT: Genetic and environmental factors are involved in the pathogenesis of type 1 diabetes mellitus (T1DM), and vitamin D (VD) deficiency appears as a candidate to risk factor for developing diabetic kidney disease (DKD). OBJECTIVE: The purpose of study was to evaluate the existence of an association between low levels of VD and the presence and degree of DKD in T1DM. PATIENTS AND METHODS: We performed a cross-sectional study, between November 2014 and December 2015. Levels of 25(OH)D and albuminuria were analyzed in 37 patients with T1DM and normal glomerular filtration rate. Thirty-six subjects were evaluated as a control group. RESULTS: Patients with T1DM and hypovitaminosis D had higher levels of albuminuria compared to those with normal VD levels [albuminuria (log10) = 1.92 vs. 1.44; p < 0.05]. When we have separated the group of patients according to stage of DKD in patients with normo, micro, and macroalbuminuria, there are lower levels of 25(OH)D in the last when compared to the first two groups (26.7 ± 6.2, 24.8 ± 7.0, and 15.9 ± 7.6 ng/ml; p < 0.05, respectively). In T1DM group, we have found correlations between VD levels and both albuminuria and DKD stages (r = -0.5; p < 0.01 and r = -0.4; p < 0.05, respectively). A simple linear regression model, with albuminuria as the dependent variable and VD as an independent variable, showed r2 = 0.2 and p < 0.01. CONCLUSION: Our data suggest an association between reduced levels of VD and the presence and severity of DKD.