In vitro and in silico studies of the acaricidal and anticholinesterase activities of Randia aculeata seeds against the southern cattle tick Rhipicephalus (Boophilus) microplus.

Rhipicephalus (Boophilus) microplus is a leading cause of significant economic losses in the livestock industry, and tick populations have developed multiple forms of resistance to acaricides; therefore, the potential of novel natural bioactive compounds that are effective for targeting ticks must be addressed. The aim of this study was to evaluate the acaricidal and anticholinesterase activities of R. aculeata seeds and to identify naturally occurring compounds that potentially inhibit anticholinesterase through in silico docking. The acaricidal activity of the extract of R. aculeata seeds against larval and adult R. microplus ticks was assessed through immersion tests. Inhibition of anticholinesterase activity was measured spectrophotometrically. Extracts of R. aculeata seeds showed activity against larvae and engorged females of R. microplus, and a reduction in the reproductive index were also observed. Rutin, chlorogenic acid, quercetin, and epicatechin exhibited noteworthy interactions with the active site residues of RmAChE. These findings could significantly contribute to the exploration of novel natural products that can potentially inhibit RmAChE and could be used in the development of new acaricides for tick control.

Production and Characterization of Cross-Linked Aggregates of Geobacillus thermoleovorans CCR11 Thermoalkaliphilic Recombinant Lipase.

Immobilization of enzymes has many advantages for their application in biotechnological processes. In particular, the cross-linked enzyme aggregates (CLEAs) allow the production of solid biocatalysts with a high enzymatic loading and the advantage of obtaining derivatives with high stability at low cost. The purpose of this study was to produce cross-linked enzymatic aggregates (CLEAs) of LipMatCCR11, a 43 kDa recombinant solvent-tolerant thermoalkaliphilic lipase from Geobacillus thermoleovorans CCR11. LipMatCCR11-CLEAs were prepared using (NH4)2SO4 (40% w/v) as precipitant agent and glutaraldehyde (40 mM) as cross-linker, at pH 9, 20 °C. A U10(56) uniform design was used to optimize CLEA production, varying protein concentration, ammonium sulfate %, pH, glutaraldehyde concentration, temperature, and incubation time. The synthesized CLEAs were also analyzed using scanning electron microscopy (SEM) that showed individual particles of <1 µm grouped to form a superstructure. The cross-linked aggregates showed a maximum mass activity of 7750 U/g at 40 °C and pH 8 and retained more than 20% activity at 100 °C. Greater thermostability, resistance to alkaline conditions and the presence of organic solvents, and better durability during storage were observed for LipMatCCR11-CLEAs in comparison with the soluble enzyme. LipMatCCR11-CLEAs presented good reusability by conserving 40% of their initial activity after 9 cycles of reuse.

Effect of Hyperbaric Oxygen Therapy (HBOT) on Insulin Resistance Associated with Abdominal Obesity in Wistar Rats with Dietary Sucrose-Induced Metabolic Syndrome.

Metabolic syndrome (MS) is a combination of risk factors related to the development of mainly type 2 diabetes mellitus, cardiovascular disease (CVD) and nonalcoholic fatty liver disease (NAFLD). Its prevalence has increased worldwide, and healthcare systems will face major challenges in addressing this problem. The aim of this work was to evaluate the effect of hyperbaric oxygen therapy (HBOT) on insulin resistance (IR) and obesity associated with MS in Wistar rats. The experimental design consisted of three groups of sucrose-induced MS rats: the MS group that consumed sucrose (MS-Suc; n=5), the MS group that ingested sucrose and HBOT (MS-Suc-HBOT; n=5), the MS group that did not consume sucrose and that received HBOT (MS-HBOT; n=5) and the control group. The rats received HBOT for 20 d at 2.4 atmospheres absolute (ATA) for 60 min. Subsequently, the rats were euthanized, and body fat weight, serum biochemical parameters and microscopic analysis of adipose tissue were determined. Rats with hyperoxia had decreased body weight, adipose tissue hypertrophy, and abdominal and epididymal fat. Likewise, markers of insulin resistance (glucose, insulin and HOMA-IR), biochemical parameters of dyslipidemia (cholesterol and triglycerides) and nonalcoholic fatty liver (AST and ALT) decreased; in contrast, compared to the control group, HBOT increased the 1/HOMA-IR, HOMA-ßCell and McAuley indexes, which were related to the improvement in insulin sensitivity (p<0.05; p<0.01). HBOT showed beneficial effects in the treatment of IR and obesity associated with sucrose-induced metabolic syndrome in Wistar rats.

Comparative Effect between Sardine Oil and Fish Oil Rich in Omega-3 Fatty Acids on Hypertension and the Membrane Composition of Adipocytes in SHR Rats.

Omega-3 polyunsaturated fatty acids, have an important role in reducing hypertriglyceridemia, these acids decrease the mortality for Coronary Heart Disease. Very important is the relationship between fatty acid biosynthesis and distribution in organs and tissues involved in insulin resistance and hypertension due to its role in the production of vasoactive eicosanoids and their effects on insulin sensitivity; which is estimated with the HOMA-IR index, which relates the physiological and metabolic behavior of glucose and insulin in the body. The aim of this project was to compare the effect of sardine oil and omega-3 oils rich in polyunsaturated fatty acids: EPA (≈30%) and DHA (≈50%) administered for 6 to 8 wk respectively; on the lipid composition of the plasma membrane of epididymal adipocytes in spontaneously hypertensive rats (SHR) and their relation to obesity, insulin resistance and hypertension. The administration of omega-3 enriched oil significantly decreased the HOMA criteria as an insulin resistance indicator compared to the sardine oil.

Improved expression and immobilization of Geobacillus thermoleovorans CCR11 thermostable recombinant lipase.

Production of recombinant thermo-alkali-stable lipase LipMatCCR11, expressed in Escherichia coli BL21 (DE3), was investigated via response surface methodology by using a face-centered design with three levels of each factor. Additionally, improvement of the catalytic performance of expressed lipase was assessed by immobilization on microporous polypropylene. Results showed that inducer (isopropyl ß-d-1-thiogalactopyranoside [IPTG]) concentration and temperature were found to be the significant factors (P < 0.05). The maximum lipase expression was obtained at IPTG 0.6 mM, 16 °C, and 18 H, with a specific lipase activity of 7.29 × 106  U/mg, which was 36.4 times higher (over 1,300-fold increase) than lipase activity measured under nonoptimized conditions. On the other hand, immobilized lipase showed a high biocatalytic activity, particularly in the synthesis of aroma esters.

Enhanced Bioavailability of Curcumin Nanoemulsions Stabilized with Phosphatidylcholine Modified with Medium Chain Fatty Acids.

BACKGROUND: Curcumin is a natural, oil-soluble polyphenolic compound with potent anticancer, anti-inflammatory, and antioxidant activities. In its free form, it is very poorly absorbed in the gut due to its very low solubility. The use of nanoemulsions as carrier is a feasible way for improving curcumin bioavailability. To this end, the choice of emulsifying agent for stabilizing the nanoemulsions is of the upmost importance for achieving a desired functionality. METHODS: Phosphatidylcholine (PC) and phosphatidycholine enriched (PCE) with medium chain fatty acids (42.5 mol %) in combination with glycerol as co-surfactant, were used for preparing oil-in water nanoemulsions coded as NEPC and NEPCE, respectively. RESULTS: NEPCE displayed significantly smaller mean droplet size (30 nm), equal entrapment efficiency (100%), better droplet stability and suffered lower encapsulation efficiency loss (3%) during storage time (120 days, 4ºC) than NEPC. Bioavailability, measured in terms of area under the curve of curcumin concentration versus time, and maximum curcumin plasma concentration, was in general terms significantly higher for NEPCE than for NEPC, and for curcumin coarse aqueous suspension (CCS). Also, NEPCE produced significantly higher curcumin concentrations in liver and lung than NEPC and CCS. CONCLUSION: These data support the role of phosphatidylcholine enriched with medium chain fatty acids to increase the bioavailability of nanoemulsions for therapeutic applications.

Gene Cloning and Characterization of the Geobacillus thermoleovorans CCR11 Carboxylesterase CaesCCR11, a New Member of Family XV.

A gene encoding a carboxylesterase produced by Geobacillus thermoleovoras CCR11 was cloned in the pET-3b cloning vector, sequenced and expressed in Escherichia coli BL21(DE3). Gene sequence analysis revealed an open reading frame of 750 bp that encodes a polypeptide of 250 amino acid residues (27.3 kDa) named CaesCCR11. The enzyme showed its maximum activity at 50 °C and pH 5-8, with preference for C4 substrates, confirming its esterase nature. It displayed good resistance to temperature, pH, and the presence of organic solvents and detergents, that makes this enzyme biotechnologically applicable in the industries such as fine and oleo-chemicals, cosmetics, pharmaceuticals, organic synthesis, biodiesel production, detergents, and food industries. A 3D model of CaesCCR11 was predicted using the Bacillus sp. monoacyl glycerol lipase bMGL H-257 structure as template (PBD code 3RM3, 99 % residue identity with CaesCCR11). Based on its canonical α/ß hydrolase fold composed of 7 ß-strands and 6 α-helices, the α/ß architecture of the cap domain, the GLSTG pentapeptide, and the formation of distinctive salt bridges, we are proposing CaesCCR11 as a new member of family XV of lipolytic enzymes.

Significant improvement of Geobacillus thermoleovorans CCR11 thermoalkalophilic lipase production using response surface methodology.

The medium optimization for the production of the Geobacillus thermoleovorans CCR11 thermoalkalophilic lipase was carried out in shake flask cultures using safflower high oleic oil. In the first step of optimization, a two level fractional factorial design allowed the identification of the concentration of nutrient broth and temperature as the main variables significantly affecting lipase production (P<0.05). In a second step, a D-optimal design was applied to determine the variables optimal values, defined as those yielding maximal lipase production in shaken flasks, thus demonstrating that the optimal concentration of nutrient broth was 3.8 g/l and the optimal culture temperature was 39.5°C. The model was experimentally validated, yielding a lipase production of 2283.70 ± 118.36 U/mL which represents a 6.7-fold increase in comparison to the non-optimized medium.