The heat-stable protein fraction from Opuntia ficus-indica seeds exhibits an enzyme protective effect against thermal denaturation and an antibacterial activity.

Desiccation tolerance in developing seeds occurs through several mechanisms among which, a common group of proteins named dehydrins has received considerable attention. So far, there is no information dealing with the accumulation of dehydrins in seeds of Opuntia ficus-indica. We have initiated here an extraction protocol based on two critical steps: heat and acid treatments, and the purity of this fraction was analyzed by FTIR spectroscopy. Western blot analysis of the heat-stable protein fraction (HSF) revealed two main bands of approximately 45 and 44 kDa, while three others of ∼40, 32, and 31 kDa were faintly visible, which were recognized by anti-dehydrin antibodies. This fraction exhibited a Cu2+ -dependent resistance to protease treatments. Next, we performed a series of assays to compare the functional properties of the HSF with those of the previously characterized wheat dehydrin (DHN-5). Antibacterial assays revealed that HSF exhibits only moderate antibacterial activities against gram-negative and gram-positive bacteria, with a minimum inhibition concentration ranging from 0.25 to 1 mg/ml. However, in vitro assays revealed that compared to DHN-5, HSF exhibits higher protective activities of the lactate dehydrogenase (LDH) when exposed to heat, freezing, and dehydration stresses. The protective role of HSF seems to be linked to its best ability to minimize protein aggregation.

Effects of Sorafenib and Quercetin Alone or in Combination in Treating Hepatocellular Carcinoma: In Vitro and In Vivo Approaches.

Sorafenib is the first drug approved to treat advanced hepatocellular carcinoma (HCC) and continues as the gold-standard therapy against HCC. However, acquired drug resistance represents a main concern about sorafenib therapy. The flavanol quercetin found in plants has shown great anti-cancer and anti-inflammatory properties. In this work, quercetin was used as a therapeutic agent alone or in combination with a sorafenib chemotherapy drug to improve the routine HCC treatment with sorafenib. The in vitro and in vivo results presented here confirm that quercetin alone or in combination with sorafenib significantly inhibited HCC growth, induced cell cycle arrest and induced apoptosis and necrosis. Further molecular data shown in this report demonstrate that quercetin alone or combined with sorafenib downregulated key inflammatory, proliferative and angiogenesis-related genes (TNF-α, VEGF, P53 and NF-κB). Combined quercetin/sorafenib treatment markedly improved the morphology of the induced liver damage and showed significant antioxidant and anti-tumor effects. The advantage of combined treatment efficacy reported here can be attributed to quercetin's prominent effects in modulating cell cycle arrest, apoptosis, oxidative stress and inflammation.

Anti-Hepatocellular Carcinoma Biomolecules: Molecular Targets Insights.

This report explores the available curative molecules directed against hepatocellular carcinoma (HCC). Limited efficiency as well as other drawbacks of existing molecules led to the search for promising potential alternatives. Understanding of the cell signaling mechanisms propelling carcinogenesis and driven by cell proliferation, invasion, and angiogenesis can offer valuable information for the investigation of efficient treatment strategies. The complexity of the mechanisms behind carcinogenesis inspires researchers to explore the ability of various biomolecules to target specific pathways. Natural components occurring mainly in food and medicinal plants, are considered an essential resource for discovering new and promising therapeutic molecules. Novel biomolecules normally have an advantage in terms of biosafety. They are also widely diverse and often possess potent antioxidant, anti-inflammatory, and anti-cancer properties. Based on quantitative structure-activity relationship studies, biomolecules can be used as templates for chemical modifications that improve efficiency, safety, and bioavailability. In this review, we focus on anti-HCC biomolecules that have their molecular targets partially or completely characterized as well as having anti-cancer molecular mechanisms that are fairly described.

Design, synthesis of novel Triazolones and bis-Triazolones derivatives under ultrasound irradiation and evaluation as potent angiotensin converting enzyme (ACE) inhibitors.

The condensation of several primary amines and diamines with various N1-ethoxycarbonyles N1-tosylhydrazonates (1a-b), triazolones (2) and bis-triazolone (3) resulted in ethanol under ultrasound irradiation. Compared with the conventional methods, the main advantages of the present procedure are milder conditions, shorter reaction time and higher yields. The newly synthesized compounds were evaluated for angiotensin I-converting enzyme (ACE) inhibition. The results were compared to Captopril as a reference drug. Compounds 3b, 2h, 3a, 2d, and 2f showed not only inhibition activity with IC50 values of 0.162, 0.253, 0.253, 0.281 and 0.382 µM, respectively, but also minimal toxicity. The docking of chemical compounds in the ACE active site showed possible inhibitory effect of all compounds on the catalytic activity of the enzyme, which would satisfactorily explain the anti-hypertensive effect of these compounds.

A newly thermoactive and detergent-stable lipase from annular sea bream (Diplodus annularis): Biochemical properties.

A lipolytic activity was located in the annular seabream pyloric caeca, from which a digestive lipase (AsDL) was purified. Pure AsDL has an apparent molecular mass of 50 kDa. The purified lipase is thermoactive as it displays its maximal activity on short- and long-chain triacylglycerols at a temperature of 50 °C. The enzyme is alkaline resistant as it retains 90% of its maximal activity when incubated during 1 H at pH 10. No colipase was detected in the annular seabream pyloric caeca. Similar results were reported for the sardine and the gray mullet digestive systems. This is in line with the idea that colipase might have evolved in mammal animals simultaneously with the appearance of an exocrine pancreas. AsDL is a serine enzyme, like all known lipases from different origins. Interestingly, the pure lipase was found to be insensitive to Triton X-100, a synthetic detergent, addition even at a concentration as high as 12 mM. The purified enzyme has potential applications in detergent and food industry because of its thermal activity and alkaline nature.

Segregation of S292F TPO gene mutation in three large Tunisian families with thyroid dyshormonogenesis: evidence of a founder effect.

UNLABELLED: We aimed to identify causal mutation(s) in 13 patients with thyroid dyshormonogenesis (TD) from three consanguineous Tunisian families. A 12-year clinical follow-up showed phenotypic variability ranging from the presence to the absence of goiter, sensorineural deafness, and mental retardation. Genetic analysis using microsatellite markers within two candidate genes (TPO and PDS) gave evidence of linkage with the TPO gene. Sequencing of its 17 exons and their flanking intron-exon junctions revealed the previously described c.875C>T (p.S292F) mutation in homozygous state. No additional mutations were found in either a 900 bp of the TPO gene promoter or PDS gene. In silico analysis showed that p.S292F mutation might reduce the catalytic cavity of the TPO which would restrict access of a potential substrate to the catalytic pocket. Using 4SNPs and one microsatellite marker in the TPO gene, an associated haplotype: G-C-G-G-214 was found, giving evidence of a founder mutation. CONCLUSION: This is the first description of a TD causing mutation in Tunisia and thus may help to develop a genetic screening protocol for congenital hypothyroidism in the studied region. Although structural modeling suggested a pathogenic effect of this mutation, functional studies are needed. Additional causing and/or modifier genes, together with late diagnosis could explain the clinical variability observed in our patients.

Optimization of marine waste based-growth media for microbial lipase production using mixture design methodology.

Lipase production by Staphylococcus xylosus and Rhizopus oryzae was investigated using a culture medium based on a mixture of synthetic medium and supernatants generated from tuna by-products and Ulva rigida biomass. The proportion of the three medium components was optimized using the simplex-centroid mixture design method (SCMD). Results indicated that the experimental data were in good agreement with predicted values, indicating that SCMD was a reliable method for determining the optimum mixture proportion of the growth medium. Maximal lipase activities of 12.5 and 23.5 IU/mL were obtained with a 50:50 (v:v) mixture of synthetic medium and tuna by-product supernatant for Staphylococcus xylosus and Rhizopus oryzae, respectively. The predicted responses from these mixture proportions were also validated experimentally.

Design, synthesis and biological evaluation of new 1,ω-Bis-(5-alkyl-3-tosyl-1,3,4,2-triazaphospholino)alkanes as in vitro α-amylase and lipase inhibitors.

A series of new 1,ω-bis-(5-alkyl-3-tosyl-1,3,4,2-triazaphospholino)alkanes 2 and 3 were obtained in excellent yields by the condensation of 1,ω-bis-(1-tosylamidrazone)alkanes 1 with two equivalent molars of Lawesson's Reagent (LR) and trisdimethylaminophosphine, respectively. All synthesized compounds were characterized by various spectroscopic techniques including IR, 1H NMR, 13C NMR and 31P NMR and elemental analysis. The newly synthesized compounds were evaluated against key enzymes related to diabetes and obesity such as α-amylase and lipase. This study showed that the compounds 3a and 2b are an excellent inhibitor of α-amylase (with IC50 = 18.8 mM) and lipase (with IC50 = 19 mM) respectively, as compared with standard, orlistat (IC50 = 22 mM). Among this series, compounds 3a and 2b with the CH3 or C2H5 group at position 6 were identified as the most potent inhibitors against α-amylase, and lipase enzymes. The remaining compounds were found to be moderately active. Further, molecular docking simulation studies were done to identify the interactions and binding mode of synthesized analogs at binding site of α-amylase and lipase enzymes.

Role of flavonoids in controlling obesity: molecular targets and mechanisms.

Obesity presents a major health challenge that increases the risk of several non-communicable illnesses, such as but not limited to diabetes, hypertension, cardiovascular diseases, musculoskeletal and neurological disorders, sleep disorders, and cancers. Accounting for nearly 8% of global deaths (4.7 million) in 2017, obesity leads to diminishing quality of life and a higher premature mortality rate among affected individuals. Although essentially dubbed as a modifiable and preventable health concern, prevention, and treatment strategies against obesity, such as calorie intake restriction and increasing calorie burning, have gained little long-term success. In this manuscript, we detail the pathophysiology of obesity as a multifactorial, oxidative stress-dependent inflammatory disease. Current anti-obesity treatment strategies, and the effect of flavonoid-based therapeutic interventions on digestion and absorption, macronutrient metabolism, inflammation and oxidative stress and gut microbiota has been evaluated. The use of several naturally occurring flavonoids to prevent and treat obesity with a long-term efficacy, is also described.

Enzymatic transesterification of palm stearin and olein blends to produce zero-trans margarine fat.

BACKGROUND: Food industries aim to replace trans fat in their products by formulations having equivalent functionality and economic viability. Enzymatic transesterification can be a technological option to produce trans free fats targeting commercial applications. RESULTS: Palm stearin and palm olein blends in different ratios were enzymatically transesterified in a solvent free system using a Rhizopus oryzae lipase immobilised onto CaCO3 to produce a suitable fat for margarine formulation. Slip melting points and triacylglycerols profiles were evaluated upon transesterification. Results indicated that all transesterified blends had lower slip melting points than their non transesterified counterparts. Furthermore, the triacylglycerols profile showed a decrease in the concentration of the high melting point triacylglycerols. The rheological analysis showed that margarine prepared with the transesterified blend showed a better spreadability than that of a control margarine prepared with non transesterified fat. Adding powder of dry bark orange to margarine preparation improved its colour and fairly affected its spreadability and rheological behaviour. The margarine prepared with transesterified fat displayed a rheological behaviour that was comparable to that of commercial sample. CONCLUSIONS: This study is an ecofriendly approach to the utilization of relatively low value bioresources like palm stearin and palm olein for making margarine free of trans fatty acids that are now implicated as risk factor for heart diseases.

Lipolytic activity levels and colipase presence in digestive glands of some marine animals.

Studies on the digestive secretions in aquatic animals can elucidate certain aspects of their nutritive physiology. The aim of the present study was to compare the digestive lipase and phospholipase activities in ten marine species belonging to four classes following the taxonomic classification of marine organisms. All aquatic digestive tissues tested are equipped with lipase and phospholipase activities, assuming the hydrolysis of fat-rich food. The lipolytic activities determined in the pancreases of cartilaginous fishes were greater than those in bony fishes, molluscs and crustaceans. This finding might be explained by the strong digestive utilization of fat-rich macronutrients by these carnivorous fishes. A trend of activities and stabilities at different pH and temperatures for crude lipases and phospholipases from these aquatic animals suggests that the optimum pH and temperature for marine lipases are species dependent. Interestingly, the sardine caecal lipase and phospholipase were found to be mostly stable in a broad range of acidic pH values. The maximum activities of lipolytic enzymes from the hepatopancreases of Hexaplex trunculus (molluscs) and Carcinus mediterranus (crustaceans) were found to be 50 and 60 °C, respectively, whereas the optimal temperature of lipolytic enzymes for the other species was classically around 40 °C. Thermoactivity of molluscs' lipolytic preparations makes them potential candidates in industrial applications. Among digestive glands studied, only pancreas (cartilaginous fish) contained the classically known colipase. Regarded as the most primitive living jawed vertebrates, cartilaginous fishes represented by sharks and rays could be considered as the oldest vertebrates possessing a complex digestive system like that of mammals.

Detection of Middle East Respiratory Syndrome Coronavirus-Specific RNA and Anti-MERS-Receptor-Binding Domain Antibodies in Camel Milk from Different Regions of Saudi Arabia.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes viral pneumonia disease in humans. The close contact with camels and drinking milk may cause MERS-CoV transfer to humans. This study was designed to detect the existence of MERS-CoV in camel milk samples collected from healthy animals of various barns located around Saudi Arabia. Camel milk samples were examined for MERS-CoV RNA by real time-quantitative polymerase chain reaction, also enzyme-linked immunosorbent assay was performed to detect IgG antibodies directed against Middle East respiratory syndrome receptor-binding domain. Among 83 camel milk samples tested, the result showed that seven samples (8.4%) were positive for MERS-CoV RNA, whereas 40.9% of camel milk samples had antibodies directed against MERS-CoV. The findings indicate that some regions (East and South part) are characterized by a high incidence of viral antibodies. The Southwestern region displayed the lowest infection rates. Among the camel breeds, the lowest positivity for detection of MERS-CoV RNA and IgG antibodies was found in Sahilia. This could be related to a higher resistance to viral infection of the breed Sahilia and/or to the geographical origin of the camels sampled in the study. This needs to be more explored to reduce spread of infection and also to understand the underlying reasons. The presence of viral RNA in camel milk samples warrants for measures to prevent possible foodborne transmission of MERS-CoV through milk consumption.

Therapeutic Effects of Crocin Alone or in Combination with Sorafenib against Hepatocellular Carcinoma: In Vivo & In Vitro Insights.

This study investigated the therapeutic effects of the phytochemical crocin alone or in combination with sorafenib both in rats chemically induced with hepatocellular carcinoma (HCC) and in human liver cancer cell line (HepG2). Male rats were randomly divided into five groups, namely, control group, HCC induced group, and groups treated with sorafenib, crocin or both crocin and sorafenib. HCC was induced in rats with a single intraperitoneal injection of diethylnitrosamine (DEN), then 2-acetylaminofluorene (2-AAF). The HCC-induced rats showed a significant decrease in body weight compared to animals treated with either or both examined drugs. Serum inflammatory markers (C-reactive protein (CRP); interleukin-6 (IL-6); lactate dehydrogenase (LDH), and oxidative stress markers were significantly increased in the HCC group and were restored upon treatment with either or both of therapeutic molecules. Morphologically, the HCC-induced rats manifested most histopathological features of liver cancer. Treatment with either or both of crocin and sorafenib successfully restored normal liver architecture. The expression of key genes involved in carcinogenesis (TNFα, p53, VEGF and NF-κB) was highly augmented upon HCC induction and was attenuated post-treatment with either or both examined drugs. Treatment with both crocin and sorafenib improved the histopathological and inflammation parameters as compared to single treatments. The in vivo anti-cancer effects of crocin and/or sorafenib were supported by their respective cytotoxicity on HepG2 cells. Crocin and sorafenib displayed an anti-tumor synergetic effect on HepG2 cells. The present findings demonstrated that a treatment regimen with crocin and sorafenib reduced liver toxicity, impeded HCC development, and improved the liver functions.

Immobilized Rhizopus oryzae lipase catalyzed synthesis of palm stearin and cetyl alcohol wax esters: optimization by response surface methodology.

BACKGROUND: Waxes are esters of long-chain fatty acids and long-chain alcohols. Their principal natural sources are animals (sperm whale oil) and vegetables (jojoba) which are expensive and not easily available. Wax esters synthesized by enzymatic transesterification, using palm stearin as raw material, can be considered as an alternative to natural ones. RESULTS: Palm stearin is a solid fraction obtained by fractionation of palm oil. Palm stearin was esterified with cetyl alcohol to produce a mixture of wax esters. A non-commercial immobilized lipase from Rhizopus oryzae was used as biocatalyst. Response surface methodology was employed to determine the effects of the temperature (30-50 °C), the enzyme concentration (33.34-300 IU/mL), the alcohol/palm stearin molar ratio (3-7 mol/mol) and the substrate concentration (0.06-0.34 g/mL) on the conversion yield of palm stearin. Under optimal conditions (temperature, 30 °C; enzyme concentration, 300 IU/mL; molar ratio 3 and substrate concentration 0.21 g/mL) a high conversion yield of 98.52% was reached within a reaction time of 2 h. CONCLUSIONS: Response surface methodology was successfully applied to determine the optimum operational conditions for synthesis of palm stearin based wax esters. This study may provide useful tools to develop economical and efficient processes for the synthesis of wax esters.

A new chymotrypsin-like serine protease involved in dietary protein digestion in a primitive animal, Scorpio maurus: purification and biochemical characterization.

BACKGROUND: Most recent works on chymotrypsins have been focused on marine animals and insects. However, no study was reported in chelicerate. RESULTS: Scorpion chymotrypsin-like protease (SCP) was purified to homogeneity from delipidated hepatopancreases. The protease NH2-terminal sequence exhibited more than 60% monoacids identity with those of insect putative peptidases. The protease displayed no sequence homology with classical proteases. From this point of view, the protease recalls the case of the scorpion lipase which displayed no sequence homology with known lipases. The scorpion amylase purified and characterized by our time, has an amino-acids sequence similar to those of mammalian amylases. The enzyme was characterized with respect its biochemical properties: it was active on a chymotrypsin substrate and had an apparent molecular mass of 25 kDa, like the classically known chymotrypsins. The dependence of the SCP activity and stability on pH and temperature was similar to that of mammalian chymotrypsin proteases. However, the SCP displayed a lower specific activity and a boarder pH activity range (from 6 to 9). CONCLUSION: Lower animal have a less evaluated digestive organ: a hepatopancreas, whereas, higher ones possess individualized pancreas and liver. A new chymotrypsin-like protease was purified for the first time from the scorpion hepatopancreas. Its biochemical characterization showed new features as compared to classical chymotrypsin-higher-animals proteases.

Nutrient composition of the marine snail (Hexaplex trunculus) from the Tunisian Mediterranean coasts.

BACKGROUND: Marine snail (Hexaplex trunculus) presents increasing nutritional, commercial and economical importance, being widely consumed in northern Africa, particularly in Mediterranean countries. From a nutritional point of view there is still limited information on the chemical composition of edible tissues (meat and hepatopancreas) of this species. Therefore, the aims of the present work were to study the proximate chemical composition, fatty acid and amino acid profiles of H. trunculus from the Tunisian Mediterranean coasts. RESULTS: Fatty acid profiles showed that the polyunsaturated fatty acids (PUFA) content is higher than the saturated fatty acids (SFA). The yields of PUFA and SFA present in the meat fat were 68.2% and 33.4% of the total fatty acids, respectively. Similar values were obtained in the hepatopancreatic lipidic fraction. Snail tissues contain valuable concentrations of PUFA, especially n-6 and n-3 with chain lengths of 20 and 22 carbons. All edible tissues were valuable sources of essential amino acids. Aspartic acid is the major amino acids present in the meat and hepatopancreas. The concentrations of nutrients were also determined in the hepatopancreas and meat of H. trunculus. Significantly high concentrations of minerals and trace elements were found in these tissues. CONCLUSION: This study suggests that H. trunculus is an important source of protein and essential amino acids. Furthermore, the snail lipidic fraction contains high proportions of polyunsaturated fatty acids benefical for human health.

Characterization of a novel ABCC2 mutation in infantile Dubin Johnson syndrome.

BACKGROUND AND AIMS: The Dubin Johnson Syndrome (DJS) occurs mostly in young adults but an early-onset of the disease has been reported in less common forms (Neonatal DJS and Infantile DJS). In this case, the clinical findings are of limit for the DJS diagnosis. Hence, the genetic testing remains the method of choice to provide an accurate diagnosis. In our study, we aimed to perform a genetic analysis for two siblings presented with an intrahepatic cholestasis before the age of 1 year to provide a molecular explanation for the developed phenotype. PATIENTS & METHODS: A Tunisian family, having two siblings, manifesting signs of a hepatopathy, was enrolled in our study. A molecular analysis was performed, using a panel-based next generation sequencing, supplying results that were the subject of computational analysis. Then, a clinical follow-up was carried out to assess the evolution of the disease. RESULTS: The genetic analysis revealed the presence of a novel missense c.4179G > T, (p.M1393I) mutation in ABCC2 gene associated with a substitution c.2789G > A (R930Q) in ATP8B1 gene. Predictive results consolidated the pathogenic effect of both variants. These results confirmed the DJS diagnosis in the studied patients. The clinical course of both patients fit well with the benign nature of DJS. CONCLUSION: We described here a novel ABCC2 mutation associated with a putative ATP8B1 modifier variant. This finding constituted the first report of a complex genotype in DJS. Hence, genetic analysis by a panel-based next generation sequencing permits an accurate diagnosis and the identification of putative variants that could influence the developed phenotype.

Dissecting the Interaction Deficiency of a Cartilaginous Fish Digestive Lipase with Pancreatic Colipase: Biochemical and Structural Insights.

A full-length cDNA encoding digestive lipase (SmDL) was cloned from the pancreas of the smooth-hound (Mustelus mustelus). The obtained cDNA was 1350 bp long encoding 451 amino acids. The deduced amino acid sequence has high similarity with known pancreatic lipases. Catalytic triad and disulphide bond positions are also conserved. According to the established phylogeny, the SmDL was grouped with those of tuna and Sparidae lipases into one fish digestive lipase cluster. The recently purified enzyme shows no dependence for bile salts and colipase. For this, the residue-level interactions between lipase-colipase are yet to be clearly understood. The structural model of the SmDL was built, and several dissimilarities were noticed when analyzing the SmDL amino acids corresponding to those involved in HPL binding to colipase. Interestingly, the C-terminal domain of SmDL which holds the colipase shows a significant role for colipase interaction. This is apt to prevent the interaction between fish lipase and the pancreatic colipase which and can provide more explanation on the fact that the classical colipase is unable to activate the SmDL.

Identification of a novel missense mutation in NIPAL4 gene: First 3D model construction predicted its pathogenicity.

BACKGROUND: The NIPAL4 gene is described to be implicated of Congenital Ichthyosiform Erythroderma (CIE). It encodes a magnesium transporter membrane-associated protein, hypothetically involved in epidermal lipid processing and in lamellar body formation. The aim of this work is to investigate the causative mutation in a consanguineous Tunisian family with a clinical feature of CIE with a yellowish severe palmoplantar keratoderma. METHODS: Four patients were dignosed with CIE. The blood samples were collected from patients and all members of their nuclear family for mutation analysis. The novel mutation of NIPAL4 gene was analysed with several software tools to predict its pathogenicity. Then, the secondary structure and the 3D model of ichthyn was generated in silico. RESULTS: The sequencing analysis of the NIPAL4 gene in patients revealed a novel homozygous missense mutation c.534A>C (p.E178D) in the exon 4. Bioinformatic tools predicted its pathogenicity. The secondary structure prediction and the 3D model construction expected the presence of 9 transmembrane helices and revealed that mutation p.E178D was located in the middle of the second transmembrane helices. Besides, the 3D model construction revealed that the p.E178D mutation is inducing a shrinking in the transport channel containing the mutated NIPA4 protein. CONCLUSION: We found a homozygous mutation in exon 4 of NIPAL4 c.534A>C (p.E178D), which was identified for the first time in our study. Bioinformatic investigations supported its involvement in the phenotype of patients with CIE. Interestingly, this mutation was located in the hypothetical transport channel cavity and leads to changes in the channel architecture, which would probably affect its transport function.