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.

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.

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.

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.

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.

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.

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.

Heterologous Expression and Functional Characterization of Sparidae Fish Digestive Phospholipase A2.

In this study, we have produced for the first time a fish phospholipase (PLA2) in heterologous system (E. coli). The Diplodus annularis PLA2 (DaPLA2) was then refolded from inclusion bodies and purified by Ni-affinity chromatography. We used the pH-stat method (with emulsified phosphatidylcholine as substrate) and the monomolecular film technique (using various glycerophospholipids substrates spread in the form of monomolecular films at the air-water interface) to access the biochemical and kinetic properties of the recombinant DaPLA2. The DaPLA2 was found to be active and stable at higher temperatures (37-50 °C) than expected. Interestingly, DaPLA2 hydrolyzes efficiently both purified phosphatidylglycerol and phosphatidylethanolamine at 20 mN/m. These analytical results corroborate with the fact that the catalytic activity of DaPLA2, measured with the pH-stat using egg yolk as substrate, is mainly due to the hydrolysis of the PE fraction present in egg yolk, whereas the phosphatidylglycerol is a hallmark substrate for the most secreted PLA2-IB.

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.

Efficient heterologous expression, functional characterization and molecular modeling of annular seabream digestive phospholipase A2.

Here we report the cDNA cloning of a phospholipase A2 (PLA2) from five Sparidae species. The deduced amino acid sequences show high similarity with pancreatic PLA2. In addition, a phylogenetic tree derived from alignment of various available sequences revealed that Sparidae PLA2 are closer to avian PLA2 group IB than to mammals' ones. In order to understand the structure-function relationships of these enzymes, we report here the recombinant expression in E.coli, the refolding and characterization of His-tagged annular seabream PLA2 (AsPLA2). A single Ni-affinity chromatography step was used to obtain a highly purified recombinant AsPLA2 with a molecular mass of 15kDa as attested by gel electrophoresis and MALDI-TOF mass spectrometry data. The enzyme has a specific activity of 400U.mg-1 measured on phosphatidylcholine at pH 8.5 and 50°C. The enzyme high thermo-activity and thermo-stability make it a potential candidate in various biological applications. The 3D structure models of these enzymes were compared with structures of phylogenetically related pancreatic PLA2. By following these models and utilizing molecular dynamics simulations, the resistance of the AsPLA2 at high temperatures was explained. Using the monomolecular film technique, AsPLA2 was found to be active on various phospholipids spread at the air/water interface at a surface pressure between 12 and 25dyncm-1. Interestingly, this enzyme was shown to be mostly active on dilauroyl-phosphatidylglycerol monolayers and this behavior was confirmed by molecular docking and dynamics simulations analysis. The discovery of a thermo-active new member of Sparidae PLA2, provides new insights on structure-activity relationships of fish PLA2.

Identification and molecular docking of novel ACE inhibitory peptides from protein hydrolysates of shrimp waste.

The effect of enzymatic hydrolysis by Savinase on the interfacial properties and antihypertensive activity of shrimp waste proteins was evaluated. The physicochemical characterization, interfacial tension, and surface characteristics of shrimp waste protein hydrolysates (SWPH) using different enzyme/substrate (E/S) (SWPH5 (SWPH using E/S = 5), SWPH15 (SWPH using E/S = 15), and SWPH40 (SWPH using E/S = 40)) were also studied. SWPH5, SWPH15, and SWPH40 had an isoelectric pH around 2.07, 2.17, and 2.54 respectively. SWPH5 exhibited the lowest interfacial tension (68.96 mN/m) followed by SWPH15 (69.36 mN/m) and SWPH40 (70.29 mN/m). The in vitro ACE inhibitory activity of shrimp waste protein hydrolysates showed that the most active hydrolysate was obtained using an enzyme/substrate of 15 U/mg (SWPH15). SWPH15 had a lower IC50 value (2.17 mg/mL) than that of SWPH5 and SWPH40 (3.65 and 5.7 mg/mL, respectively). This hydrolysate was then purified and characterized. Fraction F1 separated by Sephadex G25 column which presents the best ACE inhibition activity was then separated by reversed-phase high performance liquid chromatography. Four ACE inhibitory peptides were identified and their molecular masses and amino acid sequences were determined using ESI-MS and ESI-MS/MS, respectively. The structures of the most potent peptides were SSSKAKKMP, HGEGGRSTHE, WLGHGGRPDHE, and WRMDIDGDIMISEQEAHQR. The structural modeling of anti-ACE peptides from shrimp waste through docking simulations results showed that these peptides bound to ACE with high affinity.

The smooth-hound lipolytic system: Biochemical characterization of a purified digestive lipase, lipid profile and in vitro oil digestibility.

In order to identify fish enzymes displaying novel biochemical properties, we choose the common smooth-hound (Mustelus mustelus) as a starting biological material to characterize the digestive lipid hydrolyzing enzyme. A smooth-hound digestive lipase (SmDL) was purified from a delipidated pancreatic powder. The SmDL molecular weight was around 50kDa. Specific activities of 2200 and 500U/mg were measured at pH 9 and 40°C using tributyrin and olive oil emulsion as substrates, respectively. Unlike known mammal pancreatic lipases, the SmDL was stable at 50°C and it retained 90% of its initial activity after 15min of incubation at 60°C. Interestingly, bile salts act as an activator of the SmDL. It's worth to notice that the SmDL was also salt-tolerant since it was active in the presence of high salt concentrations reaching 0.8M. Fatty acid (FA) analysis of oil from the smooth-hound viscera showed a dominance of unsaturated ones (UFAs). Interestingly, the major n-3 fatty acids were DHA and EPA with contents of 18.07% and 6.14%, respectively. In vitro digestibility model showed that the smooth hound oil was efficiently hydrolyzed by pancreatic lipases, which suggests the higher assimilation of fish oils by consumers.

Functional and Structural Characterization of a Thermostable Phospholipase A2 from a Sparidae Fish (Diplodus annularis).

Novel phospholipase (PLA2) genes from the Sparidae family were cloned. The sequenced PLA2 revealed an identity with pancreatic PLA2 group IB. To better understand the structure/function relationships of these enzymes and their evolution, the Diplodus annularis PLA2 (DaPLA2) was overexpressed in E. coli. The refolded enzyme was purified by Ni-affinity chromatography and has a molecular mass of 15 kDa as determined by MALDI-TOF spectrometry. Interestingly, unlike the pancreatic type, the DaPLA2 was active and stable at higher temperatures, which suggests its great potential in biotechnological applications. The 3D structure of DaPLA2 was constructed to gain insights into the functional properties of sparidae PLA2. Molecular docking and dynamic simulations were performed to explain the higher thermal stability and the substrate specificity of DaPLA2. Using the monolayer technique, the purified DaPLA2 was found to be active on various phospholipids ranging from 10 to 20 mN·m-1, which explained the absence of the hemolytic activity for DaPLA2.

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.

Original tandem duplication in FXIIIA gene with splicing site modification and four amino acids insertion causes factor XIII deficiency.

: Recessive mutations of F13A gene are reported to be responsible of FXIIIA subunit deficiency (FXIIIA). In all, some intronic nucleotide changes identified in this gene were investigated by in-silico analysis and occasionally supported by experimental data or reported in some cases as a polymorphism. To determine the molecular defects responsible of congenital factor XIII deficiency in Libyan patient, molecular analysis was performed by direct DNA sequencing of the coding regions and splice junctions of the FXIIIA subunit gene (F13A). A splicing minigene assay was used to study the effect of this mutation. Bioinformatics exploration was fulfilled to conceive consequences on protein. A 12-bp duplication straddling the border of intron 9 and exon 10 leads to two 3' acceptor splice sites, resulting in silencing of the downstream wild 3' splice site. It caused an in-frame insertion of 12 nucleotides into mRNA and four amino acids into protein. Bioinformatic analysis predicts that the insertion of four amino acids affects the site 3 of calcium binding site, which disturbs the smooth function of the FXIIIA peptide causing the factor XIII deficiency. This study showed that a small duplication seems to weaken the original 3' splice site and enhance the activation of a new splice site responsible for an alternative splicing. It would be interesting to examine the underlying molecular mechanism involved in this rearrangement.

Biochemical characterization, cloning and molecular modeling of a digestive lipase from red seabream (Pagrus major): Structural explanation of the interaction deficiency with colipase and lipidic interface.

Red seabream digestive lipase (RsDL) was purified from fresh pyloric caeca. Pure RsDL has an apparent molecular mass of 50 kDa. The RsDL is more active on short-chain triacylglycerols (TC4), and enzymatic activity decreases when medium (TC8) or long-chain (olive oil) triacylglycerols were used as substrates. The specific activities of RsDL are very weak as compared to those obtained with classical pancreatic lipases. No colipase was detected in the red seabream pyloric caeca. Furthermore, the RsDL was not activated by a mammal colipase. Similar results were reported for annular seabream lipase. In order to explain structurally the discrepancies between sparidae and mammal lipases, genes encoding mature RsDL and five other lipases from sparidae fish species were cloned and sequenced. Phylogenetic studies indicated the closest homology of sparidae lipases to bird pancreatic ones. Structural models were built for annular seabream and RsDL under their closed and open forms using mammal pancreatic lipases as templates. Several differences were noticed when analyzing the amino acids corresponding to those involved in HPL binding to colipase. This is likely to prevent interaction between the fish lipase and the mammalian colipase and may explain the fact that mammalian colipase is not effective in activating sparidae lipases. In addition, several hydrophobic residues, playing a key role in anchoring pancreatic lipase onto the lipid interface, are replaced by polar residues in fish lipases. This might explain the reason why the latter enzymes display weak activity levels when compared to mammalian pancreatic lipases.