Combining Anti-Mitochondrial Antibodies, Anti-Histone, and PLA2/COX Biomarkers to Increase Their Diagnostic Accuracy for Autism Spectrum Disorders.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairments in social interaction and restricted and repetitive behaviors. Oxidative stress may be a critical link between mitochondrial dysfunction and ASD as reactive oxygen species (ROS) generated from pro-oxidant environmental toxicants and activated immune cells can result in mitochondrial failure. Recently, mitochondrial dysfunction, autoimmunity, and abnormal lipid mediators have been identified in multiple investigations as an acknowledged etiological mechanism of ASD that can be targeted for therapeutic intervention. METHODS: The relationship between lipid mediator markers linked to inflammation induction, such as phospholipase A2/cyclooxygenase-2 (PLA2/Cox-2), and the mitochondrial dysfunction marker anti-mitochondrial antibodies (AMA-M2), and anti-histone autoantibodies in the etiology of ASD was investigated in this study using combined receiver operating characteristic (ROC) curve analyses. This study also sought to identify the linear combination for a given set of markers that optimizes the partial area under ROC curves. This study included 40 age- and sex-matched controls and 40 ASD youngsters. The plasma of both groups was tested for PLA2/COX-2, AMA-M2, and anti-histone autoantibodies' levels using ELISA kits. ROC curves and logistic regression models were used in the statistical analysis. RESULTS: Using the integrated ROC curve analysis, a notable rise in the area under the curve was noticed. Additionally, the combined markers had markedly improved specificity and sensitivity. CONCLUSIONS: The current study suggested that measuring the predictive value of selected biomarkers related to mitochondrial dysfunction, autoimmunity, and lipid metabolism in children with ASD using a ROC curve analysis could lead to a better understanding of the etiological mechanism of ASD as well as its relationship with metabolism.

Effective Soybean Oil Degumming by Immobilized Phospholipases A2 from Walterinnesia aegyptia Venom.

Enzymatic degumming utilizing phospholipase enzymes could be used in ecologically friendly procedures with enhanced oil recovery yields. In this study, two phospholipases A2 of group I and II, WaPLA2-I and WaPLA2-II, from the snake venom of Saudi Walterinnesia aegyptia were evaluated for soybean oil degumming after being immobilized on three different support materials (calcium alginate (CA), CA-gelatin (CAG), and CA-chitosan (CAC), and cross-linked with glutaraldehyde). Higher yields of CAC-immobilized PLA2-I (85 ± 3%) and PLA2-II (87 ± 3.6%) compared to CAG (77.3 ± 2.1 and 79 ± 2.6%, respectively) and CA beads (55.7 ± 2.5% and 57.3 ± 3.1%, respectively) were observed. In addition, the optimal temperature of immobilized WaPLA2-I and WaPLA2-II increased from 45 to 55 °C and from 55 to 65 °C, respectively. Their stability at high temperatures was also significantly enhanced covering a larger range (70-80 °C). Likewise, the pH/activity profile of WaPLA2 was greatly expanded upon immobilization with the pH-optima being shifted by 0.5 to 1 pH unit to the basic side. Similarly, the stability of WaPLA2s in the presence of organic solvents was also significantly improved, while the affinity for calcium and bile salt was the same for both free and immobilized enzymes. Interestingly, the remaining activity of immobilized WaPLA2 onto different supports was more than 50 or 60% after eight recycles or 120 days of storage at 4 °C, respectively. CAC-WaPLA2-II was the best immobilized enzyme complex for the oil degumming process by reducing its final residual phosphorus content from 168 mg/kg to less than 10 mg/kg in only 4 h. Overall, CAC-WaPLA2-II showed the most attractive profiles of temperature, pH, and reaction duration as well as significant storage stability and reusability.

Highly Efficient Electrocarboxylation Method to Synthesize Novel Acid Derivatives of 1,4-Dihydropyridines and to Study Their Antimicrobial Activity.

1,4-Dihydropyridines (1,4-DHPs) hold a top-notch position in the pharmaceutical world due to a broader spectrum of applications, whereas the carboxylic moiety has been an integral part of the physiological world, effective food preservatives, and antimicrobial agents. Seeking the enormous potential and applications of these two classes, we worked to combine these to synthesize 2,2'-[3,5-bis(ethoxycarbonyl)-4-phenyl-1,4-dihydropyridine-2,6-diyl]diacetic acid the novel dicarboxylic derivatives of 1,4-DHP (9a-k) achieved via the electro-carboxylation of tetrasubstituted-1,4-dihydropyridines (8a-k) derivatives using Mg-Pt electrodes in an undivided cell. The targeted compounds were established by 1H, 13C NMR, IR, and ESI-MS. Further, the synthesized compounds show excellent resistance against various microbes and the activity increased 2-3 folds after the introduction of acid groups. Compound 9b (against E. coli, S. aureus, B. subtilis, A. niger, and P. glabrum), 9d (against E. coli, K. pneumonia, S. aureus, A. janus, and F. oxysporum), 9f (against E. coli and P. fluorescens), and 9k (against F. oxysporum and P. glabrum) were found to be highly active at 4 µg/mL with reference to standard amoxicillin and fluconazole. Further, the present synthetic protocol would open new gates for other researchers to develop new molecules by bioisosteres of these substrates.

Correction to: Therapeutic effects of probiotics on neurotoxicity induced by clindamycin and propionic acid in juvenile hamsters.

The original version of this article unfortunately contained a mistake. The family name of the fourth author listed in the title was incorrect, and the correct name is Nadine Moubayed, as noted in the addresses. Her name is now corrected in the author group of this article.

Therapeutic effects of probiotics on neurotoxicity induced by clindamycin and propionic acid in juvenile hamsters.

The present study investigated the therapeutic effects of probiotics on brain intoxication induced by clindamycin and propionic acid (PPA) in hamsters. Fifty golden Syrian hamsters were randomly divided into five experimental groups of ten animals each: (A) control group receiving phosphate buffered saline; (B) oral buffered PPA-treated group being administered with a neurotoxic dose of 250 mg/kg PPA during three days; (C) oral clindamycin-treated group receiving a single dose of 30 mg clindamycin/kg; and (D, E) the two therapeutic groups being administered the same doses of clindamycin and PPA followed by probiotics for three weeks at a daily dose of 0.2 g/kg. Biochemical parameters of energy metabolism and oxidative stress were examined in brain homogenates from all hamsters. The development of pathogenic bacteria was monitored on stool samples from all hamsters. Descriptive changes in fecal microbiota and overgrowth of Clostridium species in clindamycin and PPA treated hamsters were recorded. Interestingly, probiotics were shown effective to restore normal gut microbiota. Clindamycin and PPA treatments caused an elevation in lipid peroxidation and catalase activity, as oxidative stress markers, together with a reduction in GST activity and GSH level. Energy metabolism impairment was ascertained via the activation of creatine kinase and a decrease of lactate dehydrogenase. These findings suggest that bacteria overgrowth caused by PPA and clindamycin was efficient to illustrate signs of neuronal toxicity. The present study indicates that probiotic treatment can improve poor detoxification, oxidative stress, and altered gut microbiota as mechanisms implicated in the etiology of many neurological disorders.

Probiotic treatment reduces the autistic-like excitation/inhibition imbalance in juvenile hamsters induced by orally administered propionic acid and clindamycin.

Increasing evidence suggests that the gut microbiota plays a key role in the central nervous system (CNS), and alterations of the gut microbiota composition due to environmental factors can contribute to neurodevelopmental disorders. Animal modeling may help to identify drugs that can normalize the altered gut microbiota and thereby ameliorate abnormal brain signaling pathways. The purpose of the present study was to investigate the therapeutic potency of probiotics such as Bifidobacteria and Lactobacilli on glutamate excitotoxicity as a neurotoxic effect induced by clindamycin and propionic acid (PPA) in juvenile hamsters. Fifty young golden Syrian hamsters weighing between 60 and 70 g were enrolled in the study. The hamsters were randomly divided into five groups, each with ten hamsters. The hamsters in the control group only received phosphate-buffered saline orally. The PPA-treated group received a neurotoxic dose of 250 mg PPA/kg body weight (BW)/day for three days. The clindamycin-treated group received 30 mg clindamycin/kg BW as a single orogastric dose on the day the experiment started. The two therapeutic groups received the same doses of PPA and clindamycin followed by 0.2 g probiotic/kg BW for three weeks. Biochemical parameters related to glutamate excitotoxicity were investigated in brain homogenates from each group of hamsters. Additionally, the development of pathogenic bacteria was monitored in stool samples from all groups. The microbiology results of the present study revealed descriptive changes in the fecal microbiota and the appearance of Clostridium species in the hamsters treated with clindamycin and PPA. Additionally, the effectiveness of the probiotic in the restoration of the normal gut microbiota was demonstrated. Moreover, clindamycin and PPA were found to induce a significant depletion of Mg2+ and γ-aminobutyric acid (GABA) and a remarkable increase in the Na+/Mg2+ and glutamate/GABA ratios but non-significant changes in the absolute levels of K+, Na+ and glutamate. The bacteria overgrowth induced by PPA and clindamycin in the present study effectively showed signs of neuronal toxicity. The study indicates that probiotics can be used safely to ameliorate glutamate excitotoxicity mostly through increasing depleted GABA and Mg2+ and decreasing the excitatory neurotransmitter, glutamate.

Novel biomarkers of metabolic dysfunction is autism spectrum disorder: potential for biological diagnostic markers.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that is behaviorally defined by social and communication impairments and restricted interests and repetitive behaviors. There is currently no biomarkers that can help in the diagnosis. Several studies suggest that mitochondrial dysfunction is commonly involved in ASD pathophysiology, but standard mitochondrial biomarkers are thought to be very variable. In the present study we examine a wide variety of plasma biomarkers of mitochondrial metabolism and the related abnormalities of oxidative stress and apoptosis in 41 ASD patients assessed for ASD severity using the Childhood Autism Rating Scales and 41 non-related age and sex matched healthy controls. Our findings confirm previous studies indicating abnormal mitochondrial and related biomarkers in children with ASD including pyruvate, creatine kinase, Complex 1, Glutathione S-Transferase, glutathione and Caspase 7. As a novel finding, we report that lactate dehydrogenase is abnormal in children with ASD. We also identified that only the most severe children demonstrated abnormalities in Complex 1 activity and Glutathione S-Transferase. Additionally, we find that several biomarkers could be candidates for differentiating children with ASD and typically developing children, including Caspase 7, gluthatione and Glutathione S-Transferase by themselves and lactate dehydrogenase and Complex I when added to other biomarkers in combination. Caspase 7 was the most discriminating biomarker between ASD patients and healthy controls suggesting its potential use as diagnostic marker for the early recognition of ASD pathophysiology. This study confirms that several mitochondrial biomarkers are abnormal in children with ASD and suggest that certain mitochondrial biomarkers can differentiate between ASD and typically developing children, making them possibly useful as a tool to diagnosis ASD and identify ASD subgroups.

Purification and characterization of a newly serine protease inhibitor from Rhamnus frangula with potential for use as therapeutic drug.

Protease inhibitors from plants are well known to be potent inhibitors of the growth of bacteria, fungi, and even certain viruses which make them excellent candidates for use as the lead compounds for the development of novel antimicrobial agents for applications in medicine. In this study, Rhamnus frangula was selected as a protease inhibitor source. The maximum recovery of the protease inhibitor against trypsin was recorded in the crude extract made in 0.1 M phosphate buffer (pH 7.0) and isolated from the mature leaves. Then, the protease inhibitor designated as RfIP1 was purified to homogeneity by Sephadex G50 with an apparent molecular mass of 22.5 kDa and its N-terminal sequence exhibited a high degree of homology with known serine protease inhibitor sequences. The RfIP1 displayed maximal activity at pH 7 and 37 °C. It maintained almost 80% of its maximal activity through a large pH range. The thermo-stability of RfIP1 was markedly enhanced by BSA, CaCl2, and sorbitol, whereas the addition of Mg2+, Zn2+, NaTDC, SDS, DTT, and ß-ME significantly promoted inhibitory activity. The protease inhibitor displayed high inhibitory activity toward some known proteases (cathepsin B, chymotrypsin, collagenase, thrombin, and trypsin) that have more importance in pharmaceutical industry and it acted as potent inhibitor of some commercially proteases from Aspergillus oryzae, Bacillus sp, and Bacillus licheniformis. The protease inhibitor also possessed an appreciable antibacterial effect against both Gram-positive and Gram-negative bacteria.

Anti-bactericidal properties of stingray Dasyatis pastinaca groups V, IIA, and IB phospholipases A2: a comparative study.

Group IIA secreted phospholipase A2 (group IIA sPLA2) is known to display potent Gram-positive bactericidal activity in vitro and in vivo. We have analyzed the bactericidal activity of the full set of native stingray and dromedary groups V, IIA, and IB sPLA2s on several Gram-positive and Gram-negative strains. The rank order potency among both marine and mammal sPLA2s against Gram-positive bacteria is group IIA > V > IB, whereas Gram-negative bacteria exhibited a much higher resistance. There is a synergic action of the sPLA2 with lysozyme when added to the bacteria culture prior to sPLA2.The bactericidal efficiency of groups V and IIA sPLA2s was shown to be dependent upon the presence of calcium ions and to a less extent Mg(2+) ions and then a correlation could be made to its hydrolytic activity of membrane phospholipids. Importantly, we showed that stingray and dromedary groups V, IIA, and IB sPLA2s present no cytotoxicity after their incubation with MDA-MB-231cells. stingray groups V and IIA sPLA2s, like mammal ones, may be considered as future therapeutic agents against bacterial infections.

Toxicity of novel nanosized formulations used in medicine.

Nanotechnology involves the creation and manipulation of materials at nanoscale levels (1-100 nm) to create products that exhibit novel properties. While this motivation has driven nanoscience and technology in physics and engineering, it is not the main reason that nanoparticles are useful for systemic applications in the human body. The application of nanotechnology to medicine, known as nanomedicine, concerns the use of precisely engineered materials at this length scale to develop novel therapeutic and diagnostic modalities. A number of nanotherapeutic formulations are already approved for medical use and more are in the approval pipeline currently. This chapter is intended to provide an overview of the toxicity of these therapeutic nanoparticles and to summarize the current state of the field. We begin with background on the sources of exposure to nanoparticles, followed by reviewing different forms of nanosized therapeutic tools as quantum dots, nanoshells, nanocapsules, echogenic bubble, and "nanoshuttles." Moreover, cytotoxic effects of nanoparticles on cell membrane, mitochondrial function, prooxidant/antioxidant status, enzyme leakage, DNA, and other biochemical endpoints were elucidated. We highlight the need for caution during the use and disposal of such manufactured nanomaterials to prevent unintended environmental impacts. Moreover, different strategies which could be used to minimize or eliminate nanotoxicity were also discussed in detail. Understanding of how to tune size and surface properties to provide safety will permit the creation of new, more effective nanomedicines for systemic use.

Relationship between chronic lead toxicity and plasma neurotransmitters in autistic patients from Saudi Arabia.

OBJECTIVE: This study aims to clarify the relationship between blood Pb(2+) concentration as a ubiquitous environmental pollutant and plasma neurotransmitters as biochemical parameters that reflect brain function in Saudi autistic patients. METHODS: RBC's lead content together with plasma concentration of gamma aminobutyric acid (GABA), serotonin (5HT) and dopamine (DA) were measured in 25 Saudi autistic patients and compared to 16 age-matching control samples. RESULTS: The obtained data recorded that Saudi autistic patients have a remarkable higher levels of Pb(2+) and significantly elevated levels of GABA, 5HT and DA compared to healthy subjects. ROC analysis revealed satisfactory values of specificity and sensitivity of the measured parameters. CONCLUSION: This study suggests that postnatal lead toxicity in autistic patients of Saudi Arabia could represent a causative factor in the pathogenesis of autism. Elevated GABA, 5HT and DA were discussed in relation to the chronic lead toxicity recorded in the investigated autistic samples.

Purification and biochemical characterization of pancreatic phospholipase A2 from the common stingray Dasyatis pastinaca.

BACKGROUND: Mammalian sPLA2-IB are well characterized. In contrast, much less is known about aquatic ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes. RESULTS: A marine stingray phospholipase A2 (SPLA2) was purified from delipidated pancreas. Purified SPLA2, which is not glycosylated protein, was found to be monomeric protein with a molecular mass of 14 kDa. A specific activity of 750 U/mg for purified SPLA2 was measured at optimal conditions (pH 8.5 and 40 °C) in the presence of 4 mM NaTDC and 8 mM CaCl2 using PC as substrate. The sequence of the first twenty first amino-acid residues at the N-terminal extremity of SPLA2 was determined and shows a close similarity with known mammal and bird pancreatic secreted phospholipases A2. SPLA2 stability in the presence of organic solvents, as well as in acidic and alkaline pH and at high temperature makes it a good candidate for its application in food industry. CONCLUSIONS: SPLA2 has several advantageous features for industrial applications. Stability of SPLA2 in the presence of organic solvents, and its tolerance to high temperatures, basic and acidic pH, makes it a good candidate for application in food industry to treat phospholipid-rich industrial effluents, or to synthesize useful chemical compounds.

Structural homologies, importance for catalysis and lipid binding of the N-terminal peptide of a fungal and a pancreatic lipase.

Lipases share an overall alpha/beta hydrolase fold structure characteristic of serine hydrolases. Nevertheless, each lipases group possesses its characteristic 3-D structure and catalytic properties. The purified N-terminal truncated forms of a pancreatic (from ostrich) and a fungal (from Rhizopus oryzae, ROL32) (sayari et al., 2005) lipases displayed much lower activities as compared to the native proteins. The aim of this study is to explain this common functional feature on a structural basis. The molecular modelling showed that the N-terminal peptide of the fungal lipase displays an extended "V" shaped structure motif (sayari et al., 2005). We observed that the N-terminal peptide of a pancreatic lipase shares the same extended structure with that of the ROL32, despite the low sequence homology between the two peptides. Upon superimposition of the 3-D structure of the N-terminal catalytic domain of the pancreatic lipase with the model of the ROL32, we have shown that the N-terminal peptide and the open lid domain, of each lipase, are located distally within the putative interfacial binding surface. In particular, two hydrophobic residues, Leu and Ile belonging to the N-terminal peptide of each lipase are well placed to interact with the lipidic substrate. Furthermore, the N-terminal peptide of each lipase seems to be well placed to interact with the loop bearing the catalytic aspartic acid. All these observations might explain the fact that the loss of the N-terminal peptide affects the lipase activity. This work shows that the two lipases share striking structural and functional features with respect to their N-terminal peptide despite the fact that they belong to very distant kingdoms such as fungal and higher animals' ones.

A novel hepatopancreatic phospholipase A2 from Hexaplex trunculus with digestive and toxic activities.

A marine snail digestive phospholipase A2 (mSDPL) was purified from delipidated hepatopancreas. Unlike known digestive phospholipases A2, which are 14 kDa proteins, the purified mSDPL has a molecular mass of about 30 kDa. It has a specific activity of about 180 U/mg measured at 50 degrees C and pH 8.5 using phosphatidylcholine liposomes as a substrate in the presence of 4 mM NaTDC and 6mM CaCl2. The N-terminal amino-acid of the purified mSDPL does not share any homology with known phospholipases. Moreover, the mSDPL exhibits hemolytic activity in intact erythrocytes and can penetrate phospholipid monolayers at high surface pressure, comparable to snake venom PLA2. These observations suggest that mSDPL could be toxic to mammal cells. However, mSDPL can be classified as a member of a new family of enzymes. It should be situated between the class of toxic phospholipase A2 from venoms and another class of non toxic pancreatic phospholipase A2 from mammals.

Purification and characterization of a low molecular weight of beta-mannanase from Penicillium occitanis Pol6.

The highest beta-mannanase activity was produced by Penicillium occitanis Pol6 on flour of carob seed, whereas starch-containing medium gave lower enzymes titles. The low molecular weight enzyme was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography procedures. The purified beta-mannanase (ManIII) has been identified as a glycoprotein (carbohydrate content 5%) with an apparent molecular mass of 18 kDa. It was active at 40 degrees C and pH 4.0. It was stable for 30 min at 70 degrees C and has a broad pH stability (2.0-12.0). ManIII showed K (m), V (max), and K (cat) values of 17.94 mg/ml, 93.52 U/mg, and 28.13 s(-1) with locust bean gum as substrate, respectively. It was inhibited by mannose with a K (I) of 0.610(-3) mg/ml. ManIII was activated by CuSO4 and CaCl2 (2.5 mM). However, in presence of 2.5 mM Co2+, its activity dropped to 60% of the initial activity. Both N-terminal and internal amino acid sequences of ManIII presented no homology with mannanases of glycosides hydrolases. During incubation with locust bean gum and Ivory nut mannan, the enzyme released mainly mannotetraose, mannotriose, and mannobiose.

Biochemical characterization, cloning, and molecular modelling of chicken pancreatic lipase.

Chicken pancreatic lipase (CPL) was purified from delipidated pancreas. Pure CPL was obtained after ammonium sulphate fractionation, then DEAE-cellulose, Sephacryl S-200 gel filtration, and FPLC Mono-Q Sepharose columns. The pure lipase is a glycosylated monomer having a molecular mass of about 50kDa. The 23 N-terminal amino acid residues of CPL were sequenced. The sequence is similar to those of avian and mammalian pancreatic lipases. CPL presents the interfacial activation phenomenon tested with tripropionin or vinyl ester. When CPL was inhibited by synthetic detergent (TX-100) or amphipathic protein (BSA), simultaneous addition of bile salts and colipase was required to restore the full CPL activity. In the absence of colipase and bile salts, CPL was unable to hydrolyse tributyrin emulsion. This enzyme can tolerate, more efficiently than HPL, the accumulation of long-chain free fatty acids at the interface when olive oil emulsion was used as substrate in the absence of bile salts and colipase. The CPL activity, under these conditions, was linear whereas that of HPL decreased rapidly. Anti-TPL polyclonal antibodies cross-reacted specifically with CPL. The gene encoding the mature CPL was cloned and sequenced. The deduced amino acid sequence of the mature lipase shows a high degree of homology with the mammalian pancreatic lipases. A 3D structure model of CPL was built using the HPL structure as template. We have concluded that a slight increase in the exposed hydrophobic residues on the surface of CPL, as compared to HPL, could be responsible for a higher tolerance to the presence of long-chain free fatty acids at the lipid/water interface.

Purification and biochemical characterization of phospholipase A2 from dromedary pancreas.

Dromedary pancreatic PLA2 (DrPLA2) was purified from delipidated pancreases. Pure protein was obtained after heat and acidic treatment (70 degrees C; pH 3.0), precipitation by ammonium sulphate and ethanol respectively, followed by sequential column chromatographies on Sephadex G-50, MonoS Sepharose, MonoQ Sepharose and C-8 reverse phase high pressure liquid chromatography. Purified DrPLA2, which is not glycosylated protein, was found to be monomeric protein with a molecular mass of 13748.55 Da. A specific activity of 600 U/mg for purified DrPLA2 was measured at optimal conditions (pH 8.0 and 37 degrees C) in the presence of 3 mM NaTDC and 7 mM CaCl(2) using PC as substrate. The sequence of the first fourteen amino-acid residues at the N-terminal extremity of DrPLA2 was determined by automatic Edman degradation. One single sequence was obtained and shows a close similarity with all other known pancreatic secreted phospholipases A2.