Metabonomic analysis of Drosophila mutants using high resolution mass spectrometry

The availability of fully sequenced genomes of model organisms such as Drosophila and their subsequent annotation has afforded opportunities for reverse genetics in a complex model organism. Metabonomics used as an aid to functional genomics can be used to understand the functions of genes in living systems. Thus metabonomics has been employed to study Drosophila samples extracted from whole animals at different developmental stages or in response to external stimuli or genetic mutation. With unmatched mass resolution, accuracy, and detection sensitivity, linear ion trap - Fourier Transform Orbitrap Mass Spectrometry (LTQ-Orbitrap-MS) has the potential for high throughput metabonomic analysis. Five different but linked studies are reported in this work. A global metabolic profiling method based on electrospray ionisation mass spectrometry was developed for Drosophila melanogaster metabolites. The method involved optimizing the extraction of Drosophila metabolites followed by analysis using liquid chromatography coupled with high-resolution mass spectrometry. The effect of extraction conditions and storage were studied, thus 750-800 metabolites were putatively identified in order to obtain the metabolite profiles of Drosophila reference strains and mutants. Metabolic studies were carried out to elucidate gene functions using established protocols. The online resource FlyAtlas.org provides detailed microarray-based expression data for the tissues and life-stages of Drosophila. Since downstream genes, such as urate oxidase are tubule-specific, an Orbitrap technology has been used to elucidate tissue specific metabolomes. Additionally, genetic interventions using designed RNA interference were also made and validated by qPCR and metabonomics. The method produced a new opportunity for metabonomics use in validating gene expressions. The xanthine oxidase inhibitor allopurinol was used to phenocopy the rosy mutation which caused the levels of xanthine and hypoxanthine to rise while the levels of uric acid fell. In addition, many unexpected metabolic changes followed this treatment with effects on the pentose phosphate pathway and tryptophan metabolism being the most marked. The yellow (y) gene was first discovered in Drosophila, but occurs in many insect species and in some bacteria. The y protein is similar to the major royal jelly proteins produced by bees. Metabolomic profiling was carried out on Oregon R (OR) and y Drosophila larvae at the third instar. There were numerous metabolic differences between the metabolic profiles of OR and y. Phenylalanine, tyrosine and DOPA were all elevated in y, as might be expected since the mutation blocks melanin biosynthesis. In addition, there were other metabolic effects including marked effects on to lysine metabolism. The white mutation of Drosophila, which affects ABC transporters, was studied with regards to its effect on pigment biosynthesis in Drosophila. In addition to the expected effects on pigments there were interesting male/female differences possibly related to the presence of the white gene in the X chromosome. In addition the effect of salt stess on wild type and white flies was studied. Overall, LTQ-Orbitrap-MS proved suitable for metabonomic analysis of both wild-type and mutant Drosophila and had potential in the analysis of metabolomes of single tissues. The possibility of using Orbitrap-based metabonomics in combination with Drosphila for drug testing is discussed and is a goal for the future.The availability of fully sequenced genomes of model organisms such as Drosophila and their subsequent annotation has afforded opportunities for reverse genetics in a complex model organism. Metabonomics used as an aid to functional genomics can be used to understand the functions of genes in living systems. Thus metabonomics has been employed to study Drosophila samples extracted from whole animals at different developmental stages or in response to external stimuli or genetic mutation. With unmatched mass resolution, accuracy, and detection sensitivity, linear ion trap - Fourier Transform Orbitrap Mass Spectrometry (LTQ-Orbitrap-MS) has the potential for high throughput metabonomic analysis. Five different but linked studies are reported in this work. A global metabolic profiling method based on electrospray ionisation mass spectrometry was developed for Drosophila melanogaster metabolites. The method involved optimizing the extraction of Drosophila metabolites followed by analysis using liquid chromatography coupled with high-resolution mass spectrometry. The effect of extraction conditions and storage were studied, thus 750-800 metabolites were putatively identified in order to obtain the metabolite profiles of Drosophila reference strains and mutants. Metabolic studies were carried out to elucidate gene functions using established protocols. The online resource FlyAtlas.org provides detailed microarray-based expression data for the tissues and life-stages of Drosophila. Since downstream genes, such as urate oxidase are tubule-specific, an Orbitrap technology has been used to elucidate tissue specific metabolomes. Additionally, genetic interventions using designed RNA interference were also made and validated by qPCR and metabonomics. The method produced a new opportunity for metabonomics use in validating gene expressions. The xanthine oxidase inhibitor allopurinol was used to phenocopy the rosy mutation which caused the levels of xanthine and hypoxanthine to rise while the levels of uric acid fell. In addition, many unexpected metabolic changes followed this treatment with effects on the pentose phosphate pathway and tryptophan metabolism being the most marked. The yellow (y) gene was first discovered in Drosophila, but occurs in many insect species and in some bacteria. The y protein is similar to the major royal jelly proteins produced by bees. Metabolomic profiling was carried out on Oregon R (OR) and y Drosophila larvae at the third instar. There were numerous metabolic differences between the metabolic profiles of OR and y. Phenylalanine, tyrosine and DOPA were all elevated in y, as might be expected since the mutation blocks melanin biosynthesis. In addition, there were other metabolic effects including marked effects on to lysine metabolism. The white mutation of Drosophila, which affects ABC transporters, was studied with regards to its effect on pigment biosynthesis in Drosophila. In addition to the expected effects on pigments there were interesting male/female differences possibly related to the presence of the white gene in the X chromosome. In addition the effect of salt stess on wild type and white flies was studied. Overall, LTQ-Orbitrap-MS proved suitable for metabonomic analysis of both wild-type and mutant Drosophila and had potential in the analysis of metabolomes of single tissues. The possibility of using Orbitrap-based metabonomics in combination with Drosphila for drug testing is discussed and is a goal for the future

Synthesis and evaluation of some new 1,3,4-oxadiazoles bearing thiophene, thiazole, coumarin, pyridine and pyridazine derivatives as antiviral agents

In an attempt to produce heterocyclic compounds based on 1,3,4-oxadiazole derivatives with potential antiviral activity, the synthesis of compound 1 [2-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)acetonitrile] was performed through the reaction of cyanoacetic acid hydrazide with carbon disulfide in alcoholic potassium hydroxide. Compound 1 has an activating methylene group, so it was directed toward some specific reactions. Thus, aryldiazonium chlorides reacted with compound 1 affording hydrazono derivatives 2a,b,c. Also, aromatic aldehydes reacted with compound 1 to produce compounds 3a,b. Furthermore, cyclic ketones were subjected to synthesis of fused thiophene derivatives 4a,b via the reaction with compound 1 in the presence of elemental sulfur. In addition, 1,3,4-oxadiazole derivative 1 when reacted with isothiocyanates, salicylaldehyde or 1,3-dicarbonyl compounds formed thiazole derivatives 5a,b, coumarin derivative 6 and alkenyl derivatives 7a,b resp. Compound 7b underwent cyclization to afford pyridine derivative 8. Arylhydrazono derivatives 9a,b were produced through the reaction of compound 7a with aryldiazonium chlorides. The latter products 9a,b underwent cyclization to produce pyridazine derivatives 10a,b. Finally 1,3,4-oxadiazole derivative 1 was directed toward the reaction with hydrazine derivatives, bromoacetophenone and ethylchloroacetate affording compounds 11a,b, 12 and 13, resp. The fused thiophene derivatives 14a,b were produced via the reaction of compounds 4a,b with a mixture of malononitrile and ethylorthoformate. Antiviral activity of the synthesized products showed that 5-(4-amino-3-ethyl-2-thioxo-2,3-dihydrothiazol-5-yl)-1,3,4-oxadiazole-2(3H)-thione (5a) and 5-(4-amino-3-phenyl-2-thioxo-2,3-dihydrothiazol-5-yl)-1,3,4-oxadiazole-2(3H)-thione (5b) act as the most active agents against Feline herpes virus, Feline corona virus, Herpes simplex virus-1 and Herpes simplex virus-2, whereas compound 2-(5-(2-phenyl- hydrazono)-4,5-dihydro-1,3,4-oxadiazol-2-yl)acetonitrile (11b) was the most effective against Vaccinia virus, Herpes simplex virus (TK-KOS-ACVr ), Coxsackie virus B4 and Vesicular stomatitis virus

Synthesis of some novel pyrimidine, thiophene, coumarin, pyridine and pyrrole derivatives and their biological evaluation as analgesic, antipyretic and anti-inflammatory agents

Pyrimidine derivative 3 was afforded through the reaction of compound (1) with 5-ureidohydantion (2). Product 3 underwent a cyclization to produce fused pyrimidine derivative 7, although the latter product 7 was synthesized through one step via the reaction of compound (1) with 5-ureidohydantion (2) using another catalyst. Compound 3 was oriented to react with cyclic ketones 8a,b in the presence of elemental sulfur, salicylaldehyde (10), aryldiazonium chlorides 12a,b and ω-bromo-4-methoxy- acetophenone (14), which afforded, fused thiophene derivatives 9a,b, coumarin derivative 11, arylhdrazono derivatives 13a,b and 4-methoxyphenyl butenyl derivative 15, respectively. The latter product 15 was reacted with either potassium cyanide (16a) or potassium thiocyanide (16b) to form cyano and thiocyano derivatives 17a,b, respectively. Compound 17a underwent further cyclization to afford pyridopyrimidine derivative 19. Compound 15 was reacted with either hydrazine (20a) or phenylhydrazine (20b) to produce hydrazo derivatives 21a,b and these products were cyclize to produce pyrrole derivatives 23a,b. Finally, 5-ureidohydantion (2) was reacted with compounds 24a,b,c to afford pyrimidine derivatives 25a,b,c. The structures of the synthesized compounds were confirmed using IR, 1 H NMR, 13C NMR and mass spectrometry techniques. Compounds 11 and 19 have promising as analgesic and antipyretic activities

Effect of dapagliflozin on exercise capacity and cardiovascular risk in patients with heart failure

Heart failure (HF) is a serious disorder that affects millions of people worldwide, with a high rate of exercise intolerance, rehospitalization, and death. HF has many underlying causes, including type 2 diabetes mellitus (T2DM), which corresponds with high mortality and short survival among patients with HF. Numerous studies have shown the crucial role of gliflozins, a new generation of blood glucose-lowering medications, in cardiac remodeling, with beneficial impacts on exercise capacity and cardiovascular (CV) mortality, even in non-diabetic individuals. The foundational CV-protective frameworks of these agents are intricate and multifaceted. Dapagliflozin is a new widely used drug and a valuable alternative for patients with T2DM and CV risk factors. Dapagliflozin was approved by the Food and Drug Administration (FDA) in 2019 to lower the risk of HF hospitalization in patients with concurrent T2DM and CV disease or associated risk factors. However, the effects of this new drug on exercise capacity and CV risk still need to be elucidated. The primary objective of this review is to summarize the effect of dapagliflozin on exercise capacity and CV risk in patients with HF

Pathogenesis and management of traumatic brain injury (TBI): role of neuroinflammation and anti-inflammatory drugs.

Traumatic brain injury (TBI) is an important global health concern that represents a leading cause of death and disability. It occurs due to direct impact or hit on the head caused by factors such as motor vehicles, crushes, and assaults. During the past decade, an abundance of new evidence highlighted the importance of inflammation in the secondary damage response that contributes to neurodegenerative and neurological deficits after TBI. It results in disruption of the blood-brain barrier (BBB) and initiates the release of macrophages, neutrophils, and lymphocytes at the injury site. A growing number of researchers have discovered various signalling pathways associated with the initiation and progression of inflammation. Targeting different signalling pathways (NF-κB, JAK/STAT, MAPKs, PI3K/Akt/mTOR, GSK-3, Nrf2, RhoGTPase, TGF-β1, and NLRP3) helps in the development of novel anti-inflammatory drugs in the management of TBI. Several synthetic and herbal drugs with both anti-inflammatory and neuroprotective potential showed effective results. This review summarizes different signalling pathways, associated pathologies, inflammatory mediators, pharmacological potential, current status, and challenges with anti-inflammatory drugs

Polymorphism of HLA and Susceptibility of Breast Cancer

Breast cancer (BC) is the second most common malignancy in the world. Numerous studies have demonstrated the association between human leukocyte antigen (HLA) and cancer. The occurrence and development of BC are closely linked to genetic factors. Human leukocyte antigens G and E (HLA-G and HLA-E) are non-classical major histocompatibility complex (MHC) class I molecules. These molecules play an important role in immune surveillance by inhibiting the cytotoxic and natural killer T cells responsible for immune escape. The expression of HLA-G and HLA-E has been associated with several diseases, including tumors. The HLA system plays a key role in the escape of tumor cells from immune surveillance. This review aims to determine the correlation between BC susceptibility and HLA markers specific HLA alleles such as HLA-B07, HLA-DRB111, HLA-DRB113, and HLA-DRB115 are associated with an increased risk of developing BC. Furthermore, HLA-G mutations have been attributed to an elevated likelihood of metastasis in BC patients. Understanding the complex associations between the HLA system and BC development is critical for developing novel cancer prevention, detection, and treatment strategies. This review emphasizes the importance of analyzing HLA polymorphisms in the management of BC patients, as well as the urgent need for further research in this area

Mitochondrial targeting theranostic nanomedicine and molecular biomarkers for efficient cancer diagnosis and therapy.

Mitochondria play a crucial part in the cell's ability to adapt to the changing microenvironments and their dysfunction is associated with an extensive array of illnesses, including cancer. Mitochondrial dysfunction has been identified as a potential therapeutic target for cancer therapy. The objective of this article is to give an in-depth analysis of cancer treatment that targets the mitochondrial genome at the molecular level. Recent studies provide insights into nanomedicine techniques and theranostic nanomedicine for mitochondrial targeting. It also provides conceptual information on mitochondrial biomarkers for cancer treatment. Major drawbacks and challenges involved in mitochondrial targeting for advanced cancer therapy have also been discussed. There is a lot of evidence and reason to support using nanomedicine to focus on mitochondrial function. The development of a delivery system with increased selectivity and effectiveness is a prerequisite for a theranostic approach to cancer treatment. If given in large amounts, several new cancer-fighting medicines have been created that are toxic to healthy cells as well. For effective therapy, a new drug must be developed rather than an old one. When it comes to mitochondrial targeting therapy, theranostic techniques offer valuable insight

Integrating network pharmacology with molecular docking to rationalize the ethnomedicinal use of Alchornea laxiflora (Benth.) Pax & K. Hoffm. for efficient treatment of depression

Background: Alchornea laxiflora (Benth.) Pax & K. Hoffm. (A. laxiflora) has been indicated in traditional medicine to treat depression. However, scientific rationalization is still lacking. Hence, this study aimed to investigate the antidepressant potential of A. laxiflora using network pharmacology and molecular docking analysis.Materials and methods: The active compounds and potential targets of A. laxiflora and depression-related targets were retrieved from public databases, such as PubMed, PubChem, DisGeNET, GeneCards, OMIM, SwissTargetprediction, BindingDB, STRING, and DAVID. Essential bioactive compounds, potential targets, and signaling pathways were predicted using in silico analysis, including BA-TAR, PPI, BA-TAR-PATH network construction, and GO and KEGG pathway enrichment analysis. Later on, with molecular docking analysis, the interaction of essential bioactive compounds of A. laxiflora and predicted core targets of depression were verified.Results: The network pharmacology approach identified 15 active compounds, a total of 219 compound-related targets, and 14,574 depression-related targets with 200 intersecting targets between them. SRC, EGFR, PIK3R1, AKT1, and MAPK1 were the core targets, whereas 3-acetyloleanolic acid and 3-acetylursolic acid were the most active compounds of A. laxiflora with anti-depressant potential. GO functional enrichment analysis revealed 129 GO terms, including 82 biological processes, 14 cellular components, and 34 molecular function terms. KEGG pathway enrichment analysis yielded significantly enriched 108 signaling pathways. Out of them, PI3K-Akt and MAPK signaling pathways might have a key role in treating depression. Molecular docking analysis results exhibited that core targets of depression, such as SRC, EGFR, PIK3R1, AKT1, and MAPK1, bind stably with the analyzed bioactive compounds of A. laxiflora.Conclusion: The present study elucidates the bioactive compounds, potential targets, and pertinent mechanism of action of A. laxiflora in treating depression. A. laxiflora might exert an antidepressant effect by regulating PI3K-Akt and MAPK signaling pathways. However, further investigations are required to validate

Benchmarked molecular docking integrated molecular dynamics stability analysis for prediction of SARS-CoV-2 papain-like protease inhibition by olive secoiridoids

Objectives: We performed a virtual screening of olive secoiridoids of the OliveNetTM library to predict SARS-CoV-2 PLpro inhibition. Benchmarked molecular docking protocol that evaluated the performance of two docking programs was applied to execute virtual screening. Molecular dynamics stability analysis of the top-ranked olive secoiridoid docked to PLpro was also carried out. Methods: Benchmarking virtual screening used two freely available docking programs, AutoDock Vina 1.1.2. and AutoDock 4.2.1. for molecular docking of olive secoiridoids to a single PLpro structure. Screening also included benchmark structures of known active and decoy molecules from the DEKOIS 2.0 library. Based on the predicted binding energies, the docking programs ranked the screened molecules. We applied the usual performance evaluation metrices to evaluate the docking programs using the predicted ranks. Molecular dynamics of the top-ranked olive secoiridoid bound to PLpro and computation of MM-GBSA energy using three iterations during the last 50 ps of the analysis of the dynamics in Desmond supported the stability prediction. Results and discussions: Predictiveness curves suggested that AutoDock Vina has a better predictive ability than AutoDock, although there was a moderate correlation between the active molecules rankings (Kendall's correlation of rank (τ) = 0.581). Interestingly, two same molecules, Demethyloleuropein aglycone, and Oleuroside enriched the top 1 % ranked olive secoiridoids predicted by both programs. Demethyloleuropein aglycone bound to PLpro obtained by docking in AutoDock Vina when analyzed for stability by molecular dynamics simulation for 50 ns displayed an RMSD, RMSF<2 Å, and MM-GBSA energy of −94.54 ± 6.05 kcal/mol indicating good stability. Molecular dynamics also revealed the interactions of Demethyloleuropein aglycone with binding sites 2 and 3 of PLpro, suggesting a potent inhibition. In addition, for 98 % of the simulation time, two phenolic hydroxy groups of Demethyloleuropein aglycone maintained two hydrogen bonds with Asp302 of PLpro, specifying the significance of the groups in receptor binding. Conclusion: AutoDock Vina retrieved the active molecules accurately and predicted Demethyloleuropein aglycone as the best inhibitor of PLpro. The Arabian diet consisting of olive products rich in secoiridoids benefits from the PLpro inhibition property and reduces the risk of viral infection

Analytical Techniques for the Characterization and Quantification of Monoclonal Antibodies

Monoclonal antibodies (mAbs) are a fast-growing class of biopharmaceuticals. They are widely used in the identification and detection of cell makers, serum analytes, and pathogenic agents, and are remarkably used for the cure of autoimmune diseases, infectious diseases, or malignancies. The successful application of therapeutic mAbs is based on their ability to precisely interact with their appropriate target sites. The precision of mAbs rely on the isolation techniques delivering pure, consistent, stable, and safe lots that can be used for analytical, diagnostic, or therapeutic applications. During the creation of a biologic, the key quality features of a particular mAb, such as structure, post-translational modifications, and activities at the biomolecular and cellular levels, must be characterized and profiled in great detail. This implies the requirement of powerful state of the art analytical techniques for quality control and characterization of mAbs. Until now, various analytical techniques have been developed to characterize and quantify the mAbs according to the regulatory guidelines. The present review summarizes the major techniques used for the analyses of mAbs which include chromatographic, electrophoretic, spectroscopic, and electrochemical methods in addition to the modifications in these methods for improving the quality of mAbs. This compilation of major analytical techniques will help students and researchers to have an overview of the methodologies employed by the biopharmaceutical industry for structural characterization of mAbs for eventual release of therapeutics in the drug market