Gut dysbiosis, defective autophagy and altered immune responses in neurodegenerative diseases: Tales of a vicious cycle.

The human microbiota comprises trillions of symbiotic microorganisms and is involved in regulating gastrointestinal (GI), immune, nervous system and metabolic homeostasis. Recent observations suggest a bidirectional communication between the gut microbiota and the brain via immune, circulatory and neural pathways, termed the Gut-Brain Axis (GBA). Alterations in gut microbiota composition, such as seen with an increased number of pathobionts and a decreased number of symbionts, termed gut dysbiosis or microbial intestinal dysbiosis, plays a prominent role in the pathogenesis of central nervous system (CNS)-related disorders. Clinical reports confirm that GI symptoms often precede neurological symptoms several years before the development of neurodegenerative diseases (NDDs). Pathologically, gut dysbiosis disrupts the integrity of the intestinal barrier leading to ingress of pathobionts and toxic metabolites into the systemic circulation causing GBA dysregulation. Subsequently, chronic neuroinflammation via dysregulated immune activation triggers the accumulation of neurotoxic misfolded proteins in and around CNS cells resulting in neuronal death. Emerging evidence links gut dysbiosis to the aggravation and/or spread of proteinopathies from the peripheral nervous system to the CNS and defective autophagy-mediated proteinopathies. This review summarizes the current understanding of the role of gut microbiota in NDDs, and highlights a vicious cycle of gut dysbiosis, immune-mediated chronic neuroinflammation, impaired autophagy and proteinopathies, which contributes to the development of neurodegeneration in Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We also discuss novel therapeutic strategies targeting the modulation of gut dysbiosis through prebiotics, probiotics, synbiotics or dietary interventions, and faecal microbial transplantation (FMT) in the management of NDDs.

Neuroinflammation Mechanisms and Phytotherapeutic Intervention: A Systematic Review.

Neuroinflammation is indicated in the pathogenesis of several acute and chronic neurological disorders. Acute lesions in the brain parenchyma induce intense and highly complex neuroinflammatory reactions with similar mechanisms among various disease prototypes. Microglial cells in the CNS sense tissue damage and initiate inflammatory responses. The cellular and humoral constituents of the neuroinflammatory reaction to brain injury contribute significantly to secondary brain damage and neurodegeneration. Inflammatory cascades such as proinflammatory cytokines from invading leukocytes and direct cell-mediated cytotoxicity between lymphocytes and neurons are known to cause "collateral damage" in models of acute brain injury. In addition to degeneration and neuronal cell loss, there are secondary inflammatory mechanisms that modulate neuronal activity and affect neuroinflammation which can even be detected at the behavioral level. Hence, several of health conditions result from these pathogenetic conditions which are underlined by progressive neuronal function loss due to chronic inflammation and oxidative stress. In the first part of this Review, we discuss critical neuroinflammatory mediators and their pathways in detail. In the second part, we review the phytochemicals which are considered as potential therapeutic molecules for treating neurodegenerative diseases with an inflammatory component.

Probing the Antitumor Mechanism of Solanum nigrum L. Aqueous Extract against Human Breast Cancer MCF7 Cells.

Solanum nigrum L. is one of the major medicinal plants used to treat cancer. However, the functional mechanism of S. nigrum L. extract is still unknown in spite of numerous studies on its active components. In this study, we probed the potential anticancer mechanism of the aqueous extract of S. nigrum L. (AESN) towards human breast cancer cell line MCF7. At a concentration of 10 g/L, AESN caused 43% cytotoxicity, inhibited the migration, and suppressed the activities of hexokinase and pyruvate kinase by about 30% and 40%, respectively, towards the MCF7 cells. RT2-PCR analysis of a panel of 89 caner-related genes identified 13 upregulated and eight downregulated genes (>2-folds) in MCF7 cells upon AESN treatment. Gene ontology (GO) and functional disease ontology (FunDO) analyses show that the antitumor function of S. nigrum L. involves multiple genes and these genes are shared across other diseases or disorders.

Benefits of curcumin in brain disorders.

Curcumin is widely consumed in Asia either as turmeric directly or as one of the culinary ingredients in food recipes. The benefits of curcumin in different organ systems have been reported extensively in several neurological diseases and cancer. Curcumin has got its global recognition because of its strong antioxidant, anti-inflammatory, anti-cancer, and antimicrobial activities. Additionally, it is used in diabetes and arthritis as well as in hepatic, renal, and cardiovascular diseases. Recently, there is growing attention on usage of curcumin to prevent or delay the onset of neurodegenerative diseases. This review summarizes available data from several recent studies on curcumin in various neurological diseases such as Alzheimer's disease, Parkinson's disease, Multiple Sclerosis, Huntington's disease, Prions disease, stroke, Down's syndrome, autism, Amyotrophic lateral sclerosis, anxiety, depression, and aging. Recent advancements toward increasing the therapeutic efficacy of curcuma/curcumin formulation and the novel delivery strategies employed to overcome its minimal bioavailability and toxicity studies have also been discussed. This review also summarizes the ongoing clinical trials on curcumin for different neurodegenerative diseases and patent details of curcuma/curcumin in India.

Residues His172 and Lys238 are Essential for the Catalytic Activity of the Maleylacetate Reductase from Sphingobium chlorophenolicum Strain L-1.

Maleylacetate reductase (PcpE), the last enzyme in the pentachlorophenol biodegradation pathway in Sphingobium chlorophenolicum L-1, catalyzes two consecutive reductive reactions, reductive dehalogenation of 2-chloromaleylacetate (2-CMA) to maleylacetate (MA) and subsequent reduction of MA to 3-oxoadipate (3-OXO). In each reaction, one molecule of NADH is consumed. To better understand its catalytic function, we undertook a structural model-based site-directed mutagenesis and steady-state kinetics study of PcpE. Our results showed that the putative catalytic site of PcpE is located in a positively charged solvent channel at the interface of the two domains and the binding of 2-CMA/MA involves seven basic amino acids, His172, His236, His237, His241 and His251, Lys140 and Lys238. Mutagenesis studies showed that His172 and Lys238 are essential for the catalytic activity of PcpE. However, the mutation of His236 to an alanine can increase the catalytic efficiency (k cat /K m ) of PcpE by more than 2-fold, implying that PcpE is still in an early stage of molecular evolution. Similar to tetrachlorobenzoquinone reductase (PcpD), PcpE is also inhibited by pentachlorophenol in a concentration-dependent manner. Furthermore, our studies showed that PcpE exhibits an extremely low but detectable level of alcohol dehalogenase activity toward ethanol and supports the notion that it is evolved from an iron-containing alcohol dehydrogenase.

Significance of HbA1c Test in Diagnosis and Prognosis of Diabetic Patients.

Diabetes is a global endemic with rapidly increasing prevalence in both developing and developed countries. The American Diabetes Association has recommended glycated hemoglobin (HbA1c) as a possible substitute to fasting blood glucose for diagnosis of diabetes. HbA1c is an important indicator of long-term glycemic control with the ability to reflect the cumulative glycemic history of the preceding two to three months. HbA1c not only provides a reliable measure of chronic hyperglycemia but also correlates well with the risk of long-term diabetes complications. Elevated HbA1c has also been regarded as an independent risk factor for coronary heart disease and stroke in subjects with or without diabetes. The valuable information provided by a single HbA1c test has rendered it as a reliable biomarker for the diagnosis and prognosis of diabetes. This review highlights the role of HbA1c in diagnosis and prognosis of diabetes patients.

Novel phytochemical-antibiotic conjugates as multitarget inhibitors of Pseudomononas aeruginosa GyrB/ParE and DHFR.

BACKGROUND: There is a dearth of treatment options for community-acquired and nosocomial Pseudomonas infections due to several rapidly emerging multidrug resistant phenotypes, which show resistance even to combination therapy. As an alternative, developing selective promiscuous hybrid compounds for simultaneous modulation of multiple targets is highly appreciated because it is difficult for the pathogen to develop resistance when an inhibitor has activity against multiple targets. METHODS: In line with our previous work on phytochemical-antibiotic combination assays and knowledge-based methods, using a fragment combination approach we here report a novel drug design strategy of conjugating synergistic phytochemical-antibiotic combinations into a single hybrid entity for multi-inhibition of P. aeruginosa DNA gyrase subunit B (GyrB)/topoisomerase IV subunit B (ParE) and dihydrofolate reductase (DHFR) enzymes. The designed conjugates were evaluated for their multitarget specificity using various computational methods including docking and dynamic simulations, drug-likeness using molecular properties calculations, and pharmacophoric features by stereoelectronic property predictions. RESULTS: Evaluation of the designed hybrid compounds based on their physicochemical properties has indicated that they are promising drug candidates with drug-like pharmacotherapeutic profiles. In addition, the stereoelectronic properties such as HOMO (highest occupied molecular orbital), LUMO (lowest unoccupied molecular orbital), and MEP (molecular electrostatic potential) maps calculated by quantum chemical methods gave a good correlation with the common pharmacophoric features required for multitarget inhibition. Furthermore, docking and dynamics simulations revealed that the designed compounds have favorable binding affinity and stability in both the ATP-binding sites of GyrB/ParE and the folate-binding site of DHFR, by forming strong hydrogen bonds and hydrophobic interactions with key active site residues. CONCLUSION: This new design concept of hybrid "phyto-drug" scaffolds, and their simultaneous perturbation of well-established antibacterial targets from two unrelated pathways, appears to be very promising and could serve as a prospective lead in multitarget drug discovery.

Hybrid-drug design targeting Pseudomonas aeruginosa dihydropteroate synthase and dihydrofolate reductase

In this study, we successfully present the dual-target design hypothesis to inhibit both dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) enzymes using a novel scheme that integrates our previous antibiotic-phytochemical interaction data, fragment combination and knowledge-based methods. Both the enzymes are well established antibacterial targets from folate biosynthesis pathway and their synergistic modulation by a single hybrid entity may have profound therapeutic benefits. Evaluation of the designed hybrid compounds based on their physico-chemical properties has indicated them as promising drug candidates with drug-like pharmacotherapeutic profiles. In addition, the stereo-electronic properties such as HOMO, LUMO and MEP maps calculated by quantum chemical methods gave a good correlation with the common pharmacophoric features required for dual-site interactions. Furthermore, docking and dynamics simulation studies reveal that the designed hybrid compounds have favorable binding affinity and stability in both pterin-binding site of DHPS and folate-binding site of DHFR by forming strong hydrogen bonds and hydrophobic interactions with key active-site residues. Looking forward this study could serve as a prospective lead in the process of new natural-product based hybrid-drugs development.

Therapeutic implications of targeting energy metabolism in breast cancer.

PPARs are ligand activated transcription factors. PPARγ agonists have been reported as a new and potentially efficacious treatment of inflammation, diabetes, obesity, cancer, AD, and schizophrenia. Since cancer cells show dysregulation of glycolysis they are potentially manageable through changes in metabolic environment. Interestingly, several of the genes involved in maintaining the metabolic environment and the central energy generation pathway are regulated or predicted to be regulated by PPARγ. The use of synthetic PPARγ ligands as drugs and their recent withdrawal/restricted usage highlight the lack of understanding of the molecular basis of these drugs, their off-target effects, and their network. These data further underscores the complexity of nuclear receptor signalling mechanisms. This paper will discuss the function and role of PPARγ in energy metabolism and cancer biology in general and its emergence as a promising therapeutic target in breast cancer.

Coffee component hydroxyl hydroquinone (HHQ) as a putative ligand for PPAR gamma and implications in breast cancer.

BACKGROUND: Coffee contains several compounds that have the potential to influence breast cancer risk and survival. However, epidemiologic data on the relation between coffee compounds and breast cancer survival are sparse and inconsistent. RESULTS: We show that coffee component HHQ has significant apoptotic effect on MDA-MB-231 and MCF-7 cells in vitro, and that ROS generation, change in mitochondrial membrane permeability, upregulation of Bax and Caspase-8 as well as down regulation of PGK1 and PKM2 expression may be important apoptosis-inducing mechanisms. The results suggest that PPARγ ligands may serve as potential therapeutic agents for breast cancer therapy. HHQ was also validated as a ligand for PPARγ by docking procedure. CONCLUSION: This is the first report on the anti-breast cancer (in vitro) activity of HHQ.

Glycolytic enzymes PGK1 and PKM2 as novel transcriptional targets of PPARγ in breast cancer pathophysiology.

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor and plays important roles in breast cancer cell proliferation. The complexity of the underlying biochemical and molecular mechanisms of breast cancer and the involvement of PPARγ in breast cancer pathophysiology are unclear. In this study, we carried out prediction of the peroxisome proliferator response element (PPRE) motifs in 2332 genes reported to be involved in breast cancer in literature. A total of 178 genes were found to have PPRE (DR1/DR2) and/or PPAR-associated conserved motif (PACM) motifs. We further constructed protein-protein interaction network, disease gene network and gene ontology (GO) analyses to identify novel key genes for experimental validation. We identified two genes in the glycolytic pathway (phosphoglycerate kinase 1 (PGK1) and pyruvate kinase M2 (PKM2)) at the ATP production steps and experimentally validated their repression by PPARγ in two breast cancer cell lines MDA-MB-231 and MCF-7. Further analysis suggested that this repression leads to decrease in ATP levels and apoptosis. These investigations will help us in understanding the molecular mechanisms by which PPARγ regulates the cellular energy pathway and the use of its ligands in human breast cancer therapeutics.

Comparative study for the effects of variable nutrient conditions on the biodegradation of microcystin-LR and concurrent dynamics in microcystin-degrading gene abundance.

Microcystin-LR (MCLR) degradation capability of biofilm was investigated with and without additional nutrients (nitrate, ammonium, peptone and glucose) at concentrations of 100 and 1000 mg L(-1). The MCLR-degradation was stimulated with nitrate and inhibited with other nutrients, except for that glucose of low concentration had no obvious effect. Both stimulatory and inhibitory effects enhanced with increasing concentration of corresponding nutrient. Quantitative polymerase chain reaction (qPCR) indicated that enhanced inhibition in biodegradation correlated to increased inhibition in functional gene (mlrA) abundance, as nutrient concentration increased. Stimulated biodegradation under low nitrate concentration may result from more rapid increase in mlrA gene abundance. These suggested that MCLR-degradation largely depended upon responsible bacterial population, which was affected by population of other bacteria in biofilm according to 16S rDNA-targeting qPCR. However, inhibited mlrA gene abundance implied that the stimulated biodegradation under high nitrate concentration might be involved in the mechanisms not related to MCLRDB population.

Insights into antifolate activity of phytochemicals against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic drug resistant pathogen. Drug interaction studies for phytochemicals (protocatechuic acid (PA), gallic acid (GA), quercetin (QUER), and myricetin (MYR)) in combination with antifolates (sulfamethoxazole (SMX) and trimethoprim (TMP)) are presented. Our results show that the combinations of SMX and phytochemicals are synergistic, whereas TMP in combination with phytochemicals results in additive mode of interaction. Molecular docking of phytochemicals in the active site of modeled P. aeruginosa dihydrofolate reductase (DHFR), an important enzyme in the folic acid biosynthesis pathway, shows that the phytochemicals QUER and MYR dock in the active site of P. aeruginosa DHFR with promoted binding at the NADP site, PA, and GA dock in the active site of P. aeruginosa DHFR with promoted binding at the folate binding site. Possible mode of action of these phytochemicals as anti-DHFR compounds in this bacterium is suggested. Taken together, the above findings provide novel insights to mode of interactions of these phytochemicals with antibiotics and may have significance as prospective leads in the development of antipseudomonal drug developments.

Targeting multiple targets in Pseudomonas aeruginosa PAO1 using flux balance analysis of a reconstructed genome-scale metabolic network.

Constraint-based flux balance analysis (FBA) is a powerful tool for predicting target genes that can be engineered by analyzing the redistribution of metabolic fluxes on specific gene modifications. Specifically, the effects of metabolic gene deletions on flux distribution can be examined by forcing the fluxes of different reactions catalyzed by the corresponding gene product to zero. However, the target enzyme needs to be essential for survival of the organism to ensure that efficient chemical inhibition results in cell stasis or death. Here, we investigate the essentiality of enzymes in iMO1056 metabolic model of nosocomial pathogen Pseudomonas aeruginosa by performing in silico enzyme deletions using FBA. We identified 116/113 essential enzymes in rich medium in P. aeruginosa. These were then compared with human metabolic model to identify nonhomologous enzymes that could be possible drug targets. Here, we present a refined list of 41 novel potential targets for P. aeruginosa. These targets were then matched with the enzymes belonging to 97 correlated clusters through which we propose the concept of "one target per cluster." Our approach relates to the "single drug multiple target (SDMT)" concept and has potential in efficient drug target discovery.

Cloning and targeted disruption of two lipopolysaccharide biosynthesis genes, kdsA and waaG, of Pseudomonas aeruginosa PAO1 by site-directed mutagenesis.

The emergence of antibiotic resistance in bacterial pathogens poses a great challenge to public health and emphasizes the need for new antimicrobial targets. The recent development of microbial genomics and the availability of genome sequences allows for the identification of essential genes which could be novel and potential targets for antibacterial drugs. However, these predicted targets need experimental validation to confirm essentiality. Here, we report on experimental validation of a two potential targets in the lipopolysaccharide (LPS) biosynthesis pathway of the pathogen Pseudomonas aeruginosa PAO1 using insertion duplication. Two genes, kdsA and waaG, from LPS encoding proteins 2-dehydro-3-deoxyphosphooctonate aldolase and UDP-glucose (heptosyl) LPS α-1,3-glucosyltransferase were selected as putative target candidates for the gene disruption experiments using plasmid insertion mutagenesis to determine essentiality. The introduction of a selectable ampicillin and kanamycin resistance marker into the chromosome resulted in lack of recovery of antibiotic-resistant colonies suggesting the essentiality of these genes for the survival of P. aeruginosa. Several molecular analyses were carried out in order to confirm the essentiality of these genes. We propose that the above two validated drug targets are essential and can be screened for functional inhibitors for the discovery of novel therapeutic compounds against antibiotic-resistant opportunistic pathogen P. aeruginosa.

Activity and interactions of antibiotic and phytochemical combinations against Pseudomonas aeruginosa in vitro.

In this study the in vitro activities of seven antibiotics (ciprofloxacin, ceftazidime, tetracycline, trimethoprim, sulfamethoxazole, polymyxin B and piperacillin) and six phytochemicals (protocatechuic acid, gallic acid, ellagic acid, rutin, berberine and myricetin) against five P. aeruginosa isolates, alone and in combination are evaluated. All the phytochemicals under investigation demonstrate potential inhibitory activity against P. aeruginosa. The combinations of sulfamethoxazole plus protocatechuic acid, sulfamethoxazole plus ellagic acid, sulfamethoxazole plus gallic acid and tetracycline plus gallic acid show synergistic mode of interaction. However, the combinations of sulfamethoxazole plus myricetin shows synergism for three strains (PA01, DB5218 and DR3062). The synergistic combinations are further evaluated for their bactericidal activity against P. aeruginosa ATCC strain using time-kill method. Sub-inhibitory dose responses of antibiotics and phytochemicals individually and in combination are presented along with their interaction network to suggest on the mechanism of action and potential targets for the phytochemicals under investigation. The identified synergistic combinations can be of potent therapeutic value against P. aeruginosa infections. These findings have potential implications in delaying the development of resistance as the antibacterial effect is achieved with lower concentrations of both drugs (antibiotics and phytochemicals).

In vitro drug interactions of gallates with antibiotics in Staphylococcus Aureus.

The antimicrobial activities of tetracycline, mupirocin, and fusidic acid are tested in combination with Epicatechin Gallate (ECG), and Ethyl Gallate (EG) using 2 Methicillin resistant (MRSA) and 2 Methicillin sensitive (MSSA) strains of Staphylococcus aureus. Sub-inhibitory concentration of EG at 256 mg l-1 is found to be synergistic when used in combination with tetracycline, mupirocin, and fusidic acid; and a sub-inhibitory concentration of ECG at 32 mg l-1 is found to be synergistic with tetracycline in all the four Staphylococcus aureus strains tested. The synergistic combinations reduce the MICs of all the above three antibiotics by 4 fold. Combining ECG at 32 mg l-1 with mupirocin, reduces the MIC of mupirocin by four fold in MSSA C1 strain. 74 per cent of the combinations show consistent results in both time-kill assay and checkerboard method. The identified combinations may lead towards novel therapeutic interventions for treating MRSA infections.

Human genomic diversity, viral genomics and proteomics, as exemplified by human papillomaviruses and H5N1 influenza viruses.

The diversity of hosts, pathogens and host-pathogen relationships reflects the influence of selective pressures that fuel diversity through ongoing interactions with other rapidly evolving molecules in the environment. This paper discusses specific examples illustrating the phenomenon of diversity of hosts and pathogens, with special reference to human papillomaviruses and H5N1 influenza viruses. We also review the influence of diverse host-pathogen interactions that determine the pathophysiology of infections, and their responses to drugs or vaccines.

Activity of Chitosans in combination with antibiotics in Pseudomonas aeruginosa.

Chitosan and its derivative water soluble Chitosan oligosaccharide are used in a variety of applications in pharmaceutical preparations. In this study, 2 wild (ATCC 15729 and PAO1) and 2 mutant strains (PT121 and PT149) of P. aeruginosa are investigated for drug-drug interactions in vitro. 10 antimicrobial agents (antibiotics) are combined with different degree of deacetylated Chitosans and Chitosan oligosaccharide. All the chitosans show synergistic activity with sulfamethoxazole, a sulfonamide antimicrobial agent. It is interesting to observe that the MIC value for the MexEF-OprN overexpressing mutant strain of P. aeruginosa is 5 fold higher than the other strains under investigation suggesting a possible role of this efflux pump in Sulfamethoxazole efflux. The findings suggest on the use of chitosans as enhancing agent in combination with antibiotics in pharmaceutical preparations.