The restoration of hippocampal nerve de-myelination by methylcobalamin relates with the enzymatic regulation of homocysteine level in a rat model of moderate grade hepatic encephalopathy.

This article describes how methylcobalamin (MeCbl) restores nerve myelination in a moderate- grade hepatic encephalopathy (MoHE) model of ammonia neurotoxicity. The comparative profiles of myelin basic protein (MBP), homocysteine (Hcy) and methionine synthase (MS: a MeCbl- dependent enzyme) activity versus nerve myelination status were studied in the hippocampus of the control, the MoHE (developed by administering 100 mg/kg bw thioacetamide i.p. for 10 days) and the MoHE rats treated with MeCbl (500 µg/kg BW i.p.) for 7 days. Compared to those of control rats, the hippocampal CA1 and CA3 regions of the MoHE rats showed significantly lower myelinated areas and MBP immunostaining. This coincided with the deranged myelin layering in TEM images, decreased MBP protein and its transcript levels in hippocampus of MoHE rats. However, all these parameters recovered to normal levels after MeCbl treatment. MeCbl is a cofactor of MS that catalyzes the conversion of Hcy to methionine as a feeder step of methylation reactions. We observed significantly increased serum and hippocampal Hcy levels in MoHE rats, however, these levels were restored to control values with a concordant activation of MS due to MeCbl treatment. A significant recovery in neurobehavioral impairments in the MoHE rats due to MeCbl treatment was also observed. These findings suggest that MoHE pathogenesis is associated with deranged nerve myelination in the hippocampus and that MeCbl treatment is able to restore it mainly by activating MS, a MeCbl-dependent Hcy-metabolizing enzyme.

Modulation of brain energy metabolism in hepatic encephalopathy: impact of glucose metabolic dysfunction.

Cerebral function is linked to a high level of metabolic activity and relies on glucose as its primary energy source. Glucose aids in the maintenance of physiological brain activities; as a result, a disruption in metabolism has a significant impact on brain function, launching a chain of events that leads to neuronal death. This metabolic insufficiency has been observed in a variety of brain diseases and neuroexcitotoxicity disorders, including hepatic encephalopathy. It is a significant neurological complication that develops in people with liver disease, ranging from asymptomatic abnormalities to coma. Hyperammonemia is the main neurotoxic villain in the development of hepatic encephalopathy and induces a wide range of complications in the brain. The neurotoxic effects of ammonia on brain function are thought to be mediated by impaired glucose metabolism. Accordingly, in this review, we provide an understanding of deranged brain energy metabolism, emphasizing the role of glucose metabolic dysfunction in the pathogenesis of hepatic encephalopathy. We also highlighted the differential metabolic profiles of brain cells and the status of metabolic cooperation between them. The major metabolic pathways that have been explored are glycolysis, glycogen metabolism, lactate metabolism, the pentose phosphate pathway, and the Krebs cycle. Furthermore, the lack of efficacy in current hepatic encephalopathy treatment methods highlights the need to investigate potential therapeutic targets for hepatic encephalopathy, with regulating deficient bioenergetics being a viable alternative in this case. This review also demonstrates the importance of the development of glucose metabolism-focused disease diagnostics and treatments, which are now being pursued for many ailments.

Fisetin induces apoptosis in colorectal cancer cells by suppressing autophagy and down-regulating nuclear factor erythroid 2-related factor 2 (Nrf2).

Modulation of autophagy is evolving as a relevant strategy in cancer pathogenesis and therapeutic intervention and hence, needs to be examined as a target for the promising anticancer agents. Fisetin, a dietary flavanol, is emerging as a potent anticancer agent, however, its tumour-specific pharmacological targets remain largely unexplored. This article describes correlative profiles of autophagy and apoptotic markers versus nuclear factor erythroid 2-related factor 2 (Nrf2) and reactive oxygen species (ROS) in the colorectal cancer (CRC) cell line SW-480. As compared to the untreated cells, significantly less number of fluorescent detected autophagic vacuoles (AVOs) in the fisetin-treated cells coincided with a similar decline of the autophagy flux markers, Beclin 1 and microtubule-associated protein-1 light chain-3 and accumulation of p62 in those cells. The significantly increased number of annexin-V/propidium iodide (+/+) positive and acridine orange/ethidium bromide-stained apoptotic cells coincided with the enhanced signals for the cleaved caspase 3 and nuclear PARP-1 in those fisetin-treated cells. This was consistent with the collapse of mitochondrial membrane potential and release of cytochrome c. The fisetin-treated cells showed increased ROS level and a significant decline in nuclear Nrf2 immunosignal versus recovery in nuclear Nrf2 due to the treatment with curcumin and resveratrol (Nrf2 activators) and thus, suggesting a role of Nrf2 suppression in fisetin-mediated apoptosis in SW-480 cells. The effect of chloroquine, an autophagy inhibitor, resulted into declined number of AVOs and enhanced apoptosis, similar to that of the fisetin effect. Also, regaining of AVOs number and reduced apoptosis of CRC cells due to the treatment with rapamycin, an autophagy inducer, could be observed. These loss and gain of functions experiments thus suggested a correlation between fisetin-mediated autophagy suppression and apoptotic induction in a colorectal cell line.

Design and development of benzyl piperazine linked 5-phenyl-1,2,4-triazole-3-thione conjugates as potential agents to combat Alzheimer's disease.

Our present work demonstrates the molecular hybridization-assisted design, synthesis, and biological evaluation of 22 benzylpiperazine-linked 1,2,4-triazole compounds (PD1-22) as AD modifying agents. All the compounds were tested for their in vitro hChEs, hBACE-1, and Aß-aggregation inhibition properties. Among them, compound PD-08 and PD-22 demonstrated good hChE and hBACE-1 inhibition as compared to standards donepezil and rivastigmine. Both compounds displaced PI from PAS at 50 µM concentration which was comparable to donepezil and also demonstrated anti-Aß aggregation properties in self- and AChE-induced thioflavin T assay. Both compounds have shown excellent BBB permeation via PAMPA-BBB assay and were found to be non-neurotoxic at 80 µM concentration against differentiated SH-SY5Y cell lines. Compound PD-22 demonstrated an increase in rescued eye phenotype in Aß-phenotypic drosophila AD model and amelioration of behavioral deficits in the Aß-induced rat model of AD. The in-silico docking studies of compound PD-22 revealed a good binding profile towards CAS and PAS residues of AChE and the catalytic dyad of the BACE-1. The 100 ns molecular dynamics simulation studies of compound PD-22 complexed with AChE and BACE-1 enzymes suggested stable ligand-protein complex throughout the simulation run. Based on our findings compound PD-22 could further be utilized as a lead to design a promising candidate for AD therapy.

Cilostamide, a phosphodiesterase 3A inhibitor, sustains meiotic arrest of rat oocytes by modulating cyclic adenosine monophosphate level and the key regulators of maturation promoting factor.

Cilostamide, a phosphodiesterase 3A (Pde3A) inhibitor, is known to increase intraoocyte cyclic adenosine monophosphate (cAMP) level which is involved in sustaining meiotic arrest of the oocytes. To explore the mechanisms involved in the cilostamide-mediated meiotic arrest of the oocytes, the present study describes the effects of cilostamide on cAMP level and related factors involved in maturation of the oocytes at its different meiotic stages; diplotene, metaphase I (MI) and metaphase II (MII). The oocytes from these three stages were collected from rat ovary and incubated with 10 µM cilostamide for 3 h in CO2 incubator. The levels of cAMP, cyclic guanosine monophosphate (cGMP) and the key players of maintaining meiotic arrest during oocyte maturation; Emi2, Apc, Cyclin B1, and Cdk1, were analyzed in diplotene, MI and MII stages. Pde3A was found to be expressed at all three stages but with the lowest level in MI oocyte. As compared to the control sets, the cAMP concentration was found to be highest in MII whereas cGMP was highest in the diplotene stage of cilostamide-treated group. The treated group showed declined reactive oxygen species level as compared with the control counterparts. Relatively increased levels of the Emi2, Cyclin B1, and phosphorylated thr161 of Cdk1 versus declined levels of phosphorylated thr14/tyr15 of Cdk1 in diplotene and MII stage oocytes are known to be involved in maintaining meiotic arrest and all these factors were found to undergo similar pattern of change due to the treatment with cilostamide. The findings thus suggest that cilostamide treatment promotes meiotic arrest by Pde3A inhibition led increase of both cAMP and cGMP level vis-a-vis modulation of the related regulatory factors such as Emi2, CyclinB1, and phosphorylated status of Cdk1 in diplotene and MII stage oocytes. Such a mechanism of meiotic arrest could allow the oocyte to prepare itself for meiotic maturation and thereby to improve oocyte quality.

Mapping Connectome in Mammalian Brain: A Novel Approach by Bioengineering Neuro-Glia specific Vectors.

The connectome is the comprehensive map of the brain represented by wiring diagram of the full set of neuro-glia and synapses within entire brain of an organism. Some recent scientific efforts have successfully been made to visualize such map at neuro-glial networking level, however, capturing it as one unit of the entire brain have never been elucidated. Moreover, in order to derive structure-function relationship of different brain regions in response to a defined stimulus, there is a need to elucidate the connectome at single neuro-glial ensemble level after brain is challenged with the known memory function. This needs developing molecular approaches to tag neuro-glial activities in response to a conditioned brain function. Such approaches of using specific molecular tags have been tried to visualize independently neuron and glial specific events in response to a memory function, however, they could not tag the connectome together at single neuro-glia ensemble level. Therefore, there is a need to develop new methods for mapping entire connectome up to a single neuro-glial precision and resolution, with a purpose of tagging specific brain region accountable to execute a special memory formation process. The present hypothetical paper aims to propose a novel molecular method to generate the structural connectome at neuro-glial level in mice brain. Herein, we propose to tag the entire connectome at neuro-glia precision by generating a transgenic mice via transposing and recombining engineered novel "Neuro-Glia specific Vectors" (NGVs: specific to excitatory neurons, inhibitory neurons and glial cells) vis a vis "Transcriptional/ Translational Messenger (TMs: specific to metalloproteinases, MMP-9) coupled with different color protein tags, followed by the Clarity. Herein, the NGVs will be translated via Neuro-glia specific promoters, while TMs will be translated via endogenous MMP-9 promoter in all neuro-glial cells. The viability of all constructs will be verified in cortical/ hippocampal culture by inducing them to undergo chemically induced long term potentionation (cLTP) following visualization of different colored pattern. This will be further confirmed by Immunostaning, Western Blot and RT-PCR analysis. Additionally, in this approach, one can decipher the dynamics of molecular and cellular events associated with MMP-9 seretome by monitoring the trafficking of tagged endogenous MMP-9 protein after neuronal stimulation by cLTP in vitro. However, for visualizing complete connectome, the adult transgenic mice will be challenged with fear consolidation (Fear context and contextual cue) tests followed by Clarity coupled Light Sheet Microscopy to analyze neuro-glia ensemble following whole brain imaging.

Activation of oxidative stress and inflammatory factors could account for histopathological progression of aflatoxin-B1 induced hepatocarcinogenesis in rat.

Aflatoxin-B1 (AFB1) intoxication is known to develop hepatocellular carcinoma (HCC). However, pathogenesis and diagnosis of AFB1-induced HCC remain undefined. This article describes histopathological progression versus kinetics of the placental glutathione S-transferase (GST-pi) expression and profiles of the antioxidant enzymes, pro-inflammatory cytokines, and proliferative cell nuclear antigen (PCNA) in the liver from the rats administered with two doses of 1 mg AFB1/kg b.w. Histopathologically, hepatocytes necrosis could be observed after 4 weeks of the AFB1 treatment, which subsequently developed into the well-defined foci of altered hepatocytes (FAH) at 10 weeks post-treatment stage. This was consistent with an increasing trend of GST-pi immunostaining especially in the liver foci as a function of FAH progression and thus, suggesting that GST-pi over expression may serve as a marker for AFB1-induced hepatocarcinogenesis. The liver from AFB1-treated rats showed significantly enhanced level of reactive oxygen species coinciding with the declined immunostaining for superoxide dismutase-1, a committed enzyme of the antioxidant pathway, in the FAH regions and also with declined activity of the other antioxidant enzymes. Concordantly, the liver from the AFB1-treated rats showed over expression of pro-inflammatory cytokines; TNF-α & IL-1α and a cell proliferative marker PCNA. These findings present histological characterization of AFB1-induced HCC development and provide evidence for activation of oxidative stress-pro-inflammatory pathway during hepatocarcinogenesis induced by AFB1 toxicity.

Pannexin1 as a novel cerebral target in pathogenesis of hepatic encephalopathy.

Hepatic encephalopathy (HE) represents a nervous system disorder caused due to liver dysfunction. HE is broadly classified as acute/overt and moderate-minimal HE. Since HE syndrome severely affects quality of life of the patients and it may be life threatening, it is important to develop effective therapeutic strategy against HE. Mainly ammonia neurotoxicity is considered accountable for HE. Increased level of ammonia in the brain activates glutamate-NMDA (N-methyl-D-aspartate) receptor (NMDAR) pathway leading to Ca(2+) influx, energy deficit and oxidative stress in the post synaptic neurons. Moreover, NMDAR blockage has been found to be a poor therapeutic option, as this neurotransmitter receptor plays important role in maintaining normal neurophysiology of the brain. Thus, searching new molecular players in HE pathogenesis is of current concern. There is an evolving concept about roles of the trans-membrane channels in the pathogenesis of a number of neurological complications. Pannexin1 (Panx1) is one of them and has been described to be implicated in stroke, epilepsy and ischemia. Importantly, the pathogenesis of these complications relates to some extent with NMDAR over activation. Thus, it is speculated that HE pathogenesis might also involve Panx1. Indeed, some recent observations in the animal models of HE provide support to this argument. Since opening of Panx1 channel is mostly associated with the neuronal dysfunctions, down regulation of this channel could serve as a relevant therapeutic strategy without producing any serious side effects. In the review article an attempt has been made to summarize the current information on implication of Panx1 in the brain disorders and its prospects for being examined as pharmacological target in HE pathogenesis.

Restoration of hippocampal adult neurogenesis by CDRI-08 (Bacopa monnieri extract) relates with the recovery of BDNF-TrkB levels in male rats with moderate grade hepatic encephalopathy.

Modulation of in vivo adult neurogenesis (AN) is an evolving concept in managing neurodegenerative diseases. CDRI-08, a bacoside-enriched fraction of Bacopa monnieri, has been demonstrated for its neuroprotective actions, but its effect on AN remains unexplored. This article describes the status of AN by monitoring neuronal stem cells (NSCs) proliferation, differentiation/maturation markers and BDNF-TrkB levels (NSCs signalling players) vs. the level of neurodegeneration and their modulations by CDRI-08 in the hippocampal dentate gyrus (DG) of male rats with moderate grade hepatic encephalopathy (MoHE). For NSC proliferation, 10 mg/kg b.w. 5-bromo-2'-deoxyuridine (BrdU) was administered i.p. during the last 3 days, and for the NSC differentiation study, it was given during the first 3 days to the control, the MoHE (developed by 100 mg/kg b.w. of thioacetamide i.p. up to 10 days) and to the MoHE male rats co-treated with 350 mg/kg b.w. CDRI-08. Compared with the control rats, the hippocampus DG region of MoHE rats showed significant decreases in the number of Nestin+/BrdU+ and SOX2+/BrdU+ (proliferating) and DCX+/BrdU+ and NeuN+/BrdU+ (differentiating) NSCs. This was consistent with a similar decline in BDNF+/TrkB+ NSCs. However, all these NSC marker positive cells were observed to be recovered to their control levels, with a concordant restoration of total cell numbers in the DG of the CDRI-08-treated MoHE rats. The findings suggest that the restoration of hippocampal AN by CDRI-08 is consistent with the recovery of BDNF-TrkB-expressing NSCs in the MoHE rat model of neurodegeneration.

Preliminary data on cytotoxicity and functional group assessment of a herb-mineral combination against colorectal carcinoma cell line.

OBJECTIVES: The invasive screening methods and the late stage diagnosis of colorectal carcinoma (CRC) are contributing for the devastative prognosis. The gradual shift of the disease pattern among younger generations requires the implementation of phytochemicals and traditional medicines. Arkeshwara rasa (AR) is a herb-mineral combination of Tamra bhasma/incinerated copper ashes and Dwigun Kajjali/mercury sulphide levigated with Calotropis procera leaf juice, Plumbago zeylanica root decoction and the decoction of three myrobalans (Terminalia chebula, Terminalia bellerica, Emblica Officinalis decoction)/Triphala decoction. METHODS: The SW-480 cell line was checked for the cytotoxicity and the cell viability criteria with MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay. The acridine orange/ethidium bromide (AO/EtBr) assay revealed the depth of apoptosis affected cells in the fluorescent images. The FTIR analysis exhibited the graphical spectrum of functional groups within the compound AR. RESULTS: The IC50 from the 10-7 to 10-3 concentrations against SW-480 cells was 40.4 µg/mL. The staining of AO/EtBr was performed to visualize live and dead cells and it is evident from the result that number of apoptotic cells increases at increasing concentration of AR. The single bond with stretch vibrations of O-H and N-H are more concentrated in the 2,500-3,200 cm-1 and 3,700-4,000 cm-1 of the spectra whereas, the finger print region carries the O-H and S=O type peaks. CONCLUSIONS: The AR shows strong cyto-toxicity against the SW-480 cells by inducing apoptosis. It also modulates cellular metabolism with the involvement of functional groups which antagonizes the strong acids. Moreover, these effects need to be analyzed further based in the in vivo and various in vitro models.

Oral Release Kinetics, Biodistribution, and Excretion of Dopants from Barium-Containing Bioactive Glass in Rats.

Background: Inorganic biomaterials are biologically active and are used as implants and drug delivery system. They have therapeutically active elements present in their framework that are released in the physiological milieu. Release of these dopants above the supraphysiological limit may produce adverse effects and physicochemical interactions with the loaded drugs. Therefore, this necessitates evaluating the in vivo release kinetics, biodistribution, and excretion profiles of dopants from barium-doped bioglass (BaBG) that has potential anti-inflammatory, antiulcer, and regenerative properties. Methods: In vitro leaching of Ca, Si, and Ba from BaBG was analyzed in simulated body fluid. Release kinetics post single-dose oral administration (1, 5, and 10 mg/kg) was performed in rats. Blood was collected at different time points, and pharmacokinetic parameters of released elements were calculated. The routes of excretion and biodistribution in major organs were evaluated using ICP-MS. Results: Elements were released after the oral administration of BaBG into the plasma. They showed dose-dependent release kinetics and mean residence time. Cmax was observed at 24 h for all elements, followed by a downhill fall. There was also a dose-dependent increase in the volume of distribution, and the clearance of dopants was mostly through feces. Ba and Si were biodistributed significantly in the liver, spleen, and kidneys. However, by the end of day 7, there was a leveling-off effect observed for all elements. Conclusion: All of the dopants exhibited a dose-dependent increase in release kinetics and biodistribution in vital organs. This study will help in dose optimization and understanding of various physicochemical and pharmacokinetic interactions when BaBG is used for future pharmacological studies.

Design, synthesis, and biological evaluation of some 2-(3-oxo-5,6-diphenyl-1,2,4-triazin-2(3H)-yl)-N-phenylacetamide hybrids as MTDLs for Alzheimer's disease therapy.

Inspite of established symptomatic relief drug targets, a multi targeting approach is highly in demand to cure Alzheimer's disease (AD). Simultaneous inhibition of cholinesterase (ChE), ß secretase-1 (BACE-1) and Dyrk1A could be promising in complete cure of AD. A series of 18 diaryl triazine based molecular hybrids were successfully designed, synthesized, and tested for their hChE, hBACE-1, Dyrk1A and Aß aggregation inhibitory potentials. Compounds S-11 and S-12 were the representative molecules amongst the series with multi-targeted inhibitory effects. Compound S-12 showed hAChE inhibition (IC50 value = 0.486 ± 0.047 µM), BACE-1 inhibition (IC50 value = 0.542 ± 0.099 µM) along with good anti-Aß aggregation effects in thioflavin-T assay. Only compound S-02 of the series has shown Dyrk1A inhibition (IC50 value = 2.000 ± 0.360 µM). Compound S-12 has also demonstrated no neurotoxic liabilities against SH-SY5Y as compared to donepezil. The in vivo behavioral studies of the compound S-12 in the scopolamine- and Aß-induced animal models also demonstrated attanuation of learning and memory functions in rats models having AD-like characteristics. The ex vivo studies, on the rat hippocampal brain demonstrated reduction in certain biochemical markers of the AD brain with a significant increase in ACh level. The Western blot and Immunohistochemistry further revealed lower tau, APP and BACE-1 molecular levels. The drosophilla AD model also revealed improved eyephenotype after treatment with compound S-12. The molecular docking studies of the compounds suggested that compound S-12 was interacting with the ChE-PAS & CAS residues and catalytic dyad residues of the BACE-1 enzymes. The 100 ns molecular dynamics simulation studies of the ligand-protein complexed with hAChE and hBACE-1 also suggested stable ligand-protein confirmation throughout the simulation run.

Design, Synthesis, and Biological Investigation of Quinazoline Derivatives as Multitargeting Therapeutics in Alzheimer's Disease Therapy.

An efficient and promising method of treating complex neurodegenerative diseases like Alzheimer's disease (AD) is the multitarget-directed approach. Here in this work, a series of quinazoline derivatives (AV-1 to AV-21) were rationally designed, synthesized, and biologically evaluated as multitargeted directed ligands against human cholinesterase (hChE) and human ß-secretase (hBACE-1) that exhibit moderate to good inhibitory effects. Compounds AV-1, AV-2, and AV-3 from the series demonstrated balanced and significant inhibition against these targets. These compounds also displayed excellent blood-brain barrier permeability via the PAMPA-BBB assay. Compound AV-2 significantly displaced propidium iodide (PI) from the acetylcholinesterase-peripheral anionic site (AChE-PAS) and was found to be non-neurotoxic at the maximum tested concentration (80 µM) against differentiated SH-SY5Y cell lines. Compound AV-2 also prevented AChE- and self-induced Aß aggregation in the thioflavin T assay. Additionally, compound AV-2 significantly ameliorated scopolamine and Aß-induced cognitive impairments in the in vivo behavioral Y-maze and Morris water maze studies, respectively. The ex vivo and biochemical analysis further revealed good hippocampal AChE inhibition and the antioxidant potential of the compound AV-2. Western blot and immunohistochemical (IHC) analysis of hippocampal brain revealed reduced Aß, BACE-1, APP/Aß, and Tau molecular protein expressions levels. The pharmacokinetic analysis of compound AV-2 demonstrated significant oral absorption with good bioavailability. The in silico molecular modeling studies of lead compound AV-2 moreover demonstrated a reasonable binding profile with AChE and BACE-1 enzymes and stable ligand-protein complexes throughout the 100 ns run. Compound AV-2 can be regarded as the lead candidate and could be explored more for AD therapy.

Moderate grade hyperammonemia activates lactate dehydrogenase-4 and 6-phosphofructo-2-kinase to support increased lactate turnover in the brain slices.

Rapid metabolism of lactate is an important aspect of bioenergetic adaptation in the brain during non-physiological conditions. The low grade hyperammonemia (HA) is a common condition in the patients with chronic hepatic encephalopathy (HE); however, biochemistry of lactate turnover during low grade HA remains poorly defined. The present article describes profile of lactate dehydrogenase (LDH) isozymes vis-a-vis lactate level in the brain slices exposed with 0.1-0.5 mM ammonia, found to exist in the brain during chronic HE. A significant increment in LDH activity coincided with a similar increase in lactate level in the brain slices exposed with 0.5 mM ammonia. This was consistent with a selective increment of LDH-4 that synthesizes lactate from pyruvate with a concomitant decline in LDH-1 which catalyzes conversion of lactate to pyruvate; resulting into ~3-fold increase in LDH-4/LDH-1 ratio in those brain slices. The PFK2 domain of PFK2/FBPase2 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) regulates glycolysis to maintain the pyruvate pool for lactate synthesis. The PFK2 expression was also observed to be increased ~2-fold (P < 0.001) in 0.5 mM ammonia treated brain slices. These findings provide enzymatic regulation of increased lactate turnover in the brain exposed with moderate HA.

Impairments of Spatial Memory and N-methyl-d-aspartate Receptors and Their Postsynaptic Signaling Molecules in the Hippocampus of Developing Rats Induced by As, Pb, and Mn Mixture Exposure.

Exposure to metal mixtures is recognized as a real-life scenario, needing novel studies that can assess their complex effects on brain development. There is still a significant public health concern associated with chronic low levels of metal exposure. In contrast to other metals, these three metals (As, Pb, and Mn) are commonly found in various environmental and industrial contexts. In addition to additive or synergistic interactions, concurrent exposure to this metal mixture may also have neurotoxic effects that differ from those caused by exposure to single components. The NMDA receptor and several important signaling proteins are involved in learning, memory, and synaptic plasticity in the hippocampus, including CaMKII, postsynaptic density protein-95 (PSD-95), synaptic Ras GTPase activating protein (SynGAP), a negative regulator of Ras-MAPK activity, and CREB. We hypothesized that alterations in the above molecular players may contribute to metal mixture developmental neurotoxicity. Thus, the aim of this study was to investigate the effect of these metals and their mixture at low doses (As 4 mg, Pb 4 mg, and Mn 10 mg/kg bw/p.o) on NMDA receptors and their postsynaptic signaling proteins during developing periods (GD6 to PD59) of the rat brain. Rats exposed to As, Pb, and Mn individually or at the same doses in a triple-metal mixture (MM) showed impairments in learning and memory functions in comparison to the control group rats. Declined protein expressions of NR2A, PSD-95, p- CaMKII, and pCREB were observed in the metal mix-exposed rats, while the expression of SynGAP was found to be enhanced in the hippocampus as compared to the controls on PD60. Thereby, our data suggest that alterations in the NMDA receptor complex and postsynaptic signaling proteins could explain the cognitive dysfunctions caused by metal-mixture-induced developmental neurotoxicity in rats. These outcomes indicate that incessant metal mixture exposure may have detrimental consequences on brain development.

Design, Synthesis, and Biological Evaluation of Piperazine and N-Benzylpiperidine Hybrids of 5-Phenyl-1,3,4-oxadiazol-2-thiol as Potential Multitargeted Ligands for Alzheimer's Disease Therapy.

Our present work demonstrates the successful design and synthesis of a new class of compounds based upon a multitargeted directed ligand design approach to discover new agents for use in Alzheimer's disease (AD). All the compounds were tested for their in vitro inhibitory potential against human acetylcholinesterase (hAChE), human butylcholinesterase (hBChE), ß-secretase-1 (hBACE-1), and amyloid ß (Aß) aggregation. Compounds 5d and 5f have shown hAChE and hBACE-1 inhibition comparable to donepezil, while hBChE inhibition was comparable to rivastigmine. Compounds 5d and 5f also demonstrated a significant reduction in the formation of Aß aggregates through the thioflavin T assay and confocal, atomic force, and scanning electron microscopy studies and significantly displaced the total propidium iodide, that is, 54 and 51% at 50 µM concentrations, respectively. Compounds 5d and 5f were devoid of neurotoxic liabilities against RA/BDNF (RA = retinoic acid; BDNF = brain-derived neurotrophic factor)-differentiated SH-SY5Y neuroblastoma cell lines at 10-80 µM concentrations. In both the scopolamine- and Aß-induced mouse models for AD, compounds 5d and 5f demonstrated significant restoration of learning and memory behaviors. A series of ex vivo studies of hippocampal and cortex brain homogenates showed that 5d and 5f elicit decreases in AChE, malondialdehyde, and nitric oxide levels, an increase in glutathione level, and reduced levels of pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) mRNA. The histopathological examination of mice revealed normal neuronal appearance in the hippocampal and cortex regions of the brain. Western blot analysis of the same tissue indicated a reduction in Aß, amyloid precursor protein (APP)/Aß, BACE-1, and tau protein levels, which were non-significant compared to the sham group. The immunohistochemical analysis also showed significantly lower expression of BACE-1 and Aß levels, which was comparable to donepezil-treated group. Compounds 5d and 5f represent new lead candidates for developing AD therapeutics.

Moderate grade hyperammonemia induced concordant activation of antioxidant enzymes is associated with prevention of oxidative stress in the brain slices.

Acute hyperammonemia (HA) induced oxidative stress in the brain is considered to play critical roles in the neuropathology of end stage hepatic encephalopathy (HE). Moderate grade HA led minimal/moderate type HE is more common in the patients with chronic liver failure. However, implication of oxygen free radical ([Formula: see text]) based oxidative mechanisms remain to be defined during moderate grade HA. This article describes profiles of all the antioxidant enzymes Vis a Vis status of oxidative stress/damage in the brain slices exposed to 0.1-1 mM ammonia, reported to exist in the brain of animals with chronic liver failure and in liver cirrhotic patients. Superoxide dismutase catalyzes the first step of antioxidant mechanism and, with concerted activity of catalase, neutralizes [Formula: see text] produced in the cells. Both these enzymes remained unchanged up to 0.2-0.3 mM ammonia, however, with significant increments (P < 0.01-0.001) in the brain slices exposed to 0.5-1 mM ammonia. This was consistent with the similar pattern of production of reactive oxygen species in the brain slices. However, level of lipid peroxidation remained unchanged throughout the ammonia treatment. Synchronized activities of glutathione peroxidase and glutathione reductase regulate the level of glutathione to maintain reducing equivalents in the cells. The activities of both these enzymes also increased significantly in the brain slices exposed to 0.5-1 mM ammonia with concomitant increments in GSH/GSSG ratio and in the levels of total and protein bound thiol. The findings suggest resistance of brain cells from ammonia induced oxidative damage during moderate grade HA due to concordant activations of antioxidant enzymes.

Gender-specific association of oxidative stress and immune response in septic shock mortality using NMR-based metabolomics.

Background: Sepsis and septic shock are still associated with a high mortality rate. The early-stage prediction of septic shock outcomes would be helpful to clinicians for designing their treatment protocol. In addition, it would aid clinicians in patient management by understanding gender disparity in terms of clinical outcomes of septic shock by identifying whether there are sex-based differences in sepsis-associated mortality. Objective: This study aimed to test the hypothesis that gender-based metabolic heterogeneity is associated with sepsis survival and identify the biomarkers of mortality for septic shock in an Indian cohort. Method: The study was performed in an Indian population cohort diagnosed with sepsis/septic shock within 24 hours of admission. The study group was 50 patients admitted to intensive care, comprising 23 females and 27 males. Univariate and multivariate analysis were performed to identify the biomarkers for septic shock mortality and the gender-specific metabolic fingerprint in septic shock-associated mortality. Results: The energy-related metabolites, ketone bodies, choline, and NAG were found to be primarily responsible for differentiating survivors and non-survivors. The gender-based mortality stratification identified a female-specific association of the anti-inflammatory response, innate immune response, and ß oxidation, and a male-specific association of the pro-inflammatory response to septic shock. Conclusion: The identified mortality biomarkers may help clinicians estimate the severity of a case, as well as predict the outcome and treatment efficacy. The study underlines that gender is one of the most significant biological factors influencing septic shock metabolomic profiles. This understanding can be utilized to identify novel gender-specific biomarkers and innovative targets relevant for gender medicine.

Serum metabolic profiles of septic shock patients based upon co-morbidities and other underlying conditions.

Diagnosis and management of patients with septic shock is still a significant challenge for clinicians with its high mortality amongst hospitalized patients. Septic shock is a heterogeneous condition and is usually accompanied by various underlying disease conditions. Dissecting the specific metabolic changes induced by these underlying disease conditions through metabolomics has shown the potential to improve our understanding of the disease's relevant pathophysiological mechanisms, leading to improved treatment. This study has shown the metabolic alterations caused due to co-morbid conditions like diabetes, hypertension, CAD, and CKD in septic shock. It has also shown the distinct metabolic profiles of septic shock patients with underlying respiratory illnesses and encephalopathy. Metabolic profiling of sera obtained from 50 septic shock patients and 20 healthy controls was performed using high-resolution 1D 1H CPMG and diffusion-edited NMR spectra. Univariate and multivariate statistical analyses were performed to identify the potential molecular biomarkers. Noted dysregulations in amino acids, carbohydrates, and lipid metabolism were observed in septic shock patients. Further stratification within the septic shock patients based on co-morbid conditions and primary diagnosis has shown their role in causing metabolic alterations. Evaluation of these compounds during treatment will help design a personalized treatment protocol for the patients, improving therapeutics.

Mitochondrial SIRT3 and neurodegenerative brain disorders.

Sirtuins are highly conserved NAD+ dependent class III histone deacetylases and catalyze deacetylation and ADP ribosylation of a number of non-histone proteins. Since, they require NAD+ for their activity, the cellular level of Sirtuins represents redox status of the cells and thereby serves as bona fide metabolic stress sensors. Out of seven homologues of Sirtuins identified in mammals, SIRT3, 4 & 5 have been found to be localized and active in mitochondria. During recent past, clusters of protein substrates for SIRT3 have been identified in mitochondria and thereby advocating SIRT3 as the main mitochondrial Sirtuin which could be involved in protecting stress induced mitochondrial integrity and energy metabolism. As mitochondrial dysfunction underlies the pathogenesis of almost all neurodegenerative diseases, a role of SIRT3 becomes an arguable speculation in such brain disorders. Some recent findings demonstrate that SIRT3 over expression could prevent neuronal derangements in certain in vivo and in vitro models of aging and neurodegenerative brain disorders like; Alzheimer's disease, Huntington's disease, stroke etc. Similarly, loss of SIRT3 has been found to accelerate neurodegeneration in the brain challenged with excitotoxicity. Therefore, it is argued that SIRT3 could be a relevant target to understand pathogenesis of neurodegenerative brain disorders. This review is an attempt to summarize recent findings on (1) the implication of SIRT3 in neurodegenerative brain disorders and (2) whether SIRT3 modulation could ameliorate neuropathologies in relevant models.