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Neuroblastomas are pediatric tumors arising from undifferentiated cells of neural crest origin with stem cell-like characteristics. Dysregulation of Wnt/β-catenin signaling has been shown to be linked to the development of various tumors. Activated Wnt signaling results in β-catenin accumulation in the nucleus to support pro-neoplastic traits. DKK1, a secreted glycoprotein, is an inhibitor of Wnt signaling, and the addition of DKKI to the culture medium has been used to suppress the Wnt pathway. This study aimed to analyze the role of Dickopff-1 as a potential differentiating agent for the neuroblastoma cell line SH-SY5Y and neurospheres derived from it. The treatment of SH-5Y5Y derived neurospheres by DKK1 resulted in their disintegration and reduced proliferation markers like Ki67, PCNA. DKK1 treatment to the neurospheres also resulted in the loss of cancer stem cell markers like CD133, KIT and pluripotency markers like SOX2, OCT4, NANOG. DKK1 treatment caused reduction in mRNA expression of β-catenin and TCF genes like TCF4, TCF12. When the SH-SY5Y cancer cells were grown under differentiating conditions, DKKI caused neuronal differentiation by itself, and in synergy with retinoic acid. This was verified by the expression of markers like MAPT, DCX, GAP43, ENO2 and also with changes in neurite length. We concluded that Wnt inhibition, as exemplified by DKK1 treatment, is therefore a possible differentiating condition and also suppresses the proliferative and cancer stemness related properties of SH-SY5Y neuroblastoma cells.

Neurotropic viruses can cross the otherwise dynamically regulated blood-brain barrier (BBB) and affect the brain cells. Zika virus (ZIKV) is an enveloped neurotropic Flavivirus known to cause severe neurological complications, such as encephalitis and fetal microcephaly. In the present study, we employed human brain microvascular endothelial cells (hBMECs) and astrocytes derived from human progenitors to establish a physiologically relevant BBB model. We used this model to investigate the effects of ZIKV envelope (E) protein on properties of cells comprising the BBB. E protein is the principal viral protein involved in interaction with host cell surface receptors, facilitating the viral entry. Our findings show that the presence of ZIKV E protein leads to activation of both hBMECs and astrocytes. In hBMECs, we observed a decrease in the expression of crucial endothelial junction proteins such as ZO-1, Occludin and VE-Cadherin, which are vital in establishment and maintenance of the BBB. Consequently, the ZIKV E protein induced changes in BBB integrity and permeability. We also found upregulation of genes involved in leukocyte recruitment along with increased proinflammatory chemokines and cytokines upon exposure to E protein. Additionally, the E protein also led to astrogliosis, evident from the elevated expression of GFAP and Vimentin. Both cell types comprising the BBB exhibited inflammatory response upon exposure to E protein which may influence viral access into the central nervous system (CNS) and subsequent infection of other CNS cells. Overall, our study provides valuable insights into the transient changes that occur at the site of BBB upon ZIKV infection.

Mortalin is a chaperone protein that regulates physiological functions of cells. Its multifactorial role allows cells to survive pathological conditions. Pharmacological, chemical, and siRNA-mediated downregulation of mortalin increases oxidative stress, mitochondrial dysfunction leading to unregulated inflammation. In addition to its well-characterized function in controlling oxidative stress, mitochondrial health, and maintaining physiological balance, recent evidence from human brain autopsies and cell culture–based studies suggests a critical role of mortalin in attenuating the damage seen in several neurodegenerative diseases. Overexpression of mortalin provides an important line of defense against accumulated proteins, inflammation, and neuronal loss, a key characteristic feature observed in neurodegeneration. Neurodegenerative diseases are a group of progressive disorders, sharing pathological features in Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and HIV-associated neurocognitive disorder. Aggregation of insoluble amyloid beta-proteins and neurofibrillary tangles in Alzheimer’s disease are among the leading cause of neuropathology in the brain. Parkinson’s disease is characterized by the degeneration of dopamine neurons in substantia nigra pars compacta. A substantial synaptic loss leading to cognitive decline is the hallmark of HIV-associated neurocognitive disorder (HAND). Brain autopsies and cell culture studies showed reduced expression of mortalin in Alzheimer’s, Parkinson’s, and HAND cases and deciphered the important role of mortalin in brain cells. Here, we discuss mortalin and its regulation and describe how neurotoxic conditions alter the expression of mortalin and modulate its functions. In addition, we also review the neuroprotective role of mortalin under neuropathological conditions. This knowledge showcases the importance of mortalin in diverse brain functions and offers new opportunities for the development of therapeutic targets that can modulate the expression of mortalin using chemical compounds.

Oxidative stress and excitotoxicity are some of the pathophysiological abnormalities in hypoxia-induced brain injury. This study evaluated the intrinsic antioxidant property of methanol fruit extract of Tetrapleura tetraptera (TT), traditionally used for managing brain diseases such as cerebral infarction in West Africa, and its ability to protect primary astrocytes from anoxia-induced cell death. The effect of the phytochemicals present in TT on excitotoxicity was assessed in silico, through docking with human glutamate synthetase (hGS). Chromatographic and spectrophotometric analyses of TT were performed. Primary astrocytes derived from neural stem cells were treated with TT and its effect on astrocyte viability was assessed. TT-treated astrocytes were then subjected to anoxic insult and, cell viability and mitochondrial membrane potential were evaluated. Molecular docking of hGS with detected phytochemicals in TT (aridanin, naringenin, ferulic acid, and scopoletin) was performed and the number of interactions with the lead compounds, aridanin, analyzed. HPLC-DAD analysis of TT revealed the presence of various bioactive phytochemicals. TT demonstrated notable antioxidant and radical scavenging activities. TT also protected astrocytes from anoxic insult by restoring cell viability and preventing alteration to mitochondrial membrane integrity. Aridanin, naringenin, ferulic acid, and scopoletin demonstrated good binding affinities with hGS indicating that Tetrapleura tetraptera is a potential source of new plant-based bioactives relevant in the therapy of neurodegenerative diseases.

Abstract Background In human immunodeficiency virus-1 (HIV-1) infection, activation of astrocytes induces imbalance in physiological functions due to perturbed astrocytic functions that unleashes toxicity on neurons. This leads to inflammatory response finally culminating into neurocognitive dysfunction. In neuroAIDS, HIV-1 protein, transactivator of transcription (Tat) is detected in the cerebrospinal fluid of infected patients. Mortalin, a multifunctional protein, has anti-inflammatory role following its activation in various stress conditions. Recent studies demonstrate downregulation of mortalin in neurodegenerative diseases. Here, we explored the mechanisms of mortalin in modulating HIV-1 Tat-mediated neuroinflammation. Methods Expression of mortalin in autopsy section in normal and diseased individuals were examined using immunohistochemistry. To decipher the role of mortalin in HIV-1 Tat-induced activation, human fetal brain-derived astrocytes were transiently transfected with Tat and mortalin using expression vectors. HIV-1 Tat-mediated damage was analyzed using RT-PCR and western blotting. Modulatory role of mortalin was examined by coexpressing it with Tat, followed by examination of mitochondrial morphodynamics using biochemical assay and confocal and electron microscopy. Extracellular ATP release was monitored using luciferase assay. Neuroinflammation in astrocytes was examined using flow cytometry, dye based study, immunocytochemistry, immunoprecipitation, and western blotting. Indirect neuronal damage was also analyzed. Results HIV-1 Tat downregulates the expression of mortalin in astrocytes, and this is corroborated with autopsy sections of HIV-1 patients. We found that overexpression of mortalin with Tat reduced inflammation and also rescued astrocytic-mediated neuronal death. Using bioinformatics, we discovered that binding of mortalin with Tat leads to Tat degradation and rescues the cell from neuroinflammation. Blocking of proteosomal pathway rescued the Tat degradation and revealed the ubiquitination of Tat. Conclusion Overall, our data demonstrated the protective role of mortalin in combating HIV-1 Tat-mediated damage. We also showed that mortalin could degrade Tat through direct binding with HIV-1 Tat. Overexpression of mortalin in the presence of Tat could significantly reduce cytotoxic effects of Tat in astrocytes. Indirect neuronal death was also found to be rescued. Our in vitro findings were validated as we found attenuated expression of mortalin in the autopsy sections of HIV-1 patients.

Abstract Amyloid beta is a major constituent of the plaques found in the brains of patients suffering from Alzheimer’s disease (AD). A growing body of research work suggests that neuroinflammation plays important roles in the development of AD. Thus, considerable efforts are directed towards identification of compounds that can reduce or inhibit neuroinflammation. Here, we show that sinomenine, a compound present in a Chinese medicinal plant, Sinomenium acutum, inhibits oligomeric amyloid beta-induced production of reactive oxygen species (ROS), nitric oxide (NO) and inflammation-related molecules from astrocytic cells. The conditioned medium from oligomeric amyloid beta-treated astrocytic cells induces cell death in the hippocampal neuronal cells. Importantly, sinomenine inhibits this cell death. In addition, this compound has inhibitory effects on the production of ROS, NO and inflammation-related factors from oligomeric amyloid-beta treated human astrocytes. Finally, the conditioned medium from oligomeric amyloid beta-treated human astrocytes induces cell death in the primary culture of human neurons, which is inhibited by sinomenine. Thus, sinomenine inhibits amyloid beta-induced production of toxic factors from astrocytes, and confers protection to hippocampal neuronal cells as well as human neurons against indirect toxicity. The results suggest that this compound could provide beneficial effects in AD and other neurodegenerative conditions by reducing inflammation and neuronal cell death.

Glial cells perform important supporting functions for neurons through a dynamic crosstalk. Neuron–glia communication is the major phenomenon to sustain homeostatic functioning of the brain. Several interactive pathways between neurons and astrocytes are critical for the optimal functioning of neurons, and one such pathway is the ephrinA3–ephA4 signaling. The role of this pathway is essential in maintaining the levels of extracellular glutamate by regulating the excitatory amino acid transporters, EAAT1 and EAAT2 on astrocytes. Human immunodeficiency virus-1 (HIV-1) and its proteins cause glutamate excitotoxicity due to excess glutamate levels at sites of high synaptic activity. This study unravels the effects of HIV-1 transactivator of transcription (Tat) from clade B on ephrinA3 and its role in regulating glutamate levels in astrocyte–neuron co-cultures of human origin. It was observed that the expression of ephrinA3 increases in the presence of HIV-1 Tat B, while the expression of EAAT1 and EAAT2 was attenuated. This led to reduced glutamate uptake and therefore high neuronal death due to glutamate excitotoxicity. Knockdown of ephrinA3 using small interfering RNA, in the presence of HIV-1 Tat B reversed the neurotoxic effects of HIV-1 Tat B via increased expression of glutamate transporters that reduced the levels of extracellular glutamate. The in vitro findings were validated in autopsy brain sections from acquired immunodeficiency syndrome patients and we found ephrinA3 to be upregulated in the case of HIV-1-infected patients. This study offers valuable insights into astrocyte-mediated neuronal damage in HIV-1 neuropathogenesis.

Cellular metabolism has key roles in T cells differentiation and function. CD4+ T helper-1 (Th1), Th2, and Th17 subsets are highly glycolytic while regulatory T cells (Tregs) use glucose during expansion but rely on fatty acid oxidation for function. Upon uptake, glucose can enter pentose phosphate pathway (PPP) or be used in glycolysis. Here, we showed that blocking 6-phosphogluconate dehydrogenase (6PGD) in the oxidative PPP resulted in substantial reduction of Tregs suppressive function and shifts toward Th1, Th2, and Th17 phenotypes which led to the development of fetal inflammatory disorder in mice model. These in turn improved anti-tumor responses and worsened the outcomes of colitis model. Metabolically, 6PGD blocked Tregs showed improved glycolysis and enhanced non-oxidative PPP to support nucleotide biosynthesis. These results uncover critical role of 6PGD in modulating Tregs plasticity and function, which qualifies it as a novel metabolic checkpoint for immunotherapy applications.

Circulating cell-free mitochondrial DNA (cf-mtDNA) has been found in the plasma of severely ill COVID-19 patients and is now known as a strong predictor of mortality. However, the underlying mechanism of mtDNA release is unexplored. Here, we show a novel mechanism of SARS-CoV-2-mediated pro-inflammatory/pro-apoptotic mtDNA release and a rational therapeutic stem cell-based approach to mitigate these effects. We systematically screened the effects of 29 SARS-CoV-2 proteins on mitochondrial damage and cell death and found that NSP4 and ORF9b caused extensive mitochondrial structural changes, outer membrane macropore formation, and the release of inner membrane vesicles loaded with mtDNA. The macropore-forming ability of NSP4 was mediated through its interaction with BCL2 antagonist/killer (BAK), whereas ORF9b was found to inhibit the anti-apoptotic member of the BCL2 family protein myeloid cell leukemia-1 (MCL1) and induce inner membrane vesicle formation containing mtDNA. Knockdown of BAK and/or overexpression of MCL1 significantly reversed SARS-CoV-2-mediated mitochondrial damage. Therapeutically, we engineered human mesenchymal stem cells (MSCs) with a simultaneous knockdown of BAK and overexpression of MCL1 (MSCshBAK+MCL1) and named these cells IMAT-MSCs (intercellular mitochondrial transfer-assisted therapeutic MSCs). Upon co-culture with SARS-CoV-2-infected or NSP4/ORF9b-transduced airway epithelial cells, IMAT-MSCs displayed functional intercellular mitochondrial transfer (IMT) via tunneling nanotubes (TNTs). The mitochondrial donation by IMAT-MSCs attenuated the pro-inflammatory and pro-apoptotic mtDNA release from co-cultured epithelial cells. Our findings thus provide a new mechanistic basis for SARS-CoV-2-induced cell death and a novel therapeutic approach to engineering MSCs for the treatment of COVID-19.

Summary: Although T cell expansion depends on glycolysis, T effector cell differentiation requires signaling via the production of reactive oxygen species (ROS). Because the pentose phosphate pathway (PPP) regulates ROS by generating nicotinamide adenine dinucleotide phosphate (NADPH), we examined how PPP blockade affects T cell differentiation and function. Here, we show that genetic ablation or pharmacologic inhibition of the PPP enzyme 6-phosphogluconate dehydrogenase (6PGD) in the oxidative PPP results in the generation of superior CD8+ T effector cells. These cells have gene signatures and immunogenic markers of effector phenotype and show potent anti-tumor functions both in vitro and in vivo. In these cells, metabolic reprogramming occurs along with increased mitochondrial ROS and activated antioxidation machinery to balance ROS production against oxidative damage. Our findings reveal a role of 6PGD as a checkpoint for T cell effector differentiation/survival and evidence for 6PGD as an attractive metabolic target to improve tumor immunotherapy.

Over the past two decades, our understanding of Parkinson's disease (PD) has been gleaned from the discoveries made in familial and/or sporadic forms of PD in the Caucasian population. The transferability and the clinical utility of genetic discoveries to other ethnically diverse populations are unknown. The Indian population has been under-represented in PD research. The Genetic Architecture of PD in India (GAP-India) project aims to develop one of the largest clinical/genomic bio-bank for PD in India. Specifically, GAP-India project aims to: (1) develop a pan-Indian deeply phenotyped clinical repository of Indian PD patients; (2) perform whole-genome sequencing in 500 PD samples to catalog Indian genetic variability and to develop an Indian PD map for the scientific community; (3) perform a genome-wide association study to identify novel loci for PD and (4) develop a user-friendly web-portal to disseminate results for the scientific community. Our “hub-spoke” model follows an integrative approach to develop a pan-Indian outreach to develop a comprehensive cohort for PD research in India. The alignment of standard operating procedures for recruiting patients and collecting biospecimens with international standards ensures harmonization of data/bio-specimen collection at the beginning and also ensures stringent quality control parameters for sample processing. Data sharing and protection policies follow the guidelines established by local and national authorities.We are currently in the recruitment phase targeting recruitment of 10,200 PD patients and 10,200 healthy volunteers by the end of 2020. GAP-India project after its completion will fill a critical gap that exists in PD research and will contribute a comprehensive genetic catalog of the Indian PD population to identify novel targets for PD.

The lack of information on astrogenesis following stressor effect, notwithstanding the imperative roles of astroglia in normal physiology and pathophysiology, incited us to assess temporal astrogenesis and astrocyte density in an intra-generational protein malnutrition (PMN) rat model. Standard immunohistochemical procedures for glial lineage markers and their intensity measurements, and qRT-PCR studies, were performed to reveal the spatio-temporal origin and density of astrocytes. Reduced A2B5+ glia restricted precursor population in ventricles and caused poor dissemination to cortex at embryonic days (E)11-14, and low BLBP+ secondary radial glia in the subventricular zone (SVZ) of E16 low protein (LP) brains reflect compromised progenitor pooling. Contrary to large-sized BLBP+ gliospheres in high protein (HP) brains at E16, small gliospheres and discrete BLBP+ cells in LP brains evidence loss of colonization and low proliferative potential. Delayed emergence of GFAP expression, precocious astrocyte maturation and significantly reduced astrocyte number suggest impaired temporal and compromised astrogenesis within LP-F1 brains. Our findings of protein deprivation induced impairments in temporal astrogenesis, compromised density and astrocytic dysfunction, strengthen the hypothesis of astrocytes as possible drivers of neurodevelopmental disorders. This study may increase our understanding of stressor-associated brain development, opening up windows for effective therapeutic interventions against debilitating neurodevelopmental disorders.

Abstract In this study we have tested the efficacy of citrate therapy in various cancer models. We found that citrate administration inhibited A549 lung cancer growth and additional benefit accrued in combination with cisplatin. Interestingly, citrate regressed Ras-driven lung tumors. Further studies indicated that citrate induced tumor cell differentiation. Additionally, citrate treated tumor samples showed significantly higher infiltrating T-cells and increased blood levels of numerous cytokines. Moreover, we found that citrate inhibited IGF-1R phosphorylation. In vitro studies suggested that citrate treatment inhibited AKT phosphorylation, activated PTEN and increased expression of p-eIF2a. We also found that p-eIF2a was decreased when PTEN was depleted. These data suggest that citrate acts on the IGF-1R-AKT-PTEN-eIF2a pathway. Additionally, metabolic profiling suggested that both glycolysis and the tricarboxylic acid cycle were suppressed in a similar manner in vitro in tumor cells and in vivo but only in tumor tissue. We reproduced many of these observations in an inducible Her2/Neu-driven breast cancer model and in syngeneic pancreatic tumor (Pan02) xenografts. Our data suggests that citrate can inhibit tumor growth in diverse tumor types and via multiple mechanisms. Dietary supplementation with citrate may be beneficial as a cancer therapy.

ABSTRACT RNA viruses are known to modulate host microRNA (miRNA) machinery for their own benefit. Japanese encephalitis virus (JEV), a neurotropic RNA virus, has been reported to manipulate several miRNAs in neurons or microglia. However, no report indicates a complete sketch of the miRNA profile of neural stem/progenitor cells (NSPCs), hence the focus of our current study. We used an miRNA array of 84 miRNAs in uninfected and JEV-infected human neuronal progenitor cells and primary neural precursor cells isolated from aborted fetuses. Severalfold downregulation of hsa-miR-9-5p, hsa-miR-22-3p, hsa-miR-124-3p, and hsa-miR-132-3p was found postinfection in both of the cell types compared to the uninfected cells. Subsequently, we screened for the target genes of these miRNAs and looked for the biological pathways that were significantly regulated by the genes. The target genes involved in two or more pathways were sorted out. Protein-protein interaction (PPI) networks of the miRNA target genes were formed based on their interaction patterns. A binary adjacency matrix for each gene network was prepared. Different modules or communities were identified in those networks by community detection algorithms. Mathematically, we identified the hub genes by analyzing their degree centrality and participation coefficient in the network. The hub genes were classified as either provincial (P 0.4) hubs. We validated the expression of hub genes in both cell line and primary cells through qRT-PCR after JEV infection and respective miR mimic transfection. Taken together, our findings highlight the importance of specific target gene networks of miRNAs affected by JEV infection in NSPCs. IMPORTANCE JEV damages the neural stem/progenitor cell population of the mammalian brain. However, JEV-induced alteration in the miRNA expression pattern of the cell population remains an open question, hence warranting our present study. In this study, we specifically address the downregulation of four miRNAs, and we prepared a protein-protein interaction network of miRNA target genes. We identified two types of hub genes in the PPI network, namely, connector hubs and provincial hubs. These two types of miRNA target hub genes critically influence the participation strength in the networks and thereby significantly impact up- and downregulation in several key biological pathways. Computational analysis of the PPI networks identifies key protein interactions and hubs in those modules, which opens up the possibility of precise identification and classification of host factors for viral infection in NSPCs.

Tumor-induced immune tolerance permits growth and spread of malignant cells. Cancer cells have strong influence on surrounding cells and shape the hypoxic tumor microenvironment (TME) facilitating cancer progression. A dynamic change in glucose metabolism occurring in cancer cells and its influence on the TME are still poorly understood. Indeed, cancer and/or immune cells undergo rapid adaptation in metabolic pathways during cancer progression. Metabolic reprograming affects macrophages, T cells, and myeloid derived suppressor cells (MDSCs) among other immune cells. Their role in the TME depends on a nature and concentration of factors, such as cytokines, reactive oxygen species (ROS), growth factors, and most importantly, diffusible metabolites (i.e., lactate). Further, the amounts of available nutrients and oxygen as well as activity of microbiota may influence metabolic pathways in the TME. The roles of metabolites in regulating of the interaction between immune and cancer cell are highlighted in this review. Targeting metabolic reprogramming or signaling pathways controlling cell metabolism in the TME might be a potential strategy for anti-cancer therapy alone or in combination with current immunotherapies.

There has been significant progress in utilizing our immune system against cancer, mainly by checkpoint blockade and T cell-mediated therapies. The field of cancer immunotherapy is growing rapidly but durable clinical benefits occur only in a small subset of responding patients. It is currently recognized that cancer creates a suppressive metabolic microenvironment, which contributes to ineffective immune function. Metabolism is a common cellular feature, and although there has been significant progress in understanding the detrimental role of metabolic changes of the tumor microenvironment (TEM) in immune cells, there is still much to be learned regarding unique targetable pathways. Elucidation of cancer and immune cell metabolic profiles is critical for identifying mechanisms that regulate metabolic reprogramming within the TEM. Metabolic targets that mediate immunosuppression and are fundamental in sustaining tumor growth can be exploited therapeutically for the development of approaches to increase the efficacy of immunotherapies. Here, we will highlight the importance of metabolism on the function of tumor-associated immune cells and will address the role of key metabolic determinants that might be targets of therapeutic intervention for improvement of tumor immunotherapies.

Many cancer cells display the Warburg effect, that is, enhanced glycolysis followed by fermentation (conversion of pyruvate to lactate). Recently, the molecular basis for these effects has started to be elucidated, and the upregulation of the lactate dehydrogenase A (LDH-A) isoform of lactate dehydrogenase is felt to be a major molecular mediator of this phenomenon. Moreover, LDH-A expression in tumor tissue and LDH-A levels in blood portend a bad prognosis, and LDH-A blockade can lead to tumor growth inhibition in tumor transplant models. We have extended existing data (some of which were published during the time when we were carrying out our studies) in two important ways: 1) inhibition of LDH-A in a glycolytic lung cancer cell line results in reactive oxygen species– mediated apoptosis and increased sensitivity to the chemotherapeutic drug paclitaxel and 2) inhibition of fermentative glycolysis can also be accomplished by activation of the pyruvate dehydrogenase complex by the drug dichloroacetate, now undergoing clinical trials, and that this phenomenon can be monitored in vivo in a noninvasive real-time manner through magnetic resonance spectroscopy using hyperpolarized pyruvate. Collectively, these data suggest that in vivo effects of drugs that redirect the fate of pyruvate, and hence are aimed at reversing the Warburg effect, could be monitored through the use of hyperpolarized magnetic resonance spectroscopy, a method that is scalable to human use.

Background: Diabetic ketoacidosis, a well-known and major acute metabolic complication classically occurs in young patients with type 1 diabetes. However, it may occur in patients with type 2 Diabetes Mellitus too. Objective: Aim of this study was to look into the clinical profile, precipitating factors and clinical outcome in the patients presenting with Diabetic ketoacidosis in the Emergency of a Tertiary care hospital. Design: The study was a prospective study conducted over a period of two years in Kasturba Medical College, Manipal Karnataka, India. Materials and Methods: Clinical profile of 60 diabetic patients admitted in the Emergency with the diagnosis of Diabetic ketoacidosis were analysed. Results: Out of 60 patients, 12 were of Type 1 and 48 were Type 2 Diabetes Mellitus. Mean duration of diabetes was 8.65 years. Only 14 (23.3%) patients were taking regular treatment for Diabetes Mellitus whereas 32 (53.33%) patients were on irregular treatment and eight (13.33%) were not on any treatment at all. Among 12 Type 1 Diabetic patients, six patients were freshly diagnosed to be diabetic when they presented with Diabetic ketoacidosis complication. Nausea and vomiting (63.33%) were the most common symptoms of these patients. Infections (73.33%) were the most common precipitating factor for Diabetic ketoacidosis. Mean fluid requirement on first day of therapy was 3.51 liters. Mortality of 10% was seen. Conclusion: Diabetic ketoacidosis is a fatal acute metabolic complication of Diabetes Mellitus with heterogeneous clinical presentation. Early diagnosis and treatment can avoid morbidity & mortality.

The neuropathological abnormalities of human immunodeficiency virus (HIV)-1 patients abusing illicit drugs suggest extensive interactions between the two agents, thereby leading to increased rate of progression to neurodegeneration. The role of HIV-1 transactivating protein, Tat has been elucidated in mediating neuronal damage via apoptosis, a hallmark of HIV-associated dementia (HAD), however the underlying mechanisms involved in enhanced neurodegeneration by illicit drugs remain elusive. In this study, we demonstrated that morphine enhances HIV-Tat induced toxicity in human neurons and neuroblastoma cells. Enhanced toxicity by Tat and morphine was accompanied by increased numbers of TUNEL positive apoptotic neurons, elevated caspase-3 levels and decreased ratio of anti- and pro-apoptotic proteins, Bcl2/Bax. Tat and morphine together elicited high levels of reactive oxygen species that were NADPH dependent. Significant alterations in mitochondrial membrane homeostasis were also observed with co-exposure of these agents. Extensive studies of mitogen activated protein kinase (MAPK) signaling pathways revealed the involvement of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase-1/2 (ERK1/2) pathways in enhanced toxicity of Tat and morphine. In addition to this, we found that pre-treatment of cells with platelet derived growth factor (PDGF-BB) protected neurons from HIV-Tat and morphine induced damage. PDGF-BB alleviated ROS production, maintained mitochondrial membrane potential, decreased caspase-3 activation and hence protected the cells from undergoing apoptosis. PDGF-BB mediated protection against Tat and morphine involved the phosphatidylinositol-3 kinase (PI3K) pathway, as specific inhibitor of PI3K abrogated the protection conferred by PDGF-BB. This study demonstrates the mechanism of enhanced toxicity in human neurons subjected to co-exposure of HIV protein Tat and morphine, thus implying its importance in HIV positive drug abusers, where damage to the brain is reported to be more severe than non-drug abusers. We have also showed for the first time that PDGF-BB can protect against simultaneous exposure of Tat and morphine, strengthening its role as a neuroprotective agent that could be considered for therapeutic intervention.

Malic enzyme 2 (ME2) is a mitochondrial enzyme that catalyzes the conversion of malate to pyruvate and CO2 and uses NAD as a cofactor. Higher expression of this enzyme correlates with the degree of cell de-differentiation. We found that ME2 is expressed in K562 erythroleukemia cells, in which a number of agents have been found to induce differentiation either along the erythroid or the myeloid lineage. We found that knockdown of ME2 led to diminished proliferation of tumor cells and increased apoptosis in vitro. These findings were accompanied by differentiation of K562 cells along the erythroid lineage, as confirmed by staining for glycophorin A and hemoglobin production. ME2 knockdown also totally abolished growth of K562 cells in nude mice. Increased ROS levels, likely reflecting increased mitochondrial production, and a decreased NADPH/NADP+ ratio were noted but use of a free radical scavenger to decrease inhibition of ROS levels did not reverse the differentiation or apoptotic phenotype, suggesting that ROS production is not causally involved in the resultant phenotype. As might be expected, depletion of ME2 induced an increase in the NAD+/NADH ratio and ATP levels fell significantly. Inhibition of the malate-aspartate shuttle was insufficient to induce K562 differentiation. We also examined several intracellular signaling pathways and expression of transcription factors and intermediate filament proteins whose expression is known to be modulated during erythroid differentiation in K562 cells. We found that silencing of ME2 leads to phospho-ERK1/2 inhibition, phospho-AKT activation, increased GATA-1 expression and diminished vimentin expression. Metabolomic analysis, conducted to gain insight into intermediary metabolic pathways that ME2 knockdown might affect, showed that ME2 depletion resulted in high orotate levels, suggesting potential impairment of pyrimidine metabolism. Collectively our data point to ME2 as a potentially novel metabolic target for leukemia therapy.