Hypopigmentation in burns is associated with alterations in the architecture of the skin and the dendricity of the melanocytes.

Hypopigmentation is a major problem in deep dermal burns. To date, no standard treatment is available for the post burn hypopigmentation disorder. Therefore, understanding the molecular and cellular events are of benefit for therapeutic intervention. Hematoxylin and Eosin (H&E) and Fontana Masson (FM) staining of post burn hypopigmented skin (PBHS) showed an altered architectural pattern in cellular organization, cornified layer and melanin pigment as compared to the normal skin. This was confirmed by immunohistochemistry (IHC) analysis of PBHS samples using specific marker cytokeratin 5 (CK5) for keratinocytes and melanocortin 1 receptor (MCIR) for melanocytes. Validation of these observations was performed by IHC using proliferation and differentiation markers, Ki67 and Loricrin respectively and the melanocyte specific marker tyrosinase related protein 1 (TRP1). Taking a cue from the IHC study, the interaction of keratinocytes and melanocytes was studied by developing a co-culture model from PBHS and normal skin. Culture data exhibited a change of dendritic structure, reduced proliferation rate, faulty melanin synthesis and transfer of melanin from melanocytes to keratinocytes in PBHS samples. To the best of our knowledge, this is the first study showing structural and functional aberrations of melanocytes and keratinocytes, as a potential cause of hypopigmentation in burned patients. Our study, therefore, provides valuable insight for the basis of hypopigmentation in post burn patients, which may pave the way for clinical intervention in the future.

Skin banking at a regional burns centre-The way forward.

In India approximately 1 million people get burnt every year and most of them are from the lower or middle income strata. Therefore it is obligatory to find out an economic way of treatment for the affected populace. Since use of human skin allograft is the gold standard for the treatment of burn wound, in-house skin banking for a burn unit hospital is prerequisite to make the treatment procedure affordable. Although, there was one skin bank at India till 2009, but it was difficult for a single bank to cover the entire country's need. Looking at the necessities, National Burns Centre (a tertiary burn care centre) along with Rotary International and Euro Skin Bank collaborated and developed an effective cadaveric skin banking model in Mumbai, Maharashtra in 2009. Initial two to three years were formation phase; by the year 2013 the entire system was organized and started running full fledged. The model has also been replicated in other states of India to accommodate the large burn population of the country. This paper therefore, gives a step by step account of how the bank evolved and its present status.

Genetic diversity and proviral DNA load in different neural compartments of HIV-1 subtype C infection.

In India, the low prevalence of HIV-associated dementia (HAD) in the Human immunodeficiency virus type 1 (HIV-1) subtype C infection is quite paradoxical given the high-rate of macrophage infiltration into the brain. Whether the direct viral burden in individual brain compartments could be associated with the variability of the neurologic manifestations is controversial. To understand this paradox, we examined the proviral DNA load in nine different brain regions and three different peripheral tissues derived from ten human subjects at autopsy. Using a highly sensitive TaqMan probe-based real-time PCR, we determined the proviral load in multiple samples processed in parallel from each site. Unlike previously published reports, the present analysis identified uniform proviral distribution among the brain compartments examined without preferential accumulation of the DNA in any one of them. The overall viral DNA burden in the brain tissues was very low, approximately 1 viral integration per 1000 cells or less. In a subset of the tissue samples tested, the HIV DNA mostly existed in a free unintegrated form. The V3-V5 envelope sequences, demonstrated a brain-specific compartmentalization in four of the ten subjects and a phylogenetic overlap between the neural and non-neural compartments in three other subjects. The envelope sequences phylogenetically belonged to subtype C and the majority of them were R5 tropic. To the best of our knowledge, the present study represents the first analysis of the proviral burden in subtype C postmortem human brain tissues. Future studies should determine the presence of the viral antigens, the viral transcripts, and the proviral DNA, in parallel, in different brain compartments to shed more light on the significance of the viral burden on neurologic consequences of HIV infection.

Pur-alpha regulates RhoA developmental expression and downstream signaling.

Pur-alpha is an essential protein for postnatal brain development which localizes specifically to dendrites where it plays a role in the translation of neuronal RNA. Mice lacking Pur-alpha display decreased neuronogenesis and impaired neuronal differentiation. Here we examined two Rho GTPases, Rac1 and RhoA, which play opposing roles in neurite outgrowth and are critical for dendritic maturation during mouse brain development in the presence and absence of Pur-alpha. Pur-alpha is developmentally regulated in the mouse brain with expression beginning shortly after birth and rapidly increasing to peak during the third week of postnatal development. RhoA levels analyzed by Western blotting rapidly fluctuated in the wild-type mouse brain, however, in the absence of Pur-alpha, a decrease in RhoA levels shortly after birth and a delay in the cycling of RhoA regulation was observed leading to reduced basal levels of RhoA after day 10 postnatal. Immunohistochemistry of brain tissues displayed reduced RhoA levels in the cortex and cerebellum and loss of perinuclear cytoplasmic labeling of RhoA within the cortex in the knockout mouse brain. While Rac1 levels remained relatively stable at all time points during development and were similar in both wild-type and Pur-alpha knockout mice, changes in subcellular localization of Rac1 were seen in the absence of Pur-alpha. These findings suggest that Pur-alpha can regulate RhoA at multiple levels including basal protein levels, subcellular compartmentalization, as well as turnover of active RhoA in order to promote dendritic maturation.

Redox modification of Akt mediated by the dopaminergic neurotoxin MPTP, in mouse midbrain, leads to down-regulation of pAkt.

Impairment of Akt phosphorylation, a critical survival signal, has been implicated in the degeneration of dopaminergic neurons in Parkinson's disease. However, the mechanism underlying pAkt loss is unclear. In the current study, we demonstrate pAkt loss in ventral midbrain of mice treated with dopaminergic neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), when compared to ventral midbrain of control mice treated with vehicle alone. Thiol residues of the critical cysteines in Akt are oxidized to a greater degree in mice treated with MPTP, which is reflected as a 40% loss of reduced Akt. Association of oxidatively modified Akt with the phosphatase PP2A, which can lead to enhanced dephosphorylation of pAkt, was significantly stronger after MPTP treatment. Maintaining the protein thiol homeostasis by thiol antioxidants prevented loss of reduced Akt, decreased association with PP2A, and maintained pAkt levels. Overexpression of glutaredoxin, a protein disulfide oxidoreductase, in human primary neurons helped sustain reduced state of Akt and abolished MPP(+)-mediated pAkt loss. We demonstrate for the first time the selective loss of Akt activity, in vivo, due to oxidative modification of Akt and provide mechanistic insight into oxidative stress-induced down-regulation of cell survival pathway in mouse midbrain following exposure to MPTP.

Signals and pathways regulating nucleolar retention of novel putative nucleolar GTPase NGP-1(GNL-2).

NGP-1(GNL-2) is a putative GTPase, overexpressed in breast carcinoma and localized in the nucleolus. NGP-1 belongs to the MMR1-HSR1 family of large GTPases that are emerging as crucial coordinators of signaling cascades in different cellular compartments. The members of this family share very closely related G-domains, but the signals and pathways regulating their subcellular localization and their functional relevance remain unknown. To improve our understanding of the nuclear transport mechanism of NGP-1, we have identified two nucleolar localization signals (NoLS) that are independently shown to translocate NGP-1 as well the heterologous protein to the nucleolus. Site-specific mutagenesis and immunofluorescence studies suggest that the tandem repeats of positively charged amino acids are critical for NGP-1 NoLS function. Interestingly, amino-terminal (NGP-1(1-100)) and carboxyl-terminal (NGP-1(661-731)) signals independently interact with receptors importin-ß and importin-α, respectively. This investigation, for the first time, provides evidence that the interaction of importin-α with C-terminal NoLS (NGP-1(661-731)) was able to target the heterologous protein to the nucleolar compartment. Structural modeling analysis and alanine scanning mutagenesis of conserved G-domains suggest that G4 and G5 motifs are critical for GTP binding of NGP-1 and further show that the nucleolar localization of NGP-1 is regulated by a GTP gating-mediated mechanism. In addition, our data suggest that an ongoing transcription is essential for efficient localization of NGP-1 to the nucleolus. We have observed a high level of NGP-1 expression in the mitogen-activated primary human peripheral blood mononuclear cells (hPBMC) as well as in human fetal brain-derived neural precursor cells (hNPCs) in comparison to cells undergoing differentiation. Overall, the results suggest that multiple mechanisms are involved in the localization of NGP-1 to the nucleolus for the regulation of nucleolar function in cell growth and proliferation.

Tetrahydrocurcumin confers protection against amyloid ß-induced toxicity.

Amyloid plaques and neurofibrillary tangles are the hallmarks of Alzheimer's disease. Amyloid ß, a primary component of the amyloid plaques, is neurotoxic. Considerable attention has been directed toward identifying compounds with neuroprotective properties. Using rat primary hippocampal cultures, we show that tetrahydrocurcumin (THC), a metabolite of curcumin, shows a protective effect against oligomeric amyloid-ß-induced toxicity. We further show that THC reduces amyloid-ß-induced (i) increase in the level of reactive oxygen species, (ii) decrease in mitochondrial membrane potential, and (iii) caspase activation. In addition, we show that THC protects human neurons from oligomeric amyloid-ß-induced toxicity as well. Thus, THC confers protection against amyloid-ß-induced toxicity, and the antioxidant activity may contribute to its protective effect.

Human immunodeficiency virus type 1 Tat modulates proliferation and differentiation of human neural precursor cells: implication in NeuroAIDS.

Human immunodeficiency virus type 1 (HIV-1) and viral proteins affect neuronal survival and neuron-glial cell interactions, which culminate in neurological disorders. HIV-1 infects regions of neurogenesis in human adult and pediatric brain. However, little is known about the effect of HIV-1 or viral proteins on the properties of human neural precursor cells (hNPCs), particularly neurogenesis, hence a detailed investigation on these lines is warranted. Human neural precursor cells were cultured in presence and absence of HIV-1B transactivating protein Tat to investigate if HIV-1 viral protein alters the properties of human neural precursor cells. Cellular and molecular approaches were adopted to study the effect of HIV-1B transactivating protein Tat on proliferation and differentiation potential of human fetal brain-derived NPCs. Cell proliferation assays such as BrdU and Ki67 staining and pathway-specific cDNA and protein arrays were used in the study. Data reveal that HIV-1B Tat protein severely affects proliferation of hNPCs, as evident by lower incorporation of BrdU and Ki67 staining as well as neurosphere assay. HIV-1 Tat substantially attenuated neurogenesis, as evident by the smaller numbers of Tuj-1- and doublecortin-positive cells differentiated from hNPCs, without affecting their viability. These data suggest that HIV-1 Tat alters the properties of human neural precursor cells via attenuation of the cell cycle regulatory unit cyclin D1 and the mitogen-activated protein kinase (MAPK) pathway, particularly extracellular signal-related kinase 1/2 (ERK1/2). The study provides new insights into cellular and molecular mechanisms that may modulate human neural precursor cell properties in HIV/AIDS (acquired immunodeficiency syndrome) individuals. Validation with autopsy brain samples is necessary to further substantiate these important observations.

Selective activation of p38 mitogen-activated protein kinase in dopaminergic neurons of substantia nigra leads to nuclear translocation of p53 in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice.

Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNpc). Activation of the mixed lineage kinase and c-Jun N-terminal kinase (JNK) has been reported in models of PD. Our focus was to discern whether distinct pathways were activated in cell-specific manner within the SNpc. We now demonstrate the selective phosphorylation of p38 MAP kinase within the dopaminergic neurons, whereas JNK activation occurs predominantly in the microglia. p38 activation results in downstream phosphorylation of p53 and increased p53 mediated transcription of Bax and Puma in the ventral midbrain. Treatment with p38 inhibitor, SB239063 protected primary dopaminergic neurons derived from human progenitor cells from MPP(+) mediated cell death and prevented the downstream phosphorylation of p53 and its translocation to the nucleus in vivo, in the ventral midbrain. The increased staining of phosphorylated p38 in the surviving neurons of SNpc in human brain sections from patients with PD and in MPTP treated mice but not in the ventral tegmental area provides further evidence suggesting a role for p38 in the degeneration of dopaminergic neurons of SNpc. We thus demonstrate the cell specific activation of MAP kinase pathways within the SNpc after MPTP treatment emphasizing the role of multiple signaling cascades in the pathogenesis and progression of the disease. Selective inhibitors of p38 may therefore, help preserve the surviving neurons in PD and slow down the disease progression.

Clade-specific differences in neurotoxicity of human immunodeficiency virus-1 B and C Tat of human neurons: significance of dicysteine C30C31 motif.

OBJECTIVE: Human immunodeficiency virus-1 (HIV-1) causes mild to severe cognitive impairment and dementia. The transactivator viral protein, Tat, is implicated in neuronal death responsible for neurological deficits. Several clades of HIV-1 are unequally distributed globally, of which HIV-1 B and C together account for the majority of the viral infections. HIV-1-related neurological deficits appear to be most common in clade B, but not clade C prevalent areas. Whether clade-specific differences translate to varied neuropathogenesis is not known, and this uncertainty warrants an immediate investigation into neurotoxicity on human neurons of Tat derived from different viral clades METHODS: We used human fetal central nervous system progenitor cell-derived astrocytes and neurons to investigate effects of B- and C-Tat on neuronal cell death, chemokine secretion, oxidative stress, and mitochondrial membrane depolarization by direct and indirect damage to human neurons. We used isogenic variants of Tat to gain insights into the role of the dicysteine motif (C30C31) for neurotoxic potential of Tat RESULTS: Our results suggest clade-specific functional differences in Tat-induced apoptosis in primary human neurons. This study demonstrates that C-Tat is relatively less neurotoxic compared with B-Tat, probably as a result of alteration in the dicysteine motif within the neurotoxic region of B-Tat INTERPRETATION: This study provides important insights into differential neurotoxic properties of B- and C-Tat, and offers a basis for distinct differences in degree of HIV-1-associated neurological deficits observed in patients in India. Additional studies with patient samples are necessary to validate these findings.

Modulation of interleukin-1beta mediated inflammatory response in human astrocytes by flavonoids: implications in neuroprotection.

The proinflammatory cytokine interleukin-1beta (IL-1beta) contributes to inflammation and neuronal death in CNS injuries and neurodegenerative pathologies, and astrocytes have been implicated as the primary mediators of IL-1beta induced neuronal death. As astrocytes play an important role in supporting the survival and functions of neurons, we investigated the effect of plant flavonoids quercetin and luteolin, with known anti-inflammatory properties in modulating the response of human astrocytes to IL-1beta for therapeutic intervention. Flavonoids significantly decreased the release of reactive oxygen species (ROS) from astrocytes stimulated with IL-1beta. This decrease was accompanied by an increase in expression of superoxide dismutase (SOD-1) and thioredoxin (TRX1)-mediators associated with protection against oxidative stress. Flavonoids not only modulated the expression of astrocytes specific molecules such as glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), and ceruloplasmin (CP) both in the presence and absence of IL-1beta but also decreased the elevated levels of proinflammatory cytokine interleukin-6 (IL-6) and chemokines interleukin-8 (IL-8), interferon-inducible protein (IP-10), monocyte-chemoattractant protein-1 (MCP-1), and RANTES from IL-1beta activated astrocytes. Significant decrease in neuronal apoptosis was observed in neurons cultured in conditioned medium obtained from astrocytes treated with a combination of IL-1beta and flavonoids as compared to that treated with IL-1beta alone. Our result suggests that by (i) enhancing the potential of activated astrocytes to detoxify free radical, (ii) reducing the expression of proinflammatory cytokines and chemokines, and (iii) modulating expression of mediators associated with enhanced physiological activity of astrocyte in response to injury, flavonoids confer (iv) protection against IL-1beta induced astrocyte mediated neuronal damage.

Growth, yield and elemental status of rice (Oryza sativa) grown in fly ash amended soils.

Fly ash (FA) from coal in Orissa (India) was used for amending soil at levels equivalent to 0, 1, 2.5, 5, 10 and 15 metric tons per ha in which, rice was grown and elemental residues of amended soil and plant parts were enumerated. FA amendments caused significant improvement in soil quality and germination percentage of rice seeds. Growth (shoot length, leaf area and pigment composition) and yield (panicle length, seeds per panicle, seed weight and yield per plant) of rice increased with an increase in FA amendments. Catalase and peroxidase activities of young leaves increased initially in plants cultivated at lower FA levels but declined sharply at higher FA levels while the protein content of seeds improved at higher FA levels. Sodium content of rice-roots did not change with FA amendments but the contents of K, P, Mn, Ni, Co, Pb, Zn, Cu, Cr, and Cd showed a progressive increase. Seeds of plants grown in FA amended soils accumulated Cu, Pb, Cr and Cd in amounts below allowable limits. Based on the data obtained we found that flooded-rice soil amended at 10 metric tons FA per ha level of FA not only improved the physical properties of the soil but also contributed to better growth and yield of rice.