Anita Mahadevan

A draft map of the human proteome

The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides does not exist yet. Here we present a draft map of the human proteome using high-resolution Fourier-transform mass spectrometry. In-depth proteomic profiling of 30 histologically normal human samples, including 17 adult tissues, 7 fetal tissues and 6 purified primary haematopoietic cells, resulted in identification of proteins encoded by 17,294 genes accounting for approximately 84% of the total annotated protein-coding genes in humans. A unique and comprehensive strategy for proteogenomic analysis enabled us to discover a number of novel protein-coding regions, which includes translated pseudogenes, non-coding RNAs and upstream open reading frames. This large human proteome catalogue (available as an interactive web-based resource at http://www.humanproteomemap.org) will complement available human genome and transcriptome data to accelerate biomedical research in health and disease.

Tat Protein of Human Immunodeficiency Virus Type 1 Subtype C Strains Is a Defective Chemokine

ABSTRACT Human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) is correlated with increased monocyte migration to the brain, and the incidence of HAD among otherwise asymptomatic subjects appears to be lower in India than in the United States and Europe (1 to 2% versus 15 to 30%). Because of the genetic differences between HIV-1 strains circulating in these regions, we sought to identify viral determinants associated with this difference. We targeted Tat protein for these studies in view of its association with monocyte chemotactic function. Analyses of Tat sequences representing nine subtypes revealed that at least six amino acid residues are differentially conserved in subtype C Tat (C-Tat). Of these, cysteine (at position 31) was highly (>99%) conserved in non-subtype C viruses and more than 90% of subtype C viruses encoded a serine. We hypothesized a compromised chemotactic function of C-Tat due to the disruption of CC motif and tested it with the wild type C-Tat (CS) and its two isogenic variants (CC and SC) derived by site-directed mutagenesis. We found that the CS natural variant was defective for monocyte chemotactic activity without a loss in the transactivation property. While the CC mutant is functionally competent for both the functions, in contrast, the SC mutant was defective in both. Therefore, the loss of the C-Tat chemotactic property may underlie the reduced incidence of HAD; although not presenting conclusive evidence, this study provides the first evidence for a potential epidemiologic phenomenon associated with biological differences in the subtype C viruses.

NMDA Receptor Activation by HIV-Tat Protein Is Clade Dependent

In countries infected with HIV clade B, some patients develop a rapidly progressive dementia that if untreated results in death. In regions of the world infected with HIV clade C, only milder forms of cognitive impairment have been recognized. HIV-infected macrophages are the principal mediators of dementia. HIV clade C, however, efficiently infects macrophages and HIV-infected macrophages are found in the brains of clade C-infected patients. HIV-infected macrophages release Tat protein, which may act directly on neurons to cause toxicity. We found that Tat released from Tat-expressing cells was at least 1000-fold more toxic than recombinant Tat protein. We determined whether Tat could interact with NMDA receptors and whether these interactions are clade dependent. It is demonstrated that Tat binds directly to the NMDA receptor leading to excitotoxicity. The Cys 30-Cys 31 motif in Tat is critical for exciting the NMDA receptor and the Cys31Ser mutation found in clade C Tat has a significantly attenuated neurotoxic response. Through molecular modeling and site-directed mutagenesis, we predict that Cys 31 disrupts the disulfide bond between Cys 744 and Cys 798 on the NR1 subunit of the NMDA receptor by directly interacting with Cys 744 leading to a free thiol group on Cys 798 and subsequent persistent activation of the NMDA receptor.

Activation of Wnt/β-catenin/Tcf signaling pathway in human astrocytomas

Astrocytomas are the most common form of primary brain tumors. Understanding the molecular basis of development and progression of astrocytomas is required to develop more effective therapies. Although, over activation of Wnt/beta-catenin/Tcf pathway is a hallmark of several forms of cancer, little is known about its role in human astrocytomas. Here, we report the evidence that Wnt/beta-catenin/Tcf signaling pathway is constitutively activated in astrocytic tumors. In the present study, human astrocytic tumors with different clinical grades were analyzed for mRNA expression of Dvl-1, Dvl-2, Dvl-3, beta-catenin, c-myc and cyclin D1 and protein levels of beta-catenin, Lef1, Tcf4, c-Myc, N-Myc, c-jun and cyclin D1. RT-PCR analysis demonstrated the overexpression of Dvl-3, beta-catenin, c-myc and cyclin D1 in astrocytomas. Western blotting revealed upregulation of beta-catenin, Lef1, Tcf4 and their target proteins in the core tumor tissues in comparison to peritumor and normal brain tissues. The protein and mRNA levels were positively correlated with the histological malignancy. Cytoplasmic and nuclear accumulation of beta-catenin, nuclear localization of Lef1, Tcf4, c-Myc, N-Myc, c-jun and cyclin D1 were demonstrated by immunohistochemical staining. Our studies tend to suggest that Wnt/beta-catenin/Tcf signaling pathway is implicated in malignancy of astrocytomas.

Anti-brain autoantibodies and altered excitatory neurotransmitters in obsessive-compulsive disorder.

Although serum autoantibodies directed against basal ganglia (BG) implicate autoimmunity in the pathogenesis of obsessive–compulsive disorder (OCD), it is unclear whether these antibodies can cross the blood–brain barrier to bind against BG or other components of the OCD circuit. It is also unclear how they might lead to hyperactivity in the OCD circuit. We examined this by investigating the presence of autoantibodies directed against the BG or thalamus in the serum as well as CSF of 23 OCD patients compared with 23 matched psychiatrically normal controls using western blot. We further investigated CSF amino acid (glutamate, GABA, taurine, and glycine) levels and also examined the extent to which these levels were related to the presence of autoantibodies. There was evidence of significantly more binding of CSF autoantibodies to homogenate of BG as well as to homogenate of thalamus among OCD patients compared with controls. There was no significant difference in binding between patient and control sera except for a trend toward more bands to BG and thalamic protein corresponding to 43 kD among OCD patients compared with controls. CSF glutamate and glycine levels were also significantly higher in OCD patients compared with controls, and further multivariate analysis of variance showed that CSF glycine levels were higher in those OCD patients who had autoantibodies compared with those without. The results of our study implicate autoimmune mechanisms in the pathogenesis of OCD and also provide preliminary evidence that autoantibodies against BG and thalamus may cause OCD by modulating excitatory neurotransmission.

Cryptococcal meningitis: Clinical, diagnostic and therapeutic overviews

Cryptococcal meningitis has emerged as a leading cause of infectious morbidity and mortality in patients with AIDS. Among the human immunodeficiency virus (HIV)-seropositive subjects, cryptococcal meningitis is the second most common cause of opportunistic neuro-infection. Current trends are changing due to the marked improvement of quality and length of life produced by highly active antiretroviral therapy (HAART). The introduction of generic HAART in India has resulted in an increase in the number of individuals getting treatment for HIV infection, as the cost of highly active antiretroviral therapy (HAART) has decreased 20- fold. Cryptococcal meningitis occurs in non-HIV patients who are immunodeficient due to diabetes, cancer, solid organ transplants, chemotherapeutic drugs, hematological malignancies etc and rarely in healthy individuals with no obvious predisposing factors. Diagnosis of cryptococcal meningitis is fairly straightforward once the diagnosis is considered in the differential diagnosis of chronic meningitis. Treatment of a patient with cryptococcal infection is a challenge for both the physician and the patient, but rewarding, as many would recover with timely and adequate antifungal therapy.

Identification of Subtype C Human Immunodeficiency Virus Type 1 by Subtype-Specific PCR and Its Use in the Characterization of Viruses Circulating in the Southern Parts of India

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) subtype C viruses are associated with nearly half of worldwide HIV-1 infections and are most predominant in India and the southern and eastern parts of Africa. Earlier reports from India identified the preponderance of subtype C and a small proportion of subtype A viruses. Subsequent reports identifying multiple subtypes suggest new introductions and/or their detection due to extended screening. The southern parts of India constitute emerging areas of the epidemic, but it is not known whether HIV-1 infection in these areas is associated with subtype C viruses or is due to the potential new introduction of non-subtype C viruses. Here, we describe the development of a specific and sensitive PCR-based strategy to identify subtype C-viruses (C-PCR). The strategy is based on amplifying a region encompassing a long terminal repeat and gag in the first round, followed by two sets of nested primers; one amplifies multiple subtypes, while the other is specific to subtype C. The common HIV and subtype C-specific fragments are distinguishable by length differences in agarose gels and by the difference in the numbers of NF-κB sites encoded in the subtype C-specific fragment. We implemented this method to screen 256 HIV-1-infected individuals from 35 towns and cities in four states in the south and a city in the east. With the exception of single samples of subtypes A and B and a B/C recombinant, we found all to be infected with subtype C viruses, and the subtype assignments were confirmed in a subset by using heteroduplex mobility assays and phylogenetic analysis of sequences. We propose the use of C-PCR to facilitate rapid molecular epidemiologic characterization to aid vaccine and therapeutic strategies.

MicroRNA 155 Regulates Japanese Encephalitis Virus-Induced Inflammatory Response by Targeting Src Homology 2-Containing Inositol Phosphatase 1

ABSTRACT MicroRNAs (miRNAs) are single-stranded small RNA molecules that regulate various cellular processes. miRNA 155 (miR-155) regulates various aspects of innate and adaptive immune responses and plays a key role in various viral infections and the resulting neuroinflammation. The present study evaluated the involvement of miR-155 in modulating Japanese encephalitis virus (JEV)-induced neuroinflammation. We observed that miR-155 expression was upregulated during JEV infection of mouse primary microglia, the BV-2 microglia cell line, and in both mouse and human brains. In vitro and in vivo knockdown of miR-155 minimized JEV-induced inflammatory responses. In the present study, we confirmed targeting of the Src homology 2-containing inositol phosphatase 1 (SHIP1) 3′ untranslated region (UTR) by miR-155 in the context of JEV infection. Inhibition of SHIP1 by miR-155 resulted in higher beta interferon (IFN-β) and proinflammatory cytokine production through activation of TANK-binding kinase 1 (TBK-1). Based on these observations, we conclude that miR-155 modulates the neuroinflammatory response during JEV infection via negative regulation of SHIP1 expression. Thus, modulation of miR-155 could be a novel strategy to regulate JEV-induced neuroinflammation. IMPORTANCE Japanese encephalitis virus (JEV), a member of the family Flaviviridae that causes Japanese encephalitis (JE), is the most common mosquito-borne encephalitis virus in the Asia-Pacific region. The disease is feared, as currently there are no specific antiviral drugs available. JEV targets the central nervous system, leading to high mortality and neurological and psychiatric sequelae in some of those who survive. The level of inflammation correlates well with the clinical outcome in patients. Recently, microRNA (miRNA), a single-stranded noncoding RNA, has been implicated in various brain disorders. The present study investigates the role of miRNA in JEV-induced neuroinflammation. Our results show that miRNA 155 (miR-155) targets the Src homology 2-containing inositol phosphatase 1 (SHIP1) protein and promotes inflammation by regulating the NF-κB pathway, increasing the expression of various proinflammatory cytokines and the antiviral response. Thus, miR-155 is a potential therapeutic target to develop antivirals in JE and other brain disorders where inflammation plays a significant role in disease progression.

Eccentric Target Sign in Cerebral Toxoplasmosis – neuropathological correlate to the imaging feature

Cerebral toxoplasmosis remains one of the most common focal brain lesions in patients with acquired immune deficiency syndrome (AIDS). Diagnosis is a challenge because on cranial imaging it closely mimics central nervous system lymphoma, primary and metastatic central nervous system (CNS) tumors, or other intracranial infections like tuberculoma or abscesses. A magnetic resonance imaging (MRI) feature on postcontrast T1‐weighted sequences considered pathognomonic of toxoplasmosis is the “eccentric target sign.” The pathological correlate of this imaging sign has been speculative. Herein we correlate the underlying histopathology to the MR feature of eccentric target sign in a patient with autopsy‐proven HIV/AIDS‐related cerebral toxoplasmosis. The central enhancing core of the target seen on MRI was produced by a leash of inflamed vessels extending down the length of the sulcus that was surrounded by concentric zones of necrosis and a wall composed of histiocytes and proliferating blood vessels, with impaired permeability producing the peripheral enhancing rim. J. Magn. Reson. Imaging 2010;31:1469–1472.

Low prevalence of progressive multifocal leukoencephalopathy in India and Africa: Is there a biological explanation?

Infection with human immunodeficiency virus (HIV) clade C virus is the most common form of HIV infection in the world. It largely infects populations in Africa and Asia and not much is known about the neurological complications associated with the virus. Cases of progressive multifocal leukoencephalopathy (PML) have been rarely reported in the literature in the acquired immunodeficiency syndrome (AIDS) or non-AIDS populations from these regions. In this article, the authors present three recently diagnosed patients with AIDS and PML from one neurological center in India, review the diagnostic challenges faced, and speculate on the possible biological reasons, including viral strain differences as well as HIV and JC virus interactions, that may account for the low incidence.