Amyotrophic Lateral Sclerosis: Molecular Mechanisms to Diagnostics

Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive and fatal motor neuron disease, characterized by loss of motor neurons in the cortex, brainstem and spinal cord. Clinical management is plagued by a lack of biomarkers and effective treatment. In spite of numerous scientific advancements, molecular mechanisms involved in its initiation and progression remain an enigma. At the mechanistic level, ALS is considered multifactorial. Extracellular signals may modulate nuclear events with a possible consequence being the reactivation of cell cycle-related genes and protein alterations in the terminally differentiated motor neurons. In the first specific aim, we hypothesized that re-entry of post-mitotic motor neurons into the cell cycle, concurrent with altered activity or distribution of transcription factors will result in apoptosis of motor neurons during ALS. To address this hypothesis, we utilized archived human autopsy material from the cortical and spinal cord regions of ALS and age-matched control cases. We conclude that surviving ALS motor neurons in these regions exhibited increased levels of G1 to S phase regulators (Cyclin D1, CDK4, hyperphosphorylated -pRb and E2F-1). It also revealed two intriguing results: (i) E2F-1, a transcription factor, was cytoplasmic and (ii) increased nuclear p53 was noted in spinal motor neurons but absent in neurons of the motor cortex. In addition there was increased protein levels of apoptotic death markers (BAX, FAS, Caspases) and DNA fragmentation. Therefore we have identified a potential role for cell cycle proteins in an apoptotic mode of motor neuron death in ALS. In the second specific aim we hypothesized that a mass spectrometry-based proteomics approach will identify diagnostic biomarkers and molecular targets for drug discoveries. We used cerebrospinal fluid (CSF) from ALS and control subjects to identify and validate a biomarker panel specific to ALS. Furthermore, utilizing peptide map fingerprinting and tandem mass-spectrometry, we have identified three of the protein peaks to be a carboxyl-terminal fragment of neurosecretory chaperone protein 7B2 (3.44kDa), Cystatin C (13.3kDa) and monomer of transthyretin (13.78kDa).Taken together, this body of work furthers the understanding of both the mechanisms leading to selective motor neuron loss in ALS and paves the way for diagnostics and therapeutics

Transcriptional Profiling and Deriving a Seven-Gene Signature That Discriminates Active and Latent Tuberculosis: An Integrative Bioinformatics Approach

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (M.tb.). Our integrative analysis aims to identify the transcriptional profiling and gene expression signature that distinguish individuals with active TB (ATB) disease, and those with latent tuberculosis infection (LTBI). In the present study, we reanalyzed a microarray dataset (GSE37250) from GEO database and explored the data for differential gene expression analysis between those with ATB and LTBI derived from Malawi and South African cohorts. We used BRB array tool to distinguish DEGs (differentially expressed genes) between ATB and LTBI. Pathway enrichment analysis of DEGs was performed using DAVID bioinformatics tool. The protein–protein interaction (PPI) network of most upregulated genes was constructed using STRING analysis. We have identified 375 upregulated genes and 152 downregulated genes differentially expressed between ATB and LTBI samples commonly shared among Malawi and South African cohorts. The constructed PPI network was significantly enriched with 76 nodes connected to 151 edges. The enriched GO term/pathways were mainly related to expression of IFN stimulated genes, interleukin-1 production, and NOD-like receptor signaling pathway. Downregulated genes were significantly enriched in the Wnt signaling, B cell development, and B cell receptor signaling pathways. The short-listed DEGs were validated in a microarray data from an independent cohort (GSE19491). ROC curve analysis was done to assess the diagnostic accuracy of the gene signature in discrimination of active and latent tuberculosis. Thus, we have derived a seven-gene signature, which included five upregulated genes FCGR1B, ANKRD22, CARD17, IFITM3, TNFAIP6 and two downregulated genes FCGBP and KLF12, as a biomarker for discrimination of active and latent tuberculosis. The identified genes have a sensitivity of 80–100% and specificity of 80–95%. Area under the curve (AUC) value of the genes ranged from 0.84 to 1. This seven-gene signature has a high diagnostic accuracy in discrimination of active and latent tuberculosis

Ion atmosphere relaxation controlled electron transfers in cobaltocenium polyether molten salts

A room-temperature redox molten salt for the study of electron transfers in semisolid media, based on combining bis(cyclopentadienyl)cobalt with oligomeric polyether counterions, [Cp2Co](MePEG350SO3), is reported. The transport properties of the new molten salt can be varied (plasticized) by varying the polyether content. The charge transport rate during voltammetric reduction of the ionically conductive [Cp2Co](MePEG350SO3) molten salt exceeds the actual physical diffusivity of [Cp2Co]+ because of rapid [Cp2Co]+/0 electron self-exchanges. The measured [Cp2Co]+/0 electron self-exchange rate constants (kEX) are proportional to the diffusion coefficients (DCION) of the counterions in the melt. The electron-transfer activation barrier energies are also close to those of ionic diffusion but are larger than those derived from optical intervalent charge-transfer results. Additionally, the [Cp2Co]+/0 rate constant results are close to those of dissimilar redox moieties in molten salts where DCION values are similar. All of these characteristics are consistent with the rates of electron transfers of [Cp2Co]+/0 (and the other donor−acceptor pairs) being controlled not by the intrinsic electron-transfer rates but by the rate of relaxation of the ion atmosphere around the reacting pair. In the low driving force regime of mixed-valent concentration gradients, the ion atmosphere relaxation is competitive with electron transfer. The results support the generality of the recently proposed model of ionic atmosphere relaxation control of electron transfers in ionically conductive, semisolid materials

Diverse Clinical Isolates of Mycobacterium tuberculosis Develop Macrophage-Induced Rifampin Tolerance.

The Mycobacterium tuberculosis lineage 4 strains CDC1551 and H37Rv develop tolerance to multiple antibiotics upon macrophage residence. To determine whether macrophage-induced tolerance is a general feature of clinical M. tuberculosis isolates, we assessed macrophage-induced drug tolerance in strains from lineages 1-3, representing the other predominant M. tuberculosis strains responsible for tuberculosis globally. All 3 lineages developed isoniazid tolerance. While lineage 1, 3, and 4 strains developed rifampin tolerance, lineage 2 Beijing strains did not. Their failure to develop tolerance may be explained by their harboring of a loss-of-function mutation in the Rv1258c efflux pump that is linked to macrophage-induced rifampicin tolerance

Identification and Characterization of Genetic Determinants of Isoniazid and Rifampicin Resistance in Mycobacterium tuberculosis in Southern India

Abstract: Drug-resistant tuberculosis (TB), one of the leading causes of death worldwide, arises mainly from spontaneous mutations in the genome of Mycobacterium tuberculosis. There is an urgent need to understand the mechanisms by which the mutations confer resistance in order to identify new drug targets and to design new drugs. Previous studies have reported numerous mutations that confer resistance to anti-TB drugs, but there has been little systematic analysis to understand their genetic background and the potential impacts on the drug target stability and/or interactions. Here, we report the analysis of whole-genome sequence data for 98 clinical M. tuberculosis isolates from a city in southern India. The collection was screened for phenotypic resistance and sequenced to mine the genetic mutations conferring resistance to isoniazid and rifampicin. The most frequent mutation among isoniazid and rifampicin isolates was S315T in katG and S450L in rpoB respectively. The impacts of mutations on protein stability, protein-protein interactions and protein-ligand interactions were analysed using both statistical and machine-learning approaches. Drug-resistant mutations were predicted not only to target active sites in an orthosteric manner, but also to act through allosteric mechanisms arising from distant sites, sometimes at the protein-protein interface

Genome Sequencing of Polydrug-, Multidrug-, and Extensively Drug-Resistant Mycobacterium tuberculosis Strains from South India.

The genomes of 16 clinical Mycobacterium tuberculosis isolates were subjected to whole-genome sequencing to identify mutations related to resistance to one or more anti-Mycobacterium drugs. The sequence data will help in understanding the genomic characteristics of M. tuberculosis isolates and their resistance mutations prevalent in South India.This publication presents research supported by the MRC-DBT-funded partnership between the National Institute for Research in Tuberculosis, Chennai, India (Indian Council of Medical Research, New Delhi 5/2-8/LDCE/2014 for S.K., Department of Biotechnology [BT/IN/DBT-MRC (UK)/12/SS/2015-2016] for D.N., M.N., S.P.T., S.S., and U.D.R.) and the University of Cambridge (UK Medical Research Council [MR/N501864/1] for N.K. and S.P.)

A motor neuron disease–associated mutation in p150Glued perturbs dynactin function and induces protein aggregation

The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. Dynactin is ubiquitously expressed in eukaryotes, but a G59S mutation in the p150Glued subunit of dynactin results in the specific degeneration of motor neurons. This mutation in the conserved cytoskeleton-associated protein, glycine-rich (CAP-Gly) domain lowers the affinity of p150Glued for microtubules and EB1. Cell lines from patients are morphologically normal but show delayed recovery after nocodazole treatment, consistent with a subtle disruption of dynein/dynactin function. The G59S mutation disrupts the folding of the CAP-Gly domain, resulting in aggregation of the p150Glued protein both in vitro and in vivo, which is accompanied by an increase in cell death in a motor neuron cell line. Overexpression of the chaperone Hsp70 inhibits aggregate formation and prevents cell death. These data support a model in which a point mutation in p150Glued causes both loss of dynein/dynactin function and gain of toxic function, which together lead to motor neuron cell death