Cognitive and Behavioral Profile of Patients with Amyotrophic Lateral Sclerosis Spectrum in the Indian Context.

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is characterized by motor, cognitive, and behavioral impairment. There is a paucity of evidence about the cognitive/behavioral features of ALS patients from India. We aimed to investigate the cognitive/behavioral profile of ALS spectrum disorders in the Indian context. METHODS: Sixty patients with ALS spectrum and 40 age-, gender-, and education-matched healthy controls were recruited. The scales used were Addenbrooke's Cognitive Examination (ACE-III), Clinical Dementia Rating (CDR) scale, and Frontal Systems Behavior (FrSBe) Scale. RESULTS: The mean age of the overall cohort was 55 years, and male-to-female ratio was 2.5:1. The mean duration of illness of the cohort was 16 months. Patients were classified as ALS with normal cognition (ALS-cn, n = 21), mild cognitive or behavioral deficits (ALS-ci/-bi, n = 28), and frontotemporal dementia (ALS-FTD, n = 11). ALS-cn had poorer scores compared to healthy controls in global cognition, memory, and language (p < 0.05). ALS-ci/-bi performed poorer than healthy controls on all cognitive domains (p < 0.05). ALS-FTD had poorer scores than healthy controls and ALS-cn on all cognitive domains (p < 0.001). Behavioral assessment showed an increase in apathy among all subtypes. ALS-FTD showed significant worsening in disinhibition and executive function compared to ALS-cn and ALS-ci/-bi. CONCLUSION: Our findings suggest that there are key cognitive and behavior characteristics in Indian patients with ALS spectrum. This further strengthens the evidence of a cognitive continuum in ALS and FTD in a diverse context and highlights the importance of meticulous evaluation and correct diagnosis that would assist in better management.

M. tuberculosis class II apurinic/ apyrimidinic-endonuclease/3'-5' exonuclease (XthA) engages with NAD+-dependent DNA ligase A (LigA) to counter futile cleavage and ligation cycles in base excision repair.

Class-II AP-endonuclease (XthA) and NAD+-dependent DNA ligase (LigA) are involved in initial and terminal stages of bacterial DNA base excision repair (BER), respectively. XthA acts on abasic sites of damaged DNA to create nicks with 3'OH and 5'-deoxyribose phosphate (5'-dRP) moieties. Co-immunoprecipitation using mycobacterial cell-lysate, identified MtbLigA-MtbXthA complex formation. Pull-down experiments using purified wild-type, and domain-deleted MtbLigA mutants show that LigA-XthA interactions are mediated by the BRCT-domain of LigA. Small-Angle-X-ray scattering, 15N/1H-HSQC chemical shift perturbation experiments and mutational analysis identified the BRCT-domain region that interacts with a novel 104DGQPSWSGKP113 motif on XthA for complex-formation. Isothermal-titration calorimetry experiments show that a synthetic peptide with this sequence interacts with MtbLigA and disrupts XthA-LigA interactions. In vitro assays involving DNA substrate and product analogs show that LigA can efficiently reseal 3'OH and 5'dRP DNA termini created by XthA at abasic sites. Assays and SAXS experiments performed in the presence and absence of DNA, show that XthA inhibits LigA by specifically engaging with the latter's BRCT-domain to prevent it from encircling substrate DNA. Overall, the study suggests a coordinating function for XthA whereby it engages initially with LigA to prevent the undesirable consequences of futile cleavage and ligation cycles that might derail bacterial BER.

Structure based identification of first-in-class fragment inhibitors that target the NMN pocket of M. tuberculosis NAD+-dependent DNA ligase A.

NAD+-dependent DNA ligase (LigA) is the essential replicative ligase in bacteria and differs from ATP-dependent counterparts like the human DNA ligase I (HligI) in several aspects. LigA uses NAD+ as the co-factor while the latter uses ATP. Further, the LigA carries out enzymatic activity with a single divalent metal ion in the active site while ATP-dependent ligases use two metal ions. Instead of the second metal ion, LigA have a unique NMN binding subdomain that facilitates the orientation of the ß-phosphate and NMN leaving group. LigA are therefore attractive targets for new anti-bacterial therapeutic development. Others and our group have earlier identified several LigA inhibitors that mainly bind to AMP binding site of LigA. However, no inhibitor is known to bind to the unique NMN binding subdomain. We initiated a fragment inhibitor discovery campaign against the M. tuberculosis LigA based on our co-crystal structure of adenylation domain with AMP and NMN. The study identified two fragments, 4-(4-fluorophenyl)-4,5,6,7-tetrahydro-3H imidazo[4,5-c] pyridine and N-(4-methylbenzyl)-1H-pyrrole-2-carboxamide, that bind to the NMN site. The fragments inhibit LigA with IC50 of 16.9 and 28.7 µM respectively and exhibit MIC of ~20 and 60 µg/ml against a temperature sensitive E. coli GR501 ligAts strain, rescued by MtbLigA. Co-crystal structures of the fragments with the adenylation domain of LigA show that they mimic the interactions of NMN. Overall, our results suggest that the NMN binding-site is a druggable target site for developing anti-LigA therapeutic strategies.

Modification of chickpea cystatin by reactive dicarbonyl species: Glycation, oxidation and aggregation.

Reactive dicarbonyl species such as methylglyoxal (MGO) and glyoxal (GO) have recently received extensive attention due to their high reactivity and ability to modify biological substances such as proteins, phospholipids, and DNA. In case of proteins these reactive species mainly react with lysine and arginine residues to form AGEs, oxidative products, and aggregates. Chickpea cystatin (CPC) was incubated with varying concentrations of glyoxal and methylglyoxal which caused, along with altered secondary and tertiary structures, glycation, functional inactivation, altered redox state, cross-linking and high-molecular-mass aggregation. All these processes were examined and characterized by UV-Vis, fluorescence, and CD spectroscopies. Further characterization of CPC modified by reactive dicarbonyls was done by polyacrylamide gel electrophoresis which also showed alterations in the CPC molecules. Thus, in addition to describing the effects of GO and MGO on structure, conformation and function of CPC, this study also shows the relatively superior modifying effect of methylglyoxal for CPC in terms of glycation, oxidation and aggregation. This model system could shed some more light on the role of the reactive dicarbonyls in the specific alterations of proteins with different biological consequences having implications to ageing and disease such as diabetes.

Analysing Cytochrome c Aggregation and Fibrillation upon Interaction with Acetonitrile: an in Vitro Study.

The propensity of native state to form aggregated and fibrillar assemblies is a hallmark of amyloidosis. Our study was focused at analyzing the aggregation and fibrillation tendency of cytochrome c in presence of an organic solvent i.e. acetonitrile. In vitro analysis revealed that the interaction of cytochrome c with acetonitrile facilitated the oligomerization of cytochrome c via the passage through an intermediate state which was obtained at 20 % v/v concentration of acetonitrile featured by a sharp hike in the ANS fluorescence intensity with a blue shift of 20 nm compared to the native state. Oligomers and fibrils were formed at 40 and 50 % v/v concentration respectively as indicated by a significant hike in the ThT fluorescence intensity, red shift of 55 nm in congo red binding assay and an increase in absorbance at 350 nm. They possess ß-sheet structure as evident from appearance of peak at 217 nm. Finally, authenticity of oligomeric and fibrillar species was confirmed by TEM imaging which revealed bead like aggregates and a meshwork of thread like fibrils respectively. It could be suggested that the fibrillation of bovine cytchrome c could serve as a model protein to unravel the general aggregation and fibrillation pattern of heme proteins. Graphical abstract ᅟ.

Myotonic Dystrophy Type 1 (DM1): Clinical Characteristics and Disease Progression in a Large Cohort.

BACKGROUND: DM1 is a multisystem disorder caused by expansion of a CTG triplet repeat in the 3' non-coding region of DMPK. Neuropsychological consequences and sleep abnormalities are important associations in DM1. OBJECTIVE: To describe the clinical phenotype, disease progression and characterize the sleep alterations and cognitive abnormalities in a sub-set of patients. MATERIALS AND METHODS: A retrospective study on 120 genetically confirmed DM1 cases. Findings in neuropsychological assessment and multiple sleep questionnaires were compared with 14 age and sex matched healthy individuals. All 120 patients were contacted through letters/telephonic consultation/hospital visits to record their latest physical and functional disabilities. RESULTS: The mean age at symptom onset was 23.1 ± 11.4 years, M: F = 3.8:1, mean duration of illness = 14.3 ± 9.5 years. Clinically 54.2% had adult onset form, juvenile = 27.5%, infantile = 10.8%, late adult onset = 7.5%. Paternal transmission occurred more frequently. The predominant initial symptoms were myotonia (37.5%), hand weakness (21.7%), lower limb weakness (23.3%) and bulbar (10%). Twenty patients completed sleep questionnaires (SQ). Abnormal scores were noted in Epworth sleepiness scale (55%); Pittsburgh sleep quality index (45%); Berlin SQ (30%); Rapid eye movement sleep Behaviour Disorder SQ (15%); Restless leg syndrome rating scale (10%). Neuropsychological assessment of 20 patients revealed frontal executive dysfunction, attention impairment and visuospatial dysfunction. Frontal lobe was most affected (72%) followed by parietal (16%) and temporal lobe (12%). CONCLUSIONS: The current study provides a comprehensive account of the clinical characteristics in Indian patients with DM1. Hypersomnolence was most commonly seen. Excessive daytime sleepiness and Sleep disordered breathing were the most common sleep related abnormality. Cognitive impairment comprised predominantly of frontal lobe dysfunction.

Redox oscillations destabilize and mobilize colloidal soil organic carbon.

The onset of oxic-anoxic events in soil induces biogeochemical processes that may have profound influences on the fate and transport of soil organic carbon (OC). An 18-wk long laboratory soil column study was conducted to investigate the influence of static oxic (SO), static anoxic (SA), or redox oscillation (RO) events on the release of OC. Column leachate samples were collected after 9-wk and 18-wk of incubations and separated into dissolved (<2.3 nm), natural nanoparticle (2.3-100 nm), fine colloid (100-450 nm), and particulate (> 450 nm) fractions that were characterized through spectrofluorometric analyses. The concentration of released OC from different treatment columns followed the order of SA > RO > SO. After 9-wk, total OC concentrations in the leachate samples were increased by 4-fold and 54-fold in the SO and SA columns compared to the time-zero control columns, respectively. However, after 18-wk, the released amount of OC doubled in SO but decreased by 50 % in SA columns compared to the 9-wk incubation samples. The RO columns had intermediate OC concentrations between the SO and SA treatments. The RO events further led to widely varied dynamics in the release and molecular composition of the size-fractionated OC compared to static conditions, indicating the effects of redox oscillation on the organo-mineral association in soil. The wide variations in the aromaticity of OC released after the 1st and 2nd RO events further support the notion that alternating redox processes regulate OC cycling differently than the SO or SA condition. The observed increase in fine colloid and particulate OC fractions (i.e., >100 nm) from 7 % to 40 % between 1st and 2nd RO suggests the clustering of nanoaggregates and/or formation of colloidal size aggregates. The composition of the released OC as influenced by redox fluctuations provides a baseline for the size continuum of soil OC and its potential ecological and environmental roles.

Regulation of futile ligation during early steps of BER in M. tuberculosis is carried out by a ß-clamp-XthA-LigA tri-component complex.

The Class-II AP-endonuclease (XthA) is a mycobacterial DNA base excision repair (BER) pathway enzyme that functions in the initial steps. It acts on DNA substrates that contain abasic sites to create nicks with 3'-hydroxyl (OH) and 5'-deoxyribose phosphate (5'-dRP) moieties. The NAD+-dependent DNA ligase (LigA) is the terminal player in mycobacterial BER and seals such nicks efficiently. Here, we demonstrate that the Mtbß-clamp-MtbXthA complex that exists in the initial steps of BER engages with MtbLigA to form a novel tri-component BER complex. Size exclusion chromatography (SEC) experiments analysis show that the three proteins interact with equimolar stoichiometry. Small angle X-ray scattering (SAXS) analysis and associated studies reveal that the apo tri-component BER-complex adopts an extended conformation where MtbXthA is sandwiched between the Mtbß-clamp and MtbLigA. The studies support that in the apo-complex MtbXthA binds subsite-I of Mtbß-clamp through 239QLRFPKK245 motif and to MtbLigA by 104DGQPSWSGKP113 motif simultaneously. However, the complex adopts a less-extended conformation in the presence of substrate DNA, where MtbXthA interactions switch from predominantly subsite-I to subsite-II of the Mtbß-clamp. Overall, the novel tri-component complex prevents futile ligation activity of MtbLigA on the product of MtbXthA and ensures forward progression of the pathway and productive mycobacterial BER interactions.

Cryo-EM structures of Uba7 reveal the molecular basis for ISG15 activation and E1-E2 thioester transfer.

ISG15 plays a crucial role in the innate immune response and has been well-studied due to its antiviral activity and regulation of signal transduction, apoptosis, and autophagy. ISG15 is a ubiquitin-like protein that is activated by an E1 enzyme (Uba7) and transferred to a cognate E2 enzyme (UBE2L6) to form a UBE2L6-ISG15 intermediate that functions with E3 ligases that catalyze conjugation of ISG15 to target proteins. Despite its biological importance, the molecular basis by which Uba7 catalyzes ISG15 activation and transfer to UBE2L6 is unknown as there is no available structure of Uba7. Here, we present cryo-EM structures of human Uba7 in complex with UBE2L6, ISG15 adenylate, and ISG15 thioester intermediate that are poised for catalysis of Uba7-UBE2L6-ISG15 thioester transfer. Our structures reveal a unique overall architecture of the complex compared to structures from the ubiquitin conjugation pathway, particularly with respect to the location of ISG15 thioester intermediate. Our structures also illuminate the molecular basis for Uba7 activities and for its exquisite specificity for ISG15 and UBE2L6. Altogether, our structural, biochemical, and human cell-based data provide significant insights into the functions of Uba7, UBE2L6, and ISG15 in cells.

Drug targeting Nsp1-ribosomal complex shows antiviral activity against SARS-CoV-2.

The SARS-CoV-2 non-structural protein 1 (Nsp1) contains an N-terminal domain and C-terminal helices connected by a short linker region. The C-terminal helices of Nsp1 (Nsp1-C-ter) from SARS-CoV-2 bind in the mRNA entry channel of the 40S ribosomal subunit and blocks mRNA entry, thereby shutting down host protein synthesis. Nsp1 suppresses host immune function and is vital for viral replication. Hence, Nsp1 appears to be an attractive target for therapeutics. In this study, we have in silico screened Food and Drug Administration (FDA)-approved drugs against Nsp1-C-ter. Among the top hits obtained, montelukast sodium hydrate binds to Nsp1 with a binding affinity (KD) of 10.8 ± 0.2 µM in vitro. It forms a stable complex with Nsp1-C-ter in simulation runs with -95.8 ± 13.3 kJ/mol binding energy. Montelukast sodium hydrate also rescues the inhibitory effect of Nsp1 in host protein synthesis, as demonstrated by the expression of firefly luciferase reporter gene in cells. Importantly, it shows antiviral activity against SARS-CoV-2 with reduced viral replication in HEK cells expressing ACE2 and Vero-E6 cells. We, therefore, propose montelukast sodium hydrate can be used as a lead molecule to design potent inhibitors to help combat SARS-CoV-2 infection.

Disrupted structural connectome and neurocognitive functions in Duchenne muscular dystrophy: classifying and subtyping based on Dp140 dystrophin isoform.

OBJECTIVE: Neurocognitive disabilities in Duchenne muscular dystrophy (DMD) children beginning in early childhood and distal DMD gene deletions involving disruption of Dp140 isoform are more likely to manifest significant neurocognitive impairments. MRI data analysis techniques like brain-network metrics can provide information on microstructural integrity and underlying pathophysiology. METHODS: A prospective study on 95 participants [DMD = 57, and healthy controls (HC) = 38]. The muscular dystrophy functional rating scale (MDFRS) scores, neuropsychology batteries, and multiplex ligand-dependent probe amplification (MLPA) testing were used for clinical assessment, IQ estimation, and genotypic classification. Diffusion MRI and network-based statistics were used to analyze structural connectomes at various levels and correlate with clinical markers. RESULTS: Motor and executive sub-networks were extracted and analyzed. Out of 57 DMD children, 23 belong to Dp140 + and 34 to Dp140- subgroup. Motor disabilities are pronounced in Dp140- subgroup as reflected by lower MDFRS scores. IQ parameters are significantly low in all-DMD cases; however, the Dp140- has specifically lowest scores. Significant differences were observed in global efficiency, transitivity, and characteristic path length between HC and DMD. Subgroup analysis demonstrates that the significance is mainly driven by participants with Dp140- than Dp140 + isoform. Finally, a random forest classifier model illustrated an accuracy of 79% between HC and DMD and 90% between DMD- subgroups. CONCLUSIONS: Current findings demonstrate structural network-based characterization of abnormalities in DMD, especially prominent in Dp140-. Our observations suggest that participants with Dp140 + have relatively intact connectivity while Dp140- show widespread connectivity alterations at global, nodal, and edge levels. This study provides valuable insights supporting the genotype-phenotype correlation of brain-behavior involvement in DMD children.

Salt bridges at the subdomain interfaces of the adenylation domain and active-site residues of Mycobacterium tuberculosis NAD+-dependent DNA ligase A (MtbLigA) are important for the initial steps of nick-sealing activity.

NAD+-dependent DNA ligase (LigA) is the principal bacterial ligase and catalyses a multistep ligation reaction. The adenylation (AdD) domain at the N-terminus consists of subdomains 1a and 1b, where subdomain 1a is unique to LigA. Small-angle X-ray scattering and X-ray diffraction studies were used to probe changes in the relative spatial dispositions of the two subdomains during the adenylation reaction. Structural analyses of the inter-subdomain interactions of the AdD domain suggest that salt bridges formed by Glu22, Glu26 and Glu87 of subdomain 1a with Arg144, Arg315 and His240 of subdomain 1b play an important role in stabilizing the intermediate conformations of the two subdomains. E22A, E26A and E87A mutations reduce the in vitro activity by 89%, 64% and 39%, respectively, on a nicked DNA substrate, while they show no activity loss on a pre-adenylated DNA substrate, thus suggesting that the salt bridges are important in the initial steps of the ligation reaction. Furthermore, the E22A, E26A and E87A mutants exhibited extremely delayed growth in complementation assays involving the Escherichia coli GR501 strain, which harbours its own temperature-sensitive LigA. The H236A and H236Y mutants, which involve the residue that stacks against the adenine moiety of AMP, severely impact the activity and the ability to complement the growth-defective E. coli GR501 strain. Analysis of the K123A and K123R mutations in the active site rationalizes their total loss of activity and inability to rescue the growth-defective E. coli GR501 strain.

Emetine suppresses SARS-CoV-2 replication by inhibiting interaction of viral mRNA with eIF4E.

Emetine is a FDA-approved drug for the treatment of amebiasis. Previously we demonstrated the antiviral efficacy of emetine against some RNA and DNA viruses. In this study, we evaluated the in vitro antiviral efficacy of emetine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and found it to be a low nanomolar (nM) inhibitor. Interestingly, emetine exhibited protective efficacy against lethal challenge with infectious bronchitis virus (IBV; a chicken coronavirus) in the embryonated chicken egg infection model. Emetine treatment led to a decrease in viral RNA and protein synthesis without affecting other steps of viral life cycle such as attachment, entry and budding. In a chromatin immunoprecipitation (CHIP) assay, emetine was shown to disrupt the binding of SARS-CoV-2 mRNA with eIF4E (eukaryotic translation initiation factor 4E, a cellular cap-binding protein required for initiation of protein translation). Further, molecular docking and molecular dynamics simulation studies suggested that emetine may bind to the cap-binding pocket of eIF4E, in a similar conformation as m7-GTP binds. Additionally, SARS-CoV-2 was shown to exploit ERK/MNK1/eIF4E signalling pathway for its effective replication in the target cells. Collectively our results suggest that further detailed evaluation of emetine as a potential treatment for COVID-19 may be warranted.

Quantification and molecular characterization of organo-mineral associations as influenced by redox oscillations.

Organo-mineral association is one of the most important stabilization mechanisms of soil organic matter. However, few studies have been conducted to assess the retention, transformation, and transportation of colloids (1-1000 nm) and associated organic carbon (OC) in soil. Given the particularly significant role that wetland soils play in carbon storage and cycling, we quantified the dynamics of organo-mineral association within colloidal size range by conducting three consecutive 35-day redox (reduction-oxidation) oscillation experiments using a wetland soil. Molecular compositions of natural nanoparticle (NNP, 2.3-100 nm), fine colloid (100-450 nm), and particulate (450-1000 nm) fractions were measured using isotope ratio mass spectrometry (IRMS) and x-ray photoelectron spectroscopy (XPS). Results showed that NNP and fine colloids constituted up to 8.94 ± 0.50% and 22.19 ± 7.52% of bulk C concentration (2.3-1000 nm), respectively; indicating substantial contributions of these two fractions to the operationally defined "dissolved" (<450 nm) fraction. There was significant enrichment in heavier δ13C isotopes (p < 0.001) with size: NNP (-29.64 ± 0.32‰) < fine colloid (-28.81 ± 0.31‰) < particulate (-28.34 ± 0.25‰) fractions. NNP had the highest percentages of carbonyl/carboxyl C (C=O); while fine colloid and particulate fractions contained more reduced aromatic or aliphatic C (C-C, C=C, C-H). OC became more enriched (‰) in microbial-derived C (higher δ13C) with increasing particle size as well as with repeated redox oscillations. Our findings clearly demonstrate limitations of using the operationally defined "dissolved" fraction (<450 nm) to assess C cycling in ecosystems such as wetlands. Increase in colloid and OC concentrations and presence of more microbial-derived C in larger size fractions additionally imply that redox oscillations promote the formation of molecularly diverse sub-colloid sized organo-mineral associations. Being a composite unit of soil microaggregates, organic-mineral associations can thus influence the overall stability of OC in wetland soils that undergo frequent redox oscillations.

Deciphering the Nature of Caffeic Acid to Inhibit the HSA Aggregation Induced by Glyoxal.

BACKGROUND: Under certain circumstances, the path for protein folding deviates and attains an alternative path forming misfolded states, which are the key precursors for protein aggregation. Protein aggregation is associated with variety of diseases and leads to the cytotoxicity. These protein aggregate related diseases have been untreated so far. However, extensive attempts have been applied to develop anti-aggregating agents as possible approaches to overcome protein aggregation. Different types of substances have been reported to halt or decrease the formation of ordered protein aggregates both in vitro and in vivo, such as polyphenols and metal ions. OBJECTIVE: In the present study the in vitro aggregation of human serum albumin (HSA) by using a reactive dicarbonyl glyoxal has been investigated, simultaneously an attempt has been done to inhibit the glyoxal (GO) induced aggregation of (HSA) by caffeic acid (CA). METHODS: Different methods have been employed to investigate the process, fluorescence spectroscopy, circular dichroism, cango red binding assay, thioflavin T dye binding, turbidimetric analysis, docking study and transmission electron microscopy. RESULTS: Results have shown that elevated concentration of GO forms aggregates of HSA, and the activity of CA suggested the possibility of inhibiting the HSA aggregation at higher concentrations, and this compound was found to have an anti-aggregation property. CONCLUSION: The present study explained that micro molar concentrations of CA inhibits the aggregation of HSA and showed pronounced anti-aggregation effect at increasing concentrations in the presence of GO which is elevated in diabetic and hyperglycaemia conditions.

Glycation induced conformational transitions in cystatin proceed to form biotoxic aggregates: A multidimensional analysis.

Hyperglycaemic conditions facilitate the glycation of serum proteins which may have predisposition to aggregation and thus lead to complications. The current study investigates the glycation induced structural and functional modifications of chickpea cystatin (CPC) as well as biological toxicity of the modified protein forms, using CPC-glucose as a model system. Several structural intermediates were formed during the incubation of CPC with glucose (day 4, 8, 12, & 16) as revealed by circular dichroism (CD), altered intrinsic fluorescence, and high ANS binding. Further incubation of CPC with glucose (day 21) formed abundant ß structures as revealed by Fourier transform infrared spectroscopy and CD analysis which may be due to the aggregation of protein. High thioflavin T fluorescence intensity and increased Congo red absorbance together with enhanced turbidity and Rayleigh scattering by this modified form confirmed the aggregation. Electron microscopy finally provided the valid physical authentication about the presence of aggregate structures. Functional inactivation of glucose incubated CPC was also observed with time. Single cell electrophoresis of lymphocytes and plasmid nicking assays in the presence of modified CPC showed the DNA damage which confirmed its biological toxicity. Hence, our study suggests that glycation of CPC not only leads to structural and functional alterations in proteins but also to biotoxic AGEs and aggregates.

Biochemical characterization and novel inhibitor identification of Mycobacterium tuberculosis Endonuclease VIII 2 (Rv3297).

Nei2 (Rv3297) is a DNA Base Excision Repair (BER) glycosylase that is essential for survival of Mycobacterium tuberculosis in primates. We show that MtbNei2 is a bifunctional glycosylase that specifically acts on oxidized pyrimidine-containing single-stranded, double-stranded, 5'/3' fork and bubble DNA substrates. MtbNei2 possesses Uracil DNA glycosylase activity unlike E. coli Nei. Mutational studies demonstrate that Pro2 and Glu3 located in the active site are essential for glycosylase activity of MtbNei2. Mutational analysis demonstrated that an unstructured C-terminal zinc finger domain that was important for activity in E. coli Nei and Fpg, was not required for the glycosylase activity of MtbNei2. Lastly, we screened the NCI natural product compound database and identified three natural product inhibitors with IC50 values ranging between 41.8 µM-92.7 µM against MtbNei2 in in vitro inhibition assays. Surface Plasmon Resonance (SPR) experiments showed that the binding affinity of the best inhibitor, NSC31867, was 74 nM. The present results set the stage for exploiting this important target in developing new therapeutic strategies that target Mycobacterial BER.

PeMtb: A Database of MHC Antigenic Peptide of Mycobacterium tuberculosis.

BACKGROUND: For design of a subunit vaccine for tuberculosis, identification of antigenic Tcell epitope is of utmost importance. Several MHC prediction server are available that can accurately predict antigenic peptide of variable lengths. However, peptides predicted from one server not necessarily are predicted form another server, thus creating a confusing situation for scientists to choose a best epitope. METHOD: Keeping the above problem in mind, we developed a comprehensive database of peptides of Mycobacterial proteins. Each protein was taken from PubMed and was run through different MHC prediction servers, with the results being compiled into one database. RESULTS: For each protein, PeMtb generates a set of three different mers of variable lengths (12 mer or 13-mer) based on their ranking; with each mer being predicted for a plethora of MHC alleles. Researcher can choose the peptide (mers) that gives best binding affinity from most of the servers. CONCLUSION: The database relieves the investigators of the painstaking task of searching various MHC prediction servers for the right epitope (T-cell epitope) for a particular Mycobacterial antigen. We trust and anticipate that PeMtb will be a practical platform for trial and computational analyses of antigenic peptides for Mycobacterium tuberculosis. All the resources and information can be accessed by PeMtb home page www.pemtb-amu.org.

Oral administration of Nigella sativa oil ameliorates the effect of cisplatin on membrane enzymes, carbohydrate metabolism and oxidative damage in rat liver.

Cisplatin (CP) is a potent anti-cancer drug widely used against solid tumors. However, it exhibits pronounced adverse effects including hepatotoxicity. Several strategies were attempted to prevent CP hepatotoxicity but were not found suitable for therapeutic application. Nigella sativa has been shown to prevent/reduce the progression of certain type of cardiovascular, kidney and liver diseases. Present study investigates whether N. sativa oil (NSO) can prevent CP induced hepatotoxic effects. Rats were divided into four groups viz. control, CP, NSO and CPNSO. Animals in CPNSO and NSO group were administered NSO (2 ml/kg bwt, orally) with or without single hepatotoxic dose of CP (6 mg/kg bwt, i.p.) respectively. CP hepatotoxicity was recorded by increased serum ALT and AST activities. CP treatment caused oxidant/antioxidant imbalances as reflected by increased lipid peroxidation and decreased enzymatic and non-enzymatic antioxidants. Furthermore, the activities of various carbohydrate metabolism and membrane enzymes were altered by CP treatment. In contrast, NSO administration to CP treated rats, markedly ameliorated the CP elicited deleterious alterations in liver. Histopathological observations showed extensive liver damage in CP treated animals while greatly reduced tissue injury in CPNSO group. In conclusion, NSO appears to protect CP induced hepatotoxicity by improving energy metabolism and strengthening antioxidant defense mechanism.

In Vitro Elucidation of the Folding Intermediates and Aggregate Formation of Hemoglobin Induced by Acetonitrile: A Multispectroscopic Approach.

Acetonitrile is a mild solvent, which induces ß-sheet conformation in proteins. The global conformational changes in Hb in the presence of ACN were studied using intrinsic fluorescence experiments, acrylamide quenching, ANS fluorescence measurements, soret absorbance spectroscopy, fourier transform infrared spectroscopy, circular dichroism, thioflavin T and congo red assay. Molecular docking showed the binding of hydrophobic residues of Hb to ACN. Hb exists as a partially unfolded intermediate state at 30% v/v ACN. Hb aggregates were obtained at 60% v/v ACN concentration, which were further confirmed by transmission electron microscopy.