Characterization of structural, genotoxic, and immunological effects of methyl methanesulfonate (MMS) induced DNA modifications: Implications for inflammation-driven carcinogenesis.

Genotoxic DNA damaging agents are the choice of chemicals for studying DNA repair pathways and the associated genome instability. One such preferred laboratory chemical is methyl methanesulfonate (MMS). MMS, an SN2-type alkylating agent known for its ability to alkylate adenine and guanine bases, causes strand breakage. Exploring the outcomes of MMS interaction with DNA and the associated cytotoxicity will pave the way to decipher how the cell confronts methylation-associated stress. This study focuses on an in-depth understanding of the structural instability, induced antigenicity on the DNA molecule, cross-reactive anti-DNA antibodies, and cytotoxic potential of MMS in peripheral lymphocytes and cancer cell lines. The findings are decisive in identifying the hazardous nature of MMS to alter the intricacies of DNA and morphology of the cell. Structural alterations were assessed through UV-Vis, fluorescence, liquid chromatography, and mass spectroscopy (LCMS). The thermal instability of DNA was analyzed using duplex melting temperature profiles. Scanning and transmission electron microscopy revealed gross topographical and morphological changes. MMS-modified DNA exhibited increased antigenicity in animal subjects. MMS was quite toxic for the cancer cell lines (HCT116, A549, and HeLa). This research will offer insights into the potential role of MMS in inflammatory carcinogenesis and its progression.

The complex landscape of intracellular signalling in protein modification under hyperglycaemic stress leading to metabolic disorders.

Hyperglycaemia is a life-threatening risk factor that occurs in both chronic and acute phases and has been linked to causing injury to many organs. Protein modification was triggered by hyperglycaemic stress, which resulted in pathogenic alterations such as impaired cellular function and tissue damage. Dysregulation in cellular function increases the condition associated with metabolic disorders, including cardiovascular diseases, nephropathy, retinopathy, and neuropathy. Hyperglycaemic stress also increases the proliferation of cancer cells. The major areas of experimental biomedical research have focused on the underlying mechanisms involved in the cellular signalling systems involved in diabetes-associated chronic hyperglycaemia. Reactive oxygen species and oxidative stress generated by hyperglycaemia modify many intracellular signalling pathways that result in insulin resistance and ß-cell function degradation. The dysregulation of post translational modification in ß cells is clinically associated with the development of diabetes mellitus and its associated diseases. This review will discuss the effect of hyperglycaemic stress on protein modification and the cellular signalling involved in it. The focus will be on the significant molecular changes associated with severe metabolic disorders.

Nano-therapeutics: The upcoming nanomedicine to treat cancer.

Nanotechnology is considered a successful approach for cancer diagnosis and treatment. Preferentially, cancer cell recognition and drug targeting via nano-delivery system include the penetration of anticancer agents into the cell membrane to damage the cancer cell by protein modification, DNA oxidation, or mitochondrial dysfunction. The past research on nano-delivery systems and their target has proven the beneficial achievement in a malignant tumor. Modern perceptions using inventive nanomaterials for cancer management have been offered by a multifunctional platform based on various nano-carriers with the probability of imaging and cancer therapy simultaneously. Emerging nano-delivery systems in cancer therapy still lack knowledge of the biological functions behind the interaction between nanoparticles and cancer cells. Since the potential of engineered nanoparticles addresses the various challenges, limiting the success of cancer therapy subsequently, it is a must to review the molecular targeting of a nano-delivery system to enhance the therapeutic efficacy of cancer. This review focuses on using a nano-delivery system, an imaging system, and encapsulated nanoparticles for cancer therapy.

Molecular pathways and therapeutic targets linked to triple-negative breast cancer (TNBC).

Cancer is a group of diseases characterized by uncontrolled cellular growth, abnormal morphology, and altered proliferation. Cancerous cells lose their ability to act as anchors, allowing them to spread throughout the body and infiltrate nearby cells, tissues, and organs. If these cells are not identified and treated promptly, they will likely spread. Around 70% of female breast cancers are caused by a mutation in the BRCA gene, specifically BRCA1. The absence of progesterone, oestrogen and HER2 receptors (human epidermal growth factor) distinguishes the TNBC subtype of breast cancer. There were approximately 6,85,000 deaths worldwide and 2.3 million new breast cancer cases in women in 2020. Breast cancer is the most common cancer globally, affecting 7.8 million people at the end of 2020. Compared to other cancer types, breast cancer causes more women to lose disability-adjusted life years (DALYs). Worldwide, women can develop breast cancer at any age after puberty, but rates increase with age. The maintenance of mammary stem cell stemness is disrupted in TNBC, governed by signalling cascades controlling healthy mammary gland growth and development. Interpreting these essential cascades may facilitate an in-depth understanding of TNBC cancer and the search for an appropriate therapeutic target. Its treatment remains challenging because it lacks specific receptors, which renders hormone therapy and medications ineffective. In addition to radiotherapy, numerous recognized chemotherapeutic medicines are available as inhibitors of signalling pathways, while others are currently undergoing clinical trials. This article summarizes the vital druggable targets, therapeutic approaches, and strategies associated with TNBC.

Albumin from sera of rheumatoid arthritis patients share multiple biochemical, biophysical and immunological properties with in vitro generated glyco-nitro-oxidized-albumin.

The purpose of the present study is to explore the effects of endogenous stressors on structure and function of rheumatoid arthritis (RA) patients' albumin. In contrast to glycated-albumin or nitro-oxidized-albumin, high titre antibodies against glyco-nitro-oxidized-albumin were found in the sera of RA patients. Also, compared to the other two modified forms of albumin, glyco-nitro-oxidized-albumin showed highest percent inhibition. Albumin isolated from RA patients' sera displayed hyperchromicity and quenching of tyrosine and tryptophan fluorescence. Fluorescence spectroscopy studies also revealed the presence of dityrosine and advanced glycation end products in RA patient's albumin. RA patients' albumin showed weaker binding with 1-anilinonaphthalene-8-sulfonic acid dye. Secondary structure alterations were demonstrated by circular dichroism and Fourier transform infrared spectroscopy. Biochemical investigations revealed substantial decline in the availability of free side chains of amino acid residues; increased carbonyls and decreased sulfhydryls in RA patients' albumin. The functional impairment in RA patients' albumin was revealed by their low binding with bilirubin and cobalt. Liquid chromatography mass spectrometry analysis revealed the presence of Nε-(carboxymethyl) lysine and 3-nitrotyrosine in RA patients' albumin. The amyloidogenic aggregation of RA patients' albumin was confirmed by Congo red absorption and thioflavin-T fluorescence assays. The morphology of the aggregates was visualized under scanning and transmission electron microscope. From the above findings, we inferred that endogenous stress in RA patients have modified albumin and produce structural/functional abnormalities. Also, the presence of anti-glyco-nitro-oxidized-albumin antibodies along with other clinical features may be used as biomarker for the diagnosis and assessment of treatment responses in RA patients.Communicated by Ramaswamy H. Sarma.

The Diversity, Resistance Profiles and Plasmid Content of Klebsiella spp. Recovered from Dairy Farms Located around Three Cities in Pakistan.

The rise of antimicrobial resistance (AMR) in bacterial pathogens such as Klebsiella pneumoniae (Kp) is a pressing public health and economic concern. The 'One-Health' framework recognizes that effective management of AMR requires surveillance in agricultural as well as clinical settings, particularly in low-resource regions such as Pakistan. Here, we use whole-genome sequencing to characterise 49 isolates of Klebisella spp. (including 43 Kp) and 2 presumptive Providencia rettgeri isolates recovered from dairy farms located near 3 cities in Pakistan-Quetta (n = 29), Faisalabad (n = 19), and Sargodha (n = 3). The 43 Kp isolates corresponded to 38 sequence types (STs), and 35 of these STs were only observed once. This high diversity indicates frequent admixture and limited clonal spread on local scales. Of the 49 Klebsiella spp. isolates, 41 (84%) did not contain any clinically relevant antimicrobial resistance genes (ARGs), and we did not detect any ARGs predicted to encode resistance to carbapenems or colistin. However, four Kp lineages contained multiple ARGs: ST11 (n = 2), ST1391-1LV (n = 1), ST995 (n = 1) and ST985 (n = 1). STs 11, 1391-1LV and 995 shared a core set of five ARGs, including blaCTX-M-15, harboured on different AMR plasmids. ST985 carried a different set of 16 resistance genes, including blaCTX-M-55. The two presumptive P. rettgeri isolates also contained multiple ARGs. Finally, the four most common plasmids which did not harbour ARGs in our dataset were non-randomly distributed between regions, suggesting that local expansion of the plasmids occurs independently of the host bacterial lineage. Evidence regarding how dairy farms contribute to the emergence and spread of AMR in Pakistan is valuable for public authorities and organizations responsible for health, agriculture and the environment, as well as for industrial development.

Fructosylation of human insulin causes AGEs formation, structural perturbations and morphological changes: an in silico and multispectroscopic study.

Fructosylation of proteins results in the formation of advanced glycation end-products (AGEs). A diet rich in fructose along with hyperglycemia can cause fructose mediated glycation (fructosylation) of proteins, which results in AGEs formation. Insulin is a peptide hormone that is glycated when exposed to carbohydrates such as glucose. In this study, we have analysed the interaction of insulin with fructose and biophysically characterized fructose modified insulin. In silico studies performed through molecular docking and molecular dynamics simulation revealed that fructose binds to insulin with strong affinity resulting in the formation of insulin-fructose complex. Fructosylation of insulin caused hyperchromicity, loss of intrinsic fluorescence, gain in AGEs specific fluorescence and elevated the carbonyl and fructosamine content. Enhanced thioflavin T fluorescence suggested the presence of fibrillar structures at higher concentrations of fructose. Electron microscopy revealed the formation of characteristic amorphous and amyloid like aggregates at lower and higher concentrations of fructose, respectively. These findings show that fructosylation of insulin causes AGEs production, aggregation and alters its gross structural integrity. These changes may reduce the biological activity of insulin that can aggravate conditions like type II diabetes mellitus.Communicated by Ramaswamy H. Sarma.

Studies on the synergistic action of methylglyoxal and peroxynitrite on structure and function of human serum albumin.

Albumin, an important serum protein, is continuously exposed to various oxidizing/nitrating and glycating agents. Depending upon the nature/concentration of reactive species present, the protein may be glycated, oxidized/nitroxidized or glyco-nitro-oxidized. Peroxynitrite is a powerful nitroxidant and has been reported to damage a wide array of macromolecules. On the other hand, methylglyoxal is a very strong reactive dicarbonyl and a potent precursor for the formation of advanced glycation end products under pathological conditions. In certain pathological conditions albumin may be modified by peroxynitrite and methylglyoxal simultaneously. There is dearth of literature suggests that structural/conformational and functional alteration in albumin upon glycation and oxidation/nitroxidation, however the alterations produced by glyco-nitro-oxidation has not yet been explored. Therefore, in this study, simultaneous effect of glycation and nitroxidation on the structure and conformation, vis-a-vis function of albumin was explored. Glyco-nitro-oxidized albumin showed decreased free amino acid content together with decreased affinity of albumin towards cobalt. Molecular docking model and molecular dynamic simulations showed close interaction and formation of stable complexes between methylglyoxal, peroxynitrite and albumin. Formation of carboxymethyl lysine and 3-nitrotyrosine in glyco-nitro-oxidized albumin were confirmed by MALDI-TOF MS and UP-LC MS. Aggregate formation in glyco-nitro-oxidized albumin was visualized by transmission electron microscopy. On the basis of these results, it may be speculated that, albumin modified with endogenously generated methylglyoxal and peroxynitrite might be a driving factor in the progression of heightened inflammatory autoimmune responses. The work presents a ground to study the role of glyco-nitro-oxidized albumin in the pathogenesis and progression of various autoimmune diseases including rheumatoid arthritis. Communicated by Ramaswamy H. Sarma.

Calf Thymus DNA Exposed to Quinacrine at Physiological Temperatures and pH Acquires Immunogenicity: A Threat for Long Term Quinacrine Therapy.

Quinacrine is an Acridine derivative with two potentially reactive groups; a diamino butyl side chain and an Acridine ring both capable of interacting with DNA but in different ways. This is an antimalarial drug approved by FDA for long term clinical trials and for the treatment of other diseases as well. The study evaluates the physicochemical interactions of quinacrine with DNA (calf thymus DNA) through characterizations of quinacrine DNA adduct (Q-DNA) by various techniques. It was observed that quinacrine induces stability in the structure of DNA, as the onset of melting was found to be increased by 6 °C in the melting temperature profile of Q-DNA supported by other data obtained during study, deviation from the native structure of DNA was analyzed by FTIR that showed specific shifts in the region of 1707-1400 cm-1.The study also probed the antigenicity of Q-DNA compared to its non antigenic native counterpart (N-DNA), by using both as antigens in female New Zealand White rabbits. Q-DNA was found to be antigenic with antibody titer > 1:6400. IgG was isolated and characterized to check for binding specificity. These antibodies were found to be promiscuous capable of cross reacting with other cellular molecules. Analysis of the data obtained suggested that intracellular accumulation of quinacrine and its ability to cross nucleus may allow the drug to interact with DNA. This may bring about significant structural perturbations in the macromolecule triggering an immunogenic response at the site where anti Q-DNA antibody and Q-DNA complex accumulates.

Risk of Carcinogenicity Associated with Synthetic Hair Dyeing Formulations: A Biochemical View on Action Mechanisms, Genetic Variation and Prevention.

Article tries to visualize the potential for carcinogenic trigger in humans with a preference for oxidative synthetic of hair dyeing formulations, especially which belong to the category of permanent colours. According to the International Agency for Cancer, hair dyes for personal use are not strictly classified as carcinogen to humans. However, some controversy exists that requires clarification. Some epidemiological studies support the association between the risk of cancer development and personal use of hair dyes (pooled relative risk RR = 1.50. 95% CI: 1.30-1.98). The world-wide sale of hair dyeing cosmetics have exceeded 15 billion dollars by the year 2012 and has maintained an annual growth rate of 8-10%. This raises concerns and need to be addressed. The review article briefly discusses about the different hair dye components based on their chemical nature, permanence, interaction of dye components with different parts of the hair shaft, action mechanisms, health risk assessment, associated challenges and possible alternatives. There appears variability towards the pathological changes incurred in the human system upon the use of synthetic hair formulations. This probably appears due to the presence of interindividual genetic variation of enzymes handling these xenobiotics. The redox mechanism of major hair dye components appears to be involved in the carcinogenic trigger. Most of the hair dye constituents pose serious health issues. However, we do have few better alternatives to prevent the toxicity associated with hair dye constituents without compromising the need of today's fashion statement and expectations of the youth.

4-Chloro-1,2-phenylenediamine induced structural perturbation and genotoxic aggregation in human serum albumin.

4-Chloro-1,2-phenylenediamine (4-Cl-OPD) is a halogenated aromatic diamine used as a precursor in permanent hair color production. Despite its well-documented mutagenic and carcinogenic effects in various in vitro and in vivo models, its role in fibrillar aggregate formation and their genotoxic effect in therapeutic proteins has received less attention. The significance of human serum albumin (HSA) arises from its involvement in bio-regulatory and transport processes. HSA misfolding and aggregation are responsible for some of the most frequent neurodegenerative disorders. We used various complementary approaches to track the formation of amyloid fibrils and their genotoxic effect. Molecular dynamics study demonstrated the complex stability. The impact of 4-Cl-OPD on the structural dynamics of HSA was confirmed by Raman spectroscopy, X-ray diffraction, HPLC and SDS-PAGE. Fibrilllar aggregates were investigated using Congo red assay, DLS, and SEM. The genotoxic nature of 4-Cl-OPD was confirmed using plasmid nicking assay and DAPI staining, which revealed DNA damage and cell apoptosis. 4-Cl-OPD provides a model system for studying fibrillar aggregation and their genotoxic potential in the current investigation. Future studies should investigate the inhibition of the aggregation/fibrillation process, which may yield valuable clinical insights.

N-OH-AABP Modifications in Human DNA May Lead to Auto-Antibodies in Bladder Cancer Subjects.

4-Aminobiphenyl (4-ABP) and other related arylamines have emerged to be responsible for human urinary bladder tumors and cancers. Hemoglobin-ABP adducts have been recognized in the blood of smokers, and it builds up in the circulatory system over the period of years that might lead to a bladder tumor. N-hydroxy-Acetyl 4-Aminobiphenyl (N-OH-AABP) is one of the reactive forms of 4-ABP which has a potential to initiate tumor growth and causes cancer rapidly. In the present study, commercially available human DNA was modified by N-OH-AABP, and its modifications were analyzed biophysically from fluorescence spectroscopy and thermal denaturation studies. Further, Sera and IgG from bladder cancer patients' blood were assessed for affinity to native and N-OH-AABP modified human DNA using ELISA. The study showed N-OH-AABP caused damage in the structure of the DNA macromolecule and the perturbations resulting from damage leads to change in the Tm of the DNA molecule. Bladder cancer auto-antibodies, particularly in smoker group, showed preferential binding to N-OH-AABP modified human DNA. This study shows that N-OH-AABP modified DNA could be an antigenic stimulus for the generation of autoantibodies in the sera of bladder cancer patients.

Biophysical characterization of structural and conformational changes in methylmethane sulfonate modified DNA leading to the frizzled backbone structure and strand breaks in DNA.

Methyl methanesulfonate (MMS) is a highly toxic DNA-alkylating agent that has a potential to damage the structural integrity of DNA. This work employed multiple biophysical and computational methods to report the MMS mediated structural alterations in the DNA (MMS-DNA). Spectroscopic techniques and gel electrophoresis studies revealed MMS induced exposure of chromophoric groups of DNA; methylation mediated anti→syn conformational change, DNA fragmentation and reduced nucleic acid stability. MMS induced single-stranded regions in the DNA were observed in nuclease S1 assay. FT-IR results indicated MMS mediated loss of the assigned peaks for DNA, partial loss of C-O ribose, loss of deoxyribose region, C-O stretching and bending of the C-OH groups of hexose sugar, a progressive shift in the assigned guanine and adenine peaks, loss of thymine peak, base stacking and presence of C-O-H vibrations of glucose and fructose, indicating direct strand breaks in DNA due to backbone loss. Isothermal titration calorimetry showed MMS-DNA interaction as exothermic with moderate affinity. Dynamic light scattering studies pointed towards methylation followed by the generation of single-stranded regions. Electron microscopy pictured the loss of alignment in parallel base pairs and showed the formation of fibrous aggregates in MMS-DNA. Molecular docking found MMS in close contact with the ribose sugar of DNA backbone having non-bonded interactions. Molecular dynamic simulations confirmed that MMS is capable of interacting with DNA at two levels, one at the level of nitrogenous bases and another at the DNA backbone. The study offers insights into the molecular interaction of MMS and DNA.Communicated by Ramaswamy H. Sarma.

4-Chloro-orthophenylenediamine alters DNA integrity and affects cell survival: inferences from a computational, biophysical/biochemical, microscopic and cell-based study.

The deleterious impact of toxic constituents of hair dyes over the human health has gained immense attention in the recent past. Their oncogenicity, mutagenicity, role in protein modification, impact on cellular metabolism has been documented. There is little information on the mechanism of reactivity of hair dye components with the nucleic acids and its implications. This work, therefore, uses computational, biophysical/biochemical, microscopic and cell-based study to analyze the interaction of monocyclic aromatic amine and a hair dye component, 4-chloro-orthophenylenediamine (4-Cl-OPD) with the DNA, its impact on DNA structure and cell survival. The results suggest that 4-Cl-OPD binds with the DNA in minor groove of the duplex involving three base pairs preferentially the G-C residues, induces strand breaks and makes DNA thermally labile through loss of hydrogen bonding/base unstacking. 4-Cl-OPD causes fragmentation of DNA, reduction in size of the molecule, alters B-DNA conformation and disrupts its secondary structure. The modified DNA gives fragmented appearance, shows broken strands and aggregation in ultra-structural analysis. 4-Cl-OPD induces ROS generation in lymphocytes, increases the comet's average tail length and reduces the viability of lymphocytes. This study forms a base for establishing the direct toxicity of 4-Cl-OPD at the molecular and cellular level through direct production of superoxide radicalCommunicated by Ramaswamy H. Sarma.

Characterization of Glyoxal Modified LDL: Role in the Generation of Circulating Autoantibodies in Type 2 Diabetes Mellitus and Coronary Artery Disease.

AIMS: This study aims to investigate the role of glyoxal modified LDL in the immunopathology of diabetes and cardiovascular disease. BACKGROUND: Glycoxidation of proteins is widely studied in relation to diabetes and cardiovascular disease. OBJECTIVE: This study probed the glyoxal mediated modifications in LDL, analyzed the immunogenicity of the glycated LDL and ascertained the presence of circulating antibodies against modified LDL in patients with type 2 diabetes mellitus (T2DM), coronary artery disease (CAD) and patients with both (T2DM+CAD). METHODS: Glyoxal mediated modifications in LDL were studied by multiple spectroscopic techniques, high-performance liquid chromatography and electron microscopy. Immunization studies were carried in New Zealand rabbits. The presence of antibodies against glyoxal modified LDL in immunized rabbits and human subjects was analyzed by ELISA. RESULTS: Glyoxal altered the structural integrity of LDL and led to the formation of AGEs. It decreased the alpha-helix content of LDL; increased ß sheet formation, increased carbonyl content and decreased free lysine and arginine content. Modified LDL showed aggregation, generation of of Nε- (Carboxymethyl) lysine and the formation of amorphous type aggregates. It exhibited high antigenicity and generated a specific immune response that shared common antigenic determinants with other glycated proteins. Direct binding data showed the presence of anti-glyoxal modified LDL antibodies in patients with T2DM, CAD and patients with both T2DM and CAD. Further analysis in competitive binding assay revealed specific binding characteristics of auto-antibodies. Sera from patients with T2DM+CAD exhibited the highest binding with glyoxal modified LDL. CONCLUSION: Glyoxal-modified LDL has neo-antigenic determinants that cause the generation of circulating antibodies in diabetes and coronary artery disease. The study might have potential relevance in biomarker development.

Hydroxyl radical induced structural perturbations make insulin highly immunogenic and generate an auto-immune response in type 2 diabetes mellitus.

Reactive oxygen species (ROS) cause oxidative damage to proteins and generate deleterious by-products which induce a breakdown of immune tolerance and produce antibodies against host macromolecules with implication in human diseases. This study characterizes the hydroxyl radical (OH) modifications of insulin, evaluates its cytotoxicity and immunogenicity, and probes its role in type 2 diabetes (T2DM) autoimmunity. The results demonstrate susceptibility of insulin to modifications induced by OH, causing exposure of its chromophoric aromatic amino acid residues, quenching of tyrosine fluorescence intensity, loss of α-helix and gain in ß content. Modification causes re-arrangement of native interactions of the aromatic residues in insulin. It enhanced the carbonyl content in insulin, exposed its hydrophobic patches and generated non-fibrillar, amorphous type of aggregates that are cytotoxic in nature. Native insulin induced low titre antibodies in immunized rabbits, whereas OH modified insulin generated a strong immune response. Competitive ELISA studies showed high specificity of antibodies generated against OH modified insulin towards the modified protein. Cross reaction studies showed the presence of common antigenic determinants on various oxidised proteins. Since T2DM patients show increased ROS production, oxidation of insulin is expected to occur, which might amplify autoimmune reactions against insulin. True to the assumption, direct binding ELISA showed the presence of anti-OH insulin circulating antibodies in T2DM patients which are specific for the oxidized insulin. In conclusion, insulin loses structural integrity to OH, forms cytotoxic amorphous aggregates, turns highly immunogenic and elicits humoral response in T2DM patients.

Recent advances in histone glycation: emerging role in diabetes and cancer.

Ever increasing information on genome and proteome has offered fascinating details and new opportunities to understand the molecular biology. It is now known that histone proteins surrounding the DNA play a crucial role in the chromatin structure and function. Histones undergo a plethora of posttranslational enzymatic modifications that influence nucleosome dynamics and affect DNA activity. Earlier research offered insights into the enzymatic modifications of histones; however, attention has been diverted to histone modifications induced by by-products of metabolism without enzymatic engagement in the last decade. Nonenzymatic modifications of histones are believed to be crucial for epigenetic landscape, cellular fate and for role in human diseases. Glycation of histone proteins constitutes the major nonenzymatic modifications of nuclear proteins that have implications in diabetes and cancer. It has emerged that glycation damages nuclear proteins, modifies amino acids of histones at crucial locations, generates adducts affecting histone chromatin interaction, develops neo-epitopes inducing specific immune response and impacts cell function. Presence of circulating antibodies against glycated histone proteins in diabetes and cancer has shown immunological implications with diagnostic relevance. These crucial details make histone glycation an attractive focus for investigators. This review article, therefore, makes an attempt to exclusively summarize the recent research in histone glycation, its impact on structural integrity of chromatin and elaborates on its role in diabetes and cancer. The work offers insights for future scientists who investigate the link between metabolism, biomolecular structures, glycobiology, histone-DNA interactions in relation to diseases in humans.

Withdrawal Notice: Characterization of Glyoxal Modified LDL: Role in the Generation of Circulating Autoantibodies in Type 2 Diabetes Mellitus and Coronary Artery Disease

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Preferential recognition of epitopes on peroxynitrite-modified alpha-2-macroglobulin by circulating autoantibodies in rheumatoid arthritis patients.

Autoimmune responses against post-translationally modified antigens are a hallmark of several autoimmune diseases. In this work, we have studied the changes in alpha-2-macroglobulin (α2M) upon modification by peroxynitrite. Furthermore, we have evaluated the immunogenicity of modified α2M in experimental rabbits and rheumatoid arthritis (RA) patients. Peroxynitrite-modified α2M showed disturbed microenvironment and altered aromatic residues under UV and fluorescence studies. Aggregation, reduction in ß-sheet content, production of nitrotyrosine and shift in amide I and II bands were observed in the modified α2M by polyacrylamide gel electrophoresis besides CD and FTIR spectroscopic analysis. The exposure of hydrophobic clusters and changes in contact positions were observed in ANS and ThT binding assays. Immunological studies using ELISA showed peroxynitrite-modified α2M as highly immunogenic producing high titre of specific antibodies in immunized rabbits. Cross-reactivity studies revealed the polyspecificity of the elicited antibodies. Direct binding ELISA and competitive inhibition studies confirmed the presence of circulating antibodies in the sera of RA patients having high specificity towards the peroxynitrite-modified α2M as compared to the native α2M. Sera from healthy (normal) human subjects showed lower binding with the native and modified protein. This study confirms that peroxynitrite induces structural modifications in α2M and makes it immunogenic. The presence of neo-antigenic determinants on modified α2M with enhanced binding for circulating autoantibodies in RA patients could offer new possibilities for diagnosis and etiopathology of the disease. Communicated by Ramaswamy H. Sarma.