Supramolecular polyplexes from Janus peptide nucleic acids (bm-PNA-G5): self-assembled bm-PNA G-quadruplex and its tetraduplex with DNA.

Nucleic acids (DNA and RNA) can form diverse secondary structures ranging from hairpins to duplex, triplex, G4-tetraplex and C4-i-motifs. Many of the DNA analogues designed as antisense oligonucleotides (ASO) are also adept at embracing such folded structures, although to different extents with altered stabilities. One such analogue, peptide nucleic acid (PNA), which is uncharged and achiral, forms hybrids with complementary DNA/RNA with greater stability and specificity than DNA:DNA/RNA hybrids. Like DNAs, these single-stranded PNAs can form PNA:DNA/RNA duplexes, PNA:DNA:PNA triplexes, PNA-G4 tetraplexes and PNA-C4-i-motifs. We have recently designed Janus-like bimodal PNAs endowed with two different nucleobase sequences on either side of a single aminoethylglycyl (aeg) PNA backbone and shown that these can simultaneously bind to two complementary DNA sequences from both faces of PNA. This leads to the formation of supramolecular polyplexes such as double duplexes, triple duplexes and triplexes of double duplexes with appropriate complementary DNA/RNA. Herein, we demonstrate that Janus/bimodal PNA with a poly G-sequence on the triazole side of the PNA backbone and mixed bases on the t-amide side, templates the initial formation of a (PNA-G5)4 tetraplex (triazole side), followed by the formation of a PNA:DNA duplex (t-amide side). Such a polyplex shows synergistic overall stabilisation compared to the isolated duplexes/quadruplex. The assembly of polyplexes with a shared backbone for duplexes and tetraplexes is programmable and may have potential applications in the self-assembly of nucleic acid nano- and origami structures. It is also shown that Janus PNAs enter the cells better than the standard aeg-PNA oligomers, and hence have implications for in vivo applications as well.

Molecular phylodynamics of fowl adenovirus serotype 11 and 8b from inclusion body hepatitis outbreaks.

Fowl adenovirus (FAdV) serotypes are involved in a variety of clinical manifestations in poultry and has resulted in substantial economic loss to the poultry farmers. Despite the endemicity of Inclusion body hepatitis (IBH) in South Asian countries, including India, its etiology is not well studied. In western India, the rural poultry flocks obtained from the vaccinated parents were experiencing disease outbreaks with substantial economic losses due to heavy outbreaks and mortality. Therefore, the study was conducted to decipher the molecular epidemiology of the FAdV from field outbreaks in western India. A total of 37 commercial broiler poultry flocks and 29 village poultry flocks of western India were visited during 2019 to 2021. Out of these, 19.14% flocks showed incidence of IBH during the age of 15 to 35 days. The mortality ranged from 3.3 percent to 55.28 percent. The samples were subjected for amplification of partial hexon gene covering loop 1 and loop 2. The results revealed 48.28% positivity by PCR. The sequence analysis identified 14 isolates as species D serotype 11 with 0.97 to 0.99% divergence and two as species E serotype 8b with 0.99% divergence. The FAdV-11 isolates showed amino acid substitutions D195N, T399A, N417S, and N496H. The amino acids I188 and N195 were conserved in FAdV-11. The molecular clock in Bayesian methods was used to determine most common ancestor. The isolates MH379249 and MH379248 were determined the most recent common ancestor for FAdV-11 and FAdV-8b isolates. The analysis suggested evolution of 10 FAdV-11 strains in 2012, and four FAdV-11 strains and two FAdV-8b strains in 2018.

Novel fluconazole-resistant zoonotic yeast isolated from mastitis.

A hospital-based cross-sectional study was conducted during 2018-2019 to decipher the prevalence of yeast mastitis. The results indicated a 19.68% prevalence of clinical mastitis in bovines. Among them, 5.51% of samples revealed yeasts constituting 1.09% overall prevalence. Candida albicans was recorded as a significant fungal agent involved in clinical bovine mastitis. We record the association of Kodamaea ohmeri in clinical bovine mastitis. On proteomic and molecular confirmation, K. ohmeri isolates were re-identified from phenotypically identified Candida isolates associated with bovine mastitis. After conventional identification, the yeast isolates were re-identified by MALDI-TOF MS-based proteomic approaches. The D1/D2 domains of 26S-rRNA gene and 5.8S-internal transcribed spacer (ITS) rDNA regions based molecular phylogenetic analysis identified the isolates as K. ohmeri. The isolates were resistant to fluconazole. This study reports the first systemic study of K. ohmeri isolates recovered from bovine clinical mastitis, utilizing conventional, automated, proteomic, and genomic approaches followed by antifungal susceptibility. The findings suggest K. ohmeri as a potent opportunistic emerging pathogen of veterinary and public health concern, need for accurate identification of fungal agents from mycotic mastitis, and use of validated antifungal susceptibility assay because of developing resistance to antimycotic agents. Our findings suggest judicious use of fluconazole and alternative antifungal agents may be considered in case of an outbreak.

The investigation on allelic, genotypic frequencies and gene expression study in coding region of tyrosinase gene in Deoni cattle breed of western India.

An investigation was carried out on Deoni animals of western India to study the allelic and genotypic frequencies in coding region of TYR gene as well as gene expression profile. The animals were grouped according to age, gender, strain and intensity of partial albinism (low, medium and high). The present study revealed that the genotypic frequency of TYR gene across different strains, gender, age group and level of partial albinism was found to be non-significant for both exon-I and exon-II. The AB genotype in Balankya (0.70) was observed highest genotypic frequency followed by Wanera (0.55) and Shewara (0.55) strains. The genotypic frequency of AB and BB genotypes were observed highest in male and female, respectively. In exon-I, genotype frequency of AA genotype was found highest (0.55) in low level of partial albinism. The allelic frequencies in Shewara strain, male and low level of partial albinism were 0.75, 0.63 and 0.73, respectively. However, in exon-II genotype frequency of AB and BB was observed highest (0.70) in Wanera and Balankya strains followed by AA genotype in Shewara (0.50). The highest genotypic frequency of AA (0.87) and BB (0.50) were in male and female, respectively. The genotype frequency of AB genotype was found highest in all level of partial albinism. The allelic frequency was highest (0.85 for B allele) in Wanera strain, male (0.80 for A allele) and high level (0.60 for A allele) of particle albinism. The highly significant (p = 0.002) expression of tyrosinase gene was observed in young animals as compared to adult animals. The TYR gene expression was significantly (p = 0.047) higher in animals with low intensity of partial albinism followed by in the animals with medium and high intensity. Therefore, it is inferred that the TYR gene expression in young animals were high and as compared to the old animals of Deoni cattle breed.

Molecular phylodynamics of infectious bursal disease viruses.

The present study was conducted to study the molecular phylodynamics of the Indian field IBDVs. A total of 13 organized commercial poultry farms and 29 village poultry flocks were recruited in the study. The broiler flocks showed 15.25-60.18% mortality, followed by 12.4% in improved native poultry varieties and 5% in indigenous birds. The 664 bp hypervariable VP2 gene fragment of Western and Central Indian vvIBDVs showed 97.14-98.79 and 94.49-96.69% identity to Pakistani and South Indian vvIBDVs, respectively. An isolate was 99.54% identical to the Ventri-Plus vaccine strain, while three IBDVs showed maximum identity with the Georgia strain. Out of 22, 19 strains showed typical vvIBDV amino acid signature, while three strains showed substitutions specific to classical IBDVs. Central Indian vvIBDVs showed conserved substitutions at N212D and E300A, which can be used as a regional marker. Phylogenetic genogrouping placed global IBDVs into seven genogroups based upon virulence and geographical distribution. Nineteen field vvIBDVs were placed in the G3 genogroup, and the other three were grouped with classical IBDVs in G1 genogroup. A nucleotide span from 584 to 1248 covering VP2 hypervariable fragment was found suitable for correct genogrouping of field IBDVs. The Bayesian evolutionary analysis showed tMRCA of the year 2009 for 8 Western Indian vvIBDVs with vvIBDV from Pakistan. Central Indian vvIBDVs were evolved in the year 1991 from BD-3 and PY12 strains of vvIBDVs from Bangladesh and Pondicherry, respectively. An isolate showed evolution in year 2010 from the Nigerian ABIC strain, while three classical strains showed tMRCA of the year 2009 with the Georgia strain as a recent common ancestor.

Triton X-114 Fractionated Subcellular Proteome of Leptospira interrogans Shows Selective Enrichment of Pathogenic and Outer Membrane Proteins in the Detergent Fraction.

The Triton X-114-based solubilization and temperature-dependent phase separation of proteins is used for subcellular fractionation where, aqueous, detergent, and pellet fractions represents cytoplasmic, outer membrane (OM), and inner membrane proteins, respectively. Mass spectrometry-based proteomic analysis of Triton X-114 fractions of proteomic analysis of Leptospira interrogans identified 2957 unique proteins distributed across the fractions. The results are compared with bioinformatics predictions on their subcellular localization and pathogenic nature. Analysis of the distribution of proteins across the Triton X-114 fractions with the predicted characteristics is performed based on "number" of unique type of proteins, and "quantity" which represents the amount of unique protein. The highest number of predicted outer membrane proteins (OMPs) and pathogenic proteins are found in aqueous and pellet fractions, whereas detergent fraction representing the OM has the highest quantity of OMPs and pathogenic proteins though lower in number than the aqueous and pellet fractions. This leaves the possibility of an upsurge in pathogenic proteins and OMPs on the OM under pathogenic conditions suggesting their potential use to combat leptospirosis. Further, the Triton X-114 subcellular fractions are more correlated to enrichment of pathogenic proteins predicted by MP3 software than predicted localization.

N-Chlorosuccinimide (NCS)-N,N-dimethylformamide (DMF), a reagent for the oxidation of benzylic alcohols to aldehydes and ketones.

The oxidation of benzylic alcohol to corresponding aldehyde and ketone using N-chlorosuccinimide (NCS)-N,N-dimethylformamide (DMF) has been described. This method gives easy access to the corresponding carbonyl compounds under metal-free conditions, without the use of corrosive reagent at ambient temperature in good yield.

First responder's care package on management of road traffic accident victims of Udupi: Study protocol.

AIM: To evaluate the effectiveness of 'first responder's care package' on knowledge and skill on the management of road traffic accident (RTA) victims. The outcomes relate to the quality of first responder's care by autorickshaw drivers. METHODS: Autorickshaw drivers (N = 1,040) will be assessed to identify the impediments and knowledge to provide the first responder's care to RTA victims following which, 150 autorickshaw drivers will be selected based on a cut-off knowledge score to train half of them using workshops. Drivers below 55 years and willing to participate will be recruited and drivers with serious health issues, homophobia and who cannot read English or Kannada will be excluded. Randomized controlled trial with repeated measures design will be adopted. Funding for the research is by the Indian Council of Medical Research and it is registered in the Clinical Trial Registry of India. DISCUSSION: Road traffic accidents are responsible for 85% of the total global mortality and 90% of the 'Disability Adjusted Life Years' in the developing countries amounting to an annual loss of $65 billion to $100 billion. India's rate of RTA deaths is high and postcrash care is not addressed efficiently by any agencies in India. Autorickshaw drivers could be ideal candidates for teaching the first responder's care package in India as they are a constant presence on the roads and reach all the main roads and small lanes of the country. IMPACT: The research will add to knowledge on quality of first responder's care provided to accident victims. If the intervention is found to be fruitful for the accident victims of the locality, it can be recommended to be implemented all over the state.

Effect of Methylprednisolone in Periarticular Infiltration for Primary Total Knee Arthroplasty on Pain and Rehabilitation.

BACKGROUND: Optimal pain management after total knee arthroplasty (TKA) is important to ensure timely rehabilitation and patient satisfaction. This study examines the efficacy of adding corticosteroid in periarticular infiltration cocktail with relation to postoperative pain management and rehabilitation in patients undergoing simultaneous bilateral TKA. METHODS: Fifty patients with symptomatic end-stage bilateral knee osteoarthritis undergoing bilateral TKA under the same anesthetic were recruited. More painful knee was operated first, and the study solution containing ropivacaine, clonidine, epinephrine, and ketorolac with methylprednisolone was infiltrated in one knee and an identical mixture but without methylprednisolone was infiltrated in the second knee. Outcome measures included comparison of visual analogue scale on movement of each knee and range of motion achieved during the first three days after surgery. RESULTS: Differences in visual analogue scale score and range of motion at day one and three between the two groups of knees were significant (P < .05). Postoperative inflammation and the ability to straight leg raise showed better trends in the knees receiving prednisolone although this did not reach statistical significance. CONCLUSION: Addition of methylprednisolone to periarticular infiltration cocktail for patients undergoing TKA has significant influence on reduction of pain in the early postoperative period and patients are able to regain knee flexion more quickly.

Randomized Prospective Comparative Study of Adductor Canal Block vs Periarticular Infiltration on Early Functional Outcome After Unilateral Total Knee Arthroplasty.

BACKGROUND: Total knee arthroplasty (TKA) is associated with significant postoperative pain. Effective pain relief is essential for early postoperative rehabilitation. Periarticular infiltration (PAI) and adductor canal block (ACB) have become popular modes of pain management after TKA. Our aim is to compare their efficacy and impact on early functional outcome in patients undergoing TKA. METHODS: A single-blinded randomized controlled trial, 100 patients undergoing unilateral primary TKA for symptomatic osteoarthritis were allocated to either of the 2 groups (50 in each arm): postoperative ultrasound-guided single shot of ACB (group A) or intraoperative PAI (group B). All patients underwent TKA without patella resurfacing under spinal anesthesia. Preoperative workup, surgical technique, and postoperative management were standardized for all the patients. Patients were assessed for pain using visual analogue scale at 6, 12, and 24 hours after surgery; hemoglobin level preoperatively and postoperatively on day 1 to calculate blood loss; hospital stay; tourniquet time; operative time; and postoperative complications by an independent observer blinded to the group allocation. RESULTS: Patients were matched for age, gender, American Society of Anesthesiologists grade, and deformity. Visual analogue scale (scale 0-10) between PAI and ACB at 6, 12, and 24 hours were significantly different (P < .05) with higher scores seen in patients with ACB at all time points. Tourniquet time and operative time were significantly longer in the PAI than ACB. No significant difference in hospital stay was observed. No complications occurred during the study. CONCLUSION: PAI achieves better pain control as compared to ACB in patients undergoing unilateral TKA.

Limonene inhibits Candida albicans growth by inducing apoptosis.

Anti-Candida potential of limonene was evaluated against planktonic growth, biofilm (adhesion, development and maturation) and morphogenesis of Candida albicans in this study. Limonene is a major constituent of citrus oil and most frequently used terpene in food and beverage industry due to its pleasant fragrance, nontoxic, and is generally recognized as safe (GRAS) flavoring agent as well as treatment option in many gastrointestinal diseases.Limonene exhibited excellent anti-Candida activity and was equally effective against planktonic growth of C. albicans isolates differentially susceptible to FLC (N = 35). Limonene inhibited morphogenesis significantly at low concentration. However, it showed stage dependent activity against biofilm formation, that is, it was more effective against adhesion followed by development and maturation. Limonene also exhibited excellent synergy with FLC against planktonic and biofilm growth. SWATH-MS analysis led to identification of limonene responsive proteins that provided molecular insight of its anti-Candida activity. Proteomic analysis revealed upregulation of proteins involved in cell wall glucan synthesis (Kre6); oxidative stress (Rhr2, Adh7 and Ebp1); DNA damage stress (Mbf1 and Npl3); nucleolar stress (Rpl11, Rpl7, Rpl29, Rpl15) and down regulation of cytoskeleton organization (Crn1, Pin3, Cct8, Rbl2), and so forth, in response to limonene. Limonene mediated down regulation of Tps3 indicates activation of caspase (CaMca1) and induction of apoptosis in C. albicans. These results suggest that limonene inhibits C. albicans growth by cell wall/membrane damage induced oxidative stress that leads to DNA damage resulting into modulation of cell cycle and induction of apoptosis through nucleolar stress and metacaspase dependent pathway.

Synthesis and in vitro evaluations of 6-(hetero)-aryl-imidazo[1,2-b]pyridazine-3-sulfonamide's as an inhibitor of TNF-α production.

Tumor necrosis factor-α is an important pro-inflammatory cytokine having a key role in hosts defensive process of immune systems and its over expression led to a diverse range of inflammatory diseases such as Rheumatoid arthritis, Cronh's disease, psoriasis, etc. This paper describes our medicinal chemistry efforts on imidazo[1,2-b]pyridazine scaffold: design, synthesis and biological evaluation. By the introducing sulfonamide functionality at 3 positions and substituting 6 positions with (hetero)-aryl groups', a small library of compounds was prepared. All synthesized compounds were screened for lipopolysaccharide (LPS) mediated TNF-α production inhibitory activity. Biological data revealed that the majority of the compounds of this series showed moderate to potent TNF-α production inhibitory activity. Compound 5u and 5v are the most potent compounds from the series with activity of IC50 = 0.5 µM and 0.3 µM respectively. A short SAR demonstrates that 3-sulfonyl-4-arylpiperidine-4-carbonitrile moiety on imidazo[1,2-b]pyridazine showed better activity compared to the 3-(4-aryllpiperazin-1-yl) sulfonyl) in hPBMC assay. The molecular modeling studies revealed that the potent TNF-α production inhibitory activity 5v due to the extra stability of complex because of an extra pi-pi (π-π) stacking, hydrogen-bonding interactions.

Synthesis and biological evaluation of imidazo[1,2-[Formula: see text]]pyridazines as inhibitors of TNF-[Formula: see text] production.

Tumor necrosis factor-alpha (TNF-[Formula: see text] is an important pro-inflammatory cytokine responsible for a diverse range of inflammatory diseases including rheumatoid arthritis. In the present manuscript, our medicinal chemistry efforts on the design, synthesis and TNF-[Formula: see text] evaluation of a series of 3, 6-disubstituted imidazo[1,2-b]pyridazine is described. The best compounds were 3-pyridyl and (4-(methylsulfonyl)phenyl) analogs 8q and 8w, showing inhibition of TNF-[Formula: see text] production with IC[Formula: see text]values of 0.9 and 0.4 [Formula: see text]M, respectively. The identified leads have potential for further development for treatment of inflammatory diseases.

Proteomic Insight Reveals Elevated Levels of Albumin in Circulating Immune Complexes in Diabetic Plasma.

A Hyperglycemic condition in diabetes promotes formation of advanced glycation end products, which are known to elicit immune response and form complexes with immunoglobulins called circulating immune complexes. To investigate the involvement of advanced glycation end product (AGE)-modified proteins in the elicitation of an immune response, circulating immune complexes were isolated and proteins associated were identified and characterized. Label-free-based mass spectrometric analysis of circulating immune complexes in clinical plasma of prediabetic, newly diagnosed diabetes, and diabetic microalbuminurea revealed elevated levels of serum albumin in the circulating immune complexes, which were also observed to be AGE modified. Further, to examine the role of glycation, circulating immune complexeswere analyzed in the streptozotocin-induced diabetic mice treated with or without aminoguanidine, a prototype glycation inhibitor. Mass spectrometric analysis of circulating immune complexes showed elevated levels of serum albumin in plasma from diabetic mice over that of control animals. Aminoguanidine-treated diabetic mice displayed decreased AGE modification of plasma albumin, accompanied by a reduced level of albumin in the circulating immune complexes. In addition, elevated levels of proinflammatory cytokines such as IL-1b, IL-2, and TNF-alpha were observed in diabetes, which were reduced with aminoguanidine treatment, suggesting the involvement of glycation in the immune response.

Placental Proteomics Provides Insights into Pathophysiology of Pre-Eclampsia and Predicts Possible Markers in Plasma.

Pre-eclampsia is a hypertensive disorder characterized by the new onset of hypertension >140/90 mmHg and proteinuria after the 20th week of gestation. The disorder is multifactorial and originates with abnormal placentation. Comparison of the placental proteome of normotensive (n = 25) and pre-eclamptic (n = 25) patients by gel-free proteomic techniques identified a total of 2145 proteins in the placenta of which 180 were differentially expressed (>1.3 fold, p < 0.05). Gene ontology enrichment analysis of biological process suggested that the differentially expressed proteins belonged to various physiological processes such as angiogenesis, apoptosis, oxidative stress, hypoxia, and placental development, which are implicated in the pathophysiology of pre-eclampsia. Some of the differentially expressed proteins were monitored in the plasma by multiple reaction monitoring analysis, which showed an increase in apolipoproteins A-I and A-II in gestational weeks 26-30 (2-fold, p < 0.01), while haptoglobin and hemopexin decreased in gestational weeks 26-30 and week 40/at delivery (1.8 fold, p < 0.01) in pre-eclamptic patients. This study provides a proteomic insight into the pathophysiology of pre-eclampsia. Identified candidate proteins can be evaluated further for the development of potential biomarkers associated with pre-eclampsia pathogenesis.

Abundance matters: role of albumin in diabetes, a proteomics perspective.

INTRODUCTION: Human serum albumin (HSA) is a multifaceted protein with vital physiological functions. It is the most abundant plasma protein with inherent capability to bind to diverse ligands, and thus susceptible to various post-translational modifications (PTMs) which alter its structure and functions. One such PTM is glycation, a non-enzymatic reaction between reducing sugar and protein leading to formation of heterogeneous advanced glycation end products (AGEs). Glycated albumin (GA) concentration increases significantly in diabetes and is implicated in development of secondary complications. Areas covered: In this review, we discuss in depth, formation of GA and its consequences, approaches used for characterization and quantification of GA, milestones in GA proteomics, clinical relevance of GA as a biomarker, significance of maintaining abundant levels of albumin and future perspectives. Expert commentary: Elevated GA levels are associated with development of insulin resistance as well as secondary complications, in healthy and diabetic individuals respectively. Mass spectrometry (MS) based approaches aid in precise characterization and quantification of GA including early and advanced glycated peptides, which can be useful in prediction of the disease status. Thus GA has evolved to be one of the best candidates in the pursuit of diagnostic markers for prediction of prediabetes and diabetic complications.

Tubulointerstitial nephritis antigen-like 1 protein is downregulated in the placenta of pre-eclamptic women.

BACKGROUND: Tubulointerstitial nephritis antigen-like 1 protein (TINAGL1), is a matricellular protein, known to play role in cell adhesion and cell receptor interaction. Research related to TINAGL1 is limited to cell culture and animal models. Demonstration of TINAGL1 as a positive regulator of angiogenesis and its expression in the decidua of postimplantation mouse uterus, prompted us to validate its expression in human placenta during impaired angiogenesis in pre-eclamptic condition. METHODS: Placental tissue from normotensive (n = 25) and pre-eclamptic (n = 25) pregnancies were used to study the differentially expressed proteins by two-dimensional gel electrophoresis and TINAGL1 protein was validated with Western blotting. RESULTS: A total of 55 protein spots were differentially expressed (fold change >1.5, p < 0.05), of which 27 were upregulated and 28 were downregulated in the pre-eclamptic placenta. TINAGL1 was found to be downregulated in pre-eclamptic compared to normotensive pregnant women. CONCLUSION: This is the first study reporting TINAGL1 to be present in human placenta and differentially expressed in pre-eclamptic condition. The functional role of TINAGL1 in association to human pregnancy needs to be explored further.

Methylsulfonyl benzothiazoles (MSBT) derivatives: Search for new potential antimicrobial and anticancer agents.

A series of novel 4 and 5-substituted methylsulfonyl benzothiazole (MSBT) compounds having amide, alkoxy, sulfonamide, nitro and amine functionality were synthesized from sequential reactions on 5-ethoxy-2-(methylsulfonyl)benzo[d]thiazole such as nitration, reduction, sulfonation, dealkylation, etc. All synthesized compounds were screened against antimicrobial and selected screened for anticancer activity. Antimicrobial activities studies reveled that among all compounds screened, out of MSBT-07, MSBT-11, MSBT-12, MSBT-14, MSBT-19, and MSBT-27 were found to have promising antimicrobial activity at MIC range of 4-50µg/ml against selected bacterial as well as fungal species. Compounds having good antimicrobial activity were screened for cervical cancer (HeLA cell lines). Of these MSBT-07 and MSBT-12 significantly reduced the cell growth. Consequently their calculated GI50 values were found to be 0.1 or <0.1µM.

Development of Diagnostic Fragment Ion Library for Glycated Peptides of Human Serum Albumin: Targeted Quantification in Prediabetic, Diabetic, and Microalbuminuria Plasma by Parallel Reaction Monitoring, SWATH, and MSE.

Human serum albumin is one of the most abundant plasma proteins that readily undergoes glycation, thus glycated albumin has been suggested as an additional marker for monitoring glycemic status. Hitherto, only Amadori-modified peptides of albumin were quantified. In this study, we report the construction of fragment ion library for Amadori-modified lysine (AML), N(ε)-(carboxymethyl)lysine (CML)-, and N(ε)-(carboxyethyl)lysine (CEL)-modified peptides of the corresponding synthetically modified albumin using high resolution accurate mass spectrometry (HR/AM). The glycated peptides were manually inspected and validated for their modification. Further, the fragment ion library was used for quantification of glycated peptides of albumin in the context of diabetes. Targeted Sequential Window Acquisition of all THeoretical Mass Spectra (SWATH) analysis in pooled plasma samples of control, prediabetes, diabetes, and microalbuminuria, has led to identification and quantification of 13 glycated peptides comprised of four AML, seven CML, and two CEL modifications, representing nine lysine sites of albumin. Five lysine sites namely K549, K438, K490, K88, and K375, were observed to be highly sensitive for glycation modification as their respective m/z showed maximum fold change and had both AML and CML modifications. Thus, peptides involving these lysine sites could be potential novel markers to assess the degree of glycation in diabetes.

Targeted quantification of N-1-(carboxymethyl) valine and N-1-(carboxyethyl) valine peptides of ß-hemoglobin for better diagnostics in diabetes.

BACKGROUND: N-1-(Deoxyfructosyl) valine (DFV) ß-hemoglobin (ß-Hb), commonly referred as HbA1c, is widely used diagnostic marker in diabetes, believed to provide glycemic status of preceding 90-120 days. However, the turnover of hemoglobin is about 120 days, the DFV-ß-Hb, an early and reversible glycation product eventually may undergo irreversible advanced glycation modifications such as carboxymethylation or carboxyethylation. Hence quantification of N-1-(carboxymethyl) valine (CMV) and N-1-(carboxyethyl) valine (CEV) peptides of ß-Hb would be useful in assessing actual glycemic status. RESULTS: Fragment ion library for synthetically glycated peptides of hemoglobin was generated by using high resolution-accurate mass spectrometry (HR/AM). Using parallel reaction monitoring, deoxyfructosylated, carboxymethylated and carboxyethylated peptides of hemoglobin were quantified in clinical samples from healthy control, pre-diabetes, diabetes and poorly controlled diabetes. For the first time, we report N-1-ß-valine undergoes carboxyethylation and mass spectrometric quantification of CMV and CEV peptides of ß-hemoglobin. Carboxymethylation was found to be the most abundant modification of N-1-ß-valine. Both CMV-ß-Hb and CEV-ß-Hb peptides showed better correlation with severity of diabetes in terms of fasting glucose, postprandial glucose and microalbuminuria. CONCLUSIONS: This study reports carboxymethylation as a predominant modification of N-1-ß-valine of Hb, and quantification of CMV-ß-Hb and CEV-ß-Hb could be useful parameter for assessing the severity of diabetes.