Nonenzymatic glycosylation of isolated human immunoglobulin-G by D-ribose.

Glycation is vital in terms of its damaging effect on macromolecules resulting in the formation of end products, which are highly reactive and cross-linked irreversible structures, known as advanced glycation end products (AGEs). The continuous accumulation of AGEs is associated with severe diabetes and its associated ailments. Saccharides with their reducing ends can glycate amino acid side chains of proteins, among them glucose is well-known for its potent glycating capability. However, other reducing sugars can be more reactive glycating agents than glucose. The D-ribose is a pentose sugar-containing an active aldehyde group in its open form and is responsible for affecting the biological processes of the cellular system. D-ribose, a key component of many biological molecules, is more reactive than most reducing sugars. Protein glycation by reducing monosaccharides such as D-ribose promotes the accelerated formation of AGEs that could lead to cellular impairments and dysfunctions. Also, under a physiological cellular state, the bioavailability rate of D-ribose is much higher than that of glucose in diabetes, which makes this species much more active in protein glycation as compared with D-glucose. Due to the abnormal level of D-ribose in the biological system, the glycation of proteins with D-ribose needs to be analyzed and addressed carefully. In the present study, human immunoglobulin G (IgG) was isolated and purified via affinity column chromatography. D-ribose at 10 and 100 mM concentrations was used as glycating agent, for 1-12 days of incubation at 37°C. The postglycation changes in IgG molecule were characterized by UV-visible and fluorescence spectroscopy, nitroblue tetrazolium assay, and various other physicochemical analyses for the confirmation of D-ribose mediated IgG glycation.

Glyoxal induced glycative insult suffered by immunoglobulin G and fibrinogen proteins: A comparative physicochemical characterization to reveal structural perturbations.

Glycation of proteins results in structural alteration, functional deprivation, and generation of advanced glycation end products (AGEs). Reactive oxygen species (ROS) that are generated during in vivo autoxidation of glucose induces glycoxidation of intermediate glycation-adducts, which in turn give rise to aldehyde and/or ketone groups containing dicarbonyls or reactive carbonyl species (RCS). RCS further reacts non-enzymatically and starts the glycation-oxidation vicious cycle, thus exacerbating oxidative, carbonyl, and glycative stress in the physiological system. Glyoxal (GO), a reactive dicarbonyl that generates during glycoxidation and lipid peroxidation, contributes to glycation. This in vitro physicochemical characterization study focuses on GO-induced glycoxidative damage suffered by immunoglobulin G (IgG) and fibrinogen proteins. The structural alterations were analyzed by UV-vis, fluorescence, circular dichroism, and Fourier transform infrared (FT-IR) spectroscopy. Ketoamines, protein carbonyls, hydroxymethylfurfural (HMF), free lysine, free arginine, carboxymethyllysine (CML), and protein aggregation were also quantified. Structural perturbations, increased concentration of ketoamines, protein carbonyls, HMF, and malondialdehyde (MDA) were reported in glycated proteins. The experiment results also validate increased oxidative stress and AGEs formation i.e. IgG-AGEs and Fib-AGEs. Thus, we can conclude that AGEs formation during GO-mediated glycation of IgG and fibrinogen could hamper normal physiology and might play a significant role in the pathogenesis of diabetes-associated secondary complications.

Profile of Mucormycosis Cases from a Network of Hospitals in North India Amidst COVID-19 Pandemic

Incidence of mucormycosis suddenly surged in India after the second wave of COVID-19. This is a crippling disease and needs to be studied in detail to understand the disease, its course, and the outcomes. Between 1st March and 15th July 2021, our network of hospitals in North India received a total of 155 cases of COVID-associated mucormycosis cases as all of them reported affliction by COVID-19 earlier or concurrent. Their records were retrieved from the Electronic Health Records system of the hospitals and their demographics, clinical features, treatments, and outcomes were studied. More than 80% (125 cases) had proven disease and the remaining 30 were categorized as possible mucormycosis as per the EORTC criteria. More than two-thirds (69.0%) of the cases were males and the mean age was 53 years for either sex. Nearly two-thirds (64.5%) had symptoms of nose and jaws and 42.6% had eye involvement. Some had multiple symptoms. As many as 78.7% had diabetes and 91.6% gave history of use of steroids during COVID-19 treatment. The primary surgery was functional endoscopic sinus surgery (FESS) (83.9%). Overall mortality was 16.8%, which is one-and-a-half times the mortality in hospitalized COVID-19 patients in the corresponding population. Occurrence of mucormycosis was associated with diabetes and use of steroids, but mortality was not associated with either of them. Cases undergoing surgery and on antifungal had steeply lower mortality (11.9% vs. 50.0%, P < 0.001) than those who were exclusively on antifungal drugs. Treatment by different drugs did not make much of a difference in mortality.

Hydrogen sulfide-induced GAPDH sulfhydration disrupts the CCAR2-SIRT1 interaction to initiate autophagy.

The deacetylase SIRT1 (sirtuin 1) has emerged as a major regulator of nucleocytoplasmic distribution of macroautophagy/autophagy marker MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3). Activation of SIRT1 leads to the deacetylation of LC3 and its translocation from the nucleus into the cytoplasm leading to an increase in the autophagy flux. Notably, hydrogen sulfide (H2S) is a cytoprotective gasotransmitter known to activate SIRT1 and autophagy; however, the underlying mechanism for both remains unknown. Herein, we demonstrate that H2S sulfhydrates the active site cysteine of the glycolytic enzyme GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Sulfhydration of GAPDH leads to its redistribution into the nucleus. Importantly, nuclear localization of GAPDH is critical for H2S-mediated activation of autophagy as H2S does not induce autophagy in cells with GAPDH ablation or cells overexpressing a GAPDH mutant lacking the active site cysteine. Importantly, we observed that nuclear GAPDH interacts with CCAR2/DBC1 (cell cycle activator a nd apoptosis regulator 2) inside the nucleus. CCAR2 interacts with the deacetylase SIRT1 to inhibit its activity. Interaction of GAPDH with CCAR2 disrupts the inhibitory effect of CCAR2 on SIRT1. Activated SIRT1 then deacetylates MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) to induce its translocation into the cytoplasm and activate autophagy. Additionally, we demonstrate this pathway's physiological role in autophagy-mediated trafficking of Mycobacterium tuberculosis into lysosomes to restrict intracellular mycobacteria growth. We think that the pathway described here could be involved in H2S-mediated clearance of intracellular pathogens and other health benefits.Abbreviations: ATG5: autophagy related 5; ATG7: autophagy related 7; BECN1: beclin 1, autophagy related; CCAR2/DBC1: cell cycle activator and apoptosis regulator 2; CFU: colony-forming units; DLG4/PSD95: discs large MAGUK scaffold protein 4; EX-527: 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H2S: hydrogen sulfide; HEK: human embryonic kidney cells; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; Mtb: Mycobacterium tuberculosis; MTOR: mechanistic target of rapamycin kinase; MOI: multiplicity of infection; NO: nitric oxide; PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase; PLA: proximity ligation assay; PRKAA: protein kinase, AMP-activated, alpha catalytic subunit; SIAH1: siah E3 ubiquitin protein ligase 1A; SIRT1: sirtuin 1; TB: tuberculosis; TP53INP2/DOR: transformation related protein 53 inducible nuclear protein 2; TRP53/TP53: transformation related protein 53.

Quantification of the Metabolic Heterogeneity in Mycobacterial Cells Through the Measurement of the NADH/NAD+ Ratio Using a Genetically Encoded Sensor.

NADH/NAD+ levels are an indicator of the bacterial metabolic state. NAD(H) levels are maintained through coordination of pathways involved in NAD(H) synthesis and its catabolic utilization. Conventional methods of estimating NADH/NAD+ require cell disruption and suffer from low specificity and sensitivity and are inadequate in providing spatiotemporal resolution. Recently, genetically encoded biosensors of the NADH/NAD+ ratio have been developed. One of these sensors, Peredox-mCherry, was adapted for the measurement of cellular levels of NADH/NAD+ in the slow-growing Mycobacterium tuberculosis (Mtb) and the fast-growing Mycobacterium smegmatis. Importantly, the use of the engineered reporter strains of Mtb demonstrated a significantly higher heterogeneity among the bacteria residing in macrophages compared to the bacteria grown in synthetic media. Previous estimations of NADH/NAD+ levels have missed this important aspect of the biology of Mtb, which may contribute to the variable response of intracellular Mtb to different antimycobacterial agents. In this chapter, we describe the details of a method used in the generation of reporter strains for the measurement of the NADH/NAD+ ratio in mycobacteria. Importantly, once the reporter strains are created, they can be exploited with fluorescence spectroscopy, FACS, and confocal microscopy to access the dynamic changes in the NADH/NAD+ levels in intact individual bacterial cells. Although we have only described the method for the creation of reporter strains capable of measuring NADH/NAD+ in mycobacteria in this chapter, a similar method can be used for generating reporter strains for other bacterial species, as well. We believe that such reporter stains can be used in novel screens for small molecules that could alter the metabolism of bacterial cells and thus aid in the development of new class of therapeutic agents.

Bioenergetics of Mycobacterium: An Emerging Landscape for Drug Discovery.

Mycobacterium tuberculosis (Mtb) exhibits remarkable metabolic flexibility that enables it to survive a plethora of host environments during its life cycle. With the advent of bedaquiline for treatment of multidrug-resistant tuberculosis, oxidative phosphorylation has been validated as an important target and a vulnerable component of mycobacterial metabolism. Exploiting the dependence of Mtb on oxidative phosphorylation for energy production, several components of this pathway have been targeted for the development of new antimycobacterial agents. This includes targeting NADH dehydrogenase by phenothiazine derivatives, menaquinone biosynthesis by DG70 and other compounds, terminal oxidase by imidazopyridine amides and ATP synthase by diarylquinolines. Importantly, oxidative phosphorylation also plays a critical role in the survival of persisters. Thus, inhibitors of oxidative phosphorylation can synergize with frontline TB drugs to shorten the course of treatment. In this review, we discuss the oxidative phosphorylation pathway and development of its inhibitors in detail.