Identification of potential serum biomarkers associated with HbA1c levels in Indian type 2 diabetic subjects using NMR-based metabolomics.

BACKGROUND: The prevalence of type 2 diabetes mellitus (T2DM), a progressive metabolic disorder characterized by chronic hyperglycemia and the development of insulin resistance, has increased globally, with worrying statistics coming from children, adolescents, and young adults from developing countries like India. Here, we investigated unique circulating metabolic signatures associated with prediabetes and T2DM in an Indian cohort using NMR-based metabolomics. MATERIALS AND METHODS: The study subjects included healthy volunteers (N = 101), prediabetic subjects (N = 75), and T2DM patients (N = 108). Serum metabolic profiling was performed using 1H NMR spectroscopy and major perturbed metabolites were identified by multivariate analysis and receiver operating characteristic (ROC) modules. RESULTS: Of the 36 aqueous abundant metabolites, 24 showed a statistically significant difference between healthy volunteers, prediabetics, and established T2DM subjects. On performing multivariate ROC curve analysis with 5 commonly dysregulated metabolites (namely, glucose, pyroglutamate, o-phosphocholine, serine, and methionine) in prediabetes and T2DM, AUC values obtained were 0.96 (95 % confidence interval (CI) = 0.93, 0.98) for T2DM; and 0.88 (95 % CI = 0.81, 0.93) for prediabetic subjects, respectively. CONCLUSION: We propose that the identified metabolite panel can be used in the future as a biomarker for clinical diagnosis, patient surveillance, and for predicting individuals at risk for developing diabetes.

Proteomic Analysis of the Mycobacterium tuberculosis Outer Membrane for Potential Implications in Uptake of Small Molecules.

Increased resistance to current antimycobacterial agents and a potential bias toward relatively hydrophobic chemical entities highlight an urgent need to understand how current anti-TB drugs enter the tubercle bacilli. While inner membrane proteins are well-studied, how small molecules cross the impenetrable outer membrane remains unknown. Here, we employed mass spectrometry-based proteomics to show that octyl-ß-d-glucopyranoside selectively extracts the outer membrane proteins of Mycobacterium tuberculosis. Differentially expressed proteins between nutrient-replete and nutrient-depleted conditions were enriched to identify proteins involved in nutrient uptake. We demonstrate cell surface localization of seven new proteins using immunofluorescence and show that overexpression of the proteins LpqY and ProX leads to hypersensitivity toward streptomycin, while overexpression of SubI, SpmT, and Rv2041 exhibited higher membrane permeability, assessed through an EtBr accumulation assay. Further, proton NMR metabolomics suggests the role of six outer membrane proteins in glycerol uptake. This study identifies several outer membrane proteins that are involved in the permeation of small hydrophilic molecules and are potential targets for enhancing the uptake and efficacy of anti-TB drugs.

Mapping metabolic perturbations induced by glutathione activatable synthetic ion channels in human breast cancer cells.

Ion channels and transporters play key roles in various biological processes, including cell proliferation and programmed cell death. Recently, we reported that 2,4-dinitrobenzene-sulfonyl-protected N1,N3-dihexy-2-hydroxyisophthalamide (1) forms ion channels upon activation by glutathione (GSH) and results in the induction of apoptosis by depleting the intracellular GSH reservoir in cancer cells. However, the detailed molecular events leading to the induction of apoptosis by these synthetic transport systems in cancer cells still need to be uncovered. Along these lines, we investigated the alterations in cellular metabolites and the associated metabolic pathways by performing untargeted global metabolic profiling of breast cancer cells - MCF-7 - using 1H NMR-based metabolomics. The evaluation of spectral profiles from MCF-7 cells exposed to 1 and their comparison with those corresponding to untreated (control) cells identified 14 significantly perturbed signature metabolites. These metabolites belonged mostly to antioxidant defence, energy metabolism, amino acid biosynthesis, and lipid metabolism pathways and included GSH, o-phosphocholine, malate, and aspartate, to name a few. These results would help us gain deeper insights into the molecular mechanism underlying 1-mediated cytotoxicity of MCF-7 cells and eventually help identify potential novel therapeutic targets for more effective cancer management.

Water-Controlled Keto-Enol Tautomerization of a Prebiotic Nucleobase.

Barbituric acid is believed to be a proto-RNA nucleobase that was used for biological information transfer on prebiotic earth before DNA and RNA in their present forms evolved. Nucleobases have various tautomeric forms and the relative stability of these forms is critical to their biological function. It has been shown that barbituric acid has a tri-keto form in the gas phase and an enol form in the solid state. However, its dominant tautomeric form in aqueous medium that is most relevant for biology has been investigated only to a limited extent and the findings are inconclusive. We have used multiple approaches, namely, molecular dynamics, quantum chemistry, NMR, and IR spectroscopy to determine the most stable tautomer of barbituric acid in solution. We find a delicate balance in the stability of the two tautomers, tri-keto and enol, which is tipped toward the enol as the extent of solvation by water increases.

Inherent conformational plasticity in dsRBDs enables interaction with topologically distinct RNAs.

Many double-stranded RNA-binding domains (dsRBDs) interact with topologically distinct dsRNAs in biological pathways pivotal to viral replication, cancer causation, neurodegeneration, and so on. We hypothesized that the adaptability of dsRBDs is essential to target different dsRNA substrates. A model dsRBD and a few dsRNAs, slightly different in shape from each other, were used to test the systematic shape dependence of RNA on the dsRBD-binding using nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. NMR-based titrations showed a distinct binding pattern for the dsRBD with the topologically distinct dsRNAs. The line broadening upon RNA binding was observed to cluster in the residues lying in close proximity, thereby suggesting an RNA-induced conformational exchange in the dsRBD. Further, while the intrinsic microsecond dynamics observed in the apo-dsRBD were found to quench upon binding with the dsRNA, the microsecond dynamics got induced at residues spatially proximal to quench sites upon binding with the dsRNA. This apparent relay of conformational exchange suggests the significance of intrinsic dynamics to help adapt the dsRBD to target various dsRNA-shapes. The conformational pool visualized in MD simulations for the apo-dsRBD reported here has also been observed to sample the conformations seen previously for various dsRBDs in apo- and in dsRNA-bound state structures, further suggesting the conformational adaptability of the dsRBDs. These investigations provide a dynamic basis for the substrate promiscuity for dsRBD proteins.

Construction of Entropically Favored Supramolecular Metal-Ligand Trimeric Assemblies Supported by Flexible Pyridylaminophosphorus(V) Scaffolds.

The self-assembly reactions of tetratopic metal acceptors with the flexible bidentate ligands are known to yield self-assembled molecular squares of the type [M4L8], triangles of composition [M3L6], or a mixture of these two. In this work, we demonstrate the preferential formation of a trimeric cage assembly of the formula [Pd3(L1)6·(BF4)6] (1a) over the tetrameric cage [Pd4(L1)8·(BF4)8] (1b) by employing a flexible dipodal phosphoramide ligand, [PhPO(NH(3-Py))2] (L1; 3-Py = 3-aminopyridine), in a reaction with [Pd(CH3CN)4·(BF4)2]. The entropically favored trimeric self-assembly of 1a is the predominant species in the solution [dimethyl sulfoxide (DMSO)-d6] at room temperature. In fact, at higher temperatures, 1a was found to be the only product, as observed from the disappearance of the peak due to 1b in the 31P NMR spectrum. However, in a 1:1 mixture of acetonitrile (MeCN)-d3 and DMSO-d6, the tetrameric species 1b is the preferred species, as revealed by the 31P NMR and electrospray ionization mass spectral analyses. The structure of the molecular trimer 1a has been established in the solid state by using single-crystal X-ray diffraction analysis. Interestingly, treatment of an another flexible ligand, [MePO(NH(3-Py))2] (L2), with the same Pd(II) acceptor resulted in exclusive formation of the trimeric cage [Pd3(L2)6·(BF4)6] (2).

A Pyridyl-Linked Benzimidazolyl Tautomer Facilitates Prodigious H+/Cl- Symport through a Cooperative Protonation and Chloride Ion Recognition.

We report two pyridyl-linked benzimidazolyl hydrazones as HCl cotransporters that are 5 and 2 times superior to prodigiosin, a natural product whose transport efficiency has never been routed by synthetic molecules. These hydrazones provide a suitable HCl binding site through a cooperative protonation and chloride ion recognition. HCl transport by the most active compound induces lysosome deacidification. Viability assays confirmed that the compounds induce cytotoxicity toward human breast cancer MCF-7 cells but are relatively nontoxic toward noncancerous HEK293T cells.

Myricetin protects pancreatic ß-cells from human islet amyloid polypeptide (hIAPP) induced cytotoxicity and restores islet function.

The aberrant misfolding and self-assembly of human islet amyloid polypeptide (hIAPP)-a hormone that is co-secreted with insulin from pancreatic ß-cells-into toxic oligomers, protofibrils and fibrils has been observed in type 2 diabetes mellitus (T2DM). The formation of these insoluble aggregates has been linked with the death and dysfunction of ß-cells. Therefore, hIAPP aggregation has been identified as a therapeutic target for T2DM management. Several natural products are now being investigated for their potential to inhibit hIAPP aggregation and/or disaggregate preformed aggregates. In this study, we attempt to identify the anti-amyloidogenic potential of Myricetin (MYR)- a polyphenolic flavanoid, commonly found in fruits (like Syzygium cumini). Our results from biophysical studies indicated that MYR supplementation inhibits hIAPP aggregation and disaggregates preformed fibrils into non-toxic species. This protection was accompanied by inhibition of oxidative stress, reduction in lipid peroxidation and the associated membrane damage and restoration of mitochondrial membrane potential in INS-1E cells. MYR supplementation also reversed the loss of functionality in hIAPP exposed pancreatic islets via restoration of glucose-stimulated insulin secretion. Molecular dynamics simulation studies suggested that MYR molecules interact with the hIAPP pentameric fibril model at the amyloidogenic core region and thus prevents aggregation and distort the fibrils.

Palladium-Catalyzed Insertion of Ethylene and 1,1-Disubstituted Difunctional Olefins: An Experimental and Computational Study.

Insertion or coordination copolymerization of ethylene with di-substituted olefins is challenging and the choice of di-substituted mono-functional olefin versus di-substituted di-functional olefin (DDO) appears to be decisive. Here we show that DDO-inserted species are amenable to ethylene insertion and polymerization. DDOs such as 2-acetamidoacrylic acid (AAA), methyl 2-acetamidoacrylate (MAAA), and ethyl 2-cyanoacrylate (ECA) were treated with palladium complex [{P∧O}PdMe(L)] (P∧O=κ2 -P,O-Ar2 PC6 H4 SO2 O with Ar=2-MeOC6 H4 ; L=C2 H6 OS) and the existence of respective insertion intermediates in moderate yield (up to 37 %) was established. These intermediates were exposed to ethylene and corresponding ethylene-inserted products were isolated and characterized. A careful comparison with three model compounds confirmed ethylene insertion and polymerization. Thus, the combined experimental and computational investigations show that DDO-inserted species can undergo ethylene insertion and polymerization.

Cold storage reveals distinct metabolic perturbations in processing and non-processing cultivars of potato (Solanum tuberosum L.).

Cold-induced sweetening (CIS) causes considerable losses to the potato processing industry wherein the selection of potato genotypes using biochemical information has found to be advantageous. Here, 1H NMR spectroscopy was performed to identify metabolic perturbations from tubers of five potato cultivars (Atlantic, Frito Lay-1533, Kufri Jyoti, Kufri Pukhraj, and PU1) differing in their CIS ability and processing characteristics at harvest and after cold storage (4 °C). Thirty-nine water-soluble metabolites were detected wherein significantly affected metabolites after cold storage were categorized into sugars, sugar alcohols, amino acids, and organic acids. Multivariate statistical analysis indicated significant differences in the metabolic profiles among the potato cultivars. Pathway enrichment analysis revealed that carbohydrates, amino acids, and organic acids are the key players in CIS. Interestingly, one of the processing cultivars, FL-1533, exhibited a unique combination of metabolites represented by low levels of glucose, fructose, and asparagine accompanied by high citrate levels. Conversely, non-processing cultivars (Kufri Pukhraj and Kufri Jyoti) showed elevated glucose, fructose, and malate levels. Our results indicate that metabolites such as glucose, fructose, sucrose, asparagine, glutamine, citrate, malate, proline, 4-aminobutyrate can be potentially utilized for the prediction, selection, and development of potato cultivars for long-term storage, nutritional, as well as processing attributes.

NMR analysis of nucleotide π-stacking in prebiotically relevant crowded environment.

The prebiotic soup of a putative 'RNA World' would have been replete with a plethora of molecules resulting from complex chemical syntheses and exogeneous delivery. The presence of background molecules could lead to molecular crowding, potentially affecting the course of the reactions facilitated therein. Using NMR spectroscopy, we have analyzed the effect of crowding on the stacking ability of RNA monomers. Our findings corroborate that the purines stack more efficiently than the pyrimidine ribonucleotides. This competence is further enhanced in the presence of a crowding agent. This enhanced stacking could result in greater sequestration of the purine monomers, putting their ready availability for relevant nonenzymatic reactions into question. Thus, this study demonstrates the need for systematic characterization of molecular crowding in the context of prebiotically pertinent processes. Unraveling such phenomena is essential for our understanding of the transition from abiotic to biotic, during the origin of life.

Metabolomics Studies To Decipher Stress Responses in Mycobacterium smegmatis Point to a Putative Pathway of Methylated Amine Biosynthesis.

Mycobacterium smegmatis, the saprophytic soil mycobacterium, is routinely used as a surrogate system to study the human pathogen Mycobacterium tuberculosis It has also been reported as an opportunistic pathogen in immunocompromised hosts. In addition, it can exist in several ecological setups, thereby suggesting its capacity to adapt to a variety of environmental cues. In this study, we employed untargeted proton nuclear magnetic resonance (1H-NMR)-based metabolomics to identify metabolites and metabolic pathways critical for early adaptive responses to acidic stress, oxidative stress, and nutrient starvation in Mycobacterium smegmatis We identified 31, 20, and 46 metabolites that showed significant changes in levels in response to acidic, oxidative, and nutrient starvation stresses, respectively. Pathway analyses showed significant perturbations in purine-pyrimidine, amino-acid, nicotinate-nicotinamide, and energy metabolism pathways. Besides these, differential levels of intermediary metabolites involved in α-glucan biosynthesis pathway were observed. We also detected high levels of organic osmolytes, methylamine, and betaine during nutrient starvation and oxidative stress. Further, tracing the differential levels of these osmolytes through computational search tools, gene expression studies (using reverse transcription-PCR [RT-PCR]), and enzyme assays, we detected the presence of a putative pathway of biosynthesis of betaine, methylamine, and dimethylamine previously unreported in Mycobacterium smegmatisIMPORTANCE Alterations in metabolite levels provide fast and direct means to regulate enzymatic reactions and, therefore, metabolic pathways. This study documents, for the first time, the metabolic changes that occur in Mycobacterium smegmatis as a response to three stresses, namely, acidic stress, oxidative stress, and nutrient starvation. These stresses are also faced by intracellular mycobacteria during infection and therefore may be extended to frame therapeutic interventions for pathogenic mycobacteria. In addition to the purine-pyrimidine, amino acid, nicotinate-nicotinamide, and energy metabolism pathways that were found to be affected in response to different stresses, a novel putative methylamine biosynthesis pathway was identified to be present in Mycobacterium smegmatis.

Metabolic signatures suggest o-phosphocholine to UDP-N-acetylglucosamine ratio as a potential biomarker for high-glucose and/or palmitate exposure in pancreatic ß-cells.

INTRODUCTION: Chronic exposure to high-glucose and free fatty acids (FFA) alone/or in combination; and the resulting gluco-, lipo- and glucolipo-toxic conditions, respectively, have been known to induce dysfunction and apoptosis of ß-cells in Diabetes. The molecular mechanisms and the development of biomarkers that can be used to predict similarities and differences behind these conditions would help in easier and earlier diagnosis of Diabetes. OBJECTIVES: This study aims to use metabolomics to gain insight into the mechanisms by which ß-cells respond to excess-nutrient stress and identify associated biomarkers. METHODS: INS-1E cells were cultured in high-glucose, palmitate alone/or in combination for 24 h to mimic gluco-, lipo- and glucolipo-toxic conditions, respectively. Biochemical and cellular experiments were performed to confirm the establishment of these conditions. To gain molecular insights, abundant metabolites were identified and quantified using 1H-NMR. RESULTS: No loss of cellular viability was observed in high-glucose while exposure to FFA alone/in combination with high-glucose was associated with increased ROS levels, membrane damage, lipid accumulation, and DNA double-strand breaks. Forty-nine abundant metabolites were identified and quantified using 1H-NMR. Chemometric pair-wise analysis in glucotoxic and lipotoxic conditions, when compared with glucolipotoxic conditions, revealed partial overlap in the dysregulated metabolites; however, the dysregulation was more significant under glucolipotoxic conditions. CONCLUSION: The current study compared gluco-, lipo- and glucolipotoxic conditions in parallel and elucidated differences in metabolic pathways that play major roles in Diabetes. o-phosphocholine and UDP-N-acetylglucosamine were identified as common dysregulated metabolites and their ratio was proposed as a potential biomarker for these conditions.

Correction: Synthesis of barbituric acid containing nucleotides and their implications for the origin of primitive informational polymers.

Correction for 'Synthesis of barbituric acid containing nucleotides and their implications for the origin of primitive informational polymers' by Chaitanya V. Mungi et al., Phys. Chem. Chem. Phys., 2016, 18, 20144-20152.

1H, 13C and 15N resonance assignment of domain 1 of trans-activation response element (TAR) RNA binding protein isoform 1 (TRBP2) and its comparison with that of isoform 2 (TRBP1).

TAR RNA binding protein (TRBP) is a double-stranded RNA binding protein involved in various biological processes like cell growth, development, death, etc. The protein exists as two isoforms TRBP2 and TRBP1. TRBP2 contains additional 21 amino acids at its N-terminus, which are proposed to be involved in its membrane localization, when compared to TRBP1. The resonance assignment (19-228) of the double-stranded RNA binding domains (dsRBD 1 and 2) of TRBP2 has been reported earlier. Here, we report 1H, 13C and 15N resonance assignment for dsRBD1 of TRBP2 (1-105) containing the additional N-terminal residues. This assignment will provide deeper insights to understand the effect of these residues on the structure and dynamics of TRBP2 and would therefore help in further elucidating the differences in the role of these isoforms.

Imido-P(v) trianion supported enantiopure neutral tetrahedral Pd(ii) cages.

Charge-neutral chiral hosts are attractive due to their ability to recognize a wide range of guest functionalities and support enantioselective processes. However, reports on such charge-neutral cages are very scarce in the literature. Here, we report an enantiomeric pair of tetrahedral Pd(ii) cages built from chiral tris(imido)phosphate trianions and oxalate linkers, which exhibit enantioselective separation capabilities for epichlorohydrin, ß-butyrolactone, and 3-methyl- and 3-ethyl cyclopentanone.

Origin of the Substitution Mechanism for the Binding of Organic Ligands on the Surface of CsPbBr3 Perovskite Nanocubes.

Optoelectronic properties of CsPbBr3 perovskite nanocubes (NCs) depend strongly on the interaction of the organic passivating molecules with the inorganic crystal. To understand this interaction, we employed a combination of synchrotron-based X-ray photoelectron spectroscopy (XPS), nuclear magnetic resonance (NMR) spectroscopy, and first-principles density functional theory (DFT)-based calculations. Variable energy XPS elucidated the internal structure of the inorganic part in a layer-by-layer fashion, whereas NMR characterized the organic ligands. Our experimental results confirm that oleylammonium ions act as capping ligands by substituting Cs+ ions from the surface of CsPbBr3 NCs. DFT calculations shows that the substitution mechanism does not require much energy for surface reconstruction and, in contrast, stabilizes the nanocrystal by the formation of three hydrogen bonds between the -NH3+ moiety of oleylammonium and surrounding Br- on the surface of NCs. This substitution mechanism and its origin are in stark contrast to the usual adsorption of organic ligands on the surface of typical NCs.

miRNAs: Nanomachines That Micromanage the Pathophysiology of Diabetes Mellitus.

Diabetes mellitus (DM) refers to a combination of heterogeneous complex metabolic disorders that are associated with episodes of hyperglycemia and glucose intolerance occurring as a result of defects in insulin secretion, action, or both. The prevalence of DM is increasing at an alarming rate, and there exists a need to develop better therapeutics and prognostic markers for earlier detection and diagnosis. In this review, after giving a brief introduction of diabetes mellitus and microRNA (miRNA) biogenesis pathway, we first describe various in vitro and animal model systems that have been developed to study diabetes. Further, we elaborate on the significant roles played by miRNAs as regulators of gene expression in the context of development of diabetes and its secondary complications. The different approaches to quantify miRNAs and their potential to be used as therapeutic targets for alleviation of diabetes have also been discussed.

Synthesis of barbituric acid containing nucleotides and their implications for the origin of primitive informational polymers.

Given that all processes in modern biology are encoded and orchestrated by polymers, the origin of informational molecules had to be a crucial and significant step in the origin of life on Earth. An important molecule in this context is RNA that is thought to have allowed the transition from chemistry to biology. However, the RNA molecule is comprised of intramolecular bonds which are prone to hydrolysis, especially so under the harsh conditions of the early Earth. Furthermore, the formation of nucleotides with extant bases and their subsequent polymerization have both been problematic, to say the least. Alternate heterocycles, in contrast, have resulted in nucleosides in higher yields, suggesting a viable and prebiotically relevant solution to the longstanding "nucleoside problem". In the present study, we have synthesized a nucleotide using ribose 5'-monophosphate (rMP) and barbituric acid (BA), as the base analog, using dry-heating conditions that are thought to be prevalent in several regimes of the early Earth. Polymerization of the resultant monomers, i.e. BA-nucleotides, was also observed when dehydration-rehydration cycles were carried out at low pH and high temperature. The resulting RNA-like oligomers have intact bases unlike in reactions that were carried out with canonical nucleotides, which resulted in abasic sites under acidic conditions due to cleavage of the N-glycosidic linkages. Furthermore, the incorporation of BA directly into preformed sugar-phosphate backbones was also observed when rMP oligomers were subjected to heating with BA. The results from our aforementioned experiments provide preliminary evidence that BA could have been a putative precursor of modern nucleobases, which could have been incorporated into primitive informational polymers that predated the molecules of an RNA world. Moreover, they also highlight that the prebiotic soup, which would have been replete with alternate heterocycles, could have allowed the sampling of other such heterocycles, which would have had a selective advantage under pertinent selection pressures. Importantly, these kinds of processes have implications for shaping the prebiotic landscape that allowed for the emergence of primitive informational polymers of the pre-RNA world(s), prior to the emergence of a putative RNA world.

miRNAs: early prognostic biomarkers for Type 2 diabetes mellitus?

Type 2 diabetes mellitus (T2DM) has reached epidemic proportions and is associated with peripheral insulin resistance. The currently used therapies aim to delay progression of T2DM. Their efficacy could drastically be improved if implemented at earlier stages. Classical diagnostic markers (blood glucose and HbA1C) are generally detected once metabolic imbalance has already set in. Therefore, development of biomarkers for early diagnosis would help identify individuals at risk for developing T2DM. Along with genetic predisposition, epigenetics also plays a major role in T2DM development. In this review, we discuss the potential role of early diagnostic markers such as circulating miRNAs, studies done so far and challenges to be considered while taking into account the novel role of miRNAs as prognostic biomarkers.