A Hydrogen Bonded Non-Porous Organic-Inorganic Framework for Measuring Cysteine in Blood Plasma and Endogenous Cancer Cell.

An imbalance in cysteine (Cys) levels in the cells and plasma has been identified as the risk indicator for various human diseases. The structural similarity of cysteine with its congener homocysteine and glutathione offers challenges in its measurement. Herein, we report a hydrogen-bonded organic-inorganic framework of Cu(II) (HOIF) for the selective detection of cysteine over other biothiols. The non-fluorescent HOIF showed 12-fold green emission in the presence of cysteine. The monomeric unit of HOIF is stabilized via intermolecular hydrogen bonds, resulting in a non-porous network structure. Non-interference from homocysteine, glutathione, and other competitive bio-analytes revealed explicit affinity of HOIF for cysteine. Fluorimetric titration showed a wide working concentration window (650 nM-800 µM) for measuring cysteine in an aqueous medium. The mechanistic investigation involving HRMS, EPR, and UV-vis spectroscopic studies revealed the decomplexation of HOIF with Cys, resulting in a fluorescence turn-on response from the luminescent ligand. Validation using a commercial dye, "Cysteine Green", confirmed the prospect of HOIF for early diagnostic purposes. Utilizing the fluorescence turn-on property of HOIF in the presence of cysteine, we measured cysteine quantitatively in the blood plasma samples. Bio-imaging of endogenous cysteine in cancer cells indicated the ability of HOIF to monitor the intracellular cysteine.

Label-Free Detection of Unbound Bilirubin and Nitrophenol Explosives in Water by a Mechanosynthesized Dual Functional Zinc Complex: Recognition of Picric Acid in Various Common Organic Media.

High levels of unconjugated bilirubin (UB) in serum lead to asymptomatic and neonatal jaundice and brain dysfunctions. Herein, we have reported the detection of UB at as low as 1 µM in an aqueous alkaline medium using a Zn(II) complex. The specificity of the complex has been validated by the HPLC in the concentration window 6-90 µM, which is rare. The sensory response of the probe at physiological pH against nitro explosives developed it as an instant-acting fluorosensor for picric acid (PA) and 2,4-dinitrophenol (2,4-DNP). Spectroscopic titration provided a binding constant of 4×105  M-1 with PA. The naked eye detection was found to be 15 µM. The solid-state photoluminescent nature of the complex enabled it for PA sensing in the solid phase. Interestingly, the probe remained fluorescent in various volatile and non-volatile organic solvents. As a result, it can also detect PA and 2,4-DNP in a wide range of common organic media. NMR studies revealed the coordination of PA, 2,4-DNP, and UB to the Zn(II) center of the probe, which is responsible for the observed quenching of the probe with the analytes.

A Water-Soluble Wavy Coordination Polymer of Cu(II) as a Turn-On Luminescent Probe for Histidine and Histidine-Rich Proteins/Peptides.

Histidine plays an essential role in most biological systems. Changes in the homeostasis of histidine and histidine-rich proteins are connected to several diseases. Herein, we report a water-soluble Cu(II) coordination polymer, labeled CuCP, for the fluorimetric detection of histidine and histidine-rich proteins and peptides. Single-crystal structure determination of CuCP revealed a two-dimensional wavy network structure in which a carboxylate group connects the individual Cu(II) dimer unit in a syn-anti conformation. The weakly luminescent and water-soluble CuCP shows turn-on blue emission in the presence of histidine and histidine-rich peptides and proteins. The polymer can also stain histidine-rich proteins via gel electrophoresis. The limits of quantifications for histidine, glycine-histidine, serine-histidine, human serum albumin (HSA), bovine serum albumin, pepsin, trypsin, and lysozyme were found to be 300, 160, 600, 300, 600, 800, 120, and 290 nM, respectively. Utilizing the fluorescence turn-on property of CuCP, we measured HSA quantitatively in the urine samples. We also validated the present urinary HSA measurement assay with existing analytical techniques. Job's plot, 1H NMR, high-resolution mass spectrometry (HRMS), electron paramagnetic resonance (EPR), fluorescence, and UV-vis studies confirmed the ligand displacement from CuCP in the presence of histidine.

Affinity Studies of Hemicyanine Derived Water Soluble Colorimetric Probes with Reactive Oxygen/Nitrogen/Sulfur Species.

Peroxynitrite (ONOO- ) is an essential endogenous reactive oxygen species (ROS) generated in mitochondria under various pathological and physiological conditions. An increase in its level in mitochondria is related to numerous diseases. Herein, we report a series of hemicyanine-derived water-soluble colorimetric probes (1-4) and the reactivity of which was studied with various reactive oxygen, nitrogen, and sulfur species. Probes 1-4 are formed by conjugating 1,2,3,3-tetramethyl-3H-indolium iodide and 4-hydroxybenzaldehyde or its derivatives through an alkene linkage formed by the Knoevenagel reaction. Oxidative cleavage of the electron-rich double bond of the conjugated hemicyanine dye revealed a discerning affinity of probe 3 towards peroxynitrite among all reactive oxygen species. The rapid change in color of 3 provides a sensitive and selective method for detecting peroxynitrite with a low detection limit of 180 nM. Notably, the water solubility of the probe displays excellent performance for the selective detection of peroxynitrite among ROS and reactive nitrogen (RNS)/sulfur species (RSS). UV-vis, 1 H NMR, and 13 C NMR spectroscopic data and results from theoretical calculations provide further information on the interaction of peroxynitrite with probe 3.

Alpha-ketoglutarate supplementation reduces inflammation and thrombosis in type 2 diabetes by suppressing leukocyte and platelet activation.

The interplay between platelets and leukocytes contributes to the pathogenesis of inflammation, thrombosis, and cardiovascular diseases (CVDs) in type 2 diabetes (T2D). Our recent studies described alpha-ketoglutarate (αKG), a Krebs cycle intermediate metabolite as an inhibitor to platelets and leukocytes activation by suppressing phosphorylated-Akt (pAkt) through augmentation of prolyl hydroxylase-2 (PHD2). Dietary supplementation with a pharmacological concentration of αKG significantly inhibited lung inflammation in mice with either SARS-CoV-2 infection or exposed to hypoxia treatment. We therefore investigated if αKG supplementation could suppress hyperactivation of these blood cells and reduce thromboinflammatory complications in T2D. Our study describes that dietary supplementation with αKG (8 mg/100 g body wt. daily) for 7 days significantly reduced the activation of platelets and leukocytes (neutrophils and monocytes), and accumulation of IL1ß, TNFα, and IL6 in peripheral blood of T2D mice. αKG also reduced the infiltration of platelets and leukocytes, and accumulation of inflammatory cytokines in lungs by suppressing pAkt and pP65 signaling. In a cross-sectional investigation, our study also described the elevated platelet-leukocyte aggregates and pro-inflammatory cytokines in circulation of T2D patients. T2D platelets and leukocytes showed an increased aggregation and thrombus formation in vitro. Interestingly, a pre-incubation of T2D blood samples with octyl αKG significantly suppressed the activation of these blood cells and ameliorated aggregate/thrombus formation in vitro. Thus, suggesting a potential therapeutic role of αKG against inflammation, thrombosis, and CVDs in T2D.

CXCR3 antagonist rescues ER stress and reduces inflammation and JEV infection in mice brain.

The endoplasmic reticulum (ER) is crucial for maintaining cellular homeostasis, and synthesis and folding of proteins and lipids. The ER is sensitive to stresses including viral infection that perturb the intracellular energy level and redox state, and accumulating unfolded/misfolded proteins. Viruses including Japanese encephalitis virus (JEV) activates unfolded protein response (UPR) causing ER stress in host immune cells and promotes inflammation and apoptotic cell death. The chemokine receptor CXCR3 has been reported to play important role in the accumulation of inflammatory immune cells and neuronal cell death in several disease conditions. Recently we described the involvement of CXCR3 in regulating inflammation and JEV infection in mice brain. Supplementation with a CXCR3 antagonist AMG487 significantly reduced JEV infection in the mice brain in conjunction with the downregulation of UPR pathway via PERK:eIF2α:CHOP, and decreased mitochondrial ROS generation, inflammation and apoptotic cell death. Alongside, AMG487 treatment improved interferon (IFN)-α/ß synthesis in JEV-infected mice brain. Thus, suggesting a potential therapeutic role of CXCR3 antagonist against JEV infection.

Fabrication of a paper-based facile and low-cost microfluidic device and digital imaging technique for point-of-need monitoring of hypochlorite.

Lab-on-a-paper-based devices are promising alternatives to the existing arduous techniques for point-of-need monitoring. The present work reports an instant and facile method to produce a microfluidic paper-based analytical device (µPAD). The fabricated µPAD has been used to detect hypochlorite (OCl-) by incorporating newly synthesized chromo-fluorogenic ratiometric probes 1 and 2 into the sample reception zone. The probes showed high selectivity and fast response (<10 s) toward OCl- with an excellent linear relationship in the concentration range of 0-100 µM. The concentration-dependent fluorometric change driven by the reaction of 1@µPAD with OCl- has been monitored using gel-doc imaging systems, which is unprecedented. Digitizing the intensity of the colour solution with different mathematical models of colour has developed a straightforward method for monitoring OCl- without any interference from its competitors. 1@µPAD can detect OCl- at ∼10 times lower than the WHO recommended limit. The detection limit of 1@µPAD via a digital camera-based fluorescence technique was found to be better over digital camera-based cuvette assays. Therefore, 1@µPAD has been successfully utilized to monitor OCl- in actual environmental water samples with portability, ease of use, and sensitivity. The analytical RSD was found to be ≤3% based on fluorimetric detection using 1@µPAD. The chemodosimetric reaction between OCl- and the probe was evidenced by UV-vis and fluorescence spectroscopy, 1H NMR, and ESI-MS. The rapid response time, biocompatibility, low cytotoxicity, 100% aqueous solubility, ratiometric feature, and exclusive OCl- selectivity over other competitive ROS/RNS successfully lead to the application of the probes for bioimaging of exogenous as well as endogenous OCl- in normal cells (HEK293) and cancerous cells (HeLa).

Sixth Ligand Induced HNO/NO- Release by a Five-Coordinated Cobalt(II) Nitrosyl Complex Having a {CoNO}8 Configuration.

Nitroxyl (HNO) and nitroxide (NO-) anion, the one-electron-reduced form of nitric oxide (NO), have been shown to have distinct advantages over NO from pharmacological and therapeutic points of view. However, the role of nitroxyl in chemical biology has not yet been studied as extensively as that of NO. Consequently, only a few examples of HNO donors such as Angeli's salt, Piloty's acid, or acyl- and acyloxynitroso derivatives are known. However, the intrinsic limitations of all of these hinder their widespread utility. Metal nitrosyl complexes, although few examples, could serve as an efficient HNO donor. Here, a cobalt nitrosyl complex of the {CoNO}8 (1) configuration has been reported. This complex in the presence of a sixth ligand [BF4-, DTC- (diethyldithiocarbamate anion), or imidazole] releases/donates HNO/NO-. This has been confirmed using well-known HNO/NO- acceptors like [Fe(TPP)Cl] and [Fe(DTC)3]. The HNO release has been authenticated further by the detection and estimation of N2O using gas chromatography-mass spectroscopy as well as its reaction with PPh3.

Concurrent detection and treatment of cyanide-contaminated water using mechanosynthesized receptors.

The development of receptors that can detect as well as treat cyanide ions in aqueous samples is indispensable for environmental protection. Herein, we present the bulk solvent-free and instant green synthesis of a series of turn-on fluorimetric probes that can specifically detect the deadly poison cyanide among various anions and metal ions in water. Selective recognition of cyanide by the mechanosynthesized compounds is even observable by the naked eyes, which remained unaffected in the presence of various challenging species. NMR spectroscopic investigation supports the chemodosimetric sensing of cyanide by the receptors. A remarkable 55-83 fold fluorescence enhancement by the probes enabled us to reach a limit of detection (LOD) in the range of 8-26 ppb, well below the permissible limit of cyanide in drinking water. Being minuscule soluble in water, cyanide treatment studies with the ionophores showed greater than 99% reduction in the free cyanide concentration after three consecutive cycles of operation. Furthermore, the compounds can be used as sensitive probes for the estimation of cyanide in human blood serum in physiological conditions. Overall, the results presented in this article will certainly find great use in the area of cyanide pollution with regard to simultaneous sensing and treatment of free cyanide, which is heretofore unprecedented.

Elevated glycosylation of CD36 in platelets is a risk factor for oxLDL-mediated platelet activation in type 2 diabetes.

Platelet activation and related cardiovascular complications are the hallmarks of type 2 diabetes (T2D). We investigated the mechanism of platelet activation in T2D using MS-based identification of differentially expressed platelet proteins with a focus on glycosylated forms. Glycosylation is considered one of the common post-translational modifications in T2D, and N/O-linked glycosylation of glycoproteins (GPs)/integrins is known to play crucial roles in platelet activation. Our platelet proteome data revealed elevated levels of GPs GPIbα, GPIIbIIIa, GPIV (CD36), GPV and integrins in T2D patients. T2D platelets had elevated N-linked glycosylation of CD36 at asparagine (Asn)408,417 . Enrichment analysis revealed a close association of glycosylated CD36 with thrombospondin-1, fibrinogen and SERPINA1 in T2D platelets. The glycosylation of CD36 has previously been reported to increase cellular uptake of long-chain fatty acids. Our in silico molecular docking data also showed a favorable binding of cholesterol with glycosylated Asn417 CD36 compared to the non-glycosylated form. We further investigated the CD36:LDL cholesterol axis in T2D. Elevated levels of oxidized-low density lipoprotein (oxLDL) were found to cause significant platelet activation via CD36-mediated stimulation of Lyn-JNK signaling. Sulfo-N-succinimidyl oleate, an inhibitor of CD36, effectively inhibited oxLDL-mediated platelet activation and adhesion in vitro. Our study suggests increased glycosylation of CD36 in T2D platelets as a potential route for oxLDL-mediated platelet activation. The oxLDL:CD36 axis may thus be exploited as a prospective target to develop therapeutics against thrombosis in T2D.

Inhibition of cellular activation induced by platelet factor 4 via the CXCR3 pathway ameliorates Japanese encephalitis and dengue viral infections.

BACKGROUND: Activated platelets secrete platelet factor 4 (PF4), which contributes to viral pathogenesis. Recently, we reported the proviral role of PF4 in replication of closely related flaviviruses, Japanese encephalitis virus (JEV) and dengue virus (DENV). OBJECTIVES: This study aimed to investigate the detailed mechanism of PF4-mediated virus replication. METHODS: PF4-/- or wild-type (WT) mice were infected with JEV, and host defense mechanisms, including autophagic/interferon (IFN) responses, were assessed. WT mice were pretreated with the CXCR3 antagonist AMG487 that inhibits PF4:CXCR3 pathway. This pathway was tested in PF4-/- monocytes infected with DENV or in monocytes isolated from patients with DENV infection. RESULTS: PF4-/- mice infected with JEV showed reduced viral load and improved brain inflammation and survival. PF4-/- mice synthesized more IFN-α/ß with higher expression of phosphorylated IRF3 in the brain. PF4 treatment decreased IRF-3/7/9 and IFN-α/ß expression and suppressed autophagic LC3-II flux and lysosomal degradation of viral proteins in JEV-infected cells. PF4 increased the expression of P-mTOR, P-p38, and P-ULK1Ser757 and decreased expression of LC3-II. Decreased autophagosome-lysosome fusion in turn promoted DENV2 replication. The above processes were reversed by AMG487. Uninfected PF4-/- monocytes showed elevated LC3-II and autophagosome-lysosome fusion. Microglia of JEV-infected PF4-/- mice exhibited elevated LC3-II inversely related to viral load. Similarly, monocytes from PF4-/- mice showed reduced infection by DENV2. In patients with DENV infection, higher plasma PF4 and viral load were inversely correlated with LC3-II, LAMP-1, and lysosomal degradation of DENV-NS1 in monocytes during the febrile phase. CONCLUSION: These studies suggest that PF4 deficiency or inhibition of the PF4:CXCR3 pathway prevents JEV and DENV infection. The studies also highlight the PF4:CXCR3 axis as a potential target to develop treatment regimens against flaviviruses.

Reaction of a Co(III)-peroxo complex with nitric oxide: putative formation of a peroxynitrite intermediate.

A Co(II) complex, [CoII(L)2(H2O)2](ClO4)2, 1, having a bidentate ligand L [L = bis(3,5-dimethylpyrazolyl)methane] has been synthesized. Complex 1 in acetonitrile solution at -40 °C, in the presence of H2O2 and NEt3, afforded the corresponding Co(III)-peroxo species, [CoIII(L)2(O22-)]+, as the transient intermediate 1a. Thermal instability precluded its isolation and further characterization. The addition of nitric oxide (NO) gas into the freshly prepared [CoIII(L)2(O22-)]+ in acetonitrile at -40 °C resulted in the corresponding Co(II)-nitrato complex, [CoII(L)2(NO3)](ClO4) (2). The reaction is proposed to proceed through a putative Co(II)-peroxynitrite intermediate 1b. It was evidenced by the characteristic phenol ring nitration reaction.

Coumarin-Ensembled Vanadium(V) Compounds and Their Affinity Studies Toward Biological Thiols Probed by Fluorescence Spectroscopy.

Fluorescence spectroscopic studies of a pair of new oxido-vanadium(V) compounds with biological thiols, such as homocysteine (Hcy), cysteine (Cys), and glutathione (GSH), have been investigated in this article. Despite notable progress in vanadium-thiol chemistry, no attention has been paid to exploring vanadium-based optical probes to study their interaction with biothiols. For this purpose, two oxido-vanadium(V) compounds, 1 and 2, have been prepared involving a tridentate ONO donor-based luminescent coumarin-derived ligand. Single crystal X-ray diffraction analysis, NMR (1 H, 13 C, and 51 V) spectroscopy, XPS, and DFT calculations have been used to establish their identities. The vanadium center in these compounds has a distorted octahedral environment. In compound 2, a methanol molecule is coordinated to the vanadium(V) center in the trans position of the terminal oxido moiety. The latter exerts a strong trans-labilizing influence on the coordinating methanol. Both 1 and 2 are weakly fluorescent. Photophysical investigations of the vanadium complexes in aqueous media at physiological pH (7.4) in the presence of various biothiols and amino acids showed significant fluorescence enhancement (83-fold) of the vanadium complexes, specifically with Hcy. The specific affinity of the complexes for Hcy remained unchanged even in the presence of other biothiols and amino acids. Kinetic investigation reveals pseudo-first order behavior of the compound with Hcy. Mechanistic studies have manifested that Hcy-induced reduction triggers the decomplexation of the vanadium compound, followed by hydrolysis and subsequent cyclization. Time-correlated single photon counting suggested that the radiative rate constant (kr ) of 1 and 2 in the presence of Hcy serves as the prime factor for the fluorescence enhancement of the medium. Compound 1 has been tested efficiently for Hcy measurement in blood plasma rendering it suitable for practical applications.

Reaction of a nitrosyl complex of Mn(II)-porphyrinate with superoxide: NOD activity is favoured over SOD activity.

A five-coordinated {Mn(NO)}6 complex of Mn(II)-porphyrinate, [Mn(TMPP2-)(NO)], 1 {TMPPH2 = 5,10,15,20-tetrakis(4-methoxyphenyl)porphyrin}, upon reaction with two equivalents of superoxide (O2-) in THF at -40 °C results in the corresponding MnIII-OH complex [MnIII(TMPP2-)(OH)], 2, via the formation of a putative MnIII-peroxynitrite intermediate. Spectral studies and chemical analysis suggest that one equivalent of superoxide ion is consumed to oxidize the metal center of complex 1 leading to [MnIII(TMPP2-)(NO)]+, while the subsequent equivalent reacts with [MnIII(TMPP2-)(NO)]+ to form the corresponding peroxynitrite intermediate. UV-visible and X-band EPR spectroscopic studies suggest the involvement of a MnIV-oxo species in the reaction, which forms through the O-O bond cleavage of the peroxynitrite moiety with concomitant release of NO2. The formation of MnIII-peroxynitrite is further supported by the well-established phenol ring nitration experiment. The released NO2 has been trapped using TEMPO. It should be noted that in cases of MnII-porphyrin complexes, the reaction with superoxide generally proceeds through a SOD-like pathway where the first equivalent of superoxide ion oxidizes the MnII center and itself is reduced to peroxide (O22-), while the subsequent equivalent of superoxide reduces the MnIII center with the release of O2. In contrast, here the second equivalent of superoxide reacts with the MnIII-nitrosyl complex and follows a NOD-like pathway.

Studies on the interaction between oxido/dioxidovanadium(V) compounds and reactive oxygen species: Synthesis, characterization, and photophysical investigation.

Singlet oxygen (1O2) and hypochlorite (OCl-) are two principal non-radical reactive oxygen species (ROS) which, are produced in a number of biochemical processes. In cellular systems, these analytes play various important roles. In this article, we report two mononuclear oxido- and dioxidovanadium(V) compounds 1 and 2 of an intramolecularly hydrogen bonded luminescent zwitterion ligand (HL). Single crystal X-ray diffraction analysis and multinuclear (1H and 51V) NMR spectroscopy provided the identities of 1 and 2 in the solid and solution states, respectively. Both 1 and 2 are water soluble and fluorescent. Fluorescence of the ligand HL is responsible for the fluorescent nature of 1 and 2. Protonation of the hanging amine moiety of the ligand remained unchanged in the vanadium complexes 1 and 2. However, the intramolecular H-bonding is not present in 1 and 2. Hydrophilicity and luminescent nature of the vanadium complexes provided us the opportunity to study the interaction of 1 and 2 with different ROS. Excited state photophysical investigations revealed highly selective instant response of the probes 1 and 2 for singlet oxygen and hypochlorite. Specific response of the dioxidovanadium(V) complex 1 towards singlet oxygen/hypochlorite remained unchanged in presence of other challenging ROS. Spectrofluorimetric titration provided limit of detection around 180 nM for 1O2. 1H NMR and theoretical calculations provided further information on the interactions between vanadium compound and analyte.

A unique water soluble probe for measuring the cardiac marker homocysteine and its clinical validation.

A series of copper(II) compounds 1-4 were synthesized and developed as fluorogenic probes to measure the cardiac marker homocysteine (Hcy) without any interference from other bioanalytes prevalent in human blood plasma including, cysteine and glutathione. UV-vis and EPR studies have provided confirmatory evidence for reduction-induced-emission-enhancement of the probe, which is responsible for the observed "off-to-on" behaviour towards Hcy. Water solubility, remarkable fluorescence enhancement (55-111 fold), and low detection ability (nearly 2.5 µM) make the probe suitable for clinical testing of cardiac samples. Investigation of 1 against a few reductive interferents testifies its specificity for Hcy. Results from clinical examination of cardiac samples by 1 when combined with the outcome of the reliability testing involving a clinically approved commercial immunoassay kit, validates the prospect of the molecular probe for direct measurement of Hcy in human plasma, which is unprecedented.

Advances in the Development of Water-Soluble Fluorogenic Probes for Bioimaging of Hypochlorite/Hypochlorous Acid in Cells and Organisms.

This mini-review summarizes the development of intracellular fluorogenic probes for biological investigations of hypochlorous acid/hypochlorite (HOCl/OCl-) in living cells and tissues. Monitoring the formation or effects of reactive oxygen species (ROS) inside living systems is critical in determining their roles in human physiology. HOCl/OCl- is considered as an important member of the nonradical ROS family for its decisive microbicidal action in the innate immune system. Even though HOCl/OCl- plays a defensive role in human health, abnormal or overexpression may have detrimental effects on the host physiology leading to many diseases, including neurodegeneration and cancer. In recent years, progress in the development of fluorescent imaging probes for observing HOCl/OCl- levels in live cells and tissues has been made. Despite considerable advancement, challenges still exist in areas like working solvent/media, pH, response time, buffer selection, emission region, and others. In addition, this account aims to discuss the design strategies and sensing mechanisms of the representative fluorogenic probes for bioimaging of HOCl/OCl-, endogenously and exogenously. Herein, we also have tried to provide the future direction to develop HOCl/OCl- specific probes for disease diagnosis with particular attention to the requirement of the recognition group, solvent, and buffer media, which will be beneficial for those working in the domain of biomedical research.