Single-cell genomics and regulatory networks for 388 human brains.

Single-cell genomics is a powerful tool for studying heterogeneous tissues such as the brain. Yet little is understood about how genetic variants influence cell-level gene expression. Addressing this, we uniformly processed single-nuclei, multiomics datasets into a resource comprising >2.8 million nuclei from the prefrontal cortex across 388 individuals. For 28 cell types, we assessed population-level variation in expression and chromatin across gene families and drug targets. We identified >550,000 cell type-specific regulatory elements and >1.4 million single-cell expression quantitative trait loci, which we used to build cell-type regulatory and cell-to-cell communication networks. These networks manifest cellular changes in aging and neuropsychiatric disorders. We further constructed an integrative model accurately imputing single-cell expression and simulating perturbations; the model prioritized ~250 disease-risk genes and drug targets with associated cell types.

Single-cell genomics and regulatory networks for 388 human brains.

Single-cell genomics is a powerful tool for studying heterogeneous tissues such as the brain. Yet, little is understood about how genetic variants influence cell-level gene expression. Addressing this, we uniformly processed single-nuclei, multi-omics datasets into a resource comprising >2.8M nuclei from the prefrontal cortex across 388 individuals. For 28 cell types, we assessed population-level variation in expression and chromatin across gene families and drug targets. We identified >550K cell-type-specific regulatory elements and >1.4M single-cell expression-quantitative-trait loci, which we used to build cell-type regulatory and cell-to-cell communication networks. These networks manifest cellular changes in aging and neuropsychiatric disorders. We further constructed an integrative model accurately imputing single-cell expression and simulating perturbations; the model prioritized ~250 disease-risk genes and drug targets with associated cell types.

Assessment of pain-related behaviors in HIV-1 transgenic rats as a model of HIV-associated chronic pain.

Human immunodeficiency virus-1 (HIV)-associated chronic pain is a debilitating comorbid condition that affects 25-85% of people with HIV. The use of opioids to alleviate pain has given rise to opioid dependency in this cohort. Therefore, there is an urgent need to understand mechanisms and identify novel therapeutics for HIV-associated chronic pain. Several animal models have been developed to study HIV-related comorbidities. HIV-1 transgenic (Tg) rats have been shown to serve as a reliable model that mimic the deficits observed in people with HIV, such as neurological and immune system alterations. However, pain-related behavior in these animals has not been extensively evaluated. In this study, we measured evoked and spontaneous behavior in HIV-1Tg male and female rats. The results indicated that HIV-1Tg rats exhibit similar behavior to those with HIV-1-related neuropathy, specifically, cold sensitivity. Consequently, HIV-1Tg rats can serve as a model of neuropathy to study pain-related mechanisms and therapeutics targeted toward individuals living with HIV-1.

Sex-Based Disparities in Leukocyte Migration and Activation in Response to Inhalation Lung Injury: Role of SDF-1/CXCR4 Signaling.

The aim of the study was to determine whether sex-related differences exist in immune response to inhalation lung injury. C57BL/6 mice were exposed to Cl2 gas (500 ppm for 15, 20, or 30 min). Results showed that male mice have higher rates of mortality and lung injury than females. The binding of the chemokine ligand C-X-C motif chemokine 12 (CXCL12), also called stromal-derived-factor-1 (SDF-1), to the C-X-C chemokine receptor type 4 (CXCR4) on lung cells promotes the migration of leukocytes from circulation to lungs. Therefore, the hypothesis was that elevated SDF-1/CXCR4 signaling mediates exaggerated immune response in males. Plasma, blood leukocytes, and lung cells were collected from mice post-Cl2 exposure. Plasma levels of SDF-1 and peripheral levels of CXCR4 in lung cells were higher in male vs. female mice post-Cl2 exposure. Myeloperoxidase (MPO) and elastase activity was significantly increased in leukocytes of male mice exposed to Cl2. Lung cells were then ex vivo treated with SDF-1 (100 ng/mL) in the presence or absence of the CXCR4 inhibitor, AMD3100 (100 nM). SDF-1 significantly increased migration, MPO, and elastase activity in cells obtained from male vs. female mice post-Cl2 exposure. AMD3100 attenuated these effects, suggesting that differential SDF-1/CXCR4 signaling may be responsible for sex-based disparities in the immune response to inhalation lung injury.

Live-Cell Mitochondrial Targeted NIR Fluorescent Covalent Labeling of Specific Proteins Using a Dual Localization Effect.

Here, our designed water-soluble NIR fluorescent unsymmetrical Cy-5-Mal/TPP+ consists of a lipophilic cationic TPP+ subunit that can selectively target and accumulate in a live-cell inner mitochondrial matrix where a maleimide residue of the probe undergoes faster chemoselective and site-specific covalent attachment with the exposed Cys residue of mitochondrion-specific proteins. On the basis of this dual localization effect, Cy-5-Mal/TPP+ molecules remain for a longer time period even after membrane depolarization, enabling long-term live-cell mitochondrial imaging. Due to the adequate concentration of Cy-5-Mal/TPP+ reached in live-cell mitochondria, it facilitates site-selective NIR fluorescent covalent labeling with Cys-exposed proteins, which are identified by the in-gel fluorescence assay and LC-MS/MS-based proteomics and supported by a computational method. This dual targeting approach with admirable photostability, narrow NIR absorption/emission bands, bright emission, long fluorescence lifetime, and insignificant cytotoxicity has been shown to improve real-time live-cell mitochondrial tracking including dynamics and interorganelle crosstalk with multicolor imaging applications.

Heme-Induced Macrophage Phenotype Switching and Impaired Endogenous Opioid Homeostasis Correlate with Chronic Widespread Pain in HIV.

Chronic widespread pain (CWP) is associated with a high rate of disability and decreased quality of life in people with HIV-1 (PWH). We previously showed that PWH with CWP have increased hemolysis and elevated plasma levels of cell-free heme, which correlate with low endogenous opioid levels in leukocytes. Further, we demonstrated that cell-free heme impairs ß-endorphin synthesis/release from leukocytes. However, the cellular mechanisms by which heme dampens ß-endorphin production are inconclusive. The current hypothesis is that heme-dependent TLR4 activation and macrophage polarization to the M1 phenotype mediate this phenomenon. Our novel findings showed that PWH with CWP have elevated M1-specific macrophage chemokines (ENA-78, GRO-α, and IP-10) in plasma. In vitro, hemin-induced polarization of M0 and M2 macrophages to the M1 phenotype with low ß-endorphins was mitigated by treating cells with the TLR4 inhibitor, TAK-242. Similarly, in vivo phenylhydrazine hydrochloride (PHZ), an inducer of hemolysis, injected into C57Bl/6 mice increased the M1/M2 cell ratio and reduced ß-endorphin levels. However, treating these animals with the heme-scavenging protein hemopexin (Hx) or TAK-242 reduced the M1/M2 ratio and increased ß-endorphins. Furthermore, Hx attenuated heme-induced mechanical, heat, and cold hypersensitivity, while TAK-242 abrogated hypersensitivity to mechanical and heat stimuli. Overall, these results suggest that heme-mediated TLR4 activation and M1 polarization of macrophages correlate with impaired endogenous opioid homeostasis and hypersensitivity in people with HIV.

High Heme and Low Heme Oxygenase-1 Are Associated with Mast Cell Activation/Degranulation in HIV-Induced Chronic Widespread Pain.

An overwhelming number of people with HIV (PWH) experience chronic widespread pain (CWP) throughout their lifetimes. Previously, we demonstrated that PWH with CWP have increased hemolysis and attenuated heme oxygenase 1 (HO-1) levels. HO-1 degrades reactive, cell-free heme into antioxidants like biliverdin and carbon monoxide (CO). We found that high heme or low HO-1 caused hyperalgesia in animals, likely through multiple mechanisms. In this study, we hypothesized that high heme or low HO-1 caused mast cell activation/degranulation, resulting in the release of pain mediators like histamine and bradykinin. PWH who self-report CWP were recruited from the University of Alabama at Birmingham HIV clinic. Animal models included HO-1-/- mice and hemolytic mice, where C57BL/6 mice were injected intraperitoneally with phenylhydrazine hydrochloride (PHZ). Results demonstrated that plasma histamine and bradykinin were elevated in PWH with CWP. These pain mediators were also high in HO-1-/- mice and in hemolytic mice. Both in vivo and in vitro (RBL-2H3 mast cells), heme-induced mast cell degranulation was inhibited by treatment with CORM-A1, a CO donor. CORM-A1 also attenuated mechanical and thermal (cold) allodynia in hemolytic mice. Together, the data suggest that mast cell activation secondary to high heme or low HO-1 seen in cells and animals correlates with elevated plasma levels of heme, histamine, and bradykinin in PWH with CWP.

HIV Increases the Risk of Cigarette Smoke-Induced Emphysema Through MMP-9.

BACKGROUND: HIV is associated with an increased risk for emphysema. Matrix metalloproteinase 9 (MMP-9) is a lung tissue remodeling enzyme associated with emphysema. We previously found MMP-9 activity increases with increases in oxidative stress and that HIV increases alveolar oxidative stress. We hypothesized that HIV proteins would increase the risk of cigarette smoke-induced emphysema due to MMP-9. METHODS: HIV-1 transgenic rats and wild-type littermates were exposed to cigarette smoke or sham for 8 weeks. Lung compliance and histology were assessed. Bronchoalveolar lavage (BAL), primary alveolar macrophages (AM), and serum samples were obtained. A rat alveolar macrophage cell line was exposed to the HIV protein Tat, and MMP-9 levels were assessed by Western immunoblotting. MMP-9 protein expression and activity were assessed in AM from the HIV rat model by ELISA and cytoimmunofluoresence, respectively. Serum from human subjects with and without HIV and tobacco dependence was assessed for MMP-9 levels. RESULTS: MMP-9 expression was significantly increased in rat alveolar macrophages after Tat exposure. HIV-1 transgenic rats developed emphysema while wild-type littermates did not. MMP-9 expression was also increased in the serum, BAL, and AM of HIV-1 transgenic rats after exposure to cigarette smoke compared with wild-type rats. In parallel, serum samples from HIV+ smokers had higher levels of MMP-9 than subjects without HIV and those who did not smoke. CONCLUSION: The combination of HIV and cigarette smoke increases MMP-9 expression in experimental rat HIV models and human subjects. HIV and cigarette smoke both induce alveolar oxidative stress and thereby increase MMP-9 activity.

Construction of Self-Assembling Lipopeptide-Based Benign Nanovesicles to Prevent Amyloid Fibril Formation and Reduce Cytotoxicity of GxxxGxxxGxxxG Motif.

Alzheimer's disease, a progressive severe neurodegenerative disorder, has been until now incurable, in spite of serious efforts worldwide. We have designed self-assembled myristoyl-KPGPK lipopeptide-based biocompatible nanovesicles, which can inhibit amyloid fibrillation made by the transmembrane GxxxGxxxGxxxG motif of Aß-protein and human myelin protein zero as well as reduce their neurotoxicity. Various spectroscopic and microscopic investigations illuminate that the lipopeptide-based nanovesicles dramatically inhibit random coil-to-ß-sheet transformation of Aß25-37 and human myelin protein zero protein precursor, which is the prerequisite of GxxxGxxxGxxxG motif-mediated fibril formation. Förster resonance energy transfer (FRET) assay using synthesized Cy-3 (FRET donor) and Cy-5 (FRET acceptor)-conjugated Aß25-37 also exhibits that nanovesicles strongly inhibit the fibril formation of Aß25-37. The mouse neuro-2a neuroblastoma cell line is used, which revealed the GxxxGxxxGxxxG-mediated cytotoxicity. However, the neurotoxicity has been diminished by co-incubating the GxxxGxxxGxxxG motif with the nanovesicles.

Redox nanomedicine ameliorates chronic kidney disease (CKD) by mitochondrial reconditioning in mice.

Targeting reactive oxygen species (ROS) while maintaining cellular redox signaling is crucial in the development of redox medicine as the origin of several prevailing diseases including chronic kidney disease (CKD) is linked to ROS imbalance and associated mitochondrial dysfunction. Here, we have shown that a potential nanomedicine comprising of Mn3O4 nanoparticles duly functionalized with biocompatible ligand citrate (C-Mn3O4 NPs) can maintain cellular redox balance in an animal model of oxidative injury. We developed a cisplatin-induced CKD model in C57BL/6j mice with severe mitochondrial dysfunction and oxidative distress leading to the pathogenesis. Four weeks of treatment with C-Mn3O4 NPs restored renal function, preserved normal kidney architecture, ameliorated overexpression of pro-inflammatory cytokines, and arrested glomerulosclerosis and interstitial fibrosis. A detailed study involving human embryonic kidney (HEK 293) cells and isolated mitochondria from experimental animals revealed that the molecular mechanism behind the pharmacological action of the nanomedicine involves protection of structural and functional integrity of mitochondria from oxidative damage, subsequent reduction in intracellular ROS, and maintenance of cellular redox homeostasis. To the best of our knowledge, such studies that efficiently treated a multifaceted disease like CKD using a biocompatible redox nanomedicine are sparse in the literature. Successful clinical translation of this nanomedicine may open a new avenue in redox-mediated therapeutics of several other diseases (e.g., diabetic nephropathy, neurodegeneration, and cardiovascular disease) where oxidative distress plays a central role in pathogenesis.

Detecting network anomalies using Forman-Ricci curvature and a case study for human brain networks.

We analyze networks of functional correlations between brain regions to identify changes in their structure caused by Attention Deficit Hyperactivity Disorder (ADHD). We express the task for finding changes as a network anomaly detection problem on temporal networks. We propose the use of a curvature measure based on the Forman-Ricci curvature, which expresses higher-order correlations among two connected nodes. Our theoretical result on comparing this Forman-Ricci curvature with another well-known notion of network curvature, namely the Ollivier-Ricci curvature, lends further justification to the assertions that these two notions of network curvatures are not well correlated and therefore one of these curvature measures cannot be used as an universal substitute for the other measure. Our experimental results indicate nine critical edges whose curvature differs dramatically in brains of ADHD patients compared to healthy brains. The importance of these edges is supported by existing neuroscience evidence. We demonstrate that comparative analysis of curvature identifies changes that more traditional approaches, for example analysis of edge weights, would not be able to identify.

Supramolecular Dipeptide-Based Near-Infrared Fluorescent Nanotubes for Cellular Mitochondria Targeted Imaging and Early Apoptosis.

Herein, we have designed and synthesized unsymmetrical visible Cy-3 and near-infrared (NIR) Cy-5 chromophores anchoring mitochondria targeting functional group conjugated with a Phe-Phe dipeptide by a microwave-assisted Fmoc solid phase peptide synthesis method on Wang resin. These dipeptide-based Cy-3-TPP/FF as well as Cy-5-TPP/FF molecules self-assemble to form fluorescent nanotubes in solution, and it has been confirmed by TEM, SEM, and AFM. The Cy-3-TPP/FF and Cy-5-TPP/FF molecules in solution exhibit narrow excitation as well as emission bands in the visible and NIR region, respectively. These lipophilic cationic fluorescent peptide molecules spontaneously and selectively accumulate inside the mitochondria of human carcinoma cells that have been experimentally validated by live cell confocal laser scanning microscopy and display a high Pearson's correlation coefficient in a colocalization assay. Live cell multicolor confocal imaging using the NIR Cy-5-TPP/FF in combination with other organelle specific dye is also accomplished. Moreover, these lipophilic dipeptide-based cationic molecules reach the critical aggregation concentration inside the mitochondria because of the extremely negative inner mitochondrial membrane potential [(ΔΨm)cancer ≈ -220 mV] and form supramolecular nanotubes which are accountable for malignant mitochondria targeted early apoptosis. The early apoptosis is arrested using Cy-5-TPP/FF and confirmed by annexin V-FITC/PI apoptosis detection assay.

Adiponectin Genetic Variant and Expression Coupled with Lipid Peroxidation Reveal New Signatures in Diabetic Dyslipidemia.

Increasing burden of non-communicable diseases like diabetes and cardiovascular disorders has made the global health scenario more challenging. Dyslipidemia in diabetes is a compounding risk factor for cardiovascular diseases, but there is dearth of identifying appropriate signatures to address this issue. The protein, adiponectin, is actively involved in regulating glucose levels as well as fatty acid breakdown playing crucial role in dyslipidemia and vascular complications. To identify the underlying genetic and molecular profile of adiponectin metabolic pathway in diabetic dyslipidemia and to correlate it with known biochemical and oxidative stress parameters of T2DM, we performed a case-control study in a total 264 individuals belonging to three categories such as diabetes with dyslipidemia (n = 88), diabetes without dyslipidemia (n = 86) and normal healthy controls (n = 90). Expression of adiponectin (ADIPOQ) and its receptors (ADIPOR1 and ADIPOR2) were measured in visceral and subcutaneous adipose tissues. A significant downregulated expression of ADIPOQ and its receptors in adipose tissues and PBMCs were linked with diabetic dyslipidemic condition. A multiple linear regression followed by MDR analysis implicated the elevated plasma malondialdehyde and decreased adiponectin level to be correlated with diabetic dyslipidemia. More interestingly, two single nucleotide polymorphisms of ADIPOQ (rs2241766 and rs1501299) were genetically associated with the risk of developing dyslipidemia. Other important biochemical factors found to be increased in diabetic dyslipidemic conditions included plasma C-reactive protein and 4-hydroxynonenal adducts. Our results explore, a complex interplay of genetic and biochemical parameters in diabetic dyslipidemia which is significant from the perspective of risk stratification and novel therapeutic strategy development.

Spectroscopic Studies on the Biomolecular Recognition of Toluidine Blue: Key Information Towards Development of a Non-Contact, Non-Invasive Device for Oral Cancer Detection.

Molecular interaction of aromatic dyes with biological macromolecules are important for the development of minimally invasive disease diagnostic biotechnologies. In the present work, we have used Toluidine Blue (TB) as a model dye, which is a well-known staining agent for the diagnosis of oral cancer and have studied the interaction of various biological macromolecules (protein and DNA) with the dye at different pH. Our spectroscopic studies confirm that TB interacts with Human Serum Albumin (HSA), a model protein at very high pH conditions which is very hard to achieve physiologically. On the other hand, TB significantly interacts with the DNA at physiological pH value (7.4). Our molecular studies strengthen the understanding of the Toluidine Blue staining of cancer cells, where the relative ratio of the nucleic acids is higher than the normal intracellular content. We have also developed a non-invasive, non-contact spectroscopic technique to explore the possibility of quantitatively detecting oral cancer by exploiting the interaction of TB with DNA. We have also reported development of a prototype named "Oral-O-Scope" for the detection of Oral cancer and have carried out human studies using the prototype.

Nuclear factor NF-κB1 functional promoter polymorphism and its expression conferring the risk of Type 2 diabetes-associated dyslipidemia.

Type 2 diabetes mellitus (T2DM) accompanied by hyperlipidemia confers higher risk for diabetes as well as cardiovascular diseases. NF-κB is actively involved in generating low-grade inflammation and oxidative stress triggering the development of diabetic complications. In this study, we have attempted to investigate the association between NF-κB1 functional promoter polymorphism-94 ATTG insertion/deletion (rs28362491) with inflammatory markers in developing diabetes-linked dyslipidemia. We performed a case-control study in a total of 401 individuals belonging to three categories such as Type 2 diabetes with dyslipidemia, Type 2 diabetes without dyslipidemia, and normal healthy controls. Experiments were carried out using genotyping, real-time PCR, and western blot. Pearson's correlation, analysis of variance, and logistic regression were utilized for statistical analysis. As per genetic association conducted in this study the SNP rs28362491 showed significant allelic and genotypic associations (Allelic: OR = 1.374, CI 0.9797-1.927, p = 0.003, and Genotypic in dominant model: OR = 1.77, CI 1.04-2.99, p = 0.002) with the risk of diabetes and associated dyslipidemia. The -94 ATTG insertion/insertion (ins/ins) genotype was associated with significantly increased level of serum TNF-α (p = 0.002), serum IL-6 (p = 0.067) in diabetes-induced dyslipidemia. Multiple linear regression analysis identifies independent correlation of Total cholesterol, HDL, LDL, TNF-α, and rs28362491 ATTG ins/ins with triglyceride in diabetic dyslipidemic condition. T2DM with dyslipidemia having ins/ins genotype showed significant increased expression of pro-inflammatory cytokines such as TNF-α, IL-6, and activation of NF-κB. Our study reports that individuals with ATTG insertion allele and ATTG ins/ins genotype at NF-κB1 promoter regulatory gene predicts the risk and severity of T2DM-linked dyslipidemia.

Supramolecular ß-Sheet Forming Peptide Conjugated with Near-Infrared Chromophore for Selective Targeting, Imaging, and Dysfunction of Mitochondria.

Herein, conjugation of the amyloid-ß (Aß) peptide fragment, Lys-Leu-Val-Phe-Phe (KLVFF, fragment of Aß16-20), with an unsymmetrical near-infrared (NIR) cyanine-5 (Cy-5) chromophore is achieved using microwave-assisted solid phase synthesis on 2-chlorotrityl chloride resin. Selective mitochondria tracking and staining in human carcinoma cells are accomplished by the KLVFF/Cy-5 conjugate containing triphenylphosphonium functionality, and this is compared to a control molecule KLVFF/Cy-5c. Mitochondrial target specificity of KLVFF/Cy-5 is established by the colocalization assay using mitochondria selective probe MitoTracker Red, which is monitored by confocal laser scanning microscope and shows a high Pearson's correlation coefficient. The KLVFF/Cy-5 conjugate has high photostability, NIR absorption/emission, high molar extinction coefficient, narrow absorption/emission band, high fluorescence lifetime, and high fluorescence quantum yield. Moreover, mitochondria targeting KLVFF/Cy-5 conjugate reaches the critical aggregation concentration inside the mitochondria of cancer cells due to the strong negative inner mitochondrial membrane potential [(ΔΨm)cancer -220 mV] and self-assembles to form amyloid fibrils at the target site, which is responsible for the mitochondrial dysfunction and cytotoxicity. Annexin V-FITC/PI apoptosis detection assay is used to determine the signal pathway of mitochondria targeted cellular dysfunction.

Targeting and Imaging of Mitochondria Using Near-Infrared Cyanine Dye and Its Application to Multicolor Imaging.

Herein, we report water-soluble mitochondria-selective molecules that consist of a target-specific moiety conjugated with a near-infrared (NIR) imaging agent through variable spacer length. The presented NIR fluorescent cyanine-5 (Cy-5) chromophore exhibits excellent photostability, narrow NIR absorption and emission bands, high molar extinction coefficient, high fluorescence quantum yield, and long fluorescence lifetime. The biological compatibility and negligible cytotoxicity further make the dye an attractive choice for biological applications. Confocal fluorescence microscopic studies in the fixed human lung carcinoma cell line (A549) stained with the targeting NIR Cy-5 dyes (Cy-5a and Cy-5b) at 700 nM concentration show their cellular uptake and localization, which is compared with the nontargeting Cy-5c. Mitochondrial target specificity is demonstrated by colocalization experiments using the mitochondrion-tracking probe, MitoTracker Red and lysosome-tracking probe, LysoTracker Green. Multicolor imaging of cellular organelles in A549 cells is achieved in combination with suitable target-specific dyes with distinct excitation and emission, such as green emitting FM 1-43FX to selectively image the plasma membrane, blue-fluorescent DAPI to stain the nucleus, and the synthesized NIR Cy-5 to image the mitochondria. Higher accumulation of the dye inside the cancer cell mitochondria compared to the noncancerous cell is also demonstrated.

Autophagy protects peripheral blood mononuclear cells against inflammation, oxidative and nitrosative stress in diabetic dyslipidemia.

BACKGROUND: Type 2 diabetes mellitus (T2DM) results in severe oxidative and nitrosative stress and inflammation when associated with hyperlipidemia. In this study, we have attempted to explore the role of autophagy in T2DM subjects with or without dyslipidemia. METHODS: Experiments were carried out in isolated Peripheral blood mononuclear cells (PBMC) from study subjects and insulin resistant HepG2 cells utilizing flow cytometry, confocal microscopy and molecular biology techniques like western blotting, immunofluorescence and real time PCR. RESULTS: In case of T2DM with dyslipidemia, higher population of autophagy positive cell was detected compared to T2DM which may have been originated due to higher stress. Flow cytometric data indicated autophagy to be triggered by both oxidative and nitrosative stress in PBMC of diabetic dyslipidemic patients, which is a novel finding of our work. Expression of LC3 puncta, a hallmark of autophagy was observed at periphery of PBMC and Hep G2 cells in case of diabetic dyslipidemic condition. Increased expression of ATG5, LC3B and Beclin1 supports the autophagic pathway in both PBMC and Hep G2 cells. Upon blocking autophagy by 3-methyl adenine (3MA), the apoptotic cell population increased significantly. Autophagy was also been evidenced to control oxidative stress mediated up-regulation of inflammatory markers like IL-6, TNF-α. CONCLUSION: Induction of autophagy emerged to be a protective mechanism for the diabetic cells coupled with dyslipidemia. Not only Reactive oxygen species, but also reactive nitrogen species was involved in autophagy induction process. Moreover inhibition study documented autophagy to have a protective role in pro-inflammatory responses. Thus, enhancing autophagic activity may be an efficient mechanism leading to new therapeutic strategy to restore the glycemic regulation.

Spectroscopic Studies on Dual Role of Natural Flavonoids in Detoxification of Lead Poisoning: Bench-to-Bedside Preclinical Trial.

Ubiquitousness in the target organs and associated oxidative stress are the most common manifestations of heavy-metal poisoning in living bodies. While chelation of toxic heavy metals is important as therapeutic strategy, scavenging of increased reactive oxygen species, reactive nitrogen species and free radicals are equally important. Here, we have studied the lead (Pb) chelating efficacy of a model flavonoid morin using steady-state and picosecond-resolved optical spectroscopy. The efficacy of morin in presence of other flavonoid (naringin) and polyphenol (ellagic acid) leading to synergistic combination has also been confirmed from the spectroscopic studies. Our studies further reveal that antioxidant activity (2,2-diphenyl-1-picrylhydrazyl assay) of the Pb-morin complex is sustainable compared to that of Pb-free morin. The metal-morin chelate is also found to be significantly soluble compared to that of morin in aqueous media. Heavy-metal chelation and sustainable antioxidant activity of the soluble chelate complex are found to accelerate the Pb-detoxification in the chemical bench (in vitro). Considering the synergistic effect of flavonoids in Pb-detoxification and their omnipresence in medicinal plants, we have prepared a mixture (SKP17LIV01) of flavonoids and polyphenols of plant origin. The mixture has been characterized using high-resolution liquid chromatography assisted mass spectrometry. The mixture (SKP17LIV01) containing 34 flavonoids and 76 other polyphenols have been used to investigate the Pb detoxification in mouse model. The biochemical and histopathological studies on the mouse model confirm the dual action in preclinical studies.

Porphyrins to restrict progression of pancreatic cancer by stabilizing KRAS G-quadruplex: In silico, in vitro and in vivo validation of anticancer strategy.

KRAS, a frequently mutated G-quadruplex forming proto-oncogene is responsible for almost every type of cancer which can form a parallel G-quadruplex structure in the promoter region. G-quadruplex structure is one of the most important drug targets for modern cancer therapy for their unique structure and specificity. Here, we have screened several synthetic porphyrin-based compounds as potential KRAS G-quadruplex stabilizing ligands, using molecular modeling and docking studies. Two novel porphyrins: Porphyrin-1(Cobalt containing) and Porphyrin-2 (Palladium containing) evidenced high affinity towards KRAS-promoter/G-quadruplex. As KRAS mutation is prevalent in pancreatic cancer, the efficacy of these ligands against human pancreatic ductal carcinoma cell line PANC-1 and MiaPaCa2 were examined. Both the Porphyrins exhibited significant cytotoxicity and block metastasis by inhibiting Epithelial to messenchymal transition. In vivo studies confirmed both porphyrin compounds to be effective against EAC tumors along with significantly low toxicity against normal Swiss albino mice. The expression of KRAS gene in porphyrin-treated PANC-1, MiaPaCa2 and tumor-derived EAC cells were drastically reduced at both protein and RNA levels. Thus interaction of porphyrin-based ligands with G-quadruplex DNA at the promoter region of KRAS, might be utilized as a target for anticancer therapeutic strategy.