Deciphering Redox Free Radical Mechanisms of 15LOX/PEBP1-Driven Ferroptosis in Skin

This proposal will uncover critical molecular mechanisms governing a new class of death signaling pathway (ie ferroptosis) and its relevance to ultraviolet radiation (UVR)-induced skin injury and cutaneous malignancies. The team will focus on the key role played by protein 15-lipoxygenase (15-LOX) in ferroptosis using a broad interdisciplinary approach. Specifically the team will: (1) Elucidate the local structure around the catalytic site of 15-LOX as well as in a complex of 15-LOX with PEBP1 in order to understand how metal-protein interactions affect 15-LOX function and how PEBP1 binding to 15-LOX opens the active site; (2) Measure the atomic structure and molecular interactions of 15-LOX active site in the presence of newly designed specific inhibitors of 15-LOX/PEBP1 complex in order to understand the mechanism of inhibition; and (3) Characterize the previously unexplored role of 15-LOX/PEBP1 complex in triggering ferroptotic death of skin cells (melanocytes and keratinocytes) and its regulation by selective new inhibitors and melanin biosynthesis. The interdisciplinary team brings together the experts from the School of Arts and Sciences, the School of Medicine and the School of Public Health. The project builds on the expertise of the Kagan group in the discovery and understanding of the biochemical network that underpins ferroptosis. The Saxena group brings in the knowledgebase to measure at the atomic level key interactions that are essential for ferroptosis in order to aid the development of new ferroptosis inhibitors. Finally, the Bunimovich group will expand the mechanistic studies to skin cell models and will explore the relevance of ferroptosis and 15LOX/PEBP1 inhibition in dermatological diseases. Understanding the mechanisms and regulation of ferroptosis in the skin will be critical for the prevention and treatment of radiation and drug-induced skin injury, as well as inflammatory, autoimmune and neoplastic cutaneous diseases such as psoriasis, lupus and melanoma

Kloniranje, izražajnost i pročišćavanje bjelančevine 28 iz vanjske membrane Salmonelle enterice serovar Typhimurium za razvoj podjediničnog cjepiva - kratko priopćenje

Salmonella Typhimurium, a major gastrointestinal pathogen, poses a global threat to human health. Public health problems associated with this organism have increased to the extent that it has become a major issue. The bacterium is becoming resistant to the commonly available antibiotics, and vaccines also suffer from limitations such as short lived immunity. Therefore, there is an urgent need for the development of an effective vaccine. The outer membrane proteins (Omps) of Salmonella have proven their capability to be developed as a vaccine candidate for prevention of salmonellosis. With this aim, in the present study the Omp28 gene of Salmonella Typhimurium was amplified, cloned and expressed under an IPTG induction system. The recombinant protein thus produced was purified and tested for its antigenicity. The antigenicity of the purified protein was confirmed by western blotting with antiserum raised in rabbit against Omps of S. Typhimurium. The Omp28 gene was amplified as a 330bp product. The expressed protein was found to be of approximately 28kDa and it produced a strong signal in western blot analysis. This study concluded that Omp28 may be proven to be an effective candidate for the development of r-DNA vaccine against salmonellosis.Salmonella Typhimurium glavni je gastrointestinalni patogen koji je globalna prijetnja ljudskome zdravlju. Javnozdravstveni problemi povezani s ovim organizmom povećali su se do te mjere da je postao glavno pitanje na koje se traže brojni odgovori. Bakterija postaje otporna na najčešće dostupne antibiotike, a ograničavajuća uporaba cjepiva povezana je s kratkotrajnim imunitetom. Zbog toga postoji hitna potreba za razvoj učinkovitog cjepiva. Vanjske bjelančevine membrane (engl. Outer membrane proteins, Omps) salmonele dokazale su svoju sposobnost kandidata za razvoj cjepiva koje bi se koristilo u prevenciji salmoneloze. S tim je ciljem u ovom radu, pod uvjetima IPTG indukcijskog sustava, provedeno umnažanje i kloniranje te provjerena izražajnost gena za Omp28 iz Salmonelle Typhimurium. Tako dobivena rekombinantna bjelančevina pročišćena je i testirana s obzirom na antigenu sposobnost. Antigena sposobnost pročišćene bjelančevine potvrđena je uporabom Western blot metode s antiserumom protiv Omps-a iz S. Typhimurium dobivenim od zeca. Genom Omp28 umnožen je kao 330bp produkt. Bjelančevina je imala približno 28 kDa i Western blot analizom pokazala je izraženost jakog signala. Ovim je istraživanjem zaključeno da Omp28 može poslužiti kao učinkoviti kandidat za razvoj r-DNA cjepiva protiv salmoneloze

SYNTHESIS AND EVALUATION OF SOME BENZOTHIAZOLE DERIVATIVES AS ANTIDIABETIC AGENTS

Objective: The objective of the present research investigation involves synthesis and biological evaluation of antidiabetic activity of benzothiazole derivatives.Methods: A novel series of benzothiazole derivatives 7(a-l) were synthesised and synthesised compounds were characterised for different physical and chemical properties like molecular formula, molecular weight, melting point, percentage yield, Rf value, IR, 1HNMR, 13CNMR and mass spectroscopy. The newly synthesised benzothiazole derivatives were subsequently assayed in vivo to investigate their hypoglycemic activity by the alloxan-induced diabetic model in rats. Results: All the synthesised derivatives showed significant biological efficacy. The compound 7d at 350 mg/kg exerted maximum glucose lowering effects whereas 7c showed minimum glucose lowering effects. All the compounds were effective, and experimental results were statistically significant at p<0.01 and p<0.05 level.Conclusion: From the results, it is clear that compound 7d demonstrated potent anti-diabetic activity and would be of better use in drug development to combat the metabolic disorder in future

Protective Role of Ashwagandha Leaf Extract and Its Component Withanone on Scopolamine-Induced Changes in the Brain and Brain-Derived Cells

BACKGROUND:Scopolamine is a well-known cholinergic antagonist that causes amnesia in human and animal models. Scopolamine-induced amnesia in rodent models has been widely used to understand the molecular, biochemical, behavioral changes, and to delineate therapeutic targets of memory impairment. Although this has been linked to the decrease in central cholinergic neuronal activity following the blockade of muscarinic receptors, the underlying molecular and cellular mechanism(s) particularly the effect on neuroplasticity remains elusive. In the present study, we have investigated (i) the effects of scopolamine on the molecules involved in neuronal and glial plasticity both in vivo and in vitro and (ii) their recovery by alcoholic extract of Ashwagandha leaves (i-Extract). METHODOLOGY/PRINCIPAL FINDINGS:As a drug model, scopolamine hydrobromide was administered intraperitoneally to mice and its effect on the brain function was determined by molecular analyses. The results showed that the scopolamine caused downregulation of the expression of BDNF and GFAP in dose and time dependent manner, and these effects were markedly attenuated in response to i-Extract treatment. Similar to our observations in animal model system, we found that the scopolamine induced cytotoxicity in IMR32 neuronal and C6 glioma cells. It was associated with downregulation of neuronal cell markers NF-H, MAP2, PSD-95, GAP-43 and glial cell marker GFAP and with upregulation of DNA damage--γH2AX and oxidative stress--ROS markers. Furthermore, these molecules showed recovery when cells were treated with i-Extract or its purified component, withanone. CONCLUSION:Our study suggested that besides cholinergic blockade, scopolamine-induced memory loss may be associated with oxidative stress and Ashwagandha i-Extract, and withanone may serve as potential preventive and therapeutic agents for neurodegenerative disorders and hence warrant further molecular analyses

Differential gene expression profiling of esophageal adenocarcinoma

BackgroundDifferential gene expression offers an attractive means by which to study genes that may be involved in disease development and/or progression. We performed quantitative gene expression in various stages of esophageal adenocarcinoma, treated exclusively by surgery with complete 2-field lymphadenectomy, in an attempt to discern genes involved in disease progression as well as genes that may predict survival.MethodsGene expression profiling was accomplished by cDNA-mediated annealing, selection, extension, and ligation (DASL) assay. RNA was extracted from 89 archived formalin-fixed, paraffin-embedded esophageal adenocarcinoma tissues. DASL assay was performed with the Sentrix Universal Array (Illumina Corp, San Diego, Calif) of 502 known cancer-related genes. Bioinformatics tools were used to determine significant differential gene expression in T1-2 versus T3-4 tumors and tumors without lymph node involvement (N0) versus tumors with lymph node involvement (N+). Gene expression was also correlated with overall survival.ResultsTwenty-one genes were overexpressed in T1-2 compared with T3-4 tumors (false discovery rate of 0). Underexpression of 1 gene was seen in N+ compared with N0 tumors (false discovery rate of 0). For overall survival, underexpression of 9 genes correlated with long survival.ConclusionsUsing differential gene expression of 502 known cancer genes, we identified genes that may be involved at various stages in the progression of esophageal adenocarcinoma. We also identified genes that may correlate with prolonged survival and, thus, may serve as prognostic markers. These findings may provide further insight into the mechanisms of development and/or progression of esophageal adenocarcinoma. Prospective studies are needed to verify the prognostic value of these genes

Reachability in Dynamical Systems with Rounding

We consider reachability in dynamical systems with discrete linear updates, but with fixed digital precision, i.e., such that values of the system are rounded at each step. Given a matrix $M \in \mathbb{Q}^{d \times d}$, an initial vector $x\in\mathbb{Q}^{d}$, a granularity $g\in \mathbb{Q}_+$ and a rounding operation $[\cdot]$ projecting a vector of $\mathbb{Q}^{d}$ onto another vector whose every entry is a multiple of $g$, we are interested in the behaviour of the orbit $\mathcal{O}={}$, i.e., the trajectory of a linear dynamical system in which the state is rounded after each step. For arbitrary rounding functions with bounded effect, we show that the complexity of deciding point-to-point reachability---whether a given target $y \in\mathbb{Q}^{d}$ belongs to $\mathcal{O}$---is PSPACE-complete for hyperbolic systems (when no eigenvalue of $M$ has modulus one). We also establish decidability without any restrictions on eigenvalues for several natural classes of rounding functions.Comment: To appear at FSTTCS'2

DEVELOPMENT AND CHARACTERIZATION OF SRM MICROSPHERES OF REPAGLINIDE

The aim of current work to develop and evaluate sustained release mucoadhesive (SRM) microspheres of Repaglinide using emulsification solvent evaporation technique. Effects of formulation variables i.e. polymer concentration and phase volume ratio on particle size, % mucoadhesion and drug release were investigated in this study. Scanning electron microscopy of microspheres with maximum drug content (Formulation CH1:8) demonstrated smooth surface spherical particles with mean diameter of 64.78 ± 3.26 μm. The mean Particle size, % drug loading and mucoadhesion were found to vary by changing the formulation variables. Microspheres size was significantly increased as increasing the polymer concentration in the aqueous phase while size of microspheres decrease as increase in volume of continuous phase. Decrease in size of microspheres leads to decrease in mucoadhesion time, % drug loading and faster the drug release. It can be concluded that the present mucoadhesive microspheres can be an ideal system to deliver the Repaglinide in sustained release manner for management of Type II Diabetes Mellitus

Change in dysphagia and laryngeal function after radical radiotherapy in laryngo pharyngeal malignancies — a prospective observational study

Background: Intensity modulated radiotherapy (IMRT) has the perceived advantage of function preservation by reduction of toxicities in the treatment of laryngo-pharyngeal malignancies. The aim of the study was to assess changes in dysphagia from baseline (i.e. prior to start of treatment) at three and six months post treatment in patients with laryngo-pharyngeal malignancies treated with radical radiotherapy ± chemotherapy. Functional assessment of other structures involved in swallowing was also studied. Materials and methods: 40 patients were sampled consecutively. 33 were available for final analysis. Dysphagia, laryngeal edema, xerostomia and voice of patients were assessed at baseline and at three and six months after treatment. Radiation was delivered with simultaneous integrated boost (SIB) using volumetric modulated radiation therapy (VMAT). Concurrent chemotherapy was three weekly cisplatin 100 mg/m2. Results: Proportion of patients with dysphagia rose significantly from 45.5% before the start of treatment to 57.6% at three months and 60.6% at six months post treatment (p = 0.019). 67% patients received chemotherapy and addition of chemotherapy had a significant correlation with dysphagia (p = 0.05, r = –0.336). Severity of dysphagia at three and six months correlated significantly with the mean dose received by the superior constrictors (p = 0.003, r = 0.508 and p = 0.024, r = 0.391) and oral cavity (p = 0.001, r = 0.558 and p = 0.003, r = 0.501). There was a significant worsening in laryngeal edema at three and six months post treatment (p < 0.01) when compared to the pre-treatment examination findings with 60.6% of patients having grade two edema at six months. Significant fall in the mean spoken fundamental frequency from baseline was seen at 6 months (p = 0.04), mean fall was 21.3 Hz (95% CI: 1.5–41 Hz) with significant increase in roughness of voice post treatment (p = 0.01). Conclusion: There was progressive worsening in dysphagia, laryngeal edema and voice in laryngo-pharyngeal malignancies post radical radiotherapy ± chemotherapy