Studies Towards the Discovery of Novel Natural Products Through Functional Analysis of Environmental DNA-Encoded Type II Polyketide Synthases

Natural products have historically served as a major source for most of the therapeutically relevant compounds. A significant fraction of these natural product-based drugs and leads are derived from microorganisms. Although the traditional culture-based strategy has proven very successful over the course of more than a century of investigation, it excludes a significant portion of the molecules present in environmental samples because majority of the bacteria are not readily amenable to culture-based strategies. This uncultured majority is believed to represent 99% of bacterial species in the environment. Therefore, it has remained a significant challenge to access the chemical diversity present in these microorganisms using culture-based methods. Most modern natural product discovery efforts are faced with the hurdle of high rediscovery rates (\u3e99%) of known metabolites produced by easily cultured bacteria. Metagenomics has evolved as an alternative approach to conventional microbial screening. By directly cloning environmental DNA (eDNA or metagenome) in a surrogate host, one can exhaustively investigate the metagenome, independent of the culturability of the source organisms. Metagenomic approaches can thus provide access to previously untapped pools of chemical diversity. These techniques have been employed in the current thesis to search for a family of therapeutically relevant, polycyclic aromatic natural products, synthesized by Type II polyketide synthases (PKSs) in soil-borne bacteria. Through the functional characterization of soil eDNA-derived Type II PKS containing clones, herein we attempt to identify novel PKS systems that encode structurally diverse metabolites and explore their biological activity (Chapter 1). My doctoral work towards the discovery of novel type II PKS derived natural products has led to the elucidation of the mode of action of eDNA encoded antibiotics Fasamycins A and B. To identify the target of the fasamycins, fasamycin A resistant mutants were generated. Next generation sequencing of these mutants, along with in vitro biochemical assays, showed that fasamycins inhibit the FabF gene of the type II fatty acid biosynthetic system. Subsequently, candidate gene overexpression studies consolidated the mode of action of fasamycins. (Chapter 2) Insights into the genomes of uncultured microorganisms indicate that they are rich in Type II PKS gene clusters. In our attempt to identify novel aromatic polyketide natural products, we investigated the eDNA of soil samples collected from Texas region for unique Type II PKSs using homology-based metagenomic screening techniques. The eDNA-derived clones containing unique type II polyketide synthase genes identified in this screening were examined for the ability to produce clone-specific metabolites in Streptomyces. This led to the identification of hitherto unknown biosynthetic gene cluster for Seitomycin, a Type II PKS derived antibiotic. (Chapter 3) In addition to the work using homology-based metagenomic techniques outlined above, three unique Type II PKS clones were identified based on bioinformatics analysis and comparison with previously characterized PKS gene clusters. These novel pathways were functionally reconstructed from multiple overlapping clones using Transformation Associated Recombination (TAR). Upon complete reassembly, all of the three PKS pathways were demonstrated to produce clone-specific metabolites during heterologous expression studies in Streptomyces host. In future studies, characterization of these metabolites is likely to lead to novel Type II PKS derived natural products. (Chapter 4

Bi-level optimization based on fuzzy if-then rule

A bi-level programming problem has been developed where the functional relationship linking decision variables and the objective functions of leader and follower are not utterly well known to us. Because of the uncertainty in practical life decision-making situation most of the time it is inconvenient to find the veracious relationship between the objective functions of leader, follower and the decision variables. It is expected that the source of information which gives some command about the objective functions of leader and follower, is composed by a block of fuzzy if-then rules. In order to analyze the model, A dynamic programming approach with a suitable fuzzy reasoning scheme is applied to calculate the deterministic functional relationship linking the decision variables and the objective functions of leader as well as follower. Thus a bi-level programming problem is constructed from the actual fuzzy rule-based to the conventional bi-level programming problem. To solve the final problem, we use the lingo software to find the optimal of objective function of follower first and using its solution we optimize the objective function of leader. A numerical example has been solved to signify the computational procedure.</p

Retention of ß-carotene in frozen carrots under varying conditions of temperature and time of storage

Carrots were blanched and stored in a freezer to study the retention of &#946;-carotene. Retention was found to be better in 3 min blanched samples than in 5 min ones. There was an increase in &#946;-carotene content in carrots after 5 days at all storage temperatures: 0, –8, –14, and –18oC. Decrease was found to be insignificant compared to the initial value even after 80 days of storage time at –18oC and for 3 min blanch time. Key Words: Carrots, &#946;-carotene, blanching, freezing, activation energy, storage time. African Journal of Biotechnology Vol.4(1) 2005: 102-10

Modulation of macrophage mannose receptor affects the uptake of virulent and avirulent Leishmania donovani promastigotes

The effect of oxidants and the anti-inflammatory steroid dexamethasone on the attachment and internalization of virulent and avirulent Leishmania donovani promastigotes by the macrophage mannosyl fucosyl receptor was examined. Oxidants and dexamethasone are known to down- and upregulate the expression of the mannose receptor. Macrophages, when treated with 500 &#956;M H2O2 at 37 C for 30 min, stimulate about 45% inhibition in uptake of an avirulent strain (UR6), and 30 and 25% inhibition for virulent strains AG-83 and GE-I, respectively. Treatment of macrophages with dexamethasone for 20 hr resulted in a stimulation in uptake of the parasite. When UR6 was used, a 3-fold increase in uptake was observed compared with the controls. Parasite uptake was also inhibited by the H2O2-generating system, glucose/glucose oxidase; inhibition was blocked by catalase. Treatment of macrophages either with H2O2 or dexamethasone did not affect the binding of the advanced glycosylation end product-bovine serum albumin (AGE-BSA), the ligand for AGE receptor of macrophages. Similarly, indirect evidence also shows that both types 1 and 3 complement receptors (CR1, CR3) are not affected by these treatments, indicating that, besides the mannosyl fucosyl receptor, other receptors are minimally altered in the identified condition. These results suggest that the up- and downregulation of the mannose receptor of macrophages may play a role in affecting L. donovani infection

A Theoretical Development of Distance Measure for Intuitionistic Fuzzy Numbers

The objective of this paper is to introduce a distance measure for intuitionistic fuzzy numbers. Firstly the existing distance measures for intuitionistic fuzzy sets are analyzed and compared with the help of some examples. Then the new distance measure for intuitionistic fuzzy numbers is proposed based on interval difference. Also in particular the type of distance measure for triangle intuitionistic fuzzy numbers is described. The metric properties of the proposed measure are also studied. Some numerical examples are considered for applying the proposed measure and finally the result is compared with the existing ones

Dynamics of Hot QCD Matter -- Current Status and Developments

The discovery and characterization of hot and dense QCD matter, known as Quark Gluon Plasma (QGP), remains the most international collaborative effort and synergy between theorists and experimentalists in modern nuclear physics to date. The experimentalists around the world not only collect an unprecedented amount of data in heavy-ion collisions, at Relativistic Heavy Ion Collider (RHIC), at Brookhaven National Laboratory (BNL) in New York, USA, and the Large Hadron Collider (LHC), at CERN in Geneva, Switzerland but also analyze these data to unravel the mystery of this new phase of matter that filled a few microseconds old universe, just after the Big Bang. In the meantime, advancements in theoretical works and computing capability extend our wisdom about the hot-dense QCD matter and its dynamics through mathematical equations. The exchange of ideas between experimentalists and theoreticians is crucial for the progress of our knowledge. The motivation of this first conference named "HOT QCD Matter 2022" is to bring the community together to have a discourse on this topic. In this article, there are 36 sections discussing various topics in the field of relativistic heavy-ion collisions and related phenomena that cover a snapshot of the current experimental observations and theoretical progress. This article begins with the theoretical overview of relativistic spin-hydrodynamics in the presence of the external magnetic field, followed by the Lattice QCD results on heavy quarks in QGP, and finally, it ends with an overview of experiment results.Comment: Compilation of the contributions (148 pages) as presented in the `Hot QCD Matter 2022 conference', held from May 12 to 14, 2022, jointly organized by IIT Goa & Goa University, Goa, Indi

Global baryon number conservation encoded in net-proton fluctuations measured in Pb–Pb collisions at √sNN = 2.76 TeV

Experimental results are presented on event-by-event net-proton fluctuation measurements in Pb–Pb collisions at √SNN=2.76 TeV, recorded by the ALICE detector at the CERN LHC. These measurements have as their ultimate goal an experimental test of Lattice QCD (LQCD) predictions on second and higher order cumulants of net-baryon distributions to search for critical behavior near the QCD phase boundary. Before confronting them with LQCD predictions, account has to be taken of correlations stemming from baryon number conservation as well as fluctuations of participating nucleons. Both effects influence the experimental measurements and are usually not considered in theoretical calculations. For the first time, it is shown that event-by-event baryon number conservation leads to subtle long-range correlations arising from very early interactions in the collisions.publishedVersio

ϒ production in p–Pb collisions at √sNN=8.16 TeV

ϒ production in p–Pb interactions is studied at the centre-of-mass energy per nucleon–nucleon collision √sNN = 8.16 TeV with the ALICE detector at the CERN LHC. The measurement is performed reconstructing bottomonium resonances via their dimuon decay channel, in the centre-of-mass rapidity intervals 2.03 < ycms < 3.53 and −4.46 < ycms < −2.96, down to zero transverse momentum. In this work, results on the ϒ(1S) production cross section as a function of rapidity and transverse momentum are presented. The corresponding nuclear modification factor shows a suppression of the ϒ(1S) yields with respect to pp collisions, both at forward and backward rapidity. This suppression is stronger in the low transverse momentum region and shows no significant dependence on the centrality of the interactions. Furthermore, the ϒ(2S) nuclear modification factor is evaluated, suggesting a suppression similar to that of the ϒ(1S). A first measurement of the ϒ(3S) has also been performed. Finally, results are compared with previous ALICE measurements in p–Pb collisions at √sNN = 5.02 TeV and with theoretical calculations.publishedVersio

(Anti-)deuteron production in pp collisions at 1as=13TeV

The study of (anti-)deuteron production in pp collisions has proven to be a powerful tool to investigate the formation mechanism of loosely bound states in high-energy hadronic collisions. In this paper the production of (anti-)deuterons is studied as a function of the charged particle multiplicity in inelastic pp collisions at s=13 TeV using the ALICE experiment. Thanks to the large number of accumulated minimum bias events, it has been possible to measure (anti-)deuteron production in pp collisions up to the same charged particle multiplicity (d Nch/ d \u3b7 3c 26) as measured in p\u2013Pb collisions at similar centre-of-mass energies. Within the uncertainties, the deuteron yield in pp collisions resembles the one in p\u2013Pb interactions, suggesting a common formation mechanism behind the production of light nuclei in hadronic interactions. In this context the measurements are compared with the expectations of coalescence and statistical hadronisation models (SHM)