pH-dependent complex formation with TAR RNA and DNA: application towards logic gates.

Nucleic acid-based logic gates have shown great potential in biotechnology, medicine as well as diagnostics. Herein, we have constructed pH-responsive logic devices by utilizing HIV-1 TAR hairpins in combination with a thiazole peptide that exhibits turn-on fluorescence upon interacting with TAR RNA or DNA. Based on this, INHIBIT-AND and YES-INHIBIT-AND logic gates were constructed in parallel. The pH alteration leads to conformational changes of the hairpin structure, enabling the construction of a multi-reset reusable logic system which could be developed for in vitro sensing of the HIV-1 viral RNA.

Critical Review on Balanites aegyptiaca Delile: Phytoconstituents, Pharmacological Properties and Nanointerventions.

Balanites aegyptiaca Delile (BA) is an enduring xerophytic woody and spinous flowering tree and is commonly known as desert date or Ingudi (Hingot). It belongs to the family Zygophyllaceae, which is specific to be drought areas of Nigeria, Africa, South Asia and India (Rajasthan). In Ayurveda, this traditional medicinal plant is reported for the management of jaundice, syphilis, yellow fever, metabolic disorders, liver, and spleen problems. The main aim of the review is to compile its medicinal uses and further advancements to showcase the promises inherited in various parts of the plant for the benefit of mankind. As per the literature survey, various researchers have focused on the detailed investigation of BA including the phytopharmacological evidence, chemical constituents, nano-formulations, commercialized products, and clinical trials. Several remarkable scaffolds and isolated compounds like diosgenin, yamogenin, balanitin1/2, balanitin 3, bal4/5, bal6/7, rutin-3-glycosides, 3,7-diglycosides, (3, 12, 14, 16)-(12-hydroxycholest-5-ene-3,16-diyl-bis)-D-glucopyranoside and balanitoside have been identified. Additionally, this traditional plant has been scientifically proven by in vitro and in vivo. Based on the complete review of this plant, most of the compounds have been isolated from the fruit and kernel part. Additionally, based on the literature, a histogram was developed for pharmacological activity in which antidiabetic study was found to be more compared to other pharmacological activity. As a spinous desert dates, this plant needs to be explored more to bring out newer phytochemicals in the management of various diseases.

Assessment of environmental and carcinogenic health hazards from heavy metal contamination in sediments of wetlands.

Sediment contamination jeopardizes wetlands by harming aquatic organisms, disrupting food webs, and reducing biodiversity. Carcinogenic substances like heavy metals bioaccumulate in sediments and expose consumers to a greater risk of cancer. This study reports Pb, Cr, Cu, and Zn levels in sediments from eight wetlands in India. The Pb (51.25 ± 4.46 µg/g) and Cr (266 ± 6.95 µg/g) concentrations were highest in Hirakud, Cu (34.27 ± 2.2 µg/g) in Bhadrak, and Zn (55.45 ± 2.93 µg/g) in Koraput. The mean Pb, Cr, and Cu values in sediments exceeded the toxicity reference value. The contamination factor for Cr was the highest of the four metals studied at Hirakud (CF = 7.60) and Talcher (CF = 6.97). Furthermore, high and moderate positive correlations were observed between Cu and Zn (r = 0.77) and Pb and Cr (r = 0.36), respectively, across all sites. Cancer patients were found to be more concentrated in areas with higher concentrations of Pb and Cr, which are more carcinogenic. The link between heavy metals in wetland sediments and human cancer could be used to make policies that limit people's exposure to heavy metals and protect their health.

Role of abiotic factors in enhancing the capacity of mangroves in reducing ocean acidification.

The present study investigated the effects of rising carbon dioxide levels in nature and the carbon sequestration potential of dominant mangrove species for reducing the toxic effects of ocean acidification. The study was conducted on the east coast of Odisha, in the western Bay of Bengal. To determine the effect of these ambient parameters on the absorption of carbon dioxide by the mangroves, water temperature, salinity, pH levels of seawater along with soil texture and pH, salinity expressed in electrical conductivity, compactness expressed in bulk density, and soil organic carbon were simultaneously monitored. The aboveground biomass and carbon of the selected species were studied for 2 consecutive years at 10 designated stations. The total carbon calculated for the study area varied from 242.50 ± 49.00 to 1321.29 ± 445.52 tons with a mean of 626.68 ± 174.81 tons for Bhitarkanika and Mahanadi mangrove chunks. This is equivalent to 2299.92 ± 641.55 tons of CO2 absorbed from the atmosphere. A total of 27 equations were selected as the best fit models for the study area. The equations between mangrove biomass and carbon along with aquatic and edaphic factors governing the pH of water and soil strongly support the positive influence of mangrove photosynthetic activity in shifting the equilibrium toward alkalinity. This calls for conservation of mangrove ecosystem to minimize the pace of acidification of estuarine water. The results indicate that Excoecariaagallocha and Avicennia marina as are the most capable species for combatting maximum carbon dioxide toxicity from the atmosphere; which will be helpful in REDD + programs and carbon-based payments for ecosystem services (PES).

Orally Bioavailable Enzymatic Inhibitor of CD38, MK-0159, Protects against Ischemia/Reperfusion Injury in the Murine Heart.

CD38 is one of the major nicotinamide adenine dinucleotide (NAD+)- and nicotinamide adenine dinucleotide phosphate (NADP+)-consuming enzymes in mammals. NAD+, NADP+, and their reduced counterparts are essential coenzymes for numerous enzymatic reactions, including the maintenance of cellular and mitochondrial redox balance. CD38 expression is upregulated in age-associated inflammation as well as numerous metabolic diseases, resulting in cellular and mitochondrial dysfunction. Recent literature studies demonstrate that CD38 is activated upon ischemia/reperfusion (I/R), leading to a depletion of NADP+, which results in endothelial damage and myocardial infarction in the heart. Despite increasing evidence of CD38 involvement in various disease states, relatively few CD38 enzymatic inhibitors have been reported to date. Herein, we describe a CD38 enzymatic inhibitor (MK-0159, IC50 = 3 nM against murine CD38) that inhibits CD38 in in vitro assay. Mice treated with MK-0159 show strong protection from myocardial damage upon cardiac I/R injury compared to those treated with NAD+ precursors (nicotinamide riboside) or the known CD38 inhibitor, 78c.

Identification of behenic acid as medicinal food for the diabetes mellitus: structure-based computational approach and molecular dynamics simulation studies.

Vegetables, fruits, and spices have a pivotal role in maintaining the metabolism and have diversified health welfares. The food products have medicinal values (Medifood) and benefitted prophylactically and simultaneously; health experts recommend them due to their synergistic effect on medicine. Therefore, research thrusts have developed medifood and counted in the field of pharma-nutrition. Type II diabetes mellitus is one of the foremost progressive metabolic disorders, which affects several millions of people around the globe due to ignorance of food habits. Improvement in lifestyle and intake of habitual food may improve the diabetic conditions. In the present study, we performed in silico studies of fatty acids on the free fatty acid 1 receptor to identify fatty acids readily available in daily food content. Additionally, molecular dynamic simulation study to recognize the dynamic behavior of protein-fatty acid complex. The in silico results suggest that 04 fatty acids may be promising in which behenic acid was most potential and showed binding energy of - 110.857 kcal/mol. The stability of the protein-ligand complex is confirm in simulation studies, with occupancy of 95% and 84%, respectively. Behenic acid is present in different parts of the Moringa tree and may serve as medifood in type II diabetes mellitus.

Recent update on nano-phytopharmaceuticals in the management of diabetes.

Due to changed lifestyle and other reasons, diabetes has become one of the common metabolic disorder of the globe. Numerous therapeutic options are available, which controls the plasma glucose levels. However, most of the drugs are associated with some undesired side effects. Owing to the side effects and enhanced understanding of the phytochemicals, an inclination toward herbal medicine is seen in the population. These herbal products are also associated with concerns like poor aqueous solubility, compromised permeation, and a low degree of bioavailability. So, the emergence of nanotechnology in the herbal medicine is required to nullify the associated concerns of conventional antidiabetic drugs. The present review aims to compile the literature available for the nano-interventions pertinent to herbal products for diabetes management.

Drug-Drug Binary Solids of Nitrofurantoin and Trimethoprim: Crystal Engineering and Pharmaceutical Properties.

With the aim of developing multidrug solids through a tuned crystal engineering approach, we have selected two antiurinary infective drugs, namely, nitrofurantoin (NF) and trimethoprim (TMP) and isolated eight binary drug-drug solid solvates along with a nonsolvated cocrystal. Crystal structure analyses were performed for eight of these solids and rationalized in terms of known supramolecular synthons formed by pyrimidine, imide, and amine functionalities. Notably, the TMP-NF anhydrous cocrystal and its ionic cocrystal hydrate exhibit enhanced equilibrium solubilities compared to pure NF or the simple NF hydrate. Furthermore, the ionic cocrystal hydrate exhibits greater antibacterial activity against the Gram-negative bacteria, E. coli, compared to the parent TMP and NF at the lowest concentration of 3.9 µg/mL. This study indicates initial pathways using the cocrystal methodology that would help to eventually arrive at an antiurinary cocrystal with optimal properties.

Light-controlled twister ribozyme with single-molecule detection resolves RNA function in time and space.

Small ribozymes such as Oryza sativa twister spontaneously cleave their own RNA when the ribozyme folds into its active conformation. The coupling between twister folding and self-cleavage has been difficult to study, however, because the active ribozyme rapidly converts to product. Here, we describe the synthesis of a photocaged nucleotide that releases guanosine within microseconds upon photosolvolysis with blue light. Application of this tool to O. sativa twister achieved the spatial (75 µm) and temporal (≤30 ms) control required to resolve folding and self-cleavage events when combined with single-molecule fluorescence detection of the ribozyme folding pathway. Real-time observation of single ribozymes after photo-deprotection showed that the precleaved folded state is unstable and quickly unfolds if the RNA does not react. Kinetic analysis showed that Mg2+ and Mn2+ ions increase ribozyme efficiency by making transitions to the high energy active conformation more probable, rather than by stabilizing the folded ground state or the cleaved product. This tool for light-controlled single RNA folding should offer precise and rapid control of other nucleic acid systems.

Target-Directed Azide-Alkyne Cycloaddition for Assembling HIV-1 TAR RNA Binding Ligands.

The highly conserved HIV-1 transactivation response element (TAR) binds to the trans-activator protein Tat and facilitates viral replication in its latent state. The inhibition of Tat-TAR interactions by selectively targeting TAR RNA has been used as a strategy to develop potent antiviral agents. Therefore, HIV-1 TAR RNA represents a paradigmatic system for therapeutic intervention. Herein, we have employed biotin-tagged TAR RNA to assemble its own ligands from a pool of reactive azide and alkyne building blocks. To identify the binding sites and selectivity of the ligands, the in situ cycloaddition has been further performed using control nucleotide (TAR DNA and TAR RNA without bulge) templates. The hit triazole-linked thiazole peptidomimetic products have been isolated from the biotin-tagged target templates using streptavidin beads. The major triazole lead generated by the TAR RNA presumably binds in the bulge region, shows specificity for TAR RNA over TAR DNA, and inhibits Tat-TAR interactions.

G-Quadruplex-Binding Small Molecule Induces Synthetic Lethality in Breast Cancer Cells by Inhibiting c-MYC and BCL2 Expression.

Herein, a prolinamide-derived peptidomimetic that preferentially binds to c-MYC and BCL2 G-quadruplexes present in the promoter regions of apoptosis-related genes (c-MYC and BCL2) over other DNA quadruplexes are described. Biological assays, such as real-time quantitative reverse transcription, western blot, dual luciferase, and small interfering RNA knockdown assays, indicate that the ligand triggers a synthetic lethal interaction by simultaneously inhibiting the expression of c-MYC and BCL2 genes through their promoter G-quadruplexes. The ligand shows antiproliferative activity in MCF-7 cells that overexpress both MYC and BCL2 genes, in comparison to cells that overexpress either of the two. Moreover, the ligand induces S-phase cell-cycle arrest, DNA damage, and apoptosis in MCF-7 cells.

Cell penetrating thiazole peptides inhibit c-MYC expression via site-specific targeting of c-MYC G-quadruplex.

The structural differences among different G-quadruplexes provide an opportunity for site-specific targeting of a particular G-quadruplex structure. However, majority of G-quadruplex ligands described thus far show little selectivity among different G-quadruplexes. In this work, we delineate the design and synthesis of a crescent-shaped thiazole peptide that preferentially stabilizes c-MYC quadruplex over other promoter G-quadruplexes and inhibits c-MYC oncogene expression. Biophysical analysis such as Förster resonance energy transfer (FRET) melting and fluorescence spectroscopy show that the thiazole peptide TH3 can selectively interact with the c-MYC G-quadruplex over other investigated G-quadruplexes and duplex DNA. NMR spectroscopy reveals that peptide TH3 binds to the terminal G-quartets and capping regions present in the 5'- and 3'-ends of c-MYC G-quadruplex with a 2:1 stoichiometry; whereas structurally related distamycin A is reported to interact with quadruplex structures via groove binding and end stacking modes with 4:1 stoichiometry. Importantly, qRT-PCR, western blot and dual luciferase reporter assay show that TH3 downregulates c-MYC expression by stabilizing the c-MYC G-quadruplex in cancer cells. Moreover, TH3 localizes within the nucleus of cancer cells and exhibits antiproliferative activities by inducing S phase cell cycle arrest and apoptosis.

Enzyme-Regulated DNA-Based Logic Device.

Herein, we report a carbazole (Cz) ligand that displays distinct turn-on fluorescence signals upon interaction with human telomeric G-quadruplex ( h-TELO) and nuclease enzymes. Interestingly, Cz selectively binds and stabilizes the mixed hybrid topology of h-TELO G-quadruplex that withstands digestion by exonucleases and nuclease S1. The distinct fluorescence signatures of Cz-stabilized h-TELO with nucleases are used to design conceptually novel DNA devices for selectively detecting the enzymatic activity of DNase I as well as performing logic operations. An INHIBIT logic gate is constructed using h-TELO and DNase I as the inputs while the inputs of h-TELO and nuclease S1 form a YES logic gate. Furthermore, a two-input two-output reusable logic device with "multireset" function is developed by using h-TELO and DNase I as inputs. On the basis of this platform, combinatorial logic systems (INHIBIT-INHIBIT and NOR-OR) have been successfully installed using different combinations of nucleases as inputs. Moreover, this new strategy of using a synthetic dual emissive probe and enzyme/DNA inputs for constructing reusable logic device may find important applications in biological computing and information processing.

Preferential targeting of i-motifs and G-quadruplexes by small molecules.

i-Motifs and G-quadruplexes are dynamic nucleic acid secondary structures, which are believed to play key roles in gene expression. We herein report two peptidomimetic ligands (PBP1 and PBP2) that selectively target i-motifs and G-quadruplexes over double-stranded DNA. These peptidomimetics, regioisomeric with respect to the position of triazole/prolinamide motifs, have been synthesized using a modular method involving Cu(i)-catalyzed azide and alkyne cycloaddition. The para-isomer, PBP1 exhibits high selectivity for i-motifs while the meta-isomer PBP2 binds selectively to G-quadruplex structures. Interestingly, these ligands have the ability to induce G-quadruplex or i-motif structures from the unstructured single-stranded DNA conformations, as observed using single molecule Förster resonance energy transfer (smFRET) studies. The quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and dual-luciferase assays indicate that PBP1 upregulates and PBP2 downregulates BCL-2 gene expression in cancer cells.

EC-tagging allows cell type-specific RNA analysis.

Purification of cell type-specific RNAs remains a significant challenge. One solution involves biosynthetic tagging of target RNAs. RNA tagging via incorporation of 4-thiouracil (TU) in cells expressing transgenic uracil phosphoribosyltransferase (UPRT), a method known as TU-tagging, has been used in multiple systems but can have limited specificity due to endogenous pathways of TU incorporation. Here, we describe an alternative method that requires the activity of two enzymes: cytosine deaminase (CD) and UPRT. We found that the sequential activity of these enzymes converts 5-ethynylcytosine (EC) to 5-ethynyluridine monophosphate that is subsequently incorporated into nascent RNAs. The ethynyl group allows efficient detection and purification of tagged RNAs. We show that 'EC-tagging' occurs in tissue culture cells and Drosophila engineered to express CD and UPRT. Additional control can be achieved through a split-CD approach in which functional CD is reconstituted from independently expressed fragments. We demonstrate the sensitivity and specificity of EC-tagging by obtaining cell type-specific gene expression data from intact Drosophila larvae, including transcriptome measurements from a small population of central brain neurons. EC-tagging provides several advantages over existing techniques and should be broadly useful for investigating the role of differential RNA expression in cell identity, physiology and pathology.

Synergistic Effects of an Irreversible DNA Polymerase Inhibitor and DNA Damaging Agents on HeLa Cells.

DNA repair is vital to maintaining genome integrity but thwarts the effects of cytotoxic agents that target nucleic acids. Consequently, repair enzymes are potential targets for molecules that modulate cell function and anticancer therapeutics. DNA polymerase ß (Pol ß) is an attractive target because it plays a key role in base excision repair (BER), a primary pathway that repairs the effects of many DNA damaging agents. We previously identified an irreversible inhibitor of Pol ß whose design was based upon a DNA lesion that inactivates Pol ß and its back up BER enzyme, DNA polymerase λ (Pol λ). Using this molecule as a starting point, we characterized an irreversible inhibitor (13) of Pol ß (IC50 = 0.4 µM) and Pol λ (IC50 = 0.25 µM) from a 130-member library of candidates that is ∼50-fold more effective against Pol ß. Pro-13 (5 µM) is only slightly cytotoxic to human cervical cancer cells (HeLa) but potentiates the cytotoxicity of methyl methanesulfonate (MMS). DNA isolated from HeLa cells treated with MMS contains a ∼3-fold greater amount of abasic sites when pro-13 is present, consistent with inhibition of DNA repair. Proinhibitor pro-13 continues to induce cytotoxicity in DNA damaged cells following MMS removal. HeLa cell cytotoxicity is increased ∼100-fold following an 8 h incubation with pro-13 after cells were originally subjected to conditions under which 20% of the cells survive and reproduce. The potentiation of MMS cytotoxicity by pro-13 is greater than any previously reported BER enzyme repair inhibitor.

Mechanistic Studies on RNA Strand Scission from a C2'-Radical.

The C2'-carbon-hydrogen bond in ribonucleotides is significantly weaker than other carbohydrate carbon-hydrogen bonds in RNA or DNA. Independent generation of the C2'-uridine radical (1) in RNA oligonucleotides via Norrish type I photocleavage of a ketone-substituted nucleotide yields direct strand breaks via cleavage of the ß-phosphate. The reactivity of 1 in different sequences and under a variety of conditions suggests that the rate constant for strand scission is significantly greater than 106 s-1 at pH 7.2. The initially formed C2'-radical (1) is not trapped under a variety of conditions, consistent with computational studies ( Chem.-Eur. J. 2009 , 15 , 2394 ) that suggest that the barrier to strand scission is very low and that synchronous proton transfer from the 2'-hydroxyl to the departing phosphate group facilitates cleavage. The C2'-radical could be a significant contributor to RNA strand scission by the hydroxyl radical, particularly under anaerobic conditions where 1 can be produced from nucleobase radicals.

Synthesis of Fluorescent Binaphthyl Amines That Bind c-MYC G-Quadruplex DNA and Repress c-MYC Expression.

Two novel binaphthyl amines have been designed and synthesized using Buchwald amination and oxidative homocoupling as key steps. The binaphthyl amine containing two triazole rings shows higher affinity for c-MYC G-quadruplex, exhibits fluorescence "turn-on" response with c-MYC, and stains the nucleus in cells. The triazolyl binaphthyl amine shows cytotoxicity for cancer cells by inducing G2/M phase cell cycle arrest and apoptosis. Moreover, both ligands can downregulate c-MYC expression at transcriptional and translational levels.

Ribosome-Mediated Incorporation of Dipeptides and Dipeptide Analogues into Proteins in Vitro.

Plasmids containing 23S rRNA randomized at positions 2057-2063 and 2502-2507 were introduced into Escherichia coli, affording a library of clones which produced modified ribosomes in addition to the pre-existing wild-type ribosomes. These clones were screened with a derivative of puromycin, a natural product which acts as an analogue of the 3'-end of aminoacyl-tRNA and terminates protein synthesis by accepting the growing polypeptide chain, thereby killing bacterial cells. The puromycin derivative in this study contained the dipeptide p-methoxyphenylalanylglycine, implying the ability of the modified ribosomes in clones sensitive to this puromycin analogue to recognize dipeptides. Several clones inhibited by the puromycin derivative were used to make S-30 preparations, and some of these were shown to support the incorporation of dipeptides into proteins. The four incorporated species included two dipeptides (Gly-Phe (2) and Phe-Gly (3)), as well as a thiolated dipeptide analogue (4) and a fluorescent oxazole (5) having amine and carboxyl groups approximately the same distance apart as in a normal dipeptide. A protein containing both thiolated dipeptide 4 and a 7-methoxycoumarin fluorophore was found to undergo fluorescence quenching. Introduction of the oxazole fluorophore 5 into dihydrofolate reductase or green fluorescent protein resulted in quite strong enhancement of its fluorescence emission, and the basis for this enhancement was studied. The aggregate results demonstrate the feasibility of incorporating dipeptides as a single ribosomal event, and illustrate the lack of recognition of the central peptide bond in the dipeptide, potentially enabling the incorporation of a broad variety of structural analogues.