Mot1, Ino80C, and NC2 Function Coordinately to Regulate Pervasive Transcription in Yeast and Mammals.

Pervasive transcription initiates from cryptic promoters and is observed in eukaryotes ranging from yeast to mammals. The Set2-Rpd3 regulatory system prevents cryptic promoter function within expressed genes. However, conserved systems that control pervasive transcription within intergenic regions have not been well established. Here we show that Mot1, Ino80 chromatin remodeling complex (Ino80C), and NC2 co-localize on chromatin and coordinately suppress pervasive transcription in S. cerevisiae and murine embryonic stem cells (mESCs). In yeast, all three proteins bind subtelomeric heterochromatin through a Sir3-stimulated mechanism and to euchromatin via a TBP-stimulated mechanism. In mESCs, the proteins bind to active and poised TBP-bound promoters along with promoters of polycomb-silenced genes apparently lacking TBP. Depletion of Mot1, Ino80C, or NC2 by anchor away in yeast or RNAi in mESCs leads to near-identical transcriptome phenotypes, with new subtelomeric transcription in yeast, and greatly increased pervasive transcription in both yeast and mESCs.

EP400 Deposits H3.3 into Promoters and Enhancers during Gene Activation.

Gene activation in metazoans is accompanied by the presence of histone variants H2AZ and H3.3 within promoters and enhancers. It is not known, however, what protein deposits H3.3 into chromatin or whether variant chromatin plays a direct role in gene activation. Here we show that chromatin containing acetylated H2AZ and H3.3 stimulates transcription in vitro. Analysis of the Pol II pre-initiation complex on immobilized chromatin templates revealed that the E1A binding protein p400 (EP400) was bound preferentially to and required for transcription stimulation by acetylated double-variant chromatin. EP400 also stimulated H2AZ/H3.3 deposition into promoters and enhancers and influenced transcription in vivo at a step downstream of the Mediator complex. EP400 efficiently exchanged recombinant histones H2A and H3.1 with H2AZ and H3.3, respectively, in a chromatin- and ATP-stimulated manner in vitro. Our data reveal that EP400 deposits H3.3 into chromatin alongside H2AZ and contributes to gene regulation after PIC assembly.

Fragile Nucleosomes Influence Pol II Promoter Function.

In this issue of Molecular Cell, Kubik et al. (2015) describe how the RSC chromatin remodeling complex collaborates with two DNA sequence motifs and sequence-specific general regulatory factors to assemble fragile nucleosomes at highly transcribed yeast Pol II promoters, and they distinguish these from promoters bearing stable nucleosomes.

The Ino80 complex prevents invasion of euchromatin into silent chromatin.

Here we show that the Ino80 chromatin remodeling complex (Ino80C) directly prevents euchromatin from invading transcriptionally silent chromatin within intergenic regions and at the border of euchromatin and heterochromatin. Deletion of Ino80C subunits leads to increased H3K79 methylation and noncoding RNA polymerase II (Pol II) transcription centered at the Ino80C-binding sites. The effect of Ino80C is direct, as it blocks H3K79 methylation by Dot1 in vitro. Heterochromatin stimulates the binding of Ino80C in vitro and in vivo. Our data reveal that Ino80C serves as a general silencing complex that restricts transcription to gene units in euchromatin.

HFIP-Assisted Single C-F Bond Activation of Trifluoromethyl Ketones using Visible-Light Photoredox Catalysis.

A visible light photoredox catalytic method for the selective cleavage of single strong C-F bond in trifluoromethyl ketones is reported. Single electron reduction of trifluoromethyl ketones generates difluoromethyl radicals which can be engaged in intermolecular C-C bond formation with N-methyl-N-arylmethacrylamides to furnish fluorine-containing oxindole derivatives in good yields. The reaction shows excellent chemoselectivity with good functional group tolerance under mild conditions. 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP) as a solvent plays a critical role for the selective single C-F bond cleavage. High-level DFT calculations are depicted to shed light on the mechanism.

Utilization of CO2 Feedstock for Organic Synthesis by Visible-Light Photoredox Catalysis.

CO2 is a highly abundant, green, and sustainable carbon feedstock. Despite its kinetic inertness and thermodynamic stability, the development of various catalytic techniques has enabled the conversion of CO2 to value-added products such as carboxylic acids, amino acids, and heterocyclic compounds, where visible-light photocatalysis has emerged to be an efficient promoter of these processes. This Minireview covers the progress in the areas of CO2 incorporation onto organic matters based on the combined venture of renewable resources of CO2 and light energy with significant emphasis on the last three years' developments.

LC-ESI-QTOF-MS for the Profiling of the Metabolites and in Vitro Enzymes Inhibition Activity of Bryophyllum pinnatum and Oxalis corniculata Collected from Ramechhap District of Nepal.

The objective of this study was to profile the chemical components and biological activity analysis of crude extract of Bryophyllum pinnatum and Oxalis corniculata. Results revealed that the analyzed plant materials encompass the high amount of total phenolic and flavonoids content and have significant antioxidant activities. Furthermore, methanol extracts are the potential source of α-amylase, α-glucosidase, lipase, tyrosinase and elastase inhibitors. High resolution mass spectrometry revealed the presence of diverse metabolites such as quercetin 3-O-α-L-rhamnopyranoside, myricetin 3-rhamnoside, bersaldegenin 1,3,5-orthoacetate, bryophyllin C, syringic acid, caffeic acid, p-coumaric acid, and quercetin in B. pinnatum and isoorientin, swertisin, apigenin 7,4'-diglucoside, vitexin, 4-hydroxybenzoic acid, vanillic acid, ethyl gallate, 3,3',4'-trihydroxy-5,7-dimethoxyflavone, and diosmetin-7-O-ß-D-glucopyranoside in O. corniculata. Our finding suggested that these two plant species have high medicinal importance and are potential source of inhibitors for modern pharmaceuticals, nutraceuticals and cosmetics industries.

Spectroscopic and magnetic investigations of a spin-frustrated Mn-doped CoAl2O4 spinel.

Single-phase polycrystalline spin-frustrated spinel oxides Co1-xMnxAl2O4 (0 ≤ x ≤ 0.3) have been prepared to investigate the optical and magnetic properties. Linear variation of the lattice parameter along with the characteristic hyperfine electron paramagnetic resonance (EPR) signal establish the fact that the Mn2+ ions are incorporated at Co2+ sites of the CoAl2O4 lattice. Optical absorption spectra reveal three absorption features in wavelength regions: 250-400 nm, 500-700 nm and 1000-1700 nm. The optical band gap associated with the d-d transition increases from 1.84 eV to 1.88 eV with 30% Mn substitution. Temperature dependent magnetization measurements indicate a clear transformation of the magnetic ground state from the collinear antiferromagnetic state (for x = 0) to the spin-glass-like state (for x = 0.1) to the cluster-glass-like state (for x = 0.2 and 0.3) with the increase of Mn concentration. In addition, our time dependent isothermal remanent magnetization (IRM) study further fortifies the above transformation of the magnetic ground state. The value of the magnetic frustration parameter moderately decreases with Mn substitution, but the compositional variation is not monotonous.

A survey of chiral hypervalent iodine reagents in asymmetric synthesis.

The recent years have witnessed a remarkable growth in the area of chiral hypervalent iodine chemistry. These environmentally friendly, mild and economic reagents have been used in catalytic or stoichiometric amounts as an alternative to transition metals for delivering enantioenriched molecules. Varieties of different chiral reagents and their use for demanding asymmetric transformations have been documented over the last 25 years. This review highlights the contribution of different chiral hypervalent iodine reagents in diverse asymmetric conversions.

AB5 Preassembly Is Not Required for Shiga Toxin Activity.

UNLABELLED: Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of foodborne illness, including the life-threatening complication hemolytic-uremic syndrome. The German outbreak in 2011 resulted in nearly 4,000 cases of infection, with 54 deaths. Two forms of Stx, Stx1 and Stx2, differ in potency, and subtype Stx2a is most commonly associated with fatal human disease. Stx is considered to be an AB5 toxin. The single A (enzymatically active) subunit inhibits protein synthesis by cleaving a catalytic adenine from the eukaryotic rRNA. The B (binding) subunit forms a homopentamer and mediates cellular association and toxin internalization by binding to the glycolipid globotriaosylceramide (Gb3). Both subunits are essential for toxicity. Here we report that unlike other AB5 toxin family members, Stx is produced by STEC as unassembled A and B subunits. A preformed AB5 complex is not required for cellular toxicity or in vivo toxicity to mice, and toxin assembly likely occurs at the cell membrane. We demonstrate that disruption of A- and B-subunit association by use of A-subunit peptides that lack enzymatic activity can protect mice from lethal doses of toxin. Currently, no treatments have been proven to be effective for hemolytic-uremic syndrome. Our studies demonstrate that agents that interfere with A- and B-subunit assembly may have therapeutic potential. Shiga toxin (Stx) produced by pathogenic Escherichia coli is considered to be an AB5 heterohexamer; however, no known mechanisms ensure AB5 assembly. Stx released by E. coli is not in the AB5 conformation and assembles at the receptor interface. Thus, unassembled Stx can impart toxicity. This finding shows that preventing AB5 assembly is a potential treatment for Stx-associated illnesses. IMPORTANCE: Complications due to Shiga toxin are frequently fatal, and at present, supportive care is the only treatment option. Furthermore, antibiotic treatment is contraindicated due to the ability of antibiotics to amplify bacterial expression of Shiga toxin. We report, contrary to prevailing assumptions, that Shiga toxin produced by STEC circulates as unassembled A and B subunits at concentrations that are lethal to mice. Similar to the case for anthrax toxin, assembly occurs on receptors expressed on the surfaces of mammalian target cells. Disruption of Shiga toxin assembly by use of A-subunit peptides that lack enzymatic activity protects mice from lethal challenge with Shiga toxin, suggesting a new approach for development of therapeutics.

Molecular basis of recognition of quadruplexes human telomere and c-myc promoter by the putative anticancer agent sanguinarine.

BACKGROUND: Interaction of putative anticancer agent sanguinarine with two quadruplex forming sequences, human telomeric DNA (H24) and NHE III1 upstream of the P1 promoter of c-myc (Pu27), has been studied to understand the structural basis of the recognition. METHODS: Absorption, fluorescence and circular dichroism spectroscopy have been employed to characterize the association. Energetics of the interaction was studied by isothermal titration and differential scanning calorimetry. TRAP assay was done to assess the inhibitory potential of sanguinarine. RESULTS: Absorption and fluorescence studies show that sanguinarine has high binding affinity of ~10(5)M(-1) for both sequences. Binding stoichiometry is 2:1 for H24 and 3:1 for Pu27. Results suggest stacking interaction between planar sanguinarine moiety and G-quartets. Circular dichroism spectra show that sanguinarine does not cause structural perturbation in the all-parallel Pu27 but causes a structural transition from mixed hybrid to basket form at higher sanguinarine concentration in case of H24. The interaction is characterized by total enthalpy-entropy compensation and high heat capacity values. Differential scanning calorimetry studies suggest that sanguinarine binding increases the melting temperature and also the total enthalpy of transition of both quadruplexes. TRAP results show that sanguinarine effectively blocks telomerase activity in a concentration dependent manner in cell extracts from MDAMB-231 breast cancer cell lines. CONCLUSION: These results suggest that there is a difference in the structural modes of association of sanguinarine to the quadruplexes. GENERAL SIGNIFICANCE: It helps to understand the role of quadruplex structures as a target of small molecule inhibitors of telomerase.

Straightforward synthesis of functionalized γ-Lactams using impure CO2 stream as the carbon source.

Direct utilization of CO2 into organic synthesis finds enormous applications to synthesize pharmaceuticals and fine chemicals. However, pure CO2 gas is essential to achieve these transformations, and the purification of CO2 is highly cost and energy intensive. Considering this, we describe a straightforward synthetic route for the synthesis of γ-lactams, a pivotal core structure of bioactive molecules, by using commercially available starting materials (alkenes and amines) and impure CO2 stream (exhaust gas is collected from the car) as the carbon source. This blueprint features a broad scope, excellent functional group compatibility and application to the late-stage transformation of existing pharmaceuticals and natural products to synthesize functionalized γ-lactams. We believe that our strategy will provide direct access to γ-lactams in a very sustainable way and will also enhance the Carbon Capture and Utilization (CCU) strategy.

A combined study on structural, magnetic and specific heat on double perovskite iridates Ln2CoIrO6[Ln = Pr, Nd].

We report rich magnetic behavior for Co-Ir based double perovskites consisting of different rare earth cations Pr and Nd: Pr2CoIrO6(PCIO) and Nd2CoIrO6(NCIO). Both oxides show an antisite disorder of 10% and a ferrimagnetic transition,TFiMaround 96 K and 98 K respectively. The long range magnetic ordering is arising from the canted antiferromagnetic ordering between the Co2+and Ir4+ions. A prominent peak around 27 K in magnetization data of NCIO indicates that the total moment of Nd ion is antiferromagnetically coupled to the Co-Ir sublattice. The long range order of the Nd sublattice is corroborated by the evidence of an anomaly in specific heat at very low temperature. The compounds exhibit a maximum change of magnetic entropy of 0.57 (0.48) J kg.K-1atTFiMin a magnetic field of 5 T. The strong spin-orbit coupling in 5dstates of Ir and cation disorder lead to the Mott insulating phase as found from the analysis of temperature dependent resistivity. These unique behaviors suggest an interesting interplay between localized Pr/Nd-4f, itinerant Co-3dand Ir-5delectrons.

From seed to sprout: Unveiling the potential of non-thermal plasma for optimizing cucumber growth.

Background and aims: Numerous strategies for enhancing seed germination and growth have been employed over the decades. Despite these advancements, there continues to be a demand for more effective techniques, driven by the growing global population. Recently, various forms of non-thermal atmospheric pressure plasma have garnered attention as environmentally friendly, safe, and cost-effective methods to enhance the agricultural and food sectors. This study explores the remarkable impact of non-thermal plasma (NTP) treatment on cucumber (Cucumis sativus L.) seed germination. Methods: A cost-effective, custom-designed power supply operating at line frequency was used for treating seeds, with exposure times ranging from 1 to 7 min. Various germination parameters, including water contact angle measurements, mass loss, water imbibition rate, and seedling length, were evaluated to assess the impact of plasma treatment on seed germination. Results: Cucumber seeds exposed to NTP treatment for 3 min and 5 min durations showed significant germination improvements, notably a 57.9 ± 4.25 % higher final germination percentage, 14.5 ± 3.75 % reduced mean germination time, and a remarkable 90.6 ± 4.64 % increase in germination index compared to the control. These results suggest that NTP treatment enhanced seed coat permeability, triggered essential biochemical processes, and expedited water absorption and nutrient assimilation, ultimately fostering faster and more synchronized germination. Conclusions: Our findings underscore the potential of NTP as an innovative approach to improving seed germination in agricultural practices.

Antioxidant and Enzyme Inhibitory Potential of Streptomyces sp. G-18 Grown in Various Media.

Streptomyces are bacteria well known for producing bioactive secondary metabolites which are commonly found in diverse habitats. The biosynthesis of metabolites from Streptomyces is influenced by various factors such as the growth medium, environmental conditions, and gene regulation. This study aimed to investigate the influence of different growth media on biomass production and the antioxidant and enzyme inhibitory potential of a crude extract obtained from Streptomyces sp. G-18 isolated from high altitudinal soil of Nepal. The highest dry weight growth was observed in R2YE medium (184 mg/L), followed by R5 (144 mg/L), YEME (38 mg/L), and R5M media (30 mg/L). The crude extract showed notable antioxidant activities against free radicals. The highest alpha-amylase inhibition was observed in the R2YE medium, and worthy lipase and tyrosinase inhibition was observed in the YEME medium. However, only the R2YE medium exhibited inhibitory potential against elastase and acetylcholinesterase, while crude extracts from R5, YEME, and R5 modified did not show any such activity. Overall, our findings suggest that the production of bioactive secondary metabolites in Streptomyces sp. G-18 was significantly influenced by the growth medium. This strain may be a promising source of enzyme inhibitors with potential applications in the pharmaceutical and cosmetic industries.

Replication and Injury Associated With SARS-CoV-2 in Cultured Hepatocytes.

Background: Liver dysfunction is one of the hallmarks of SARS-CoV-2 infection. The mechanism(s) of hepatic injury in SARS-CoV-2 infection remains controversial with some reporting viral replication and cellular injury and others suggesting lack of replication and injury due to non-cytopathogenic etiologies. To investigate this further, we evaluated SARS-CoV-2 replication in immortalized hepatic cell lines and primary hepatocytes, examined whether cell injury was associated with apoptotic pathways, and also determined the effect of the antiviral remdesivir on these processes. Methods: Immortalized hepatocyte cell lines (HepG2 and Huh7.5), as well as primary human hepatocytes, were exposed to SARS-CoV-2 at a multiplicity of infection of 0.1 PFU/mL. Viral replication was evaluated by plaque assays, immunohistochemical staining for the viral spike protein, and caspase-3 expression evaluated with and without exposure to remdesivir. Results: All hepatocyte cell lines and primary hepatocytes supported active replication of SARS-CoV-2. Significant cytopathic effect was observed by light microscopy, and caspase-3 staining supported activation of apoptotic pathways. Remdesivir abrogated infection in a dose-dependent fashion and was not independently associated with hepatocyte injury. Conclusion: Hepatocytes appear to be highly permissive of SARS-CoV-2 replication which leads to rapid cell death associated with activation of apoptotic pathways. Viral replication and hepatocytes injury are abrogated with remdesivir. We conclude that active viral replication is most likely a key contributor to liver enzyme abnormalities observed in the setting of acute SARS-CoV-2 infection.

Development of functional resident macrophages in human pluripotent stem cell-derived colonic organoids and human fetal colon.

Most organs have tissue-resident immune cells. Human organoids lack these immune cells, which limits their utility in modeling many normal and disease processes. Here, we describe that pluripotent stem cell-derived human colonic organoids (HCOs) co-develop a diverse population of immune cells, including hemogenic endothelium (HE)-like cells and erythromyeloid progenitors that undergo stereotypical steps in differentiation, resulting in the generation of functional macrophages. HCO macrophages acquired a transcriptional signature resembling human fetal small and large intestine tissue-resident macrophages. HCO macrophages modulate cytokine secretion in response to pro- and anti-inflammatory signals and were able to phagocytose and mount a robust response to pathogenic bacteria. When transplanted into mice, HCO macrophages were maintained within the colonic organoid tissue, established a close association with the colonic epithelium, and were not displaced by the host bone-marrow-derived macrophages. These studies suggest that HE in HCOs gives rise to multipotent hematopoietic progenitors and functional tissue-resident macrophages.

Transition-Metal-Free Regioselective Intermolecular Hydroamination of Conjugated 1,3-Dienes with Heterocyclic Amines.

The unique property of hexafluoroisopropanol (HFIP) enables the regioselective hydroamination of 1,3-dienes with nitrogen heterocycles in a Markovnikov manner in the presence of catalytic Brønsted acid. This transition-metal-free intermolecular hydroamination protocol is achieved under mild reaction conditions. The aggregation by HFIP and Brønsted acid helps to activate the terminal double bond regioselectively. Following the protonation of diene, the C-N bond formation is accomplished upon the involvement of heterocyclic amines.

Identification of a novel inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1)-mediated methylation of histone H3 Arg-17.

Methylation of the arginine residues of histones by methyltransferases has important consequences for chromatin structure and gene regulation; however, the molecular mechanism(s) of methyltransferase regulation is still unclear, as is the biological significance of methylation at particular arginine residues. Here, we report a novel specific inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1; also known as PRMT4) that selectively inhibits methylation at arginine 17 of histone H3 (H3R17). Remarkably, this plant-derived inhibitor, called TBBD (ellagic acid), binds to the substrate (histone) preferentially at the signature motif, "KAPRK," where the proline residue (Pro-16) plays a critical role for interaction and subsequent enzyme inhibition. In a promoter-specific context, inhibition of H3R17 methylation represses expression of p21, a p53-responsive gene, thus implicating a possible role for H3 Arg-17 methylation in tumor suppressor function. These data establish TBBD as a novel specific inhibitor of arginine methylation and demonstrate substrate sequence-directed inhibition of enzyme activity by a small molecule and its physiological consequence.

Mechanism of interaction of small transcription inhibitors with DNA in the context of chromatin and telomere.

Small molecules from natural and synthetic sources have long been employed as human drugs. The transcription inhibitory potential of one class of these molecules has paved their use as anticancer drugs. The principal mode of action of these molecules is via reversible interaction with genomic DNA, double and multiple stranded. In this article we have revisited the mechanism of the interaction in the context of chromatin and telomere. The established modes of association of these molecules with double helical DNA provide a preliminary mechanism of their transcription inhibitory potential, but the scenario assumes a different dimension when the genomic DNA is associated with proteins in the transcription apparatus of both prokaryotic and eukaryotic organisms. We have discussed this altered scenario as a prelude to understand the chemical biology of their action in the cell. For the telomeric quadruplex DNA, we have reviewed the mechanism of their association with the quadruplex and resultant cellular consequence.