A new method for the robust expression and single-step purification of dCas9 for CRISPR interference/activation (CRISPRi/a) applications.

CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated enzyme 9) is known for its simplicity, versatility, and scalability in genome editing applications. In vitro Cas9, when complexed with sgRNA, binds and cleaves the complementary target sequences with almost perfect precision. The enzyme is exploited for various applications in understanding and changing gene function. dCas9 (deactivated or dead Cas9) is a double mutated version of Cas9 that bears mutations in the nuclease domains of the enzyme and thus cannot cleave the target DNA. dCas9 is equally advantageous since it can alter gene expression using various transcriptional activators CRISPRa and repressors CRISPRi. Additionally, dCas9 can bind to the desired target gene without cleaving it, making it a unique reagent to study the kinetics and stability of RNA-protein-DNA interactions required to design more efficient and specific gene-editing nucleases. An appreciable quantity of pure and homogeneous protein is needed to characterise dCas9 for its structural and functional understanding. This study used an N-terminal acidic tag to express the dCas9 in an E. coli-bacterial host. A simple single-step protocol for robust and efficient production of dCas9 has been described. The study and methods are distinctive as the purification is performed in a single step using inexpensive multi-modal hydroxyapatite chromatography. The purified protein can be used in different in vitro and in vivo studies.

Protein targeting to starch 1, a functional protein of starch biosynthesis in wheat (Triticum aestivum L.).

KEY MESSAGE: TaPTST1, a wheat homolog of AtPTST1 containing CBM can interact with GBSSI and regulate starch metabolism in wheat endosperm. In cereal endosperm, native starch comprising amylose and amylopectin is synthesized by the coordinated activities of several pathway enzymes. Amylose in starch influences its physio-chemical properties resulting in several human health benefits. The Granule-Bound Starch Synthase I (GBSSI) is the most abundant starch-associated protein. GBSSI lacks dedicated Carbohydrate-binding module (CBM). Previously, Protein Targeting To Starch 1 (PTST1) was identified as a crucial protein for the localization of GBSSI to the starch granules in Arabidopsis. The function of its homologous protein in the wheat endosperm is not known. In this study, TaPTST1, an AtPTST1 homolog, containing a CBM and a coiled-coil domain was identified in wheat. Protein-coding nucleotide sequence of TaPTST1 from Indian wheat variety 'C 306' was cloned and characterized. Homology modelling and molecular docking suggested the potential interaction of TaPTST1 with glucans and GBSSI. The TaPTST1 expression was higher in wheat grain than the other tissues, suggesting a grain-specific function. In vitro binding assays demonstrated different binding affinities of TaPTST1 for native starch, amylose, and amylopectin. Furthermore, the immunoaffinity pull-down assay revealed that TaPTST1 directly interacts with GBSSI, and the interaction is mediated by a coiled-coil domain. The direct protein-protein interaction was further confirmed by bimolecular fluorescence complementation assay (BiFC) in planta. Based on our findings we postulate a functional role for TaPTST1 in starch metabolism by targeting GBSSI to starch granules in wheat endosperm.

Mechanistic insights into granule-bound starch synthase I (GBSSI.L539P) allele in high amylose starch biosynthesis in wheat (Triticum aestivum L.).

Amylose fraction of grain starch is correlated with a type of resistant starch with better nutritional quality. Granule-bound starch synthase I (GBSSI) is the known starch synthase, responsible for elongation of linear amylose chains. GBSSI expression, activity, and binding to starch and other proteins are the key factors that can affect amylose content. Previously, a QTL, qhams7A.1 carrying GBSSI mutant allele, was identified through QTL mapping using F2 population of the high amylose mutant line, 'TAC 75'. This high amylose mutant line has >2-fold higher amylose content than wild variety 'C 306'. In this study, we characterized this novel mutant allele, GBSSI.L539P. In vitro starch synthase activity of GBSSI.L539P showed improved activity than the wild type (GBSSI-wt). When expressed in yeast glycogen synthase mutants (Δgsy1gsy2), GBSSI-wt and GBSSI.L539P partially complemented the glycogen synthase (gsy1gsy2) activity in yeast. Structural analysis by circular dichroism (CD) and homology modelling showed no significant structural distortion in the mutant enzyme. Molecular docking studies suggested that the residue Leu539 is distant from the catalytic active site (ADP binding pocket) and had no detectable conformational changes in active site. Both wild and mutant enzymes were assayed for starch binding in vitro, and demonstrating higher affinity of the GBSSI.L539P mutant for starch than the wild type. The present study indicated that distant residue (L539P) influenced GBSSI activity by affecting its starch-binding ability. Therefore, it may be a potential molecular target for enhanced amylose content in grain.

Wheat inositol pyrophosphate kinase TaVIH2-3B modulates cell-wall composition and drought tolerance in Arabidopsis.

BACKGROUND: Inositol pyrophosphates (PP-InsPs) are high-energy derivatives of inositol, involved in different signalling and regulatory responses of eukaryotic cells. Distinct PP-InsPs species are characterized by the presence of phosphate at a variable number of the 6-carbon inositol ring backbone, and two distinct classes of inositol phosphate kinases responsible for their synthesis have been identified in Arabidopsis, namely ITPKinase (inositol 1,3,4 trisphosphate 5/6 kinase) and PP-IP5Kinase (diphosphoinositol pentakisphosphate kinases). Plant PP-IP5Ks are capable of synthesizing InsP8 and were previously shown to control defense against pathogens and phosphate response signals. However, other potential roles of plant PP-IP5Ks, especially towards abiotic stress, remain poorly understood. RESULTS: Here, we characterized the physiological functions of two Triticum aestivum L. (hexaploid wheat) PPIP5K homologs, TaVIH1 and TaVIH2. We demonstrate that wheat VIH proteins can utilize InsP7 as the substrate to produce InsP8, a process that requires the functional VIH-kinase domains. At the transcriptional level, both TaVIH1 and TaVIH2 are expressed in different wheat tissues, including developing grains, but show selective response to abiotic stresses during drought-mimic experiments. Ectopic overexpression of TaVIH2-3B in Arabidopsis confers tolerance to drought stress and rescues the sensitivity of Atvih2 mutants. RNAseq analysis of TaVIH2-3B-expressing transgenic lines of Arabidopsis shows genome-wide reprogramming with remarkable effects on genes involved in cell-wall biosynthesis, which is supported by the observation of enhanced accumulation of polysaccharides (arabinogalactan, cellulose, and arabinoxylan) in the transgenic plants. CONCLUSIONS: Overall, this work identifies a novel function of VIH proteins, implicating them in modulation of the expression of cell-wall homeostasis genes, and tolerance to water-deficit stress. This work suggests that plant VIH enzymes may be linked to drought tolerance and opens up the possibility of future research into using plant VIH-derived products to generate drought-resistant plants.

Understanding the regulatory relationship of abscisic acid and bZIP transcription factors towards amylose biosynthesis in wheat.

Starch is biosynthesized during seed development and this process is regulated by many bZIP proteins in bread wheat. Abscisic acid (ABA), an important phyto-hormone involved in various physiological processes mediated by bZIPs in plants including seed development. The 'Group A' TabZIP transcription factors play important roles in the ABA signaling pathway in Arabidopsis, rice and other cereal crops but their role in regulation of amylose biosynthesis in wheat is limited. In this study 83 'Group A' TabZIPs were characterized by gene expression analysis in wheat amylose mutants. A set of 17 TabZIPs was selected on the basis of differential expression (> 2 fold) in low and high amylose mutants from RNA-seq data and validated by qRT PCR. Based on qRT PCR and correlation analysis out of the 17 TabZIPs six differentially expressed candidate TabZIPs were identified, involving in high amylose biosynthesis. The TabZIP175.2, identified as upregulated in all high amylose lines and TabZIP90.2, TabZIP129.1, TabZIP132.2, TabZIP143 and TabZIP159.2 were found downregulated in all low amylose lines, after exogenous supply of ABA. Proximal promoter analysis of starch pathway genes revealed the presence of ABA-responsive elements (ABREs) that are putative binding sites for bZIPs. Collectively, these findings indicated the involvement of putative six candidate TabZIPs as transcriptional regulators of amylose related genes via an ABA-dependent pathway in wheat. This study could help the investigators to make an informed decision to edit wheat genome for high/low amylose content using gene-editing technologies.

Genome-wide Methylation Dynamics and Context-dependent Gene Expression Variability in Differentiating Preadipocytes.

Obesity, characterized by the accumulation of excess fat, is a complex condition resulting from the combination of genetic and epigenetic factors. Recent studies have found correspondence between DNA methylation and cell differentiation, suggesting a role of the former in cell fate determination. There is a lack of comprehensive understanding concerning the underpinnings of preadipocyte differentiation, specifically when cells are undergoing terminal differentiation (TD). To gain insight into dynamic genome-wide methylation, 3T3 L1 preadipocyte cells were differentiated by a hormone cocktail. The genomic DNA was isolated from undifferentiated cells and 4 hours, 2 days postdifferentiated cells, and 15 days TD cells. We employed whole-genome bisulfite sequencing (WGBS) to ascertain global genomic DNA methylation alterations at single base resolution as preadipocyte cells differentiate. The genome-wide distribution of DNA methylation showed similar overall patterns in pre-, post-, and terminally differentiated adipocytes, according to WGBS analysis. DNA methylation decreases at 4 hours after differentiation initiation, followed by methylation gain as cells approach TD. Studies revealed novel differentially methylated regions (DMRs) associated with adipogenesis. DMR analysis suggested that though DNA methylation is global, noticeable changes are observed at specific sites known as "hotspots." Hotspots are genomic regions rich in transcription factor (TF) binding sites and exhibit methylation-dependent TF binding. Subsequent analysis indicated hotspots as part of DMRs. The gene expression profile of key adipogenic genes in differentiating adipocytes is context-dependent, as we found a direct and inverse relationship between promoter DNA methylation and gene expression.

CpG methylation of half-CRE sequences creates C/EBPalpha binding sites that activate some tissue-specific genes.

DNA methylation of the cytosine in the CpG dinucleotide is typically associated with gene silencing. Genomic analyses have identified low CpG promoters that are both methylated and transcriptionally active, but the mechanism underlying the activation of these methylated promoters remains unclear. Here we show that CpG methylation of the CRE sequence (TGACGTCA) enhances the DNA binding of the C/EBPα transcription factor, a protein critical for activation of differentiation in various cell types. Transfection assays also show that C/EBPα activates the CRE sequence only when it is methylated. The biological significance of this observation was seen in differentiating primary keratinocyte cultures from newborn mice where certain methylated promoters are both bound by C/EBPα and activated upon differentiation. Experimental demethylation by either 5-azacytidine treatment or DNMT1 depletion diminished both C/EBPα binding and activation of the same methylated promoters upon differentiation suggesting that CpG methylation can localize C/EBPα. Transfection studies in cell cultures using methylated tissue-specific proximal promoters identified half-CRE (CGTCA) and half-C/EBP (CGCAA) sequences that need to be methylated for C/EBPα mediated activation. In primary dermal fibroblasts, C/EBPα activates a different set of methylated tissue-specific promoters upon differentiation into adipocytes. These data identify a new function for methyl CpGs: producing DNA binding sites at half-CRE and half-C/EBP sequences for C/EBPα that are needed to activate tissue-specific genes.

Disruption of histone acetylation homeostasis triggers cognitive dysfunction in experimental diabetes.

Epigenetic mechanisms related to diabetes-afflicted CNS complications are largely unknown. The present study investigated the role of histone acetylation mechanisms triggering cognitive dysfunction in the Type 1 and 2 diabetic mice model. Dynamic changes in diabetic parameters like fasting blood glucose levels, glucose tolerance test, and insulin levels were observed after the induction of diabetes. Cognitive performance was significantly diminished in T1D and T2D mice examined by the Morris water maze, novel object recognition test, and Y Maze as compared to controls. Histone profiling revealed a significant reduction in H3K9/14 and H4K12 acetylation in the cortex and hippocampus of T1D and T2D mice vs Controls. While histone deacetylase (HDAC) activity was significantly elevated in brain regions of T1D and T2D mice, the histone acetyltransferase (HAT) activity remain unchanged. Significantly increased HDAC 2, HDAC 3 protein and mRNA expression observed in T1D and T2D brain regions may corroborate for increased HDAC activity. No significant change was observed in protein and mRNA expression of HDAC 1, 5, 6, and 7 in diabetic brains. Reduced H3K9/14 and H4K12 acetylation paralleled transcriptional repression of memory-related markers BDNF, SYP, and PSD-95 in the cortex and hippocampus of T1D and T2D. Pharmacological inhibition of HDAC activity by Trichostatin A enhanced the cognitive changes observed in T1D and T2D by ameliorating BDNF, SYP, Psd-95. The present study provides a better insight into molecular mechanisms related to diabetes-dependent memory changes that can help to generate new advances for therapeutics to be developed in this area.

Salmonella typhimurium detection and ablation using OmpD specific aptamer with non-magnetic and magnetic graphene oxide.

Foodborne diseases have increased in the last few years due to the increased consumption of packaged and contaminated food. Major foodborne bacteria cause diseases such as diarrhea, vomiting, and sometimes death. So, there is a need for early detection of foodborne bacteria as pre-existing detection techniques are time-taking and tedious. Aptamer has gained interest due to its high stability, specificity, and sensitivity. Here, aptamer has been developed against Salmonella Typhimurium through the Cell-Selex method, and to further find the reason for specificity and sensitivity, OmpD protein was isolated, and binding studies were done. Single molecular FRET experiment using aptamer and graphene oxide studies has also been done to understand the mechanism of FRET and subsequently used for target bacterial detection. Using this assay, Salmonella Typhimurium can be detected up to 10 CFU/mL. Further, Magnetic Graphene oxide was used to develop an assay to separate and ablate bacteria using 808 nm NIR where temperature increase was more than 60 °C within 30 s and has been shown by plating as well as a confocal live dead assay. Thus, using various techniques, bacteria can be detected and ablated using specific aptamer and Graphene oxide.

Multiepitope glycan based laser assisted fluorescent nanocomposite with dual functionality for sensing and ablation of Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) infection is becoming a severe health hazard and needs early diagnosis with high specificity. However, the non-specific binding of a biosensor is a challenge to the current bacterial detection system. For the first time, we chemically synthesized a galactose tripod (GT) as a P. aeruginosa-specific ligand. We conjugated GT to a photothermally active fluorescent nanocomposite (Au@SiO2-TCPP). P. aeruginosa can be detected using Au@SiO2-TCPP-GT, and additionally ablated as well using synergistic photothermal and photodynamic therapy. Molecular dynamics and simulation studies suggested better binding of GT (binding energy = -6.6 kcal mol-1) with P. aeruginosa lectin than that of galactose monopod (GM) (binding energy = -5.9 kcal mol-1). Furthermore, a binding study was extended to target P. aeruginosa, which has a galactose-binding carbohydrate recognition domain receptor. The colorimetric assay confirmed a limit of detection (LOD) of 104 CFU mL-1. We also looked into the photosensitizing property of Au@SiO2-TCPP-GT, which is stimulated by laser light (630 nm) and causes photoablation of bacteria by the formation of singlet oxygen in the surrounding media. The cytocompatibility of Au@SiO2-TCPP-GT was confirmed using cytotoxicity assays on mammalian cell lines. Moreover, Au@SiO2-TCPP-GT also showed non-hemolytic activity. Considering the toxicity analysis and efficacy of the synthesized glycan nanocomposites, these can be utilized for the treatment of P. aeruginosa-infected wounds. Furthermore, the current glycan nanocomposites can be used for bacterial detection and ablation of P. aeruginosa in contaminated food and water samples as well.

P6981, an arylstibonic acid, is a novel low nanomolar inhibitor of cAMP response element-binding protein binding to DNA.

Several basic leucine zipper (B-ZIP) transcription factors have been implicated in cancer, substance abuse, and other pathological conditions. We previously identified arylstibonic acids that bind to B-ZIP proteins and inhibit their interaction with DNA. In this study, we used electrophoretic mobility shift assay to analyze 46 arylstibonic acids for their activity to disrupt the DNA binding of three B-ZIP [CCAAT/enhancer-binding protein α, cyclic AMP-response element-binding protein (CREB), and vitellogenin gene-binding protein (VBP)] and two basic helix-loop-helix leucine zipper (B-HLH-ZIP) [USF (upstream stimulating factor) and Mitf] proteins. Twenty-five arylstibonic acids showed activity at micromolar concentrations. The most active compound, P6981 [2-(3-stibonophenyl)malonic acid], had half-maximal inhibition at ~5 nM for CREB. Circular dichroism thermal denaturation studies indicated that P6981 binds both the B-ZIP domain and the leucine zipper. The crystal structure of an arylstibonic acid, NSC13778, bound to the VBP leucine zipper identified electrostatic interactions between both the stibonic and carboxylic acid groups of NSC13778 [(E)-3-(3-stibonophenyl)acrylic acid] and arginine side chains of VBP, which is also involved in interhelical salt bridges in the leucine zipper. P6981 induced GFP-B-ZIP chimeric proteins to partially localize to the cytoplasm, demonstrating that it is active in cells. P6981 inhibited the growth of a patient-derived clear cell sarcoma cell line whose oncogenic potential is driven by a chimeric protein EWS-ATF1 (Ewing's sarcoma protein-activating transcription factor 1), which contains the DNA binding domain of ATF1, a B-ZIP protein. NSC13778 inhibited the growth of xenografted clear cell sarcoma, and no toxicity was observed. These experiments suggest that antimony containing arylstibonic acids are promising leads for suppression of DNA binding activities of B-ZIP and B-HLH-ZIP transcription factors.

Ubiquitin tagged dominant negative induces degradation of B-ZIP proteins.

B-ZIP transcription factors heterodimerize with dominant negative designs, termed A-ZIPs, in a dimerization specific manner and inhibit its ability to bind DNA. Different A-ZIPs produce unique phenotypes in vivo suggesting that they have distinct B-ZIP heterodimerization partners. However, the identification of the in vivo heterodimerization partners of different A-ZIPs remains problematic. To identify the in vivo heterodimerization partners, a chimeric protein containing two ubiquitin motifs at the N-terminal of the A-ZIP domain was designed. The presence of ubiquitin reduced the concentration of specific co-transfected B-ZIP proteins. The ubiquitin enhanced degradation of the B-ZIP heterodimeric partner is inhibited by the proteasome inhibitor MG-132. These ubiquitin tagged A-ZIP dominant negatives may be more active in vivo because their endogenous heterodimerization partners are degraded more efficiently. This may be a general strategy to identify protein interaction partners.

Biosynthesis of natural aroma compounds using recombinant whole-cell tomato hydroperoxide lyase biocatalyst.

Green leaf volatiles impart characteristic aroma and flavour to a variety of natural foods due to their inherent grassy note contributed by aldehydes. Hydroperoxide lyase (HPL) is an enzyme that helps in the cleavage of fatty acid hydroperoxides to short-chain aldehydes and ω-oxo-acids. A tomato hydroperoxide lyase gene was successfully expressed in E. coli BL21 (DE3) cells and used in the subsequent production of (Z)-3-hexenal. Biochemical characterization of the HPL activity exhibited by these whole cells enabled the development of a suitable one-pot reaction process for conversion of the hydroperoxide substrate to the corresponding aldehyde, (Z)-3-hexenal, and finally to (Z)-3-hexenol, a high-value flavour and fragrance ingredient.

Effect of hepcidin antagonists on anemia during inflammatory disorders.

Iron is an essential element for the mammalian body however, its homeostasis must be regulated accurately for appropriate physiological functioning. Alterations in physiological iron levels can lead to moderate to severe iron disorders like chronic and acute iron deficiency (anemia) or iron overload. Hepcidin plays an important role in regulating homeostasis between circulating iron and stored iron in the cells as well as the absorption of dietary iron in the intestine. Inflammatory disorders restrict iron absorption from food due to increased circulating levels of hepcidin. Increased production of hepcidin causes ubiquitination of ferroportin (FPN) leading to its degradation, thereby retaining iron in the spleen, duodenal enterocytes, macrophages, and hepatocytes. Hepcidin inhibitors and antagonists play a consequential role to ameliorate inflammation-associated anemia. Many natural and synthesized compounds, able to reduce hepcidin expression during inflammation have been identified in recent years. Few of which are currently at various phases of clinical trial. This article comprises a comprehensive review of therapeutic approaches for the efficient treatment of anemia associated with inflammation. Many strategies have been developed targeting the hepcidin-FPN axis to rectify iron disorders. Hepcidin modulation with siRNAs, antibodies, chemical compounds, and plant extracts provides new insights for developing advanced therapeutics for iron-related disorders. Hepcidin antagonist's treatment has a high potential to improve iron status in patients with iron disorders, but their clinical success needs further recognition along with the identification and application of new therapeutic approaches.

12 Arylstibonic acids that inhibit the DNA binding of five B-ZIP dimers.

Previously, we identified an arylstibonic acid, NSC13778 that specifically binds to the basic region of the C/EBPalpha B-ZIP domain and disrupts DNA binding. We now examine a panel of 14 additional arylstibonic acid derivatives of NSC13778 for their ability to inhibit the DNA binding of five B-ZIP dimers (c-Fos|JunD, VBP, C/EBPalpha, C/EBPbeta, and CREB). They show various specificities at inhibiting the DNA binding of five B-ZIP domains. NSC13746 inhibits the DNA binding of C/EBPbeta and CREB at 100nM and promiscuously inhibiting the DNA binding of all five proteins in the 1muM range. Dialysis experiments indicate that NSC 13746 binding to the B-ZIP domain is reversible. Thermal denaturation studies indicate that NSC13746 binds the B-ZIP domain. Some compounds specifically inhibit DNA binding, with VBP and c-Fos|JunD being most easily disrupted. These compounds inhibit, with similar specificities to the pure B-ZIP domains, the DNA binding of nuclear extract to the AP1 DNA sequence but no inhibition is observed to SP1 containing oligonucleotide. Transient transfection assays indicate that NSC13746 can inhibit the TPA induced activation of two B-ZIP dependent reporters. These experiments suggest that arylstibonic acids are promising leads for inhibiting the DNA binding of a group of B-ZIP proteins in cells.

Inhibition of CREB function in mouse epidermis reduces papilloma formation.

We used a double transgenic tetracycline system to conditionally express A-CREB, a dominant negative protein that prevents the DNA binding and function of cAMP-responsive element binding protein (CREB) family members, in mouse basal epidermis using the keratin 5 promoter. There was no phenotype in the adult. However, following a 7,12-dimethylbenz(a)anthracene (DMBA)/phorbol-12-myristate-13-acetate two-stage skin carcinogenesis experiment, A-CREB-expressing epidermis develop 5-fold fewer papillomas than wild-type controls. However, A-CREB expression one month after DMBA treatment does not prevent papilloma formation, suggesting that CREB functions at an early stage of papilloma formation. Oncogenic H-Ras genes with A-->T mutations in codon 61 were found in wild-type skin but not in A-CREB-expressing skin 2 days after DMBA treatment, suggesting that A-CREB either prevents DMBA mutagenesis or kills oncogenic H-Ras cells. In primary keratinocyte cultures, A-CREB expression induced apoptosis of v-Ras(Ha)-infected cells and suppressed the expression of cell cycle proteins cyclin B1 and cyclin D1. These results suggest that inhibiting CREB function is a valuable cancer prevention strategy.

Biolistic Delivery of Programmable Nuclease (CRISPR/Cas9) in Bread Wheat.

The discovery of site-specific programmable nucleases has led to a major breakthrough in the area of genome editing. In the past few years, CRISPR/Cas system has been utilized for genome editing of a large number of crops including cereals like wheat, rice, maize, and barley. In terms of consumption, wheat is second only to rice as the most important crop of the world. In the present chapter, we describe biolistic delivery method of ribonucleoprotein (RNP) complexes of programmable nuclease (CRISPR/Cas9) for targeted genome editing and selection-free screening of transformants in wheat. The method not only overcomes the problem of random integration into the genome but also reduces the off-targets. Besides the step-by-step protocol, plausible challenges and ways to overcome them are also discussed. By using the described method of biolistic delivery of CRISPR/Cas9 in plant systems, genome-edited plants can be identified within 11 weeks.

Quercetin alleviates cognitive decline in ovariectomized mice by potentially modulating histone acetylation homeostasis.

The synergism between estrogen and histone tail acetylation-mediated memory formation is not clearly understood. Here, we attempt to study the altered histone acetylation homeostasis mediated changes in cognition following ovariectomy and evaluate the protective effect of quercetin. A significant reduction in estradiol levels with subsequent depletion in spatial memory and learning functions assessed by Morris water maze, novel object recognition test and elevated plus maze was observed in ovariectomized (OVX) mice. Correspondingly, a significant decline in neuroplasticity markers like brain-derived neurotrophic factor (BDNF), synaptophysin (SYP) and postsynaptic density-95 (PSD-95) was observed in cortex and hippocampus of OVX animals. Notably, histone acetyltransferase (HAT)/histone deacetylase (HDAC) balance was significantly disrupted in cortex and hippocampus of OVX mice. Lowered extracellular signal regulated kinases (ERK) and cAMP response element binding protein activation observed in OVX brain regions might account for HAT/HDAC imbalance. Altered HAT/HDAC homeostasis results in lowered histone 3 acetylation in OVX brain that suppressed transcriptional activation of neuroplasticity-related genes. Quercetin supplementation to OVX mice for 4 weeks was able to ameliorate cognitive impairment by restoring HAT/HDAC homeostasis through ERK activation and reversing alterations in neuroplasticity markers in cortex and hippocampus of OVX mice. Taken together, our results suggest that quercetin alleviates ovariectomy-induced cognitive decline by modulating histone acetylation homeostasis.

Mobilization of storage lipid reserve and expression analysis of lipase and lipoxygenase genes in rice (Oryza sativa var. Pusa Basmati 1) bran during germination.

Storage lipid mobilization by lipases and lipoxygenases (LOXs) in response to developmental cues take place during seed germination. After rice grain milling, the endogenous lipases and LOXs present in the bran fraction come in contact with the storage lipid reserve or triacylglycerol (TAG). Lipases catalyze the hydrolysis of TAGs to non-esterified fatty acids (NEFAs) and glycerol. The NEFAs, especially linoleic acid (18:2) produced, are further subjected to oxidative rancidity via peroxidation reaction catalyzed by LOXs. This results in the production of conjugated hydroperoxides of 18:2 that influence the off-flavors in rice bran lipids. The aim of this study is to understand how lipid mobilization and expression of lipase and LOX genes occur in the bran of germinating rice grains (Oryza sativavar. Pusa Basmati 1). Our results show that the primary source of storage lipids in bran is TAG, and its mobilization starts at 4 days after imbibition (4 DAI). Using publically available RNA-seq data and phylogeny analyses, we selected a total of 18 lipase and 16 LOX genes in rice for their expression profiles during onset of lipid mobilization. Gene expression analyses revealed OsLip1, OsLip9, and OsLip13; and OsLOX3 and OsLOX14 as the predominantly expressed genes in bran of germinating rice grains. This study explores two important events in the germinating rice grains, namely, mobilization of storage lipids and expression pattern of lipase and LOX genes. The information generated in this study can be used to efficiently manipulate the genes to enhance the shelf-stability of bran lipid reserve.

Transient Delivery of A-C/EBP Protein Perturbs Differentiation of 3T3-L1 Cells and Induces Preadipocyte Marker Genes.

Transformation of committed 3T3-L1 preadipocytes to lipid-laden adipocytes involves the timely appearance of numerous transcription factors (TFs); foremost among them, C/EBPß is expressed during the early phases of differentiation. Here, we describe liposome-mediated protein transfection approach to rapidly downregulate C/EBPß by A-C/EBP protein inhibitor. Signals from EGFP-tagged A-C/EBP protein were observed in 3T3-L1 cells within 2 h of transfections, whereas for A-C/EBP gene transfections, equivalent signals appeared in 48 h. Following transient transfections, the expression profiles of 24 marker genes belonging to pro- and anti-adipogenic, cell cycle, and preadipocyte pathways were analyzed. Expectedly, the mRNA and protein expression profiles of adipocyte marker genes showed lower expression in both A-C/EBP protein- and gene-transfected samples. Interestingly, for preadipocytes and cell fate determinant genes, striking differences were observed between A-C/EBP protein- and A-C/EBP gene-transfected samples. Preadipocyte differentiation factors Stat5a and Creb were downregulated in A-C/EBP protein samples. Five preadipocyte markers, namely, Pdgfrα, Pdgfrß, Ly6A, CD34, and Itgb1, showed high expression in A-C/EBP protein samples, whereas only Ly6A and CD34 were expressed in A-C/EBP gene-transfected samples. Pdgfrα and Pdgfrß, two known cell fate markers, were expressed in A-C/EBP protein-transfected samples, suggesting a possible reversal of differentiation. Our study provides evidences for the immediate and efficient knockdown of C/EBPß protein to understand time-dependent preadipocytes differentiation.