• Volume 45, All articles

Continuous Article Publishing mode

• Resveratrol protects H9c2 cells against hypoxia-induced apoptosis through miR-30d-5p/SIRT1/NF-κB axis

Previous studies have demonstrated the cardioprotective role of resveratrol (Res). However, the underlyingmolecular mechanisms involved in the protective role of Res are still largely unknown. H9c2 cells weredistributed into five groups: normal condition (Control), DMSO, 20 mMRes (dissolved with DMSO), hypoxia(Hyp), and Res+Hyp. Cell apoptosis was evaluated using flow cytometry and protein analysis of cleavedcaspase 3 (cle-caspase 3). qRT-PCR assay was performed to measure the expression of microRNA-30d-5p(miR-30d-5p). MTT assay was performed to evaluate the cell proliferation. The relationship between miR-30d-5p and silent information regulator 1 (SIRT1) was confirmed by luciferase reporter, RNA immunoprecipitation(RIP), and western blot assays. Western blot was performed to analyze NF-κB/p65 and I-κBa expressions. Ourdata showed that hypoxia enhanced apoptosis and NF-κB signaling pathway, which was alleviated by Restreatment. Hypoxia increased the expression of miR-30d-5p while decreased the SIRT1 expression, which wasalso attenuated by Res treatment. Furthermore, miR-30d-5p depletion inhibited the proliferation, reducedapoptosis and decreased the expression of cle-caspase 3 in H9c2 cells with hypoxia treatment. Luciferasereporter, RIP, and western blot assays further confirmed that miR-30d-5p negatively regulated the expression ofSIRT1. Interestingly, the rescue-of-function experiments further indicated that knockdown of SIRT1 attenuatedthe effect of miR-30d-5p depletion on proliferation, apoptosis NF-κB signaling pathway inH9c2 cells withhypoxia treatment. In addition, the suppression of NF-κB signaling pathway increased cell viability whiledecreased cell apoptosis in hypoxia-mediatedH9c2 cells. Our data suggested Res mayprotectH9c2 cells againsthypoxia-induced apoptosis through miR-30d-5p/SIRT1/NF-κB axis.

• Transcriptome-wide identification and profiling of miRNAs in a stress-tolerant conifer Sabina chinensis

miRNAs are important regulatory components involving in many biological processes, including plantdevelopment, vegetative and reproductive growth, and stress response. However, identification and characterizationof miRNAs still remain limited for conifer species. In this study, with deep sequencing, we obtained1,314,450 unique reads with 18–30 nt length from a stress-tolerant conifer, Sabina chinensis. We identified 37conserved and 103 novel miRNAs, their unique characteristics were further analyzed, and 10 randomlyselected were validated by qRT-PCR. Through miRNA target predictions and annotations, we found miRNAmay have several targets as well a target could be regulated by several miRNAs, and a total of 2,397 mRNAswere predicted to be targets of the 140 miRNAs. These targets included not only important transcription factorssuch as auxin response factors, but also indispensable non-transcriptional factor proteins. Pathway-basedanalysis showed that S. chinensis miRNAs are involved in 172 metabolic pathways, of which 3 were discoveredin adaptation-related pathways, indicating their possible relevance to the species’ stress-tolerancecharacteristics. This study is expected to lay the foundation for exploring the regulative roles of miRNAs indevelopment, growth, and response to environmental stresses of S. chinensis.

• Silencing of HOXB9 suppresses cellular proliferation, angiogenesis, migration and invasion of prostate cancer cells

The Homeobox B9 (HOXB9) is a homeodomain-containing transcription factor that participates in the progressionof various malignancies. Nevertheless, the functional role of HOXB9 in prostate cancer cells is largelyunknown. Hence, we aimed to address the effect of HOXB9 on the progression of prostate cancer cells. Smallinterfering RNA (siRNA) against HOXB9 was used to downregulate HOXB9 expression in PC3 and DU145cells. Western blotting was performed to detect the expression levels of HOXB9 and other related proteins. Cellproliferation was tested by the Cell Counting Kit-8 (CCK-8) and cell cycle and apoptosis were investigated byflow cytometry. Angiogenesis was examined using tube formation assays The Transwell assays were carriedout to assess the migratory and invasive capacities of cells. Here, we found that HOXB9 knockdown significantlyreduced cell proliferation via inducing cell cycle arrest at G1 phase. This treatment also reducedangiogenesis, migration and invasion abilities of PC3 and DU145 cells in vitro. We also found that HOXB9knockdown inhibits the activation of the PI3K/AKT signaling pathway in prostate cancer cells. In conclusion,our findings revealed that HOXB9 promotes prostate cancer progression and might be a novel and effectivetherapeutic target for human prostate cancer.

• Yeast glutaredoxin, GRX4, functions as a glutathione S-transferase required for red ade pigment formation in Saccharomyces cerevisiae

The adenine biosynthetic mutants ade1 and ade2 of Saccharomyces cerevisiae accumulate a characteristic redpigment in their vacuoles under adenine limiting conditions. This red pigmentation phenotype, widely used in avariety of genetic screens and assays, is the end product of a glutathione-mediated detoxification pathway,where the glutathione conjugates are transported into the vacuole. The glutathione conjugation step, however,has still remained unsolved. We show here, following a detailed analysis of all the members of the thioredoxinfoldsuperfamily, the involvement of the monothiol glutaredoxin GRX4 as essential for pigmentation. GRX4plays multiple roles in the cell, and we show that the role in ade pigmentation does not derive from itsregulatory role of the iron transcription factor, Aft1p, but a newly identified GST activity of the protein that wecould demonstrate using purified Grx4p. Further, we demonstrate that the GRX domain of GRX4 and its activesite cysteine C171 is critical for this activity. The findings thus solve a decades old enigma on a critical step inthe formation of this red pigmentation.

• Interactome of vertebrate GAF/ThPOK reveals its diverse functions in gene regulation and DNA repair

GAGA associated factor (GAF) is a sequence-specific DNA binding transcription factor that is evolutionarilyconserved from flies to humans. Emerging evidence shows a context-dependent function of vertebrate GAF(vGAF, a.k.a. ThPOK) in multiple processes like gene activation, repression, and enhancer-blocking. Wehypothesize that context-dependent interaction of vGAF with a diverse set of proteins forms the basis for themultifunctional nature of vGAF. To this end, we deciphered the protein–protein interactome of vGAF andshow that vGAF interacts with chromatin remodelers, RNA metabolic machinery, transcriptional activators/repressors, and components of DNA repair machinery. We further validated the biological significance of ourprotein–protein interaction data with functional studies and established a novel role of vGAF in DNA repairand cell-survival after UV-induced DNA damage. One of the major risk factors for skin cutaneous melanoma isprolonged exposure of UV and subsequent DNA damage. vGAF is highly expressed in normal skin tissue.Interestingly, our analysis of high-throughput RNA-sequencing data shows that vGAF is heavily downregulatedacross all major stages of skin cutaneous melanoma suggesting its potential as a diagnostic biomarker.Taken together, our study provides a plausible explanation for the diverse gene regulatory functions of vGAFand unravels its novel role in DNA repair.

• MicroRNA-143–5p modulates pulmonary artery smooth muscle cells functions in hypoxic pulmonary hypertension through targeting HIF-1α

This paper explores the potential mechanism of microRNA-143–5p regulation effects on pulmonary arterysmooth muscle cells (PASMCs) functions in hypoxic pulmonary hypertension (HPH) via targeting HIF-1α,which may offer a new idea for HPH therapy. PASMCs were transfected with mimics control/miR-143–5pmimics or inhibitor control/miR-143–5p inhibitor. We used Western blotting and RT-qPCR to detect the proteinand mRNA expressions, CCK-8 assay to detect cellular viability, Annexin V-FITC/PI staining and caspase-3/cleaved caspase-3 protein to evaluate cellular apoptosis, transwell migration experiment for cellularmigration measurement and Dual luciferase reporter gene assay to prove the target of miR-143–5p. Cells underhypoxic condition presented the decreased protein and mRNA expressions of a-smooth muscle actin (SM-α-actin),Myocardin, smooth muscle myosin heavy chain (SMMHC), and smooth muscle-22α (SM22α),Calponin1 and Hypoxia-inducible factor-1α (HIF-1α), the increased cell viability and miR-143–5p level; Overexpressionof miR-143–5p obviously reduced vascular smooth muscle-specific contraction marker proteinlevels and cellular apoptosis, increased cellular migration of PASMCs with hypoxia stimulation; Low-expressionof miR-143–5p caused the opposite changes, while co-transfected with Si HIF-1a blocked thebeneficial effects of miR-143–5p inhibition on PASMCs under hypoxia. MicroRNA-143–5p can promote thephenotype conversion, proliferation and migration of pulmonary artery smooth muscle cells under hypoxiccondition through direct targeting of HIF-1α.

• Aleurothrixus trachoides (Back) can transmit begomovirus from Duranta to potato, tomato and bell pepper

Solanum whitefly, Aleurothrixus trachoides (Back). (Hemiptera: Aleyrodidae) was considered as a non-virusvector by European and Mediterranean Plant Protection Organization (EPPO) reports. However, in the presentstudy it was found to transmit Duranta leaf curl virus (DLCV) to tomato, bell pepper and potato. A. trachoidesinfested field samples of Duranta sp (100%) and tomato (20%) tested positive for begomovirus by PCR usingbegomovirus degenerate primers and primers specific to Tomato leaf curl New Delhi virus showing ampliconof 520 bp and 2.7 Kb respectively. The DNA samples of A. trachoides collected from virus positive durantaand tomato plants also tested positive for the virus. Virulent whiteflies from duranta could successfully transmitDLCV to bell pepper (26%) and tomato (13%) plants as confirmed by Rolling Circle Amplification. The rate ofvirus transmission by A. trachoides from DLCV inoculated tomato to bell pepper and tomato to potato was100% and tomato to tomato was 80%. The results suggest whitefly A. trachoides as the vector for DLCVand tothe best of our knowledge, this is the first report for A. trachoides as vector of begomovirus. These findingssuggest need for reconsideration of A. trachoides as a virus-vector. This will have great impact on solanaceousvegetable cultivation in India and other parts of the world.

• MicroRNA-20b-5p regulates propofol-preconditioning-induced inhibition of autophagy in hypoxia-and-reoxygenation-stimulated endothelial cells

Ischemia-reperfusion (IR) injury is a major cause of clinical emergencies during and after surgical procedures.Propofol protects the heart from cardiovascular IR injury by inhibiting autophagy. MicroRNAs (miRNAs)participate in anesthetic-regulated cardiovascular injury. MiR-20b-5p targets unc-51-like autophagy activatingkinase 1 (ULK1). Its role in propofol-modulated cardiovascular IR injury remains unclear, however. In thisstudy, we used an in vitro model of hypoxia-reoxygenation (HR)-induced injury to human umbilical veinendothelial cells (HUVECs) to determine the protective effect of miR-20b-5p in cells preconditioned withpropofol. We found that miR-20b-5p was significantly higher and ULK1 was lower in propofol-preconditionedHUVECs with HR injury than in HUVECs with HR injury only. Additionally, miR-20b-5p overexpressionincreased cell viability and repressed autophagy and apoptosis more in propofol-preconditioned HUVECs withHR injury than in HUVECs with HR injury only. A luciferase reporter assay confirmed the target reactionbetween miR-20b-5p and ULK1. Overexpression of ULK1 restrained the protective effect of miR-20b-5p inpropofol-preconditioned HUVECs with HR injury. In conclusion, our results indicate that propofol inhibitsautophagic cell death via the miR-20b-5p-ULKI axis and that ULK1 may be a therapeutic target for cardiovascularIR injury.

• β-Actin facilitates etoposide-induced p53 nuclear import

As a tumor suppressor, p53 preserves genomic integrity in eukaryotes. However, limited evidence is availablefor the p53 shuttling between the cytoplasm and nucleus. Previous studies have shown that β-actin polymerizationnegatively regulates p53 nuclear import through its interaction with p53. In this study, we found thatDNA damage induces both b-actin and p53 accumulation in the nucleus. b-actin knockdown impaired thenuclear transport of p53. Additionally, b-actin could interact with p53 which was enhanced in response togenotoxic stress. Furthermore, N terminal deletion mutants of p53 shows reduced levels of association with β-actin.We further identified Ser15, Thr18 and Ser20 of p53 are critical to the β-actin: p53 interaction, whichupon mutation into alanine abrogates the binding. Taken together, this study reveals that β-actin regulates thenuclear import of p53 through protein–protein interaction.

• X-Module: A novel fusion measure to associate co-expressed gene modules from condition-specific expression profiles

A gene co-expression network (CEN) is of biological interest, since co-expressed genes share commonfunctions and biological processes or pathways. Finding relationships among modules can reveal inter-modularpreservation, and similarity in transcriptome, functional, and biological behaviors among modules of the sameor two different datasets. There is no method which explores the one-to-one relationships and one-to-manyrelationships among modules extracted from control and disease samples based on both topological andsemantic similarity using both microarray and RNA seq data. In this work, we propose a novel fusion measureto detect mapping between modules from two sets of co-expressed modules extracted from control and diseasestages of Alzheimer’s disease (AD) and Parkinson’s disease (PD) datasets. Our measure considers bothtopological and biological information of a module and is an estimation of four parameters, namely, semanticsimilarity, eigengene correlation, degree difference, and the number of common genes. We analyze the consensusmodules shared between both control and disease stages in terms of their association with diseases. Wealso validate the close associations between human and chimpanzee modules and compare with the state-ofthe-art method. Additionally, we propose two novel observations on the relationships between modules forfurther analysis.

• Glyoxal modification mediates conformational alterations in silk fibroin: Induction of fibrillation with amyloidal features

Silkwormsilk protein fibroin is widely exploited to develop novel silk-based biomaterials due to its stable b-sheetstructure, providing high crystallinity and tensile strength. The polymorphic behaviour of silk fibroin provides awindow to modulate its structural transitions during self-assembly for different functional outcomes. Most studiesare therefore mainly focused on formation of well-developed beta-sheet structure and self-assembly of silk fibroinwhich are regulated by many parameters. Glyoxal, a highly reactive alpha-oxoaldehyde, reacts with different proteinsto form advanced glycation end products (AGEs) following Maillard-like reaction. Considering the significanceof protein modification by glyoxal-derived AGEs, in the present study the effect of glyoxal (250, 500 and1000 micro-M) on the structure of silk fibroin has been investigated. CD and fluorescence studies reveal that higherconcentrations of the a-oxoaldehyde induce considerable alterations of secondary and tertiary structure of theprotein leading to aggregation following incubation with for 3 weeks. The aggregates exhibit fibrillar morphologywith amyloidal nature as evident from SEM, FTIR and XRD experiments. The findings highlight that glycationinducedmodification can be a possible approach for modulating the conformation of the silk protein which may berelevant in connection to clinical, biomedical or synthetic biology based applications.

• Development of transgenic cotton (Narasimha) using triple gene Cry2Ab-Cry1F-Cry1Ac construct conferring resistance to lepidopteran pest

High-yielding Indian cotton varieties are not amenable for regeneration and transformation because they arerecalcitrant in nature. In this work, we have developed Narasimha (NA1325) cotton variety by introducing three Crygenes driven by three different promoters conferring insect resistance. The meristematic region of embryo axisexplants were infected and co-cultivated with Agrobacterium tumefacience (LBA4404) harbouring pMDC100vector with three Cry gene cassettes (a-globulin : Cry2Ab, DECaMV35s : Cry1F and nodulin : Cry1Ac) with Npt IIas a selectable marker gene. Out of 1010 embryo axes explants infected, 121 (T0) regenerated under two rounds ofkanamycin selectionmedium.About 2551T1 seedswere collected from111T0 plants and these seeds screened againwith kanamycin at seedling stage. The transgenic plants were characterized by PCR, real time quantitative PCR,lateral flow strip protein assay and insect bioassay. Out of 145 kanamycin resistant plants (T1), twelve showedamplification of all four transgenes: Npt II, Cry2Ab, Cry1F and Cry1Ac through PCR with expected amplicons as395, 870, 840 and 618 bp, respectively. Further, lateral flow strip test revealed Cry1F and Cry1Ac proteinsaccumulated in 12 plants, whereas Cry2Ab protein was detected in eight only. The transcripts of all three Cry geneswere accumulated significantly higher in transgenic plants at T2 generation. The transgenic lines showed effectiveresistance againstHelicoverpa armigera and Spodoptera litura larvae. The T2 line L-3 exhibited highest percentageof insect mortality, in which transcripts of all cry genes were accumulated higher than other plants. The transgeniccotton plants carrying triple Cry genes could be an excellent germplasmresource for the breeders for introgressions.

• Target-specific gene delivery in plant systems and their expression: Insights into recent developments

In order to improve crop plants in terms of their yield, drought resistance, pest resistance, nutritional value, etc.,modern agriculture has relied upon plant genetic engineering. Since the advent of recombinant DNA technology,several tools have been used for genetic transformations in plants such as Agrobacterium tumefaciens,virus-mediated gene transfer, direct gene transfer systems such as electroporation, particle gun, microinjectionand chemical methods. All these traditional methods lack specificity and the transgenes are integrated atrandom sites in the plant DNA. Recently novel techniques for gene targeting have evolved such as engineerednucleases such as Zinc Finger Nucleases, Transcription Activator like effector nucleases, Clustered regularinterspaced short palindromic repeats. Other advances include improvement in tools for delivery of geneediting components which include carrier proteins, and carbon nanotubes. The present review focuses on thelatest techniques for target specific gene delivery in plants, their expression and future directions in plantbiotechnology.

• Dynamic conformational flexibility and molecular interactions of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) are highly flexible and undergo disorder to order transition uponbinding. They are highly abundant in human proteomes and play critical roles in cell signaling and regulatoryprocesses. This review mainly focuses on the dynamics of disordered proteins including their conformationalheterogeneity, protein–protein interactions, and the phase transition of biomolecular condensates that arecentral to various biological functions. Besides, the role of RNA-mediated chaperones in protein folding andstability of IDPs were also discussed. Finally, we explored the dynamic binding interface of IDPs as noveltherapeutic targets and the effect of small molecules on their interactions.

• Activation of RelA by pppGpp as the basis for its differential toxicity over ppGpp in Escherichia coli

The nucleotide derivatives (p)ppGpp, comprising ppGpp and pppGpp, are important signalling molecules thatcontrol various facets of gene regulation and protein synthesis in Escherichia coli. Their synthesis is catalysedby RelA (in response to amino acid limitation) and by SpoT (in response to the limitation of carbon source orfatty acids). SpoT is also a hydrolase for degradation of both ppGpp and pppGpp, while GppA catalyses theconversion of pppGpp to ppGpp. Here we provide evidence to show that pppGpp exerts heightened toxicitycompared to that by ppGpp. Thus, gppA spoT double mutants exhibited lethality under conditions in which thesingle mutants were viable. The extent of RelA-catalysed (p)ppGpp accumulation in the gppA spoT strain wassubstantially greater than that in its isogenic gppA? derivative. The data is interpreted in terms of a model inwhich toxicity of pppGpp in the gppA spoT mutants is mediated by its activation of RelA so as to result in avicious cycle of (p)ppGpp synthesis.

• Comprehensive molecular insights into the stress response dynamics of rice (Oryza sativa L.) during rice tungro disease by RNA-seq-based comparative whole transcriptome analysis

Rice tungro is a serious viral disease of rice resulting from infection by two viruses, Rice tungro bacilliformvirus and Rice tungro spherical virus. To gain molecular insights into the global gene expression changes inrice during tungro, a comparative whole genome transcriptome study was performed on healthy and tungroaffectedrice plants using Illumina Hiseq 2500. About 10 GB of sequenced data comprising about 50 millionpaired end reads per sample were then aligned on to the rice genome. Gene expression analysis revealedaround 959 transcripts, related to various cellular pathways concerning stress response and hormonal homeostasisto be differentially expressed. The data was validated through qRT-PCR. Gene ontology and pathwayanalyses revealed enrichment of transcripts and processes similar to the differentially expressed genes categories.In short, the present study is a comprehensive coverage of the differential gene expression landscapeand provides molecular insights into the infection dynamics of the rice-tungro virus system.

• Computational prediction of active sites and ligands in different AHL quorum quenching lactonases and acylases

With the emergence of multidrug-resistant ‘superbug’, conventional treatments become obsolete. Quorumquenching (QQ), enzyme-dependent alteration of quorum sensing (QS), is now considered as a promisingantimicrobial therapy because of its potentiality to impede virulence gene expression without resulting ingrowth inhibition and antibiotic resistance. In our study, we intended to compare between two major QQenzyme groups (i.e., AHL lactonases and AHL acylases) in terms of their structural and functional aspects.The amino acid composition-based principal component analysis (PCA) suggested that probably there is nostructural and functional overlapping between the two groups of enzymes as well as within the lactonaseenzymes but the acylases may functionally be affected by one another. In subcellular localization analysis,we also found that most lactonases are cytoplasmic while acylases are periplasmic. Investigation on thesecondary structural features showed random coil dominates over a-helix and b-sheet in all evaluatedenzymes. For structural comparison, the tertiary structures of the selected proteins were modelled andsubmitted to the PMDB database (Accession ID: PM0081007 to PM0081018). Interestingly, sequencealignment revealed the presence of several conserved domains important for functions in both proteingroups. In addition, three amino acid residues, namely aspartic acid, histidine, and isoleucine, were commonin the active sites of all protein models while most frequent ligands were found to be 3C7, FEO, and PAC.Importantly, binding interactions of predicted ligands were similar to that of native QS signal molecules.Furthermore, hydrogen bonds analysis suggested six proteins are more stable than others. We believe thatthe knowledge of this comparative study could be useful for further research in the development of QSbaseduniversal antibacterial strategies.

• Effects of biotic and abiotic factors on biofilm growth dynamics and their heterogeneous response to antibiotic challenge

Over the last couple of decades, with the crisis of new antimicrobial arsenal, multidrug-resistant clinicalpathogens have been observed extensively. In clinical and medical settings, these persistent pathogens predominantlygrow as complex heterogeneous structures enmeshed in a self-produced exopolysaccharide matrix,termed as biofilms. Since biofilms can rapidly form by adapting new environmental surroundings and havepotential effect on human health, it is critical to study them promptly and consistently. Biofilm infections arechallenging in the contamination of medical devices and implantations, food processing and pharmaceuticalindustrial settings, and in dental area caries, periodontitis and so on. The persistence of infections associatedwith biofilms has been mainly attributed to the increased antibiotic resistance offered by the cells growing inbiofilms. In fact, it is well known that this recalcitrance of bacterial biofilms is multifactorial, and there areseveral resistance mechanisms that may act in parallel in order to provide an enhanced level of resistance to thebiofilm. In combination, distinct resistance mechanisms significantly decrease our ability to control anderadicate biofilm-associated infections with current antimicrobial arsenal. In addition, various factors areknown to influence the process of biofilm formation, growth dynamics, and their heterogeneous responsetowards antibiotic therapy. The current review discusses the contribution of cellular and physiochemical factorson the growth dynamics of biofilm, especially their role in antibiotic resistance mechanisms of bacterialpopulation living in surface attached growth mode. A systematic investigation on the effects and treatment ofbiofilms may pave the way for novel therapeutic strategies to prevent and treat biofilms in healthcare andindustrial settings.

• A transmission ratio distortion and the ‘max-4’ ascus phenotype: Do both reflect the same Bateson-Dobzhansky-Muller Incompatibility emerging during trans-species introgression of translocations in Neurospora?

The ${T(EB4)}^{Nt}a$, ${T(IBj5)}^{Nt}a$, and ${T(B362i)}^{Nt}A$ strains were constructed by introgressing the insertional translocations EB4, IBj5, and B362i from Neurospora crassa into the related species N. tetrasperma. Theprogeny from crosses of ${T(IBj5)}^{Nt}a$ and ${T(B362i)}^{Nt}A$ with opposite mating-type derivatives of the standard N. tetrasperma strain 85 exhibited a unique and unprecedented transmission ratio distortion (TRD) that disfavored homokaryons produced following alternate segregation relative to those produced following adjacent-1 segregation. The TRD was not evident among the [mat A + mat a] dikaryons produced following either segregation. Further, crosses of the ${T(IBj5)}^{Nt}a$ and ${T(B362i)}^{Nt}A$ strains with the Eight spore (E) mutant showed an unusual ascus phenotype called ‘max-4’. We propose that the TRD and the max-4 phenotype are manifestations of the same Bateson-Dobzhansky-Muller incompatibility (BDMI). Since the TRD selects against 2/3 ofthe homokaryotic progeny from each introgression cross, the BDMI would have enriched for the dikaryotic progeny in the viable ascospores, and thus, paradoxically, facilitated the introgressions.

• Acid-functionalized single-walled carbon nanotubes alter epithelial tight junctions and enhance paracellular permeability

Due to their unique properties, carbon nanotubes (CNTs) are being widely explored for industrial and medicalapplications. This has necessitated a thorough assessment of the effect of CNTs on human and animalphysiology and health. Impact of CNTs on epithelial tight junctions has not been evaluated in the context oftheir toxic effects in many biological systems. In the present study, we examined the effect of acid functionalizedsingle-walled carbon nanotubes (AF-SWCNTs) on the function and expression of two tight junctionproteins (ZO-1 and occludin) in the Madin-Darby canine kidney (MDCK) cell line. Treatment of MDCK cellswith AF-SWCNT resulted in a downregulation of tight junction proteins, decreased trans-epithelial electricalresistance (TER), increased paracellular permeability, and disruption of tight junctions. Taken together, ourdata demonstrate that AF-SWCNT disrupts tight junction barrier by downregulating tight junction proteins inMDCK epithelial cells.

• Do males bond? A study of male-male relationships in Nicobar long-tailed macaques Macaca fascicularis umbrosus

In primates, males compete for a mate, which is a non-sharable resource. This makes the conditions lessconducive for males to have stable relationships. One such special kind of relationship is a bond where theinteractions are reciprocated, equitable and differentiated. Bonds in macaque societies are based on the degreeof within-group contest competition for mates which is dependent on the synchronization of female fertilephase and reliability of fertility signals. Species of the Fascicularis group, including Nicobar subspecies, showintermediate reliability in the signals with mild peaks, and studies have shown reciprocity but no differentiation.We conducted a study on a group of wild Nicobar long-tailed macaques Macaca fascicularis umbrosusto understand the existing patterns of male-male relationships. We examined whether there is reciprocity inaffiliation among the individuals and whether the rate of affiliation is balanced. We also measured the dominancelinearity and steepness in the group to understand the monopolizability of females. We used socialnetwork analysis to understand whether the relations are differentiated based on hierarchical position andwhether the high-ranking individuals are the most central individuals in the distribution of grooming in thegroup. We found that there is reciprocity among the males although that is not equitable. There was no rank relateddifferentiation of affiliation among the males of the group. Instead, the identities of individualsinfluenced affiliation patterns. Our results correspond to the existent strong relationships but lack of social bondotherwise found in the Fascicularis group of macaques.

• TET methylcytosine oxidases: new insights from a decade of research

In mammals, DNA methyltransferases transfer a methyl group from S-adenosylmethionine to the 5 position ofcytosine in DNA. The product of this reaction, 5-methylcytosine (5mC), has many roles, particularly insuppressing transposable and repeat elements in DNA. Moreover, in many cellular systems, cell lineagespecification is accompanied by DNA demethylation at the promoters of genes expressed at high levels in thedifferentiated cells. However, since direct cleavage of the C-C bond connecting the methyl group to the 5position of cytosine is thermodynamically disfavoured, the question of whether DNA methylation wasreversible remained unclear for many decades. This puzzle was solved by our discovery of the TET (Ten-Eleven Translocation) family of 5-methylcytosine oxidases, which use reduced iron, molecular oxygen and thetricarboxylic acid cycle metabolite 2-oxoglutarate (also known as alpha-ketoglutarate) to oxidise the methyl groupof 5mC to 5-hydroxymethylcytosine (5hmC) and beyond. TET-generated oxidised methylcytosines areintermediates in at least two pathways of DNA demethylation, which differ in their dependence on DNAreplication. In the decade since their discovery, TET enzymes have been shown to have important roles inembryonic development, cell lineage specification, neuronal function and cancer. We review these findings anddiscuss their implications here.

• Role of genomic imprinting in mammalian development

Non-mendelian inheritance refers to the group of phenomena and observations related to the inheritance ofgenetic information that cannot be merely explained by Mendel’s laws of inheritance. Phenomenon includingGenomic imprinting, X-chromosome Inactivation, Paramutations are some of the best studied examples ofnon-mendelian inheritance. Genomic imprinting is a process that reversibly marks one of the two homologousloci, chromosome or chromosomal sets during development, resulting in functional non-equivalence of geneexpression. Genomic imprinting is known to occur in a few insect species, plants, and placental mammals.Over the years, studies on imprinted genes have contributed immensely to highlighting the role of epigeneticmodifications and the epigenetic circuitry during gene expression and development. In this review, we discussthe phenomenon of genomic imprinting in mammals and the role it plays especially during fetoplacentalgrowth and early development.

• Cellular environment controls the dynamics of histone H3 lysine 56 acetylation in response to DNA damage in mammalian cells

Epigenetic changes play a crucial role in sensing signals and responding to fluctuations in the extracellularenvironment. How the cellular micro-environment affects DNA damage response signalling in chromatincontext is not extensively studied. Histone acetylation is dynamic and very sensitive to changes in theextracellular environment. Existing literature on H3 lysine 56 acetylation (H3K56ac) levels upon DNA damagein mammals presents a conflicting picture. The occurrence of both increased and decreased H3K56ac uponDNA damage in our experiments led us to investigate the role of the micro-environment on H3K56ac. Here,we show that the global levels of H3K56ac increase as cells grow from low density to high density whileSIRT1 and SIRT6 expression decrease. Additionally, rising lactic acid levels increase H3K56ac. Our resultsshow that cell density and accumulation of metabolites affect dynamics of H3K56ac in response to DNAdamage. Upon DNA damage, H3K56ac increases in low density cells with low initial acetylation, whileacetylation decreases in high cell density cells. These results highlight that H3K56ac levels upon DNA damageare dependent on the metabolites in the extracellular milieu which impact chromatin structure by regulatingchromatin modifying enzymes. Accumulation of lactic acid at high cell density reflects conditions similar to thetumour micro-environment. As H3K56ac increases in tumours, lactic acid and low pH might alter H3K56ac intumours, leading to deregulated gene expression, contributing to tumour progression.

• Recent advances in the spatial organization of the mammalian genome

The mammalian genome is complex and presents a dynamic structural organization that reflects function.Organization of the genome inside the mammalian nucleus impacts all nuclear processes including but notlimited to transcription, replication and repair, and in many biological contexts such as early development,differentiation and physiological adaptations. However, there is limited understating of how 3D organization ofthe mammalian genome regulates different nuclear processes. Recent advances in microscopy and a myriad ofgenomics methods—propelled by next-generation sequencing—have advanced our knowledge of genomeorganization to a great extent. In this review, we discuss nuclear compartments in general and recent advancesin the understanding of how mammalian genome is organized in these compartments with an emphasis ondynamics at the nuclear periphery.

• When histones are under glucose starvation

Under nutritional stress, cells undergo metabolic rewiring that results in changes of various cellular processesthat include gene transcription. This transcriptional regulation requires dynamic chromatin remodeling thatinvolves histone post-translational modifications. There are several histone marks that may act as switchesupon starvation for stress-response pathways.

• The CBX family of proteins in transcriptional repression and memory

For mammals to develop properly, master regulatory genes must be repressed appropriately in a heritablemanner. This review concerns the Polycomb Repressive Complex 1 (PRC1) family and the relationshipbetween the establishment of repression and memory of the repressed state. The primary focus is on the CBXfamily of proteins in PRC1 complexes and their role in both chromatin compaction and phase separation.These two activities are linked and might contribute to both repression and memory.

• Regulation of epigenetic state by non-histone chromatin proteins and transcription factors: Implications in disease

Besides the fundamental components of the chromatin, DNA and octameric histone, the non-histone chromatinproteins and non-coding RNA play a critical role in the organization of functional chromatin domains. Thenon-histone chromatin proteins therefore regulate the transcriptional outcome in both physiological andpathophysiological state as well. They also help to maintain the epigenetic state of the genome indirectly.Several transcription factors and histone interacting factors also contribute in the maintenance of the epigeneticstates, especially acetylation by the induction of autoacetylation ability of p300/CBP. Alterations of KATactivity have been found to be causally related to disease manifestation, and thus could be potential therapeutictarget.

• Role of nucleosome positioning in 3D chromatin organization and loop formation

We present a physics-based polymer model that can investigate 3D organization of chromatin accounting for DNA elasticity,DNA-bending due to nucleosomes, and 1D organization of nucleosomes along DNA. We find that the packing density ofchromatin oscillates between densities corresponding to highly folded and extended configurations as we change thenucleosome organization (length of linker DNA). We compute the looping probability of chromatin and show that thepresence of nucleosomes increases the looping probability of the chain compared to that of a bare DNA. We also show thatlooping probability has a large variability depending on the nature of nucleosome organization and density of linker histones.

• Picking a nucleosome lock: Sequence- and structure-specific recognition of the nucleosome

The nucleosome presents a formidable barrier to DNA-templated transcription by the RNA polymerase IImachinery. Overcoming this transcriptional barrier in a locus-specific manner requires sequence-specificrecognition of nucleosomal DNA by ‘pioneer’ transcription factors (TFs). Cell fate decisions, in turn, dependon the coordinated action of pioneer TFs at cell lineage-specific gene regulatory elements. Although it isalready appreciated that pioneer factors play a critical role in cell differentiation, our understanding of thestructural and biochemical mechanisms by which they act is still rapidly expanding. Recent research hasrevealed novel insight into modes of nucleosome-TF binding and uncovered kinetic principles by whichnucleosomal DNA compaction affects both TF binding and residence time. Here, we review progress and arguethat these structural and kinetic studies suggest new models of gene regulation by pioneer TFs.

• Genomic organization of Polycomb Response Elements and its functional implication in Drosophila and other insects

The epigenetic memory is an essential aspect of multicellular organisms to maintain several cell types and their geneexpression pattern. This complex process uses a number of protein factors and specific DNA elements within thedevelopmental cues to achieve this. The protein factors involved in the process are the Polycomb group (PcG)members, and, accordingly, the DNA sequences that interact with these proteins are called Polycomb ResponseElements (PREs). Since the PcG proteins are highly conserved among higher eukaryotes, including insects, andfunction at thousands of sites in the genomes, it is expected that PREs may also be present across the genome.However,the studies on PREs in insect species, other thanDrosophila, is currently lacking.We took a bioinformatics approach todevelop an inclusive PRE prediction tool, ‘PRE Mapper’, to address this need. By applying this tool on the Drosophilamelanogaster genome, we predicted more than 20,000 PREs.When compared with the available PRE prediction methods, thistool shows far better performance by correctly identifying the in vivo binding sites of PcG proteins, identified bygenome-scale ChIP experiments. Further analysis of the predicted PREs shows their cohabitation with chromatindomain boundary elements at several places in the Drosophila genome, possibly defining a composite epigeneticmodule.We analysed 10 insect genomes in this context and find several conserved features in PREs across the insectspecies with some variations in their occurrence frequency. These analyses leading to the identification of PREin insectgenomes contribute to our understanding of epigenetic mechanisms in these organisms.

• Role of PfGCN5 in nutrient sensing and transcriptional regulation in Plasmodium falciparum

Malaria is a deadly, infectious disease caused by the parasite Plasmodium, leading to millions of deathsworldwide. Plasmodium requires a coordinated pattern of sequential gene expression for surviving in bothinvertebrate and vertebrate host environments. As parasites largely depend on host resources, they also developefficient mechanisms to sense and adapt to variable nutrient conditions in the environment and modulate theirvirulence. Earlier we have shown that PfGCN5, a histone acetyltransferase, binds to the stress-responsive andvirulence-related genes in a poised state and regulates their expression under temperature and artemisinintreatment conditions in P. falciparum. In this study, we show upregulation of PfGCN5 upon nutrient stresscondition. With the help of chromatin immunoprecipitation coupled high-throughput sequencing (ChIP-seq)and transcriptomic (RNA-sequencing) analyses, we show that PfGCN5 is associated with the genes that areimportant for the maintenance of parasite cellular homeostasis upon nutrient stress condition. Furthermore, weidentified various metabolic enzymes as interacting partners of PfGCN5 by immunoprecipitation coupled withmass spectroscopy, possibly acting as a sensor of nutrient conditions in the environment. We also demonstratedthat PfGCN5 interacts and acetylates PfGAPDH in vitro. Collectively, our data provides important insights intotranscriptional deregulation upon nutrient stress condition and elucidate the role of PfGCN5 during nutrientstress condition.

• The conserved aspartate in motif III of b family AdoMet-dependent DNA methyltransferase is important for methylation

S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases (MTases) are involved in diverse cellularfunctions. These enzymes show little sequence conservation but have a conserved structural fold. The DNAMTases have characteristic motifs that are involved in AdoMet binding, DNA target recognition and catalysis.Motif III of these MTases have a highly conserved acidic residue, often an aspartate, whose functionalsignificance is not clear. Here, we report a mutational study of the residue in the beta family MTase of the Type IIIrestriction-modification enzyme EcoP15I. Replacement of this residue by alanine affects its methylationactivity. We propose that this residue contributes to the affinity of the enzyme for AdoMet. Analysis of thestructures of DNA, RNA and protein MTases reveal that the acidic residue is conserved in all of them, andinteracts with N6 of the adenine moiety of AdoMet. Interestingly, in the SET-domain protein lysine MTases,which have a fold different from other AdoMet-dependent MTases, N6 of the adenine moiety is hydrogenbonded to the main chain carbonyl group of the histidine residue of the highly conserved motif III. Our studyreveals the evolutionary conservation of a carbonyl group in DNA, RNA and protein AdoMet-dependentMTases for specific interaction by hydrogen bond with AdoMet, despite the lack of overall sequenceconservation.

• Mining histone methyltransferases and demethylases from whole genome sequence

Epigenetic regulation through post-translational modification of histones, especially methylation, is wellconserved in evolution. Although there are several insect genomes sequenced, an analysis with a focus on theirepigenetic repertoire is limited. We have utilized a novel work-flow to identify one or more domains as highprioritydomain (HPD), if present in at least 50% of the genes of a given functional class in the referencegenome, namely, that of Drosophila melanogaster. Based on this approach, we have mined histone methyltransferasesand demethylases from the whole genome sequence of Aedes aegypti (Diptera), the pea aphidAcyrthosiphon pisum, the triatomid bug Rhodnius prolixus (Hemiptera), the honeybee Apis mellifera (Hymenoptera),the silkworm Bombyx mori (Lepidoptera) and the red flour beetle Tribolium castaneum(Coleoptera). We identified 38 clusters consisting of arginine methyltransferases, lysine methyltransferases anddemethylases using OrthoFinder, and the presence of HPD was queried in these sequences using InterProScan.This approach led us to identify putative novel members and currently inaccurate ones. Other than the highprioritydomains, these proteins contain shared and unique domains that can mediate protein–protein interaction.Phylogenetic analysis indicates that there is different extent of protein sequence similarity; averagesimilarity between histone lysine methyltransferases varies from 41% (for active mark) to 48% (for repressivemark), arginine methyltransferases is 51%, and demethylases is 52%. The method utilized here facilitatesreliable identification of desired functional class in newly sequenced genomes.

• Origin of RNA Polymerase II pause in eumetazoans: Insights from Hydra

Multicellular organisms have evolved sophisticated mechanisms for responding to various developmental,environmental and physical stimuli by regulating transcription. The correlation of distribution of RNAPolymerase II (RNA Pol II) with transcription is well established in higher metazoans, however genome-wideinformation about its distribution in early metazoans, such as Hydra, is virtually absent. To gain insights intoRNA Pol II-mediated transcription and chromatin organization in Hydra, we performed chromatin immunoprecipitation(ChIP)-coupled high-throughput sequencing (ChIP-seq) for RNA Pol II and Histone H3. Strikingly,we found that Hydra RNA Pol II is uniformly distributed across the entire gene body, as opposed to itscounterparts in bilaterians such as human and mouse. Furthermore, correlation with transcriptome datarevealed that the levels of RNA Pol II correlate with the magnitude of gene expression. Strikingly, thecharacteristic peak of RNA Pol II pause typically observed in bilaterians at the transcription start sites (TSSs)was not observed in Hydra. The RNA Pol II traversing ratio in Hydra was found to be intermediate to yeastand bilaterians. The search for factors involved in RNA Pol II pause revealed that RNA Pol II pausingmachinery was most likely acquired first in Cnidaria. However, only a small subset of genes exhibited thepromoter proximal RNP Pol II pause. Interestingly, the nucleosome occupancy is highest over the subset ofpaused genes as compared to total Hydra genes, which is another indication of paused RNA Pol II at thesegenes. Thus, this study provides evidence for the molecular basis of RNA Pol II pause early during theevolution of multicellular organisms.

• Metabolic choreography of gene expression: nutrient transactions with the epigenome

Eukaryotic complexity and thus their ability to respond to diverse cues are largely driven by varyingexpression of gene products, qualitatively and quantitatively. Protein adducts in the form of post-translationalmodifications, most of which are derived from metabolic intermediates, allow fine tuning of gene expressionat multiple levels. With the advent of high-throughput and high-resolution mapping technologies there hasbeen an explosion in terms of the kind of modifications on chromatin and other factors that govern geneexpression. Moreover, even the classical notion of acetylation and methylation dependent regulation oftranscription is now known to be intrinsically coupled to biochemical pathways, which were otherwiseregarded as ‘mundane’. Here we have not only reviewed some of the recent literature but also havehighlighted the dependence of gene regulatory mechanisms on metabolic inputs, both direct and indirect. Wehave also tried to bring forth some of the open questions, and how our understanding of gene expression haschanged dramatically over the last few years, which has largely become metabolism centric. Finally,metabolic regulation of epigenome and gene expression has gained much traction due to the increasedincidence of lifestyle and age-related diseases.

• The role of histone modifications in leukemogenesis

Histone modifications play a critical role in coordinating accurate gene expression. Aside from geneticmutations which cause altered DNA sequence, it has become increasingly clear that aberrant post-translationalmodifications of histone tails are also associated with leukemogenesis. The functional roles of specific histonemarks has informed the basis of our understanding for underlying mechanisms of leukemia, while globalanalyses of interacting histone modifications has begun to distinguish subtypes of leukemia with prognosticand therapeutic implications. In this current era of personalized and precision medicine, it will be necessary tonot only identify the specific genetic mutations present in a patient’s leukemia but to also appreciate thedynamic chromatin states which are driven by histone modifications that can aid our diagnostic and therapeuticstrategies for improved management of leukemia.

• Phase-separation in chromatin organization

The organization of chromatin into different types of compact versus open states provides a means to fine tune generegulation. Recent studies have suggested a role for phase-separation in chromatin compaction, raising new possibilitiesfor regulating chromatin compartments. This perspective discusses some specific molecular mechanismsthat could leverage such phase-separation processes to control the functions and organization of chromatin.

• Histone H2A isoforms: Potential implications in epigenome plasticity and diseases in eukaryotes

Epigenetic mechanisms including the post-translational modifications of histones, incorporation of histonevariants and DNA methylation have been suggested to play an important role in genome plasticity by allowingthe cellular environment to define gene expression and the phenotype of an organism. Studies over the pastdecade have elucidated how these epigenetic mechanisms are significant in orchestrating various biologicalprocesses and contribute to different pathophysiological states. However, the role of histone isoforms and theirimpact on different phenotypes and physiological processes associated with diseases are not fully clear. Thisreview is focussed on the recent advances in our understanding of the complexity of eukaryotic H2A isoformsand their roles in defining nucleosome organization. We elaborate on their potential roles in genomic complexityand regulation of gene expression, and thereby on their overall contribution towards cellular phenotypeand development of diseases.

• Hypoxia-induced changes in intragenic DNA methylation correlate with alternative splicing in breast cancer

The tumor microenvironment is marked by gradients in the level of oxygen and nutrients, with oxygen levelsreaching a minimum at the core of the tumor, a condition known as tumor hypoxia. Mediated by members of theHIF family of transcription factors, hypoxia leads to a more aggressive tumor phenotype by transactivation ofseveral genes as well as reprogramming of pre-mRNA splicing. Intragenic DNA methylation, which is known toaffect alternative splicing in cancer, could be one of several reasons behind the changes in splicing patterns underhypoxia. Here, we have tried to establish a correlation between intragenicDNA methylation and alternative usageof exons in tumor hypoxia. First, we have generated a customhypoxia signature consisting of 34 genes that are upregulatedunder hypoxia and are direct targets of HIF-1alpha. Using this gene expression signature, we have successfullystratified publicly available breast cancer patient samples into hypoxia positive and hypoxia negativegroups followed by mining of differentially spliced isoforms between these groups. The Hypoxia Hallmarksignature from MSigDB was also used independently to stratify the same tumor samples into hypoxic andnormoxic.We found that 821 genes were showing differential splicing between samples stratified using a customsignature, whereas, 911 genes were showing differential splicing between samples stratified using the MSigDBsignature. Finally, we performed multiple correlation tests between the methylation levels (beta) of microarrayprobes located within 1 kilo base pairs of isoform-specific exons using those exons’ expression levels in the samepatient samples in which the methylation level was recorded. We found that the expression level of one of theexons ofDHX32 and BICD2 significantly correlated with the methylation levels, and we were also able to predictpatient survival (p-value: 0.02 for DHX32 and 0.0024 for BICD2). Our findings provide new insights into thepotential functional role of intragenic DNA methylation in modulating alternative splicing during hypoxia.

• A novel role of tumor suppressor ZMYND8 in inducing differentiation of breast cancer cells through its dual-histone binding function

Accumulating evidences indicate the involvement of epigenetic deregulations in cancer. While some epigeneticregulators with aberrant functions in cancer are targeted for improving therapeutic outcome in patients,reinstating the functions of tumor-suppressor-like epigenetic regulators might further potentiate anti-cancertherapies. Epigenetic reader zinc-finger MYND-type-containing 8 (ZMYND8) has been found to be endowedwith multiple anti-cancer functions like inhibition of tumor cell migration and proliferation. Here, we reportanother novel tumor suppressor role of ZMYND8 as an inducer of differentiation in breast cancer cells, byupregulating differentiation genes. Interestingly, we also demonstrated that ZMYND8 mediates all its antitumorroles through a common dual-histone mark binding to H4K16Ac and H3K36Me2. We validated thesefindings by both biochemical and biophysical analyses. Furthermore, we also confirmed the differentiationinducingpotential of ZMYND8 in vivo, using 4T1 murine breast cancer model in Balb/c mice. Differentiationtherapy holds great promise in cancer therapy, since it is non-toxic and makes the cancer cells therapysensitive.In this scenario, we propose epigenetic reader ZMYND8 as a potential therapeutic candidate fordifferentiation therapy in breast cancer.

• Editorial Topical collection on Chromatin Biology and Epigenetics

• # Journal of Biosciences

Volume 45, 2020
All articles
Continuous Article Publishing mode

• # Editorial Note on Continuous Article Publication

Posted on July 25, 2019