• Issue front cover thumbnail

      Volume 43, Issue 2

      June 2018,   pages  225-419

    • Inconclusive studies on possible CRISPR-Cas off-targets should moderate expectations about enzymes that have evolved to be non-specific


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    • What history tells us XLV. The ‘instability’ of messenger RNA


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    • A cross-eyed geneticist’s view I. Making sense of the lamin B receptor, a chimeric protein


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      The vertebrate inner nuclear membrane protein, lamin B receptor, has an N-terminal *200 residue nucleoplasmic domain(NTD), and a *420 residue C-terminal domain (CTD) that anchors the NTD to the INM. Chen et al (2016) showed theNTD interacts with Xist long noncoding RNA to effect X chromosome inactivation in female mammals. Tsai et al (2016)showed the CTD has sterol reductase activity that is essential for viability. And Nikolakaki et al (2017) proposed a model tointerconnect these disparate functions of this chimeric protein. It amuses me now to think back to 24 years ago, when I wasconcerned that these domains might have come together in a cloning artifact.

    • Evolution of genome organization and epigenetic machineries


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      The unit content of DNA in a living organism containing all of its genes is referred to as ‘genome’, which forms the basis oflife and heredity. In the path of evolution from a single-cellular prokaryotic life to multi-cellular eukaryotic system, thegenome has become more and more complex not only in the context of size but also in sequence and content. Although thesize of the genome does not directly correlate with the complexity and hierarchy of a living organism, in the eukaryoticsystem with restricted nuclear size, disproportionately higher size of DNA is packed into a highly ordered manner (Kumariet al., in Kundu (eds) Epigenetics: development and disease. Subcellular biochemistry, Springer, Dordrecht, 2013). Thispackaging of genome with the assistance of protein and RNA is not a unique feature of the eukaryotic system alone. In fact,even in a tiny prokaryotic cell, to reduce the volume and to restrict the signal dependent availability of the genomic material(DNA), packing of genome is essential. In this brief article, we shall try to put forward a concept of genome organizationand epigenetic machineries to assist the functional ability of the genomic material in the evolutionary perspective.

    • Loss of mitochondrial SIRT4 shortens lifespan and leads to a decline in physical activity


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      Mitochondrial mechanisms and pathways have recently emerged as critical determinants of organismal aging. Whilenuclear sirtuins have been shown to regulate aging, whether mitochondrial sirtuins do so is still unclear. Here, we report thatmitochondrial dSirt4 mediates organismal aging. We establish that absence of dSirt4 leads to reduced lifespan independentof dietary inputs. Further by assaying locomotion, a key correlate of aging, we demonstrate that dSirt4 null flies are severelyphysically impaired with a significant reduction in locomotion. In summary, we report that mitochondrial dSirt4 is a keydeterminant of longevity and its loss leads to early aging.

    • Lupeol induces S-phase arrest and mitochondria-mediated apoptosis in cervical cancer cells


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      Cervical cancer is fourth most common fatal cancer in women worldwide. Lupeol is a dietary triterpenoid and has shown itsanticancer efficacy against various cancer types with selectivity in targeting cancer cells. In the present study, anticancerefficacy and mechanism of action of a phytochemical, lupeol, in human cervical carcinoma (HeLa) cells has been examined.The anticancer efficacy of lupeol was assessed by trypan blue cell counting, annexin Vassay, cell cycle analysis, expressionof apoptotic proteins by RT-PCR and Western blotting and assessment of mitochondrial ROS generation by mitosox andmitotracker assays. Our results demonstrated that lupeol decreased cell proliferation and viability of HeLa cells significantly(p\0.001). Lupeol induced S-phase cell cycle arrest and also decreased the expression of S-phase Cyclins and CDKs andincreased the expression of cyclin-dependent kinase inhibitors, p21 at transcriptional and translational level. Further, lupeolinduced apoptosis and increased the expression of apoptosis markers such as cleaved PARP and Bax:Bcl-2 ratio. Furthermore,mitosox and mitotracker dye incubation followed by FACS analysis showed an increase in mitochondrialsuperoxide generation and reduction in healthy mitochondrial mass. These results suggest that lupeol could be an effectivechemotherapeutic agent against cervical carcinoma due to its growth inhibitory activity through induction of S-phase cellcycle arrest and apoptosis.

    • Electromagnetic radiation 2450 MHz exposure causes cognition deficit with mitochondrial dysfunction and activation of intrinsic pathway of apoptosis in rats


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      Electromagnetic radiation (EMR) can induce or modulate several neurobehavioral disorders. Duration and frequency ofexposure of EMR is critical to develop cognitive disorders. Even though EMR-2450 is widely used, its effects on cognitionin relation to mitochondrial function and apoptosis would provide better understanding of its pathophysiological effects.Therefore, a comparative study of different frequencies of EMR exposure would give valuable information on effects ofdiscrete frequencies of EMR on cognition. Male rats were exposed to EMR (900, 1800 and 2450 MHz) every day for 1 hfor 28 consecutive days. The cognitive behavior in terms of novel arm entries in Y-maze paradigm was evaluated everyweek after 1 h to last EMR exposure. Animals exposed to EMR-2450 MHz exhibited significant cognitive deficits. EMR-2450 MHz caused loss of mitochondrial function and integrity, an increase in amyloid beta expression. There was release ofcytochrome-c and activation of apoptotic factors such as caspase-9 and -3 in the hippocampus. Further, there was decreasein levels of acetylcholine, and increase in activity of acetyl cholinesterase, indicating impairment of cholinergic system.Therefore, exposure of EMR-2450 in rats caused cognitive deficit with related pathophysiological changes in mitochondrialand cholinergic function, and amyloidogenesis.

    • Maslinic acid modulates secreted phospholipase A2-IIA (sPLA2-IIA)-mediated inflammatory effects in macrophage foam cells formation


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      Secretory phospholipase A2-IIA (sPLA2-IIA) is one of the key enzymes causing lipoprotein modification and vascularinflammation. Maslinic acid is a pentacyclic triterpene which has potential cardioprotective and anti-inflammatory properties.Recent research showed that maslinic acid interacts with sPLA2-IIA and inhibits sPLA2-IIA-mediated monocytedifferentiation and migration. This study elucidates the potential of maslinic acid in modulating sPLA2-IIA-mediatedinflammatory effects in THP-1 macrophages. We showed that maslinic acid inhibits sPLA2-IIA-mediated LDL modificationand suppressed foam cell formation. Further analysis revealed that sPLA2-IIA only induced modest LDL oxidation and thatinhibitory effect of maslinic acid on sPLA2-IIA-mediated foam cells formation occurred independently of its anti-oxidativeproperties. Interestingly, maslinic acid was also found to significantly reduce lipid accumulation observed in macrophagestreated with sPLA2-IIA only. Flow cytometry analysis demonstrated that the effect observed in maslinic acid might becontributed in part by suppressing sPLA2-IIA-induced endocytic activity, thereby inhibiting LDL uptake. The study furthershowed that maslinic acid suppresses sPLA2-IIA-induced up-regulation of PGE2 levels while having no effects on COX-2activity. Other pro-inflammatory mediators TNF-a and IL-6 were not induced in sPLA2-IIA-treated THP-1 macrophages.The findings of this study showed that maslinic acid inhibit inflammatory effects induced by sPLA2-IIA, including foamcells formation and PGE2 production.

    • Chlorogenic acid alleviates autophagy and insulin resistance by suppressing JNK pathway in a rat model of nonalcoholic fatty liver disease


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      Non-alcoholic fatty liver disease (NAFLD) is one of the leading causes of chronic liver diseases around the world andcommonly associated with insulin resistance and hyperlipidemia. Chlorogenic acid (CG) was reported to have insulinsensitizingactivity and exert hypocholesterolemic and hypoglycemic effect. However, the involvement of CG in NAFLDremains far from being addressed. In this study, a high-fat diet-induced NAFLD rat model was used to investigate thebiological roles and underlying mechanism of CG in NAFLD. The results showed that high-fat diet-fed rats exhibited anincrease in body weight, glucose tolerance, liver injury, insulin resistance, as well as autophagy and C-Jun N-terminalkinase (JNK) pathway. Nevertheless, all these effects were alleviated by CG treatment. Moreover, angiotensin treatment inCG group activated the JNK pathway, and promoted autophagy, insulin resistance, and liver injury. In conclusion, ourfindings demonstrated that CG ameliorated liver injury and insulin resistance by suppressing autophagy via inactivation ofJNK pathway in a rat model of NAFLD. Therefore, CG might be a potential application for the treatment of NAFLD.

    • In silico prediction of ErbB signal activation from receptor expression profiles through a data analytics pipeline


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      The ErbB signalling pathway has been studied extensively owing to its role in normal physiology and its dysregulation incancer. Reverse engineering by mathematical models use the reductionist approach to characterize the network components.For an emergent, system-level view of the network, we propose a data analytics pipeline that can learn from the datagenerated by reverse engineering and use it to re-engineer the system with an agent-based approach. Data from a kineticmodel that estimates the parameters by fitting to experiments on cell lines, were encoded into rules, for the interactions ofthe molecular species (agents) involved in biochemical reactions. The agent model, a digital representation of the cell linesystem, tracks the activation of ErbB1-3 receptors on binding with ligands, resulting in their dimerization, phosphorylation,trafficking and stimulation of downstream signalling through P13-Akt and Erk pathways. The analytics pipeline has beenused to mechanistically link HER expression profile to receptor dimerization and activation of downstream signallingpathways. When applied to drug studies, the efficacy of a drug can be investigated in silico. The anti-tumour activity ofPertuzumab, a monoclonal antibody that inhibits HER2 dimerization, was simulated by blocking 80% of the cellular HER2available, to observe the effect on signal activation.

    • Effect of PEDOT:PSS in tissue engineering composite scaffold on improvement and maintenance of endothelial cell function


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      According to recent research, smart polymers can affect different kind of mammalian cells such as endothelial cells. It isknown that conductive polymers have great features, e.g. electrical conductivity, and can help increase electrical cellcommunication. To clarify the effect of one of these smart materials on endothelial cells, which are not inherentlyelectrically dependent, poly(3, 4-ethylene dioxythiophene) polystyrene sulfonate (PEDOT:PSS) was chosen. Scaffolds werecomposed of gelatin, alginate, and PEDOT:PSS and made through solvent casting. Human umbilical vein endothelial cells(HUVECs) were cultured on the scaffold with different PEDOT:PSS concentrations. SEM, MTT assay, cell attachment,nitric oxide measurement, real-time PCR and immunohistochemistry analysis were employed to assess endothelial cellresponses. Although there was no significant difference in swelling ratio, mass loss, and cell attachment when PEDOT:PSSconcentration increased in scaffold construction, cell proliferation noticeably increased after seven days. The cells showed asignificant increase in proliferation and NO release to the scaffold with 1% PEDOT:PSS concentration. The resultsindicated increases in the amount of expression of platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31), kinaseinsert domain receptor (KDR), vascular-endothelial Cadherin (VE. Cadherin), and von Will brand factor (vWf) in the groupwhich contained a conductive polymer in comparison with the non-conductive scaffold. Therefore, as a conductivepolymer, PEDOT:PSS can affect the endothelial cell behaviours.

    • Human cathelicidin LL-37 – Does it influence the homeostatic imbalance in mental disorders?


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      In addition to killing pathogens and influencing immunological processes, cathelicidin LL-37 is a multifunctional hostdefense peptide with a role in homeostasis. It has been suggested that imbalances in homeostatic signaling from inflammatory/immune, endocrine and metabolic cascades and oxidative stress may partially contribute to the pathogenesis ofmental disorders. The purpose of the study was to identify any differences in LL-37 levels between patients withschizophrenia, euthymic bipolar disorder, bacterial pneumonia, pulmonary tuberculosis, and healthy controls. Thirty-fivepatients with chronic paranoid schizophrenia, 40 patients with chronic, euthymic bipolar disorder, 30 patients with bacterialpneumonia, 32 patients with pulmonary tuberculosis, and 38 healthy volunteers were included in this study. The patientswith schizophrenia demonstrated a significantly lower level of LL-37 than those with bipolar disorder (p=0.006) and thosewith pulmonary TB (p<0.001). Significant differences in LL-37 levels were found between patients with bipolar disorder,bacterial pneumonia (p<0.001) and pulmonary TB (p=0.004). Our findings suggest that changes observed in the serumlevel of LL-37 in psychiatric patients enrolled in this study could be a result of homeostatic imbalance.

    • Gender-divergent expression of lipid and bile acid metabolismrelated genes in adult mice offspring of dams fed a high-fat diet


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      Maternal high-fat diet (HFD) consumption during pregnancy and lactation affects metabolic outcomes and lipid metabolismof offspring in later life in a gender-specific manner. However, it is not known whether maternal HFD alters bile acidmetabolism in adult mice offspring. The purpose of this study was to elucidate the relationship between maternal HFDinducedmetabolic diseases and bile acid metabolism in male and female adult mice offspring. Female mice were fed eitherstandard chow (C) or HFD (H) for 10 weeks pre-pregnancy until lactation. After weaning, offspring were fed a chow dietuntil 11 weeks of age, then challenged with either C or H diet for 4 weeks, and divided into eight groups in accordance withmother’s and offspring’s diets: male(M) CC, MHC, MCH, MHH, female(F) CC, FHC, FCH, and FHH. MHH showedgreater weight gain compared to FHH. Liver weight was higher in MHH than in FHH. Serum total cholesterol levels werehigher in MHH than in MHC, and tended to be higher in MHH than in FHH. Serum glucose levels were higher in MHHthan in MHC. Hepatic triglyceride levels were higher in MHH than in MHC. Hepatic mRNA expression of bile acid uptaketransporters Oatp1a1 and Oatp1b2 was increased in MHH, compared to MCH. Hepatic mRNA expression of HMGCoAR,Cyp7a1, Sult2a1, and Oatp1a4 was increased in FHH, compared to FCH. In conclusion, maternal HFD consumption maypromote bile acid synthesis, sulfation and excretion in female offspring fed a HFD, which may confer resistance to HFDinducedmetabolic phenotypes.

    • Putative role of invariant water molecules in the X-ray structures of family G fungal endoxylanases


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      Fungal endo-1,4-b-xylanases (EC3.2.1.8), because of their widespread industrial applications have become one of the mostresearched industrial enzymes in recent times. Despite its significance, the role of conserved water molecules in the catalyticactivities and structural stability of these enzymes from the fungi have not been studied to a great extent. Our computationalstructural bioinformatics and MD simulation studies have identified the existence of seven invariant water molecules (IW1–IW7) and reveals the stereo-chemical and electronic consequences of those conserved water molecules in G-xylanaseenzyme from eight different fungi. The buried water molecules IW1 and IW2 may have decisive roles in catalysis and mayalso be associated with ligand binding process of the enzyme, whereas IW3, IW4, IW5, IW6 and IW7 may be involved instabilizing the important (H144/R145) residues through H-bonds. Possibly they are also involved in the stabilization ofsecondary structures and anchor to maintain its stereo-chemical architecture. Moreover, a distorted ‘W’ shaped signaturegeometry that is observed at the surface of the enzyme can be used to identify the hydrophilic centers in the electron densitymap of other unknown members of the family G xylanases. The results from this computational investigation could be ofinterest to a large number of researchers working with the xylanases.

    • Deletion of Dictyostelium discoideum Sir2A impairs cell proliferation and inhibits autophagy


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      Sirtuins are a family of deacetylases (Class III histone deacetylases) with evolutionarily conserved functions in cellularmetabolism and chromatin regulation. Out of the seven human Sirtuins, the function of Sirt2 is the least understood. Thepurpose of the present study was to investigate the role of Sir2A, a homolog of human Sirt2 in Dictyostelium discoideum(Dd), a lower eukaryote. We created both overexpressing and deletion strains of Ddsir2A to analyse its functions. Weobserved sir2A mRNA expression throughout development and the transcript was present in the prespore/spore region ofmulticellular structures developed. They show a preference towards prestalk/stalk pathway when co-developed with wildtypecells during chimera formation. Deletion strain showed a multi-tipped phenotype, decrease in cell proliferation andinhibition of autophagy. In conclusion, our results show low cAMP levels, reduced cell-adhesion, weak cell migration andimpaired autophagy to be responsible for the phenotype shown by the null cells. This study provides new insights into thefunctions of Ddsir2A.

    • Inhibition of miR-1247 on cell proliferation and invasion in bladder cancer through its downstream target of RAB36


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      Recently, microRNA-1247 (miR-1247) has been reported to function as tumour suppressor in several cancer types,including pancreatic cancer, hepatocellular cancer and lung cancer. However, the biological function of miR-1247 inbladder cancer and the underlying mechanisms have remained largely uncovered. In this study, the expression of miR-1247was significantly downregulated, while RAB36 protein was remarkably upregulated in bladder cancer tissues and cell linescompared with that in paired adjacent normal tissues or normal cell line (SU-HUC-1). The function of miR-1247 andRAB36 in the cell viability, proliferation and invasion of bladder cancer cells (T24 and J82) was assessed by CCK-8,colony formation and Transwell assay, respectively. Gain of function studies showed that upregulation of miR-1247significantly inhibited cell proliferation and invasion capacity of bladder cancer cells. Consistently, downregulation ofRAB36 mimicked the suppressive effects of miR-1247 overexpression in bladder cancer cells. Importantly, miR-1247 wasconfirmed to target the 30untranslated region (UTR) of RAB36 and downregulated its expression using luciferase reporterassay and Western blot assays. In conclusion, these results provide the first clues regarding the role of miR-1247 might be apotential therapeutic agent and diagnostic marker of bladder cancer by inhibiting RAB36 expression.

    • Spiral waves and vertebrate embryonic handedness


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      During early embryonic development, the vertebrate main body axis is segmented from head-to-tail into somites. Somitesemerge sequentially from the presomitic mesoderm (PSM) as a consequence of oscillatory waves of genetic activity, calledsomitogenesis waves. Here, we discuss the implications of the dynamic patterns of early X-Delta-2 expression in theprospective somites (somitomeres) of Xenopus laevis. We report that right somitomeres normally emerge before left to formchiral structures (i.e. structures having clockwise or counter-clockwise handedness). From our observations, we infer thatsomitogenesis waves are normally counter-clockwise spirals, a novel dynamic mechanism for the control of handednessdevelopment in Xenopus. We propose that the same mechanism could control handedness development in all vertebrateembryos, providing a dynamical basis for the current asymmetric molecular transport model for generating left–rightasymmetry.

    • Spatiotemporal oscillations of morphinan alkaloids in opium poppy


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      Here, a comprehensive endeavor is made to simultaneously scrutinize spatiotemporal oscillations of three imperativemorphinan alkaloids (i.e. thebaine, codeine, and morphine) alongside dynamic transcriptional patterns of TYDC, SalAT,COR, T6ODM, and CODM genes in different tissues of Papaver somniferum (i.e. root, bottom part of stem, upper part ofstem, leaf, capsule wall, and capsule content) over five distinguished ontogenic stages (i.e. rosette, bud initiation, pendulousbud, flowering, and lancing). Apart from bottom stem and leaf, the maximum thebaine content occurred in lancing stage,while its minimum content did not follow a systematic rhythm, either among six tissues or five various sampling times.Regarding codeine, excepting upper stem, the highest ratios of codeine were observed at flowering and lacing stages, whilenegligible amounts were overall detected at early stages of plant growth like rosette. Considering morphine, apart fromupper stem, it appears that late ontogeneic times including lancing and flowering are the most appropriate phases to achievehigh amounts of morphine, while at early stages the aforesaid alkaloid possessed lower accumulation. Furthermore, all thefive genes under study, overall, exhibited a variety of transcript levels either among six tissues or five various samplingtimes. Interestingly, a connection occurred between transcript ratio of SalAT and thebaine content, suggesting that thebainebiosynthesis is coordinated tightly by the enzymatic function of SalAT enzyme. Meanwhile, despite low magnitudes ofT6ODM and CODM transcripts in the root-harvested samples at pendulous bud and flowering stages, both codeine andmorphine were surprisingly in acceptable quantities, plausibly owing to the translocation of both alkaloids from theproducing (source) tissues to the roots (sink), known as a phenomenon of ‘source-to-sink transportation’. The results,altogether, could provide us enough information in acquiring new insights towards potential impacts of spatiotemporaloscillations on the magnitudes of all the above-mentioned alkaloids alongside transcription ratios of the key genes in opiumpoppy.

    • A parasporin from Bacillus thuringiensis native to Peninsular India induces apoptosis in cancer cells through intrinsic pathway


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      Parasporins, a class of non-insecticidal crystal proteins of Bacillus thuringiensis (Bt) are being explored as promisinganticancer agents due to their specific toxicity to cancer cells. The present study has identified 25 Bt isolates harbouringparasporin genes from Western Ghats region, the hotspot of biodiversity in India. Among these, the isolate, KAU 41(Kerala Agricultural University isolate 41) contained non-hemolytic homogenous crystals showing specific cytotoxicitytowards cancer cells. SDS-PAGE analysis of this crystal, isolated by aqueous biphasic separation, revealed a 31 kDa sizedpeptide. The N-terminal sequence deciphered in BLAST analysis showed homology to a hypothetical Bt protein. Uponproteolysis, a 29 kDa active peptide was generated which exhibited heterogenic cytotoxic spectrum on various cancer cells.HeLa cells were highly susceptible to this peptide with IC50 1 lg/mL and showed characteristics of apoptosis. RT-qPCRanalysis revealed the overexpression of APAF1, caspase 3 and 9 by 14.9, 8 and 7.4 fold, respectively which indicates theactivation of intrinsic pathway of apoptosis. However, at higher concentrations of peptide ([3 lg/mL), necrotic death wasprominent. The results suggest that the 31 kDa protein from Bt isolate, KAU 41 is a parasporin that may have hightherapeutic potential.

    • Genome 3D-architecture: Its plasticity in relation to function


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      The genome of higher eukaryotes is non-randomly organized in the interphase nucleus. However, notwithstanding theabsence of membrane bound sub-compartments, the nucleus coordinates a number of functions largely by organizingchromatin in a non-random but dynamic manner. The plasticity of chromatin structure and function relies on epigeneticmodifications as well as its association with nuclear landmarks such as the nuclear envelope, nuclear lamina, nuclear porecomplex and nuclear bodies such as the nucleolus among others. In the absence of membrane-bound compartments, cellsand the nucleus, in particular, employ phase-separation, which unmixes phases that constrain biochemical reactions incomplex non-membranous sub-compartments such as the nucleolus or even the heterochromatin. This review attempts toprovide a glimpse into the microcosm of phase-separated nuclear sub-compartments, that regulate nuclear structure–function relationships.

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