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      Volume 42, Issue 4

      December 2017,   pages  523-707

    • Irregular designs and Darwinism in biology: Genomes as the test case

      B J RAO

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    • What history tells us XLIV: The construction of the zinc finger nucleases

      MICHEL MORANGE

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    • Antifolate drug resistance: Novel mutations and haplotype distribution in dhps and dhfr from Northeast India

      NP SARMAH K SARMA DR BHATTACHARYYA AA SULTAN D BANSAL N SINGH PK BHARTI R SEHGAL PK MOHAPATRA P PARIDA J MAHANTA

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      Malaria is a major public health concern in Northeast India with a preponderance of drug-resistant strains. Until recently thepartner drug for artemisinin combination therapy (ACT) was sulphadoxine pyrimethamine (SP). Antifolate drug resistancehas been associated with the mutations at dihydropteroate synthase (dhps) and dihydrofolatereductase (dhfr) genes. Thisstudy investigated antifolate drug resistance at the molecular level. A total of 249 fever cases from Arunachal Pradesh, NEIndia, were screened for malaria, and of these, 75 were found to be positive for Plasmodium falciparum. Samples weresequenced and analysed with the help of BioEdit and ClustalW. Three novel point mutations were found in the dhps genewith 10 haplotypes along with the already reported mutations. A single haplotype having quadruple mutation was found inthe dhfr gene. The study reports higher degree of antifolate drug resistance as evidenced by the presence of multiple pointmutations in dhps and dhfr genes. The findings of this study strongly discourage the use SP as a partner drug in ACT.

    • Specific mutation of transglutaminase gene from Streptomyces hygroscopicus H197 and characterization of microbial transglutaminase

      WENJIE WAN DONGLAN HE ZHIJUN XUE ZEWEN ZHANG

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      Microbial transglutaminase (MTG) gene (mtg) from Streptomyces hygroscopicus H197 strain was cloned by PCR andmutated by deleting a specific 84 bp fragment using overlapping extension PCR. The mutant MTG and the wild MTGgenes expressed by recombinant plasmid pET32a+-mutant mtg and pET32a+-mtg, respectively, and were harvested byalternating freeze–thaw steps and purified by Ni column. The purified mutant MTG and the wild MTG exhibited 0.22 U/mgand 0.16 U/mg activity, respectively, and 0.69 U/mg and 0.54 U/mg activity, respectively, after activated by trypsin. Themolecular weight of mutant MTG was estimated as 67 kDa by SDS-PAGE. Both MTGs showed optimum activity at pH6–8 for hydroxamate formation from N-CBZ-Gln-Gly and hydroxylamine, and exhibited higher stability at 40 deg C and 1–3%salinity. The two types of MTG were not stable in the presence of Zn(II), Cu(II), Hg(II), Pb(II), Fe(III), and Ag(I),suggesting that they could possess a thiol group. In addition, the mutant MTG and the wild MTG were strongly affected byethanol. Furthermore, the mutant MTG was obviously (P < 0.05 or P < 0.01) more stable than the wild MTG at 50 deg C and60 deg C, at pH 4, 5, and 9, at 7% and 9% salinity, 30% and 35% ethanol concentration, and in the presence of Li(I) and Ag(I).The polyhydroxy compounds as protein stabilizers could elevate MTG stability.

    • Thymoquinone induces cytotoxicity and reprogramming of EMT in gastric cancer cells by targeting PI3K/Akt/mTOR pathway

      LI-MIN FENG XUE-FENG WANG QING-XIAN HUANG

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      Gastric cancer is one of the lethal causes of cancer-related deaths worldwide. The incidence and mortality rates of thisdisease is comparatively higher in China. In the current study, we evaluated the anticancer effects of Thymoquinone (TQ)against gastric cancer cells (MGC80-3 and SGC-7901) and normal noncancerous GES-1 cells and attempted to investigatethe underlying mechanism. Our results indicated that TQ exhibited significant growth inhibitory effects on gastric cancercells (MGC80-3 and SGC-7901). However, lower cytotoxicity was observed against normal GES-1 cells. Moreover, TQcould inhibit the colony formation potential of MGC80-3 and SGC-7901 cells in a dose-dependent manner. TQ alsoinhibited cell migration ability of the gastric cancer cells and down-regulated the expression of the mesenchymal genes suchas N-cadherin, Vimentin, and TWIST. However, the epithelial markers such as E-cadherin and cytokeratin-19 were distinctlyup-regulated in TQ-treated gastric cancer cells. Since PI3K/Akt/ mTOR plays an important role in progression andtumorigenesis, we also investigated the effect of TQ on PI3K/Akt/mTOR signalling pathway in gastric cancer cells. It wasobserved that TQ down-regulated the expression of some of the key proteins of this pathway. Taken together, we concludethat TQ may prove lead molecule for the treatment of gastric cancer.

    • Induction of morphological and functional differentiation of human neuroblastoma cells by miR-124

      SAMANEH SHARIF MOHAMMAD HOSSEIN GHAHREMANI MASOUD SOLEIMANI

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      Neuroblastoma is the most common extracranial solid tumour in children, and differentiation is considered its mostappropriate therapy. In this work, we studied effects of miR-124 overexpression on differentiation in M17 cell line as amodel of neuroblastoma cancer. Influence of miR-124 overexpression on differentiation in M17 cells was studied. M17cells were infected with lentivirus that contained miR-124 precursor sequence and followed for 2 weeks to differentiate.Ectopic expression of miR-124 in M17 cells changed the shape of spherical undifferentiated cells to cells with extendedneurites that formed neuronal networks. Overexpression of MiR-124 respectively increased the expression level of markersof beta-Tubulin III, MAP2, SYN, NF-M and Nestin by 16-, 5-, 4-, 2.3- and 2-folds at the messenger RNA level. MiR-124overexpression also increased the protein levels of beta-Tubulin III and MAP2. Moreover, exogenous expression of miR-124significantly increased the intracellular calcium in differentiated M17 cells. Since miR-124 is naturally expressed inneuronal cells and is downregulated in neuroblastoma cancer cells, differentiation with this type of microRNA can be anovel treatment for neuroblastoma cancer.

    • Re-engineering the two-component systems as light-regulated in Escherichia coli

      SIYA MA SIWEI LUO LI WU ZHI LIANG JIA-RUI WU

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      Bacteria live in environments with dynamic changes. To sense and respond to different external stimuli, bacteria make useof various sensor-response circuits, called two-component systems (TCSs). A TCS comprises a histidine protein kinase(HK) sensing environmental stimuli and a response regulator protein (RR) regulating downstream genes. The two componentsare coupled via a phosphorylation control mechanism. In a recent study, we adopted an optogenetics approach tore-engineer the sensor HKs in Escherichia coli as a light-sensing fusion protein. We constructed a light-controllable HK byreplacing the original signal-specific sensing domain of HK with the light-sensing domain of Cph1 from CyanobacteriaSynechocystis, so that HK can be investigated by red light. Here, we extended the study to other 16 HK-RR TCSs andconstructed a library of light-responsible HK-Cph1 chimeras. By taking the NarX-NarL system as an example, wedemonstrated the light responsiveness of the constructed chimera and investigated the frequency response of the NarXNarLsystem. The constructed library serves as a toolkit for future TCS study using optogenetics approach.

    • MicroRNA-486-5p suppresses TGF-b2-induced proliferation, invasion and epithelial–mesenchymal transition of lens epithelial cells by targeting Smad2

      BEI LIU JIANHUA SUN XIAOQIN LEI ZHONGQIAO ZHU CHENG PEI LI QIN

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      The pathological development of lens epithelial cells (LECs) leads to posterior capsular opacification (PCO). This studywas undertaken to investigate the effects of microRNA-486-5p (miR-486-5p) on TGF-b2-induced proliferation, invasionand epithelial-mesenchymal transition (EMT) in the lens epithelial cell line SRA01/04, and to explore the underlyingmolecular mechanisms. The expression of miR-486-5p in TGF-b2-induced SRA01/04 cells was down-regulated, and theexpression of Smad2, p-Smad2 and p-Smad3 was up-regulated. A dual-luciferase reporter assay revealed that miR-486-5pdirectly targets the 30-UTR of Smad2. MiR-486-5p mimic transfection markedly down-regulated the expression levels ofSmad2, thus inhibiting the expression of p-Smad2 and p-Smad3. MiR-486-5p overexpression in SRA01/04 cells markedlysuppressed TGF-b2-induced proliferation and invasion, inhibited protein expression of CDK2 and CDK4, down-regulatedfibronectin, a-SMA and vimentin and up-regulated E-cadherin; these effects were partly reversed by Smad2 overexpression.In short, these data show that miR-486-5p overexpression can inhibit TGF-b2-induced proliferation, invasion andEMT in SRA01/04 cells by repressing Smad2/Smad3 signalling, implying that miR-486-5p may be an effective target tointerfere in the progression of PCO.

    • Fermentative metabolism impedes p53-dependent apoptosis in a Crabtree-positive but not in Crabtree-negative yeast

      ABHAY KUMAR JASWANDI UJWAL DANDEKAR PAIKE JAYADEVA BHAT

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      Tumour cells distinguish from normal cells by fermenting glucose to lactate in presence of sufficient oxygen and functionalmitochondria (Warburg effect). Crabtree effect was invoked to explain the biochemical basis of Warburg effect by suggestingthat excess glucose suppresses mitochondrial respiration. It is known that the Warburg effect and Crabtree effect aredisplayed by Saccharomyces cerevisiae, during growth on abundant glucose. Beyond this similarity, it was also demonstratedthat expression of human pro-apoptotic proteins in S. cerevisiae such as Bax and p53 caused apoptosis. Here, wedemonstrate that p53 expression in S. cerevisiae (Crabtree-positive yeast) causes increase in ROS levels and apoptosis whencells are growing on non-fermentable carbon sources but not on fermentable carbon sources, a feature similar to tumourcells. In contrast, in Kluyveromyces lactis (Crabtree-negative yeast) p53 causes increase in ROS levels and apoptosisregardless of the carbon source. Interestingly, the increased ROS levels and apoptosis are correlated to increased oxygenuptake in both S. cerevisiae and K. lactis. Based on these results, we suggest that at least in yeast, fermentation per se doesnot prevent the escape from apoptosis. Rather, the Crabtree effect plays a crucial role in determining whether the cellsshould undergo apoptosis or not.

    • Uptake of phenolic compounds from plant foods in human intestinal Caco-2 cells

      GAVIRANGAPPA HITHAMANI DHANYA KIZHAKAYIL KRISHNAPURA SRINIVASAN

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      In continuation of our studies on the bioaccessibility of phenolic compounds from food grains as influenced by domesticprocessing, we examined the uptake of phenolics from native/sprouted finger millet (Eleucine coracana) and green gram(Vigna radiata) and native/heat-processed onion (Allium cepa) in human Caco-2 cells. Absorption of pure phenoliccompounds, as well as the uptake of phenolic compounds from finger millet, green gram, and onion, was investigated inCaco-2 monolayer model. Transport of individual phenolic compounds from apical compartment to the basolateral compartmentacross Caco-2 monolayer was also investigated. Sprouting enhanced the uptake of syringic acid from both thesegrains. Open-pan boiling reduced the uptake of quercetin from the onion. Among pure phenolic compounds, syringic acidwas maximally absorbed, while the flavonoid isovitexin was least absorbed. Apparent permeability coefficient P(app) ofphenolic compounds from their standard solutions was 2.02 X 10-6 cm/s to 8.94 X 10-6 cm/s. Sprouting of grainsenhanced the uptake of syringic acid by the Caco-2 cells. Open-pan boiling drastically reduced the uptake of quercetin fromthe onion. The permeability of phenolic acids across Caco-2 monolayer was higher than those of flavonoids.

    • Small phosphatidate phosphatase (TtPAH2) of Tetrahymena complements respiratory function and not membrane biogenesis function of yeast PAH1

      ANOOP NARAYANA PILLAI SHUKLA SUSHMITA SUDHANSHU GAUTAM ABDUR RAHAMAN

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      Phosphatidate phosphatases (PAH) play a central role in lipid metabolism and intracellular signaling. Herein, we report thepresence of a low-molecular-weight PAH homolog in the single-celled ciliate Tetrahymena thermophila. In vitro phosphataseassay showed that TtPAH2 belongs to the magnesium-dependent phosphatidate phosphatase (PAP1) family. Loss offunction of TtPAH2 did not affect the growth of Tetrahymena. Unlike other known PAH homologs, TtPAH2 did not regulatelipid droplet number and ER morphology. TtPAH2 did not rescue growth and ER/nuclear membrane defects of the pah1Dyeast cells, suggesting that the phosphatidate phosphatase activity of the protein is not sufficient to perform these cellularfunctions. Surprisingly, TtPAH2 complemented the respiratory defect in the pah1D yeast cells indicating a specific role ofTtPAH2 in respiration. Overall, our results indicate that TtPAH2 possesses the minimal function of PAH protein family inrespiration. We suggest that the amino acid sequences absent from TtPAH2 but present in all other known PAH homologsare critical for lipid homeostasis and membrane biogenesis.

    • Klebsiella pneumoniae antibiotic resistance identified by atomic force microscopy

      VINCENZO IERARDI PAOLO DOMENICHINI SILVIA REALI GIAN MARCO CHIAPPARA GIANLUIGI DEVOTO UGO VALBUSA

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      In the last decade the detection of the resistance of bacteria to antibiotics treatment, developed by different kind of bacteria,is becoming a huge problem. We hereby present a different approach to the current problem of detection of bacteriaresistance to antibiotics. Our aims were to use the atomic force microscopy (AFM) to investigate bacteria morphologicalchanges in response to antibiotics treatment and explore the possibility of reducing the time required to obtain informationon their resistance. In particular, we studied Klebsiella pneumoniae bacteria provided by the Lavagna Hospital ASL4Liguria (Italy), where there are cases linked with antibiotics resistance of the Klebsiella pneumoniae. By comparing AFMimages of bacteria strains treated with different antibiotics is possible to identify unambiguously the Klebsiella pneumoniaestrains resistant to antibiotics. In fact, the analysis of the AFM images of the antibiotic-sensitive bacteria shows clearly thepresence of morphological alterations in the cell wall. While in the case of the antibiotic-resistant bacteria morphologicalalterations are not present. This approach is based on an easy and potentially rapid AFM analysis.

    • MicroRNA-146 protects A549 and H1975 cells from LPS-induced apoptosis and inflammation injury

      QIANG WANG DAGANG LI YUQUAN HAN XIAOQIAN DING TAO XU BINGJIAN TANG

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      Pneumonia is an inflammatory condition affecting the lungs, in which pro-inflammatory cytokines are secreted. It has beenshown that microRNA-146 (miR-146) is involved in the regulation of immune and inflammatory responses. The presentstudy explored the protective effects of miR-146 overexpression on lipopolysaccharide (LPS)-mediated injury in A549 andH1975 cells. In this study, A549 and H1975 cells were transfected with miR-146 mimic or inhibitor, and then weresubjected with LPS. Thereafter, cell viability, colony formation capacity, apoptosis, the release of proinflammatory factors,Sirt1 expression, and the expression of NF-kB and Notch pathway proteins were respectively assessed. As a result, miR-146 overexpression exerted protective functions on LPS-damaged A549 and H1975 cells, as evidenced by the increases incell viability and colony number, the decrease in apoptotic cell rate, as well as the down-regulations of IL-1, IL-6, and TNFa.Sirt1 can be positively regulated by miR-146. Furthermore, miR-146 overexpression blocked NF-kB and Notch pathways,while these blocking effects were abolished when Sirt1 was silenced. The findings in the current study indicated thatmiR-146 protected A549 and H1975 cells from LPS-induced apoptosis and inflammation injury. miR-146 exerted protectivefunctions might be via up-regulation of Sirt1 and thereby blocking NF-kB and Notch pathways.

    • IGF1 stimulates differentiation of primary follicles and their growth in ovarian explants of zebrafish (Danio rerio) cultured in vitro

      PANCHARATNA A KATTI SHEETAL S NARVEKAR BASAVARAJ B GOUNDADKAR PRASAD A DESHPANDE

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      The present study is an attempt to elucidate the involvement of insulin-like growth factor (IGF1) in the differentiation andgrowth of primary follicles in ovarian explant cultures of zebrafish. Ovaries from adult females were cultured in triplicatesets/treatment group for 15 days at 22 deg C in the laboratory. Culture medium was supplemented with either insulin (1 ng/mL)or IGF1 (1 ng/mL) or insulin ? IGF1 (Experiment 1) or 0.1 or 1.0 or 10 ng/mL of IGF1 (Experiment 2). Ovaries culturedin medium alone served as controls and those fixed at the beginning of the culture as initial controls. Experiments were repeated. On the 16th day ovarian explants were fixed in Bouin’s fluid and processed for paraffin embedding, sections(3 lm) were cut and stained with hematoxylin-eosin. Follicles were classified into 6 stages and atretic follicles (AF).Previtellogenic, vitellogenic and total follicle number was calculated. At the start of the culture, ovaries contained all stagesof growing and degenerating follicles. In in vitro cultured control ovaries, vitellogenic follicles underwent atresia, while,primary follicles remained unaffected. Insulin or insulin ? IGF1 treated ovaries did not differ significantly while IGF1exposed ovarian explants had greater (P\0.05) number of primary follicles compared to controls. IGF1 also caused anincrease in the number and growth of primary follicles in a dose dependent manner although; cultures were not supplementedwith gonadotrophic hormones. Results suggest that locally derived intra-ovarian IGF1 may have a role in thedifferentiation and growth of primary follicles in zebrafish ovary.

    • In vitro leishmanicidal, antibacterial and antitumour potential of anhydrocochlioquinone A obtained from the fungus Cochliobolus sp.

      FERNANDA F CAMPOS JONAS P RAMOS DJALMA M DE OLIVEIRA TAˆ NIA M A ALVES ELAINE M DE SOUZA-FAGUNDES CARLOS L ZANI FA´ BIO C SAMPAIO ATTILIO CONVERTI BETANIA B COTA

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      The bioassay-guided fractionation of the ethyl acetate extract of the fungus Cochliobolus sp. highlighted leishmanicidalactivity and allowed for anhydrocochlioquinone A (ANDC-A) isolation. MS, 1D and 2D NMR spectra of this compoundwere in agreement with those published in the literature. ANDC-A exhibited leishmanicidal activity with EC50 value of22.4 lg/mL (44 lM) and, when submitted to the microdilution assay against Gram-positive and Gram-negative bacteria,showed a minimal inhibitory concentration against Staphylococcus aureus ATCC 25295 of 128 lg/mL (248.7 lM). It wasalso active against five human cancer cell lines, showing IC50 values from 5.4 to 20.3 lM. ANDC-A demonstrated adifferential selectivity for HL-60 (SI 5.5) and THP-1 (SI 4.3) cell lines in comparison with Vero cells and was moreselective than cisplatin and doxorubicin against MCF-7 cell line in comparison with human peripheral blood mononuclearcells. ANDC-A was able to eradicate clonogenic tumour cells at concentrations of 20 and 50 lM and induced apoptosis inall tumour cell lines at 20 lM. These results suggest that ANDC-A might be used as a biochemical tool in the study oftumour cells biochemistry as well as an anticancer agent with durable effects on tumours.

    • Enthalpy-entropy compensation and the isokinetic temperature in enzyme catalysis

      ATHEL CORNISH-BOWDEN

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      Enthalpy-entropy compensation supposes that differences in activation enthalpy delta-H-++ for different reactions (or, typically inbiochemistry, the same reaction catalysed by enzymes obtained from different species) may be compensated for bydifferences in activation entropy delta-S-++. At the isokinetic temperature the compensation is exact, so that all samples have thesame activity. These ideas have been controversial for several decades, but examples are still frequently reported asevidence of a real phenomenon, nearly all of the reports ignoring or discounting the possibility of a statistical artefact. Evenfor measurements in pure chemistry artefacts occur often, and they are almost inescapable in enzyme kinetics and otherfields that involve biological macromolecules, on account of limited stability and the fact that kinetic equations are normallyvalid only over a restricted range of temperature. Here I review the current status and correct an error in a recent bookchapter.

    • MiR-876-5p suppresses epithelial-mesenchymal transition of lung cancer by directly down-regulating bone morphogenetic protein 4

      LIANG BAO LEI LV JINPING FENG YUYU CHEN XINHUA WANG SHUGUANG HAN HONGQING ZHAO

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      Lung cancer is the leading cause of cancer-related death throughout the world. We aimed to investigate the role of a novelmicroRNA-876-5p and its potential molecular target bone morphogenetic protein 4 (BMP-4), in the epithelial–mesenchymaltransition (EMT) of lung cancer. Expressions of microRNA-876-5p and its potential target BMP-4 were analysedin lung cancer cells and patient tissues. Luciferase activity assay was conducted to verify direct targeting of microRNA-876-5p to the 30-UTR of BMP-4 mRNA. Migration, invasion capacities of lung cancer cells expressing microRNA-876-5pwere analysed, and characteristics of lung cancer EMT protein markers were also evaluated. A xenograft tumour mousemodel was established to address the roles of microRNA-876-5p and BMP-4 in lung cancer EMT in vivo. MicroRNA-876-5p was decreased while BMP-4 was increased in lung cancer cells and tissues. MicroRNA-876-5p directly targeted 30-UTRof BMP-4 mRNA to inhibit its expression. MicroRNA-876-5p expression significantly inhibited the migration, invasionand EMT of lung cancer cells in vitro, as well as metastasis in vivo, which required BMP-4 expression. MicroRNA-876-5psuppresses EMT of lung cancer by directly down-regulating BMP-4, both of which could serve as potential therapeutictargets in the treatment of lung cancer.

    • Criticality in cell differentiation

      INDRANI BOSE MAINAK PAL

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      Cell differentiation is an important process in living organisms. Differentiation is mostly based on binary decisions with theprogenitor cells choosing between two specific lineages. The differentiation dynamics have both deterministic andstochastic components. Several theoretical studies suggest that cell differentiation is a bifurcation phenomenon, well-knownin dynamical systems theory. The bifurcation point has the character of a critical point with the system dynamics exhibitingspecific features in its vicinity. These include the critical slowing down, rising variance and lag-1 autocorrelation function,strong correlations between the fluctuations of key variables and non-Gaussianity in the distribution of fluctuations. Recentexperimental studies provide considerable support to the idea of criticality in cell differentiation and in other biologicalprocesses like the development of the fruit fly embryo. In this review, an elementary introduction is given to the concept ofcriticality in cell differentiation. The correspondence between the signatures of criticality and experimental observations onblood cell differentiation in mice is further highlighted.

    • Multiple oncogenic roles of nuclear beta-catenin

      RAJU KUMAR MURALI D BASHYAM

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      Beta-Catenin is essential for embryonic development and required for cell renewal/regeneration in adult life. Cellular beta-cateninexists in three different pools: membranous, cytoplasmic and nuclear. In this review, we focus on functions of the nuclearpool in relation to tumorigenesis. In the nucleus, beta-catenin functions as both activator and repressor of transcription ina context-dependent manner. It promotes cell proliferation and supports tumour growth by enhancing angiogenesis.Beta-Catenin-mediated signalling regulates cancer cell metabolism and is associated with tumour-initiating cells in multiplemalignancies. In addition, it functions as both pro- and anti-apoptotic factor besides acting to inhibit recruitment ofinflammatory anti-tumour T-cells. Thus, beta-catenin appears to possess a multifaceted nuclear function that may significantlyimpact tumour initiation and progression.

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