Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-beta

Actions of transforming growth factor-beta are largely context dependent. For instance, TGF-beta is growth inhibitory to epithelial cells and many tumor cell-lines while it stimulates the growth of mesenchymal cells. TGF-beta also activates fibroblast cells to a myofibroblastic phenotype. In order to understand how the responsiveness of fibroblasts to TGF-beta would change in the context of transformation, we have compared the differential gene regulation by TGF-beta in immortal fibroblasts (hFhTERT), transformed fibroblasts (hFhTERT-LTgRAS) and a human fibrosarcoma cell-line (HT1080). The analysis revealed regulation of 6735, 4163, and 3478 probe-sets by TGF-beta in hFhTERT, hFhTERT-LTgRAS and HT1080 cells respectively. Intriguingly, 5291 probe-sets were found to be either regulated in hFhTERT or hFhTERT-LTgRAS cells while 2274 probe-sets were regulated either in hFhTERT or HT1080 cells suggesting that the response of immortal hFhTERT cells to TGF-beta is vastly different compared to the response of both the transformed cells hFhTERT-LTgRAS and HT1080 to TGF-beta. Strikingly, WNT pathway showed enrichment in the hFhTERT cells in Gene Set Enrichment Analysis. Functional studies showed induction of WNT4 by TGF-beta in hFhTERT cells and TGF-beta conferred action of these cells was mediated by WNT4. While TGF-beta activated both canonical and non-canonical WNT pathways in hFhTERT cells, Erk1/2 and p38 Mitogen Activated Protein Kinase pathways were activated in hFhTERT-LTgRAS and HT1080 cells. This suggests that transformation of immortal hFhTERT cells by SV40 large T antigen and activated RAS caused a switch in their response to TGF-beta which matched with the response of HT1080 cells to TGF-beta. These data suggest context dependent activation of non-canonical signaling by TGF-beta. (C) 2015 Published by Elsevier Inc.

The nuclease activities of both the Smr domain and an additional LDLK motif are required for an efficient anti-recombination function of Helicobacter pyloriMutS2

Helicobacter pylori, a human pathogen, is a naturally and constitutively competent bacteria, displaying a high rate of intergenomic recombination. While recombination events are essential for evolution and adaptation of H.pylori to dynamic gastric niches and new hosts, such events should be regulated tightly to maintain genomic integrity. Here, we analyze the role of the nuclease activity of MutS2, a protein that limits recombination during transformation in H.pylori. In previously studied MutS2 proteins, the C-terminal Smr domain was mapped as the region responsible for its nuclease activity. We report here that deletion of Smr domain does not completely abolish the nuclease activity of HpMutS2. Using bioinformatics analysis and mutagenesis, we identified an additional and novel nuclease motif (LDLK) at the N-terminus of HpMutS2 unique to Helicobacter and related epsilon-proteobacterial species. A single point mutation (D30A) in the LDLK motif and the deletion of Smr domain resulted in approximate to 5-10-fold loss of DNA cleavage ability of HpMutS2. Interestingly, the mutant forms of HpMutS2 wherein the LDLK motif was mutated or the Smr domain was deleted were unable to complement the hyper-recombination phenotype of a mutS2(-) strain, suggesting that both nuclease sites are indispensable for an efficient anti-recombinase activity of HpMutS2.

SUB1 Plays a Negative Role during Starvation Induced Sporulation Program in Saccharomyces cerevisiae

Saccharomyces cerevisiae Sub1 is involved in several cellular processes such as, transcription initiation, elongation, mRNA processing and DNA repair. It has also been reported to provide cellular resistance during conditions of oxidative DNA damage and osmotic stress. Here, we report a novel role of SUB1 during starvation stress-induced sporulation, which leads to meiosis and spore formation in diploid yeast cells. Deletion of SUB1 gene significantly increased sporulation efficiency as compared to the wild-type cells in S288c genetic background. Whereas, the sporulation functions of the sub1(Y66A) missense mutant were similar to Sub1. SUB1 transcript and protein levels are downregulated during sporulation, in highly synchronized and sporulation proficient wild-type SK1 cells. The changes in Sub1 levels during sporulation cascade correlate with the induction of middle sporulation gene expression. Deletion of SUB1 increased middle sporulation gene transcript levels with no effect on their induction kinetics. In wild-type cells, Sub1 associates with chromatin at these loci in a temporal pattern that correlates with their enhanced gene expression seen in sub1. cells. We show that SUB1 genetically interacts with HOS2, which led us to speculate that Sub1 might function with Set3 repressor complex during sporulation. Positive Cofactor 4, human homolog of Sub1, complemented the sub1. sporulation phenotype, suggesting conservation of function. Taken together, our results suggest that SUB1 acts as a negative regulator of sporulation.

Novel Alternative Splice Variants of Mouse Cdk5rap2

Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability. A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science. Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH. To further characterize the pathomechanism underlying MCPH, we generated a conditional Cdk5rap2 LoxP/hCMV Cre mutant mouse. Further analysis, initiated on account of a lack of a microcephaly phenotype in these mutant mice, revealed the presence of previously unknown splice variants of the Cdk5rap2 gene that are at least in part accountable for the lack of microcephaly in the mice.

Genome-wide analysis correlates Ayurveda Prakriti

The practice of Ayurveda, the traditional medicine of India, is based on the concept of three major constitutional types (Vata, Pitta and Kapha) defined as ``Prakriti''. To the best of our knowledge, no study has convincingly correlated genomic variations with the classification of Prakriti. In the present study, we performed genome-wide SNP (single nucleotide polymorphism) analysis (Affymetrix, 6.0) of 262 well-classified male individuals (after screening 3416 subjects) belonging to three Prakritis. We found 52 SNPs (p <= 1 x 10(-5)) were significantly different between Prakritis, without any confounding effect of stratification, after 10(6) permutations. Principal component analysis (PCA) of these SNPs classified 262 individuals into their respective groups (Vata, Pitta and Kapha) irrespective of their ancestry, which represent its power in categorization. We further validated our finding with 297 Indian population samples with known ancestry. Subsequently, we found that PGM1 correlates with phenotype of Pitta as described in the ancient text of Caraka Samhita, suggesting that the phenotypic classification of India's traditional medicine has a genetic basis; and its Prakriti-based practice in vogue for many centuries resonates with personalized medicine.

Mapping Key Residues of ISD11 Critical for NFS1-ISD11 Subcomplex Stability IMPLICATIONS IN THE DEVELOPMENT OF MITOCHONDRIAL DISORDER, COXPD19

Biogenesis of the iron-sulfur (Fe-S) cluster is an indispensable process in living cells. In mammalian mitochondria, the initial step of the Fe-S cluster assembly process is assisted by the NFS1-ISD11 complex, which delivers sulfur to scaffold protein ISCU during Fe-S cluster synthesis. Although ISD11 is an essential protein, its cellular role in Fe-S cluster biogenesis is still not defined. Our study maps the important ISD11 amino acid residues belonging to putative helix 1 (Phe-40), helix 3 (Leu-63, Arg-68, Gln-69, Ile-72, Tyr-76), and C-terminal segment (Leu-81, Glu-84) are critical for in vivo Fe-S cluster biogenesis. Importantly, mutation of these conserved ISD11 residues into alanine leads to its compromised interaction with NFS1, resulting in reduced stability and enhanced aggregation of NFS1 in the mitochondria. Due to altered interaction with ISD11 mutants, the levels of NFS1 and Isu1 were significantly depleted, which affects Fe-S cluster biosynthesis, leading to reduced electron transport chain complex (ETC) activity and mitochondrial respiration. In humans, a clinically relevant ISD11 mutation (R68L) has been associated in the development of a mitochondrial genetic disorder, COXPD19. Our findings highlight that the ISD11 R68A/R68L mutation display reduced affinity to form a stable subcomplex with NFS1, and thereby fails to prevent NFS1 aggregation resulting in impairment of the Fe-S cluster biogenesis. The prime affected machinery is the ETC complex, which showed compromised redox properties, causing diminished mitochondrial respiration. Furthermore, the R68L ISD11 mutant displayed accumulation of mitochondrial iron and reactive oxygen species, leading to mitochondrial dysfunction, which correlates with the phenotype observed in COXPD19 patients.

Disorder, oscillatory dynamics and state switching: the role of c-Myc

In this paper, using the intrinsically disordered oncoprotein Myc as an example, we present a mathematical model to help explain how protein oscillatory dynamics can influence state switching. Earlier studies have demonstrated that, while Myc overexpression can facilitate state switching and transform a normal cell into a cancer phenotype, its downregulation can reverse state-switching. A fundamental aspect of the model is that a Myc threshold determines cell fate in cells expressing p53. We demonstrate that a non-cooperative positive feedback loop coupled with Myc sequestration at multiple binding sites can generate bistable Myc levels. Normal quiescent cells with Myc levels below the threshold can respond to mitogenic signals to activate the cyclin/cdk oscillator for limited cell divisions but the p53/Mdm2 oscillator remains nonfunctional. In response to stress, the p53/Mdm2 oscillator is activated in pulses that are critical to DNA repair. But if stress causes Myc levels to cross the threshold, Myc inactivates the p53/Mdm2 oscillator, abrogates p53 pulses, and pushes the cyclin/cdk oscillator into overdrive sustaining unchecked proliferation seen in cancer. However, if Myc is downregulated, the cyclin/cdk oscillator is inactivated and the p53/Mdm2 oscillator is reset and the cancer phenotype is reversed. (C) 2015 Elsevier Ltd. All rights reserved.

Quantitative characterization of adhesion and stiffness of corneal lens of Drosophila melanogaster using atomic force microscopy

Atomic force Microscopy (AFM) has become a versatile tool in biology due to its advantage of high-resolution imaging of biological samples close to their native condition. Apart from imaging, AFM can also measure the local mechanical properties of the surfaces. In this study, we explore the possibility of using AFM to quantify the rough eye phenotype of Drosophila melanogaster through mechanical properties. We have measured adhesion force, stiffness and elastic modulus of the corneal lens using AFM. Various parameters affecting these measurements like cantilever stiffness and tip geometry are systematically studied and the measurement procedures are standardized. Results show that the mean adhesion force of the ommatidial surface varies from 36 nN to 16 nN based on the location. The mean stiffness is 483 +/- 5 N/m, and the elastic modulus is 3.4 +/- 0.05 GPa (95% confidence level) at the center of ommatidia. These properties are found to be different in corneal lens of eye expressing human mutant tau gene (mutant). The adhesion force, stiffness and elastic modulus are decreased in the mutant. We conclude that the measurement of surface and mechanical properties of D. melanogaster using AFM can be used for quantitative evaluation of `rough eye' surface. (C) 2015 Elsevier Ltd. All rights reserved.

Rab9A is required for delivery of cargo from recycling endosomes to melanosomes

Melanosomes are a type of lysosome-related organelle that is commonly defective in Hermansky-Pudlak syndrome. Biogenesis of melanosomes is regulated by BLOC-1, -2, -3, or AP-1, -3 complexes, which mediate cargo transport from recycling endosomes to melanosomes. Although several Rab GTPases have been shown to regulate these trafficking steps, the precise role of Rab9A remains unknown. Here, we found that a cohort of Rab9A associates with the melanosomes and its knockdown in melanocytes results in hypopigmented melanosomes due to mistargeting of melanosomal proteins to lysosomes. In addition, the Rab9A-depletion phenotype resembles Rab38/ 32-inactivated or BLOC-3-deficient melanocytes, suggesting that Rab9A works in line with BLOC-3 and Rab38/ 32 during melanosome cargo transport. Furthermore, silencing of Rab9A, Rab38/ 32 or its effector VARP, or BLOC-3-deficiency in melanocytes decreased the length of STX13-positive recycling endosomal tubules and targeted the SNARE to lysosomes. This result indicates a defect in directing recycling endosomal tubules to melanosomes. Thus, Rab9A and its co-regulatory GTPases control STX13-mediated cargo delivery to maturing melanosomes.