Application of Whole Exome Sequencing in Elucidating the Phenotype and Genotype Spectrum of Junctional Epidermolysis Bullosa: A Preliminary Experience of a Tertiary Care Centre in India

Publication date: Available online 29 December 2016
Source:Journal of Dermatological Science
Author(s): Vamsi K Yenamandra, Shamsudheen K Vellarikkal, Manoj Kumar, Madhumita R Chowdhury, Rijith Jayarajan, Ankit Verma, Vinod Scaria, Sridhar Sivasubbu, Subrata B. Ray, Amit K Dinda, Madhulika Kabra, Punit Kaur, Vinod K Sharma, Gomathy Sethuraman
BackgroundJunctional epidermolysis bullosa (JEB) is a diverse group of genodermatoses associated with extreme skin fragility. Despite several well-characterized genetic studies, molecular diagnosis of this heterogeneous group is still challenging. Recent advances in the field of genomics have seen the successful implementation of whole exome sequencing (WES) as a fast and efficient diagnostic strategy in several genodermatoses.ObjectiveIn view of the scarcity and need of molecular studies for JEB in India, we sought to explore the potential of WES in understanding the mutational spectrum of this rare, in certain subtypes lethal, sub-group of EB.MethodsWES was performed using genomic DNA from each case of EB, followed by massively parallel sequencing. Resulting reads were mapped to the human reference genome hg19. Sanger sequencing subsequently confirmed the potentially pathogenic mutations.ResultsOverall, four unrelated families (6 patients) of JEB with a highly variable clinical presentation including a rare case of LOC syndrome were studied. WES revealed 4 variations in 3 genes (LAMA3, LAMB3 and COL17A1) that are implicated in JEB. None of the variations were recurrent. In addition we proposed the probable molecular consequence of a missense mutation on the structure-function relationship of lamininβ3 protein through computational modeling studies.ConclusionsBeing the first report documenting the phenotype-genotype correlations of JEB patients from India, our preliminary experience with WES is clearly encouraging and serves as a nidus for future large-scale molecular studies to actively identify and understand JEB patients in Indian population.



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Serum level of interleukin-6 is increased in nivolumab-associated psoriasiform dermatitis and tumor necrosis factor-α is a biomarker of nivolumab recativity

Publication date: Available online 29 December 2016
Source:Journal of Dermatological Science
Author(s): Ryota Tanaka, Naoko Okiyama, Mari Okune, Yosuke Ishitsuka, Rei Watanabe, Junichi Furuta, Mikio Ohtsuka, Atsushi Otsuka, Hiroshi Maruyama, Yasuhiro Fujisawa, Manabu Fujimoto




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Nick-initiated homologous recombination: Protecting the genome, one strand at a time

Publication date: Available online 29 December 2016
Source:DNA Repair
Author(s): Lianne E.M. Vriend, Przemek M. Krawczyk
Homologous recombination (HR) is an essential, widely conserved mechanism that utilizes a template for accurate repair of DNA breaks. Some early HR models, developed over five decades ago, anticipated single-strand breaks (nicks) as initiating lesions. Subsequent studies favored a more double-strand break (DSB)-centered view of HR initiation and at present this pathway is primarily considered to be associated with DSB repair. However, mounting evidence suggests that nicks can indeed initiate HR directly, without first being converted to DSBs. Moreover, recent studies reported on novel branches of nick-initiated HR (^nickHR) that rely on single-, rather than double-stranded repair templates and that are characterized by mechanistically and genetically unique properties. The physiological significance of ^nickHR is not well documented, but its high-fidelity nature and low mutagenic potential are relevant in recently developed, precise gene editing approaches. Here, we review evidence for stimulation of HR by nicks, as well as the data on the interactions of ^nickHR with other DNA repair pathways and mechanistic properties. We conclude that ^nickHR is a bona-fide pathway for nick repair, sharing the molecular machinery with the canonical HR but nevertheless characterized by unique properties that secure its inclusion in DNA repair models and warrant future investigations.



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Polarity Reversal by Centrosome Repositioning Primes Cell Scattering during Epithelial-to-Mesenchymal Transition

Publication date: Available online 29 December 2016
Source:Developmental Cell
Author(s): Mithila Burute, Magali Prioux, Guillaume Blin, Sandrine Truchet, Gaëlle Letort, Qingzong Tseng, Thomas Bessy, Sally Lowell, Joanne Young, Odile Filhol, Manuel Théry
During epithelial-to-mesenchymal transition (EMT), cells lining the tissue periphery break up their cohesion to migrate within the tissue. This dramatic reorganization involves a poorly characterized reorientation of the apicobasal polarity of static epithelial cells into the front-rear polarity of migrating mesenchymal cells. To investigate the spatial coordination of intracellular reorganization with morphological changes, we monitored centrosome positioning during EMT in vivo, in developing mouse embryos and mammary gland, and in vitro, in cultured 3D cell aggregates and micropatterned cell doublets. In all conditions, centrosomes moved from their off-centered position next to intercellular junctions toward extracellular matrix adhesions on the opposite side of the nucleus, resulting in an effective internal polarity reversal. This move appeared to be supported by controlled microtubule network disassembly. Sequential release of cell confinement using dynamic micropatterns, and modulation of microtubule dynamics, confirmed that centrosome repositioning was responsible for further cell disengagement and scattering.

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During epithelial-to-mesenchymal transition, epithelial cells migrate into the underlying interstitial tissues. Burute et al. show that this movement is enabled by a reversal of cell polarity. This reversal results from centrosome displacement from intercellular junctions to the cell center in response to controlled microtubule disassembly.


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Exosomal MicroRNA Transport from Salivary Mesenchyme Regulates Epithelial Progenitor Expansion during Organogenesis

Publication date: Available online 29 December 2016
Source:Developmental Cell
Author(s): Toru Hayashi, Isabelle M.A. Lombaert, Belinda R. Hauser, Vaishali N. Patel, Matthew P. Hoffman
Epithelial-mesenchymal interactions involve fundamental communication between tissues during organogenesis and are primarily regulated by growth factors and extracellular matrix. It is unclear whether RNA-containing exosomes are mobile genetic signals regulating epithelial-mesenchymal interactions. Here we identify that exosomes loaded with mesenchyme-specific mature microRNA contribute mobile genetic signals from mesenchyme to epithelium. The mature mesenchymal miR-133b-3p, loaded into exosomes, was transported from mesenchyme to the salivary epithelium, which did not express primary miR-133b-3p. Knockdown of miR-133b-3p in culture decreased endbud morphogenesis, reduced proliferation of epithelial KIT⁺ progenitors, and increased expression of a target gene, Disco-interacting protein 2 homolog B (Dip2b). DIP2B, which is involved in DNA methylation, was localized with 5-methylcytosine in the prophase nucleus of a subset of KIT⁺ progenitors during mitosis. In summary, exosomal transport of miR-133b-3p from mesenchyme to epithelium decreases DIP2B, which may function as an epigenetic regulator of genes responsible for KIT⁺ progenitor expansion during organogenesis.

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Epithelial-mesenchymal interactions are critical for salivary gland development. Hayashi et al. show that miRNA-containing exosomes produced by mesenchyme are transported to the epithelium, where they regulate KIT⁺ progenitor cell proliferation. A specific mesenchymal miRNA (miR-133b-3p) targets DIP2B, which is involved in DNA methylation, thus regulating progenitor cell expansion during organogenesis.


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The APC/C Coordinates Retinal Differentiation with G1 Arrest through the Nek2-Dependent Modulation of Wingless Signaling

Publication date: Available online 29 December 2016
Source:Developmental Cell
Author(s): Torcato Martins, Francesco Meghini, Francesca Florio, Yuu Kimata
The cell cycle is coordinated with differentiation during animal development. Here we report a cell-cycle-independent developmental role for a master cell-cycle regulator, the anaphase-promoting complex or cyclosome (APC/C), in the regulation of cell fate through modulation of Wingless (Wg) signaling. The APC/C controls both cell-cycle progression and postmitotic processes through ubiquitin-dependent proteolysis. Through an RNAi screen in the developing Drosophila eye, we found that partial APC/C inactivation severely inhibits retinal differentiation independently of cell-cycle defects. The differentiation inhibition coincides with hyperactivation of Wg signaling caused by the accumulation of a Wg modulator, Drosophila Nek2 (dNek2). The APC/C degrades dNek2 upon synchronous G1 arrest prior to differentiation, which allows retinal differentiation through local suppression of Wg signaling. We also provide evidence that decapentaplegic signaling may posttranslationally regulate this APC/C function. Thus, the APC/C coordinates cell-fate determination with the cell cycle through the modulation of developmental signaling pathways.

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Martins et al. discovered a cell-cycle-independent function of the APC/C ubiquitin ligase in Drosophila eye development. They show that the APC/C controls retinal differentiation by locally suppressing Wingless signaling through the destruction of Nek2. Thus, the APC/C coordinates developmental signaling activity with the cell cycle.


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APC/CFzr/Cdh1-Dependent Regulation of Planar Cell Polarity Establishment via Nek2 Kinase Acting on Dishevelled

Publication date: Available online 29 December 2016
Source:Developmental Cell
Author(s): Ursula Weber, Marek Mlodzik
The Anaphase-Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase, well known for its role in cell-cycle progression. However, it has been linked to additional functions, mainly in neuronal contexts, when using the co-activator Cdh1/Fzr. Here, our data indicate a post-mitotic requirement for the APC/C^Fzr/Cdh1 in epithelial cell patterning and planar cell polarity (PCP) in Drosophila. PCP signaling is critical for development by establishing cellular asymmetries and orientation within the plane of an epithelium, via differential localization of distinct complexes of core PCP factors. Loss of APC/C function leads to reduced levels of Dishevelled (Dsh), a core PCP factor. The effect of APC/C on Dsh is mediated by Nek2 kinase, which can phosphorylate Dsh and is a direct APC/C^Fzr/Cdh1 substrate. We have thus uncovered a pathway of regulation whereby APC/C^Fzr/Cdh1 negatively regulates Nek2, which negatively regulates Dsh, to ensure its proper stoichiometric requirement and localization during PCP establishment.

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Anaphase-Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase known for its role in cell-cycle progression. Weber and Mlodzik identify a post-mitotic APC/C function in planar cell polarity establishment in Drosophila. The APC/C regulates levels of the Nek2 kinase, which regulates Dsh localization/levels via phosphorylation and targeting to the proteasome.


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Long-Term High-Resolution Imaging of Developing C. elegans Larvae with Microfluidics

Publication date: Available online 29 December 2016
Source:Developmental Cell
Author(s): Wolfgang Keil, Lena M. Kutscher, Shai Shaham, Eric D. Siggia
Long-term studies of Caenorhabditis elegans larval development traditionally require tedious manual observations because larvae must move to develop, and existing immobilization techniques either perturb development or are unsuited for young larvae. Here, we present a simple microfluidic device to simultaneously follow development of ten C. elegans larvae at high spatiotemporal resolution from hatching to adulthood (∼3 days). Animals grown in microchambers are periodically immobilized by compression to allow high-quality imaging of even weak fluorescence signals. Using the device, we obtain cell-cycle statistics for C. elegans vulval development, a paradigm for organogenesis. We combine Nomarski and multichannel fluorescence microscopy to study processes such as cell-fate specification, cell death, and transdifferentiation throughout post-embryonic development. Finally, we generate time-lapse movies of complex neural arborization through automated image registration. Our technique opens the door to quantitative analysis of time-dependent phenomena governing cellular behavior during C. elegans larval development.

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Keil et al. present a microfluidics setup, enabling long-term, high-resolution, time-lapse microscopy of up to ten C. elegans larvae simultaneously. They collect vulval cell-cycle timing statistics, measure intensities of fluorescent transcriptional reporters during cell-fate specification, transdifferentiation, and cell death, and visualize complex neurite outgrowth in automatically registered z stacks.


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Kaempferol slows intervertebral disc degeneration by modifying LPS-induced osteogenesis/adipogenesis imbalance and inflammation response in BMSCs

Publication date: February 2017
Source:International Immunopharmacology, Volume 43
Author(s): Jun Zhu, Haoyu Tang, Zhenhua Zhang, Yong Zhang, Chengfeng Qiu, Ling Zhang, Pinge Huang, Feng Li
Intervertebral disc (IVD) degeneration is a common disease that represents a significant cause of socio-economic problems. Bone marrow-derived mesenchymal stem cells (BMSCs) are a potential autologous stem cell source for the nucleus pulposus regeneration. Kaempferol has been reported to exert protective effects against both osteoporosis and obesity. This study explored the effect of kaempferol on BMSCs differentiation and inflammation. The results demonstrated that kaempferol did not show any cytotoxicity at concentrations of 20, 60 and 100μM. Kaempferol enhanced cell viability by counteracting the lipopolysaccharide (LPS)-induced cell apoptosis and increasing cell proliferation. Western blot analysis of mitosis-associated nuclear antigen (Ki67) and proliferation cell nuclear antigen (PCNA) further confirmed the increased effect of kaempferol on LPS-induced decreased viability of BMSCs. Besides, kaempferol elevated LPS-induced reduced level of chondrogenic markers (SOX-9, Collagen II and Aggrecan), decreased the level of matrix-degrading enzymes, i.e., matrix metalloprotease (MMP)-3 and MMP-13, suggesting the osteogenesis of BMSC under kaempferol treatment. On the other hand, kaempferol enhanced LPS-induced decreased expression of lipid catabolism-related genes, i.e., carnitine palmitoyl transferase-1 (CPT-1). Kaempferol also suppressed the expression of lipid anabolism-related genes, i.e., peroxisome proliferators-activated receptor-γ (PPAR-γ). The Oil red O staining further convinced the inhibition effect of kaempferol on BMSCs adipogenesis. In addition, kaempferol alleviated inflammatory by reducing the level of pro-inflammatory cytokines (i.e., interleukin (IL)-6) and increasing anti-inflammatory cytokine (IL-10) via inhibiting the nucleus translocation of nuclear transcription factor (NF)-κB p65. Taken together, our research indicated that kaempferol may serve as a novel target for treatment of IVD degeneration.

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Liver 5-HT7 receptors: A novel regulator target of fibrosis and inflammation-induced chronic liver injury in vivo and in vitro

Publication date: February 2017
Source:International Immunopharmacology, Volume 43
Author(s): Beyzagul Polat, Zekai Halici, Elif Cadirci, Emre Karakus, Yasin Bayir, Abdulmecit Albayrak, Deniz Unal
Background and aimHepatocellular cancer (HCC) is the sixth most common cancer and liver fibrosis is strongly associated with HCC. Treatment options are limited, and preventive strategies should be developed. An important step in the beginning of liver fibrosis is a strong inflammatory response. 5-HT7 is the last recognized member of the serotonin receptor family and is expressed in both central nerve system and peripheral system and have a lot of functions like learning, memory, smooth muscular relaxation, in the control of circadian rhythms and thermoregulation, pain and migraine, schizophrenia, anxiety, cognitive disturbances, and even inflammation.MethodsWe therefore examined the biochemical, histopathological and molecular effects of the 5-HT7 receptor agonist and antagonist on inflammatory liver fibrogenesis in animal models of progressive cirrhosis: a mouse model induced by carbon tetrachloride (CCl4) and in Hep3b cells.Results5-HT7 expression was observed in the liver in vivo and in vitro in CCl4-induced damage. 5-HT7 receptor agonist but not the antagonist reduced liver markers in mice and in Hep3b cells in carbon tetrachloride (CCl4) induced damage. 5-HT7 agonist, but not antagonist, protected liver tissue from oxidative stress in fibrosis. 5-HT7 agonist but not antagonist induces anti-inflammatory, anti-fibrinotic and anti-cytokine features in liver fibrosis in vivo and in vitro.Conclusions5-HT7 receptors have modulatory function and are an up-and-coming pharmacological target in the inflammatory fibrotic process. 5-HT7 receptor agonist LP-44 showed significant hepatoprotective effects against liver fibrosis, and LP-44 might become a useful therapeutic target for chronic liver inflammation and fibrosis.



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Metal-phenolic networks as a versatile platform to engineer nanomaterials and biointerfaces

Publication date: Available online 29 December 2016
Source:Nano Today
Author(s): Hirotaka Ejima, Joseph J. Richardson, Frank Caruso
Surface modification is crucial for conferring novel functionalities to objects and interfaces. However, simple yet versatile strategies for the surface modification of multiple classes of nanomaterials, including biointerfaces, are rare, as the chemical interactions between the surface modifiers and the substrates need to be tailored on a case-by-case basis. Recently, metal-phenolic networks (MPNs) have emerged as a versatile surface modifier based on the universal adherent properties of phenolic molecules, namely the constituent gallol and catechol groups. Additionally, the dynamic interactions between metal ions and phenolic molecules confer additional functionalities to the MPNs, such as stimuli-responsiveness. Given the interest in MPNs for nanomaterial and biointerface engineering, this review aims to provide an overview of the assembly process, physicochemical properties and applications of MPN coatings.

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miR-29c is implicated in the cardioprotective activity of Panax notoginseng saponins against isoproterenol-induced myocardial fibrogenesis

Publication date: 23 February 2017
Source:Journal of Ethnopharmacology, Volume 198
Author(s): Li Liu, Bingbing Ning, Jingang Cui, Teng Zhang, Yu Chen
Ethnopharmacological relevancePanax notoginseng (Burkill) F.H. Chen (Araliaceae) has a long history of clinical application in China for the treatment of cardiovascular diseases. Panax notoginseng saponins (PNS) have been proven to be the major cardioprotective substances of Panax notoginseng (Burkill) F.H. Chen (Araliaceae).Aim of the studyThe current study further investigated the molecular mechanisms associated with the cardioprotective effect of PNS.Materials and methodsC57BL/6J mice were subject to isoproterenol (ISO)-induced myocardial injury in the absence or presence of PNS treatment. Histological, immunohistochemical and molecular biological approaches were taken to assess the effects of PNS treatment on ISO-induced myocardial injury and ensuing fibrogenesis.ResultsPNS treatment significantly attenuated ISO-induced myocardial injury and fibrosis. The expression of an anti-fibrotic microRNA, miR-29c, was significantly decreased in ISO-challenged mouse hearts. In contrast, PNS treatment resulted in increased cardiac expression of miR-29c. The expression of miR-29c target genes including Collagen (Col) 1a1, Col1a2, Col3a1 and Col5a1, fibrillin 1 (Fbn1) as well as TGFβ1 was significantly increased by ISO, which exhibited decreased expression by PNS intervention.ConclusionsOur results demonstrate for the first time that the cardioprotective effects of PNS could in part implicate increased expression of miR-29c in the heart, which may help increase the understanding of the pharmacological activities of PNS in treating cardiovascular disorders.

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Preparing to Pass the FRCA. C. Whymark

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Associate Editorial Board

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Basic Anesthesiology Examination Review. G. W. Williams and E. S. Williams

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