Mary Lyon Centre at MRC Harwell

AUTORES: Daniel J Klionsky1745,1749*, Kotb Abdelmohsen840, Akihisa Abe1237, Md Joynal Abedin1762, Hagai Abeliovich425,
\nAbraham Acevedo Arozena789, Hiroaki Adachi1800, Christopher M Adams1669, Peter D Adams57, Khosrow Adeli1981,
\nPeter J Adhihetty1625, Sharon G Adler700, Galila Agam67, Rajesh Agarwal1587, Manish K Aghi1537, Maria Agnello1826,
\nPatrizia Agostinis664, Patricia V Aguilar1960, Julio Aguirre-Ghiso784,786, Edoardo M Airoldi89,422, Slimane Ait-Si-Ali1376,
\nTakahiko Akematsu2010, Emmanuel T Akporiaye1097, Mohamed Al-Rubeai1394, Guillermo M Albaiceta1294,
\nChris Albanese363, Diego Albani561, Matthew L Albert517, Jesus Aldudo128, Hana Alg€ul1164, Mehrdad Alirezaei1198,
\nIraide Alloza642,888, Alexandru Almasan206, Maylin Almonte-Beceril524, Emad S Alnemri1212, Covadonga Alonso544,
\nNihal Altan-Bonnet848, Dario C Altieri1205, Silvia Alvarez1497, Lydia Alvarez-Erviti1395, Sandro Alves107,
\nGiuseppina Amadoro860, Atsuo Amano930, Consuelo Amantini1554, Santiago Ambrosio1458, Ivano Amelio756,
\nAmal O Amer918, Mohamed Amessou2089, Angelika Amon726, Zhenyi An1538, Frank A Anania291, Stig U Andersen6,
\nUsha P Andley2079, Catherine K Andreadi1690, Nathalie Andrieu-Abadie502, Alberto Anel2027, David K Ann58,
\nShailendra Anoopkumar-Dukie388, Manuela Antonioli832,858, Hiroshi Aoki1791, Nadezda Apostolova2007,
\nSaveria Aquila1500, Katia Aquilano1876, Koichi Araki292, Eli Arama2098, Agustin Aranda456, Jun Araya591,
\nAlexandre Arcaro1472, Esperanza Arias26, Hirokazu Arimoto1225, Aileen R Ariosa1749, Jane L Armstrong1930,
\nThierry Arnould1773, Ivica Arsov2120, Katsuhiko Asanuma675, Valerie Askanas1924, Eric Asselin1867, Ryuichiro Atarashi794,
\nSally S Atherton369, Julie D Atkin713, Laura D Attardi1131, Patrick Auberger1787, Georg Auburger379, Laure Aurelian1727,
\nRiccardo Autelli1992, Laura Avagliano1029,1755, Maria Laura Avantaggiati364, Limor Avrahami1166, Suresh Awale1986,
\nNeelam Azad404, Tiziana Bachetti568, Jonathan M Backer28, Dong-Hun Bae1933, Jae-sung Bae677, Ok-Nam Bae409,
\nSoo Han Bae2117, Eric H Baehrecke1729, Seung-Hoon Baek17, Stephen Baghdiguian1368,
\nAgnieszka Bagniewska-Zadworna2, Hua Bai90, Jie Bai667, Xue-Yuan Bai1133, Yannick Bailly884,
\nKithiganahalli Narayanaswamy Balaji473, Walter Balduini2002, Andrea Ballabio316, Rena Balzan1711, Rajkumar Banerjee239,
\nG abor B anhegyi1052, Haijun Bao2109, Benoit Barbeau1363, Maria D Barrachina2007, Esther Barreiro467, Bonnie Bartel997,
\nAlberto Bartolom e222, Diane C Bassham550, Maria Teresa Bassi1046, Robert C Bast Jr1273, Alakananda Basu1798,
\nMaria Teresa Batista1578, Henri Batoko1336, Maurizio Battino970, Kyle Bauckman2085, Bradley L Baumgarner1909,
\nK Ulrich Bayer1594, Rupert Beale1553, Jean-Fran¸cois Beaulieu1360, George R. Beck Jr48,294, Christoph Becker336,
\nJ David Beckham1595, Pierre-Andr e B edard749, Patrick J Bednarski301, Thomas J Begley1135, Christian Behl1419,
\nChristian Behrends757, Georg MN Behrens406, Kevin E Behrns1627, Eloy Bejarano26, Amine Belaid490,
\nFrancesca Belleudi1041, Giovanni B enard497, Guy Berchem706, Daniele Bergamaschi983, Matteo Bergami1401,
\nBen Berkhout1441, Laura Berliocchi714, Am elie Bernard1749, Monique Bernard1354, Francesca Bernassola1880,
\nAnne Bertolotti791, Amanda S Bess272, S ebastien Besteiro1351, Saverio Bettuzzi1828, Savita Bhalla913,
\nShalmoli Bhattacharyya973, Sujit K Bhutia838, Caroline Biagosch1159, Michele Wolfe Bianchi520,1378,1381,
\nMartine Biard-Piechaczyk210, Viktor Billes298, Claudia Bincoletto1314, Baris Bingol350, Sara W Bird1128, Marc Bitoun1112,
\nIvana Bjedov1258, Craig Blackstone843, Lionel Blanc1183, Guillermo A Blanco1496, Heidi Kiil Blomhoff1812,
\nEmilio Boada-Romero1297, Stefan B€ockler1464, Marianne Boes1423, Kathleen Boesze-Battaglia1835, Lawrence H Boise286,287,
\nAlessandra Bolino2063, Andrea Boman693, Paolo Bonaldo1823, Matteo Bordi897, J€urgen Bosch608, Luis M Botana1308,
\nJoelle Botti1375, German Bou1405, Marina Bouch e1038, Marion Bouchecareilh1331, Marie-Jos ee Boucher1901,
\nMichael E Boulton481, Sebastien G Bouret1926, Patricia Boya133, Micha€el Boyer-Guittaut1345, Peter V Bozhkov1141,
\nNathan Brady374, Vania MM Braga469, Claudio Brancolini1997, Gerhard H Braus353, Jos e M Bravo-San Pedro299,393,508,1374,
\nLisa A Brennan322, Emery H Bresnick2022, Patrick Brest490, Dave Bridges1939, Marie-Agn es Bringer124, Marisa Brini1822,
\nGlauber C Brito1311, Bertha Brodin631, Paul S Brookes1872, Eric J Brown352, Karen Brown1690, Hal E Broxmeyer480,
\nAlain Bruhat486,1339, Patricia Chakur Brum1893, John H Brumell446, Nicola Brunetti-Pierri315,1171,
\nRobert J Bryson-Richardson781, Shilpa Buch1777, Alastair M Buchan1819, Hikmet Budak1022, Dmitry V Bulavin118,505,1789,
\nScott J Bultman1792, Geert Bultynck665, Vladimir Bumbasirevic1470, Yan Burelle1356, Robert E Burke216,217,
\nMargit Burmeister1750, Peter B€utikofer1473, Laura Caberlotto1987, Ken Cadwell896, Monika Cahova112, Dongsheng Cai24,
\nJingjing Cai2099, Qian Cai1018, Sara Calatayud2007, Nadine Camougrand1343, Michelangelo Campanella1700,
\nGrant R Campbell1525, Matthew Campbell1249, Silvia Campello556,1876, Robin Candau1769, Isabella Caniggia1983,
\nLavinia Cantoni560, Lizhi Cao116, Allan B Caplan1656, Michele Caraglia1051, Claudio Cardinali1043, Sandra Morais Cardoso1579, Jennifer S Carew208, Laura A Carleton874, Cathleen R Carlin101, Silvia Carloni2002,
\nSven R Carlsson1267, Didac Carmona-Gutierrez1643, Leticia AM Carneiro312, Oliana Carnevali971, Serena Carra1318,
\nAlice Carrier120, Bernadette Carroll900, Caty Casas1324, Josefina Casas1116, Giuliana Cassinelli324, Perrine Castets1462,
\nSusana Castro-Obregon214, Gabriella Cavallini1841, Isabella Ceccherini568, Francesco Cecconi253,555,1884,
\nArthur I Cederbaum459, Valent ın Ce~na199,1281, Simone Cenci1323,2064, Claudia Cerella444, Davide Cervia1996,
\nSilvia Cetrullo1478, Hassan Chaachouay2028, Han-Jung Chae187, Andrei S Chagin634, Chee-Yin Chai626,628,
\nGopal Chakrabarti1502, Georgios Chamilos1601, Edmond YW Chan1142, Matthew TV Chan181, Dhyan Chandra1003,
\nPallavi Chandra548, Chih-Peng Chang818, Raymond Chuen-Chung Chang1653, Ta Yuan Chang345, John C Chatham1434,
\nSaurabh Chatterjee1910, Santosh Chauhan527, Yongsheng Che62, Michael E Cheetham1263, Rajkumar Cheluvappa1783,
\nChun-Jung Chen1153, Gang Chen598,1676, Guang-Chao Chen9, Guoqiang Chen1078, Hongzhuan Chen1077, Jeff W Chen1514,
\nJian-Kang Chen370,371, Min Chen249, Mingzhou Chen2104, Peiwen Chen1823, Qi Chen1674, Quan Chen172,
\nShang-Der Chen138, Si Chen325, Steve S-L Chen10, Wei Chen2125, Wei-Jung Chen829, Wen Qiang Chen979, Wenli Chen1113,
\nXiangmei Chen1133, Yau-Hung Chen1157, Ye-Guang Chen1250, Yin Chen1447, Yingyu Chen953,955, Yongshun Chen2135,
\nYu-Jen Chen712, Yue-Qin Chen1145, Yujie Chen1208, Zhen Chen339, Zhong Chen2123, Alan Cheng1702,
\nChristopher HK Cheng184, Hua Cheng1728, Heesun Cheong814, Sara Cherry1836, Jason Chesney1703,
\nChun Hei Antonio Cheung817, Eric Chevet1359, Hsiang Cheng Chi140, Sung-Gil Chi656, Fulvio Chiacchiera308,
\nHui-Ling Chiang958, Roberto Chiarelli1826, Mario Chiariello235,567,577, Marcello Chieppa835, Lih-Shen Chin290,
\nMario Chiong1285, Gigi NC Chiu878, Dong-Hyung Cho676, Ssang-Goo Cho650, William C Cho982, Yong-Yeon Cho105,
\nYoung-Seok Cho1064, Augustine MK Choi2095, Eui-Ju Choi656, Eun-Kyoung Choi387,400,685, Jayoung Choi1563,
\nMary E Choi2093, Seung-Il Choi2116, Tsui-Fen Chou412, Salem Chouaib395, Divaker Choubey1574, Vinay Choubey1936,
\nKuan-Chih Chow822, Kamal Chowdhury730, Charleen T Chu1856, Tsung-Hsien Chuang827, Taehoon Chun657,
\nHyewon Chung652, Taijoon Chung978, Yuen-Li Chung1194, Yong-Joon Chwae18, Valentina Cianfanelli254,
\nRoberto Ciarcia1775, Iwona A Ciechomska886, Maria Rosa Ciriolo1876, Mara Cirone1042, Sofie Claerhout1694,
\nMichael J Clague1698, Joan Cl aria1457, Peter GH Clarke1687, Robert Clarke361, Emilio Clementi1045,1398, C edric Cleyrat1781,
\nMiriam Cnop1366, Eliana M Coccia574, Tiziana Cocco1459, Patrice Codogno1375, J€orn Coers271, Ezra EW Cohen1533,
\nDavid Colecchia235,567,577, Luisa Coletto25, N uria S Coll123, Emma Colucci-Guyon516, Sergio Comincini1829,
\nMaria Condello578, Katherine L Cook2073, Graham H Coombs1929, Cynthia D Cooper2076, J Mark Cooper1395,
\nIsabelle Coppens601, Maria Tiziana Corasaniti1387, Marco Corazzari485,1884, Ramon Corbalan1566,
\nElisabeth Corcelle-Termeau251, Mario D Cordero1899, Cristina Corral-Ramos1289, Olga Corti507,1109, Andrea Cossarizza1767,
\nPaola Costelli1993, Safia Costes1518, Susan L Cotman721, Ana Coto-Montes946, Sandra Cottet566,1688, Eduardo Couve1301,
\nLori R Covey1015, L Ashley Cowart762, Jeffery S Cox1536, Fraser P Coxon1427, Carolyn B Coyne1846, Mark S Cragg1919,
\nRolf J Craven1679, Tiziana Crepaldi1995, Jose L Crespo1300, Alfredo Criollo1285, Valeria Crippa558, Maria Teresa Cruz1576,
\nAna Maria Cuervo26, Jose M Cuezva1277, Taixing Cui1907, Pedro R Cutillas987, Mark J Czaja27, Maria F Czyzyk-Krzeska1572,
\nRuben K Dagda2068, Uta Dahmen1404, Chunsun Dai800, Wenjie Dai1187, Yun Dai2059, Kevin N Dalby1940,
\nLuisa Dalla Valle1822, Guillaume Dalmasso1340, Marcello D’Amelio557, Markus Damme188, Arlette Darfeuille-Michaud1340,
\nCatherine Dargemont950, Victor M Darley-Usmar1433, Srinivasan Dasarathy205, Biplab Dasgupta202, Srikanta Dash1254,
\nCrispin R Dass242, Hazel Marie Davey8, Lester M Davids1560, David D avila227, Roger J Davis1731, Ted M Dawson604,
\nValina L Dawson606, Paula Daza1898, Jackie de Belleroche470, Paul de Figueiredo1180,1182,
\nRegina Celia Bressan Queiroz de Figueiredo135, Jos e de la Fuente1023, Luisa De Martino1775,
\nAntonella De Matteis1171, Guido RY De Meyer1443, Angelo De Milito631, Mauro De Santi2002,

BACKGROUND: The success of the CRISPR/Cas9 genome editing technique depends on the choice of the guide RNA sequence, which is facilitated by various websites. Despite the importance and popularity of these algorithms, it is unclear to which extent their predictions are in agreement with actual measurements. RESULTS: We conduct the first independent evaluation of CRISPR/Cas9 predictions. To this end, we collect data from eight SpCas9 off-target studies and compare them with the sites predicted by popular algorithms. We identify problems in one implementation but found that sequence-based off-target predictions are very reliable, identifying most off-targets with mutation rates superior to 0.1 %, while the number of false positives can be largely reduced with a cutoff on the off-target score. We also evaluate on-target efficiency prediction algorithms against available datasets. The correlation between the predictions and the guide activity varied considerably, especially for zebrafish. Together with novel data from our labs, we find that the optimal on-target efficiency prediction model strongly depends on whether the guide RNA is expressed from a U6 promoter or transcribed in vitro. We further demonstrate that the best predictions can significantly reduce the time spent on guide screening. CONCLUSIONS: To make these guidelines easily accessible to anyone planning a CRISPR genome editing experiment, we built a new website ( http://crispor.org ) that predicts off-targets and helps select and clone efficient guide sequences for more than 120 genomes using different Cas9 proteins and the eight efficiency scoring systems evaluated here.

An analysis of nearest neighbour dinucleotide frequencies and the level of DNA methylation in animals strongly supports the suggestion that 5-methylcytosine (5mC) tends to mutate abnormally frequently to T. This tendency is the likely cause of the CpG deficiency in heavily methylated genomes.

Neurodegenerative disorders, such as Alzheimer's disease, are a global public health burden with poorly understood aetiology. Neuroinflammation and oxidative stress (OS) are undoubtedly hallmarks of neurodegeneration, contributing to disease progression. Protein aggregation and neuronal damage result in the activation of disease-associated microglia (DAM) via damage-associated molecular patterns (DAMPs). DAM facilitate persistent inflammation and reactive oxygen species (ROS) generation. However, the molecular mechanisms linking DAM activation and OS have not been well-defined; thus targeting these cells for clinical benefit has not been possible. In microglia, ROS are generated primarily by NADPH oxidase 2 (NOX2) and activation of NOX2 in DAM is associated with DAMP signalling, inflammation and amyloid plaque deposition, especially in the cerebrovasculature. Additionally, ROS originating from both NOX and the mitochondria may act as second messengers to propagate immune activation; thus intracellular ROS signalling may underlie excessive inflammation and OS. Targeting key kinases in the inflammatory response could cease inflammation and promote tissue repair. Expression of antioxidant proteins in microglia, such as NADPH dehydrogenase 1 (NQO1), is promoted by transcription factor Nrf2, which functions to control inflammation and limit OS. Lipid droplet accumulating microglia (LDAM) may also represent a double-edged sword in neurodegenerative disease by sequestering peroxidised lipids in non-pathological ageing but becoming dysregulated and pro-inflammatory in disease. We suggest that future studies should focus on targeted manipulation of NOX in the microglia to understand the molecular mechanisms driving inflammatory-related NOX activation. Finally, we discuss recent evidence that therapeutic target identification should be unbiased and founded on relevant pathophysiological assays to facilitate the discovery of translatable antioxidant and anti-inflammatory therapeutics.

The BioMart Community Portal (www.biomart.org) is a community-driven effort to provide a unified interface to biomedical databases that are distributed worldwide. The portal provides access to numerous database projects supported by 30 scientific organizations. It includes over 800 different biological datasets spanning genomics, proteomics, model organisms, cancer data, ontology information and more. All resources available through the portal are independently administered and funded by their host organizations. The BioMart data federation technology provides a unified interface to all the available data. The latest version of the portal comes with many new databases that have been created by our ever-growing community. It also comes with better support and extensibility for data analysis and visualization tools. A new addition to our toolbox, the enrichment analysis tool is now accessible through graphical and web service interface. The BioMart community portal averages over one million requests per day. Building on this level of service and the wealth of information that has become available, the BioMart Community Portal has introduced a new, more scalable and cheaper alternative to the large data stores maintained by specialized organizations.

Parkinson's disease (PD) is characterized pathologically by intraneuronal inclusions called Lewy bodies, largely comprised of α-synuclein. Multiplication of the α-synuclein gene locus increases α-synuclein expression and causes PD. Thus, overexpression of wild-type α-synuclein is toxic. In this study, we demonstrate that α-synuclein overexpression impairs macroautophagy in mammalian cells and in transgenic mice. Our data show that α-synuclein compromises autophagy via Rab1a inhibition and Rab1a overexpression rescues the autophagy defect caused by α-synuclein. Inhibition of autophagy by α-synuclein overexpression or Rab1a knockdown causes mislocalization of the autophagy protein, Atg9, and decreases omegasome formation. Rab1a, α-synuclein, and Atg9 all regulate formation of the omegasome, which marks autophagosome precursors.

Resident macrophages are an integral component of many tissues and are important in homeostasis and repair. This study examines the contribution of resident tissue macrophages to bone physiology. Using immunohistochemistry, we showed that a discrete population of resident macrophages, OsteoMacs, was intercalated throughout murine and human osteal tissues. OsteoMacs were distributed among other bone lining cells within both endosteum and periosteum. Furthermore, OsteoMacs were coisolated with osteoblasts in murine bone explant and calvarial preparations. OsteoMacs made up 15.9% of calvarial preparations and persisted throughout standard osteoblast differentiation cultures. Contrary to previous studies, we showed that it was OsteoMacs and not osteoblasts within these preparations that responded to pathophysiological concentrations of LPS by secreting TNF. Removal of OsteoMacs from calvarial cultures significantly decreased osteocalcin mRNA induction and osteoblast mineralization in vitro. In a Transwell coculture system of enriched osteoblasts and macrophages, we demonstrated that macrophages were required for efficient osteoblast mineralization in response to the physiological remodeling stimulus, elevated extracellular calcium. Notably, OsteoMacs were closely associated with areas of bone modeling in situ, forming a distinctive canopy structure covering >75% of mature osteoblasts on diaphyseal endosteal surfaces in young growing mice. Depletion of OsteoMacs in vivo using the macrophage-Fas-induced apoptosis (MAFIA) mouse caused complete loss of osteoblast bone-forming surface at this modeling site. Overall, we have demonstrated that OsteoMacs are an integral component of bone tissues and play a novel role in bone homeostasis through regulating osteoblast function. These observations implicate OsteoMacs, in addition to osteoclasts and osteoblasts, as principal participants in bone dynamics.

A series of nine Ce(iv)-based metal organic frameworks with the UiO-66 structure containing linker molecules of different sizes and functionalities were obtained under mild synthesis conditions and short reaction times. Thermal and chemical stabilities were determined and a Ce-UiO-66-BDC/TEMPO system was successfully employed for the aerobic oxidation of benzyl alcohol.

SUMMARY Dimensional measurements have been made on a small number of tracings of low magnification scanning electron micrographs of trabecular bone sections, using the Quantimet Image Analysing Computer, with a view to expressing the properties of the local bone pattern in a quantitative manner. Measurements of the orthogonally projected length of the boundary profile of the interface between bone and marrow were made as a function of the direction of projection, and these showed that the majority of the patterns were distinctly anisotropic. A formula is proposed for calculating the mean widths of the trabecular bands as they appear on the surfaces of the sections, from the mean area of bone and the boundary profile length. As the validity of this formula is not theoretically evident, the results have been compared with direct experimental measurements, and found to agree very well. The mean intercept lengths in the bone and in the marrow spaces have been calculated as a function of direction across the section. The polar diagrams of these lengths are found to have the shapes of ellipses, to an unexpected degree of accuracy. This makes it possible to express the departure from isotropy of the pattern by a single number, for instance by the ratio of the axes of the ellipse. The essential limitations of the methods used in these experiments are briefly discussed, in particular the impossibility of finding the exact symmetry properties of the surface patterns by experiments of this type.

ABSTRACT Primitive-streak-stage mouse embryos were treated with Mitomycin C injected intraperitoneally into pregnant females at 6·75–7·0 days post coitum. The newborn mice developed poorly and mortality was high during the suckling period. Many weaned survivors showed impaired fertility and poor breeding performance. Histological examination revealed a paucity of germ cells in the adult gonads. The deficiency was mainly caused by a severe reduction of the primordial germ cell population in early embryonic life, which was not fully compensated for during the compensatory growth phase of the Mitomycin C-treated embryo. Also contributing to such impaired fertility were retarded migration of the primordial germ cells into the genital ridges, poor development of the foetal gonad and secondary loss of the germ cells during gametogenesis in males.

BACKGROUND: The mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms. RESULTS: We undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems. CONCLUSIONS: Comparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains.

One component of the circadian clock in mammals is the Clock-Bmal1 heterodimeric transcription factor. Among its downstream targets, two genes, Cry1 and Cry2, encode inhibitors of the Clock-Bmal1 complex that establish a negative-feedback loop. We found that both Cry1 and Cry2 proteins are ubiquitinated and degraded via the SCF(Fbxl3) ubiquitin ligase complex. This regulation by SCF(Fbxl3) is a prerequisite for the efficient and timely reactivation of Clock-Bmal1 and the consequent expression of Per1 and Per2, two regulators of the circadian clock that display tumor suppressor activity. Silencing of Fbxl3 produced no effect in Cry1-/-;Cry2-/- cells, which shows that Fbxl3 controls clock oscillations by mediating the degradation of CRY proteins.

To study the characteristics of molecular damage induced by ionizing radiation at the DNA level, Monte Carlo track simulation of energetic electrons and ions in liquid water, a canonical model of B-DNA, and a comprehensive classification of DNA damage in terms of the origin and complexity of damage were used to calculate the frequencies of simple and complex strand breaks. A threshold energy of 17.5 eV was used to model the damage by direct energy deposition, and a probability of 0.13 was applied to model the induction of a single-strand break produced in DNA by OH radical reactions. For preliminary estimates, base damage was assumed to be induced by the same direct energy threshold deposition or by the reaction of an OH radical with the base, with a probability of 0.8. Computational data are given on the complexity of damage, including base damage by electrons with energies of 100-4500 eV and ions with energies of 0.3-4.0 MeV/nucleon (59-9 keV microm(-1) protons and 170-55 keV microm(-1) alpha particles). Computational data are presented on the frequencies of single- and double-strand breaks induced as a function of the LET of the particles, and on the relative frequencies of complex single- and double-strand breaks for electrons. The modeling and calculations of strand breaks show that: (1) The yield of strand breaks per unit absorbed dose is nearly constant over a wide range of LET. (2) The majority of DNA damage is of a simple type, but the majority of the simple single-strand breaks are accompanied by at least one base damage. (3) For low-energy electrons, nearly 20-30% of the double-strand breaks are of a complex type by virtue of additional breaks. The proportion of this locally clustered damage increases with LET, reaching about 70% for the highest-LET alpha particles modeled, with the complexity of damage increasing further, to about 90%, when base damage is considered. (4) The extent of damage in the local hit region of the DNA duplex is mostly limited to a length of a few base pairs. (5) The frequency of base damage when no strand breaks are present in the hit segment of DNA varies between 20-40% as a function of LET for protons and alpha particles.

By screening N-ethyl-N-nitrosourea-mutagenized animals for alterations in rhythms of wheel-running activity, we identified a mouse mutation, after hours (Afh). The mutation, a Cys(358)Ser substitution in Fbxl3, an F-box protein with leucine-rich repeats, results in long free-running rhythms of about 27 hours in homozygotes. Circadian transcriptional and translational oscillations are attenuated in Afh mice. The Afh allele significantly affected Per2 expression and delayed the rate of Cry protein degradation in Per2::Luciferase tissue slices. Our in vivo and in vitro studies reveal a central role for Fbxl3 in mammalian circadian timekeeping.

Two fragments cloned from purified human satellite III DNA do not cross-react with each other. One fragment, for which a partial sequence is reported, hybridises to satellite II as well as III and is shown to originate on chromosome 1. The other cloned fragment originates from the Y chromosome. This fragment has undergone considerable changes in size when cloned in lambda gt WES lambda B. Human satellite III is shown to consist of a number of non-cross-reacting sequences which nevertheless are related by the presence of closely spaced Hin F1 sites.

Given the anthropometric differences between men and women and previous evidence of sex-difference in genetic effects, we conducted a genome-wide search for sexually dimorphic associations with height, weight, body mass index, waist circumference, hip circumference, and waist-to-hip-ratio (133,723 individuals) and took forward 348 SNPs into follow-up (additional 137,052 individuals) in a total of 94 studies. Seven loci displayed significant sex-difference (FDR<5%), including four previously established (near GRB14/COBLL1, LYPLAL1/SLC30A10, VEGFA, ADAMTS9) and three novel anthropometric trait loci (near MAP3K1, HSD17B4, PPARG), all of which were genome-wide significant in women (P<5×10(-8)), but not in men. Sex-differences were apparent only for waist phenotypes, not for height, weight, BMI, or hip circumference. Moreover, we found no evidence for genetic effects with opposite directions in men versus women. The PPARG locus is of specific interest due to its role in diabetes genetics and therapy. Our results demonstrate the value of sex-specific GWAS to unravel the sexually dimorphic genetic underpinning of complex traits.

The International Mouse Phenotyping Consortium (IMPC; https://www.mousephenotype.org/) web portal makes available curated, integrated and analysed knockout mouse phenotyping data generated by the IMPC project consisting of 85M data points and over 95,000 statistically significant phenotype hits mapped to human diseases. The IMPC portal delivers a substantial reference dataset that supports the enrichment of various domain-specific projects and databases, as well as the wider research and clinical community, where the IMPC genotype-phenotype knowledge contributes to the molecular diagnosis of patients affected by rare disorders. Data from 9,000 mouse lines and 750 000 images provides vital resources enabling the interpretation of the ignorome, and advancing our knowledge on mammalian gene function and the mechanisms underlying phenotypes associated with human diseases. The resource is widely integrated and the lines have been used in over 4,600 publications indicating the value of the data and the materials.

The development of the mammalian brain is dependent on extensive neuronal migration. Mutations in mice and humans that affect neuronal migration result in abnormal lamination of brain structures with associated behavioral deficits. Here, we report the identification of a hyperactive N-ethyl-N-nitrosourea (ENU)-induced mouse mutant with abnormalities in the laminar architecture of the hippocampus and cortex, accompanied by impaired neuronal migration. We show that the causative mutation lies in the guanosine triphosphate (GTP) binding pocket of alpha-1 tubulin (Tuba1) and affects tubulin heterodimer formation. Phenotypic similarity with existing mouse models of lissencephaly led us to screen a cohort of patients with developmental brain anomalies. We identified two patients with de novo mutations in TUBA3, the human homolog of Tuba1. This study demonstrates the utility of ENU mutagenesis in the mouse as a means to discover the basis of human neurodevelopmental disorders.

Type 2 diabetes mellitus is the result of a combination of impaired insulin secretion with reduced insulin sensitivity of target tissues. There are an estimated 150 million affected individuals worldwide, of whom a large proportion remains undiagnosed because of a lack of specific symptoms early in this disorder and inadequate diagnostics. In this study, NMR-based metabolomic analysis in conjunction with multivariate statistics was applied to examine the urinary metabolic changes in two rodent models of type 2 diabetes mellitus as well as unmedicated human sufferers. The db/db mouse and obese Zucker (fa/fa) rat have autosomal recessive defects in the leptin receptor gene, causing type 2 diabetes. 1H-NMR spectra of urine were used in conjunction with uni- and multivariate statistics to identify disease-related metabolic changes in these two animal models and human sufferers. This study demonstrates metabolic similarities between the three species examined, including metabolic responses associated with general systemic stress, changes in the TCA cycle, and perturbations in nucleotide metabolism and in methylamine metabolism. All three species demonstrated profound changes in nucleotide metabolism, including that of N-methylnicotinamide and N-methyl-2-pyridone-5-carboxamide, which may provide unique biomarkers for following type 2 diabetes mellitus progression.

There are as yet no licensed therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric spike whose receptor binding domain (RBD) recognizes angiotensin-converting enzyme 2, initiating conformational changes that drive membrane fusion. We find that the monoclonal antibody CR3022 binds the RBD tightly, neutralizing SARS-CoV-2, and report the crystal structure at 2.4 Å of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilizing CR3022 epitope is inaccessible in the prefusion spike, suggesting that CR3022 binding facilitates conversion to the fusion-incompetent post-fusion state. Cryogenic electron microscopy (cryo-EM) analysis confirms that incubation of spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope could be useful therapeutically, possibly in synergy with an antibody that blocks receptor attachment.