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Construction of Synthetic Antibody Phage-Display Libraries.

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Construction of Synthetic Antibody Phage-Display Libraries.

Methods Mol Biol. 2018;1701:45-60

Authors: Nilvebrant J, Sidhu SS

Abstract
Synthetic antibody libraries provide a vast resource of renewable antibody reagents that can rival or exceed those of natural antibodies and can be rapidly isolated through controlled in vitro selections. Use of highly optimized human frameworks enables the incorporation of defined diversity at positions that are most likely to contribute to antigen recognition. This protocol describes the construction of synthetic antibody libraries based on a single engineered human autonomous variable heavy domain scaffold with diversity in all three complementarity-determining regions. The resulting libraries can be used to generate recombinant domain antibodies for a wide range of protein antigens using phage display. Furthermore, analogous methods can be used to construct antibody libraries based on larger antibody fragments or second-generation libraries aimed to fine-tune antibody characteristics including affinity, specificity, and manufacturability. The procedures rely on standard reagents and equipment available in most molecular biology laboratories.

PMID: 29116499 [PubMed - in process]



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A computational approach for designing D-proteins with non-canonical amino acid optimised binding affinity.

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A computational approach for designing D-proteins with non-canonical amino acid optimised binding affinity.

PLoS One. 2017;12(11):e0187524

Authors: Garton M, Sayadi M, Kim PM

Abstract
Redesigning protein surface topology to improve target binding holds great promise in the search for highly selective therapeutics. While significant binding improvements can be achieved using natural amino acids, the introduction of non-canonical residues vastly increases sequence space and thus the chance to significantly out-compete native partners. The potency of protein inhibitors can be further enhanced by synthesising mirror image, D-amino versions. This renders them non-immunogenic and makes them highly resistant to proteolytic degradation. Current experimental design methods often preclude the use of D-amino acids and non-canonical amino acids for a variety of reasons. To address this, we build an in silico pipeline for D-protein designs featuring non-canonical amino acids. For a test scaffold we use an existing D-protein inhibitor of VEGF: D-RFX001. We benchmark the approach by recapitulating previous experimental optimisation with canonical amino acids. Subsequent incorporation of non-canonical amino acids allows designs that are predicted to improve binding affinity by up to -7.18 kcal/mol.

PMID: 29108013 [PubMed - indexed for MEDLINE]



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The influence of microRNAs and poly(A) tail length on endogenous mRNA-protein complexes.

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The influence of microRNAs and poly(A) tail length on endogenous mRNA-protein complexes.

Genome Biol. 2017 Oct 31;18(1):211

Authors: Rissland OS, Subtelny AO, Wang M, Lugowski A, Nicholson B, Laver JD, Sidhu SS, Smibert CA, Lipshitz HD, Bartel DP

Abstract
BACKGROUND: All mRNAs are bound in vivo by proteins to form mRNA-protein complexes (mRNPs), but changes in the composition of mRNPs during posttranscriptional regulation remain largely unexplored. Here, we have analyzed, on a transcriptome-wide scale, how microRNA-mediated repression modulates the associations of the core mRNP components eIF4E, eIF4G, and PABP and of the decay factor DDX6 in human cells.
RESULTS: Despite the transient nature of repressed intermediates, we detect significant changes in mRNP composition, marked by dissociation of eIF4G and PABP, and by recruitment of DDX6. Furthermore, although poly(A)-tail length has been considered critical in post-transcriptional regulation, differences in steady-state tail length explain little of the variation in either PABP association or mRNP organization more generally. Instead, relative occupancy of core components correlates best with gene expression.
CONCLUSIONS: These results indicate that posttranscriptional regulatory factors, such as microRNAs, influence the associations of PABP and other core factors, and do so without substantially affecting steady-state tail length.

PMID: 29089021 [PubMed - in process]



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Combined delivery of chondroitinase ABC and human induced pluripotent stem cell-derived neuroepithelial cells promote tissue repair in an animal model of spinal cord injury.

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Combined delivery of chondroitinase ABC and human induced pluripotent stem cell-derived neuroepithelial cells promote tissue repair in an animal model of spinal cord injury.

Biomed Mater. 2017 Oct 30;:

Authors: Führmann T, Anandakumaran P, Payne S, Pakulska M, Varga BV, Nagy A, Tator C, Shoichet MS

Abstract
The lack of tissue regeneration after traumatic spinal cord injury (SCI) in animal models is largely attributed to the local inhibitory microenvironment. To overcome this inhibitory environment while promoting tissue regeneration, we investigated the combined delivery of chondroitinase ABC (chABC) with human induced pluripotent stem cell-derived neuroepithelial stem cells (NESCs). ChABC was delivered to the injured spinal cord at the site of injury by affinity release from a crosslinked methylcellulose hydrogel by injection into the intrathecal space. NESCs were distributed in a hydrogel comprised of hyaluronan and methylcellulose and injected into the spinal cord tissue both rostral and caudal to the site of injury. Cell transplantation led to reduced cavity formation, but did not improve motor function. While few surviving cells were found 2 weeks post injury, the majority of live cells were neurons, with only few astrocytes, oligodendrocytes, and progenitor cells. At 9 weeks post injury, there were more progenitor cells and a more even distribution of cell types compared to those at 2 weeks post injury, suggesting preferential survival and differentiation. Interestingly, animals that received cells and chABC had more neurons than animals that received cells alone, suggesting that chABC influenced the injury environment such that neuronal differentiation or survival was favoured.

PMID: 29083317 [PubMed - as supplied by publisher]



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Adult skin-derived precursor Schwann cell grafts form growths in the injured spinal cord of Fischer rats.

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Adult skin-derived precursor Schwann cell grafts form growths in the injured spinal cord of Fischer rats.

Biomed Mater. 2017 Oct 25;:

Authors: May Z, Kumar R, Führmann T, Tam R, Vulic K, Forero J, Lucas-Osma AM, Fenrich K, Assinck P, Lee MJ, Moulson A, Shoichet MS, Tetzlaff W, Biernaskie J, Fouad K

Abstract
In this study, GFP+ skin-derived precursor Schwann cells (SKP-SCs) from adult rats were grafted into the injured spinal cord of immunosuppressed rats. Our goal was to improve grafted cell survival in the injured spinal cord, which is typically low. Cells were grafted in hyaluronan-methylcellulose hydrogel (HAMC) or hyaluronan-methylcellulose modified with laminin- and fibronectin-derived peptide sequences (eHAMC). The criteria for selection of hyaluronan was for its shear-thinning properties, making the hydrogel easy to inject, methylcellulose for its inverse thermal gelation, helping to keep grafted cells in situ, and fibronectin and laminin to improve cell attachment and, thus, prevent cell death due to dissociation from substrate molecules (i.e., anoikis). Post-mortem examination revealed large masses of GFP+ SKP-SCs in the spinal cords of rats that received cells in HAMC (5 out of n = 8) and eHAMC (6 out of n = 8). Cell transplantation in eHAMC caused significantly greater spinal lesions compared to lesion and eHAMC only control groups. A parallel study showed similar masses in the contused spinal cord of rats after transplantation of adult GFP+ SKP-SCs without a hydrogel or immunosuppression. These findings suggest that adult GFP+ SKP-SCs, cultured/transplanted under the conditions described here, have a capacity for uncontrolled proliferation. Growth-formation in pre-clinical research has also been documented after transplantation of: human induced pluripotent stem cell-derived neural stem cells (Itakura et al., 2015), embryonic stem cells and embryonic stem cell-derived neurons (Brederlau et al., 2006; Dressel et al., 2008), bone marrow derived mesenchymal stem cells (Jeong et al., 2011) and rat nerve-derived SCs following in vitro expansion for ˃11 passages (Funk et al., 2007; Langford et al., 1988; Morrissey et al., 1991). It is of upmost importance to define the precise culture/transplantation parameters for maintenance of normal cell function and safe and effective use of cell therapy.

PMID: 29068322 [PubMed - as supplied by publisher]



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Anti-CRISPR: discovery, mechanism and function.

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Anti-CRISPR: discovery, mechanism and function.

Nat Rev Microbiol. 2018 Jan;16(1):12-17

Authors: Pawluk A, Davidson AR, Maxwell KL

Abstract
CRISPR-Cas adaptive immune systems are widespread among bacteria and archaea. Recent studies have shown that these systems have minimal long-term evolutionary effects in limiting horizontal gene transfer. This suggests that the ability to evade CRISPR-Cas immunity must also be widespread in phages and other mobile genetic elements. In this Progress article, we discuss recent discoveries that highlight how phages inactivate CRISPR-Cas systems by using anti-CRISPR proteins, and we outline evolutionary and biotechnological implications of their activity.

PMID: 29062071 [PubMed - indexed for MEDLINE]



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Association analysis identifies 65 new breast cancer risk loci.

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Association analysis identifies 65 new breast cancer risk loci.

Nature. 2017 11 02;551(7678):92-94

Authors: Michailidou K, Lindström S, Dennis J, Beesley J, Hui S, Kar S, Lemaçon A, Soucy P, Glubb D, Rostamianfar A, Bolla MK, Wang Q, Tyrer J, Dicks E, Lee A, Wang Z, Allen J, Keeman R, Eilber U, French JD, Qing Chen X, Fachal L, McCue K, McCart Reed AE, Ghoussaini M, Carroll JS, Jiang X, Finucane H, Adams M, Adank MA, Ahsan H, Aittomäki K, Anton-Culver H, Antonenkova NN, Arndt V, Aronson KJ, Arun B, Auer PL, Bacot F, Barrdahl M, Baynes C, Beckmann MW, Behrens S, Benitez J, Bermisheva M, Bernstein L, Blomqvist C, Bogdanova NV, Bojesen SE, Bonanni B, Børresen-Dale AL, Brand JS, Brauch H, Brennan P, Brenner H, Brinton L, Broberg P, Brock IW, Broeks A, Brooks-Wilson A, Brucker SY, Brüning T, Burwinkel B, Butterbach K, Cai Q, Cai H, Caldés T, Canzian F, Carracedo A, Carter BD, Castelao JE, Chan TL, David Cheng TY, Seng Chia K, Choi JY, Christiansen H, Clarke CL, NBCS Collaborators, Collée M, Conroy DM, Cordina-Duverger E, Cornelissen S, Cox DG, Cox A, Cross SS, Cunningham JM, Czene K, Daly MB, Devilee P, Doheny KF, Dörk T, Dos-Santos-Silva I, Dumont M, Durcan L, Dwek M, Eccles DM, Ekici AB, Eliassen AH, Ellberg C, Elvira M, Engel C, Eriksson M, Fasching PA, Figueroa J, Flesch-Janys D, Fletcher O, Flyger H, Fritschi L, Gaborieau V, Gabrielson M, Gago-Dominguez M, Gao YT, Gapstur SM, García-Sáenz JA, Gaudet MM, Georgoulias V, Giles GG, Glendon G, Goldberg MS, Goldgar DE, González-Neira A, Grenaker Alnæs GI, Grip M, Gronwald J, Grundy A, Guénel P, Haeberle L, Hahnen E, Haiman CA, Håkansson N, Hamann U, Hamel N, Hankinson S, Harrington P, Hart SN, Hartikainen JM, Hartman M, Hein A, Heyworth J, Hicks B, Hillemanns P, Ho DN, Hollestelle A, Hooning MJ, Hoover RN, Hopper JL, Hou MF, Hsiung CN, Huang G, Humphreys K, Ishiguro J, Ito H, Iwasaki M, Iwata H, Jakubowska A, Janni W, John EM, Johnson N, Jones K, Jones M, Jukkola-Vuorinen A, Kaaks R, Kabisch M, Kaczmarek K, Kang D, Kasuga Y, Kerin MJ, Khan S, Khusnutdinova E, Kiiski JI, Kim SW, Knight JA, Kosma VM, Kristensen VN, Krüger U, Kwong A, Lambrechts D, Le Marchand L, Lee E, Lee MH, Lee JW, Neng Lee C, Lejbkowicz F, Li J, Lilyquist J, Lindblom A, Lissowska J, Lo WY, Loibl S, Long J, Lophatananon A, Lubinski J, Luccarini C, Lux MP, Ma ESK, MacInnis RJ, Maishman T, Makalic E, Malone KE, Kostovska IM, Mannermaa A, Manoukian S, Manson JE, Margolin S, Mariapun S, Martinez ME, Matsuo K, Mavroudis D, McKay J, McLean C, Meijers-Heijboer H, Meindl A, Menéndez P, Menon U, Meyer J, Miao H, Miller N, Taib NAM, Muir K, Mulligan AM, Mulot C, Neuhausen SL, Nevanlinna H, Neven P, Nielsen SF, Noh DY, Nordestgaard BG, Norman A, Olopade OI, Olson JE, Olsson H, Olswold C, Orr N, Pankratz VS, Park SK, Park-Simon TW, Lloyd R, Perez JIA, Peterlongo P, Peto J, Phillips KA, Pinchev M, Plaseska-Karanfilska D, Prentice R, Presneau N, Prokofyeva D, Pugh E, Pylkäs K, Rack B, Radice P, Rahman N, Rennert G, Rennert HS, Rhenius V, Romero A, Romm J, Ruddy KJ, Rüdiger T, Rudolph A, Ruebner M, Rutgers EJT, Saloustros E, Sandler DP, Sangrajrang S, Sawyer EJ, Schmidt DF, Schmutzler RK, Schneeweiss A, Schoemaker MJ, Schumacher F, Schürmann P, Scott RJ, Scott C, Seal S, Seynaeve C, Shah M, Sharma P, Shen CY, Sheng G, Sherman ME, Shrubsole MJ, Shu XO, Smeets A, Sohn C, Southey MC, Spinelli JJ, Stegmaier C, Stewart-Brown S, Stone J, Stram DO, Surowy H, Swerdlow A, Tamimi R, Taylor JA, Tengström M, Teo SH, Beth Terry M, Tessier DC, Thanasitthichai S, Thöne K, Tollenaar RAEM, Tomlinson I, Tong L, Torres D, Truong T, Tseng CC, Tsugane S, Ulmer HU, Ursin G, Untch M, Vachon C, van Asperen CJ, Van Den Berg D, van den Ouweland AMW, van der Kolk L, van der Luijt RB, Vincent D, Vollenweider J, Waisfisz Q, Wang-Gohrke S, Weinberg CR, Wendt C, Whittemore AS, Wildiers H, Willett W, Winqvist R, Wolk A, Wu AH, Xia L, Yamaji T, Yang XR, Har Yip C, Yoo KY, Yu JC, Zheng W, Zheng Y, Zhu B, Ziogas A, Ziv E, ABCTB Investigators, ConFab/AOCS Investigators, Lakhani SR, Antoniou AC, Droit A, Andrulis IL, Amos CI, Couch FJ, Pharoah PDP, Chang-Claude J, Hall P, Hunter DJ, Milne RL, García-Closas M, Schmidt MK, Chanock SJ, Dunning AM, Edwards SL, Bader GD, Chenevix-Trench G, Simard J, Kraft P, Easton DF

Abstract
Breast cancer risk is influenced by rare coding variants in susceptibility genes, such as BRCA1, and many common, mostly non-coding variants. However, much of the genetic contribution to breast cancer risk remains unknown. Here we report the results of a genome-wide association study of breast cancer in 122,977 cases and 105,974 controls of European ancestry and 14,068 cases and 13,104 controls of East Asian ancestry. We identified 65 new loci that are associated with overall breast cancer risk at P < 5 × 10-8. The majority of credible risk single-nucleotide polymorphisms in these loci fall in distal regulatory elements, and by integrating in silico data to predict target genes in breast cells at each locus, we demonstrate a strong overlap between candidate target genes and somatic driver genes in breast tumours. We also find that heritability of breast cancer due to all single-nucleotide polymorphisms in regulatory features was 2-5-fold enriched relative to the genome-wide average, with strong enrichment for particular transcription factor binding sites. These results provide further insight into genetic susceptibility to breast cancer and will improve the use of genetic risk scores for individualized screening and prevention.

PMID: 29059683 [PubMed - indexed for MEDLINE]



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Identification of ten variants associated with risk of estrogen-receptor-negative breast cancer.

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Identification of ten variants associated with risk of estrogen-receptor-negative breast cancer.

Nat Genet. 2017 Dec;49(12):1767-1778

Authors: Milne RL, Kuchenbaecker KB, Michailidou K, Beesley J, Kar S, Lindström S, Hui S, Lemaçon A, Soucy P, Dennis J, Jiang X, Rostamianfar A, Finucane H, Bolla MK, McGuffog L, Wang Q, Aalfs CM, ABCTB Investigators, Adams M, Adlard J, Agata S, Ahmed S, Ahsan H, Aittomäki K, Al-Ejeh F, Allen J, Ambrosone CB, Amos CI, Andrulis IL, Anton-Culver H, Antonenkova NN, Arndt V, Arnold N, Aronson KJ, Auber B, Auer PL, Ausems MGEM, Azzollini J, Bacot F, Balmaña J, Barile M, Barjhoux L, Barkardottir RB, Barrdahl M, Barnes D, Barrowdale D, Baynes C, Beckmann MW, Benitez J, Bermisheva M, Bernstein L, Bignon YJ, Blazer KR, Blok MJ, Blomqvist C, Blot W, Bobolis K, Boeckx B, Bogdanova NV, Bojesen A, Bojesen SE, Bonanni B, Børresen-Dale AL, Bozsik A, Bradbury AR, Brand JS, Brauch H, Brenner H, Bressac-de Paillerets B, Brewer C, Brinton L, Broberg P, Brooks-Wilson A, Brunet J, Brüning T, Burwinkel B, Buys SS, Byun J, Cai Q, Caldés T, Caligo MA, Campbell I, Canzian F, Caron O, Carracedo A, Carter BD, Castelao JE, Castera L, Caux-Moncoutier V, Chan SB, Chang-Claude J, Chanock SJ, Chen X, Cheng TD, Chiquette J, Christiansen H, Claes KBM, Clarke CL, Conner T, Conroy DM, Cook J, Cordina-Duverger E, Cornelissen S, Coupier I, Cox A, Cox DG, Cross SS, Cuk K, Cunningham JM, Czene K, Daly MB, Damiola F, Darabi H, Davidson R, De Leeneer K, Devilee P, Dicks E, Diez O, Ding YC, Ditsch N, Doheny KF, Domchek SM, Dorfling CM, Dörk T, Dos-Santos-Silva I, Dubois S, Dugué PA, Dumont M, Dunning AM, Durcan L, Dwek M, Dworniczak B, Eccles D, Eeles R, Ehrencrona H, Eilber U, Ejlertsen B, Ekici AB, Eliassen AH, EMBRACE, Engel C, Eriksson M, Fachal L, Faivre L, Fasching PA, Faust U, Figueroa J, Flesch-Janys D, Fletcher O, Flyger H, Foulkes WD, Friedman E, Fritschi L, Frost D, Gabrielson M, Gaddam P, Gammon MD, Ganz PA, Gapstur SM, Garber J, Garcia-Barberan V, García-Sáenz JA, Gaudet MM, Gauthier-Villars M, Gehrig A, GEMO Study Collaborators, Georgoulias V, Gerdes AM, Giles GG, Glendon G, Godwin AK, Goldberg MS, Goldgar DE, González-Neira A, Goodfellow P, Greene MH, Alnæs GIG, Grip M, Gronwald J, Grundy A, Gschwantler-Kaulich D, Guénel P, Guo Q, Haeberle L, Hahnen E, Haiman CA, Håkansson N, Hallberg E, Hamann U, Hamel N, Hankinson S, Hansen TVO, Harrington P, Hart SN, Hartikainen JM, Healey CS, HEBON, Hein A, Helbig S, Henderson A, Heyworth J, Hicks B, Hillemanns P, Hodgson S, Hogervorst FB, Hollestelle A, Hooning MJ, Hoover B, Hopper JL, Hu C, Huang G, Hulick PJ, Humphreys K, Hunter DJ, Imyanitov EN, Isaacs C, Iwasaki M, Izatt L, Jakubowska A, James P, Janavicius R, Janni W, Jensen UB, John EM, Johnson N, Jones K, Jones M, Jukkola-Vuorinen A, Kaaks R, Kabisch M, Kaczmarek K, Kang D, Kast K, kConFab/AOCS Investigators, Keeman R, Kerin MJ, Kets CM, Keupers M, Khan S, Khusnutdinova E, Kiiski JI, Kim SW, Knight JA, Konstantopoulou I, Kosma VM, Kristensen VN, Kruse TA, Kwong A, Lænkholm AV, Laitman Y, Lalloo F, Lambrechts D, Landsman K, Lasset C, Lazaro C, Le Marchand L, Lecarpentier J, Lee A, Lee E, Lee JW, Lee MH, Lejbkowicz F, Lesueur F, Li J, Lilyquist J, Lincoln A, Lindblom A, Lissowska J, Lo WY, Loibl S, Long J, Loud JT, Lubinski J, Luccarini C, Lush M, MacInnis RJ, Maishman T, Makalic E, Kostovska IM, Malone KE, Manoukian S, Manson JE, Margolin S, Martens JWM, Martinez ME, Matsuo K, Mavroudis D, Mazoyer S, McLean C, Meijers-Heijboer H, Menéndez P, Meyer J, Miao H, Miller A, Miller N, Mitchell G, Montagna M, Muir K, Mulligan AM, Mulot C, Nadesan S, Nathanson KL, NBSC Collaborators, Neuhausen SL, Nevanlinna H, Nevelsteen I, Niederacher D, Nielsen SF, Nordestgaard BG, Norman A, Nussbaum RL, Olah E, Olopade OI, Olson JE, Olswold C, Ong KR, Oosterwijk JC, Orr N, Osorio A, Pankratz VS, Papi L, Park-Simon TW, Paulsson-Karlsson Y, Lloyd R, Pedersen IS, Peissel B, Peixoto A, Perez JIA, Peterlongo P, Peto J, Pfeiler G, Phelan CM, Pinchev M, Plaseska-Karanfilska D, Poppe B, Porteous ME, Prentice R, Presneau N, Prokofieva D, Pugh E, Pujana MA, Pylkäs K, Rack B, Radice P, Rahman N, Rantala J, Rappaport-Fuerhauser C, Rennert G, Rennert HS, Rhenius V, Rhiem K, Richardson A, Rodriguez GC, Romero A, Romm J, Rookus MA, Rudolph A, Ruediger T, Saloustros E, Sanders J, Sandler DP, Sangrajrang S, Sawyer EJ, Schmidt DF, Schoemaker MJ, Schumacher F, Schürmann P, Schwentner L, Scott C, Scott RJ, Seal S, Senter L, Seynaeve C, Shah M, Sharma P, Shen CY, Sheng X, Shimelis H, Shrubsole MJ, Shu XO, Side LE, Singer CF, Sohn C, Southey MC, Spinelli JJ, Spurdle AB, Stegmaier C, Stoppa-Lyonnet D, Sukiennicki G, Surowy H, Sutter C, Swerdlow A, Szabo CI, Tamimi RM, Tan YY, Taylor JA, Tejada MI, Tengström M, Teo SH, Terry MB, Tessier DC, Teulé A, Thöne K, Thull DL, Tibiletti MG, Tihomirova L, Tischkowitz M, Toland AE, Tollenaar RAEM, Tomlinson I, Tong L, Torres D, Tranchant M, Truong T, Tucker K, Tung N, Tyrer J, Ulmer HU, Vachon C, van Asperen CJ, Van Den Berg D, van den Ouweland AMW, van Rensburg EJ, Varesco L, Varon-Mateeva R, Vega A, Viel A, Vijai J, Vincent D, Vollenweider J, Walker L, Wang Z, Wang-Gohrke S, Wappenschmidt B, Weinberg CR, Weitzel JN, Wendt C, Wesseling J, Whittemore AS, Wijnen JT, Willett W, Winqvist R, Wolk A, Wu AH, Xia L, Yang XR, Yannoukakos D, Zaffaroni D, Zheng W, Zhu B, Ziogas A, Ziv E, Zorn KK, Gago-Dominguez M, Mannermaa A, Olsson H, Teixeira MR, Stone J, Offit K, Ottini L, Park SK, Thomassen M, Hall P, Meindl A, Schmutzler RK, Droit A, Bader GD, Pharoah PDP, Couch FJ, Easton DF, Kraft P, Chenevix-Trench G, García-Closas M, Schmidt MK, Antoniou AC, Simard J

Abstract
Most common breast cancer susceptibility variants have been identified through genome-wide association studies (GWAS) of predominantly estrogen receptor (ER)-positive disease. We conducted a GWAS using 21,468 ER-negative cases and 100,594 controls combined with 18,908 BRCA1 mutation carriers (9,414 with breast cancer), all of European origin. We identified independent associations at P < 5 × 10-8 with ten variants at nine new loci. At P < 0.05, we replicated associations with 10 of 11 variants previously reported in ER-negative disease or BRCA1 mutation carrier GWAS and observed consistent associations with ER-negative disease for 105 susceptibility variants identified by other studies. These 125 variants explain approximately 16% of the familial risk of this breast cancer subtype. There was high genetic correlation (0.72) between risk of ER-negative breast cancer and breast cancer risk for BRCA1 mutation carriers. These findings may lead to improved risk prediction and inform further fine-mapping and functional work to better understand the biological basis of ER-negative breast cancer.

PMID: 29058716 [PubMed - indexed for MEDLINE]



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A General Strategy for Discovery of Inhibitors and Activators of RING and U-box E3 Ligases with Ubiquitin Variants.

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A General Strategy for Discovery of Inhibitors and Activators of RING and U-box E3 Ligases with Ubiquitin Variants.

Mol Cell. 2017 Oct 19;68(2):456-470.e10

Authors: Gabrielsen M, Buetow L, Nakasone MA, Ahmed SF, Sibbet GJ, Smith BO, Zhang W, Sidhu SS, Huang DT

Abstract
RING and U-box E3 ubiquitin ligases regulate diverse eukaryotic processes and have been implicated in numerous diseases, but targeting these enzymes remains a major challenge. We report the development of three ubiquitin variants (UbVs), each binding selectively to the RING or U-box domain of a distinct E3 ligase: monomeric UBE4B, phosphorylated active CBL, or dimeric XIAP. Structural and biochemical analyses revealed that UbVs specifically inhibited the activity of UBE4B or phosphorylated CBL by blocking the E2∼Ub binding site. Surprisingly, the UbV selective for dimeric XIAP formed a dimer to stimulate E3 activity by stabilizing the closed E2∼Ub conformation. We further verified the inhibitory and stimulatory functions of UbVs in cells. Our work provides a general strategy to inhibit or activate RING/U-box E3 ligases and provides a resource for the research community to modulate these enzymes.

PMID: 29053960 [PubMed - indexed for MEDLINE]



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Reporter-Based Synthetic Genetic Array Analysis: A Functional Genomics Approach for Investigating Transcript or Protein Abundance Using Fluorescent Proteins in Saccharomyces cerevisiae.

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Reporter-Based Synthetic Genetic Array Analysis: A Functional Genomics Approach for Investigating Transcript or Protein Abundance Using Fluorescent Proteins in Saccharomyces cerevisiae.

Methods Mol Biol. 2018;1672:613-629

Authors: Göttert H, Mattiazzi Usaj M, Rosebrock AP, Andrews BJ

Abstract
Fluorescent reporter genes have long been used to quantify various cell features such as transcript and protein abundance. Here, we describe a method, reporter synthetic genetic array (R-SGA) analysis, which allows for the simultaneous quantification of any fluorescent protein readout in thousands of yeast strains using an automated pipeline. R-SGA combines a fluorescent reporter system with standard SGA analysis and can be used to examine any array-based strain collection available to the yeast community. This protocol describes the R-SGA methodology for screening different arrays of yeast mutants including the deletion collection, a collection of temperature-sensitive strains for the assessment of essential yeast genes and a collection of inducible overexpression strains. We also present an alternative pipeline for the analysis of R-SGA output strains using flow cytometry of cells in liquid culture. Data normalization for both pipelines is discussed.

PMID: 29043651 [PubMed - in process]



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