De novo design of bioactive protein switches.

Allosteric regulation of protein function is widespread in biology, but is challenging for de novo protein design as it requires the explicit design of multiple states with comparable free energies. Here we explore the possibility of designing switchable protein systems de novo, through the modulation of competing inter- and intramolecular interactions. We design a static, five-helix 'cage' with a single interface that can interact either intramolecularly with a terminal 'latch' helix or intermolecularly with a peptide 'key'. Encoded on the latch are functional motifs for binding, degradation or nuclear export that function only when the key displaces the latch from the cage. We describe orthogonal cage-key systems that function in vitro, in yeast and in mammalian cells with up to 40-fold activation of function by key. The ability to design switchable protein functions that are controlled by induced conformational change is a milestone for de novo protein design, and opens up new avenues for synthetic biology and cell engineering

“Horrible—But Worth It”: Exploring Weight Cutting Practices, Eating Behaviors, and Experiences of Competitive Female Taekwon-Do Athletes. A Mixed Methods Study

This mixed methods study aimed to investigate weight cutting practices of female taekwon-do athletes internationally and explore their experiences of “making weight.” A survey of weight loss practices and eating behaviors was completed by 103 taekwon-do athletes from 12 countries, which illustrated that 72.5% of athletes engage in both acute and chronic weight loss practices prior to competition and that there were higher levels of disordered eating within this athletic population than nonweight cutting athletes. Semistructured interviews were conducted with five international-level competitors; thematic analysis of the interviews identified that the women in general felt weight cutting was “horrible—but worth it” and the women believed that (a) weight cutting is unpleasant, difficult, and challenging; and (b) weight cutting provides a competitive advantage. The implications of this study are that weight cutting is widespread among high-level competitive female taekwon-do athletes and this is unlikely to change given the perceived advantages. Efforts are needed to make sure that the women are knowledgeable of the risks and are provided with safe and effective means of making weight

Generation of ordered protein assemblies using rigid three-body fusion

Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A longstanding design approach uses genetic fusion to join protein homo-oligomer subunits via α-helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method that performs rigid three-body fusion of homo-oligomer and spacer building blocks to generate user-defined architectures, while at the same time significantly increasing the number of geometric solutions over typical symmetric fusion. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies from a set of designed homo-dimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from DARPins (designed ankyrin repeat proteins), anchored on one end by α-helical fusion and on the other by a designed homo-dimer interface, and we explored their use for cryo-EM structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects, small scaffold size, and the low-order symmetry of these dihedral scaffolds, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins

Generation of ordered protein assemblies using rigid three-body fusion

Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A longstanding design approach uses genetic fusion to join protein homo-oligomer subunits via α-helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method that performs rigid three-body fusion of homo-oligomer and spacer building blocks to generate user-defined architectures, while at the same time significantly increasing the number of geometric solutions over typical symmetric fusion. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies from a set of designed homo-dimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from DARPins (designed ankyrin repeat proteins), anchored on one end by α-helical fusion and on the other by a designed homo-dimer interface, and we explored their use for cryo-EM structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects, small scaffold size, and the low-order symmetry of these dihedral scaffolds, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins

Developmental phosphoproteomics identifies the kinase CK2 as a driver of Hedgehog signaling and a therapeutic target in medulloblastoma

A major limitation of targeted cancer therapy is the rapid emergence of drug resistance, which often arises through mutations at or downstream of the drug target or through intrinsic resistance of subpopulations of tumor cells. Medulloblastoma (MB), the most common pediatric brain tumor, is no exception, and MBs that are driven by sonic hedgehog (SHH) signaling are particularly aggressive and drug-resistant. To find new drug targets and therapeutics for MB that may be less susceptible to common resistance mechanisms, we used a developmental phosphoproteomics approach in murine granule neuron precursors (GNPs), the developmental cell of origin of MB. The protein kinase CK2 emerged as a driver of hundreds of phosphorylation events during the proliferative, MB-like stage of GNP growth, including the phosphorylation of three of the eight proteins commonly amplified in MB. CK2 was critical to the stabilization and activity of the transcription factor GLI2, a late downstream effector in SHH signaling. CK2 inhibitors decreased the viability of primary SHH-type MB patient cells in culture and blocked the growth of murine MB tumors that were resistant to currently available Hh inhibitors, thereby extending the survival of tumor-bearing mice. Because of structural interactions, one CK2 inhibitor (CX-4945) inhibited both wild-type and mutant CK2, indicating that this drug may avoid at least one common mode of acquired resistance. These findings suggest that CK2 inhibitors may be effective for treating patients with MB and show how phosphoproteomics may be used to gain insight into developmental biology and pathology

Are pediatric Open Access journals promoting good publication practice? An analysis of author instructions

<p>Abstract</p> <p>Background</p> <p>Several studies analyzed whether conventional journals in general medicine or specialties such as pediatrics endorse recommendations aiming to improve publication practice. Despite evidence showing benefits of these recommendations, the proportion of endorsing journals has been moderate to low and varied considerably for different recommendations. About half of pediatric journals indexed in the Journal Citation Report referred to the Uniform Requirements for Manuscripts of the International Committee of Medical Journal Editors (ICMJE) but only about a quarter recommended registration of trials. We aimed to investigate to what extent pediatric open-access (OA) journals endorse these recommendations. We hypothesized that a high proportion of these journals have adopted recommendations on good publication practice since OA electronic publishing has been associated with a number of editorial innovations aiming at improved access and transparency.</p> <p>Methods</p> <p>We identified 41 journals publishing original research in the subject category "Health Sciences, Medicine (General), Pediatrics" of the Directory of Open Access Journals <url>http://www.doaj.org</url>. From the journals' online author instructions we extracted information regarding endorsement of four domains of editorial policy: the Uniform Requirements for Manuscripts, trial registration, disclosure of conflicts of interest and five major reporting guidelines such as the CONSORT (Consolidated Standards of Reporting Trials) statement. Two investigators collected data independently.</p> <p>Results</p> <p>The Uniform Requirements were mentioned by 27 (66%) pediatric OA journals. Thirteen (32%) required or recommended trial registration prior to publication of a trial report. Conflict of interest policies were stated by 25 journals (61%). Advice about reporting guidelines was less frequent: CONSORT was referred to by 12 journals (29%) followed by other reporting guidelines (MOOSE, PRISMA or STARD) (8 journals, 20%) and STROBE (3 journals, 7%). The EQUATOR network, a platform of several guideline initiatives, was acknowledged by 4 journals (10%).</p> <p>Journals published by OA publishing houses gave more guidance than journals published by professional societies or other publishers.</p> <p>Conclusions</p> <p>Pediatric OA journals mentioned certain recommendations such as the Uniform Requirements or trial registration more frequently than conventional journals; however, endorsement is still only moderate. Further research should confirm these exploratory findings in other medical fields and should clarify what the motivations and barriers are in implementing such policies.</p

The evolution of reproductive isolation in a simultaneous hermaphrodite, the freshwater snail Physa

<p>Abstract</p> <p>Background</p> <p>The cosmopolitan freshwater snail <it>Physa acuta </it>has recently found widespread use as a model organism for the study of mating systems and reproductive allocation. Mitochondrial DNA phylogenies suggest that <it>Physa carolinae</it>, recently described from the American southeast, is a sister species of <it>P. acuta</it>. The divergence of the <it>acuta/carolinae </it>ancestor from the more widespread <it>P. pomilia </it>appears to be somewhat older, and the split between a hypothetical <it>acuta/carolinae/pomilia </it>ancestor and <it>P. gyrina </it>appears older still.</p> <p>Results</p> <p>Here we report the results of no-choice mating experiments yielding no evidence of hybridization between <it>gyrina </it>and any of four other populations (<it>pomilia, carolinae</it>, Philadelphia <it>acuta</it>, or Charleston <it>acuta</it>), nor between <it>pomilia </it>and <it>carolinae</it>. Crosses between <it>pomilia </it>and both <it>acuta </it>populations yielded sterile F1 progeny with reduced viability, while crosses between <it>carolinae </it>and both <it>acuta </it>populations yielded sterile F1 hybrids of normal viability. A set of mate-choice tests also revealed significant sexual isolation between <it>gyrina </it>and all four of our other <it>Physa </it>populations, between <it>pomilia </it>and <it>carolinae</it>, and between <it>pomilia </it>and Charleston <it>acuta</it>, but not between <it>pomilia </it>and the <it>acuta </it>population from Philadelphia, nor between <it>carolinae </it>and either <it>acuta </it>population. These observations are consistent with the origin of hybrid sterility prior to hybrid inviability, and a hypothesis that speciation between <it>pomilia </it>and <it>acuta </it>may have been reinforced by selection for prezygotic reproductive isolation in sympatry.</p> <p>Conclusions</p> <p>We propose a two-factor model for the evolution of postzygotic reproductive incompatibility in this set of five <it>Physa </it>populations consistent with the Dobzhansky-Muller model of speciation, and a second two-factor model for the evolution of sexual incompatibility. Under these models, species trees may be said to correspond with gene trees in American populations of the freshwater snail, <it>Physa</it>.</p

A continental-scale hydro-economic model for integrating water-energy-land nexus solutions

This study presents the development of a new bottom‐up large‐scale hydro‐economic model, Extended Continental‐scale Hydro‐economic Optimization (ECHO), that works at a sub‐basin scale over a continent. The strength of ECHO stems from the integration of a detailed representation of local hydrological and technological constraints with regional and global policies, while accounting for the feedbacks between water, energy and agricultural sectors. In this study, ECHO has been applied over Africa as a case study with the aim of demonstrating the benefits of this integrated hydro‐economic modeling framework. Results of this framework are overall consistent with previous findings evaluating the cost of water supply and adaptation to global changes in Africa. Moreover, results provide critical assessments of future investment needs in both supply and demand side water management options, economic implications of contrasting future socio‐economic and climate change scenarios, and the potential tradeoffs among economic and environmental objectives. Overall, this study demonstrates the capacity of ECHO to address challenging research questions examining the sustainability of water supply, and the impacts of water management on energy and food sectors and vice versa. As such, we propose ECHO as useful tool for water‐related scenario analysis and management options evaluation

Combinatorial hydrogel library enables identification of materials that mitigate the foreign body response in primates

The foreign body response is an immune-mediated reaction that can lead to the failure of implanted medical devices and discomfort for the recipient. There is a critical need for biomaterials that overcome this key challenge in the development of medical devices. Here we use a combinatorial approach for covalent chemical modification to generate a large library of variants of one of the most widely used hydrogel biomaterials, alginate. We evaluated the materials in vivo and identified three triazole-containing analogs that substantially reduce foreign body reactions in both rodents and, for at least 6 months, in non-human primates. The distribution of the triazole modification creates a unique hydrogel surface that inhibits recognition by macrophages and fibrous deposition. In addition to the utility of the compounds reported here, our approach may enable the discovery of other materials that mitigate the foreign body response.Leona M. and Harry B. Helmsley Charitable Trust (3-SRA-2014-285-M-R)United States. National Institutes of Health (EB000244)United States. National Institutes of Health (EB000351)United States. National Institutes of Health (DE013023)United States. National Institutes of Health (CA151884)United States. National Institutes of Health (P41EB015871-27)National Cancer Institute (U.S.) (P30-CA14051

Doxycycline versus prednisolone as an initial treatment strategy for bullous pemphigoid: a pragmatic non-inferiority randomised controlled trial

Background: Bullous pemphigoid (BP) is a blistering skin disorder with increased mortality. We tested whether a strategy of starting treatment with doxycycline conveys acceptable short-term blister control whilst conferring long-term safety advantages over starting treatment with oral corticosteroids. Methods: Pragmatic multi-centre parallel-group randomised controlled trial of adults with BP (≥3 blisters ≥2 sites and linear basement membrane IgG/C3) plus economic evaluation. Participants were randomised to doxycycline (200 mg/day) or prednisolone (0·5 mg/kg/day). Localised adjuvant potent topical corticosteroids (<30 g/week) was permitted weeks 1-3. The non-inferiority primary effectiveness outcome was the proportion of participants with ≤3 blisters at 6 weeks. We assumed that doxycycline would be 25% less effective than corticosteroids with a 37% acceptable margin of noninferiority. The primary safety outcome was the proportion with severe, life-threatening or fatal treatment-related adverse events by 52 weeks. Analysis used a regression model adjusting for baseline disease severity, age and Karnofsky score, with missing data imputed. Results: 132 patients were randomised to doxycycline and 121 to prednisolone from 54 UK and 7 German dermatology centres. Mean age was 77·7 years and 68.4% had moderate to severe baseline disease. For those starting doxycycline, 83/112 (74·1%) had ≤3 blisters at 6 weeks compared with 92/101 (91·1%) for prednisolone, a difference of 18·6% favouring prednisolone (upper limit of 90% CI, 26·1%, within the predefined 37% margin). Related severe, life-threatening and fatal events at 52 weeks were 18·5% for those starting doxycycline and 36·6% for prednisolone (mITT analysis), an adjusted difference of 19·0% (95% CI, 7·9%, 30·1%, p=0·001). Conclusions: A strategy of starting BP patients on doxycycline is non-inferior to standard treatment with oral prednisolone for short-term blister control and significantly safer long-term