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Data driven flexible backbone protein design.

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Data driven flexible backbone protein design.

PLoS Comput Biol. 2017 Aug;13(8):e1005722

Authors: Sun MGF, Kim PM

Abstract
Protein design remains an important problem in computational structural biology. Current computational protein design methods largely use physics-based methods, which make use of information from a single protein structure. This is despite the fact that multiple structures of many protein folds are now readily available in the PDB. While ensemble protein design methods can use multiple protein structures, they treat each structure independently. Here, we introduce a flexible backbone strategy, FlexiBaL-GP, which learns global protein backbone movements directly from multiple protein structures. FlexiBaL-GP uses the machine learning method of Gaussian Process Latent Variable Models to learn a lower dimensional representation of the protein coordinates that best represent backbone movements. These learned backbone movements are used to explore alternative protein backbones, while engineering a protein within a parallel tempered MCMC framework. Using the human ubiquitin-USP21 complex as a model we demonstrate that our design strategy outperforms current strategies for the interface design task of identifying tight binding ubiquitin variants for USP21.

PMID: 28837553 [PubMed - indexed for MEDLINE]



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Human pluripotent stem cell-derived TSC2-haploinsufficient smooth muscle cells recapitulate features of Lymphangioleiomyomatosis.

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Human pluripotent stem cell-derived TSC2-haploinsufficient smooth muscle cells recapitulate features of Lymphangioleiomyomatosis.

Cancer Res. 2017 Aug 22;:

Authors: Julian LM, Delaney SP, Wang Y, Goldberg AA, Doré C, Yockell-Lelièvre J, Tam RY, Giannikou K, McMurray F, Shoichet MS, Harper ME, Henske EP, Kwiatkowski DJ, Darling TN, Moss J, Kristof AS, Stanford WL

Abstract
Lymphangioleiomyomatosis (LAM) is a progressive destructive neoplasm of the lung associated with inactivating mutations in the TSC1 or TSC2 tumor suppressor genes. Cell or animal models that accurately reflect the pathology of LAM have been challenging to develop. Here we generated a robust human cell model of LAM by reprogramming TSC2 mutation-bearing fibroblasts from a patient with both Tuberous Sclerosis Complex (TSC) and LAM (TSC-LAM) into induced pluripotent stem cells (iPSCs), followed by selection of cells that resemble those found in LAM tumors by unbiased in vivo differentiation. We established expandable cell lines under smooth muscle cell (SMC) growth conditions that retained a patient-specific genomic TSC2+/- mutation and recapitulated the molecular and functional characteristics of pulmonary LAM cells. These include multiple indicators of hyperactive mTORC1 signaling, presence of specific neural crest and SMC markers, expression of VEGF-D and female sex hormone receptors, reduced autophagy, and metabolic reprogramming. Intriguingly, the LAM-like features of these cells suggest that haploinsufficiency at the TSC2 locus contributed to LAM pathology, and demonstrated that iPSC reprogramming and SMC lineage differentiation of somatic patient cells with germline mutations was a viable approach to generate LAM-like cells. The patient-derived SMC lines we have developed thus represent a novel cellular model of LAM which can advance our understanding of disease pathogenesis and develop therapeutic strategies against LAM.

PMID: 28830860 [PubMed - as supplied by publisher]



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Exercise-responsive phosphoproteins in the heart.

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Exercise-responsive phosphoproteins in the heart.

J Mol Cell Cardiol. 2017 Aug 04;:

Authors: Guo H, Isserlin R, Emili A, Burniston JG

Abstract
Endurance exercise improves cardiac performance and affords protection against cardiovascular diseases but the signalling events that mediate these benefits are largely unexplored. Phosphorylation is an widely studied post-translational modification involved in intracellular signalling, and to discover novel phosphorylation events associated with exercise we have profiled the cardiac phosphoproteome response to a standardised exercise test to peak oxygen uptake (VO2peak). Male Wistar rats (346±18g) were assigned to 3 independent groups (n=6, in each) that were familiarised with running on a motorised treadmill within a metabolic chamber. Animals performed a graded exercise test and were killed either immediately (0h) after or 3h after terminating the test at a standardised physiological end point (i.e. peak oxygen uptake; VO2peak). Control rats were killed at a similar time of day to the exercised animals, to minimise possible circadian effects. Cardiac proteins were digested with trypsin and phosphopeptides were enriched by selective binding to titanium dioxide (TiO2). Phosphopeptides were analysed by liquid chromatography and high-resolution tandem mass spectrometry, and phosphopeptides were quantified by MS1 intensities and identified against the UniProt knowledgebase using MaxQuant (data are available via ProteomeXchange, ID PXD006646). The VO2peak of rats in the 0h and 3h groups was 66±5mlkg(-1)min(-1) and 69.8±5mlkg(-1)min(-1), respectively. Proteome profiling detected 1169 phosphopeptides and one-way ANOVA found 141 significant (P<0.05 with a false discovery rate of 10%) differences. Almost all (97%) of the phosphosites that were responsive to exercise are annotated in the PhosphoSitePlus database but, importantly, the majority of these have not previously been associated with the cardiac response to exercise. More than two-thirds of the exercise-responsive phosphosites were different from those identified in previous phosphoproteome profiling of the cardiac response to β1-adrenergic receptor stimulation. Moreover, we report entirely new phosphorylation sites on 4 cardiac proteins, including S81 of muscle LIM protein, and identified 7 exercise-responsive kinases, including myofibrillar protein kinases such as obscurin, titin and the striated-muscle-specific serine/threonine kinase (SPEG) that may be worthwhile targets for future investigation.

PMID: 28826663 [PubMed - as supplied by publisher]



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Mechanism of bidirectional thermotaxis in Escherichia coli.

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Mechanism of bidirectional thermotaxis in Escherichia coli.

Elife. 2017 Aug 03;6:

Authors: Paulick A, Jakovljevic V, Zhang S, Erickstad M, Groisman A, Meir Y, Ryu WS, Wingreen NS, Sourjik V

Abstract
In bacteria various tactic responses are mediated by the same cellular pathway, but sensing of physical stimuli remains poorly understood. Here, we combine an in-vivo analysis of the pathway activity with a microfluidic taxis assay and mathematical modeling to investigate the thermotactic response of Escherichia coli. We show that in the absence of chemical attractants E. coli exhibits a steady thermophilic response, the magnitude of which decreases at higher temperatures. Adaptation of wild-type cells to high levels of chemoattractants sensed by only one of the major chemoreceptors leads to inversion of the thermotactic response at intermediate temperatures and bidirectional cell accumulation in a thermal gradient. A mathematical model can explain this behavior based on the saturation-dependent kinetics of adaptive receptor methylation. Lastly, we find that the preferred accumulation temperature corresponds to optimal growth in the presence of the chemoattractant serine, pointing to a physiological relevance of the observed thermotactic behavior.

PMID: 28826491 [PubMed - as supplied by publisher]



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Assessing predictions of fitness effects of missense mutations in SUMO-conjugating enzyme UBE2I.

Assessing predictions of fitness effects of missense mutations in SUMO-conjugating enzyme UBE2I.

Hum Mutat. 2017 Sep;38(9):1051-1063

Authors: Zhang J, Kinch LN, Cong Q, Weile J, Sun S, Cote AG, Roth FP, Grishin NV

Abstract
The exponential growth of genomic variants uncovered by next-generation sequencing necessitates efficient and accurate computational analyses to predict their functional effects. A number of computational methods have been developed for the task, but few unbiased comparisons of their performance are available. To fill the gap, The Critical Assessment of Genome Interpretation (CAGI) comprehensively assesses phenotypic predictions on newly collected experimental datasets. Here, we present the results of the SUMO conjugase challenge where participants were predicting functional effects of missense mutations in human SUMO-conjugating enzyme UBE2I. The performance of the predictors is similar to each other and is far from perfection. Evolutionary information from sequence alignments dominates the success: deleterious mutations at conserved positions and benign mutations at variable positions are accurately predicted. Prediction accuracy of other mutations remains unsatisfactory, and this fast-growing field of research is yet to learn the use of spatial structure information to improve the predictions significantly.

PMID: 28817247 [PubMed - in process]



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Reply to the 'Comment on "Towards a personalized approach to aromatase inhibitor therapy: a digital microfluidic platform for rapid analysis of estradiol in core-needle-biopsies"' by P. E. Lønning, Lab Chip, 2017, 17, DOI: 10.1039/C7LC00617A.

Reply to the 'Comment on "Towards a personalized approach to aromatase inhibitor therapy: a digital microfluidic platform for rapid analysis of estradiol in core-needle-biopsies"' by P. E. Lønning, Lab Chip, 2017, 17, DOI: 10.1039/C7LC00617A.

Lab Chip. 2017 Aug 17;:

Authors: Casper RF, Wheeler AR

Abstract
This article provides our response to a comment on our article that appeared in Lab on a Chip (S. Abdulwahab, A. H. C. Ng, M. D. Chamberlain, H. Ahmado, L.-A. Behan, H. Gomaa, R. F. Casper and A. R. Wheeler, Lab Chip, 2017, 17, 1594).

PMID: 28816309 [PubMed - as supplied by publisher]



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Modular tissue engineering for the vascularization of subcutaneously transplanted pancreatic islets.

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Modular tissue engineering for the vascularization of subcutaneously transplanted pancreatic islets.

Proc Natl Acad Sci U S A. 2017 Aug 16;:

Authors: Vlahos AE, Cober N, Sefton MV

Abstract
The transplantation of pancreatic islets, following the Edmonton Protocol, is a promising treatment for type I diabetics. However, the need for multiple donors to achieve insulin independence reflects the large loss of islets that occurs when islets are infused into the portal vein. Finding a less hostile transplantation site that is both minimally invasive and able to support a large transplant volume is necessary to advance this approach. Although the s.c. site satisfies both these criteria, the site is poorly vascularized, precluding its utility. To address this problem, we demonstrate that modular tissue engineering results in an s.c. vascularized bed that enables the transplantation of pancreatic islets. In streptozotocin-induced diabetic SCID/beige mice, the injection of 750 rat islet equivalents embedded in endothelialized collagen modules was sufficient to restore and maintain normoglycemia for 21 days; the same number of free islets was unable to affect glucose levels. Furthermore, using CLARITY, we showed that embedded islets became revascularized and integrated with the host's vasculature, a feature not seen in other s.c.
STUDIES: Collagen-embedded islets drove a small (albeit not significant) shift toward a proangiogenic CD206(+)MHCII(-)(M2-like) macrophage response, which was a feature of module-associated vascularization. While these results open the potential for using s.c. islet delivery as a treatment option for type I diabetes, the more immediate benefit may be for the exploration of revascularized islet biology.

PMID: 28814629 [PubMed - as supplied by publisher]



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Inhibiting MYC binding to the E-box DNA motif by ME47 decreases tumour xenograft growth.

Inhibiting MYC binding to the E-box DNA motif by ME47 decreases tumour xenograft growth.

Oncogene. 2017 Aug 14;:

Authors: Lustig LC, Dingar D, Tu WB, Lourenco C, Kalkat M, Inamoto I, Ponzielli R, Chan WCW, Shin JA, Penn LZ

Abstract
Developing therapeutics to effectively inhibit the MYC oncoprotein would mark a key advance towards cancer patient care as MYC is deregulated in over 50% of human cancers. MYC deregulation is correlated with aggressive disease and poor patient outcome. Despite strong evidence in mouse models that inhibiting MYC would significantly impact tumour cell growth and patient survival, traditional approaches have not yet yielded the urgently needed therapeutic agents that directly target MYC. MYC functions through its interaction with MAX to regulate gene transcription by binding to E-box DNA response elements of MYC target genes. Here we used a structure-based strategy to design ME47, a small minimalist hybrid protein (MHP) able to disrupt the MAX:E-box interaction/binding and block transcriptional MYC activity. We show that inducing ME47 expression in established tumour xenografts inhibits tumour growth and decreases cellular proliferation. Mechanistically, we show by chromatin immunoprecipitation that ME47 binds to E-box binding sites of MYC target genes. Moreover, ME47 occupancy decreases MYC:DNA interaction at its cognate E-box binding sites. Taken together, ME47 is a prototypic MHP inhibitor that antagonizes tumour cell growth in vitro and in vivo and inhibits the interaction of MYC with DNA E-box elements. These results support ME47's role as a MYC inhibitor and suggest that MHPs provide an alternative therapeutic targeting system that can be used to target transcription factors important in human diseases, including cancer.Oncogene advance online publication, 14 August 2017; doi:10.1038/onc.2017.275.

PMID: 28806396 [PubMed - as supplied by publisher]



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Adult High Grade B-cell Lymphoma with Burkitt Lymphoma Signature: Genomic features and Potential Therapeutic Targets.

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Adult High Grade B-cell Lymphoma with Burkitt Lymphoma Signature: Genomic features and Potential Therapeutic Targets.

Blood. 2017 Aug 11;:

Authors: Bouska A, Bi C, Lone W, Zhang W, Kedwaii A, Heavican T, Lachel CM, Yu J, Ferro R, Eldorghamy N, Greiner TC, Vose J, Weisenburger DD, Gascoyne RD, Rosenwald A, Ott G, Campo E, Rimsza LM, Jaffe ES, Braziel RM, Siebert R, Miles RR, Dave S, Reddy A, Delabie J, Staudt LM, Song JY, McKeithan TW, Fu K, Green M, Chan WC, Iqbal J

Abstract
The adult high-grade B-cell lymphomas sharing molecular features with Burkitt lymphoma (BL) are highly aggressive lymphomas with poor clinical outcome. High-resolution structural and functional genomic analysis of adult Burkitt lymphoma (BL) and high-grade B-cell lymphoma with BL gene-signature (adult-mBL), revealed the MYC-ARF-p53 axis as the primary deregulated pathway. Adult-mBL had either unique or more frequent genomic aberrations (del13q14, del17p, gain8q24 and gain18q21) compared with pediatric-mBL, but shared commonly mutated genes. Mutations in genes promoting the tonic B-cell receptor (BCR)→PI3K pathway (TCF3 and ID3) did not differ by age, whereas effectors of chronic BCR→NF-κB signaling were associated with adult-mBL. A subset of adult-mBL had BCL2 translocation and mutation, and elevated BCL2 mRNA and protein expression, but had a mutation profile similar to mBL. These double-hit lymphomas may have arisen from a tumor precursor that acquired both BCL2 and MYC translocations and/or KMT2D (MLL2) mutation. Gain/amplification of MIR17HG and its paralogue loci was observed in 50% of adult-mBL. In vitro studies suggested miR-17~92's role in constitutive activation of BCR signaling and sensitivity to ibrutinib. Overall integrative analysis identified an interrelated gene network affected by CN and mutation, leading to disruption of the p53 pathway and the BCR→PI3K/ or NF-κB activation which can be further exploited in vivo by small-molecule inhibitors for effective therapy in adult-mBL.

PMID: 28801451 [PubMed - as supplied by publisher]



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Home sweet home: the neural stem cell niche throughout development and after injury.

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Home sweet home: the neural stem cell niche throughout development and after injury.

Cell Tissue Res. 2017 Aug 03;:

Authors: Ruddy RM, Morshead CM

Abstract
Neural stem cells and their progeny reside in two distinct neurogenic niches within the mammalian brain: the subventricular zone and the dentate gyrus. The interplay between the neural stem cells and the niche in which they reside can have significant effects on cell kinetics and neurogenesis. A comprehensive understanding of the changes to the niche that occur through postnatal development and aging, as well as following injury, is relevant for developing therapeutics and interventions to promote neural repair. We discuss changes that occur within the neural stem and progenitor cell populations, the vasculature, extracellular matrix, microglia, and secreted proteins through aging which impact cell behavior within the neurogenic niches. We examine neural precursor cell and niche responses to injury in neonatal hypoxia-ischemia, juvenile cranial irradiation, and adult stroke. This review examines the interplay between the niche and stem cell behavior through aging and following injury as a means to understand intrinsic and extrinsic factors that regulate neurogenesis in vivo.

PMID: 28776186 [PubMed - as supplied by publisher]



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