scholarly journals Barcoded Rational AAV Vector Evolution enables systematicin vivomapping of peptide binding motifs

2018 ◽  
Author(s):  
Marcus Davidsson ◽  
Gang Wang ◽  
Patrick Aldrin-Kirk ◽  
Tiago Cardoso ◽  
Sara Nolbrant ◽  
...  
Keyword(s):  
Selection Process ◽  
Retrograde Transport ◽  
Aav Vector ◽  
Binding Motifs ◽  
Model Based Clustering ◽  
Cell Type Specific ◽  
Novel Method ◽  
Protein Libraries ◽  
And Function

Engineering of Adeno-associated viral (AAV) vector capsids through directed evolution has been used to generate novel capsids with altered tropism and function1-9. This approach, however, involves a selection process that requires multiple generations of screenings to identify real functional capsids2-4. Due to the random nature of this process, it is also inherently unreproducible, and the resulting capsid variants provide little mechanistic insights into the molecular targets engaged. To overcome this, we have developed a novel method for rational capsid evolution named Barcoded Rational AAV Vector Evolution (BRAVE). The key to this method is a novel viral production approach where each virus particle displays a protein-derived peptide on the surface which is linked to a unique barcode in the packaged genome10. Through hidden Markov model-based clustering11, we were able to identify novel consensus motifs for cell-type specific retrograde transport in neurons in vivo in the brain. The BRAVE approach enables the selection of novel capsid structures using only a single-generation screening. Furthermore, it can be used to map, with high resolution, the putative binding sequences of large protein libraries.

Barcoded Rational AAV Vector Evolution Enables Systematic In Vivo Mapping of Peptide Binding Motifs

2018 ◽  
Author(s):  
Marcus Davidsson ◽  
Gang Wang ◽  
Patrick Aldrin-Kirk ◽  
Tiago Cardoso ◽  
Sara Nolbrant ◽  
...  
Keyword(s):  
Peptide Binding ◽  
Aav Vector ◽  
Binding Motifs

scholarly journals Profound functional and molecular diversity of mitochondria revealed by cell type-specific profiling in vivo

2018 ◽  
Author(s):  
Caroline Fecher ◽  
Laura Trovò ◽  
Stephan A. Müller ◽  
Nicolas Snaidero ◽  
Jennifer Wettmarshausen ◽  
...  
Keyword(s):  
Molecular Diversity ◽  
Molecular Level ◽  
Cell Types ◽  
Long Chain Fatty Acids ◽  
Cell Type ◽  
Mitochondrial Proteome ◽  
Novel Approach ◽  
Cell Type Specific ◽  
And Function

AbstractMitochondria vary in morphology and function in different tissues, however little is known about their molecular diversity among cell types. To investigate mitochondrial diversity in vivo, we developed an efficient protocol to isolate cell type-specific mitochondria based on a new MitoTag mouse. We profiled the mitochondrial proteome of three major neural cell types in cerebellum and identified a substantial number of differential mitochondrial markers for these cell types in mice and humans. Based on predictions from these proteomes, we demonstrate that astrocytic mitochondria metabolize long-chain fatty acids more efficiently than neurons. Moreover, we identified Rmdn3 as a major determinant of ER-mitochondria proximity in Purkinje cells. Our novel approach enables exploring mitochondrial diversity on the functional and molecular level in many in vivo contexts.

scholarly journals Functional partitioning of a liquid-like organelle during assembly of axonemal dyneins

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Chanjae Lee ◽  
Rachael M Cox ◽  
Ophelia Papoulas ◽  
Amjad Horani ◽  
Kevin Drew ◽  
...  
Keyword(s):  
Affinity Purification ◽  
Human Genetic Disease ◽  
Interaction Partners ◽  
A Cell ◽  
Cell Type Specific ◽  
Axonemal Dynein ◽  
And Function ◽  
Affinity Purification Mass Spectrometry ◽  
Functional Partitioning

Ciliary motility is driven by axonemal dyneins that are assembled in the cytoplasm before deployment to cilia. Motile ciliopathy can result from defects in the dyneins themselves or from defects in factors required for their cytoplasmic pre-assembly. Recent work demonstrates that axonemal dyneins, their specific assembly factors, and broadly-acting chaperones are concentrated in liquid-like organelles in the cytoplasm called DynAPs (Dynein Axonemal Particles). Here, we use in vivo imaging in Xenopus to show that inner dynein arm (IDA) and outer dynein arm (ODA) subunits are partitioned into non-overlapping sub-regions within DynAPs. Using affinity- purification mass-spectrometry of in vivo interaction partners, we also identify novel partners for inner and outer dynein arms. Among these, we identify C16orf71/Daap1 as a novel axonemal dynein regulator. Daap1 interacts with ODA subunits, localizes specifically to the cytoplasm, is enriched in DynAPs, and is required for the deployment of ODAs to axonemes. Our work reveals a new complexity in the structure and function of a cell-type specific liquid-like organelle that is directly relevant to human genetic disease.

scholarly journals Large-Scale Computational Discovery of Binding Motifs in tRNA Fragments

2021 ◽  
Vol 8 ◽  
Author(s):  
Lingyu Guan ◽  
Vincent Lam ◽  
Andrey Grigoriev
Keyword(s):  
Large Scale ◽  
Potential Interaction ◽  
Binding Motifs ◽  
Argonaute Proteins ◽  
Potential Binding ◽  
Computational Discovery ◽  
And Function ◽  
Trna Halves ◽  
Binding Mechanisms

Accumulating evidence has suggested that tRNA-derived fragments (tRFs) could be loaded to Argonaute proteins and function as regulatory small RNAs. However, their mode of action remains largely unknown, and investigations of their binding mechanisms have been limited, revealing little more than microRNA-like seed regions in a handful of tRFs and a few targets. Here, we identified such regions of potential interaction on a larger scale, using in vivo formed hybrids of guides and targets in crosslinked chimeric reads in two orientations. We considered “forward pairs” (with guides located on the 5′ ends and targets on the 3′ ends of hybrids) and “reverse pairs” (opposite orientation) and compared them as independent sets of biological constructs. We observed intriguing differences between the two chimera orientations, including the paucity of tRNA halves and abundance of polyT-containing targets in forward pairs. We found a total of 197 quality-ranked motifs supported by ∼120,000 tRF–mRNA chimeras, with 103 interacting motifs common in forward and reverse pairs. By analyzing T→C conversions in human and mouse PAR-CLIP datasets, we detected Argonaute crosslinking sites in tRFs, conserved across species. We proposed a novel model connecting the formation of asymmetric pairs in two sets to the potential binding mechanisms of tRFs, involving the identified interaction motifs and crosslinking sites to Argonaute proteins. Our results suggest the way forward for further experimental elucidation of tRF-binding mechanisms.

scholarly journals Learning from mistakes: Accurate prediction of cell type-specific transcription factor binding

2017 ◽  
Author(s):  
Jens Keilwagen ◽  
Stefan Posch ◽  
Jan Grau
Keyword(s):  
Transcription Factor ◽  
Cell Types ◽  
Transcription Factor Binding ◽  
Ensemble Prediction ◽  
Training Procedure ◽  
Cell Type ◽  
Binding Motifs ◽  
Factor Binding ◽  
Cell Type Specific

Computational prediction of cell type-specific, in-vivo transcription factor binding sites is still one of the central challenges in regulatory genomics, and a variety of approaches has been proposed for this purpose.Here, we present our approach that earned a shared first rank in the “ENCODE-DREAM in vivo Transcription Factor Binding Site Prediction Challenge” in 2017. This approach employs features derived from chromatin accessibility, binding motifs, gene expression, genomic sequence and annotation to train classifiers using a supervised, discriminative learning principle. Two further key aspects of this approach are learning classifier parameters in an iterative training procedure that successively adds additional negative examples to the training set, and creating an ensemble prediction by averaging over classifiers obtained for different training cell types.In post-challenge analyses, we benchmark the influence of different feature sets and find that chromatin accessiblity and binding motifs are sufficient to yield state-of-the-art performance for in-vivo binding site predictions. We also show that the iterative training procedure and the ensemble prediction are pivotal for the final prediction performance.To make predictions of this approach readily accessible, we predict 682 peak lists for a total of 31 transcription factors in 22 primary cell types and tissues, which are available for download at https://www.synapse.org/#!Synapse:syn11526239, and we demonstrate that these may help to yield biological conclusions. Finally, we provide a user-friendly version of our approach as open source software at http://jstacs.de/index.php/[email protected]

scholarly journals Functional partitioning of a liquid-like organelle during assembly of axonemal dyneins

2020 ◽  
Author(s):  
Chanjae Lee ◽  
Rachael M. Cox ◽  
Ophelia Papoulas ◽  
Amjad Horani ◽  
Kevin Drew ◽  
...  
Keyword(s):  
Affinity Purification ◽  
Human Genetic Disease ◽  
Interaction Partners ◽  
A Cell ◽  
Cell Type Specific ◽  
Axonemal Dynein ◽  
And Function ◽  
Affinity Purification Mass Spectrometry ◽  
Functional Partitioning

AbstractCiliary motility is driven by axonemal dyneins that are assembled in the cytoplasm before deployment to cilia. Motile ciliopathy can result from defects in the dyneins themselves or from defects in factors required for their cytoplasmic pre-assembly. Recent work demonstrates that axonemal dyneins, their specific assembly factors, and broadly acting chaperones are concentrated in liquid-like organelles in the cytoplasm called DynAPs (Dynein Axonemal Particles). Here, we use in vivo imaging to show that inner dynein arm (IDA) and outer dynein arm (ODA) subunits are partitioned into non-overlapping sub-regions within DynAPs. Using affinity purification mass-spectrometry of in vivo interaction partners, we also identify novel partners for inner and outer dynein arms. Among these, we identify C16orf71/Daap1 as a novel axonemal dynein regulator. Daap1 interacts with ODA subunits, localizes specifically to the cytoplasm, is enriched in DynAPs, and is required for the deployment of ODAs to axonemes. Our work reveals a new complexity in the structure and function of a cell-type specific liquid-like organelle that is directly relevant to human genetic disease.

scholarly journals A trans-eQTL network regulates osteoclast multinucleation and bone mass

2020 ◽  
Author(s):  
Marie Pereira ◽  
Jeong-Hun Ko ◽  
John Logan ◽  
Hayley Protheroe ◽  
Kee-Beom Kim ◽  
...  
Keyword(s):  
Bone Mass ◽  
Gene Network ◽  
Regulatory Networks ◽  
Causal Link ◽  
Hub Gene ◽  
Functional Characterisation ◽  
Skeletal Homeostasis ◽  
Cell Type Specific ◽  
And Function

AbstractFunctional characterisation of cell-type specific regulatory networks is key to establish a causal link between genetic variation and phenotype. The osteoclast offers a unique model for interrogating the contribution of co-regulated genes to in vivo phenotype as its multinucleation and resorption activities determine quantifiable skeletal traits. Here we took advantage of a trans-regulated gene network (MMnet, macrophage multinucleation network) which we found to be significantly enriched for GWAS variants associated with bone-related phenotypes. We found that the network hub gene Bcat1 and seven other co-regulated MMnet genes out of 13, regulate bone function. Specifically, global (Pik3cb−/−, Atp8b2+/−, Igsf8−/−, Eml1−/−, Appl2−/−, Deptor−/−) and myeloid-specific Slc40a1ΔLysMCre knockout mice displayed abnormal bone phenotypes. We report antagonizing effects of MMnet genes on bone mass in mice and osteoclast multinucleation/resorption in humans with strong correlation between the two. These results identify MMnet as a functionally conserved network that regulates osteoclast fusion and bone mass.Impact statementWe took advantage of the osteoclast whose multinucleation properties correlate with bone mass. We show that a trans-regulated gene network (MMnet) controls skeletal homeostasis through osteoclast multinucleation and function.

scholarly journals The golgin protein Coy1 functions in intra-Golgi retrograde transport and interacts with the COG complex and Golgi SNAREs

2017 ◽  
Vol 28 (20) ◽  
pp. 2686-2700 ◽  
Author(s):  
Nadine S. Anderson ◽  
Indrani Mukherjee ◽  
Christine M. Bentivoglio ◽  
Charles Barlowe
Keyword(s):  
Protein Interactions ◽  
Coiled Coil ◽  
Retrograde Transport ◽  
Snare Proteins ◽  
Growth Defects ◽  
Cog Complex ◽  
Physical Interactions ◽  
And Function

Extended coiled-coil proteins of the golgin family play prominent roles in maintaining the structure and function of the Golgi complex. Here we further investigate the golgin protein Coy1 and document its function in retrograde transport between early Golgi compartments. Cells that lack Coy1 displayed a reduced half-life of the Och1 mannosyltransferase, an established cargo of intra-Golgi retrograde transport. Combining the coy1Δ mutation with deletions in other putative retrograde golgins (sgm1Δ and rud3Δ) caused strong glycosylation and growth defects and reduced membrane association of the conserved oligomeric Golgi (COG) complex. In contrast, overexpression of COY1 inhibited the growth of mutant strains deficient in fusion activity at the Golgi (sed5-1 and sly1-ts). To map Coy1 protein interactions, coimmunoprecipitation experiments revealed an association with the COG complex and with intra-Golgi SNARE proteins. These physical interactions are direct, as Coy1 was efficiently captured in vitro by Lobe A of the COG complex and the purified SNARE proteins Gos1, Sed5, and Sft1. Thus our genetic, in vivo, and biochemical data indicate a role for Coy1 in regulating COG complex-dependent fusion of retrograde-directed COPI vesicles.

Osseointegration interface of calcified bone and endosseous implants

1992 ◽  
Vol 50 (2) ◽  
pp. 1096-1097
Author(s):  
K.E. Krizan ◽  
J.E. Laffoon ◽  
M.J. Buckley
Keyword(s):  
Structure And Function ◽  
Titanium Implants ◽  
En Bloc ◽  
Endosseous Implants ◽  
Ultrastructural Studies ◽  
The Past ◽  
Cacodylate Buffer ◽  
One Year ◽  
And Function

With increase use of tissue-integrated prostheses in recent years it is a goal to understand what is happening at the interface between haversion bone and bulk metal. This study uses electron microscopy (EM) techniques to establish parameters for osseointegration (structure and function between bone and nonload-carrying implants) in an animal model. In the past the interface has been evaluated extensively with light microscopy methods. Today researchers are using the EM for ultrastructural studies of the bone tissue and implant responses to an in vivo environment. Under general anesthesia nine adult mongrel dogs received three Brånemark (Nobelpharma) 3.75 × 7 mm titanium implants surgical placed in their left zygomatic arch. After a one year healing period the animals were injected with a routine bone marker (oxytetracycline), euthanized and perfused via aortic cannulation with 3% glutaraldehyde in 0.1M cacodylate buffer pH 7.2. Implants were retrieved en bloc, harvest radiographs made (Fig. 1), and routinely embedded in plastic. Tissue and implants were cut into 300 micron thick wafers, longitudinally to the implant with an Isomet saw and diamond wafering blade [Beuhler] until the center of the implant was reached.

Functional characterization of human brown adipose tissue metabolism

2020 ◽  
Vol 477 (7) ◽  
pp. 1261-1286 ◽  
Author(s):  
Marie Anne Richard ◽  
Hannah Pallubinsky ◽  
Denis P. Blondin
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Functional Characterization ◽  
Human Infants ◽  
Positron Emission ◽  
Brown Adipose ◽  
Treatment Of Obesity ◽  
And Function

Brown adipose tissue (BAT) has long been described according to its histological features as a multilocular, lipid-containing tissue, light brown in color, that is also responsive to the cold and found especially in hibernating mammals and human infants. Its presence in both hibernators and human infants, combined with its function as a heat-generating organ, raised many questions about its role in humans. Early characterizations of the tissue in humans focused on its progressive atrophy with age and its apparent importance for cold-exposed workers. However, the use of positron emission tomography (PET) with the glucose tracer [18F]fluorodeoxyglucose ([18F]FDG) made it possible to begin characterizing the possible function of BAT in adult humans, and whether it could play a role in the prevention or treatment of obesity and type 2 diabetes (T2D). This review focuses on the in vivo functional characterization of human BAT, the methodological approaches applied to examine these features and addresses critical gaps that remain in moving the field forward. Specifically, we describe the anatomical and biomolecular features of human BAT, the modalities and applications of non-invasive tools such as PET and magnetic resonance imaging coupled with spectroscopy (MRI/MRS) to study BAT morphology and function in vivo, and finally describe the functional characteristics of human BAT that have only been possible through the development and application of such tools.

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