Publications

YAP and TAZ Mediate Osteocyte Perilacunar/Canalicular Remodeling

Anonymous — Mon, 14 Oct 2019 00:00:00 +0000

YAP and TAZ Mediate Osteocyte Perilacunar/Canalicular Remodeling Anonymous (not verified) Sun, 10/13/2019 - 18:00

Christopher Kegelman , Jennifer Coulombe , Kelsey Jordan , Daniel Horan , Ling Qin , Alexander Robling , Virginia Ferguson , Teresita Bellido , Joel Boerckel

Bone fragility fractures are caused by low bone mass or impaired bone quality. Osteoblast/osteoclast coordination determines bone mass, but the factors that control bone quality are poorly understood. Osteocytes regulate osteoblast and osteoclast activity on bone surfaces but can also directly reorganize the bone matrix to improve bone quality through perilacunar/canalicular remodeling; however, the molecular mechanisms remain unclear. We previously found that deleting the transcriptional regulators Yes‐associated protein (YAP) and transcriptional co‐activator with PDZ‐motif (TAZ) from osteoblast‐lineage cells caused lethality in mice due to skeletal fragility. Here, we tested the hypothesis that YAP and TAZ regulate osteocyte‐mediated bone remodeling by conditional ablation of both YAP and TAZ from mouse osteocytes using 8 kb‐DMP1‐Cre. Osteocyte‐conditional YAP/TAZ deletion reduced bone mass and dysregulated matrix collagen content and organization, which together decreased bone mechanical properties. Further, YAP/TAZ deletion impaired osteocyte perilacunar/canalicular remodeling by reducing canalicular network density, length, and branching, as well as perilacunar flourochrome‐labeled mineral deposition. Consistent with recent studies identifying TGF‐β as a key inducer of osteocyte expression of matrix‐remodeling enzymes, YAP/TAZ deletion in vivo decreased osteocyte expression of matrix proteases MMP13, MMP14, and CTSK. In vitro, pharmacologic inhibition of YAP/TAZ transcriptional activity in osteocyte‐like cells abrogated TGF‐β‐induced matrix protease gene expression. Together, these data show that YAP and TAZ control bone matrix accrual, organization, and mechanical properties by regulating osteocyte‐mediated bone remodeling. Elucidating the signaling pathways that control perilacunar/canalicular remodeling may enable future therapeutic targeting of bone quality to reverse skeletal fragility. © 2019 American Society for Bone and Mineral Research.

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Linking long noncoding RNA to drug resistance

Anonymous — Thu, 10 Oct 2019 00:00:00 +0000

Linking long noncoding RNA to drug resistance Anonymous (not verified) Wed, 10/09/2019 - 18:00

Michael Smallegan , John Rinn

As we move into the era of personalized medicine, it may not be too far off that a visit to our doctor includes a number of genome-scale “seq” experiments to diagnose disease and estimate our likelihood of response to an array of possible treatments. One such experiment measures the cell’s total RNA abundance (its transcriptome) which provides a snapshot of a cellular activity or state. For decades global gene expression levels and changes in expression have been used to identify disease and drug response markers. This has been enabled by technological advances such as “whole-exome” sequencing which can home in on biomarker genes that define cellular states. These approaches were first designed to survey protein-coding gene (PCG) levels of expression and have uncovered numerous PCG biomarkers, led to advances in how drugs are screened, and generated insights into how gene pathways are misregulated in disease.

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Leveraging whole genome sequencing data for demographic inference with approximate Bayesian computation

Anonymous — Thu, 12 Sep 2019 00:00:00 +0000

Leveraging whole genome sequencing data for demographic inference with approximate Bayesian computation Anonymous (not verified) Wed, 09/11/2019 - 18:00

Chris Smith , Sam Flaxman

Accounting for historical demographic features, such as the strength and timing of gene flow and divergence times between closely related lineages, is vital for many inferences in evolutionary biology. Approximate Bayesian Computation (ABC) is one method commonly used to estimate demographic parameters. However, the DNA sequences used as input for this method, often microsatellites or RADseq loci, usually represent a small fraction of the genome. Whole genome sequencing (WGS) data, on the other hand, have been used less often with ABC, and questions remain about the potential benefit of, and how to best implement, this type of data; we used pseudo observed datasets to explore such questions. Specifically, we addressed the potential improvements in parameter estimation accuracy that could be associated with WGS data in multiple contexts; namely, we quantified the effects of (1) more data, (2) haplotype‐based summary statistics, and (3) locus length. Compared with a hypothetical RADseq dataset with 2.5Mbp of data, using a 1Gbp dataset consisting of 100Kbp sequences led to substantial gains in the accuracy of parameter estimates, which was mostly due to haplotype statistics and increased data. We also quantified the effects of including (i) locus‐specific recombination rates, and (ii) background selection information in ABC analyses. Importantly, assuming uniform recombination or ignoring background selection had a negative effect on accuracy in many cases. Software and results from this method validation study should be useful for future demographic history analyses.

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Transcatheter aortic valve replacements alter circulating serum factors to mediate myofibroblast deactivation

Anonymous — Wed, 11 Sep 2019 00:00:00 +0000

Transcatheter aortic valve replacements alter circulating serum factors to mediate myofibroblast deactivation Anonymous (not verified) Tue, 09/10/2019 - 18:00

Brian Aguado , Katherine Schuetze , Joseph Grim , Cierra Walker , Anne Cox , Tova Ceccato , Aik-Choon Tan , Carmen Sucharov , Leslie Leinwand , Matthew Taylor , Timothy , Kristi Anseth

The transcatheter aortic valve replacement (TAVR) procedure has emerged as a minimally invasive treatment for patients with aortic valve stenosis (AVS). However, alterations in serum factor composition and biological activity after TAVR remain unknown. Here, we quantified the systemic inflammatory effects of the TAVR procedure and hypothesized that alterations in serum factor composition would modulate valve and cardiac fibrosis. Serum samples were obtained from patients with AVS immediately before their TAVR procedure (pre-TAVR) and about 1 month afterward (post-TAVR). Aptamer-based proteomic profiling revealed alterations in post-TAVR serum composition, and ontological analysis identified inflammatory macrophage factors implicated in myofibroblast activation and deactivation. Hydrogel biomaterials used as valve matrix mimics demonstrated that post-TAVR serum reduced myofibroblast activation of valvular interstitial cells relative to pre-TAVR serum from the same patient. Transcriptomics and curated network analysis revealed a shift in myofibroblast phenotype from pre-TAVR to post-TAVR and identified p38 MAPK signaling as one pathway involved in pre-TAVR–mediated myofibroblast activation. Post-TAVR serum deactivated valve and cardiac myofibroblasts initially exposed to pre-TAVR serum to a quiescent fibroblast phenotype. Our in vitro deactivation data correlated with patient disease severity measured via echocardiography and multimorbidity scores, and correlations were dependent on hydrogel stiffness. Sex differences in cellular responses to male and female sera were also observed and may corroborate clinical observations regarding sex-specific TAVR outcomes. Together, alterations in serum composition after TAVR may lead to an antifibrotic fibroblast phenotype, which suggests earlier interventions may be beneficial for patients with advanced AVS to prevent further disease progression.

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Mimicking the heart's microenvironment

Anonymous — Wed, 11 Sep 2019 00:00:00 +0000

Mimicking the heart's microenvironment Anonymous (not verified) Tue, 09/10/2019 - 18:00

Trent Knoss

CU ��«��Ƶ engineers and faculty from the Consortium for Fibrosis Research & Translation (CFReT) at the CU Anschutz Medical Campus have teamed up to develop biomaterial-based “mimics” of heart tissues to measure patients’ responses to an aortic valve replacement procedure, offering new insight into the ways that cardiac tissue re-shapes itself post-surgery.

Aortic valve stenosis (AVS), a progressive disease characterized by heart valve tissue stiffening and obstructed blood flow from the heart, is known as a “silent killer,” affecting 12.4 percent of the population over 75 years old with a mortality range of 2-5 years if left untreated. Transcatheter aortic valve replacement (TAVR) procedures, which place an artificial valve at the site of the blockage, have been widely and successfully adopted as a remedy in recent decades.

Details of the broader biological reaction to the valve replacement have remained largely unknown, but nevertheless hold significant ramifications for quantifying the quality of recovery, the risk of complications and the assessment of overall patient outcomes.

During AVS disease progression, tissue-specific cells known as fibroblasts transition into myofibroblasts, which promote tissue stiffening. The researchers were interested in understanding how and why, post TAVR, myofibroblasts revert to the more benign fibroblasts.

“Previous studies have shown significant remodeling of cardiac tissues post-intervention,” said Dr. Brian Aguado, lead author of the study and a post-doctoral researcher in CU ��«��Ƶ’s Department of Chemical and Biological Engineering. “Our hypothesis was that perhaps there are biochemical cues in a patient’s blood that may revert myofibroblasts back to fibroblasts.”

Modeling such a transformation in the lab is one thing, Aguado said, but the key to the new study was obtaining blood samples from real AVS patients and then using biomaterials to replicate the microenvironment of the heart.

“The heart is not made of plastic like a petri dish is,” he said. “We needed to engineer materials that could reflect the various stiffnesses of both healthy and diseased valve and cardiac tissue.”

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A Global Survey of Mycobacterial Diversity in Soil

Anonymous — Wed, 14 Aug 2019 00:00:00 +0000

A Global Survey of Mycobacterial Diversity in Soil Anonymous (not verified) Tue, 08/13/2019 - 18:00

Corinne Walsh , Matthew Gebert , Manuel Delgado-Baquerizo , Fernando Maestre , Noah Fierer

Mycobacteria are a diverse bacterial group ubiquitous in many soil and aquatic environments. Members of this group have been associated with human and other animal diseases, including the nontuberculous mycobacteria (NTM), which are of growing relevance to public health worldwide. Although soils are often considered an important source of environmentally acquired NTM infections, the biodiversity and ecological preferences of soil mycobacteria remain largely unexplored across contrasting climates and ecosystem types. Using a culture-independent approach by combining 16S rRNA marker gene sequencing with mycobacterium-specific hsp65gene sequencing, we analyzed the diversity, distributions, and environmental preferences of soil-dwelling mycobacteria in 143 soil samples collected from a broad range of ecosystem types. The surveyed soils harbored highly diverse mycobacterial communities that span the full extent of the known mycobacterial phylogeny, with most soil mycobacteria (97% of mycobacterial clades) belonging to previously undescribed lineages. While mycobacteria tended to have higher relative abundances in cool, wet, and acidic soil environments, several individual mycobacterial clades had contrasting environmental preferences. We identified the environmental preferences of many mycobacterial clades, including the clinically relevant Mycobacterium avium complex that was more commonly detected in wet and acidic soils. However, most of the soil mycobacteria detected were not closely related to known pathogens, calling into question previous assumptions about the general importance of soil as a source of NTM infections. Together, this work provides novel insights into the diversity, distributions, and ecological preferences of soil mycobacteria and lays the foundation for future efforts to link mycobacterial phenotypes to their distributions.

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A taxonomically broad metagenomic survey of 339 species spanning 57 families suggests cystobasidiomycete yeasts are not ubiquitous across all lichens

Anonymous — Fri, 09 Aug 2019 00:00:00 +0000

A taxonomically broad metagenomic survey of 339 species spanning 57 families suggests cystobasidiomycete yeasts are not ubiquitous across all lichens Anonymous (not verified) Thu, 08/08/2019 - 18:00

James Lendemer , Kyle Keepers , Erin Tripp , Cloe Pogoda , Christy McCain , Nolan Kane

Premise

Lichens are fungi that enter into obligate symbioses with photosynthesizing organisms (algae, cyanobacteria). Traditional narratives of lichens as binary symbiont pairs have given way to their recognition as dynamic metacommunities. Basidiomycete yeasts, particularly of the genus Cyphobasidium, have been inferred to be widespread and important components of lichen metacommunities. Yet, the presence of basidiomycete yeasts across a wide diversity of lichen lineages has not previously been tested.

Methods

We searched for lichen‐associated cystobasidiomycete yeasts in newly generated metagenomic data from 413 samples of 339 lichen species spanning 57 families and 25 orders. The data set was generated as part of a large‐scale project to study lichen biodiversity gradients in the southern Appalachian Mountains Biodiversity Hotspot of southeastern North America.

Results

Our efforts detected cystobasidiomycete yeasts in nine taxa (Bryoria nadvornikiana,Heterodermia leucomelos, Lecidea roseotincta, Opegrapha vulgata, Parmotrema hypotropum,P. subsumptum, Usnea cornuta, U. strigosa, and U. subgracilis), representing 2.7% of all species sampled. Seven of these taxa (78%) are foliose (leaf‐like) or fruticose (shrubby) lichens that belong to families where basidiomycete yeasts have been previously detected. In several of the nine cases, cystobasidiomycete rDNA coverage was comparable to, or greater than, that of the primary lichen fungus single‐copy nuclear genomic rDNA, suggesting sampling artifacts are unlikely to account for our results.

Conclusions

Studies from diverse areas of the natural sciences have led to the need to reconceptualize lichens as dynamic metacommunities. However, our failure to detect cystobasidiomycetes in 97.3% (330 species) of the sampled species suggests that basidiomycete yeasts are not ubiquitous in lichens.

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The ATPase cycle of human muscle myosin II Isoforms: adaptation of a single mechanochemical cycle for different physiological roles

Anonymous — Tue, 06 Aug 2019 00:00:00 +0000

The ATPase cycle of human muscle myosin II Isoforms: adaptation of a single mechanochemical cycle for different physiological roles Anonymous (not verified) Mon, 08/05/2019 - 18:00

Chloe Johnson , Jonathan Walklate , Marina Svicevic , Srbolujub Mijailovich , Carlos Vera , Anastasia Karabina , Leslie Leinwand , Michael Geeves

Striated muscle myosins are encoded by a large gene family in all mammals, including human. These isoforms define several of the key characteristics of the different striated muscle fiber types including maximum shortening velocity. We have previously used recombinant isoforms of the motor domains of seven different human myosin isoforms to define the actin.myosin cross-bridge cycle in solution. Here we present data on an eighth isoform the perinatal, which has not previously been charaterized. The perinatal is distinct from the embryonic isoforms appearing to have features in common with the adult fast muscle isoform, including weak affinity of ADP for A.M and fast ADP release. We go on to use a recently developed modeling approach MUSICO to explore how well the experimentally defined cross-bridge cycles for each isoform in solution can predict the characteristics of muscle fiber contraction including duty ratio, shortening velocity, ATP economy and the load dependence of these parameters. The work shows that the parameters of the cross-bridge cycle predict many of the major characteristics of each muscle fiber type and raises the question of what sequence changes are responsible for these characteristics.

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Three-Dimensional Microscale Imaging and Measurement of Soft Material Contact Interfaces under Quasi-Static Normal Indentation and Shear

Anonymous — Wed, 10 Jul 2019 00:00:00 +0000

Three-Dimensional Microscale Imaging and Measurement of Soft Material Contact Interfaces under Quasi-Static Normal Indentation and Shear Anonymous (not verified) Tue, 07/09/2019 - 18:00

Karl Johannes , Kristin Calahan , Yuan Qi , Rong Long , Mark Rentschler

Understanding the contact and friction between soft materials is vital to a wide variety of engineering applications including soft sealants and medical devices such as catheters and stents. Although the mechanisms of friction between stiff materials have been extensively studied, the mechanisms of friction between soft materials are much less understood. Time-dependent material responses, large deformations, and fluid layers at the contact interface, common in soft materials, pose new challenges toward understanding the friction between soft materials. This article aims to characterize the three-dimensional (3D) contact interfaces in soft materials under large deformations and complex contact conditions. Specifically, we introduce a microindentation and visualization (MIV) system capable of investigating soft material contact interfaces with combined normal and shear loading. When combined with a laser scanning confocal microscope, the MIV system enables the acquisition of 3D image stacks of the deformed substrate and the indenter under fixed normal and shear displacements. The 3D imaging data allows us to quantify the 3D contact profiles and correlate them with the applied normal and shear displacements. Using a spherical indenter and a hydrogel substrate as a model system, we demonstrate that the MIV system and the associated analysis techniques accurately measure the contact area under combined normal and shear loading. Although the limited speed of confocal scanning implies that this method is most suitable for quasi-static loading conditions, potential methods to increase the imaging speed and the corresponding trade-off in image resolution are discussed. The method presented here will be useful for the future investigation of soft material contact and friction involving complex surface geometries.

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Extensive chloroplast genome rearrangement amongst three closely related Halamphora spp. (Bacillariophyceae), and evidence for rapid evolution as compared to land plants

Anonymous — Wed, 03 Jul 2019 00:00:00 +0000

Extensive chloroplast genome rearrangement amongst three closely related Halamphora spp. (Bacillariophyceae), and evidence for rapid evolution as compared to land plants Anonymous (not verified) Tue, 07/02/2019 - 18:00

Kyle Keepers , Cloe Pogoda

Diatoms are the most diverse lineage of algae, but the diversity of their chloroplast genomes, particularly within a genus, has not been well documented. Herein, we present three chloroplast genomes from the genus Halamphora (H. americana, H. calidilacuna, and H. coffeaeformis), the first pennate diatom genus to be represented by more than one species. Halamphorachloroplast genomes ranged in size from ~120 to 150 kb, representing a 24% size difference within the genus. Differences in genome size were due to changes in the length of the inverted repeat region, length of intergenic regions, and the variable presence of ORFs that appear to encode as-yet-undescribed proteins. All three species shared a set of 161 core features but differed in the presence of two genes, serC and tyrC of foreign and unknown origin, respectively. A comparison of these data to three previously published chloroplast genomes in the non-pennate genus Cyclotella (Thalassiosirales) revealed that Halamphora has undergone extensive chloroplast genome rearrangement compared to other genera, as well as containing variation within the genus. Finally, a comparison of Halamphora chloroplast genomes to those of land plants indicates diatom chloroplast genomes within this genus may be evolving at least ~4–7 times faster than those of land plants. Studies such as these provide deeper insights into diatom chloroplast evolution and important genetic resources for future analyses.

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