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    CIRCADIAN CLOCK-ASSOCIATED1 Controls Resistance to Aphids by Altering Indole Glucosinolate Production
    (Oxford University Press, 2019) Lei, Jiaxin; Jayaprakasha, Guddadarangavvanahally K.; Singh, Jashbir; Uckoo, Rammohan; Borrego, Eli J.; Finlayson, Scott; Kolomiets, Mike; Patil, Bhimanagouda S.; Braam, Janet; Zhu-Salzman, Keyan
    CIRCADIAN CLOCK-ASSOCIATED1 (CCA1), a well-known central circadian clock regulator, coordinates plant responses to environmental challenges. Its daily rhythmic expression in Arabidopsis (Arabidopsis thaliana) confers host resistance to the caterpillar Trichoplusia ni. However, it is unclear whether CCA1 plays a role in defense against phloem sap-feeding aphids. In this study, we showed that green peach aphid (Myzus persicae) displayed an intrinsic circadian feeding rhythm. Under constant light, wild-type Columbia-0 (Col-0) Arabidopsis plants coentrained with aphids in the same light/dark cycles exhibited greater antixenotic activity than plants preentrained in the opposite cycle from the aphids. Consistently, circadian mutants cca1-1, cca1-11, lhy-21, ztl-1, ztl-4, and lux-2 suffered more severe damage than Col-0 plants when infested by aphids, suggesting that the Arabidopsis circadian clock plays a defensive role. However, the arrhythmic CCA1 overexpression line (CCA1-OX) displayed strong antixenotic and antibiotic activities despite its loss of circadian regulation. Aphids feeding on CCA1-OX plants exhibited lower reproduction and smaller body size and weight than those on Col-0. Apparently, CCA1 regulates both clock-dependent and -independent defense responses. Systematic investigation based on bioinformatics analyses indicated that resistance to aphids in CCA1-OX plants was due primarily to heightened basal indole glucosinolate levels. Interestingly, aphid feeding induced alternatively spliced intron-retaining CCA1a/b transcripts, which are normally expressed at low levels, whereas expression of the major fully spliced CCA1 transcript remained largely unchanged. We hypothesize that posttranscriptional modulation of CCA1 expression upon aphid infestation maximizes the potential of circadian-mediated defense and stress tolerance while ensuring normal plant development.
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    CLN8 is an endoplasmic reticulum cargo receptor that regulates lysosome biogenesis
    (Springer Nature, 2018) di Ronza, Alberto; Bajaj, Lakshya; Sharma, Jaiprakash; Sanagasetti, Deepthi; Lotfi, Parisa; Adamski, Carolyn Joy; Collette, John; Palmieri, Michela; Amawi, Abdallah; Popp, Lauren; Chang, Kevin Tommy; Meschini, Maria Chiara; Leung, Hon-Chiu Eastwood; Segatori, Laura; Simonati, Alessandro; Sifers, Richard Norman; Santorelli, Filippo Maria; Sardiello, Marco
    Organelle biogenesis requires proper transport of proteins from their site of synthesis to their target subcellular compartment1,2,3. Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and traffic through the Golgi complex before being transferred to the endolysosomal system4,5,6, but how they are transferred from the ER to the Golgi is unknown. Here, we show that ER-to-Golgi transfer of lysosomal enzymes requires CLN8, an ER-associated membrane protein whose loss of function leads to the lysosomal storage disorder, neuronal ceroid lipofuscinosis 8 (a type of Batten disease)7. ER-to-Golgi trafficking of CLN8 requires interaction with the COPII and COPI machineries via specific export and retrieval signals localized in the cytosolic carboxy terminus of CLN8. CLN8 deficiency leads to depletion of soluble enzymes in the lysosome, thus impairing lysosome biogenesis. Binding to lysosomal enzymes requires the second luminal loop of CLN8 and is abolished by some disease-causing mutations within this region. Our data establish an unanticipated example of an ER receptor serving the biogenesis of an organelle and indicate that impaired transport of lysosomal enzymes underlies Batten disease caused by mutations in CLN8.
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    OncomiR-10b hijacks the small molecule inhibitor linifanib in human cancers
    (Springer Nature, 2018) Monroig-Bosque, Paloma del C.; Shah, Maitri Y.; Fu, Xiao; Fuentes-Mattei, Enrique; Ling, Hui; Ivan, Cristina; Nouraee, Nazila; Huang, Beibei; Chen, Lu; Pileczki, Valentina; Redis, Roxana S.; Jung, Eun-Jung; Zhang, Xin; Lehrer, Michael; Nagvekar, Rahul; Mafra, Ana Carolina P.; Monroig-Bosque, Maria del Mar; Irimie, Alexandra; Rivera, Carlos; Dumitru, Calin Dan; Berindan-Neagoe, Ioana; Nikonowicz, Edward P.; Zhang, Shuxing; Calin, George A.
    The pervasive role of microRNAs (miRNAs) in cancer pathobiology drives the introduction of new drug development approaches such as miRNA inhibition. In order to advance miRNA-therapeutics, meticulous screening strategies addressing specific tumor targets are needed. Small molecule inhibitors represent an attractive goal for these strategies. In this study, we devised a strategy to screen for small molecule inhibitors that specifically inhibit, directly or indirectly, miR-10b (SMIRs) which is overexpressed in metastatic tumors. We found that the multi-tyrosine kinase inhibitor linifanib could significantly inhibit miR-10b and reverse its oncogenic function in breast cancer and liver cancer both in vitro and in vivo. In addition, we showed that the efficacy of linifanib to inhibit tyrosine kinases was reduced by high miR-10b levels. When the level of miR-10b is high, it can "hijack" the linifanib and reduce its kinase inhibitory effects in cancer resulting in reduced anti-tumor efficacy. In conclusion, our study describes an effective strategy to screen for small molecule inhibitors of miRNAs. We further propose that miR-10b expression levels, due to the newly described "hijacking" effect, may be used as a biomarker to select patients for linifanib treatment.
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    Understanding molecular recognition of promiscuity of thermophilic methionine adenosyltransferase sMAT from Sulfolobus solfataricus
    (Wiley, 2014) Wang, Fengbin; Singh, Shanteri; Zhang, Jianjun; Huber, Tyler D.; Helmich, Kate E.; Sunkara, Manjula; Hurley, Katherine A.; Goff, Randal D.; Bingman, Craig A.; Morris, Andrew J.; Thorson, Jon S.; Phillips, George N.Jr.
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    Deficiency in Perlecan/HSPG2 During Bone Development Enhances Osteogenesis and Decreases Quality of Adult Bone in Mice
    (Springer, 2014) Lowe, Dylan A.; Lepori-Bui, Nadia; Fomin, Peter V.; Sloofman, Laura G.; Zhou, Xiaozhou; Farach-Carson, Mary C.; Wang, Liyun; Kirn-Safran, Catherine B.
    Perlecan/HSPG2 (Pln) is a large heparan sulfate proteoglycan abundant in the extracellular matrix of cartilage and the lacunocanalicular space of adult bones. Although Pln function during cartilage development is critical, evidenced by deficiency disorders including Schwartz–Jampel Syndrome and dyssegmental dysplasia Silverman-Handmaker type, little is known about its function in development of bone shape and quality. The purpose of this study was to understand the contribution of Pln to bone geometric and mechanical properties. We used hypomorph mutant mice that secrete negligible amount of Pln into skeletal tissues and analyzed their adult bone properties using micro-computed tomography and three-point-bending tests. Bone shortening and widening in Pln mutants was observed and could be attributed to loss of growth plate organization and accelerated osteogenesis that was reflected by elevated cortical thickness at older ages. This effect was more pronounced in Pln mutant females, indicating a sex-specific effect of Pln deficiency on bone geometry. Additionally, mutant females, and to a lesser extent mutant males, increased their elastic modulus and bone mineral densities to counteract changes in bone shape, but at the expense of increased brittleness. In summary, Pln deficiency alters cartilage matrix patterning and, as we now show, coordinately influences bone formation and calcification.
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    Hyaluronan: A simple polysaccharide with diverse biological functions
    (Elsevier, 2014) Dicker, Kevin T.; Gurski, Lisa A.; Pradhan-Bhatt, Swati; Witt, Robert L.; Farach-Carson, Mary C.; Jia, Xinqiao
    Hyaluronan (HA) is a linear polysaccharide with disaccharide repeats of d-glucuronic acid and N-acetyl-d-glucosamine. It is evolutionarily conserved and abundantly expressed in the extracellular matrix (ECM), on the cell surface and even inside cells. Being a simple polysaccharide, HA exhibits an astonishing array of biological functions. HA interacts with various proteins or proteoglycans to organize the ECM and to maintain tissue homeostasis. The unique physical and mechanical properties of HA contribute to the maintenance of tissue hydration, the mediation of solute diffusion through the extracellular space and the lubrication of certain tissues. The diverse biological functions of HA are manifested through its complex interactions with matrix components and resident cells. Binding of HA with cell surface receptors activates various signaling pathways, which regulate cell function, tissue development, inflammation, wound healing and tumor progression and metastasis. Taking advantage of the inherent biocompatibility and biodegradability of HA, as well as its susceptibility to chemical modification, researchers have developed various HA-based biomaterials and tissue constructs with promising and broad clinical potential. This paper illustrates the properties of HA from a matrix biology perspective by first introducing the principles underlying the biosynthesis and biodegradation of HA, as well as the interactions of HA with various proteins and proteoglycans. It next highlights the roles of HA in physiological and pathological states, including morphogenesis, wound healing and tumor metastasis. A deeper understanding of the mechanisms underlying the roles of HA in various physiological processes can provide new insights and tools for the engineering of complex tissues and tissue models.
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    Structure, Dynamics, and Specificity of Endoglucanase D from Clostridium cellulovorans
    (Elsevier, 2013) Bianchetti, Christopher M.; Brumm, Phillip; Smith, Robert W.; Dyer, Kevin; Hura, Greg L.; Rutkoski, Thomas J.; Phillips, George N.Jr.
    The enzymatic degradation of cellulose is a critical step in the biological conversion of plant biomass into an abundant renewable energy source. An understanding of the structural and dynamic features that cellulases utilize to bind a single strand of crystalline cellulose and hydrolyze the β-1,4-glycosidic bonds of cellulose to produce fermentable sugars would greatly facilitate the engineering of improved cellulases for the large-scale conversion of plant biomass. Endoglucanase D (EngD) from Clostridium cellulovorans is a modular enzyme comprising an N-terminal catalytic domain and a C-terminal carbohydrate-binding module, which is attached via a flexible linker. Here, we present the 2.1-Å-resolution crystal structures of full-length EngD with and without cellotriose bound, solution small-angle X-ray scattering (SAXS) studies of the full-length enzyme, the characterization of the active cleft glucose binding subsites, and substrate specificity of EngD on soluble and insoluble polymeric carbohydrates. SAXS data support a model in which the linker is flexible, allowing EngD to adopt an extended conformation in solution. The cellotriose-bound EngD structure revealed an extended active-site cleft that contains seven glucose-binding subsites, but unlike the majority of structurally determined endocellulases, the active-site cleft of EngD is partially enclosed by Trp162 and Tyr232. EngD variants, which lack Trp162, showed a significant reduction in activity and an alteration in the distribution of cellohexaose degradation products, suggesting that Trp162 plays a direct role in substrate binding.
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    SCHEMA Computational Design of Virus Capsid Chimeras: Calibrating How Genome Packaging, Protection, and Transduction Correlate with Calculated Structural Disruption
    (American Chemical Society, 2013) Ho, Michelle L.; Adler, Benjamin A.; Torre, Michael L.; Silberg, Jonathan J.; Suh, Junghae
    Adeno-associated virus (AAV) recombination can result in chimeric capsid protein subunits whose ability to assemble into an oligomeric capsid, package a genome, and transduce cells depends on the inheritance of sequence from different AAV parents. To develop quantitative design principles for guiding site-directed recombination of AAV capsids, we have examined how capsid structural perturbations predicted by the SCHEMA algorithm correlate with experimental measurements of disruption in seventeen chimeric capsid proteins. In our small chimera population, created by recombining AAV serotypes 2 and 4, we found that protection of viral genomes and cellular transduction were inversely related to calculated disruption of the capsid structure. Interestingly, however, we did not observe a correlation between genome packaging and calculated structural disruption; a majority of the chimeric capsid proteins formed at least partially assembled capsids and more than half packaged genomes, including those with the highest SCHEMA disruption. These results suggest that the sequence space accessed by recombination of divergent AAV serotypes is rich in capsid chimeras that assemble into 60-mer capsids and package viral genomes. Overall, the SCHEMA algorithm may be useful for delineating quantitative design principles to guide the creation of libraries enriched in genome-protecting virus nanoparticles that can effectively transduce cells. Such improvements to the virus design process may help advance not only gene therapy applications but also other bionanotechnologies dependent upon the development of viruses with new sequences and functions.
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    A promoter-level mammalian expression atlas
    (Nature Publishing Group, 2014) FANTOM Consortium; RIKEN PMI; CLST (DGT)
    Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly ‘housekeeping’, whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.
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    Perlecan-Containing Pericellular Matrix Regulates Solute Transport and Mechanosensing Within the Osteocyte Lacunar-Canalicular System
    (Wiley, 2014) Wang, Bin; Lai, Xiaohan; Price, Christopher; Thompson, William R.; Li, Wen; Quabili, Tonima R.; Tseng, Wei-Ju; Liu, Xiaowei Sherry; Zhang, Hong-Yi; Pan, Jun; Kirn-Safran, Catherine B.; Farach-Carson, Mary C.; Wang, Liyun
    The pericellular matrix (PCM), a thin coating surrounding nearly all mammalian cells, plays a critical role in many cell-surface phenomena. In osteocytes, the PCM is believed to control both “outside-in” (mechanosensing) and “inside-out” (signaling molecule transport) processes. However, the osteocytic PCM is challenging to study in situ because it is thin (∼100 nm) and enclosed in mineralized matrix. To this end, we recently developed a novel tracer velocimetry approach that combined fluorescence recovery after photobleaching (FRAP) imaging with hydrodynamic modeling to quantify the osteocytic PCM in young murine bone. In this study, we applied the technique to older mice expressing or deficient for perlecan/HSPG2, a large heparan-sulfate proteoglycan normally secreted in osteocytic PCM. The objectives were (1) to characterize transport within an altered PCM; (2) to test the sensitivity of our approach in detecting the PCM alterations; and (3) to dissect the roles of the PCM in osteocyte mechanosensing. We found that: (1) solute transport increases in the perlecan-deficient (hypomorphic [Hypo]) mice compared with control mice; (2) PCM fiber density decreases with aging and perlecan deficiency; (3) osteocytes in the Hypo bones are predicted to experience higher shear stress (+34%), but decreased fluid drag force (−35%) under 3-N peak tibial loading; and (4) when subjected to tibial loading in a preliminary in vivo experiment, the Hypo mice did not respond to the anabolic stimuli as the CTL mice did. These findings support the hypothesis that the PCM fibers act as osteocyte's sensing antennae, regulating load-induced cellular stimulations and thus bone's sensitivity and in vivo bone adaptation. If this hypothesis is further confirmed, osteocytic PCM could be new targets to develop osteoporosis treatments by modulating bone's intrinsic sensitivity to mechanical loading and be used to design patient-specific exercise regimens to promote bone formation.
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    The crystal structure of BlmI as a model for nonribosomal peptide synthetase peptidyl carrier proteins
    (Wiley, 2014) Lohman, Jeremy R.; Ma, Ming; Cuff, Marianne E.; Bigelow, Lance; Bearden, Jessica; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.Jr.; Shen, Ben
    Carrier proteins (CPs) play a critical role in the biosynthesis of various natural products, especially in nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymology, where the CPs are referred to as peptidyl-carrier proteins (PCPs) or acyl-carrier proteins (ACPs), respectively. CPs can either be a domain in large multifunctional polypeptides or standalone proteins, termed Type I and Type II, respectively. There have been many biochemical studies of the Type I PKS and NRPS CPs, and of Type II ACPs. However, recently a number of Type II PCPs have been found and biochemically characterized. In order to understand the possible interaction surfaces for combinatorial biosynthetic efforts we crystallized the first characterized and representative Type II PCP member, BlmI, from the bleomycin biosynthetic pathway from Streptomyces verticillus ATCC 15003. The structure is similar to CPs in general but most closely resembles PCPs. Comparisons with previously determined PCP structures in complex with catalytic domains reveals a common interaction surface. This surface is highly variable in charge and shape, which likely confers specificity for interactions. Previous nuclear magnetic resonance (NMR) analysis of a prototypical Type I PCP excised from the multimodular context revealed three conformational states. Comparison of the states with the structure of BlmI and other PCPs reveals that only one of the NMR states is found in other studies, suggesting the other two states may not be relevant. The state represented by the BlmI crystal structure can therefore serve as a model for both Type I and Type II PCPs.
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    Hyaluronan (HA) Interacting Proteins RHAMM and Hyaluronidase Impact Prostate Cancer Cell Behavior and Invadopodia Formation in 3D HA-Based Hydrogels
    (Public Library of Science, 2012) Gurski, Lisa A.; Xu, Xian; Labrada, Lyana N.; Nguyen, Ngoc T.; Xiao, Longxi; van Golen, Kenneth L.; Jia, Xinqiao; Farach-Carson, Mary C.
    To study the individual functions of hyaluronan interacting proteins in prostate cancer (PCa) motility through connective tissues, we developed a novel three-dimensional (3D) hyaluronic acid (HA) hydrogel assay that provides a flexible, quantifiable, and physiologically relevant alternative to current methods. Invasion in this system reflects the prevalence of HA in connective tissues and its role in the promotion of cancer cell motility and tissue invasion, making the system ideal to study invasion through bone marrow or other HA-rich connective tissues. The bio-compatible cross-linking process we used allows for direct encapsulation of cancer cells within the gel where they adopt a distinct, cluster-like morphology. Metastatic PCa cells in these hydrogels develop fingerlike structures, “invadopodia”, consistent with their invasive properties. The number of invadopodia, as well as cluster size, shape, and convergence, can provide a quantifiable measure of invasive potential. Among candidate hyaluronan interacting proteins that could be responsible for the behavior we observed, we found that culture in the HA hydrogel triggers invasive PCa cells to differentially express and localize receptor for hyaluronan mediated motility (RHAMM)/CD168 which, in the absence of CD44, appears to contribute to PCa motility and invasion by interacting with the HA hydrogel components. PCa cell invasion through the HA hydrogel also was found to depend on the activity of hyaluronidases. Studies shown here reveal that while hyaluronidase activity is necessary for invadopodia and inter-connecting cluster formation, activity alone is not sufficient for acquisition of invasiveness to occur. We therefore suggest that development of invasive behavior in 3D HA-based systems requires development of additional cellular features, such as activation of motility associated pathways that regulate formation of invadopodia. Thus, we report development of a 3D system amenable to dissection of biological processes associated with cancer cell motility through HA-rich connective tissues.
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    Recreating the tumor microenvironment in a bilayer, hyaluronic acid hydrogel construct for the growth of prostate cancer spheroids
    (Elsevier, 2012) Xu, Xian; Gurski, Lisa A.; Zhang, Chu; Harrington, Daniel Anton; Farach-Carson, Mary C.; Jia, Xinqiao
    Cancer cells cultured in physiologically relevant, three-dimensional (3D) matrices can recapture many essential features of native tumor tissues. In this study, a hyaluronic acid (HA)-based bilayer hydrogel system that not only supports the tumoroid formation from LNCaP prostate cancer (PCa) cells, but also simulates their reciprocal interactions with the tumor-associated stroma was developed and characterized. HA hydrogels were prepared by mixing solutions of HA precursors functionalized with acrylate groups (HA-AC) and reactive thiols (HA-SH) under physiological conditions. The resultant viscoelastic gels have an average elastic modulus of 234 ± 30 Pa and can be degraded readily by hyaluronidase. The orthogonal and cytocompatible nature of the crosslinking chemistry permits facile incorporation of cytokine-releasing particles and PCa cells. In our bilayer hydrogel construct, the top layer contains heparin (HP)-decorated, HA-based hydrogel particles (HGPs) capable of releasing heparin-binding epidermal growth factor-like growth factor (HB-EGF) in a sustained manner at a rate of 2.5 wt%/day cumulatively. LNCaP cells embedded in the bottom layer receive the growth factor signals from the top, and in response form enlarging tumoroids with an average diameter of 85 μm by day 7. Cells in 3D hydrogels assemble into spherical tumoroids, form close cellular contacts through E-cadherin, and show cortical organization of F-actin, whereas those plated as 2D monolayers adopt a spread-out morphology. Compared to cells cultured on 2D, the engineered tumoroids significantly increased the expression of two pro-angiogenic factors, vascular endothelial growth factor-165 (VEGF(165)) and interleukin-8 (IL-8), both at mRNA and protein levels. Overall, the HA model system provides a useful platform for the study of tumor cell responses to growth factors and for screening of anticancer drugs targeting these pathways.
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    Transcriptional Activation by NFκB Increases Perlecan/HSPG2 Expression in the Desmoplastic Prostate Tumor Microenvironment
    (Wiley, 2014) Warren, Curtis R.; Grindel, Brian J.; Francis, Lewis L.W.; Carson, Daniel D.; Farach-Carson, Mary C.
    Perlecan/HSPG2, a heparan sulfate proteoglycan typically found at tissue borders including those separating epithelia and connective tissue, increases near sites of invasion of primary prostatic tumors as previously shown for other proteins involved in desmoplastic tissue reaction. Studies of prostate cancer cells and stromal cells from both prostate and bone, the major site for prostate cancer metastasis, showed that cancer cells and a subset of stromal cells increased production of perlecan in response to cytokines present in the tumor microenvironment. In silico analysis of the HSPG2 promoter revealed two conserved NFκB binding sites, in addition to the previously reported SMAD3 binding sites. By systematically transfecting cells with a variety of reporter constructs including sequences up to 2.6 kb from the start site of transcription, we identified an active cis element in the distal region of the HSPG2 promoter, and showed that it functions in regulating transcription of HSPG2. Treatment with TNF-α and/or TGFβ1 identified TNF-α as a major cytokine regulator of perlecan production. TNF-α treatment also triggered p65 nuclear translocation and binding to the HSPG2 regulatory region in stromal cells and cancer cells. In addition to stromal induction of perlecan production in the prostate, we identified a matrix-secreting bone marrow stromal cell type that may represent the source for increases in perlecan in the metastatic bone marrow environment. These studies implicate perlecan in cytokine-mediated, innate tissue responses to cancer cell invasion, a process we suggest reflects a modified wound healing tissue response co-opted by prostate cancer cells.
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    A novel in vivo model for evaluating functional restoration of a tissue-engineered salivary gland
    (Wiley, 2014) Pradhan-Bhatt, Swati; Harrington, Daniel Anton; Duncan, Randall L.; Farach-Carson, Mary C.; Jia, Xinqiao; Witt, Robert L.
    Objectives/Hypothesis: To create a novel model for development of a tissue-engineered salivary gland from human salivary gland cells that retains progenitor cell markers useful for treatment of radiation-induced xerostomia. Study Design: A three-dimensional (3D) hyaluronic acid (HA)-based hydrogel scaffold was used to encapsulate primary human salivary gland cells and to obtain organized acini-like spheroids. Hydrogels were implanted into rat models, and cell viability and receptor expression were evaluated. Methods: A parotid gland surgical resection model for xenografting was developed. Salivary cells loaded in HA hydrogels formed spheroids and in vitro were implanted in the three-fourths resected parotid bed of athymic rats. Implants were removed after 1 week and analyzed for spheroid viability and phenotype retention. Results: Spheroids in 3D stained positive for HA receptors CD168/RHAMM and CD44, which is also a progenitor cell marker. The parotid gland three-fourths resection model was well-tolerated by rodent hosts, and the salivary cell/hydrogel scaffolds were adherent to the remaining parotid gland, with no obvious signs of inflammation. A majority of human cells in the extracted hydrogels demonstrated robust expression of CD44. Conclusions: A 3D HA-based hydrogel scaffold that supported long-term culture of salivary gland cells into organized spheroids was established. An in vivo salivary gland resection model was developed that allowed for integration of the 3D HA hydrogel scaffold with the existing glandular parenchyma. The expression of CD44 among salivary cultures may partially explain their regenerative potential, and the expression of CD168/RHAMM along with CD44 may aid the development of these 3D spheroids into regenerated salivary glands.
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    p62/SQSTM1 is required for cell survival of apoptosis-resistant bone metastatic prostate cancer cell lines
    (Wiley, 2014) Chang, Megan A.; Morgado, Micaela; Warren, Curtis R.; Hinton, Cimona V.; Farach-Carson, Mary C.; Delk, Nikki A.
    BACKGROUND: Bone marrow stromal cell (BMSC) paracrine factor(s) can induce apoptosis in bone metastatic prostate cancer (PCa) cell lines. However, the PCa cells that escape BMSC-induced apoptosis can upregulate cytoprotective autophagy. METHODS: C4-2, C4-2B, MDA PCa 2a, MDA PCa 2b, VCaP, PC3, or DU145 PCa cell lines were grown in BMSC conditioned medium and analyzed for mRNA and/or protein accumulation of p62 (also known as sequestome-1/SQSTM1), Microtubule-associated protein 1 light chain 3B (LC3B), or lysosomal-associated membrane protein 1 (LAMP1) using quantitative polymerase chain reaction (QPCR), Western blot, or immunofluorescence. Small interfering RNA (siRNA) was used to determine if p62 is necessary PCa cell survival. RESULTS: BMSC paracrine signaling upregulated p62 mRNA and protein in a subset of the PCa cell lines. The PCa cell lines that were insensitive to BMSC-induced apoptosis and autophagy induction had elevated basal p62 mRNA and protein. In the BMSC-insensitive PCa cell lines, siRNA knockdown of p62 was cytotoxic and immunostaining showed peri-nuclear clustering of autolysosomes. However, in the BMSC-sensitive PCa cell lines, p62 siRNA knockdown was not appreciably cytotoxic and did not affect autolysosome subcellular localization. CONCLUSIONS: A pattern emerges wherein the BMSC-sensitive PCa cell lines are known to be osteoblastic and express the androgen receptor, while the BMSC-insensitive PCa cell lines are characteristically osteolytic and do not express the androgen receptor. Furthermore, BMSC-insensitive PCa may have evolved a dependency on p62 for cell survival that could be exploited to target and kill these apoptosis-resistant PCa cells in the bone.
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    Redox properties of human hemoglobin in complex with fractionated dimeric and polymeric human haptoglobin
    (Elsevier, 2014) Mollan, Todd L.; Jia, Yiping; Banerjee, Sambuddha; Wu, Gang; Kreulen, R.Timothy; Tsai, Ah-Lim; Olson, John S.; Crumbliss, Alvin L.; Alayash, Abdu I.
    Haptoglobin (Hp) is an abundant and conserved plasma glycoprotein, which binds acellular adult hemoglobin (Hb) dimers with high affinity and facilitates their rapid clearance from circulation after hemolysis. Humans possess three main phenotypes of Hp, designated Hp 1-1, Hp 2-1, and Hp 2-2. These variants exhibit diverse structural configurations and have been reported to be functionally nonequivalent. We have investigated the functional and redox properties of Hb–Hp complexes prepared using commercially fractionated Hp and found that all forms exhibit similar behavior. The rate of Hb dimer binding to Hp occurs with bimolecular rate constants of ~0.9 μM−1 s−1, irrespective of the type of Hp assayed. Although Hp binding does accelerate the observed rate of HbO2 autoxidation by dissociating Hb tetramers into dimers, the rate observed for these bound dimers is three- to fourfold slower than that of Hb dimers free in solution. Co-incubation of ferric Hb with any form of Hp inhibits heme loss to below detectable levels. Intrinsic redox potentials (E1/2) of the ferric/ferrous pair of each Hb–Hp complex are similar, varying from +54 to +59 mV (vs NHE), and are essentially the same as reported by us previously for Hb–Hp complexes prepared from unfractionated Hp. All Hb–Hp complexes generate similar high amounts of ferryl Hb after exposure to hydrogen peroxide. Electron paramagnetic resonance data indicate that the yields of protein-based radicals during this process are approximately 4 to 5% and are unaffected by the variant of Hp assayed. These data indicate that the Hp fractions examined are equivalent to one another with respect to Hb binding and associated stability and redox properties and that this result should be taken into account in the design of phenotype-specific Hp therapeutics aimed at countering Hb-mediated vascular disease.
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    Modeling delay in genetic networks: From delay birth-death processes to delay stochastic differential equations
    (AIP Publishing, 2014) Gupta, Chinmaya; López, José Manuel; Azencott, Robert; Bennett, Matthew R.; Josić, Krešimir; Ott, William; Institute of Biosciences and Bioengineering
    Delay is an important and ubiquitous aspect of many biochemical processes. For example, delay plays a central role in the dynamics of genetic regulatory networks as it stems from the sequential assembly of first mRNA and then protein. Genetic regulatory networks are therefore frequently modeled as stochastic birth-death processes with delay. Here, we examine the relationship between delay birth-death processes and their appropriate approximating delay chemical Langevin equations. We prove a quantitative bound on the error between the pathwise realizations of these two processes. Our results hold for both fixed delay and distributed delay. Simulations demonstrate that the delay chemical Langevin approximation is accurate even at moderate system sizes. It captures dynamical features such as the oscillatory behavior in negative feedback circuits, cross-correlations between nodes in a network, and spatial and temporal information in two commonly studied motifs of metastability in biochemical systems. Overall, these results provide a foundation for using delay stochastic differential equations to approximate the dynamics of birth-death processes with delay.
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    The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome
    (Public Library of Science, 2015) Hurst, Laurence D.; Ghanbarian, Avazeh T.; Forrest, Alistair R.R.; FANTOM Consortium; Huminiecki, Lukasz
    X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X’s gene content, gene expression, and evolution.
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    The PRE-Derived NMR Model of the 38.8-kDa Tri-Domain IsdH Protein from Staphylococcus aureus Suggests That It Adaptively Recognizes Human Hemoglobin
    (Elsevier, 2016) Sjodt, Megan; Macdonald, Ramsay; Spirig, Thomas; Chan, Albert H.; Dickson, Claire F.; Fabian, Marian; Olson, John S.; Gell, David A.; Clubb, Robert T.
    Staphylococcus aureus is a medically important bacterial pathogen that, during infections, acquires iron from human hemoglobin (Hb). It uses two closely related iron-regulated surface determinant (Isd) proteins to capture and extract the oxidized form of heme (hemin) from Hb, IsdH and IsdB. Both receptors rapidly extract hemin using a conserved tri-domain unit consisting of two NEAT (near iron transporter) domains connected by a helical linker domain. To gain insight into the mechanism of extraction, we used NMR to investigate the structure and dynamics of the 38.8-kDa tri-domain IsdH protein (IsdHN2N3, A326–D660 with a Y642A mutation that prevents hemin binding). The structure was modeled using long-range paramagnetic relaxation enhancement (PRE) distance restraints, dihedral angle, small-angle X-ray scattering, residual dipolar coupling and inter-domain NOE nuclear Overhauser effect data. The receptor adopts an extended conformation wherein the linker and N3 domains pack against each other via a hydrophobic interface. In contrast, the N2 domain contacts the linker domain via a hydrophilic interface and, based on NMR relaxation data, undergoes inter-domain motions enabling it to reorient with respect to the body of the protein. Ensemble calculations were used to estimate the range of N2 domain positions compatible with the PRE data. A comparison of the Hb-free and Hb-bound forms reveals that Hb binding alters the positioning of the N2 domain. We propose that binding occurs through a combination of conformational selection and induced-fit mechanisms that may promote hemin release from Hb by altering the position of its F helix.