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Engineering a humanized bone organ model in mice to study bone metastases.

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Engineering a humanized bone organ model in mice to study bone metastases.

Nat Protoc. 2017 Apr;12(4):639-663

Authors: Martine LC, Holzapfel BM, McGovern JA, Wagner F, Quent VM, Hesami P, Wunner FM, Vaquette C, De-Juan-Pardo EM, Brown TD, Nowlan B, Wu DJ, Hutmacher CO, Moi D, Oussenko T, Piccinini E, Zandstra PW, Mazzieri R, Lévesque JP, Dalton PD, Taubenberger AV, Hutmacher DW

Abstract
Current in vivo models for investigating human primary bone tumors and cancer metastasis to the bone rely on the injection of human cancer cells into the mouse skeleton. This approach does not mimic species-specific mechanisms occurring in human diseases and may preclude successful clinical translation. We have developed a protocol to engineer humanized bone within immunodeficient hosts, which can be adapted to study the interactions between human cancer cells and a humanized bone microenvironment in vivo. A researcher trained in the principles of tissue engineering will be able to execute the protocol and yield study results within 4-6 months. Additive biomanufactured scaffolds seeded and cultured with human bone-forming cells are implanted ectopically in combination with osteogenic factors into mice to generate a physiological bone 'organ', which is partially humanized. The model comprises human bone cells and secreted extracellular matrix (ECM); however, other components of the engineered tissue, such as the vasculature, are of murine origin. The model can be further humanized through the engraftment of human hematopoietic stem cells (HSCs) that can lead to human hematopoiesis within the murine host. The humanized organ bone model has been well characterized and validated and allows dissection of some of the mechanisms of the bone metastatic processes in prostate and breast cancer.

PMID: 28253234 [PubMed - indexed for MEDLINE]



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Combinatorial Therapies After Spinal Cord Injury: How Can Biomaterials Help?

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Combinatorial Therapies After Spinal Cord Injury: How Can Biomaterials Help?

Adv Healthc Mater. 2017 Mar 01;:

Authors: Führmann T, Anandakumaran PN, Shoichet MS

Abstract
Traumatic spinal cord injury (SCI) results in an immediate loss of motor and sensory function below the injury site and is associated with a poor prognosis. The inhibitory environment that develops in response to the injury is mainly due to local expression of inhibitory factors, scarring and the formation of cystic cavitations, all of which limit the regenerative capacity of endogenous or transplanted cells. Strategies that demonstrate promising results induce a change in the microenvironment at- and around the lesion site to promote endogenous cell repair, including axonal regeneration or the integration of transplanted cells. To date, many of these strategies target only a single aspect of SCI; however, the multifaceted nature of SCI suggests that combinatorial strategies will likely be more effective. Biomaterials are a key component of combinatorial strategies, as they have the potential to deliver drugs locally over a prolonged period of time and aid in cell survival, integration and differentiation. Here we summarize the advantages and limitations of widely used strategies to promote recovery after injury and highlight recent research where biomaterials aided combinatorial strategies to overcome some of the barriers of spinal cord regeneration.

PMID: 28247563 [PubMed - as supplied by publisher]



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Optimization of Satellite Cell Culture Through Biomaterials.

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Optimization of Satellite Cell Culture Through Biomaterials.

Methods Mol Biol. 2017;1556:329-341

Authors: Davoudi S, Gilbert PM

Abstract
Hydrogels, a type of biomaterial, are an invaluable part of biomedical research as they are highly hydrated and properties such as elasticity, porosity, and ligand density can be tuned to desired values. Recently, culture substrate stiffness was found to be an important regulator of muscle stem cell self-renewal. Polyethylene glycol (PEG), a synthetic polymer, can be fabricated into hydrogels that match the softness of skeletal muscle tissue, thereby providing a culture surface that is optimal for maintaining muscle stem cell self-renewal potential ex vivo. In this Chapter, we describe a method to produce flat PEG hydrogels across a range of stiffnesses, including a formulation that matches the bulk stiffness of healthy skeletal muscle (12 kPa), while maintaining a constant ligand density. Since PEG is inert to protein adsorption, the steps required to surface functionalize the hydrogel with an adhesive interface (e.g., laminin) are also described.

PMID: 28247359 [PubMed - in process]



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Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR): a novel antimicrobial resistance multilocus typing scheme for tracking the global dissemination of N. gonorrhoeae strains.

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Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR): a novel antimicrobial resistance multilocus typing scheme for tracking the global dissemination of N. gonorrhoeae strains.

J Clin Microbiol. 2017 Feb 22;:

Authors: Demczuk W, Sidhu S, Unemo M, Whiley DM, Allen VG, Dillon JR, Cole M, Seah C, Trembizki E, Trees DL, Kersh EN, Abrams AJ, de Vries HJ, van Dam AP, Medina I, Bharat A, Mulvey MR, Van Domselaar G, Martin I

Abstract
A curated web-based user-friendly sequence typing tool based on antimicrobial resistance determinants in Neisseria gonorrhoeae was developed and is publicly accessible at https://ngstar.canada.ca The N. gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) molecular typing scheme uses the DNA sequences of 7 genes (penA, mtrR, porB, ponA, gyrA, parC, 23S rRNA) associated with resistance to β-lactam antimicrobials, macrolides, or fluoroquinolones. NG-STAR uses the entire penA sequence combining the historical nomenclature for penA types I-XXXVIII with novel nucleotide sequence designations; the full mtrR sequence and a portion of its promoter region; portions of ponA, porB, gyrA and parC; and 23S rRNA sequences. NG-STAR grouped 768 isolates into 139 sequence types (STs) (n=660) consisting of 29 CCs having a maximum of a single locus variation; and 76 NG-STAR STs (n=109) were identified as unrelated singletons. NG-STAR had a high Simpson's diversity index of 96.5% (CI 95%=0.959-0.969). The most common STs were NG-STAR: ST-90 (n=100, 13.0%), ST-42 and ST-91 (n=45, 5.9%), ST-64 (n=44, 5.72%), and ST-139 (n=42, 5.5%). Decreased susceptibility to azithromycin was associated with NG-STAR ST-58, ST-61, ST-64, ST-79, ST-91 and ST-139(n=156, 92.3%); decreased susceptibility to cephalosporins with NG-STAR ST-90, ST-91 and ST-97 (n=162, 94.2%); and ciprofloxacin resistance with NG-STAR ST-26, ST-90, ST-91, ST-97, ST-150 and ST-158 (n=196, 98.0%). All isolates of NG-STAR ST-42, ST-43, ST-63, ST-81, and ST-160 (n=106) were susceptible to all four antimicrobials. The standardization of nomenclature associated with antimicrobial resistance determinants through an internationally available database will facilitate the monitoring of the global dissemination of antimicrobial resistant N. gonorrhoeae strains.

PMID: 28228492 [PubMed - as supplied by publisher]



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Automated cell tracking identifies mechanically-oriented cell divisions during Drosophila axis elongation.

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Automated cell tracking identifies mechanically-oriented cell divisions during Drosophila axis elongation.

Development. 2017 Feb 17;:

Authors: Wang MF, Hunter M, Wang G, McFaul C, Yip CM, Fernandez-Gonzalez R

Abstract
Embryos extend their anterior-posterior (AP) axis in the conserved process of axis elongation. Drosophila axis elongation occurs in an epithelial monolayer, the germband, and is driven by cell intercalation, cell shape changes, and oriented cell divisions at the posterior germband. Anterior germband cells also divide during axis elongation. We developed image analysis and pattern recognition methods to track dividing cells from confocal microscopy movies in a generally-applicable approach. Mesectoderm cells, forming the ventral midline, divided parallel to the AP axis, while lateral cells displayed a uniform distribution of division orientations. Mesectoderm cells did not intercalate and sustained increased AP strain before cell division. After division, mesectoderm cell density increased along the AP axis, thus relieving strain. We used laser ablation to isolate mesectoderm cells from other tissues. Uncoupling the mesectoderm from intercalating cells did not affect cell division orientation. Conversely, separating the mesectoderm from the anterior and posterior poles of the embryo resulted in uniformly-oriented divisions. Our data suggest that mesectoderm cells align their division angle to reduce strain caused by mechanical forces along the AP axis of the embryo.

PMID: 28213553 [PubMed - as supplied by publisher]



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CX-5461 is a DNA G-quadruplex stabilizer with selective lethality in BRCA1/2 deficient tumours.

CX-5461 is a DNA G-quadruplex stabilizer with selective lethality in BRCA1/2 deficient tumours.

Nat Commun. 2017 Feb 17;8:14432

Authors: Xu H, Di Antonio M, McKinney S, Mathew V, Ho B, O'Neil NJ, Santos ND, Silvester J, Wei V, Garcia J, Kabeer F, Lai D, Soriano P, Banáth J, Chiu DS, Yap D, Le DD, Ye FB, Zhang A, Thu K, Soong J, Lin SC, Tsai AH, Osako T, Algara T, Saunders DN, Wong J, Xian J, Bally MB, Brenton JD, Brown GW, Shah SP, Cescon D, Mak TW, Caldas C, Stirling PC, Hieter P, Balasubramanian S, Aparicio S

Abstract
G-quadruplex DNAs form four-stranded helical structures and are proposed to play key roles in different cellular processes. Targeting G-quadruplex DNAs for cancer treatment is a very promising prospect. Here, we show that CX-5461 is a G-quadruplex stabilizer, with specific toxicity against BRCA deficiencies in cancer cells and polyclonal patient-derived xenograft models, including tumours resistant to PARP inhibition. Exposure to CX-5461, and its related drug CX-3543, blocks replication forks and induces ssDNA gaps or breaks. The BRCA and NHEJ pathways are required for the repair of CX-5461 and CX-3543-induced DNA damage and failure to do so leads to lethality. These data strengthen the concept of G4 targeting as a therapeutic approach, specifically for targeting HR and NHEJ deficient cancers and other tumours deficient for DNA damage repair. CX-5461 is now in advanced phase I clinical trial for patients with BRCA1/2 deficient tumours (Canadian trial, NCT02719977, opened May 2016).

PMID: 28211448 [PubMed - in process]



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Stem cells in the adult CNS revealed: examining their regulation by myelin basic protein.

Stem cells in the adult CNS revealed: examining their regulation by myelin basic protein.

Neural Regen Res. 2016 Dec;11(12):1916-1917

Authors: Xu W, Lakshman N, Morshead CM

PMID: 28197181 [PubMed - in process]



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LUMIER: A Discovery Tool for Mammalian Protein Interaction Networks.

LUMIER: A Discovery Tool for Mammalian Protein Interaction Networks.

Methods Mol Biol. 2017;1550:137-148

Authors: Barrios-Rodiles M, Ellis JD, Blencowe BJ, Wrana JL

Abstract
Protein-protein interactions (PPIs) play an essential role in all biological processes. In vivo, PPIs occur dynamically and depend on extracellular cues. To discover novel protein-protein interactions in mammalian cells, we developed a high-throughput automated technology called LUMIER (LUminescence-based Mammalian IntERactome). In this approach, we co-express a Luciferase (LUC)-tagged fusion protein along with a Flag-tagged protein in an efficiently transfectable cell line such as HEK-293T cells. The interaction between the two proteins is determined by co-immunoprecipitation using an anti-Flag antibody, and the presence of the LUC-tagged interactor in the complex is subsequently detected via its luciferase activity. LUMIER can easily detect transmembrane protein partners, interactions that are signaling- or splice isoform-dependent, as well as those that may occur only in the presence of posttranslational modifications. Using various collections of Flag-tagged proteins, we have generated protein interaction networks for several TGF-β family receptors, Wnt pathway members, and have systematically analyzed the effect of neural-specific alternative splicing on protein interaction networks. The results have provided important insights into the physiological and functional relevance of some of the novel interactions found. LUMIER is highly scalable and can be used for both low- and high-throughput strategies. LUMIER is thus a valuable tool for the identification and characterization of dynamically regulated PPIs in mammalian systems. Here, we describe a manual version of LUMIER in a 96-well format that can be easily implemented in any laboratory.

PMID: 28188528 [PubMed - in process]



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SMYD2 lysine methyltransferase regulates leukemia cell growth and regeneration after genotoxic stress.

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SMYD2 lysine methyltransferase regulates leukemia cell growth and regeneration after genotoxic stress.

Oncotarget. 2017 Feb 06;:

Authors: Zipin-Roitman A, Aqaqe N, Yassin M, Biechonski S, Amar M, van Delft MF, Gan OI, McDermott SP, Buzina A, Ketela T, Shlush L, Xie S, Voisin V, Moffat J, Minden MD, Dick JE, Milyavsky M

Abstract
The molecular determinants governing escape of Acute Myeloid Leukemia (AML) cells from DNA damaging therapy remain poorly defined and account for therapy failures. To isolate genes responsible for leukemia cells regeneration following multiple challenges with irradiation we performed a genome-wide shRNA screen. Some of the isolated hits are known players in the DNA damage response (e.g. p53, CHK2), whereas other, e.g. SMYD2 lysine methyltransferase (KMT), remains uncharacterized in the AML context. Here we report that SMYD2 knockdown confers relative resistance to human AML cells against multiple classes of DNA damaging agents. Induction of the transient quiescence state upon SMYD2 downregulation correlated with the resistance. We revealed that diminished SMYD2 expression resulted in the upregulation of the related methyltransferase SET7/9, suggesting compensatory relationships. Indeed, pharmacological targeting of SET7/9 with (R)-PFI2 inhibitor preferentially inhibited the growth of cells expressing low levels of SMYD2.Finally, decreased expression of SMYD2 in AML patients correlated with the reduced sensitivity to therapy and lower probability to achieve complete remission. We propose that the interplay between SMYD2 and SET7/9 levels shifts leukemia cells from growth to quiescence state that is associated with the higher resistance to DNA damaging agents and rationalize SET7/9 pharmacological targeting in AML.

PMID: 28187429 [PubMed - as supplied by publisher]



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Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds.

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Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds.

Front Immunol. 2017;8:38

Authors: Liu H, Saxena A, Sidhu SS, Wu D

Abstract
Therapeutic monoclonal antibodies have become molecules of choice to treat autoimmune disorders, inflammatory diseases, and cancer. Moreover, bispecific/multispecific antibodies that target more than one antigen or epitope on a target cell or recruit effector cells (T cell, natural killer cell, or macrophage cell) toward target cells have shown great potential to maximize the benefits of antibody therapy. In the past decade, many novel concepts to generate bispecific and multispecific antibodies have evolved successfully into a range of formats from full bispecific immunoglobulin gammas to antibody fragments. Impressively, antibody fragments such as bispecific T-cell engager, bispecific killer cell engager, trispecific killer cell engager, tandem diabody, and dual-affinity-retargeting are showing exciting results in terms of recruiting and activating self-immune effector cells to target and lyse tumor cells. Promisingly, crystallizable fragment (Fc) antigen-binding fragment and monomeric antibody or half antibody may be particularly advantageous to target solid tumors owing to their small size and thus good tissue penetration potential while, on the other hand, keeping Fc-related effector functions such as antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cell-mediated phagocytosis, and extended serum half-life via interaction with neonatal Fc receptor. This review, therefore, focuses on the progress of Fc engineering in generating bispecific molecules and on the use of small antibody fragment as scaffolds for therapeutic development.

PMID: 28184223 [PubMed - in process]



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