EphA4-dependent Brachyury expression is required for dorsal mesoderm involution in the Xenopus gastrula.

Xenopus provides a well-studied model of vertebrate gastrulation, but a central feature, the movement of the mesoderm to the interior of the embryo, has received little attention. Here, we analyze mesoderm involution at the Xenopus dorsal blastopore lip. We show that a phase of rapid involution - peak involution - is intimately linked to an early stage of convergent extension, which involves differential cell migration in the prechordal mesoderm and a new movement of the chordamesoderm, radial convergence. The latter process depends on Xenopus Brachyury, the expression of which at the time of peak involution is controlled by signaling through the ephrin receptor, EphA4, its ligand ephrinB2 and its downstream effector p21-activated kinase. Our findings support a conserved role for Brachyury in blastopore morphogenesis.

Characterization of SHP-1 protein tyrosine phosphatase transcripts, protein isoforms and phosphatase activity in epithelial cancer cells.

We identified 7 SHP-1 (PTPN6) transcripts using epithelial cancer-derived cell lines. Four were shown to utilize the epithelial promoter 1 to transcribe a full-length, a partial (exon 3) or complete (exons 3 & 4) deletion of the N-SH2 domain, and also a non-coding transcript having a stop codon caused by a frame shift due to intron 2 retention. Three additional transcripts were shown to utilize the hematopoietic promoter 2 to transcribe a full-length, a partial (exon 3) deletion of the N-SH2 domain and a non-coding transcript with intron 2 retention. We show that endogenous proteins corresponding to the open-reading-frame (ORF) transcripts are produced. Using GST-fusion proteins we show that each product of the ORF SHP-1 transcripts has phosphatase activity and isoforms with an N-SH2 deletion have increased phosphatase activity and novel protein-protein interactions. This study is the first to document utilization of promoter 2 by SHP-1 transcripts and a noncoding transcript in human epithelial cells.

Immunohistochemical examination of the mTORC1 pathway in high grade prostatic intraepithelial neoplasia (HGPIN) and prostatic adenocarcinomas (PCa): a tissue microarray study (TMA).

BACKGROUND: The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase and a key regulator of protein synthesis and growth and is upregulated in many cancers. mTOR is activated by AKT phosphorylation (p-mTOR). p-mTOR associates with regulatory-associated protein of TOR (RAPTOR), forming the mTORC1 complex. mTORC1 promotes the activation of p70 ribosomal protein s6 kinase 1 (p70(S6K1)) and ribosomal protein s6 (RPS6). Upregulation of this pathway can lead to an aberrant increase in cell growth and metabolism characteristic of malignant transformation. METHODS: This study presents the immunohistochemical (IHC) expression of the mTORC1 pathway in prostate neoplasia. The expression of p-mTOR and RAPTOR and p-p70(S6K1) and p-RPS6 were examined in HGPIN and PCa using tissue microarrays (TMA). Since each case in our TMAs was represented by three tissue cores, we quantified the IHC intratumoral heterogeneity of mTOR expression. This extensive analysis is the first detailed assessment documenting the IHC heterogeneity of mTOR expression in HGPIN and prostate cancer and represents the first IHC description of the mTORC1 pathway in HGPIN and PCa. RESULTS: A Cochran-Armitage analysis demonstrated decreasing p-mTOR activity progressing from PIN through GL6 and GL7 to HG PCa. There was considerable intratumoral IHC heterogeneity within an individual patient. However, a statistically significant correlation was observed between p-mTOR, p-p70(S6K1), and p-RPS6 in each representative core. CONCLUSION: mTOR inhibitors may be an effective treatment for HGPIN and PCa. The extent of mTOR expression in an individual patient would determine the effective use of mTOR inhibitors as a potential therapeutic strategy.

Urinary bladder tissue engineering using natural scaffolds in a porcine model: role of Toll-like receptors and impact of biomimetic molecules.

INTRODUCTION: Natural scaffolds have been shown to induce T helper 2 (TH2)-specific immune responses in host tissues; however, the precise mechanisms that underlie this immune response are unknown. Using a porcine animal model, we evaluated the role of Toll-like receptors (TLRs) and matrix remodelling in the implantation of bladder acellular matrix (ACM) grafts and ACMs fortified with biomimetic materials. MATERIALS AND METHODS: Bladders were decellularized with detergent and treated in 3 different ways prior to implantation: ACM alone, hyaluronic acid (HA)-ACM and HA-vascular endothelial growth factor (VEGF)-ACM. Animals were sacrificed at 4 or 10 weeks post-implantation and total gene expressions for TH2 (IL-4), TH1 (IFN-γ), TLR2, TLR4, and TGF-ß1 were analyzed using real-time RT-PCR. Using histology (H&E and Masson's trichrome) and immunohistochemistry (uroplakin, α-smooth muscle actin, CD31 and factor VIII) the regenerative capacity was correlated with the gene expression of different proteins. RESULTS: IL-4, TLR2, and TLR4 gene expression were markedly decreased at 4 and 10 weeks in both the HA-ACM group and the HA-VEGF-ACM group compared to ACM alone. IFN-γ expression was negligible in all groups and time periods. TGF-ß1 expression was highest in the HA- and VEGF-treated grafts. Recellularization was inversely proportional to TLR and TH2 expression but proportional to TGF-ß1. CONCLUSION: ACM alone grafts demonstrated stronger TLR4 expression which may promote a distinct TH2 immune response and a reduced regenerative capacity in grafts. Treatment of grafts with HA and VEGF may help regulate host immune responses by reducing TLR4 and IL-4 and increasing TGF-ß1.

Breast Implant-Associated Anaplastic Large Cell Lymphoma (ALCL): A Case Report.

BACKGROUND Anaplastic large cell lymphomas (ALCL) are a rare type of primary breast lymphoma. The association between breast implants and ALCL was first described in 1997 and since then 34-173 cases have been presented. The annual incidence of breast implant-associated ALCL (BI-ALCL) is 0.1-0.3 per 100 000 women who undergo breast reconstruction, and cases are often underreported due to the rarity of these tumors. BI-ALCL arises from the inflammatory T cells surrounding the fibrous capsule, and most tumors are in situ. CASE REPORT Here, we present the case of a 51-year-old woman with ALCL following bilateral silicone breast implants. The patient presented with breast enlargement and tenderness 9 years following reconstructive surgery. Imagining studies showed fluid collection surrounding the affected breast implant. Staging studies and histocytopathology examination confirmed the presence BI-ALCL without capsular invasion or metastasis. Complete surgical excision was performed. The patient continues to be in complete remission. CONCLUSIONS Due to the rarity of these tumors, establishing the diagnosis of BI-ALCL can be challenging and requires a multidisciplinary approach. Clinicians should be aware of the relationship between breast implants and BI-ALCL.

Blood Vessel Maturation in Health and Disease and its Implications for Vascularization of Engineered Tissues.

Engineered blood vessels have often been found to be immature and unstable. Similarly, numerous pathologies such as diabetic retinopathy and cancer are characterized by highly abnormal, defective, hypervascular networks, consisting of immature, leaky, and irregular vessels with a marked loss of perivascular cell coverage. An emerging therapeutic concept in treatment of such vascular diseases and their management is the potential to normalize blood vessels by strengthening the cellular components that form the vascular network. Vessel normalization is characterized by the reduction in the number and size of immature vessels, a decrease in interstitial fluid pressure, and increase in perivascular cell coverage. Understanding the molecular and cellular defects associated with abnormal blood vessels will allow us to find appropriate treatment options that can promote normal blood vessel development. These, in turn, can be applied to improve vessel maturation in engineered tissues. In this review, we describe the major perivascular abnormalities associated with various human diseases and engineered vasculatures and the major advances in obtaining mature vasculatures for translational applications.

Canadian DOs: International Expansion of Osteopathic Medical Education North of the Border.

CONTEXT: The growth of osteopathic medicine in the United States has led to a vibrant expansion of the profession internationally. Canadian students represent the majority of international applicants and matriculants to US colleges of osteopathic medicine (COMs); however, to our knowledge, no studies have explored this population. OBJECTIVE: To gain a better understanding of Canadian students attending US colleges of osteopathic medicine by examining their residency training preference, visa preference, intent to practice in the United States or Canada, receptiveness to incorporating osteopathic manipulative medicine into practice, specialty preference, estimated debt incurred, and effect of debt on specialty choice. METHODS: A 10-question electronic survey was sent to Canadian osteopathic medical students in the 17 COMs and branch campuses that accept international applicants. The initial survey pool consisted of frst-, second-, third-, and fourth-year medical students (classes of 2014-2017) compiled from a database managed by the Canadian Osteopathic Medical Student Association. RESULTS: Of the 102 students contacted, 66 (65%) completed the survey. Respondents had a strong desire to practice in Canada (44 [67%]) but were considering an Accreditation Council on Graduate Medical Education (ACGME) or dually accredited residency program in the United States (46 [70%] and 15 [23%], respectively) that would sponsor an H1B visa. Respondents were receptive to incorporating osteopathic manipulative medicine into practice (44 [67%]). Most respondents chose non-primary care specialties (40 [61%]) and incurred a debt of more than $200,000 (44 of 65 [68%]); however, debt had a limited infuence on respondents' choice of specialty (χ23=1.911; P=.591). CONCLUSION: Most respondents planned to complete ACGME training, to return to Canada to practice medicine, and to practice in a non-primary care specialty. As a growing population that will play a large role in the expansion and reception of the profession internationally, Canadian osteopathic medical students and US-trained Canadian DOs merit further examination.

Doctors of osteopathic medicine (DO): a Canadian perspective.

BACKGROUND: Doctors of osteopathic medicine (DO) are one of the fastest growing segments of health care professionals in the United States. Although Canada has taken significant leaps in the acknowledgment of US trained DOs, there continues to be a lack of understanding of the profession by Canadian trained physicians. In this article, we provide a brief overview of osteopathic medical education and training in the United States. METHOD: Current information of osteopathic training by American Association of Colleges of Osteopathic Medicine (AACOM) and American Osteopathic Association (AOA) was presented. Data pertaining to Canadians enrolled in osteopathic colleges was compared with allopathic (MD) and international medical graduates (IMGs). RESULTS: Doctors of osteopathic medicine programs provide an additional pathway for students interested in pursuing a medical education. Canadian applications to osteopathic colleges are expected to grow due to successful post-graduate US residency matching, increased difficulty of matriculating at Canadian medical schools, and a greater awareness of the profession in Canada. CONCLUSIONS: Given the increasing enrollment of Canadian students in US osteopathic medical schools, we expect that Canadian DOs will play a significant role in shaping health care in both the US and Canada.

SHP-1 protein tyrosine phosphatase associates with the adaptor protein CrkL.

SHP-1, encoded by the PTPN6 gene, is a protein tyrosine phosphatase with two src-homology-2 (SH2) domains that is implicated as providing suppression of hematopoietic malignancies. A number of reports have shown protein-protein interactions between SHP-1 SH2 domains and tyrosine-phosphorylated proteins. However, despite its having three proline-rich, potential SH3-binding motifs, no reports of protein-protein interactions through src-homology-3 (SH3)-binding domains with SHP-1 have been described. Herein we show that the SH3 domain-containing CT10 regulator of kinase-like (CrkL) adaptor protein associates with SHP-1. We also provide results that suggest this association is due to CrkL binding to PxxP domains located at amino acid residues 158-161 within the SHP-1 C-terminal SH2 domain, and amino acid residues 363-366 within its phosphatase domain. This study is the first to identify and define an interaction between SHP-1 and an SH3 domain-containing protein. Our findings provide an alternative way that SHP-1 can be linked to potential substrates.

mTOR-RAPTOR and 14-3-3σ immunohistochemical expression in high grade prostatic intraepithelial neoplasia and prostatic adenocarcinomas: a tissue microarray study.

BACKGROUND: The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase which associates with regulatory-associated protein of TOR (RAPTOR), forming the mTORC1 complex, which is necessary for activation of the mTOR pathway. 14-3-3σ belongs to a family of proteins known to regulate the mTOR-RAPTOR interaction and signalling of this cascade. The mTOR pathway is a key regulator of protein synthesis and growth and is up-regulated in many cancers. The correlation of mTOR, RAPTOR and 14-3-3σ in high grade prostatic intraepithelial neoplasia (HGPIN) and prostate cancer has not previously been investigated. AIMS: To examine the immunohistochemical expression of phosphorylated mTOR (p-mTOR), RAPTOR and 14-3-3σ in HGPIN and prostatic adenocarcinoma (PCa) using tissue microarrays. METHODS AND RESULTS: There were contrasting immunohistochemical patterns of expression for mTOR and 14-3-3σ in HGPIN and PCa. Cochran-Armitage analysis demonstrated decreasing p-mTOR and increasing 14-3-3σ expression, progressing from PIN through GL6 and GL7 to high grade PCa. In cores with coexistent staining for 14-3-3σ and p-mTOR, the expression of each marker was restricted to different geographical areas of an individual core. CONCLUSION: The inverse correlation of p-mTOR and 14-3-3σ expression supports the role of 14-3-3σ as an inhibitor of p-mTOR activity in the prostate. The extent of 14-3-3σ and mTOR expression in an individual patient with prostate cancer would determine how effective the use of mTOR inhibitors would be as potential therapeutic agents.