Low-density lipoprotein promotes microvascular thrombosis by enhancing von Willebrand factor self-association.

von Willebrand factor (VWF) mediates primary hemostasis and thrombosis in response to hydrodynamic forces. We previously showed that high shear promoted self-association of VWF into hyperadhesive strands, which can be attenuated by high-density lipoprotein (HDL) and apolipoprotein A-I. In this study, we show that low-density lipoprotein (LDL) binds VWF under shear and enhances self-association. Vortexing VWF in tubes resulted in its loss from the solution and deposition onto tube surfaces, which was prevented by HDL. At a stabilizing HDL concentration of 1.2 mg/mL, increasing concentrations of LDL progressively increased VWF loss, the effect correlating with the LDL-to-HDL ratio and not the absolute concentration of the lipoproteins. Similarly, HDL diminished deposition of VWF in a post-in-channel microfluidic device, whereas LDL increased both the rate and extent of strand deposition, with both purified VWF and plasma. Hypercholesterolemic human plasma also displayed accelerated VWF accumulation in the microfluidic device. The initial rate of accumulation correlated linearly with the LDL-to-HDL ratio. In Adamts13-/- and Adamts13-/-LDLR-/- mice, high LDL levels enhanced VWF and platelet adhesion to the myocardial microvasculature, reducing cardiac perfusion, impairing systolic function, and producing early signs of cardiomyopathy. In wild-type mice, high plasma LDL concentrations also increased the size and persistence of VWF-platelet thrombi in ionophore-treated mesenteric microvessels, exceeding the accumulation seen in similarly treated ADAMTS13-deficient mice that did not receive LDL infusion. We propose that targeting the interaction of VWF with itself and with LDL may improve the course of thrombotic microangiopathies, atherosclerosis, and other disorders with defective microvascular circulation.

Specific electrostatic interactions between charged amino acid residues regulate binding of von Willebrand factor to blood platelets.

The plasma protein von Willebrand factor (VWF) is essential for hemostasis initiation at sites of vascular injury. The platelet-binding A1 domain of VWF is connected to the VWF N-terminally located D'D3 domain through a relatively unstructured amino acid sequence, called here the N-terminal linker. This region has previously been shown to inhibit the binding of VWF to the platelet surface receptor glycoprotein Ibα (GpIbα). However, the molecular mechanism underlying the inhibitory function of the N-terminal linker has not been elucidated. Here, we show that an aspartate at position 1261 is the most critical residue of the N-terminal linker for inhibiting binding of the VWF A1 domain to GpIbα on platelets in blood flow. Through a combination of molecular dynamics simulations, mutagenesis, and A1-GpIbα binding experiments, we identified a network of salt bridges between Asp1261 and the rest of A1 that lock the N-terminal linker in place such that it reduces binding to GpIbα. Mutations aimed at disrupting any of these salt bridges activated binding unless the mutated residue also formed a salt bridge with GpIbα, in which case the mutations inhibited the binding. These results show that interactions between charged amino acid residues are important both to directly stabilize the A1-GpIbα complex and to indirectly destabilize the complex through the N-terminal linker.

High-density lipoprotein modulates thrombosis by preventing von Willebrand factor self-association and subsequent platelet adhesion.

The ability of von Willebrand factor (VWF) to initiate platelet adhesion depends on the number of monomers in individual VWF multimers and on the self-association of individual VWF multimers into larger structures. VWF self-association is accelerated by shear stress. We observed that VWF self-association occurs during adsorption of VWF onto surfaces, assembly of secreted VWF into hyperadhesive VWF strings on the endothelial surface, and incorporation of fluid-phase VWF into VWF fibers. VWF adsorption under static conditions increased with increased VWF purity and was prevented by a component of plasma. We identified that component as high-density lipoprotein (HDL) and its major apolipoprotein ApoA-I. HDL and ApoA-I also prevented VWF on the endothelium from self-associating into longer strands and inhibited the attachment of fluid-phase VWF onto vessel wall strands. Platelet adhesion to VWF fibers was reduced in proportion to the reduction in self-associated VWF. In a mouse model of thrombotic microangiopathy, HDL also largely prevented the thrombocytopenia induced by injection of high doses of human VWF. Finally, a potential role for ApoA-I in microvascular occlusion associated with thrombotic thrombocytopenic purpura and sepsis was revealed by the inverse relationship between the concentration of ApoA-I and that of hyperadhesive VWF. These results suggest that interference with VWF self-association would be a new approach to treating thrombotic disorders.

Cold temperature induces a TRPM8-independent calcium release from the endoplasmic reticulum in human platelets.

The detection of temperature by the human sensory system is life-preserving and highly evolutionarily conserved. Platelets are sensitive to temperature changes and are activated by a decrease in temperature, akin to sensory neurons. However, the molecular mechanism of this temperature-sensing ability is unknown. Yet, platelet activation by temperature could contribute to numerous clinical sequelae, most importantly to reduced quality of ex vivo-stored platelets for transfusion. In this multidisciplinary study, we present evidence for the expression of the temperature-sensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in human platelets and precursor cells. We found the TRPM8 mRNA and protein in MEG-01 cells and platelets. Inhibition of TRPM8 prevented temperature-induced platelet activation and shape change. However, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. When exposing platelets to below-normal body temperature, we detected a cytosolic calcium increase which was independent of TRPM8 but was completely dependent on the calcium release from the endoplasmic reticulum. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies. Our study suggests that the cold response of platelets is complex and TRPM8 appears to play a role in early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response.

Evidence-Based Lifestyle Guidelines and Self-Management Strategies Utilized by Women with Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a complex endocrine disorder, affecting 13% of reproductive-aged women. While lifestyle management is the first-line treatment for improving complications, women experience challenges with implementation. This cross-sectional study aims to identify the types and sources of dietary and physical activity (PA) interventions implemented by women with PCOS and understand how they use self-management strategies to support lifestyle change. An online questionnaire was disseminated via a consumer-based PCOS website (May 2015-2016). Women (n = 1167) were aged 18-45 years and primarily born within the United States (70%). A quarter or less of women (diet 25%, PA 14%) sought lifestyle advice from health professionals (medical clinicians or dietitians) compared to over half (diet 59%, PA 67%) using alternative sources, namely from online platforms. While only 33% and 16% of women reported following formal dietary or PA guidelines, respectively, 57% had implemented a 'special diet' to manage their condition, many of which were inconsistent with evidence-based practice in PCOS. Participants also displayed a low level of engagement with important self-management behaviors, including goal setting and positive self-talk. These findings suggest that online information may promote inaccurate and ineffective lifestyle advice and emphasize the need to increase engagement with qualified health professionals.

Cold temperature induces a TRPM8-independent calcium release from the endoplasmic reticulum in human platelets.

Platelets are sensitive to temperature changes and akin to sensory neurons, are activated by a decrease in temperature. However, the molecular mechanism of this temperature-sensing ability is unknown. Yet, platelet activation by temperature could contribute to numerous clinical sequelae, most importantly to reduced quality of ex vivo-stored platelets for transfusion. In this interdisciplinary study, we present evidence for the expression of the temperature-sensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in human platelets and precursor cells. We found the TRPM8 mRNA and protein in MEG-01 cells and platelets. Inhibition of TRPM8 prevented temperature-induced platelet activation and shape change. However, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. When exposing platelets to below-normal body temperature, we detected a cytosolic calcium increase which was independent of TRPM8 but was completely dependent on the calcium release from the endoplasmic reticulum. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies. Our study suggests that the cold response of platelets is complex and TRPM8 appears to play a role in early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response.

The primary care nutrition training program: an approach to communication on behavior change.

The New York City Department of Health has designed a Primary Care Nutrition Training program for implementation in high-need neighborhoods that face growing diet-related epidemics of diabetes and obesity and a heavy burden of cardiovascular disease. Seven hundred fifty-six primary care team members complete pretest surveys and 665 complete posttest surveys at 45 training sessions between January and July 2007. Skills-building sessions center on the innovative application of visual aids and manipulatives and the provision of specific language for addressing nutritional issues with patients in busy primary care settings. Program evaluation data indicate that the training was well received by participants of all education levels, including medical assistants, physicians, nurses, and others, with 91% noting that the training content was pitched at about the right comprehension level for them. The Primary Care Nutrition Training Program offers a practical approach to continuing education for health professionals that may help to address the dearth of nutrition services currently in urban primary care.

Helium in diamonds unravels over a billion years of craton metasomatism.

Chemical events involving deep carbon- and water-rich fluids impact the continental lithosphere over its history. Diamonds are a by-product of such episodic fluid infiltrations, and entrapment of these fluids as microinclusions in lithospheric diamonds provide unique opportunities to investigate their nature. However, until now, direct constraints on the timing of such events have not been available. Here we report three alteration events in the southwest Kaapvaal lithosphere using U-Th-He geochronology of fluid-bearing diamonds, and constrain the upper limit of He diffusivity (to D ≈ 1.8 × 10-19 cm2 s-1), thus providing a means to directly place both upper and lower age limits on these alteration episodes. The youngest, during the Cretaceous, involved highly saline fluids, indicating a relationship with late-Mesozoic kimberlite eruptions. Remnants of two preceding events, by a Paleozoic silicic fluid and a Proterozoic carbonatitic fluid, are also encapsulated in Kaapvaal diamonds and are likely coeval with major surface tectonic events (e.g. the Damara and Namaqua-Natal orogenies).

GRADE guidelines: 21 part 1. Study design, risk of bias, and indirectness in rating the certainty across a body of evidence for test accuracy.

OBJECTIVES: This article provides updated GRADE guidance about how authors of systematic reviews and health technology assessments and guideline developers can assess the results and the certainty of evidence (also known as quality of the evidence or confidence in the estimates) of a body of evidence addressing test accuracy (TA). STUDY DESIGN AND SETTING: We present an overview of the GRADE approach and guidance for rating certainty in TA in clinical and public health and review the presentation of results of a body of evidence regarding tests. Part 1 of the two parts in this 21st guidance article about how to apply GRADE focuses on understanding study design issues in test accuracy, provide an overview of the domains, and describe risk of bias and indirectness specifically. RESULTS: Supplemented by practical examples, we describe how raters of the evidence using GRADE can evaluate study designs focusing on tests and how they apply the GRADE domains risk of bias and indirectness to a body of evidence of TA studies. CONCLUSION: Rating the certainty of a body of evidence using GRADE in Cochrane and other reviews and World Health Organization and other guidelines dealing with in TA studies helped refining our approach. The resulting guidance will help applying GRADE successfully for questions and recommendations focusing on tests.

GRADE guidelines: 21 part 2. Test accuracy: inconsistency, imprecision, publication bias, and other domains for rating the certainty of evidence and presenting it in evidence profiles and summary of findings tables.

OBJECTIVES: This article provides updated GRADE guidance about how authors of systematic reviews and health technology assessments and guideline developers can rate the certainty of evidence (also known as quality of the evidence or confidence in the estimates) of a body of evidence addressing test accuracy (TA) on the domains imprecision, inconsistency, publication bias, and other domains. It also provides guidance for how to present synthesized information in evidence profiles and summary of findings tables. STUDY DESIGN AND SETTING: We present guidance for rating certainty in TA in clinical and public health and review the presentation of results of a body of evidence regarding tests. RESULTS: Supplemented by practical examples, we describe how raters of the evidence can apply the GRADE domains inconsistency, imprecision, and publication bias to a body of evidence of TA studies. CONCLUSION: Using GRADE in Cochrane and other reviews as well as World Health Organization and other guidelines helped refining the GRADE approach for rating the certainty of a body of evidence from TA studies. Although several of the GRADE domains (e.g., imprecision and magnitude of the association) require further methodological research to help operationalize them, judgments need to be made on the basis of what is known so far.

The private partners of public health: public-private alliances for public good.

OBJECTIVE: We sought to convey lessons learned by the Centers for Disease Control and Prevention's (CDC's) Prevention Research Centers (PRCs) about the value and challenges of private-sector alliances resulting in innovative health promotion strategies. Several PRCs based in a variety of workplace and community settings contributed. METHODS: We conducted interviews with principal investigators, a literature review, and a review of case studies of private-sector alliances in a microbusiness model, a macrobusiness model, and as multiparty partnerships supporting public health research, implementation, and human resource services. RESULTS: Private-sector alliances provide many advantages, particularly access to specialized skills generally beyond the expertise of public health entities. These skills include manufacturing, distribution, marketing, business planning, and development. Alliances also allow ready access to employee populations. Public health entities can offer private-sector partners funding opportunities through special grants, data gathering and analysis skills, and enhanced project credibility and trust. Challenges to successful partnerships include time and resource availability and negotiating the cultural divide between public health and the private sector. Critical to success are knowledge of organizational culture, values, mission, currency, and methods of operation; an understanding of and ability to articulate the benefits of the alliance for each partner; and the ability and time to respond to unexpected changes and opportunities. CONCLUSION: Private-public health alliances are challenging, and developing them takes time and resources, but aspects of these alliances can capitalize on partners' strengths, counteract weaknesses, and build collaborations that produce better outcomes than otherwise possible. Private partners may be necessary for program initiation or success. CDC guidelines and support materials may help nurture these alliances.

Opto-acoustic imaging of relative blood oxygen saturation and total hemoglobin for breast cancer diagnosis.

Opto-acoustic imaging involves using light to produce sound waves for visualizing blood in biological tissue. By using multiple optical wavelengths, diagnostic images of blood oxygen saturation and total hemoglobin are generated using endogenous optical contrast, without injection of any external contrast agent and without using any ionizing radiation. The technology has been used in recent clinical studies for diagnosis of breast cancer to help distinguish benign from malignant lesions, potentially reducing the need for biopsy through improved diagnostic imaging accuracy. To enable this application, techniques for mapping oxygen saturation differences within tissue are necessary. Using biologically relevant opto-acoustic phantoms, we analyze the ability of an opto-acoustic imaging system to display colorized parametric maps that are generated using a statistical mapping approach. To mimic breast tissue, a material with closely matching properties for optical absorption, optical scattering, acoustic attenuation, and speed of sound is used. The phantoms include two vessels filled with whole blood at oxygen saturation levels determined using a sensor-based approach. A flow system with gas-mixer and membrane oxygenator adjusts the oxygen saturation of each vessel independently. Datasets are collected with an investigational Imagio® breast imaging system. We examine the ability to distinguish vessels as the oxygen saturation level and imaging depth are varied. At depth of 15 mm and hematocrit of 42%, a sufficient level of contrast to distinguish between two 1.6-mm diameter vessels was measured for an oxygen saturation difference of ∼4.6 % . In addition, an oxygenated vessel was visible at a depth of 48 mm using an optical wavelength of 1064 nm, and a deoxygenated vessel was visible to a depth of 42 mm with 757 nm. The results provide insight toward using color mapped opto-acoustic images for diagnosing breast cancer.

Archaean and Proterozoic diamond growth from contrasting styles of large-scale magmatism.

Precise dating of diamond growth is required to understand the interior workings of the early Earth and the deep carbon cycle. Here we report Sm-Nd isotope data from 26 individual garnet inclusions from 26 harzburgitic diamonds from Venetia, South Africa. Garnet inclusions and host diamonds comprise two compositional suites formed under markedly different conditions and define two isochrons, one Archaean (2.95 Ga) and one Proterozoic (1.15 Ga). The Archaean diamond suite formed from relatively cool fluid-dominated metasomatism during rifting of the southern shelf of the Zimbabwe Craton. The 1.8 billion years younger Proterozoic diamond suite formed by melt-dominated metasomatism related to the 1.1 Ga Umkondo Large Igneous Province. The results demonstrate that resolving the time of diamond growth events requires dating of individual inclusions, and that there was a major change in the magmatic processes responsible for harzburgitic diamond formation beneath Venetia from the Archaean to the Proterozoic.Dating of inclusions within diamonds is used to reconstruct Earth's geodynamic history. Here, the authors report isotope data on individual garnet inclusions within diamonds from Venetia, South Africa, showing that two suites of diamonds define two isochrons, showing the importance of dating individual inclusions.

Efficacy of EZH2 inhibitory drugs in human papillomavirus-positive and human papillomavirus-negative oropharyngeal squamous cell carcinomas.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide with rates of HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) dramatically increasing. The overexpression of enhancer of zeste homolog 2 (EZH2), a histone methyltransferase responsible for the trimethylation at lysine 27 of histone 3 (H3K27me3), is associated with a poor clinical prognosis and aggressive HPV-positive phenotypes. METHODS: We utilized three EZH2 pathway inhibitors, GSK-343, DZNeP, and EPZ-5687, and tested their efficacy in two HPV-positive and two HPV-negative OPSCC cell lines. RESULTS: Treatment with GSK-343 decreased H3K27me3 in all cell lines and treatment with DZNeP decreased H3K27me3 in only HPV-negative cell lines as determined by Western blot. Cells treated with EPZ-5687 displayed no appreciable change in H3K27me3. Epigenetic effect on gene expression was measured via ddPCR utilizing 11 target probes. Cells treated with DZNeP showed the most dramatic expressional changes, with decreased EGFR in HPV-positive cell lines and an overall increase in proliferation markers in HPV-negative cell lines. GSK-343-treated cells displayed moderate expressional changes, with CCND1 increased in HPV-positive cell lines and decreased TP53 in HPV-negative SCC-1. EPZ-5687-treated cell lines displayed few expressional changes overall. Only DZNeP-treated cells displayed anti-proliferative characteristics shown in wound-healing assays. CONCLUSIONS: Our findings suggest that EZH2 inhibitors are a viable therapeutic option for the role of epigenetic effect, potentially sensitizing tumors to current chemotherapies or limiting cell differentiation.

Prevention of radiation induced xerostomia by surgical transfer of submandibular salivary gland into the submental space.

BACKGROUND AND PURPOSE: Xerostomia is a significant morbidity of radiation treatment in the management of head and neck cancers. We hypothesized that the surgical transfer of one submandibular salivary gland to the submental space, where it can be shielded from radiation treatment (XRT), would prevent xerostomia. MATERIALS AND METHODS: We conducted a prospective Phase II clinical trial and the patients were followed clinically with salivary flow studies and the University of Washington Quality of Life questionnaire. RESULTS: We report the results on 76 evaluable patients. The salivary gland transfer was done in 60 patients. Nine patients (of 60) did not have postoperative XRT and in eight patients (of 60) the transferred gland was not shielded from XRT due to proximity of disease. The median follow up is 14 months. Of the 43 patients with the salivary gland transfer and post-operative XRT with protection of the transferred gland, 81% have none or minimal xerostomia, and 19% developed moderate to severe xerostomia. Three patients (6.9%) developed local recurrence, five patients (11.6%) developed distant metastases and five patients (11.6%) have died. There were no complications attributed to the surgical procedure. CONCLUSION: Surgical transfer of a submandibular salivary gland to the submental space preserves its function and prevents the development of radiation induced xerostomia.

Plexiform fibrohistiocytic tumor: a rare low-grade malignancy of children and young adults.

Plexiform fibrohistiocytic tumor (PFT) is a low-grade, superficial, soft tissue neoplasm with a limited but significant ability to metastasize. This type of tumor only rarely presents in the skin of the head and neck. Clinicians first encountering young patients with facial neoplasia, such as a PFT, might be unaware of its exact oncologic potential and instead be primarily concerned with the cosmetic outcome. We treated a 17-year-old boy with a PFT on his cheek who was initially treated only by shave biopsy. The tumor subsequently recurred and metastasized to the cervical lymph nodes 3 years after the initial biopsy. Therefore, appropriate initial therapy for PFT requires complete excision with negative resection margins.

How medical students' behaviors and attitudes affect the impact of a brief curriculum on nutrition counseling.

OBJECTIVE: To evaluate a nutrition curriculum and explore the influence of medical students' own nutrition practices on its impact. METHODS: An anonymous survey was given to first-year medical students attending a required course immediately prior to and 2 weeks after a 2-hour interactive nutrition curriculum intervention in a large private urban medical school in New York, New York. Main outcomes included self-reported nutrition counseling confidence, ability to assess diet, and nutrition knowledge measured using 4-point Likert scales. RESULTS: One hundred eleven students completed surveys pre-curriculum (69%) and 121 completed them post-curriculum (75%). The authors found overall pre-post differences in dietary assessment ability (2.65 vs 3.05, P < .001) and counseling confidence (1.86 vs 2.22, P < .001). In addition to the curricular impact, students' nutrition-related behaviors and attitudes were positively associated with outcomes. CONCLUSIONS AND IMPLICATIONS: A nutrition curriculum for medical students improves students' nutrition counseling-related confidence, knowledge, and skills even when controlling for personal nutrition-related behaviors.

Formation of diamond in the Earth's mantle.

The principal sources of natural diamonds are peridotitic (about 2/3 of diamonds) and eclogitic (1/3) domains located at 140-200 km depth in the subcratonic lithospheric mantle. There, diamonds probably form during redox reactions in the presence of melt (likely for eclogitic and lherzolitic diamonds) or under subsolidus conditions in the presence of CHO fluids (likely for harzburgitic diamonds). Co-variations of δ(13)C and the nitrogen content of diamonds suggest that two modes of formation may have been operational in peridotitic sources: (1) reduction of carbonates, that during closed system fractionation drives diamond compositions to higher δ(13)C values and lower nitrogen concentrations and (2) oxidation of methane, that in a closed system leads to a trend of decreasing δ(13)C with decreasing nitrogen. The present day redox state of subcratonic lithospheric mantle is generally too reduced to allow for methane oxidation to be a widespread process. Therefore, reduction of carbonate dissolved in melts and fluids is likely the dominant mode of diamond formation for the Phanerozoic (545 Ma-present) and Proterozoic (2.5 Ga-545 Ma). Model calculations indicate, however, that for predominantly Paleoarchean (3.6-3.2 Ga) to Mesoarchean (3.2-2.8 Ga) harzburgitic diamonds, methane reduction is the principal mode of precipitation. This suggests that the reduced present day character (oxygen fugacity below carbonate stability) of peridotitic diamond sources may be a secondary feature, possibly acquired during reducing Archean (>2.5 Ga) metasomatism. Recycling of biogenic carbonates back into the mantle through subduction only became an important process in the Paleoproterozoic (2.5-1.6 Ga) and diamonds forming during carbonate reduction, therefore, may predominantly be post-Archean in age. For eclogitic diamonds, open system fractionation processes involving separation of a CO(2) fluid appear to dominate, but in principal the same two modes of formation (methane oxidation, carbonate reduction) may have operated. Direct conversion of graphitized subducted organic matter is not considered to be an important process for the formation of eclogitic diamonds. The possible derivation of (12)C enriched carbon in eclogitic diamonds from remobilized former organic matter is, however, feasible in some cases and seems likely involved, for example, in the formation of sublithospheric eclogitic diamonds from the former Jagersfontein Mine (South Africa).

Regulated expression of active biotinylated G-protein coupled receptors in mammalian cells.

We have developed a mammalian expression system suitable for the production of enzymatically biotinylated integral membrane proteins. The key feature of this system is the doxycycline (dox)-regulated co-expression of a secreted variant of Escherichia coli biotin ligase (BirA) and a target protein with a 13-residue biotin acceptor peptide (BioTag) appended to its extracellular domain. Here we describe the expression and functional analysis of three G-protein coupled receptors (GPCRs): protease-activated receptors (PARs) 1 and 2, and the platelet ADP receptor, P2Y(12). Clonal Chinese hamster ovary (CHO) Tet-On cell lines that express biotinylated GPCRs were rapidly isolated by fluorescence-activated cell sorting following streptavidin-FITC staining, thereby circumventing the need for manual colony picking. Analysis by Western blotting with streptavidin-HRP following endoglycosidase treatment revealed that all three GPCRs undergo N-linked glycosylation. The expression of biotinylated GPCRs on the cell surface was regulated by the concentration of dox in the medium, reaching a maximum at approximately 1 microg/mL dox. Similarly, the extent of GPCR biotinylation was dependent on biotin concentration, with maximum and complete biotinylation achieved upon supplementation with 50 microM biotin. Biotinylated PAR1 and PAR2 were readily and specifically cleaved on the surface of intact cells by their cognate proteases, and were capable of transducing extracellular stimuli, resulting in the downstream phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Notably, P2Y(12) mediated agonist-induced ERK phosphorylation only when it was expressed at low levels on the cell surface, highlighting the utility of regulated expression for the production of functionally active GPCRs in mammalian cells.

A versatile system for site-specific enzymatic biotinylation and regulated expression of proteins in cultured mammalian cells.

We have developed a system for producing biotinylated recombinant proteins in mammalian cells. The expression construct consists of an inducible tetracycline response element (TRE) that drives expression of a bicistronic cassette comprising a biotin acceptor peptide (BioTag) fused to either terminus of the target protein, the gene for Escherichia coli biotin ligase (BirA), and an intervening internal ribosome entry site (IRES). By either transient or stable transfection of Chinese hamster ovary (CHO) Tet-On cells, we successfully expressed, detected, and immobilized biotinylated human Itch, a pleiotropic multi-domain ubiquitin-protein ligase, as well as Gla-RTK, a putative vitamin K-dependent receptor tyrosine kinase. The biotinylation of recombinant Itch in transiently transfected CHO Tet-On cells required biotin supplementation and coexpression of BirA, occurred quantitatively and specifically on the lysine residue of the BioTag, and enabled detection of Itch by Western blot in as little as 10ng of total lysate protein. Stably selected clones were rapidly pre-screened for doxycycline (dox)-inducible BirA expression by ELISA, and subsequently screened for dox-inducible expression of biotinylated Itch. Biotinylated Gla-RTK was detectable in as little as 5ng of total lysate protein from transiently transfected CHO Tet-On cells, and exhibited pronounced tyrosine phosphorylation. In stable clones however, constitutive phosphorylation was prevented by reducing the expression level of Gla-RTK through the titration of dox. These results demonstrate the utility of this system for the expression of 'difficult' proteins, particularly those that are cytotoxic or those that may require lower expression levels to ensure appropriate post-translational modification.