A double-spike MC TIMS measurement procedure for low-amount Ca isotopic analysis of limited biological tissue samples.

The application of Ca isotopic analysis in biomedical studies has great potential to identify changes in Ca metabolism and bone metabolism. Reliable measurement of Ca isotope-amount ratios is challenging considering limited Ca amounts and significant procedural blank levels. In this study, Ca purification was performed using the DGA Resin, optimized for low procedural blanks and separation of Ca from matrix elements and isobaric interferences (Na, Mg, K, Ti, Fe, Ba), while maintaining quasi-quantitative recoveries which are sufficient since a 42Ca-48Ca double-spike (DS) was applied. Ca isotopic analysis was performed using multicollector thermal ionization mass spectrometry (MC TIMS). The obtained procedural Ca blank of ≤10 ng enables processing of limited Ca amounts down to 670 ng. Data reduction of the measured Ca isotope-amount ratios was performed using an in-house developed software solving the DS algorithm. Data quality was improved by extension of equilibration time of the sample-DS mixture and implementation of a normalization strategy for raw isotopic data. The reported δ(44Ca/40Ca)NIST SRM 915a of NIST SRM 915a processed as a sample was found to be 0.01 ‰ ± 0.08 ‰ (2 SD, n = 15). Ca isotope-amount ratios of the reference material NIST SRM 1400 (bone ash), NIST SRM 1486 (bone meal), GBW07601 (human hair), and IAPSO (seawater) were in good agreement within uncertainty with literature data. Novel data on additional reference materials for biological tissues (hair) is presented, which might indicate a potential fractionation of Ca incorporated into hair tissue when compared to the blood pool.

Physician attitudes about non-medical switching to biosimilars: results from an online physician survey in the United States.

OBJECTIVE: This study was designed to understand the level of familiarity of US rheumatologists, gastroenterologists and dermatologists with biosimilar therapies, their experience with non-medical switching (switching medications for reasons unrelated to patient health) of patients between biologics and their attitudes towards switching from a biologic to a biosimilar. METHODS: A total of 297 US physicians who currently prescribe biologics for their patients completed a 15-minute online survey. Rheumatologists, dermatologists and gastroenterologists were included. RESULTS: The majority of physicians (84%) did not want stable patients undergoing a non-medical switch to a biosimilar. While 60% of physicians believed non-medical switching to biosimilars may have a positive impact on healthcare system costs, multiple negative impacts were also expected. A majority of physicians anticipated a negative impact on patient mental health (59%), treatment efficacy (57%), patient safety (53%) and physician office management (60%). CONCLUSIONS: The majority of physicians had concerns regarding non-medical switching to biosimilars and the impact such switching would have on patient care and physician practice.

Patient attitudes about non-medical switching to biosimilars: results from an online patient survey in the United States.

OBJECTIVE: To evaluate patient attitudes regarding non-medical switching (NMS) to biosimilars among patients with autoimmune disease currently receiving a biologic. METHODS: An online survey was conducted among patients meeting the following criteria: ≥18 years of age; residing in the US; diagnosis of rheumatoid arthritis, Crohn's disease, ulcerative colitis, psoriasis or psoriatic arthritis; currently taking a biologic; and consenting to participate. Patients answered questions about their attitudes and experiences related to NMS. Descriptive statistics were used to summarize responses. RESULTS: A total of 1696 patients completed the 20-min survey. Eighty-five per cent of patients were concerned that biosimilars wouldn't treat their disease as well; 85% didn't want to switch to a biosimilar if their current biologic was helping their disease; and 83% were concerned that switching may cause more side-effects. Twenty per cent of patients had previously received notification about a potential NMS to another biologic (that was not a biosimilar) from their insurance company. Of these, 79% took at least one action to avoid the NMS and 45% ultimately switched. Of these patients (n = 150), 67% indicated that their previous biologic worked well for them and 70% didn't want to switch to another biologic. Most patients who switched (67%) did so to avoid paying a higher cost. More than half (56%) went without therapy for administrative reasons during the period of transition from the old biologic to the other treatment. CONCLUSIONS: Patients reported multiple concerns about NMS that might impact treatment outcomes, and many of the patients who non-medically switched in this survey missed treatments. Future studies should be conducted on patient expectations and experiences with NMS to understand the impact on healthcare delivery, treatment persistency, and patient outcomes. The patient perspective and experience should be considered by decision-makers when developing coverage policies for biosimilar medications and associated communication strategies.

The Impact of Nonphysician Providers on Diagnostic and Interventional Radiology Practices: Operational and Educational Implications.

The numbers of nurse practitioners (NPs) and physician assistants (PAs) are increasing throughout the entire health care enterprise, and a similar expansion continues within radiology. The use of radiologist assistants is growing in some radiology practices as well. The increased volume of services rendered by this growing nonphysician provider subset of the health care workforce within and outside radiology departments warrants closer review, particularly with regard to their potential influence on radiology education and medical imaging resource utilization. In this article (the second in a two-part series), the authors review recent literature and offer recommendations for radiology practices regarding the impact NPs, PAs, and radiologist assistants may have on interventional and diagnostic radiology practices. Their potential impact on medical education is also discussed. Finally, staffing for radiology departments, as a result of an enlarging nonradiology NP and PA workforce ordering diagnostic imaging, is considered.

The impact of nonphysician providers on diagnostic and interventional radiology practices: regulatory, billing, and compliance perspectives.

The numbers of nurse practitioners and physician assistants are increasing throughout the entire health care enterprise, and a similar expansion continues within radiology. Some practices have instead embraced radiologist assistants. The increased volume of services rendered by this growing nonphysician provider subset of the health care workforce within and outside of radiology departments warrants closer review. The authors evaluate the recent literature and offer recommendations to radiology practices regarding both regulatory and scope-of-practice issues related to these professionals. Additionally, billing and compliance issues for care provided by nurse practitioners, physician assistants, and radiologist assistants are detailed. An analysis of the integration of these professionals into interventional and diagnostic radiology practices, as well as potential implications for medical education, is provided in the second part of this series.

Expanding roles of nurse practitioners and physician assistants as providers of nonvascular invasive radiology procedures.

PURPOSE: To evaluate national trends in nonvascular invasive radiology procedures performed by advanced practice providers (APPs), focusing specifically on nurse practitioners and physician assistants. METHODS: Nonvascular invasive radiology procedures commonly performed by APPs at our 2 largest hospitals were used to identify procedure groups for national trends analysis. We mapped categories of services annually to then-current Current Procedural Terminology codes from 1994 to 2012 and identified national Medicare Part B beneficiary paid claims frequency using Physician Supplier Procedure Summary Master Files. Trends were studied for APPs, radiologists, and all providers nationally for 7 categories of service: paracentesis, thoracentesis, fine-needle aspiration (FNA), superficial lymph node biopsy, abdominal biopsy, thoracic biopsy, and abdominal drainage. RESULTS: Of 1,352 nonvascular invasive procedures performed by APPs at our facilities over a 1-year period through August 2013, a total of 1,161 (85.9%) fell into the 7 defined categories. Between 1994 and 2012, national Medicare claims by APPs increased dramatically for all of these categories: paracentesis from 0 to 17,967; thoracentesis from 119 to 4,141 (+3,379%); FNA from 0 to 3,921; superficial lymph node biopsy from 0 to 251; abdominal biopsy from 1 to 1,819 (+1,818%); thoracic biopsy from 0 to 552; and abdominal drainage from 37 to 410 (+1,008%). Overall, volumes increased for both radiologists and all providers, with the total fraction of national services performed by APPs increasing from 0% to 10.7% for paracentesis, 0.1% to 5.7% for thoracentesis, 0% to 2.1% for FNA, 0% to 1.4% for superficial lymph node biopsy, 0% to 1.7% for abdominal biopsy, 0% to 1.0% for thoracic biopsy, and 0.1% to 1.2% for abdominal drainage. CONCLUSIONS: Although APPs perform a relatively small portion of commonly performed nonvascular invasive radiology procedures nationally, paid Medicare claims for those services have increased dramatically over nearly 2 decades, and at a faster pace than that for all providers as a whole. Given the multiple hurdles involved in obtaining Medicare reimbursement, that growth indicates increasing acceptance of APPs as procedure service providers at the institutional credentialing, state licensure, and payer policy levels.

Optimization of endometrial preparation results in a normal endometrial function test (EFT) and good reproductive outcome in donor ovum recipients.

PURPOSE: Numerous studies have investigated potential markers of endometrial receptivity as predictors of successful implantation. Cyclin E and p27 have recently been studied using the endometrial function test (EFT). Our objective is to determine the correlation between the expression of cyclin E and p27 and the adequacy of uterine preparation of recipients using donor oocytes. METHODS: Twenty recipients undergoing preparatory cycles with leuprolide acetate, estrogen, and progesterone. Endometrial biopsies were obtained 10-12 days after progesterone supplementation following the course of estrogen. The tissue was prepared for histological analysis and immunohistochemical staining for cyclin E assessment. The outcome of their subsequent ovum donation cycle was blinded to the reviewer of the EFT. RESULTS: All recipients showed normal luteal transformation. Nineteen (95%) of the recipients had a normal EFT. This is significantly higher than what we demonstrated, previously, in unexplained infertility patients, where only 40% of such patients had a normal EFT. Thirteen recipients with a normal EFT had a clinical pregnancy, while 6 did not become pregnant in their subsequent transfer cycles. The sole patient with an abnormal EFT did not conceive on 2 subsequent cycles. CONCLUSIONS: While a normal EFT does not guarantee a successful pregnancy, an abnormal EFT appears to be associated with pregnancy failure. This may be useful in identifying women who need adjustments to their stimulation protocols prior to progressing to a physically, emotionally, and financially costly cycle.

Characterization of the structure and composition of gecko adhesive setae.

The ability of certain reptiles to adhere to vertical (and hang from horizontal) surfaces has been attributed to the presence of specialized adhesive setae on their feet. Structural and compositional studies of such adhesive setae will contribute significantly towards the design of biomimetic fibrillar adhesive materials. The results of electron microscopy analyses of the structure of such setae are presented, indicating their formation from aggregates of proteinaceous fibrils held together by a matrix and potentially surrounded by a limiting proteinaceous sheath. Microbeam X-ray diffraction analysis has shown conclusively that the only ordered protein constituent in these structures exhibits a diffraction pattern characteristic of beta-keratin. Raman microscopy of individual setae, however, clearly shows the presence of additional protein constituents, some of which may be identified as alpha-keratins. Electrophoretic analysis of solubilized setal proteins supports these conclusions, indicating the presence of a group of low-molecular-weight beta-keratins (14-20 kDa), together with alpha-keratins, and this interpretation is supported by immunological analyses.

A rapid and sensitive PCR-based diagnostic assay to detect bovine herpesvirus 1 in routine diagnostic submissions.

We describe a rapid, sensitive and specific polymerase chain reaction (PCR) assay for the detection of BHV1 DNA in a range of routine diagnostic submissions without the need for prior virus isolation. The assay, which is based on the selected amplification of a portion of the viral tk gene, detected both BHV1.1 and BHV1.2 subtypes in a panel of 15 characterised field isolates, and its sensitivity was estimated to be <0.125 TCID(50). BHV2, alcephaline herpesvirus, BHV4, equine herpesvirus 1 (EHV1), EHV4 and pseudorabies virus were not detected confirming the specificity of the assay. One hundred and five diagnostic submissions, including tissues, nasal secretions and nasal swabs were taken from cattle with respiratory disease and tested using the routine methods of virus isolation (VI) and the fluorescent antibody test (FAT), and the results were compared with those obtained by PCR. The PCR assay detected BHV1 DNA in all samples that were positive by VI. BHV1 DNA was also detectable by PCR in raw and extended semen samples at a sensitivity of 1 TCID(50) per 50microl. The assay also detected BHV5, permitting differentiation between it and BHV1 by virtue of the size of the amplified PCR product. The PCR assay is more sensitive and independent of sample quality than either virus isolation or FAT, and it is faster than virus isolation. The sample preparation method is simple with few steps involved. There are no extra post-amplification blotting/hybridisation steps and the assay is not based on a nested PCR strategy that might otherwise exacerbate the problem of oversensitivity/contamination in the routine use of such a test in a diagnostic laboratory. This assay would permit discrimination between those animals naturally infected with wild type BHV1 and those vaccinated with tk-BHV1 strains.

The bfl-1 gene is transcriptionally upregulated by the Epstein-Barr virus LMP1, and its expression promotes the survival of a Burkitt's lymphoma cell line.

The recently identified bfl-1 gene (also known as A1 or GRS), a homologue of bcl-2, encodes an antiapoptotic protein that suppresses apoptosis induced by the p53 tumor suppressor protein and exhibits proliferative and potent cooperative transforming activities. We show that elevated levels of bfl-1 mRNA are a feature of Epstein-Barr virus (EBV)-immortalized B-cell lines and Burkitt's lymphoma cell lines expressing the full spectrum of EBV latent proteins. Using an EBV-negative Burkitt's lymphoma cell line in which the expression of EBV latent membrane protein 1 (LMP1) is inducibly regulated by tetracycline, we demonstrate that LMP1 expression coincides with a dramatic increase in the level of bfl-1 mRNA. Also in this system, an increase in the level of Bcl-2 protein was seen to occur earlier than that of bcl-2 mRNA, suggesting that both transcriptional and translational mechanisms are involved in the control of Bcl-2 expression by LMP-1. We show that elevated bfl-1 mRNA stability can contribute to this effect of LMP-1, thus providing evidence of a novel mechanism of gene regulation by this EBV protein. Upregulation of bfl-1 by LMP1 was not observed in the T-cell line Jurkat or the epithelial cell line C33A. Ectopic expression of Bfl-1 in an EBV-positive cell line exhibiting a latency type I infection protects against apoptosis induced by growth factor deprivation, thereby providing a functional role for Bfl-1 in this cellular context and adding Bfl-1 to the list of antiapoptotic proteins whose expression is modulated by EBV. This is the first report of the regulation of bfl-1 expression by a viral protein, and this novel finding may thus represent an important link between the EBV oncoprotein LMP1 and its cellular growth-transforming properties.

The Epstein-Barr virus determined nuclear antigens EBNA-3A, -3B, and -3C repress EBNA-2-mediated transactivation of the viral terminal protein 1 gene promoter.

The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) has been shown to transactivate both cellular and viral gene promoters including the promoter for the viral terminal protein 1 gene (TP-1). We investigated whether three other EBV nuclear antigens EBNA-3A, -3B, and -3C (which themselves share a degree of primary sequence homology) could also play a role in TP-1 gene regulation. The TP-1 promoter sequence was linked to the chloramphenicol acetyltransferase (CAT) gene and used in cotransfection experiments in an EBV negative cell line with various combinations of vectors expressing individual EBNA-3s. In the absence of other EBV proteins, the EBNA-3s did not stimulate TP-1 promoter activity. In the presence of EBNA-2, the EBNA-3s were shown to be capable of reducing the level of TP-1 promoter-driven CAT activity. The EBNA-3s had no effect on a panel of heterologous promoters, indicating that EBNA-2 and/or transcription elements specific to the TP-1 promoter are essential for the observed activity of the EBNA-3s. The functional antagonism between the EBNA-2 and EBNA-3 proteins may be important in the overall viral strategy.

Repression of the viral latent promoter BC-R2 in Epstein-Barr virus negative cell lines.

The regulation of the latent promoter BC-R2 of Epstein-Barr virus (EBV) was examined using the chloramphenicol acetyl transferase (CAT) gene reporter system. A 5' deletion analysis of BC-R2 promoter sequences has been used to characterize a region, described previously as a transcriptional enhancer in EBV positive cell lines, which can repress the BC-R2 transcriptional activity in EBV negative cell lines.

The identification of new transcription elements active in Epstein-Barr virus latent gene expression.

The immortalization in vitro of human B cells by the Epstein-Barr virus (EBV) is associated with the expression of a restricted group of nuclear proteins (EBNA). EBNA transcription is driven from either of two mutually exclusive promoters whose modulation is likely to involve an important host/virus interaction. Our experiments on the EBNA transcriptional domain have led us to identify several new transcription elements which are located on the DNA sequence between the two promoters. These elements stimulate the activity of the upstream EBNA promoter by a factor of at least one hundred. One of these elements may also be involved in the in vivo reactivation of EBV in certain cases.