Blocking the ZZ domain of sequestosome1/p62 suppresses myeloma growth and osteoclast formation in vitro and induces dramatic bone formation in myeloma-bearing bones in vivo.

We reported that p62 (sequestosome 1) serves as a signaling hub in bone marrow stromal cells (BMSCs) for the formation of signaling complexes, including NFκB, p38MAPK and JNK, that are involved in the increased osteoclastogenesis and multiple myeloma (MM) cell growth induced by BMSCs that are key contributors to multiple myeloma bone disease (MMBD), and demonstrated that the ZZ domain of p62 (p62-ZZ) is required for BMSC enhancement of MMBD. We recently identified a novel p62-ZZ inhibitor, XRK3F2, which inhibits MM cell growth and BMSC growth enhancement of human MM cells. In the current study, we evaluate the relative specificity of XRK3F2 for p62-ZZ, characterize XRK3F2's capacity to inhibit growth of primary MM cells and human MM cell lines, and test the in vivo effects of XRK3F2 in the immunocompetent 5TGM1 MM model. We found that XRK3F2 induces dramatic cortical bone formation that is restricted to MM containing bones and blocked the effects and upregulation of tumor necrosis factor alpha (TNFα), an osteoblast (OB) differentiation inhibitor that is increased in the MM bone marrow microenvironment and utilizes signaling complexes formed on p62-ZZ, in BMSC. Interestingly, XRK3F2 had no effect on non-MM bearing bone. These results demonstrate that targeting p62 in MM models has profound effects on MMBD.

The impact of domain knowledge on structured data collection and templated note design.

OBJECTIVE: The objective of this case report is to evaluate the importance of specialized domain knowledge when designing and using structured templated notes in a clinical environment. METHODS: To analyze the impact of specialization on structured note generation we compared notes generated for three scenarios: 1) We compared the templated history of present illness (HPI) for patients presenting with a dermatology concern to the dermatologist versus the emergency department. 2) We compared the evaluation of chest pain by ED physicians versus cardiologists. 3) Finally, we compared the data elements asked for in the evaluation of the gastrointestinal system between cardiologists and the liver transplant service (LTS). We used the SNOMED CT representation via BioPortal to evaluate specificity and grouping between data elements and specialized physician groups. RESULTS: We found few similarities in structured data elements designed by and for the specific physician groups. The distinctness represented both differences in granularity as well as fundamental differences in data elements requested. When compared to ED physicians, dermatologists had different and more granular elements while cardiologists requested much more granular data. Comparing cardiologists and LTS, there were differences in the data elements requested. CONCLUSION: This case study supports the importance of domain knowledge in EHR design and implementation. That different specialities should want and use different information is well supported by cognitive science literature. Despite this, it is rare for domain knowledge to be considered in EHR implementation. Physicians with correct domain knowledge should be involved in the design process of templated notes.

Autophagy promotes T-cell survival through degradation of proteins of the cell death machinery.

Autophagy is implicated in regulating cell death in activated T cells, but the underlying mechanism is unclear. Here, we show that inhibition of autophagy via Beclin 1 gene deletion in T cells leads to rampant apoptosis in these cells upon TCR stimulation. Beclin 1-deficient mice fail to mount autoreactive T-cell responses and are resistant to experimental autoimmune encephalomyelitis. Compared with Th17 cells, Th1 cells are much more susceptible to cell death upon Beclin 1 deletion. Cell death proteins are highly increased in Beclin 1-deficient T cells and inhibition of caspases and genetic deletion of Bim reverse apoptosis. In addition, p62/sequestosome 1 binds to caspase-8 but does not control levels of procaspase-8 or other cell death-related proteins. These results establish a direct role of autophagy in inhibiting the programmed cell death through degradation of apoptosis proteins in activated T cells.

Adoption of electronic health records: a qualitative study of academic and private physicians and health administrators.

OBJECTIVE: Less than 20% of hospitals in the US have an electronic health record (EHR). In this qualitative study, we examine the perspectives of both academic and private physicians and administrators as stakeholders, and their alignment, to explore their perspectives on the use of technology in the clinical environment. METHODS: Focus groups were conducted with 74 participants who were asked a series of open-ended questions. Grounded theory was used to analyze the transcribed data and build convergent themes. The relevance and importance of themes was constructed by examining frequency, convergence, and intensity. A model was proposed that represents the interactions between themes. RESULTS: Six major themes emerged, which include the impact of EHR systems on workflow, patient care, communication, research/outcomes/billing, education/learning, and institutional culture. Academic and private physicians were confident of the future benefits of EHR systems, yet cautious about the current implementations of EHR, and its impact on interactions with other members of the healthcare team and with patients, and the amount of time necessary to use EHR's. Private physicians differed on education and were uneasy about the steep learning curve necessary for use of new systems. In contrast to physicians, university and hospital administrators are optimistic, and value the availability of data for use in reporting. CONCLUSION: The results of our study indicate that both private and academic physicians concur on the need for features that maintain and enhance the relationship with the patient and the healthcare team. Resistance to adoption is related to insufficient functionality and its potential negative impact on patient care. Integration of data collection into clinical workflows must consider the unexpected costs of data acquisition.

Electronic Health Record Adoption - Maybe It's not about the Money: Physician Super-Users, Electronic Health Records and Patient Care.

OBJECTIVE: The slow adoption of electronic health record (EHR) systems has been linked to physician resistance to change and the expense of EHR adoption. This qualitative study was conducted to evaluate benefits, and clarify limitations of two mature, robust, comprehensive EHR Systems by tech-savvy physicians where resistance and expense are not at issue. METHODS: Two EHR systems were examined - the paperless VistA / Computerized Patient Record System used at the Veterans' Administration, and the General Electric Centricity Enterprise system used at an academic medical center. A series of interviews was conducted with 20 EHR-savvy multiinstitutional internal medicine (IM) faculty and house staff. Grounded theory was used to analyze the transcribed data and build themes. The relevance and importance of themes were constructed by examining their frequency, convergence, and intensity. RESULTS: Despite eliminating resistance to both adoption and technology as drivers of acceptance, these two robust EHR's are still viewed as having an adverse impact on two aspects of patient care, physician workflow and team communication. Both EHR's had perceived strengths but also significant limitations and neither were able to satisfactorily address all of the physicians' needs. CONCLUSION: Difficulties related to physician acceptance reflect real concerns about EHR impact on patient care. Physicians are optimistic about the future benefits of EHR systems, but are frustrated with the non-intuitive interfaces and cumbersome data searches of existing EHRs.

The maximum A posteriori approach to the inverse problem of electrocardiography.

In this paper we investigate the previously proposed maximum a posteriori (MAP) approach to the problem of determining epicardial potentials from measured body surface potentials, a form of the inverse problem of electrocardiography. The MAP inverse approach uses a priori knowledge of the covariances between epicardial electrograms in its estimate of epicardial potentials. However, in practice, this information is not generally available. In this paper we examined the effectiveness of this method when the covariances are estimated using one depolarization sequence and the MAP method is used with these covariances to estimate the epicardial potentials for a different depolarization sequence.

A new method for incorporating weighted temporal and spatial smoothing in the inverse problem of electrocardiography.

In this paper, we present a method for incorporating temporal smoothing (TS) into the estimate of epicardial potentials from body surface potential data. Our algorithm employs a different spatial smoothing parameter, chosen by the composite residual error and smoothing operator criteria, at each time step in the sequence. The total spatial smoothing term is then simply partitioned between temporal and spatial smoothing. The algorithm appears to be quite robust with regard to this partitioning. The new method was evaluated in the setting of additive Gaussian noise, but otherwise realistic conditions of body geometry and reference epicardial potentials. In examining the match between estimated and measured electrograms, or the match between estimated isopotential maps and measured isopotential maps, the estimates constructed using the new TS algorithm produced consistently smaller relative errors than those constructed using a quasi-static (QS) algorithm or those constructed by postprocessing the QS estimate with a moving average filter.

Osteoclasts formed by measles virus-infected osteoclast precursors from hCD46 transgenic mice express characteristics of pagetic osteoclasts.

Pagetic osteoclasts (OCLs) are abnormal in size and contain paramyxoviral-like nuclear inclusions that cross-react with antibodies to measles virus (MV). However, the role that MV infection plays in Paget's disease is unknown, because no animal model of Paget's disease is available. Therefore, we targeted a cellular MV receptor, human CD46 (hCD46), to cells in the OCL lineage in transgenic mice using the mouse tartrate-resistant acid phosphatase (TRAP) gene promoter. In vitro infection of OCL precursors from hCD46 transgenic mice with MV significantly increased OCL formation in bone marrow cultures. The numbers of TRAP-positive mononuclear cells and CFU-GM, the earliest identifiable OCL precursor, were also significantly increased. MV-infected OCLs formed from hCD46 marrow were increased in size, contained markedly increased numbers of nuclei, and had increased bone-resorbing capacity per OCL compared with OCLs formed from marrow of nontransgenic littermates. Furthermore, IL-6 and 24-hydroxylase messenger RNA expression levels were increased in MV-infected hCD46 transgenic mouse bone marrow cultures. Treatment of MV-infected hCD46 marrow cultures with a neutralizing antibody to IL-6 blocked the increased OCL formation seen in these cultures. These data demonstrate that MV infection of OCL precursors results in OCLs that have many features of pagetic OCLs, that the enhanced OCL formation is in part mediated by increased IL-6 expression induced by MV infection, and suggest that the hCD46 transgenic mouse may be a useful model for examining the effects of MV infection on OCL formation in vivo.

Parameter choice methods and temporal filtering for the generalized eigensystem method applied to the inverse problem of electrocardiography.

We have previously proposed the generalized eigensystem (GES) method as a modal expansion method for estimating electrical potentials on the heart outer surface from measurements of electrical potentials on the body surface. In this paper, we present an alternative formulation of GES more like that of classical Tikhonov regularization where a single continuous parameter needs to be chosen. We then compare this formulation of GES with zero order Tikhonov regularization on data collected from a swine experiment with the swine heart paced from six different sites. Although the inverse problems are solved at each time instant independently, we also incorporate temporal information by moving average filtering the estimates, and this is more effective for the GES methods than for Tikhonov.

Normalization of ventricular repolarization with flecainide in long QT syndrome patients with SCN5A:DeltaKPQ mutation.

BACKGROUND: The Long QT Syndrome (LQTS) is a genetic channelopathy with life-threatening implications. The LQT3 form of this disease is caused by mutations of the SCN5A sodium-channel gene. A specific mutation, SCN5A:DeltaKPQ, is associated with repetitive reopenings of the sodium channel and prolonged inward current. This dominant inward current is manifest on the electrocardiogram as QT prolongation. Flecainide is a potent blocker of the open sodium channel. METHODS AND RESULTS: The effect of flecainide on the duration of the QT-interval and the T-wave morphology was systematically evaluated in five male patients age 2-64 years having the SCN5A:DeltaKPQ mutation. After baseline electrocardiograms were obtained, low-dose oral flecainide was administered for 48 hours. Serial electrocardiograms and blood flecainide levels were obtained during flecainide therapy. The QTc interval decreased on average by 104 ms, from a baseline value of 565 +/- 60 ms to 461 +/- 23 ms (P < 0.04) at a mean flecainide level of 0.28 +/- 0.08 mg/L, with shortening of the QTonset interval (P < 0.003) and normalization of T-wave morphology. The effects of flecainide were compared with oral mexiletine in two patients, with flecainide showing greater QTc shortening and more complete normalization of repolarization. No adverse side effects or proarrhythmia were observed with flecainide in this study. CONCLUSION: Low-dose, oral flecainide consistently shortened the QTc interval and normalized the repolarization T-wave pattern in five LQT3 patients with SCN5A:DeltaKPQ mutation. This preliminary study indicates that low-dose flecainide is a promising therapeutic agent for LQTS patients with the SCN5A:DeltaKPQ sodium channel mutation.

Potential risk of vasovagal syncope for motor vehicle driving.

Vasovagal syncope is the most common cause of syncope, but its risk for driving remains uncertain. We analyzed the clinical characteristics of patients who had syncope during driving and subsequently underwent the head-up tilt test (HUTT). Of the 245 consecutive patients undergoing HUTT, 23 (9%) had > or =1 episode of syncope during driving. HUTT was positive in 19 (group A) and negative in 4 (group B) patients. No patient had structural heart disease. In group A, the driving incident occurred on the first syncope in 3 patients, and the other 16 patients had 1 to 4 episodes of prior syncope not associated with driving. In group B, the driving incident occurred on the first syncope in 1 patient, and the other 3 patients had prior syncope (3 episodes in each) not associated with driving. Seven group A and 1 group B patients had 2 syncope-related driving incidents, and the remaining patients had only 1 syncope-related driving incident. The syncope-related driving incidents caused personal injury in 7 group A and 2 group B patients. One incident in 1 group A patient caused the death of another driver. After HUTT, all but 1 patient in group A received medical treatment and only 1 patient in group B received empirical beta-blocker therapy. During the follow-up of 51+/-26 months, 1 patient died and another was lost to follow-up. Of the remaining patients, 4 patients had recurrence of syncope and 2 patients had presyncope in group A. One of these patients had another syncope-related driving incident. No group B patient had syncope recurrence. A second etiology of syncope was never found in any patient. We conclude that vasovagal syncope during driving is not uncommon in patients referred for syncope evaluation. Early medical attention to patients with vasovagal syncope may help reduce syncope-related driving incidents.

Employing a transgenic animal model to obtain cementoblasts in vitro.

BACKGROUND: Proper formation of cementum, a mineralized tissue lining the tooth root surface, is required for development of a functional periodontal ligament. Further, the presence of healthy cementum is considered to be an important criterion for predictable restoration of periodontal tissues lost as a consequence of disease. Despite the significance of cementum to general oral health, the mechanisms controlling development and regeneration of this tissue are not well understood and research has been hampered by the lack of adequate in vitro experimental models. METHODS: In an effort to establish cementoblast cell populations, without the trappings of a heterogeneous population containing periodontal ligament (PDL) cells, cells were obtained from the root surface of first mandibular molars of OC-TAg transgenic mice. These mice contain the SV40 large T-antigen (TAg) under control of the osteocalcin (OC) promoter. Therefore, only cells that express OC also express TAg and are immortalized in vitro. Based on results of prior in situ studies, OC is expressed by cementoblasts during root development, but not by cells within the PDL. Consequently, when populations are isolated from developing molars using collagenase/trypsin digestion, only cementoblasts, not PDL cells, are immortalized and thus, will survive in culture. RESULTS: The resulting immortalized cementoblast population (OC/CM) expressed bone sialoprotein (BSP), osteopontin (OPN), and OC, markers selective to cells lining the root surface. These cells also expressed type I and XII collagen and type I PTH/PTHrP receptor (PTH1R). In addition to expression of genes associated with cementoblasts, OC/CM cells promoted mineral nodule formation and exhibited a PTHrP mediated cAMP response. CONCLUSIONS: This approach for establishing cementoblasts in vitro provides a model to study cementogenesis as required to enhance our knowledge of the mechanisms controlling development, maintenance, and regeneration of periodontal tissues.

Genetic determinants of response to chemotherapy in transgenic mouse mammary and salivary tumors.

Several transgenic mouse tumor models were utilized to explore how specific genetic alterations affect the tumor cell response to chemotherapeutic agents in vivo. Specifically, MMTV-ras transgenic mice were interbred to p53 knock-out mice to create a model for assessing the role of p53 in chemotherapeutic responses. In addition, MMTV-ras tumors were compared to MMTV-myc and MMTV-ras/myc tumors. Mice of each genotype reproducibly develop mammary and/or salivary tumors, but tumor growth dynamics vary considerably between genotypes. MMTV-ras/p53-/- tumors exhibit higher S phase fractions than MMTV-ras/p53+/+ tumors, although both tumor types display very low apoptosis levels. In contrast, MMTV-myc tumors exhibit both high S phase fractions and spontaneous apoptosis levels. Tumor-bearing mice of each genotype were treated with either doxorubicin or paclitaxel, and effects on overall tumor growth, cell cycle distribution and apoptosis were evaluated. Surprisingly, neither agent efficiently induced apoptosis in any of the tumor models, including those with wildtype p53. Rather, tumor responses were mediated primarily by changes in cell cycle distribution. However, the spontaneous apoptosis levels did serve as a predictor of tumor growth response, in that only those tumors with high pretreatment apoptosis levels underwent significant regression following treatment with either agent.

Ras protein farnesyltransferase: A strategic target for anticancer therapeutic development.

Ras proteins are guanine nucleotide-binding proteins that play pivotal roles in the control of normal and transformed cell growth and are among the most intensively studied proteins of the past decade. After stimulation by various growth factors and cytokines, Ras activates several downstream effectors, including the Raf-1/mitogen-activated protein kinase pathway and the Rac/Rho pathway. In approximately 30% of human cancers, including a substantial proportion of pancreatic and colon adenocarcinomas, mutated ras genes produce mutated proteins that remain locked in an active state, thereby relaying uncontrolled proliferative signals. Ras undergoes several posttranslational modifications that facilitate its attachment to the inner surface of the plasma membrane. The first-and most critical-modification is the addition of a farnesyl isoprenoid moiety in a reaction catalyzed by the enzyme protein farnesyltransferase (FTase). It follows that inhibiting FTase would prevent Ras from maturing into its biologically active form, and FTase is of considerable interest as a potential therapeutic target. Different classes of FTase inhibitors have been identified that block farnesylation of Ras, reverse Ras-mediated cell transformation in human cell lines, and inhibit the growth of human tumor cells in nude mice. In transgenic mice with established tumors, FTase inhibitors cause regression in some tumors, which appears to be mediated through both apoptosis and cell cycle regulation. FTase inhibitors have been well tolerated in animal studies and do not produce the generalized cytotoxic effects in normal tissues that are a major limitation of most conventional anticancer agents. There are ongoing clinical evaluations of FTase inhibitors to determine the feasibility of administering them on dose schedules like those that portend optimal therapeutic indices in preclinical studies. Because of the unique biologic aspects of FTase, designing disease-directed phase II and III evaluations of their effectiveness presents formidable challenges.

Usefulness of ibutilide in facilitating successful external cardioversion of refractory atrial fibrillation.

We repeated direct-current cardioversion of atrial fibrillation after ibutilide injection in patients who failed conventional cardioversion. Eleven of 12 patients (92%) had successful cardioversion and avoided the need for internal cardioversion.

Quality-of-life six months after CABG surgery in patients randomized to ICD versus no ICD therapy: findings from the CABG Patch Trial.

UNLABELLED: ICDs can affect a patient's perceived quality-of-life (QOL). This article describes the QOL in patients who participated in The CABG Patch Trial. This trial evaluated the potential benefit of empiric ICD implantation in patients with an increased risk of arrhythmic cardiac death as determined by reduced ejection fraction (<0.36) and an abnormal signal-averaged ECG. Patients were randomized to control (no ICD) or treatment (ICD) limbs. QOL was measured using the SF-36 and other measures among 490 (68%) of 719 patients available at 6-month follow-up. Analysis was performed on 228 control patients (those without ICDs) and 262 patients with ICDs. RESULTS: Six months after having CABG surgery, patients in the ICD group had lower levels of psychological well-being than those in the control group. In addition, compared to controls, patients whose ICDs had delivered therapy reported feeling less healthy, had reduced physical and emotional role functioning, and had lower levels of psychological well-being. CONCLUSION: Strategies aimed at easing patients' adjustment to ICDs should be developed and tested for efficacy in the setting of ICD prophylaxis.

Retinoblastoma in transgenic mice: models of hereditary retinoblastoma.

Retinoblastoma, the most common intraocular malignancy ill childhood, has served as a paradigm for the study of genetic mechanisms of oncogenesis. The retinoblastoma susceptibility gene RB1 was the first tumor suppressor gene to be cloned, and genetic and molecular biologic studies of this tumor have greatly expanded the understanding of the mechanics of tumorigenesis. Human retinoblastoma has essentially no naturally occuring animal counterpart. The development of transgenic murine models of retinoblastoma have created an experimental tool for manipulation of a tumor gene system in vivo. These models have also enabled studies of new therapeutic modalities. This review outlines the development of the transgenic murine models of retinoblastoma, together with the genetic mechanisms of retinoblastoma origin. Current therapeutic innovations developed by means of the transgenic models are described.