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Objective To assess the efficacy of an intern surgical skills curriculum involving a boot camp for core open and laparoscopic skills, self-guided practice with positive and negative incentives, and semiannual performance evaluations. Design Longitudinal cohort study. Setting Academic tertiary care center. Participants Intervention group (n = 15): residents who completed the intern surgical skills curriculum and had performance evaluations in fall of intern year, spring of intern year, and fall of second year. Control group (n = 8): second-year residents who were 1 year ahead of the intervention group in the same residency program, did not participate in the curriculum, and had performance evaluations in fall of second year. Results In fall of second year of residency, the intervention group had better performance (presented as median values with interquartile ranges) than the control group on one-hand ties (left hand: 9.1 [6.3-10.1] vs 14.6 [13.5-15.4] seconds, p = 0.007; right hand: 8.7 [8.5-9.6] vs 11.5 [9.9-16.8] seconds, p = 0.039). The intervention group also had better performance on all open suturing skills, including mattress suturing (vertical: 33.4 [30.0-40.0] vs 55.8 [50.0-67.6] seconds, p = 0.001; horizontal: 28.7 [27.3-39.9] vs 52.7 [40.7-57.8] seconds, p = 0.003), and a water-filled glove clamp, divide, and ligate task (28.0 [25.0-31.0] vs 59.1 [53.0-93.0] seconds, p < 0.001). Finally, the intervention group had better performance on all laparoscopic skills, including peg transfer (66.0 [59.0-82.0] vs 95.2 [87.5-101.5] seconds, p = 0.018), circle cut (82.0 [69.0-124.0] seconds vs 191.8 [155.5-231.5] seconds, p = 0.002), and intracorporeal suturing (195.0 [117.0-200.0] seconds vs 359.5 [269.0-450.0] seconds, p = 0.002). Conclusions Implementation of a comprehensive surgical skills curriculum was associated with improved performance on core open and laparoscopic skills. Further research is needed to understand and optimize motivational factors for deliberate practice and surgical skill acquisition.

OBJECTIVE The index for mortality prediction after cardiac transplantation (IMPACT) risk score incorporates 12 preoperative recipient-specific variables, and has been validated as an accurate predictor of short- and long-term mortality after orthotopic heart transplantation (OHTx). We believe it can also be used to predict hospital costs, and we hypothesize that higher preoperative IMPACT risk scores are associated with increased hospital resource consumption. METHODS All OHTx patients ≥18 years of age at our institution were reviewed from 1 January 2000 to 31 December 2014. Total index hospitalization costs post-transplant were extracted and presented in 2014 consumer price index inflation-adjusted US dollars. Patients were stratified into quartiles (Q) according to IMPACT risk scores. Logarithmic transformation normalized cost data, and linear regression assessed for correlation. A comparison of cost between Q of IMPACT risk score was performed using rank-sum and Kruskal-Wallis tests. Survival was estimated using the Kaplan-Meier method. RESULTS Three hundred fifty-six (n = 356) OHTx were performed during the study period. The median IMPACT score for the cohort was five (interquartile range [IQR] 3-6). Eight (2.2%) patients died within 30-days and 1-year Kaplan-Meier survival was 88.3%. The median length of stay (LOS) was 16 (IQR 14-24) days. The median hospital cost for index admission was $222 200 (IQR:$169 200-$313 700). Median LOS was longer in Q4 vs Q1 (18 days vs 15 days, P = .01) and index hospital costs in Q4 were significantly higher compared to Q1 patients ($280 400 vs $205 000, P

Background: Increasing opioid-related deaths have heightened focus on combating the opioid epidemic. The impact of surgical trainees on opioid-related deaths is unclear, and there is little data examining the association between trainee pain management education and opioid prescribing practices. Methods: An anonymous, online survey was distributed to members of the Resident and Associate Society of the American College of Surgeons. The survey covered five themes: education and knowledge, prescribing practices, clinical case scenarios, policy, and beliefs and attitudes. Linear mixed models were used to evaluate the influence of respondent characteristics on reported morphine milligram equivalents (MME) prescribed for common general surgery clinical scenarios. Results: Of 427 respondents, 54 percent indicated receiving training in postoperative pain management during medical school and 66 percent during residency. Only 35 percent agreed that they had received adequate training in prescribing opioids. There was a significant association between undergoing formal pain management training in medical school and prescribing fewer MME for common outpatient general surgery scenarios (94 ± 15.2 vs 108 ± 15.0; p = 0.003). Similarly, formal pain management training in residency was associated with prescribing fewer MME in the survey scenarios (92.6 ± 15.2 vs 109 ± 15.2; p = 0.002). Conclusion: In this survey, nearly two-thirds of surgical residents felt that they were inadequately trained in opioid prescribing. Our findings additionally suggest that improving education may result in increased resident comfort with managing surgical pain, potentially leading to more responsible opioid prescribing. Further work will facilitate residency programs’ development of educational curricula for opioid prescribing best practices.

Increasing mortality from opioid overdoses has prompted increased focus on prescribing practices of physicians. Unfortunately, resident physicians rarely receive formal education in effective opioid prescribing practices or postoperative pain management. Data to inform surgical training programs regarding the utility and feasibility of formal training are lacking.Following Institutional Review Board approval, a single institution's resident physicians who had completed at least one surgical rotation were surveyed to assess knowledge of pain management and evaluate opioid prescribing practices.Fifty-three respondents (68% males and 32% females) completed the survey. Most respondents denied receiving formal instruction in opioid pain medication prescribing practices during either medical school (62.3%) or residency (56.6%); however, nearly all respondents stated they were aware of the side effects of opioid pain medications, and a majority felt confident in their knowledge of opioid pharmacokinetics and pharmacodynamics. Of the respondents, 47% either "agreed" or "strongly agreed" that they prescribed more opioid medications than necessary to patients being discharged following a surgical procedure. Individual case scenario responses demonstrated variability in the number of morphine milligram equivalents prescribed across scenarios (P 0.001). Male and nonsurgical specialty respondents reported prescribing significantly fewer overall morphine milligram equivalents in these scenarios.This pilot study shows wide variability in opioid prescribing practices and attitudes toward pain management among surgical trainees, illustrating the potential utility of formal education in pain management and effective prescribing of these medications. A broader assessment of surgical trainees' knowledge and perception of opioid prescribing practices is warranted to facilitate the development of such a program.

Several aspects of medical training may contribute to the ultimate goal of producing excellent physicians whose patients will have the best possible outcomes. However, the relative importance of education, evaluation and feedback, duty hours, practice structure, and program culture in achieving this goal is unclear. This study assessed associations among in-training exam performance, Accreditation Council for Graduate Medical Education (ACGME) Resident Survey responses, and American Board of Medical Specialties (ABMS) national board exam performance. Residency training programs at a university teaching hospital were classified as having 5-year first-time ABMS pass rates above (n=12) or below (n=3) the national average for their specialty. These groups were compared by ACGME Resident Survey data and in-training exam performance. Surveys were collected from 484/543 eligible residents (89%), including 177 surveys from programs with below-average board pass rates and 307 surveys from programs with aboveaverage board pass rates. In-training exam performance was similar between groups. Aboveaverage programs had stronger agreement with statements that their culture reinforced patient safety (4.72 vs. 4.30, p=0.006) and that information was not lost during transitions of care (4.14 vs. 3.63, p=0.001). Although the occurrence of interprofessional teamwork was similar between groups, above-average programs had stronger agreement with the statement that interprofessional teamwork was effective (4.60 vs. 4.17, p=0.003). Residency programs emphasizing patient safety and effective interprofessional teamwork had above-average first-time national board pass rates.

Quantitative objective measures to determine fusion achievement further enable the comparison of new technologies, such as interbody cage surface enhancement. Our aims were to compare in vivo biomechanical responses of ovine L4/5 lumbar motion segments with two cages: 1) Polyetheretherketone or 2) Polyetheretherketone with a nanosurfaced titanium porous scaffold from Nanovis, Inc.Fourteen Merino sheep randomly received either 1) standard Polyetheretherketone cage or 2) Nanocoated Polyetheretherketone cage at L4/L5 with autologous bone graft. At baseline and one-year follow-up, dynamic spinal stiffness was quantified in vivo using a validated mechanical assessment at 2 Hz, 6 Hz, and 12 Hz. The dorsoventral secant stiffness (kBoth implants increased spinal stiffness at 2 Hz (21 and 39%, respectively, p .005), and at 6 Hz (12 and 27%, p .0001). Significantly greater spinal stiffness was observed with Nanocoated Polyetheretherketone at one-year for both frequencies (p .05). No significant differences were observed at 12 Hz within or between groups. L4-L5 dorsoventral accelerations were significantly decreased one year following cage placement only with Nanocoated Polyetheretherketone (p .05) and greater reductions in acceleration were observed with Nanocoated Polyetheretherketone compared to standard Polyetheretherketone (p .05).Both cages increased spinal stiffness, yet, nanosurfaced cages resulted in greater spinal stiffness changes and decreases in L4-L5 accelerations. These findings may assist in clinical decision making and post-operative recovery strategies.

OBJECTIVE The Ages and Stages Questionnaire (ASQ) and Modified Checklist for Autism in Toddlers (M-CHAT) are commonly used screening tools for developmental delay and autism, respectively. This study aimed to evaluate the effectiveness of implementing 18-month developmental and autism screening by mail, with a standardized follow-up process for abnormal screen results. METHODS Within a prospective cohort study design, parents of 892 children received by mail the 18-month ASQ and M-CHAT questionnaires between December 2008 and September 2009. A registered nurse scored the questionnaires and, if needed, administered follow-up screening or set up a referral to Early Intervention or subspecialty pediatrics. Medical record reviews determined clinical outcomes of children who required intervention after initial screening through September 2010. Additionally, demographic factors were evaluated for association with responding to the questionnaires. RESULTS The ASQ and M-CHAT questionnaires were returned by 529 (59.3%) of the parents. Parents of White children (390/575 [67.8%]) and those with private insurance (457/660 [69.2%]) were significantly more likely to return screening questionnaires than parents of non-White (64/171 [37.4%]; P < .001) and government-insured children (58/169 [34.3%]; P < .001), respectively. Of the 529 children with returned surveys, 109 (20.6%) did not pass at least 1 of the initial screens and 12 (2.3%) were referred after not passing the follow-up screening process. CONCLUSIONS Developmental and autism screening by mail is not a sufficient method to comprehensively screen a general pediatric population. A nurse-completed, standardized follow-up process after an initial failed developmental screen may increase the yield of appropriate Early Intervention or subspecialty referrals.

Molecular imaging with a fluorescent version of Tilmanocept may permit an accurate and facile detection of sentinel nodes of endometrial cancer. Tilmanocept accumulates in sentinel lymph nodes (SLN) by binding to a cell surface receptor unique to macrophages and dendritic cells. Four female Yorkshire pigs underwent cervical stromal injection of IRDye800-Tilmanocept, a molecular imaging agent tagged with near-infrared fluorescent dye and radiolabeled with gallium-68 and technetium-99m. PET/CT scans 1.5 hours post-injection provided pre-operative SLN mapping. Robotic-assisted lymphadenectomy was performed two days after injection, using the FireFly imaging system to identify nodes demonstrating fluorescent signal. After removal of fluorescent nodes, pelvic and periaortic node dissections were performed. Nodes were assayed for technetium-99m activity, and SLNs were established using the "10%-rule", requiring that the radioactivity of additional SLNs be greater than 10% of the "hottest" SLN. Thirty-four nodal samples were assayed ex vivo for radioactivity. All the SLNs satisfying the "10%-rule" were detected using the FireFly system. Five fluorescent nodes were detected, corresponding with preoperative PET/CT scan. Three pigs had one SLN and one pig had two SLNs, with 100% concordance between fluorescence and radioactivity. Fluorescent-labeled Tilmanocept permits real-time intraoperative detection of SLNs during robotic-assisted lymphadenectomy for endometrial cancer in a porcine model. When radiolabeled with gallium-68, Tilmanocept allows for preoperative localization of SLNs using PET/CT, and shows specificity to SLNs with persistent fluorescent signal, detectable using the FireFly system, for two days post-injection. In conclusion, these findings suggest that a phase I trial in human subjects is warranted, and that a long-term goal of an intra-operative administration of non-radioactive fluorescent-labeled Tilmanocept is possible.

The development of new medicines holds particular fascination for chemistry, biochemistry, and biology students interested in a career in medicine or the life sciences. The identification and refinement of lead compounds to treat diseases requires researchers to be facile in a number of different disciplines including organic synthesis, biochemistry, cell biology, and molecular biology. We have developed an interdisciplinary, inquiry-based laboratory spanning both organic chemistry and biochemistry classes that acquaints students with research in medicinal chemistry. The first part of the exercise takes place in the second semester of organic chemistry, where pairs of students design and execute their own multistep synthesis of a novel compound with anti-inflammatory and/or antimalarial potential. Later, in first semester biochemistry, many of the same students then test these synthesized compounds for cytotoxicity, inhibition of the enzyme nitric oxide synthase, and inhibition of the transcription factor NF-kB. Learning outcomes, measured by the Classroom Undergraduate Research Experience (CURE) survey, suggest that students participating in both classes had higher gains than an average student. © 2018 International Union of Biochemistry and Molecular Biology, 46(5):424-434, 2018.

PurposeTo summarize data on computed tomographic (CT) radiation doses collected from consecutive CT examinations performed at 12 facilities that can contribute to the creation of reference levels.Materials and methodsThe study was approved by the institutional review boards of the collaborating institutions and was compliant with HIPAA. Radiation dose metrics were prospectively and electronically collected from 199 656 consecutive CT examinations in 83 181 adults and 3871 consecutive CT examinations in 2609 children at the five University of California medical centers during 2013. The median volume CT dose index (CTDIvol), dose-length product (DLP), and effective dose, along with the interquartile range (IQR), were calculated separately for adults and children and stratified according to anatomic region. Distributions for DLP and effective dose are reported for single-phase examinations, multiphase examinations, and all examinations.ResultsFor adults, the median CTDIvol was 50 mGy (IQR, 37-62 mGy) for the head, 12 mGy (IQR, 7-17 mGy) for the chest, and 12 mGy (IQR, 8-17 mGy) for the abdomen. The median DLPs for single-phase, multiphase, and all examinations, respectively, were as follows: head, 880 mGy · cm (IQR, 640-1120 mGy · cm), 1550 mGy · cm (IQR, 1150-2130 mGy · cm), and 960 mGy · cm (IQR, 690-1300 mGy · cm); chest, 420 mGy · cm (IQR, 260-610 mGy · cm), 880 mGy · cm (IQR, 570-1430 mGy · cm), and 550 mGy · cm (IQR 320-830 mGy · cm); and abdomen, 580 mGy · cm (IQR, 360-860 mGy · cm), 1220 mGy · cm (IQR, 850-1790 mGy · cm), and 960 mGy · cm (IQR, 600-1460 mGy · cm). Median effective doses for single-phase, multiphase, and all examinations, respectively, were as follows: head, 2 mSv (IQR, 1-3 mSv), 4 mSv (IQR, 3-8 mSv), and 2 mSv (IQR, 2-3 mSv); chest, 9 mSv (IQR, 5-13 mSv), 18 mSv (IQR, 12-29 mSv), and 11 mSv (IQR, 6-18 mSv); and abdomen, 10 mSv (IQR, 6-16 mSv), 22 mSv (IQR, 15-32 mSv), and 17 mSv (IQR, 11-26 mSv). In general, values for children were approximately 50% those for adults in the head and 25% those for adults in the chest and abdomen.ConclusionThese summary dose data provide a starting point for institutional evaluation of CT radiation doses.

In this Communication, we describe an enzyme-responsive, paclitaxel-loaded nanoparticle and assess its safety and efficacy in vivo in a human fibrosarcoma murine xenograft. The material was generated via graft-through block copolymerization of norbornene monomers with hydrophilic targeting peptides together with hydrophobic paclitaxel prodrugs. This work represents a proof-of-concept study demonstrating the utility of enzyme-responsive nanoscale drug carriers capable of targeted accumulation and retention in tumor tissue in response to overexpressed endogenous enzymes. Critically, we observed low systemic toxicity in healthy mice following intravenous administration, with the maximum tolerated dose (MTD) exceeding 240 mg/kg with respect to paclitaxel. Furthermore, we observed efficacy against tumorigenesis paralleling that of paclitaxel at equivalent intravenous dosing, and near complete tumor growth suppression when administered intratumorally. This work represents a significant departure from traditional targeted drug delivery systems and presents a new avenue of exploration for nanomedicine.

Importance Radiation doses for computed tomography (CT) vary substantially across institutions. Objective To assess the impact of institutional-level audit and collaborative efforts to share best practices on CT radiation doses across 5 University of California (UC) medical centers. Design, Setting, and Participants In this before/after interventional study, we prospectively collected radiation dose metrics on all diagnostic CT examinations performed between October 1, 2013, and December 31, 2014, at 5 medical centers. Using data from January to March (baseline), we created audit reports detailing the distribution of radiation dose metrics for chest, abdomen, and head CT scans. In April, we shared reports with the medical centers and invited radiology professionals from the centers to a 1.5-day in-person meeting to review reports and share best practices. Main Outcomes and Measures We calculated changes in mean effective dose 12 weeks before and after the audits and meeting, excluding a 12-week implementation period when medical centers could make changes. We compared proportions of examinations exceeding previously published benchmarks at baseline and following the audit and meeting, and calculated changes in proportion of examinations exceeding benchmarks. Results Of 158 274 diagnostic CT scans performed in the study period, 29 594 CT scans were performed in the 3 months before and 32 839 CT scans were performed 12 to 24 weeks after the audit and meeting. Reductions in mean effective dose were considerable for chest and abdomen. Mean effective dose for chest CT decreased from 13.2 to 10.7 mSv (18.9% reduction; 95% CI, 18.0%-19.8%). Reductions at individual medical centers ranged from 3.8% to 23.5%. The mean effective dose for abdominal CT decreased from 20.0 to 15.0 mSv (25.0% reduction; 95% CI, 24.3%-25.8%). Reductions at individual medical centers ranged from 10.8% to 34.7%. The number of CT scans that had an effective dose measurement that exceeded benchmarks was reduced considerably by 48% and 54% for chest and abdomen, respectively. After the audit and meeting, head CT doses varied less, although some institutions increased and some decreased mean head CT doses and the proportion above benchmarks. Conclusions and Relevance Reviewing institutional doses and sharing dose-optimization best practices resulted in lower radiation doses for chest and abdominal CT and more consistent doses for head CT.

Purpose To determine patient, vendor, and institutional factors that influence computed tomography (CT) radiation dose. Materials and Methods The relevant institutional review boards approved this HIPAA-compliant study, with waiver of informed consent. Volume CT dose index (CTDIvol) and effective dose in 274 124 head, chest, and abdominal CT examinations performed in adult patients at 12 facilities in 2013 were collected prospectively. Patient, vendor, and institutional characteristics that could be used to predict (a) median dose by using linear regression after log transformation of doses and (b) high-dose examinations (top 25% of dose within anatomic strata) by using modified Poisson regression were assessed. Results There was wide variation in dose within and across medical centers. For chest CTDIvol, overall median dose across all institutions was 11 mGy, and institutional median dose was 7-16 mGy. Models including patient, vendor, and institutional factors were good for prediction of median doses (R2 = 0.31-0.61). The specific institution where the examination was performed (reflecting the specific protocols used) accounted for a moderate to large proportion of dose variation. For chest CTDIvol, unadjusted median CTDIvol was 16.5 mGy at one institution and 6.7 mGy at another (adjusted relative median dose, 2.6 mGy [95% confidence interval: 2.5, 2.7]). Several variables were important predictors that a patient would undergo high-dose CT. These included patient size, the specific institution where CT was performed, and the use of multiphase scanning. For example, while 49% of patients (21 411 of 43 696) who underwent multiphase abdominal CT had a high-dose examination, 8% of patients (4977 of 62 212) who underwent single-phase CT had a high-dose examination (adjusted relative risk, 6.20 [95% CI: 6.17, 6.23]). If all patients had been examined with single-phase CT, 69% (18 208 of 26 388) of high-dose examinations would have been eliminated. Patient size, institutional-specific protocols, and multiphase scanning were the most important predictors of dose (change in R2 = 8%-32%), followed by manufacturer and iterative reconstruction (change in R2, 0.2%-15.0%). Conclusion CT doses vary considerably within and across facilities. The primary factors that influenced dose variation were multiphase scanning and institutional protocol choices. It is unknown if the variation in these factors influenced diagnostic accuracy. © RSNA, 2016.

BACKGROUND Transanal endoscopic microsurgery is used in the surgical management of advanced rectal polyps and early rectal cancers. There are case reports of transanal endoscopic microsurgery colorectal anastomoses being performed with laparoscopic assistance in humans. METHODS The concept of a transanal endoscopic microsurgery colorectal anastomosis without laparoscopic assistance has been discussed and trialed on animal and cadaveric specimens; however, to date, there have been no technical reports of this particular procedure in the literature. RESULTS We present a technical note describing a transanal endoscopic microsurgery intraperitoneal colorectal anastomosis in a live human without laparoscopic assistance.

The goal of targeted therapeutics and molecular diagnostics is to accumulate drugs or probes at the site of disease in higher quantities relative to other locations in the body. To achieve this, there is tremendous interest in the development of nanomaterials capable of acting as carriers or reservoirs of therapeutics and diagnostics in vivo.[1] Generally, nanoscale particles are favored for this task[2] as they can be large enough to function as carriers of multiple copies of a given small mole cule, can display multiple targeting functionalities, and can be small enough to be safely injected into the blood stream.[3] The general goal is that particles will either target passively via the enhanced permeability and retention (EPR) effect, actively by incorporation of targeting groups, or by a combination of both.[3b,4] Nanoparticle targeting strategies have largely relied on the use of surface conjugated ligands designed to bind overexpressed cell-membrane receptors associated with a given cell-type.[5] We envisioned an alternative targeting strategy that would lead to an active accumulation of nanoparticles by virtue of a supramolecular assembly event specific to tumor tissue, occurring in response to a specific signal (Figure 1). The most desirable approach to stimuli-induced targeting would be to utilize an endogenous signal, specific to the diseased tissue itself, capable of actively targeting materials introduced via intravenous (IV) injection. Such an approach is in contrast to efforts to develop systems capable of targeting and release via the local application of external stimuli such as light[6] or magnetic fields.[7] With respect to viable endogenous signals, one could reasonably consider materials that accumulate in response to stimuli including pH changes,[8] temperature variation,[9] or redox reactions. [10] However, we aim to develop nanoparticles capable of assembling in vivo in response to selective, endogenous, biomolecular signals.[11] For this purpose, we aim to utilize enzymes as stimuli, rather than other recognition events, because they are uniquely capable of propagating a signal via catalytic amplification in vivo as in enzyme-prodrug therapy strategies.[12]

While an increasing number of (hexacarbonyldicobalt)alkynes have been found to possess antiproliferative activity against a number of cancer cell lines, the role of the organometallic moiety in this bioactivity is not well understood. To gain a better understanding of cobalt's role in the medicinal chemistry of these compounds, several simplified analogs of a known organocobalt anticancer compound were synthesized and assessed for antiproliferative activity against MDA-MB-231 human breast cancer cells. These compounds, mostly (hexacarbonyldicobalt)propargyl aryl ethers, caused 45-93% growth inhibition of that cell line at 40μM in a 72h crystal violet staining assay. The most active analog was the organocobalt nitroaromatic ether 3a, with an IC(50) of 3.3±0.9μM. Flow cytometric assays on the same cell line demonstrated that 3a strongly induces apoptosis, arrests the cell cycle at the S phase, increases cellular oxidative stress levels, and induces permeability of the mitochondrial membrane. While the non-cobalt-containing precursor to 3a also caused an increase in mitochondrial membrane permeability, it did not produce an increase in oxidative stress levels, nor did it have apoptosis-inducing or antiproliferative effects. The induction of oxidative stress in the cell may be responsible for some of the antiproliferative activity of compound 3a against this cell line.

Calciphylaxis, or calcific uremic arteriolopathy, is a rare but particularly morbid condition involving systemic medial calcification of arterioles causing ischemia and subsequent tissue necrosis. Although most commonly occurring over the abdomen and proximal extremities, calciphylaxis can present on nearly any skin surface with a tendency toward areas of increased adiposity. We report a case of a 53-year-old female with end-stage renal disease who presented with bilateral palpable breast masses and overlying skin changes. Diagnostic mammography and percutaneous biopsy of the lesion facilitated the diagnosis of calciphylaxis and she was treated with medical therapy, local wound care, and eventual tissue extirpation. Due to the morbidity attributed to calciphylaxis and associated wound complications, surgical extirpation is at times unavoidable. Once malignancy has been excluded, we recommend nonoperative management with prompt referral to Nephrology for medical optimization, reserving surgical debridement for nonhealing wounds and superinfection.

Lung retransplantation (ReTx) comprises an increasing share of lung transplants and recently has shown improved outcomes. The aim of this study was to identify risk factors affecting overall survival after pulmonary ReTx.The United Network for Organ Sharing database was used to identify patients undergoing lung transplantation at our institution from 1995 to 2014. Of the total 542 lung transplants performed, 87 (16.1%) were ReTxs. The primary outcome was overall survival. Multivariate Cox regression models were used to assess the effect of recipient and donor characteristics on survival.Of the patients who underwent ReTx, median survival was 2 years. Predictors of worse survival include recipient age between 50 and 60 years (relative risk, 4.3; p = 0.02) or older than 60 years (relative risk, 10.2; p 0.001), and time to ReTx of less than 2 years (relative risk, 3.8; p = 0.01). ReTx for bronchiolitis obliterans syndrome had longer median survival than for restrictive chronic lung allograft dysfunction (2.7 years vs 0.9 years; p = 0.055). Overall survival of ReTx patients after initiation of the lung allocation score was not significantly different (p = 0.21).Lung ReTx outcomes are significantly worse than for primary transplantation but may be appropriate in well-selected patients with certain diagnoses. Lung ReTx in patients older than 50 years or within 2 years of primary lung transplantation was associated with decreased survival. Further work is warranted to identify patients who benefit most from ReTx.

Although rhinoviral infections, a major cause of asthma exacerbations, occur predominantly in upper airway bronchial epithelial cells, monocytic-lineage cells are implicated in establishing the inflammatory microenvironment observed during the disease. Human rhinovirus (HRV) is unique in that nearly genetically identical viruses bind either the ICAM-1 or low-density lipoprotein receptor (LDL-R). Within minutes of binding, HRV is capable of eliciting a signaling response in both epithelial cells and monocyte-derived macrophages. It is unclear whether this signaling response is important to the subsequent release of inflammatory mediators, particularly in cells not capable of supporting viral replication. We show here that the small molecular mass G-protein Rac is activated following exposure of macrophages to HRV serotypes known to be ICAM-1- and LDL-R-tropic. We demonstrate that inhibiting Rac resulted in the upregulation of TLR3 in macrophages exposed to major- and minor-group HRV, and resulted in increased release of IFN-α. Furthermore, inhibiting Rac in HRV-exposed macrophages attenuated activation of the stress kinase p38 and release of the pro-inflammatory cytokine CCL2, but inhibiting Rac did not affect release of the pro-inflammatory cytokine CCL5. These findings suggest that Rac is an important regulator in establishing the inflammatory microenvironment that is initiated in the human airway upon exposure to rhinovirus.

Human rhinovirus (HRV) is one of the most common human respiratory pathogens and is responsible for the majority of upper respiratory illnesses. Recently, a phylogeny was constructed from all known American Type Culture Collection (ATCC) HRV sequences. From this study, three HRV classifications (HRVA, HRVB, and HRVC) were determined and techniques for classifying new isolates of HRV were reported. The genetic change of this virus in specific populations over time is of great interest to understand the evolution and epidemiology of viruses. To facilitate the collections of HRV sequences over a number of years, a virology experiment was designed in which students test nasal lavage samples to look for HRV infection. Students will learn a variety of techniques including RNA isolation, cDNA synthesis, qPCR, and agarose gel electrophoresis as well as bioinformatic skills though examination of sequences from the HRV‐field isolates. Furthermore, students can look at symptom data from subjects to investigate correlations between symptom severity and factors such as stress and sleep patterns. Such information can be used to examine hypotheses regarding HRV mutation, symptom severity and epidemiology. BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Vol. 39, No. 6, pp. 457–458, 2011

Osteoarthritis (OA) is the most common degenerative disease affecting articular cartilage and is characterized by disrupted cartilage extracellular matrix (ECM) homeostasis, ultimately resulting in loss of cartilage without effective replacement. OA is caused in part by exposure of chondrocytes to inflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) (1–4). IL-1β and TNFα have long been known to induce matrix metalloproteinase (MMP) expression in chondrocytes, thereby leading to ECM degradation and cartilage breakdown (3–6). The action of the inflammatory cytokines disrupts the delicate balance between ECM synthesis and degradation in articular cartilage and leads to the destruction of cartilage and the onset of arthritis. Since OA is usually evident in the fourth to fifth decade of life, it is considered an age-associated disease (1,2). It is therefore likely that gene products regulating lifespan and aging would have an impact on OA. One such protein is SirT1, a lysine deacetylase that is responsible for lifespan extension under conditions of caloric restriction (7–9). SirT1 is an NAD-dependent protein deacetylase that targets both chromatin (histones) and nonchromatin proteins. While SirT1 has been shown to play an important role in a variety of age-related diseases, such as diabetes, cancer, osteoporosis, and neurodegeneration (9–12), little is known of the role it plays in either cartilage biology or OA. Recently, it was demonstrated that SirT1 enhances cartilage-specific ECM gene expression (13). SirT1 appears to accomplish this function, at least for α2(I) collagen, by enhancing SOX9-mediated transcription via the recruitment of a number of transcription activators (i.e., histone acetyltransferases) to the promoter and enhancer sites (13). It has also recently been demonstrated that SirT1 blocks apoptosis in chondrocytes, and that it accomplishes this by multiple mechanisms (14,15). Additionally, evidence indicates that SirT1 levels are reduced in chondrocytes from OA cartilage compared to normal cartilage, suggesting that the altered pattern of gene expression and apoptosis evident in OA is correlated with a reduction in SirT1 levels (13,14). While OA is generally not considered to be an inflammatory disease, it is nevertheless influenced by inflammatory cytokines, such as IL-1β and TNFα (2,4,16). Interestingly, SirT1 demonstrates a broad anti-inflammatory function in a variety of tissues (8,9). SirT1 likely accomplishes this in part by the deacetylation of the p65 subunit of NF-κB, blocking its ability to bind DNA, thereby inhibiting transcription of proinflammatory genes (17). While it appears that SirT1 can inhibit inflammation, there is no evidence to date suggesting that the opposite is true, that mediators of inflammation can interfere with SirT1 function. In the present study, we explored the idea that an inflammatory cytokine modulates the activity of SirT1. We found that in cells treated with a nonapoptotic dose of TNFα, SirT1 undergoes a cathepsin B–mediated cleavage event, generating a stable 75-kd fragment with reduced enzymatic activity. This novel modification to SirT1 is discussed in terms of the pathologic conditions of arthritis.

The potential for contaminated dietary supplements to result in a failed doping test remains a concern for athletes, trainers, and sporting authorities despite improvements to regulatory guidelines. Previous surveys of readily available supplements confirm that many are contaminated with steroids and stimulants prohibited for use in elite sport. Suggested responses to this issue include the complete avoidance of all supplements. Many athletes, however, use nutritional supplements to achieve effective training and also to ensure that daily nutritional requirements are met (e.g. recommended levels of vitamins and minerals). This ensures that the use of supplements is and will remain the norm for a range of sports. As a result, an alternative approach of rigorous testing of materials destined for use by elite athletes has been introduced in several countries. While the testing of final product for banned substances may help mitigate the problem, it will not help to remove the underlying issue of contamination. In this article we describe an alternative approach that uses appropriate quality assurance procedures backed up by testing to remove sources of contamination. The decrease in the incidence of contamination amongst supplement companies adopting such a system is explained, and contrasted with the relatively high incidences of contamination found in products that are not part of a quality system. These findings are of key importance to both supplement manufacturers and those involved in advising athletes about supplement use. Copyright © 2010 John Wiley & Sons, Ltd.

The role of histone deacetylase 1 and 2 (HDAC1 and HDAC2) in regulating cartilage-specific gene expression was explored in primary human chondrocytes. HDAC1 and HDAC2 protein levels were elevated in chondrocytes from osteoarthritic patients, consistent with a down-regulation of some cartilage marker genes. When expressed in these cells, HDAC1 and HDAC2 repressed aggrecan and collagen 2(α1) expression but differed in their repression of collagen 9(α1), collagen 11(α1), dermatopontin, and cartilage oligomeric matrix protein (COMP). To identify the basis of these differences between HDAC1 and HDAC2, their carboxy-terminal domains (CTDs) were deleted, which led to proteins that retained enzymatic activity but were unable to repress cartilage gene expression. Further, exchange of the CTDs between HDAC1 and HDAC2 led to proteins that were enzymatically active but displayed altered target gene specificity, indicating that these CTDs can function independently of HDAC enzymatic activity, to target the HDACs to specific genes. The Snail transcription factor was identified as a mediator of HDAC1 and HDAC2 repression of the collagen 2(α1) gene, via its interaction with the HDAC1 and 2 CTDs. The data indicate that the CTD serves a novel function within HDAC1 and HDAC2, to mediate repression of cartilage-specific gene expression in human chondrocytes.—Hong, S., Derfoul, A., Pereira-Mouries, L., Hall, D. J. A novel domain in histone deacetylase 1 and 2 mediates repression of cartilage-specific genes in human chondrocytes.

Osteoarthritis (OA) is a multifactorial disease. Different risk factors have been identified such as aging and obesity and different models have been used to study the impact of obesity and overweight in this pathology. The field the more studied is in vitro cartilage submitted to mechanical stresses. Four different stresses can be applied on this tissue: shear stress, loading, tensile stress (stretching) and hydrostatic pressure. The signal transduction to the chondrocyte and to the nucleus of the cell is a large field of investigation named mechano-transduction. The response of cartilage depends on quality of subchondral bone as well. So, more and more teams are studying the impact of mechanical stresses on bone, mainly by stretching osteoblasts or by submitting them to a fluid shear stress. Recently, a new model of bone compression has been set up, with osteoblasts in their own extracellular matrix. Finally the third field studied is the role of adipokines, mediators playing a key role in obesity, on the aetiology of OA. Adipokines like leptin, resistin, adiponectin and visfatin, seems to play a pro-inflammatory role in arthritis. Studying the role of obesity in OA could be more complex than expected. The link between OA and obesity may not simply be due to high mechanical stresses applied on the tissues, but soluble mediators may play an important role in the onset of OA in obese patients.

Cartilage oligomeric matrix protein (COMP) is a component of cartilage, synovium, ligament, and tendon, yet its normal function is largely unknown. To identify its function we have expressed it in 293 and HeLa cell lines and in primary human chondrocytes. We find that COMP protects these cells against death, either in the presence or absence of tumor necrosis factor α and is able to block activation of caspase 3, a critical effector caspase. This effect appears to be mediated by the IAP (inhibitor of apoptosis protein) family of anti-apoptotic proteins because the levels of XIAP, survivin, cIAP1 and cIAP2 are significantly elevated in the COMP-expressing cells and down-regulation of survivin and XIAP protein levels by small interfering RNAs blocks the ability of COMP to enhance survival. The mRNAs for most of the IAP family members were not increased by COMP, indicating that a translational/post-translational mechanism was involved in their induction. However, in both HeLa cells and chondrocytes, COMP induced survivin mRNA by 5-fold. Thus survivin is the first gene identified to be up-regulated transcriptionally by COMP. The carboxyl-terminal half of the protein comprising the type 3 repeats and the RGD sequence (CaCTD domain) was sufficient to promote survival and to elevate the IAPs. Further, an RGD peptide was able to block the prosurvival effect of COMP and the induction of XIAP and survivin, indicating that survival is likely mediated through integrin signaling. These data point to a new role for COMP in protecting cells against death.

In the adult human, mesenchymal stem cells (hMSCs) resident in the bone marrow retain the capacity to proliferate and differentiate along multiple connective tissue lineages, including cartilage. Glucocorticoids (GCs) are required for chondrogenic differentiation of hMSCs in vitro; however, the exact role of GCs in this process is not known. In this study, we examined the effects of dexamethasone (DEX) on chondrogenic differentiation of hMSCs in the presence or absence of DEX, transforming growth factor-β (TGF-β), or DEX plus TGF-β. GC treatment upregulated gene expression of cartilage matrix components aggrecan, dermatopontin, and collagen type XI; enhanced TGF-β-mediated upregulation of collagen type II and cartilage oligomeric matrix protein; and increased aggrecan and collagen type II production as well as cartilage matrix-sulfated proteoglycans as assessed by immunohistochemistry and alcian blue staining. Inclusion of an antagonist of GCs inhibited expression of chondrogenic differentiation markers, suggesting that the GC effects during chondrogenesis are mediated by the GC receptor (GR). Steady levels of the major active form of GR, GRα, were detected in both undifferentiated and differentiating hMSCs, whereas the dominant-negative isoform GRβ, present at low levels in undifferentiated hMSCs, was downregulated during chondrogenesis. In the presence of DEX and TGF-β, expression of a collagen type II gene promoter luciferase reporter construct in hMSCs was upregulated. However, coexpression of GRβ dramatically inhibited promoter activity, suggesting that GRα is required for GC-mediated modulation of chondrogenesis and that GCs may play an important role in the maintenance of cartilage homeostasis.

STUDY DESIGN: A prospective, randomized, double-blind, placebo-controlled trial of intradiscal electrothermal therapy (IDET) for the treatment of chronic discogenic low back pain (CDLBP). OBJECTIVES: To test the safety and efficacy of IDET compared with a sham treatment (placebo). SUMMARY OF BACKGROUND DATA: In North America alone, more than 40,000 intradiscal catheters have been used to treat CDLBP. The evidence for efficacy of IDET is weak coming from retrospective and prospective cohort studies providing only Class II and Class III evidence. There is one study published with Class I evidence. This demonstrates statistically significant improvements following IDET; however, the clinical significance of these improvements is questionable. METHODS: Patients with CDLBP who failed to improve following conservative therapy were considered for this study. Inclusion criteria included the presence of one- or two-level symptomatic disc degeneration with posterior or posterolateral anular tears as determined by provocative computed tomography (CT) discography. Patients were excluded if there was greater than 50% loss of disc height or previous spinal surgery. Fifty-seven patients were randomized with a 2:1 ratio: 38 to IDET and 19 to sham procedure (placebo). In all cases, the IDET catheter was positioned to cover at least 75% of the annular tear as defined by the CT discography. An independent technician connected the catheter to the generator and then either delivered electrothermal energy (active group) or did not (sham group). Surgeon, patient, and independent outcome assessor were all blinded to the treatment. All patients followed a standard postprocedural rehabilitation program. Independent statistical analysis was performed. OUTCOME MEASURES: Low Back Outcome Score (LBOS), Oswestry Disability Index (ODI), Short Form 36 questionnaire (SF-36), Zung Depression Index (ZDI), and Modified Somatic Perceptions Questionnaire (MSPQ) were measured at baseline and 6 months. Successful outcome was defined as: no neurologic deficit, improvement in LBOS of greater then 7 points, and improvement in SF-36 subsets (physical function and bodily pain) of greater than 1 standard deviation. RESULTS: Baseline demographic data, initial LBOS, ODI, SF-36, ZDI, and MSPQ were similar for both groups. No neurologic deficits occurred. No subject in either arm showed improvement of greater than 7 points in LBOS or greater than 1 standard deviation in the specified domains of the SF-36. Mean ODI was 41.42 at baseline and 39.77 at 6 months for the IDET group, compared with 40.74 at baseline and 41.58 at 6 months for the placebo group. There was no significant change in ZDI or MSPQ scores for either group. CONCLUSIONS: The IDET procedure appeared safe with no permanent complications. No subject in either arm met criteria for successful outcome. Further detailed analyses showed no significant change in outcome measures in either group at 6 months. This study demonstrates no significant benefit from IDET over placebo.

The utilization of adult stem cells in tissue engineering is a promising solution to the problem of tissue or organ shortage. Adult bone marrow derived mesenchymal stem cells (MSCs) are undifferentiated, multipotential cells which are capable of giving rise to chondrocytes when maintained in a three-dimensional culture and treated with members of the transforming growth factor-beta (TGF-beta) family of growth factors. In this study, we fabricated a nanofibrous scaffold (NFS) made of a synthetic biodegradable polymer, poly(-caprolactone) (PCL), and examined its ability to support in vitro chondrogenesis of MSCs. The electrospun PCL porous scaffold was constructed of uniform, randomly oriented nanofibers with a diameter of 700 nm, and structural integrity of this scaffold was maintained over a 21-day culture period. MSCs cultured in NFSs in the presence of TGF-beta1 differentiated to a chondrocytic phenotype, as evidenced by chondrocyte-specific gene expression and synthesis of cartilage-associated extracellular matrix (ECM) proteins. The level of chondrogenesis observed in MSCs seeded within NFSs was comparable to that observed for MSCs maintained as cell aggregates or pellets, a widely used culture protocol for studying chondrogenesis of MSCs in vitro. Due to the physical nature and improved mechanical properties of NFSs, particularly in comparison to cell pellets, the findings reported here suggest that the PCL NFS is a practical carrier for MSC transplantation, and represents a candidate scaffold for cell-based tissue engineering approaches to cartilage repair.

With the advent of recent protocols to isolate multipotent human mesenchymal stem cells (MSCs), there is a need for efficient transfection methodologies for these cells. Most standard transfection methods yield poor transfection efficiencies for MSCs (1%). Here we have optimized a high-efficiency transfection technique for low passage MSCs derived from adult human bone marrow. This technique is an extension of electroporation, termed amaxa Nucleofection, where plasmid DNA is transfected directly into the cell nucleus, independent of the growth state of the cell. With this technique, we demonstrate up to 90% transfection efficiency of the viable population of MSCs, using plasmid construct containing a standard cytomegalovirus (CMV) early promoter driving expression of green fluorescent protein (GFP). Although little variation in transfection efficiency was observed between patient samples, a 2-fold difference in transfection efficiency and a 10-fold difference in expression levels per cell were seen using two distinct CMV-GFP expression plasmids. By fluorescence-activated cell sorting, the GFP expressing cells were sorted and subcultured. At 2 wk posttransfection, approx 25% of the population of sorted cells were GFP positive, and by 3 wk, nearly 10% of the cells still retained GFP expression. Transfection of these cells with plasmid containing either the collagen type I (Col1a1) promoter or the cartilage oligomeric matrix protein (COMP) promoter, each driving expression of GFP, produced a somewhat lower transfection efficiency (approx 40%), due in part to the lower activity of transcription from these promoters compared to that of CMV. Transfection with the collagen type II (Col2a1) promoter linked to GFP exhibited low expression, due to the fact that collagen type II is not expressed in these cells. Upon culturing of the Col2a1-GFP transfected cells in a transforming growth factor-beta3-containing medium known to induce mesenchymal chondrogensis, a significant enhancement of GFP level was seen, indicating the ability of the transfected cells to differentiate into chondrocytes and express cartilage-specific genes, such as Col2a1. Taken together, these data provide evidence of the applicability of this technique for the efficient transfection of MSCs.

The multilineage differentiation potential of adult tissue-derived mesenchymal progenitor cells (MPCs), such as those from bone marrow and trabecular bone, makes them a useful model to investigate mechanisms regulating tissue development and regeneration, such as cartilage. Treatment with transforming growth factor-beta (TGF-beta) superfamily members is a key requirement for the in vitro chondrogenic differentiation of MPCs. Intracellular signaling cascades, particularly those involving the mitogen-activated protein (MAP) kinases, p38, ERK-1, and JNK, have been shown to be activated by TGF-betas in promoting cartilage-specific gene expression. MPC chondrogenesis in vitro also requires high cell seeding density, reminiscent of the cellular condensation requirements for embryonic mesenchymal chondrogenesis, suggesting common chondro-regulatory mechanisms. Prompted by recent findings of the crucial role of the cell adhesion protein, N-cadherin, and Wnt signaling in condensation and chondrogenesis, we have examined here their involvement, as well as MAP kinase signaling, in TGF-beta1-induced chondrogenesis of trabecular bone-derived MPCs. Our results showed that TGF-beta1 treatment initiates and maintains chondrogenesis of MPCs through the differential chondro-stimulatory activities of p38, ERK-1, and to a lesser extent, JNK. This regulation of MPC chondrogenic differentiation by the MAP kinases involves the modulation of N-cadherin expression levels, thereby likely controlling condensation-like cell-cell interaction and progression to chondrogenic differentiation, by the sequential up-regulation and progressive down-regulation of N-cadherin. TGF-beta1-mediated MAP kinase activation also controls WNT-7A gene expression and Wnt-mediated signaling through the intracellular beta-catenin-TCF pathway, which likely regulates N-cadherin expression and subsequent N-cadherin-mediated cell-adhesion complexes during the early steps of MPC chondrogenesis.

The most frequent complication of total joint arthroplasty is periprosthetic osteolysis initiated by an inflammatory response to orthopaedic wear debris, which if left untreated, can result in implant instability and failure, eventually requiring revision surgery. We have previously reported that osteogenic differentiation of human marrow stroma-derived mesenchymal stem cells (hMSCs) is suppressed upon exposure to titanium particles, accompanied by reduced bone sialoprotein (BSP) gene expression, diminished production of collagen type I and BSP, decreased cellular viability and proliferation, and inhibition of extracellular matrix mineralization. In this study, we have further investigated hMSC cytotoxicity upon exposure to submicron particles of commercially pure titanium (cpTi) and zirconium oxide (ZrO(2)). Our results showed that direct exposure to cpTi and ZrO(2) particles compromises cell viability through the induction of apoptosis, eliciting increased levels of the tumor suppressor proteins p53 and p73, in a manner dependent on material composition, particle dosage, and time. Additionally, conditioned medium collected from hMSCs exposed to cpTi particles, but not to ZrO(2) particles, is cytotoxic to hMSCs, inducing apoptosis in the absence of particles. These findings demonstrate that exposure to orthopaedically derived wear particles can compromise hMSC viability through the direct and indirect induction of apoptosis. Thus, prolonged in vivo exposure of marrow-derived hMSCs to implant-derived wear debris is likely to reduce the population of viable osteoprogenitor cells, and may contribute to poor periprosthetic bone quality and implant loosening.

Several proteins with important roles in oncogenesis have been shown to regulate the function of the E2F-1 transcription factor, which is known to activate the expression of genes required for proliferation and apoptosis. Here we identify the MDMX oncoprotein as an E2F-1-binding factor, from a yeast-two hybrid screen using a portion of the E2F-1 protein as “bait.” We demonstrate that the region within MDMX needed for the E2F-1:MDMX interaction is located in the central part of the protein, C-terminal of the p53-binding domain. The region within E2F-1 needed for this association is adjacent to the DNA binding domain. Further, when expressed in vivo or in vitro the MDMX protein migrates as two isoforms on SDS-PAGE, the faster migrating isoform having the stronger affinity for the E2F-1 proteins. It appears that this interaction reduces the ability of E2F-1 to bind DNA. Expression of MDMX along with E2F-1 and Dp-1 in Saos2 cells reduces the ability of E2F-1 to bind to its consensus DNA sequence, without altering E2F-1 protein levels. These data indicate that the MDMX protein is capable of associating with E2F-1 and negatively regulating its DNA binding ability. © 2002 Wiley-Liss, Inc.

Here we show that distinct subpopulations of cells exist within traumatic human extremity wounds, each having the ability to differentiate into multiple cells types in vitro. A crude cell suspension derived from traumatized muscle was positively sorted for CD29, CD31, CD34, CD56 or CD91. The cell suspension was also simultaneously negatively sorted for either CD45 or CD117 to exclude hematopoietic stem cells. These subpopulations varied in terms their total numbers and their abilities to grow, migrate, differentiate and secrete cytokines. While all five subpopulations demonstrated equal abilities to undergo osteogenesis, they were distinct in their ability to undergo adipogenesis and vascular endotheliogenesis. The most abundant subpopulations were CD29+ and CD34+, which overlapped significantly. The CD29+ and CD34+ cells had the greatest proliferative and migratory capacity while the CD56+ subpopulation produced the highest amounts of TGFß1 and TGFß2. When cultured under endothelial differentiation conditions the CD29+ and CD34+ cells expressed VE-cadherin, Tie2 and CD31, all markers of endothelial cells. These data indicate that while there are multiple cell types within traumatized muscle that have osteogenic differentiation capacity and may contribute to bone formation in post-traumatic heterotopic ossification (HO), the major contributory cell types are CD29+ and CD34+, which demonstrate endothelial progenitor cell characteristics.

Major- and minor-group human rhinoviruses (HRV) enter their host by binding to the cell surface molecules ICAM-1 and LDL-R, respectively, which are present on both macrophages and epithelial cells. Although epithelial cells are the primary site of productive HRV infection, previous studies have implicated macrophages in establishing the cytokine dysregulation that occurs during rhinovirus-induced asthma exacerbations. Analysis of the transcriptome of primary human macrophages exposed to major- and minor-group HRV demonstrated differential gene expression. Alterations in gene expression were traced to differential mitochondrial activity and signaling pathway activation between two rhinovirus serotypes, HRV16 (major-group) and HRV1A (minor-group), upon initial HRV binding. Variances in phosphorylation of kinases (p38, JNK, ERK5) and transcription factors (ATF-2, CREB, CEBP-alpha) were observed between the major- and minor-group HRV treatments. Differential activation of signaling pathways led to changes in the production of the asthma-relevant cytokines CCL20, CCL2, and IL-10. This is the first report of genetically similar viruses eliciting dissimilar cytokine release, transcription factor phosphorylation, and MAPK activation from macrophages, suggesting that receptor use is a mechanism for establishing the inflammatory microenvironment in the human airway upon exposure to rhinovirus.

E2F-1-activated transcription promotes cell cycle progression and apoptosis. These functions are regulated by several factors including the E2F-1-binding protein MDM2 and the retinoblastoma protein pRb. Using a yeast two-hybrid screen we have identified the MDM2-related protein, MDMX, as an E2F-1-binding protein. In these studies we find that coexpression of MDMX with E2F-1 results in degradation of the MDMX protein. Although this proteolytic degradation can be blocked by the protease inhibitors bafilomycin A(1), N-acetyl-Leu-Leu-Norleu-AL, and N-acetyl-Leu-Leu-Met-AL, MDMX degradation is not inhibited by lactacystin, suggesting that degradation occurs by a proteasome-independent mechanism. Using an E2F-1 deletion mutant (E2F-1(180-437)) we show that E2F-1-targeted degradation of MDMX does not require the E2F-1 DNA binding domain and therefore is independent of E2F-1-driven transcription. We also find that this transcriptionally inactive E2F-1 mutant is capable of degrading the MDMX-related protein MDM2 and the MDMX isoform MDMX-S. Mapping of the E2F-1 C terminus reveals that neither a previously characterized C-terminal MDM2 binding domain nor the pRb binding domain on E2F-1 is required for MDMX and MDM2 degradation.

Encephalomyocarditis virus (EMCV) is the prototype member of the cardiovirus genus of picornaviruses. For cardioviruses and the related aphthoviruses, the first protein segment translated from the plus-strand RNA genome is the Leader protein. The aphthovirus Leader (173-201 amino acids) is an autocatalytic papain-like protease that cleaves translation factor eIF-4G to shut off cap-dependent host protein synthesis during infection. The less characterized cardioviral Leader is a shorter protein (67-76 amino acids) and does not contain recognizable proteolytic motifs. Instead, these Leaders have sequences consistent with N-terminal zinc-binding motifs, centrally located tyrosine kinase phosphorylation sites, and C-terminal, acid-rich domains. Deletion mutations, removing the zinc motif, the acid domain, or both domains, were engineered into EMCV cDNAs. In all cases, the mutations gave rise to viable viruses, but the plaque phenotypes in HeLa cells were significantly smaller than for wild-type virus. RNA transcripts containing the Leader deletions had reduced capacity to direct protein synthesis in cell-free extracts and the products with deletions in the acid-rich domains were less effective substrates at the L/P1 site, for viral proteinase 3Cpro. Recombinant EMCV Leader (rL) was expressed in bacteria and purified to homogeneity. This protein bound zinc stoichiometrically, whereas protein with a deletion in the zinc motif was inactive. Polyclonal mouse sera, raised against rL, immunoprecipitated Leader-containing precursors from infected HeLa cell extracts, but did not detect significant pools of the mature Leader. However, additional reactions with antiphosphotyrosine antibodies show that the mature Leader, but not its precursors, is phosphorylated during viral infection. The data suggest the natural Leader may play a role in regulation of viral genome translation, perhaps through a triggering phosphorylation event.

The multipotential murine embryonic C3H10T1/2 mesenchymal cell line is able to undergo chondrogenesis in vitro, in a high density micromass environment, following treatment with soluble human bone morphogenetic protein-2 (BMP-2). To enhance this process, the human BMP-2 cDNA was cloned into a retroviral expression vector and a high titer, infectious retrovirus (replication defective) was generated. Infection of C3HIOT1/2 cells with this retroviral construct resulted in an infection efficiency of 90-95% and was highly effective in converting cells in micromass culture to a chondrocyte phenotype, as assessed by positive Alcian blue staining for extracellular matrix proteoglycans, increased sulfate incorporation, increased expression of the cartilage marker genes collagen type II and aggrecan, and decreased expression of collagen type I. Interestingly, BMP-2 expression in the micromass cultures also induced the expression of the cell cycle inhibitory protein/differentiation factor p21/WAF1, suggesting its functional involvement in chondrogenesis. The chondrogenic effect of retrovirally expressed BMP-2 in these high-density cultures was limited to the infected cells, since uninfected cells did not chondrify when co-cultured as a nonoverlapping micromass adjacent to BMP-2 expressing cells. These data indicate that retrovirally expressed BMP-2 is highly effective at inducing a chondrocyte phenotype in a multipotential mesenchymal cell line in vitro, and its action is restricted to the infected cell population. These findings should provide a framework for the optimization of chondrogenesis in culture using mesenchymal stem cells and retroviral gene transfer.

Matrix metalloproteinase enzymes, overexpressed in HT-1080 human fibrocarcinoma tumors, were used to guide the accumulation and retention of an enzyme-responsive nanoparticle in a xenograft mouse model. The nanoparticles were prepared as micelles from amphiphilic block copolymers bearing a simple hydrophobic block and a hydrophilic peptide brush. The polymers were end-labeled with Alexa Fluor 647 dyes leading to the formation of labeled micelles upon dialysis of the polymers from DMSO/DMF to aqueous buffer. This dye-labeling strategy allowed the presence of the retained material to be visualized via whole animal imaging in vivo and in ex vivo organ analysis following intratumoral injection into HT-1080 xenograft tumors. We propose that the material is retained by virtue of an enzyme-induced accumulation process whereby particles change morphology from 20 nm spherical micelles to micrometer-scale aggregates, kinetically trapping them within the tumor. This hypothesis is tested here via an unprecedented super-resolution fluorescence analysis of ex vivo tissue slices confirming a particle size increase occurs concomitantly with extended retention of responsive particles compared to unresponsive controls.

Mineralocorticoid and glucocorticoid hormones activate the expression of the Na/K ATPase beta1 through direct binding of the mineralocorticoid receptor (MR) and glucocorticoid receptors (GR) to a mineralocorticoid- and glucocorticoid-responsive element in the beta1 promoter region, but activation of the beta1 promoter is inhibited by coexpression of both receptors. Here, using a series of mutated and chimeric receptors, we show that the N-terminal region of MR mediates an inhibitory effect on MR and GR activation from the beta1 promoter, in CV-1 cells. Deletion of the N-terminal region of MR (1-603) enhanced MR activation four-fold. Activation by chimeric MR, in which the N-terminus of GR replaces the N-terminal region of MR, was threefold that of wild-type MR. In addition, whereas coexpression of wild-type MR and GR was inhibitory, coexpression of chimeric MR and wild-type GR was nearly equal to that of MR. By contrast, mutated GR lacking its N-terminal region (1-420) was less efficient than the wild type in activating this promoter. These results demonstrate that the N-terminal domains of MR and GR have opposite transactivation properties and that MR region 1-603 is indeed inhibitory for both MR- and GR-mediated regulation of the Na/K ATPase beta1 gene promoter.

Caspases (aspartate-specific cysteine proteases) play a critical role in the execution of the mammalian apoptotic program. To address the regulation of human caspase activation, we used the yeast Saccharomyces cerevisiae, which is devoid of endogenous caspases. The apical procaspases, -8beta and -10, were efficiently processed and activated in yeast. Although protease activity, per se, was insufficient to drive cell death, caspase-10 activity had little effect on cell viability, whereas expression of caspase-8beta was cytotoxic. This lethal phenotype was abrogated by co-expression of the pan-caspase inhibitor, baculovirus p35, and by mutation of the active site cysteine of procaspase-8beta. In contrast, autoactivation of the executioner caspase-3 and -6 zymogens was not detected. Procaspase-3 activation required co-expression of procaspase-8 or -10. Surprisingly, activation of procaspase-6 required proteolytic activities other than caspase-8, -10, or -3. Caspase-8beta or -10 activity was insufficient to catalyze the maturation of procaspase-6. Moreover, a constitutively active caspase-3, although cytotoxic in its own right, was unable to induce the processing of wild-type procaspase-6 and vice versa. These results distinguish sequential modes of activation for different caspases in vivo and establish a yeast model system to examine the regulation of caspase cascades. Moreover, the distinct terminal phenotypes induced by various caspases attest to differences in the cellular targets of these apoptotic proteases, which may be defined using this system.

The E2F element is a cis-acting DNA sequence within the P2 promoter of c-myc proto-oncogene. While it is required for optimal transcription, the multiprotein complexes formed on this site have not been well characterized. We show that in extracts of human glioblastoma cells and NIH3T3 fibroblasts, significant E2F transcription factor binding to the c-myc E2F site occurs as a both a monomer (the active form) and as only two mutually exclusive complexes with the retinoblastoma gene product (pRb) or the cyclin A protein. The E2F protein monomer was found predominantly in the cytosolic fraction of the cellular extracts while the pRb and cyclin A complexes in the nuclear fraction, indicating that the monomer has novel physical properties. Thus, protein complex formation on the c-myc E2F site appears to contribute in a unique way to transcriptional activation.

Fluorescence imaging is one of the most versatile and widely used visualization methods in biomedical research. However, tissue autofluorescence is a major obstacle confounding interpretation of in vivo fluorescence images. The unusually long emission lifetime (5-13 μs) of photoluminescent porous silicon nanoparticles can allow the time-gated imaging of tissues in vivo, completely eliminating shorter-lived (< 10 ns) emission signals from organic chromophores or tissue autofluorescence.Here, using a conventional animal imaging system not optimized for such long-lived excited states, we demonstrate improvement of signal to background contrast ratio by > 50-fold in vitro and by > 20-fold in vivo when imaging porous silicon nanoparticles. Time-gated imaging of porous silicon nanoparticles accumulated in a human ovarian cancer xenograft following intravenous injection is demonstrated in a live mouse. The potential for multiplexing of images in the time domain by using separate porous silicon nanoparticles engineered with different excited state lifetimes is discussed.

Site-specific mutations within the proteinase 3C-dependent P3 region cleavage sequences of encephalomyocarditis virus have been constructed. The mutations altered the normal QG cleavage site dipeptide pairs of the 2C/3A, 3A/3B, 3B/3C, and 3C/3D junctions into QV, QC, QF, QY, and RG sequences. When translated in vitro in the context of full-length viral polyproteins, all mutations blocked endogenous 3C-mediated processing at their engineered sites and produced stable forms of the expected viral P3 precursors that were also resistant to cleavage by exogenously added recombinant 3C. Relative to wild-type viral sequences, each mutant form of P3 had a somewhat different ability to mediate overall polyprotein processing. Mutations at the 2C/3A, 3A/3B, and 3B/3C sites, for example, were generally less impaired than 3C/3D mutations, when the cleavage reactions were quantitated with cotranslated L-P1-2A precursors. A notable exception was mutant 3B3C(QG3RG), which provedfarlessactivethansiblingmutants3B3C(QG3QF)and3B3C(QG3QV),afindingthatpossiblyimplicates thissegmentintheproperfoldingofanactive3C.WhentransfectedintoHeLacells,allmutantsequenceswere lethal, presumably because of the reduced L-P1-2A processing levels or reduced RNA synthesis capacity. However, when specifically tested for the latter activity, all mutations except those at the 3C/3D cleavage site were indeed able to initiate and perpetuate viral RNA replication in transfected cells, albeit to RNA accumulationlevelslowerthanthoseproducedbywild-typesequences.Thetransfectioneffectscouldbemimickedwith cell-free synthesized proteins, in that translation samples containing locked 3CD polymerase precursors were catalyticallyinactiveinpoly(A)-oligo(U)-dependentassays,whileallothermutantprocessingsamplesinitiated detectable RNA synthesis. Surprisingly, not only did the 3B/3C mutant sequences prove capable of directing RNA synthesis, but the viral RNA thus synthesized could be immunolabeled and precipitated with 3C-specific monoclonal antibody reagents, indicating an unexpected covalent attachment of the proteinase to the RNA product whenever this cleavage site was blocked. Picornavirus RNA genomes encode a large polyprotein which is proteolytically processed in cotranslational and posttranslationalreactionsbyseveralself-encodedreactivecenters. The majority of cleavages are catalyzed by one specific proteinase, 3C, an unusual cysteine-reactive, chymotrypsin-like sequence located in the carboxyl third of the polyprotein. During translation, 3C is synthesized in an enzymatically active form, and even while still part of the nascent polyprotein, it rapidly and efficiently catalyzes an ordered series of monomolecular and bimolecular scissions within its parental molecule to release mature viral proteins and precursor combinations. The consensus 3C cleavage sites in most picornavirus polyproteins are at Gln-Gly dipeptide pairs, but not all conforming viral sequences are always cleaved, and in many virus strains, alternative combinations like Gln-Ser, Gln-Ala, GluGly,orGlu-Seroccuratthenaturalsites(6).Experimentswith

Previously, we described three mengovirus mutants derived from cDNA plasmids, containing shortened poly(C) tracts (C8, C12, and C13UC10), that exhibited strong attenuation for virulence in mice yet grew like wild-type virus in HeLa cells. Thirteen additional mutants hav now been constructed and characterized. Five of these differ only in poly(C) length, including one with a precise deletion of the tract. The other mutants bear deletions into the regions juxtaposing poly(C). Studies with HeLa cells confirm the essential dispensability of mengovirus's poly(C) tract but reveal a subtle, measurable correlation between poly(C) length and plaque diameter. Virulence studies with mice also revealed a strong correlation between poly(C) length and virulence. For the poly(C)-flanking mutations, the 15 bases directly 5' of the tract proved dispensable for virus viability, whereas the 20 to 30 bases 3' of poly(C) were critical for growth, thus implicating this region in the basal replication of the virus.

The amino terminus of the E2F1 transcription factor is a protein-protein interaction domain since it associates with cyclin A/cdk2. Here, the two-hybrid yeast screen was used to clone genes whose products associate with the amino terminus of E2F1. The amino-terminal 121 amino acids of E2F1 were fused to the Lex A binding domain while a partial length cDNA library from the embryo of a 12 day old mouse was fused to the VP16 activation domain. Following coexpression of these fusions in yeast, two novel genes were cloned that code for proteins that associate with E2F1. In an in vitro assay, these E2F1 Binding Proteins (EBP1 and EBP2) associate with residues 1-121 of E2F1 or with the full-length protein; however, they do not associate with its carboxy terminus (residues 88-437). When EBP1 or EBP2 were expressed in COS cells along with E2F1 and the target promoter DNA polymerase alpha, repression of transcription was observed. However, no repression of DNA polymerase alpha was seen if the cells expressed a nonassociating mutant E2F1 (residues 88-437), along with EBP1 or EBP2. Finally, expression of the EBP2 gene is up-regulated in growing NIH3T3 fibroblasts, relative to serum-starved cells. However, this up-regulation of EBP2 expression is not seen in fibroblasts constitutively expressing E2F1.

Mengo virus 3C proteinase was cloned and expressed to high levels in a bacterial vector system. The protein was solubilized from inclusion bodies then purified to homogeneity (>95%) by ion exchange chromatography. The recombinant enzyme was proteolytically active in cell-free processing assays with a mengo capsid precursor substrate, L-P1-2A, correctly and proficiently cleaving it into L, 1AB, 1C, 1D and 2A protein products. Further analyses with synthetic peptide substrates encompassing the Mengo or rhinovirus-14 2C/3A cleavage sequences, showed the Mengo 3C could recognize and process specific glutamine-glycine sites within these peptides. The reactivity with the rhinovirus peptide was unexpected, because cross-reactivity between a picornavirus 3C enzyme and a protein substrate from different genus of this family has otherwise never been observed. In reciprocal reactions, a rhinovirus-14 3C preparation was unable to cleave the Mengo-derived synthetic peptide substrate. The recombinant Mengo 3C reactions were also characterized with regard to substrate Km, optimum pH and temperature. The protein was additionally used to raise monoclonal antibodies (mAbs) in mice, which in turn localized natural 3C, 3ABC, 3CD and P3 in immunoblots, immunoprecipitations and indirect immunofluorescence assays of Mengo-infected HeLa cells. The monoclonals showed cross-reactivity with 3C and 3C-containing precursors from encephalomyocarditis virus (EMCV), but did not react with 3C proteins from rhinovirus-14 or poliovirus-1M.

Since surgical fusion of the spine was first described in 1911, multiple methods have been used to assess it. Although open surgical exploration remains the standard of care for determination of fusion, it is impractical in most clinical situations. Static radiographs have long been used as a practical method of fusion assessment, but they tend to significantly overestimate the presence of a solid fusion. Dynamic radiographs improve accuracy but limitations include measurement reliability, disagreement on allowable motion, and the two-dimensional nature of radiographs. Ultimately, lack of movement at a fused segment does not confirm fusion. Radiostereometric analysis further improves accuracy; however, methodological demands make it largely impractical for routine use. CT is now widely accepted as the standard for noninvasive assessment of spinal fusion. Fine-cut imaging, multiplanar reconstruction, and metal artifact reduction have increased the ability to assess fusion on CT. However, significant concerns remain regarding the effects of high radiation exposure. Although MRI is appealing, its utility in assessing fusion remains unproven. Understanding the limitations of each technique allows judicious use of radiology in the assessment of spinal fusion.

Analysis of the regulatory promoter region of the human α1 (I) collagen-encoding gene (COL1A1) gene indicated the presence of G + C-rich sequence elements that are potential binding sites for the transcription factor Sp1. As a step toward understanding transcriptional regulation of the human COL1A1, we examined Spl binding in the promoter region using DNase I footprinting, and analyzed the effect of Spl expression on COL1A1 promoter activity in transiently transfected Drosophila melanogaster cells in vivo. The results indicated that recombinant human Spl interacted specifically with two G + C-rich sequences within the COL1A1 promoter. Binding of factors in nuclear extracts prepared from human dermal fibroblasts to a 22-nucleotide deoxyribonucleotide (oligo) spanning the 5′ G + C-rich sequence required Zn2+, and was abolished by excess Spl consensus binding site oligos, or by anti-Spl antibodies. Studies in which a series of progressively 5′-deleted COL1A1 promoter::cat constructs were co-expressed with an Spl expression plasmid in a cellular background devoid of Spl homology demonstrated that Spl markedly enhanced the COL1A1 promoter activity. These results suggest that the transcriptional activity of the human COL1A1 can be positively regulated by Spl.

Optical imaging methods are being explored as a potential means of screening for breast cancer. Previous investigations of time-resolved imaging techniques have suggested that due to the lack of photons with sufficiently small pathlengths, the spatial resolution achievable through a human breast would be unlikely to be better than a centimeter. Experimental results presented here indicate, however, that higher resolution may be achieved by extrapolating the measured temporal distribution of transmitted photons. This is performed using a least-squares fit between data and an analytic model of photon transport. The spatial resolution of a time-resolved imaging system was evaluated by measuring the edge response produced by an opaque mask embedded in the center of a 51-mm-thick, very highly scattering medium. The limiting spatial resolution was improved from about 13 mm to about 5 mm.