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Study region: The Lower Flint River Basin (LFRB): a karst catchment in southwestern Georgia, USA. Study focus: A year-long field campaign from July 2018 to July 2019 measured steady and dynamic stream-aquifer exchanges on Ichawaynochaway Creek (INC), a fifth-order tributary to the karstic Flint River in Georgia. The net change in discharge along a 24-km Creek segment was determined between two US Geological Survey gaging stations with no surface-water inflows. Stream-aquifer interactions were measured along a 120-m piezometer transect perpendicular to the bank. Hydraulic parameters to aid future modeling efforts were estimated using the method of moments applied to changes in bank flow direction from Hurricane Michael. New hydrological insights for the region: Results display the effects of local water use on streamflow as most channel storage gains occurred in the fall and winter. Negligible net channel gains and some losses occurred during the spring and summer months. Approximately 150 million m3 of net channel gains were observed during the study period. Initial stormflow contributed to aquifer recharge as indicated by reversals in the hydraulic gradient. Diffusivity and transmissivity increased along the transect (9–420 m2/d) and (3–150 m2/d), respectively, and hydraulic conductivity equaled 2.85 m/d. Stream-reach orientation with respect to regional groundwater gradients explains increased stream-aquifer interactions. Surface-groundwater interactions in karst systems are better understood after characterizing variation of hydraulic processes.

Background: Disability-related microaggressions are unique among microaggressions in the assumptions they reflect and the verbal and non-verbal forms they take. They impact patients and providers alike. Yet, medical and dental students are not routinely educated about disability-related microaggressions. A medical school student-faculty team harnessed Kern's six-step curriculum design process to co-produce a novel 90-min educational intervention centered on recognizing and responding to disability-related microaggressions. The session was piloted in February 2022 as a required element of the school's mandatory professional development training for first-year medical and dental students., Objective: This mixed-methods study examined session impact on student-reported learning pertinent to addressing and mitigating disability-related microaggressions., Methods: Voluntary surveys were distributed to all first-year students to capture pre-/post-session self-assessment of knowledge, skills, and comfort, as well as post-session reflection on lessons learned. Quantitative data was analyzed using summary statistics, unpaired t-tests, and Mann Whitney U tests; qualitative data was analyzed using a hybrid inductive-deductive approach., Results: Survey response rate was 61 % (100/164) pre-session and 25 % (41/164) post-session. Post-session, there was significantly increased student agreement with statements addressing microaggressions knowledge, comfort teaching others, and strategies to support disability-sensitive workplaces. Post-session narrative reflections revealed learning within five themes: nature of microaggressions, identifying microaggressions, preventing microaggressions, responding to microaggressions, and medical student empowerment. Ninety-three percent of post-session respondents (38/41) agreed the session empowered them to actively contribute to anti-ableist learning environments., Conclusions: The pilot educational intervention provides a promising strategy to foster anti-ableism and advocacy among first-year medical/dental students. While limited by low response rate, small sample size, participant self-selection for survey participation, and unlinked individual pre- and post-session responses, mixed methods analysis suggests the session had a positive impact on student action-oriented knowledge and empowerment., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cardiac in silico clinical trials can virtually assess the safety and efficacy of therapies using human-based modelling and simulation. These technologies can provide mechanistic explanations for clinically observed pathological behaviour. Designing virtual cohorts for in silico trials requires exploiting clinical data to capture the physiological variability in the human population. The clinical characterisation of ventricular activation and the Purkinje network is challenging, especially non-invasively. Our study aims to present a novel digital twinning pipeline that can efficiently generate and integrate Purkinje networks into human multiscale biventricular models based on subject-specific clinical 12-lead electrocardiogram and magnetic resonance recordings. Essential novel features of the pipeline are the human-based Purkinje network generation method, personalisation considering ECG R wave progression as well as QRS morphology, and translation from reduced-order Eikonal models to equivalent biophysically-detailed monodomain ones. We demonstrate ECG simulations in line with clinical data with clinical image-based multiscale models with Purkinje in four control subjects and two hypertrophic cardiomyopathy patients (simulated and clinical QRS complexes with Pearson's correlation coefficients > 0.7). Our methods also considered possible differences in the density of Purkinje myocardial junctions in the Eikonal-based inference as regional conduction velocities. These differences translated into regional coupling effects between Purkinje and myocardial models in the monodomain formulation. In summary, we demonstrate a digital twin pipeline enabling simulations yielding clinically consistent ECGs with clinical CMR image-based biventricular multiscale models, including personalised Purkinje in healthy and cardiac disease conditions., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Julia Camps reports financial support was provided by Engineering and Physical Sciences Research Council. Blanca Rodriguez reports financial support was provided by Wellcome Trust Fellowship in Basic Biomedical Sciences. Zhinuo Jenny Wang reports financial support was provided by CompBioMed 2 Centre of Excellence in Computational Biomedicine. Kevin Burrage reports financial support was provided by Australian Research Council Centre of Excellence for Mathematical and Statistical Frontiers. Kevin Burrage reports financial support was provided by Australian Research Council Discovery Project. Leto Luana Riebel reports financial support was provided by BBSRC PhD scholarship in collaboration with AstraZeneca. Rodrigo Weber dos Santos reports financial support was provided by Coordination of Higher Education Personnel Improvement. Rodrigo Weber dos Santos reports financial support was provided by National Council for Scientific and Technological Development. Rafael Sebastian reports financial support was provided by Generalitat Valenciana., (Copyright © 2024. Published by Elsevier B.V.)

Purpose: The use of systemic immune checkpoint blockade before surgery is increasing in patients with metastatic renal cell carcinoma, however, the safety and feasibility of performing consolidative cytoreductive nephrectomy after the administration of systemic therapy are not well described., Patients and Methods: A retrospective review of patients undergoing nephrectomy was performed using our prospectively maintained institutional database. Patients who received preoperative systemic immunotherapy were identified, and the risk of postoperative complications were compared to those who underwent surgery without upfront systemic treatment. Perioperative characteristics and surgical complications within 90 days following surgery were recorded., Results: Overall, we identified 220 patients who underwent cytoreductive nephrectomy from April 2015 to December 2022, of which 46 patients (21%) received systemic therapy before undergoing surgery. Unadjusted rates of surgical complications included 20% (n = 35) in patients who did not receive upfront systemic therapy and 20% (n = 9) in those who received upfront systemic immunotherapy. In our propensity score analysis, there was no statistically significant association between receipt of upfront immunotherapy and 90-day surgical complications [odds ratio (OR): 1.82, 95% confidence interval (CI): 0.59-5.14; P = 0.3]. This model, however, demonstrated an association between receipt of upfront immunotherapy and an increased odds of requiring a blood transfusion [OR: 4.53, 95% CI: 1.83-11.7; P = 0.001]., Conclusion: In our cohort, there was no significant difference in surgical complications among patients who received systemic therapy before surgery compared to those who did not receive upfront systemic therapy. Cytoreductive nephrectomy is safe and with low rates of complications following the use of systemic therapy., Competing Interests: Declaration of Competing Interest Maria Carlo has no conflicts of interest to disclose. Jonathan Coleman has no conflicts of interest to disclose. Lennert Eismann has no conflicts of interest to disclose. Darren Feldman reports research funding from Telix, BioNTech, Astellas, and Decibel Therapeutics; is a consultant for Telix and BioNTech; and reports royalties from UpToDate. Alvin Goh is a consultant for Medtronic. A. Ari Hakimi is on the Merck Advisory board. Sari Khaleel (KhaleelS@mskcc.org) has no conflicts of interest to disclose. Ritesh Kotecha reports advisory board consultation for Eisai; receives institutional research funding from Pfizer, Novartis, Takeda, and Allogene Therapeutics; and is supported in part by the Academy of Kidney Cancer Investigators of the CDMRP/DOD (KC200127). Chung-Han Lee reports honoraria from Ideology Health, Intellisphere, Research to Practice, American Institute of Continuing Medical Education, and Medscape; has a consulting or advisory role for Aveo, Cardinal Health, Eisai, Bristol Myers Squibb, Merck, Pfizer/EMD Serono, and Exelixis; and reports research funding from Eisai, Bristol Myers Squibb, Calithera Biosciences, Exelixis, Merck, and AstraZeneca. Richard Matulewicz has no conflicts of interest to disclose. Robert Motzer reports grants from Bristol Myers Squibb and Pfizer; grants and personal fees from Eisai, Exelixis, Merck, and Genentech/Roche; and personal fees from EMD Serono Research. Irina Ostrovnaya has no conflicts of interest to disclose. Stephen Reese has no conflicts of interest to disclose. Katiana Vazquez-Rivera has no conflicts of interest to disclose. Paul Russo has no conflicts of interest to disclose. Juan Arroyave Villada has no conflicts of interest to disclose. Martin Voss reports receiving commercial research grants from Bristol Myers Squibb, Pfizer, and Genentech/Roche; honoraria from Novartis and Bristol Myers Squibb; and travel/accommodation from AstraZeneca, Eisai, Novartis, and Takeda; and serving as a consultant/advisory board member for Alexion Pharmaceuticals, Aveo, Bayer, Calithera Biosciences, Corvus Pharmaceuticals, Exelixis, Eisai, Genentech, GlaxoSmithKline, Merck, Natera, Onquality Pharmaceuticals, Novartis, and Pfizer. Charlie White has no conflicts of interest to disclose., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Purpose: While most small renal masses (SRM) < 4 cm have an excellent prognosis following resection, the impact of adverse T3a pathologic features on oncologic outcomes of SRMs remains unclear. We sought to compare clinical outcomes for surgically resected pT3a versus pT1a SRMs at our institution., Materials and Methods: We retrospectively reviewed records of patients who underwent radical or partial nephrectomy (RN, PN) for renal tumors <4 cm at our institution between 2010 and 2020. We compared features and outcomes of pT3a vs pT1a SRMs. Continuous and categorical variables were compared using Student's t and Pearson's chi-squared tests, respectively. Postoperative outcomes of interest including overall, cancer-specific, and recurrence-free survival (OS, CSS, and RFS) were analyzed using Kaplan-Meier method, Cox proportional hazard regression, and competing risk analysis. Analyses were performed using R statistical package (R Foundation, v4.0)., Results: We identified 1,837 patients with malignant SRMs. Predictors of postoperative pT3a upstaging included higher renal score, larger tumor size, and presence of radiologic features concerning for T3a disease (odds ratio [OR] = 5.45, 95% confidence interval [CI] 3.92-7.59, P < 0.001). On univariable modeling, pT3a SRMs had higher positive margin rates (9.6% vs 4.1%, P < 0.001), worse OS (hazard ratio [HR] = 2.9, 95% CI 1.6-5.3, P = 0.002), RFS (HR 9.32, 95% CI 2-40.1, P = 0.003), and CSS (HR = 3.6, 95% CI 1.5-8.2, P = 0.003). On multivariable modeling, pT3a status remained associated with worse RFS (HR = 2.7, 95% CI 1.04-7, P = 0.04), but not OS (HR 1.6, 95% CI = 0.83-3.1, P = 0.2); multivariable modeling was deferred for CSS due to low event rates., Conclusions: Adverse T3a pathologic features portend worse outcomes for SRMs, highlighting the crucial role of pre-operative planning and case selection. These patients have relatively poor prognosis, and should be monitored more closely and counseled for consideration of adjuvant therapy or clinical trials., Competing Interests: Declaration of Competing Interest The authors do not have any direct conflicts of interest to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Leadership skills of team leaders can impact the functioning of their teams. It is unknown whether attending surgeons' leadership skills impact residents' physiological stress. This study sought to (1) assess the relationship between attending surgeons' leadership skills and residents' physiological stress and (2) to characterize lifestyle behaviors associated with resident physiological stress. We hypothesized that strong attending leadership skills would be associated with low resident physiological stress., Study Design: This prospective observational cohort study was conducted at a single urban, academic medical center in the US, over 12 months. Residents were enrolled during their rotation of 1 to 2 months on the Trauma and ICU services. The primary predictor was the attending surgeons' leadership skills that were measured using a weekly survey filled out by residents, using the Surgeons' Leadership Inventory (SLI). The SLI uses a 4-point Likert scale to measure surgeons' leadership skills across eight domains. The primary outcome was residents' physiological stress, which was measured by their Heart Rate Variability (HRV). We recorded the residents' HRV with a WHOOP strap that was continuously worn on the wrist or the bicep. We used multivariate repeated measures gamma regression to assess the relationship between attending leadership skills and residents' physiological stress, adjusting for hours of sleep, age, and service., Results: Sixteen residents were enrolled over 12 months. The median attending surgeons' leadership score was 3.8 (IQR: 3.2-4.0). The median residents' percent of maximal HRV was 70.8% (IQR: 56.7-83.7). Repeated measure gamma regression model demonstrated a minimal nonsignificant increase of 1.6 % (95% CI: -5.6, 8.9; p-value = 0.65) in the percent of maximal HRV (less resident physiological stress) for every unit increase in leadership score. There was an increase of 2.9% (95% CI= 1.6, 4.2; p-value < 0.001) in the percent of maximal HRV per hour increase in sleep and a significant decrease of 10.9% (95% CI= -16.8, -5.2; < 0.001) in the percent of HRV when working in the ICU compared to the Trauma service., Conclusion: This study revealed that more residents' sleep was associated with lower physiological stress. Attending surgeons' leadership skills were not associated with residents' physiological stress., (Copyright © 2023 Association of Program Directors in Surgery. Published by Elsevier Inc. All rights reserved.)

High-throughput quantification of the first- and second-phase insulin secretion dynamics is intractable with current methods. The fact that independent secretion phases play distinct roles in metabolism necessitates partitioning them separately and performing high-throughput compound screening to target them individually. We developed an insulin-nanoluc luciferase reporter system to dissect the molecular and cellular pathways involved in the separate phases of insulin secretion. We validated this method through genetic studies, including knockdown and overexpression, as well as small-molecule screening and their effects on insulin secretion. Furthermore, we demonstrated that the results of this method are well correlated with those of single-vesicle exocytosis experiments conducted on live cells, providing a quantitative reference for the approach. Thus, we have developed a robust methodology for screening small molecules and cellular pathways that target specific phases of insulin secretion, resulting in a better understanding of insulin secretion, which in turn will result in a more effective insulin therapy through the stimulation of endogenous glucose-stimulated insulin secretion., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

US physicians in multiple specialties who order or conduct radiological procedures lack formal radiation science education and thus sometimes order procedures of limited benefit or fail to order what is necessary. To this end, a multidisciplinary expert group proposed an introductory broad-based radiation science educational program for US medical schools. Suggested preclinical elements of the curriculum include foundational education on ionizing and nonionizing radiation (eg, definitions, dose metrics, and risk measures) and short- and long-term radiation-related health effects as well as introduction to radiology, radiation therapy, and radiation protection concepts. Recommended clinical elements of the curriculum would impart knowledge and practical experience in radiology, fluoroscopically guided procedures, nuclear medicine, radiation oncology, and identification of patient subgroups requiring special considerations when selecting specific ionizing or nonionizing diagnostic or therapeutic radiation procedures. Critical components of the clinical program would also include educational material and direct experience with patient-centered communication on benefits of, risks of, and shared decision making about ionizing and nonionizing radiation procedures and on health effects and safety requirements for environmental and occupational exposure to ionizing and nonionizing radiation. Overarching is the introduction to evidence-based guidelines for procedures that maximize clinical benefit while limiting unnecessary risk. The content would be further developed, directed, and integrated within the curriculum by local faculties and would address multiple standard elements of the Liaison Committee on Medical Education and Core Entrustable Professional Activities for Entering Residency of the Association of American Medical Colleges., (Published by Elsevier Inc.)

Successfully replacing damaged cartilage with tissue-engineered constructs requires integration with the host tissue and could benefit from leveraging the native tissue's intrinsic healing capacity; however, efforts are limited by a poor understanding of how cartilage repairs minor defects. Here, we investigated the conditions that foster natural cartilage tissue repair to identify strategies that might be exploited to enhance the integration of engineered/grafted cartilage with host tissue. We damaged porcine articular cartilage explants and using a combination of pulsed SILAC-based proteomics, ultrastructural imaging, and catabolic enzyme blocking strategies reveal that integration of damaged cartilage surfaces is not driven by neo-matrix synthesis, but rather local depletion of proteoglycans. ADAMTS4 expression and activity are upregulated in injured cartilage explants, but integration could be reduced by inhibiting metalloproteinase activity with TIMP3. These observations suggest that catabolic enzyme-mediated proteoglycan depletion likely allows existing collagen fibrils to undergo cross-linking, fibrillogenesis, or entanglement, driving integration. Catabolic enzymes are often considered pathophysiological markers of osteoarthritis. Our findings suggest that damage-induced upregulation of metalloproteinase activity may be a part of a healing response that tips towards tissue destruction under pathological conditions and in osteoarthritis, but could also be harnessed in tissue engineering strategies to mediate repair. STATEMENT OF SIGNIFICANCE: Cartilage tissue engineering strategies require graft integration with the surrounding tissue; however, how the native tissue repairs minor injuries is poorly understood. We applied pulsed SILAC-based proteomics, ultrastructural imaging, and catabolic enzyme blocking strategies to a porcine cartilage explant model and found that integration of damaged cartilage surfaces is driven by catabolic enzyme-mediated local depletion of proteoglycans. Although catabolic enzymes have been implicated in cartilage destruction in osteoarthritis, our findings suggest that damage-induced upregulation of metalloproteinase activity may be a part of a healing response that tips towards tissue destruction under pathological conditions. They also suggest that this natural cartilage tissue repair process could be harnessed in tissue engineering strategies to enhance the integration of engineered cartilage with host tissue., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Ltd.)

Background: Rapid response (RR) systems' impact on clinical outcomes is influenced by institutional social factors. This study applied the realist evaluation (context-mechanism-outcomes) framework to review significant RRs defined as REACT (Rapid Escalation After Critical Transfer) events for appraising a pediatric RR system., Methods: REACT events included all RRs with cardiopulmonary arrest (CPA) and/or ventilation and/or hemodynamic support instituted within 24 hours after RR. A continuous quality improvement process was employed to identify, debrief, and review REACT events to recognize and act on RR mechanistic and contextual deficiencies. The aim was to decrease REACT events with mechanistic/contextual gaps categorized into crisis resource management (CRM) themes by 25% over three years while ensuring process sustainability., Results: From 2015 to 2019, 5,581 RR events occurred, of which 67.2% transferred to ICU, and 1,392 (24.9%) were identified as REACTs. In the first two years, 100% identification and review within three months of 90% REACTs was accomplished. One hundred percent of the 17 providers ascertained that the process was safe and transparent, and 80.0% of respondents expressed their commitment from perceived benefit to patient care. Over five years, the proportion of REACTs with CRM gaps decreased from 62.3% to 26.5%, those with multiple deficiencies reduced from 72.5% to 23.2%, and CPAs outside ICUs decreased from 15 to 3 per year. Improvement actions included modifications to RR system (activation, process, and management), hospital (resources and policies), dedicated RR training, and sharing of positive feedback., Conclusion: The realist evaluation framework facilitated holistic assessment of an RR system. Review of REACTs was feasible, sustainable, and yielded useful information to guide systemwide improvement., (Copyright © 2022 The Joint Commission. Published by Elsevier Inc. All rights reserved.)

Objective: To assess the validity, reliability, and acceptability of self-collected human papillomavirus (HPV) tests in women living with HIV (WLHIV) in the United States., Methods: WLHIV ≥30 years of age underwent self-collected (clinic and home) and clinician-collected HPV tests. Sensitivity and specificity analyses were performed using the clinician-collected HPV tests as the comparator. The unweighted kappa statistic was used to evaluate the validity and reliability of self-collected HPV testing, and the level of agreement between the clinician-collected mRNA test and a DNA test that was used for routine care. A 13-question survey was used to assess acceptability., Results: Among the 70 participants, the median age was 50 years, 75% had an undetectable HIV RNA, and 11% had a CD4 count of <200 cells/μl. Nearly 63% had at least one positive HPV test. The sensitivity and specificity of the self-collected HPV test were 84.6% (95% confidence interval (CI) 65.1-95.6%) and 62.9% (95% CI 44.9-78.5%), respectively (κ=0.5, 95% CI 0.2-0.7). The agreement between the two self-collected tests was good (κ=0.8, 95% CI 0.5-1.0). There was good agreement between clinician-collected mRNA tests and DNA tests (κ=0.8, 95% CI 0.7-1.0). Self-collection was highly acceptable., Conclusions: Among WLHIV, self-collected HPV tests had good sensitivity and moderate specificity compared to clinician-collected HPV tests. The reliability between self-collected testing locations was good. Self-collected HPV testing had high acceptability., (Copyright © 2020. Published by Elsevier Ltd.)

Our study determines the selectivity of graphene oxide (GO) to recognize its ligands (e.g. flavonoids) in facilitating the binding with their respective cellular targets. The polyhydroxy phenolic compounds, flavonoids, have a broad spectrum of therapeutic activities with high potency and low systemic toxicity. Despite the vast medicinal importance, their bioavailability is low. In this exploratory study, GO has been used as the transporter of three flavonols fisetin (3, 7, 3', 4'-OH flavone), quercetin (3, 5, 7, 3', 4'-OH flavone), and morin (3, 5, 7, 2', 4'-OH flavone) for the physiological target DNA. Calf thymus DNA is chosen as the model physiological target. Characterization of GO is performed using FTIR, Raman and dynamic light scattering (DLS) spectroscopy. The strong absorption peak at 1730 cm -1 indicated the presence of carbonyl groups (C=O) at the edges of GO. The presence of sp 3 carbons due to oxidation of sp 2 carbons in GO is further proved by Raman spectroscopy. DLS provided the average size of the GO particles to be ~9 μm. The dual luminescence behavior of the flavonols has been used in this study for the noninvasive sensing of the GO-flavonol and GO-flavonol-DNA interactions; as well as for the selectivity of GO for one flavonol over other in transferring the ligand to DNA. Furthermore, circular dichroism (CD) indicated that the optical activity of GO undergoes drastic change when conjugated with flavonols. Molecular modeling corroborated the findings from the binding studies. GO provides high promise as facilitators for drug delivery., (Copyright © 2019. Published by Elsevier B.V.)

Introduction: Small renal masses (SRMs) with evidence of clear cell renal cell carcinoma (ccRCC) are understudied. Current algorithms for the management of SRMs include surgical resection, ablation, and active surveillance. We sought to identify genomic biomarkers that could potentially refine the management of ccRCC in SRMs, especially in patients being evaluated for active surveillance., Methods: We identified patients who had SRMs (4cm or less) at time of surgery, had sequencing performed on their primary tumor and had a diagnosis of ccRCC. Patients were selected from 3 publicly available cohorts, The Cancer Genome Atlas (n = 110), University of Tokyo (n = 37), The International Cancer Genome Consortium (n = 31), and from our own institutional prospective database (n = 25). Among this cohort we analyzed mutations present in at least 5% of tumors, assessing for the enrichment of mutations and progression-free survival using the composite endpoint of recurrence or death of disease. Analysis was adjusted for multiple testing. A Cox regression model was used to assess clinical variables with significant mutations., Results: In total, 203 patients were available for analysis. Median follow-up was 43.1 months among survivors. Mutations in VHL, PBRM1, SETD2, BAP1, KDM5C, and MTOR were present in more than 5% of tumors. Twenty-three patients (11.3%) had recurrence or died of their disease. Mutations in KDM5C were associated with inferior survival from either recurrence or death from disease, adjusted P 0.033., Conclusions: We identified mutations in SRMs in ccRCC that are associated with recurrence and lethality. The strongest association was seen in those with KDM5C mutations. Use of these genomic biomarkers may improve stratification of patients with SRMs and for those who may be appropriate for active surveillance. Prospective evaluation of these markers is needed., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: The safety and feasibility of active surveillance in comorbid patients with renal masses ≥4.0cm is uncertain. The aim of this study is to describe our institutional experience with the observation of large renal masses., Materials and Methods: One hundred patients were identified with renal masses ≥ 4.0cm that were followed on observation for at least 6 months without surgical intervention between 1994 and 2016. Linear regression was conducted to determine predictors for renal mass growth and competing risk methods were used to estimate the probability of progression in the setting of death from other causes., Results: Median age at diagnosis was 73 years and 73% of patients had a Charlson Comorbidity index ≥ 4. At presentation, the median mass size was 4.9cm. The median growth rate was 0.4cm/y and there were no significant predictors of growth. Surveillance was discontinued in 34 patients who underwent delayed intervention. Median follow up for metastasis-free survivors was 4 years. In total, 10 patients developed metastatic disease, 3 died from kidney cancer and 30 patients died from other causes. The 5-year probability of other cause mortality was 22% (95% CI: 14%-32%) compared to 6% (95% CI: 2%-13%) for metastatic progression of kidney cancer., Conclusion: In highly comorbid patients, the observation of large renal masses has low likelihood for metastatic progression relative to the risk of nonkidney cancer related death. This data supports the use of surveillance as an acceptable strategy for highly selected patients with competing risks from other serious illnesses., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Synaptic exocytosis relies on assembly of three soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins into a parallel four-helix bundle to drive membrane fusion. SNARE assembly occurs by stepwise zippering of the vesicle-associated SNARE (v-SNARE) onto a binary SNARE complex on the target plasma membrane (t-SNARE). Zippering begins with slow N-terminal association followed by rapid C-terminal zippering, which serves as a power stroke to drive membrane fusion. SNARE mutations have been associated with numerous diseases, especially neurological disorders. It remains unclear how these mutations affect SNARE zippering, partly due to difficulties to quantify the energetics and kinetics of SNARE assembly. Here, we used single-molecule optical tweezers to measure the assembly energy and kinetics of SNARE complexes containing single mutations I67T/N in neuronal SNARE synaptosomal-associated protein of 25kDa (SNAP-25B), which disrupt neurotransmitter release and have been implicated in neurological disorders. We found that both mutations significantly reduced the energy of C-terminal zippering by ~10 k B T, but did not affect N-terminal assembly. In addition, we observed that both mutations lead to unfolding of the C-terminal region in the t-SNARE complex. Our findings suggest that both SNAP-25B mutations impair synaptic exocytosis by destabilizing SNARE assembly, rather than stabilizing SNARE assembly as previously proposed. Therefore, our measurements provide insights into the molecular mechanism of the disease caused by SNARE mutations., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Aims: To evaluate the accuracy of contrast-enhanced ultrasound (CEUS) for monitoring tumor necrosis following WST-11 vascular targeted photodynamic therapy (VTP) using imaging-pathology correlation., Methods: Renal adenocarcinoma cells were injected into the hindlimb of 13 BalB/c mice resulting in tumors ranging from 9.8 to 194.3mm 3 . US guidance was used to place a laser fiber into the tumor, and VTP was performed. CEUS was performed prior to animal sacrifice, 24h post-VTP. Whole tumors were extracted for histopathologic analysis using H&E and TUNEL staining. Pathology samples corresponding to the CEUS imaging plane were prepared in order to compare the size and extents of tumor necrosis., Results: Tumor necrosis following VTP appeared as a central region of non-enhancement on CEUS, while viable tumor appeared as patchy regions of enhancement in the tumor periphery. The region of tumor necrosis measured in mean 66% and 64.8% of total tumor area on CEUS and pathology respectively (p=0.2). The size and location of the necrosis on CEUS images and pathology samples were found correlative with no inter-observer difference (weighted kappa of 0.771 and 0.823, respectively)., Conclusion: CEUS allows accurate monitoring of VTP induced tumor necrosis in a small animal model., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Kinase gene fusions are important drivers of oncogenic transformation and can be inhibited with targeted therapies. Clinical grade diagnostics using RNA sequencing to detect gene rearrangements in solid tumors are limited, and the few that are available require prior knowledge of fusion break points. To address this, we have analytically validated a targeted RNA sequencing assay (OSU-SpARKFuse) for fusion detection that interrogates complete transcripts from 93 kinase and transcription factor genes. From a total of 74 positive and 36 negative control samples, OSU-SpARKFuse had 93.3% sensitivity and 100% specificity for fusion detection. Assessment of repeatability and reproducibility revealed 96.3% and 94.4% concordance between intrarun and interrun technical replicates, respectively. Application of this assay on prospective patient samples uncovered OLFM4 as a novel RET fusion partner in a small-bowel cancer and led to the discovery of a KLK2-FGFR2 fusion in a patient with prostate cancer who subsequently underwent treatment with a pan-fibroblast growth factor receptor inhibitor. Beyond fusion detection, OSU-SpARKFuse has built-in capabilities for discovery research, including gene expression analysis, detection of single-nucleotide variants, and identification of alternative splicing events., (Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Introduction and Objectives: Identifying patients with prostate cancer (CaP) who will ultimately develop bone metastasis (BM) or die of disease is essential. Alkaline phosphatase velocity (APV) has been shown to predict overall survival (OS) and bone metastasis-free survival (BMFS) in an earlier study of an equal access military patient cohort of patients with castrate-resistant prostate cancer (CRPC). To confirm these findings, we examined a cohort of patients from a high-volume cancer center to validate a previous observation that faster alkaline phosphatase (AP) kinetics are predictive of OS and BMFS in this second cohort of patients., Materials and Methods: A retrospective cohort study was conducted of patients with CRPC treated at Memorial Sloan Kettering Cancer Center between 1989 and 2010. All patients who received androgen deprivation therapy (ADT) as primary treatment in response to a rising PSA after definitive surgery for CaP were eligible. For those who received primary ADT or surgery followed by ADT, CRPC was defined as one rising PSA value after a PSA nadir ≤4ng/ml, and confirmed by a second rising PSA value, with concurrently documented testosterone levels <50ng/dl. APV was computed as the slope of the linear regression line of all AP values (>2 values per patient) plotted against time. Study outcome included BMFS and OS. Univariable Kaplan-Meier analysis was used to examine time-to-event outcomes. Multivariable Cox proportional hazards regression analysis was used to model time to BMFS and OS., Results: Of 89 patients with CRPC with evaluable data and CRPC, 17 (19%) experienced BM and 26 (29%) died. APV was dichotomized at the uppermost quartile split of all observed APV values:≥5.42U/l/y vs. the lower 3 quartiles combined,<5.42U/l/y. Patients with faster APV had significantly worse outcomes, including faster progression to BM and poorer OS when compared with those with slower APV (P = 0.0451 and P = 0.0109, respectively). There was strong correlation between PSA doubling time (PSADT) (<10,≥10mo) and APV (≥5.42U/l/y vs.<5.42U/l/y) (P = 0.0289), preventing simultaneous evaluation of both factors in multivariable analysis. Kaplan-Meier analysis showed that PSADT was also predictive of BM and OS (log-rank P<0.0001). Separate multivariable Cox proportional hazards regression models were used to examine PSADT and APV, as predictors of each study outcome (BMFS and OS). Both PSADT and APV were strongly predictive of BMFS and OS (respectively)., Conclusions: APV and PSADT were predictors of BM and OS in patients with CRPC, respectively. These data are additional evidence of the potential value of AP kinetics in patients with advanced CaP. Prospective studies will be required to clarify these associations. However, given the restrictions on the current patient population in excluding metastatic disease within 12 months of ADT and a PSA nadir >4ng/ml, the findings are not inappropriately generalized to other men., (Published by Elsevier Inc.)

Background: Polygenic risk scores (PRSs) have successfully summarized genome-wide effects of genetic variants in schizophrenia with significant predictive power. In a clinical sample of first-episode psychosis (FEP) patients, we estimated the ability of PRSs to discriminate case-control status and to predict the development of schizophrenia as opposed to other psychoses., Methods: The sample (445 case and 265 control subjects) was genotyped on the Illumina HumanCore Exome BeadChip with an additional 828 control subjects of African ancestry genotyped on the Illumina Multi-Ethnic Genotyping Array. To calculate PRSs, we used the results from the latest Psychiatric Genomics Consortium schizophrenia meta-analysis. We examined the association of PRSs with case-control status and with schizophrenia versus other psychoses in European and African ancestry FEP patients and in a second sample of 248 case subjects with chronic psychosis., Results: PRS had good discriminative ability of case-control status in FEP European ancestry individuals (9.4% of the variance explained, p < 10 -6 ), but lower in individuals of African ancestry (R 2 = 1.1%, p = .004). Furthermore, PRS distinguished European ancestry case subjects who went on to acquire a schizophrenia diagnosis from those who developed other psychotic disorders (R 2 = 9.2%, p = .002)., Conclusions: PRS was a powerful predictor of case-control status in a European sample of patients with FEP, even though a large proportion did not have an established diagnosis of schizophrenia at the time of assessment. PRS was significantly different between those case subjects who developed schizophrenia from those who did not, although the discriminative accuracy may not yet be sufficient for clinical utility in FEP., (Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

This paper deals with two aspects, namely a historical analysis of the use of unmanned vehicles (UAVs ROVs, AUVs) in maritime spill incidents and a detailed description of a multi-agency oil and HNS incident response exercise involving the integration and analysis of unmanned vehicles environmental sensing equipment. The exercise was a first in terms of the level of robotic systems deployed to assist in survey, surveillance and inspection roles for oil spills and harmful and noxious substances., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: To analyze patients with kidney cancer referred for evaluation at a high-volume genetics service at a comprehensive cancer center and identify factors associated with positive tests for hereditary cancer syndromes., Methods: A retrospective review of patients referred to the Clinical Genetics Service at Memorial Sloan-Kettering Cancer Center was performed, and patients with a personal history of kidney cancer were identified. Patient and disease characteristics were reviewed. In all, 4 variables including age at diagnosis of kidney tumor, presence of syndromic manifestations, family history of kidney cancer, and number of primary malignancies were evaluated for association with positive test results in 2 groups: patients tested for renal cell carcinoma syndromes and Lynch syndrome. Guidance for genetic testing strategy in patients with kidney cancer is provided., Results: Between 1999 and 2012, 120 patients with a history of kidney cancer were evaluated by the Clinical Genetics Service. The mean age at kidney cancer diagnosis was 52 years (interquartile range: 42-63), with 57% being women. A family history of kidney cancer was reported by 39 patients (33%). Time between diagnosis of first cancer and genetic consultation was <1 year in 54%, 2 to 5 years in 23%, and>5 years in the remaining 23%. Overall, 95 patients were tested for genetic abnormalities with 27 (28%) testing positive. Testing for renal cell carcinoma (RCC)-related syndromes was performed on 43 patients, with 13 testing positive (30%). Lynch syndrome testing was positive in 9 patients (32%) after 28 were tested. In RCC-associated syndromes, young age of diagnosis was associated with positive test results. Conversely, syndromic manifestations and increasing number of primary malignancies were associated with positive Lynch testing., Conclusions: The discovery of inherited kidney cancer syndromes has provided a unique opportunity to identify patients at increased risk for cancer. Factors associated with positive genetic testing are unique to different syndromes. These data suggest that in kidney cancer patients evaluated for hereditary cancer syndromes, young age is associated with diagnosis of RCC syndromes, whereas syndromic manifestations and multiple primaries are found in Lynch syndrome. These results, along with clinical awareness, may be useful for practicing urologists to select patients with kidney cancer to refer for genetic counseling., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Weight loss and metformin are hypothesized to improve breast cancer outcomes; however the joint impacts of these treatments have not been investigated. Reach for Health is a randomized trial using a 2 × 2 factorial design to investigate the effects of weight loss and metformin on biomarkers associated with breast cancer prognosis among overweight/obese postmenopausal breast cancer survivors. This paper describes the trial recruitment strategies, design, and baseline sample characteristics. Participants were randomized in equal numbers to (1) placebo, (2) metformin, (3) weight loss intervention and placebo, or (4) weight-loss intervention and metformin. The lifestyle intervention was a personalized, telephone-based program targeting a 7% weight-loss in the intervention arm. The metformin dose was 1500 mg/day. The duration of the intervention was 6 months. Main outcomes were biomarkers representing 3 metabolic systems putatively related to breast cancer mortality: glucoregulation, inflammation, and sex hormones. Between August 2011 and May 2015, we randomized 333 breast cancer survivors. Mass mailings from the California Cancer Registry were the most successful recruitment strategy with over 25,000 letters sent at a cost of $191 per randomized participant. At baseline, higher levels of obesity were significantly associated with worse sleep disturbance and impairment scores, lower levels of physical activity and higher levels of sedentary behavior, hypertension, hypercholesterolemia, and lower quality of life (p<0.05 for all). These results illustrate the health burden of obesity. Results of this trial will provide mechanistic data on biological pathways and circulating biomarkers associated with lifestyle and pharmacologic interventions to improve breast cancer prognosis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: For children with sickle cell anaemia and high transcranial doppler (TCD) flow velocities, regular blood transfusions can effectively prevent primary stroke, but must be continued indefinitely. The efficacy of hydroxycarbamide (hydroxyurea) in this setting is unknown; we performed the TWiTCH trial to compare hydroxyurea with standard transfusions., Methods: TWiTCH was a multicentre, phase 3, randomised, open-label, non-inferiority trial done at 26 paediatric hospitals and health centres in the USA and Canada. We enrolled children with sickle cell anaemia who were aged 4-16 years and had abnormal TCD flow velocities (≥ 200 cm/s) but no severe vasculopathy. After screening, eligible participants were randomly assigned 1:1 to continue standard transfusions (standard group) or hydroxycarbamide (alternative group). Randomisation was done at a central site, stratified by site with a block size of four, and an adaptive randomisation scheme was used to balance the covariates of baseline age and TCD velocity. The study was open-label, but TCD examinations were read centrally by observers masked to treatment assignment and previous TCD results. Participants assigned to standard treatment continued to receive monthly transfusions to maintain 30% sickle haemoglobin or lower, while those assigned to the alternative treatment started oral hydroxycarbamide at 20 mg/kg per day, which was escalated to each participant's maximum tolerated dose. The treatment period lasted 24 months from randomisation. The primary study endpoint was the 24 month TCD velocity calculated from a general linear mixed model, with the non-inferiority margin set at 15 cm/s. The primary analysis was done in the intention-to-treat population and safety was assessed in all patients who received at least one dose of assigned treatment. This study is registered with ClinicalTrials.gov, number NCT01425307., Findings: Between Sept 20, 2011, and April 17, 2013, 159 patients consented and enrolled in TWiTCH. 121 participants passed screening and were then randomly assigned to treatment (61 to transfusions and 60 to hydroxycarbamide). At the first scheduled interim analysis, non-inferiority was shown and the sponsor terminated the study. Final model-based TCD velocities were 143 cm/s (95% CI 140-146) in children who received standard transfusions and 138 cm/s (135-142) in those who received hydroxycarbamide, with a difference of 4·54 (0·10-8·98). Non-inferiority (p=8·82 × 10(-16)) and post-hoc superiority (p=0·023) were met. Of 29 new neurological events adjudicated centrally by masked reviewers, no strokes were identified, but three transient ischaemic attacks occurred in each group. Magnetic resonance brain imaging and angiography (MRI and MRA) at exit showed no new cerebral infarcts in either treatment group, but worsened vasculopathy in one participant who received standard transfusions. 23 severe adverse events in nine (15%) patients were reported for hydroxycarbamide and ten serious adverse events in six (10%) patients were reported for standard transfusions. The most common serious adverse event in both groups was vaso-occlusive pain (11 events in five [8%] patients with hydroxycarbamide and three events in one [2%] patient for transfusions)., Interpretation: For high-risk children with sickle cell anaemia and abnormal TCD velocities who have received at least 1 year of transfusions, and have no MRA-defined severe vasculopathy, hydroxycarbamide treatment can substitute for chronic transfusions to maintain TCD velocities and help to prevent primary stroke., Funding: National Heart, Lung, and Blood Institute, National Institutes of Health., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: Vascular surgeons may aid in primarily nonvascular procedures. Such activity has not been quantified, and hospital administrators may be unaware of the importance of vascular surgeons to support other hospital-based surgical programs. This study reviewed intraoperative consultations by vascular surgeons to support other surgical services., Methods: Intraoperative vascular consultations were reviewed from January 2006 to January 2014 for consulting service, indication, and whether consultation occurred with advanced notice. Patient demographics, operative times, estimated blood loss, length of stay, and relative value units (RVUs) assigned for each consultation were also assessed. Consultations for trauma and iatrogenic injuries occurring outside the operating theater were excluded., Results: Vascular surgeons performed 225 intraoperative consultations in support of procedures by nonvascular surgeons. Requesting services were surgical oncology (46%), orthopedics (17%), urology (11%), otolaryngology (7%), and others (19%). Reasons for consultation overlapped and included vascular reconstruction (53%), control of hemorrhage (39%), and assistance with difficult dissections (43%). Seventy-four percent were for intra-abdominal procedures, and venous (53%) and arterial (50%) problems were encountered equally with some overlap. Most patients were male (59%), overweight (56%; body mass index ≥25 kg/m(2)), had previous surgery (72%) and were undergoing elective procedures (89%). Mean total procedural anesthesia time was 9.4 hours, mean procedural operating time was 7.9 hours, and mean total and vascular-related estimated blood loss was 1702 mL and 327 mL, respectively. Mean length of stay was 14.7 days, mean intensive care unit stay was 2.9 days, and 30-day mortality was 6.2%. Mean nonvascular RVUs per operation were 46.0, and mean vascular RVUs per operation were 30.9., Conclusions: Unexpected intraoperative need for vascular surgical expertise occurs often enough that vascular surgeons should be regarded as an essential operating room resource to the general operating room, nonvascular surgeons, and their patients. Intraoperative vascular surgical consultation in support of other surgeons requires a high level of open technical operative skills and is time and labor intensive., (Copyright © 2015. Published by Elsevier Inc.)

The use of Insensitive Munitions eXplosives (IMX) is increasing as the Army seeks to replace certain conventional munitions constituents, such as 2,4,6-trinitrotolene (TNT), for improved safety. The IMX formulations are more stable and therefore less prone to accidental detonation while designed to match the performance of legacy materials. Two formulations, IMX 101 and 104 are being investigated as a replacement for TNT in artillery rounds and composition B Army mortars, respectively. The chemical formulations of IMX-101 and 104 are comprised of four constituents;2,4-dinitroanisole (DNAN), 3-nitro-1,2,4-triazol-5-one (NTO), 1-nitroguanidine (NQ), and Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) which are mixed in various ratios to achieve the desired performance. The current work details the analysis of the IMX constituents by single column HPLC-UV-ESI-MS. Detection limits determined are in agreement with similar HPLC analysis of compounds, ranging from 7 to 9μg/L. Gradient mobile phases are used to allow separation of the 4 target compounds in more complex mixture of other concomitant compounds. Mass spectra are used to confirm analyte identity with chromatographic retention time., (Published by Elsevier B.V.)

Objectives: There is debate about the optimal clinical target volume (CTV) expansion and prophylactic nodal dose (PND) in head and neck IMRT. We evaluated our patterns-of-failure (POF) after helical tomotherapy-based concurrent chemoradiotherapy (CCRT) to assess the oncologic safety of reducing the CTV, PND, and bilateral parotid sparing (BPS)., Materials and Methods: All patients with locally advanced squamous cell carcinoma of the head and neck treated with curative intent CCRT between January 2007 and April 2013 at a single institution were included in this retrospective study. Locoregional recurrences (LRR) were overlaid on the treatment plan, and POF was determined relative to planned dose., Results: One hundred and fourteen patients treated with CCRT were evaluated, 74% of whom underwent BPS. The median follow-up for surviving patients was 29.3 months. The 3-year cumulative incidence of locoregional failure, distant metastasis, progression-free and overall survival were 20%, 20%, 56% and 73% respectively. The local failures (n = 12) were either entirely contained within or centered on the original gross tumor volume (GTV), and all but 2 regional recurrences were in GTV. There were no nodal failures in the low-dose or peri-parotid neck (including ipsilateral neck)., Discussion: Nearly all LRR were located within the GTV suggesting that minimal-to-zero margin is required for CTV 70. The nodal recurrence pattern suggests the safety of routine bilateral parotid sparing and relatively low biologically equivalent dose (54 Gy in 33fx) to the low-risk neck., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Transplant surgeons have historically traveled to donor hospitals, performing complex, time-sensitive procedures with unfamiliar personnel. This often involves air travel, significant delays, and frequently occurs overnight.In 2001, we established the nation's first organ recovery center. The goal was to increase efficiency,reduce costs and reduce surgeon travel. Liver donors and recipients, donor costs, surgeon hours and travel time, from April 1,2001 through December 31,2011 were analyzed. Nine hundred and fifteen liver transplants performed at our center were analyzed based on procurement location (living donors and donation after cardiac death donors were excluded). In year 1, 36% (9/25) of donor procurements occurred at the organ procurement organization (OPO) facility, rising to 93%(56/60) in the last year of analysis. Travel time was reduced from 8 to 2.7 h (p<0.0001), with a reduction of surgeon fly outs by 93% (14/15) in 2011. Liver organ donor charges generated by the donor were reduced by37% overall for donors recovered at the OPO facility versus acute care hospital. Organs recovered in this novel facility resulted in significantly reduced surgeon hours, air travel and cost. This practice has major implications for cost containment and OPO national policy and could become the standard of care.

Background: The use of alcohol by pediatric patients has not been thoroughly examined in the United States (US). Patients with complaints related to alcohol use frequently present to the Emergency Department initially., Objective: Our aim was to determine the number of pediatric patients (ages 17 years and younger) presenting to Emergency Departments (EDs) in the US from 2006 to 2008 for alcohol-related disorders and examine selected clinical and demographic features of this population., Methods: This was a retrospective cohort study using 3 years (2006-2008) of data from the Nationwide Emergency Department Sample. This database was used to identify patients younger than 18 years of age with an alcohol-related ED visit, and clinical and demographic features were examined., Results: From 2006 to 2008, a total of 218,514 pediatric patients presented to US EDs and received a subsequent diagnosis of an alcohol-related disorder. Mean age of patients was 15.61 years. Most patients were male and tended to be from higher-income communities., Conclusions: There were 218,514 visits to US EDs by patients younger than 18 years of age for alcohol-related disorders, accounting for >$850 million dollars in charges. ED-based brief alcohol interventions shown to work in adult populations should be explored for use in pediatric patients., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The rapid improvements in DNA synthesis technology hold the potential to revolutionize biosciences in the near future. Traditional genetic engineering methods are template dependent and make extensive but laborious use of site-directed mutagenesis to explore the impact of small variations on an existing sequence "theme." De novo gene and genome synthesis frees the investigator from the restrictions of the pre-existing template and allows for the rational design of any conceivable new sequence theme. Viruses, being among the simplest replicating entities, have been at the forefront of the advancing biosciences since the dawn of molecular biology. Viral genomes, especially those of RNA viruses, are relatively short, often less than 10,000 bases long, making them amenable to whole genome synthesis with the currently available technology. For this reason viruses are once again poised to lead the way in the budding field of synthetic biology--for better or worse.

Computer display height and desk design are believed to be important workstation features and are included in international standards and guidelines. However, the evidence base for these guidelines is lacking a comparison of neck/shoulder muscle activity during computer and paper tasks and whether forearm support can be provided by desk design. This study measured the spinal and upper limb muscle activity in 36 young adults whilst they worked in different computer display, book and desk conditions. Display height affected spinal muscle activity with paper tasks resulting in greater mean spinal and upper limb muscle activity. A curved desk resulted in increased proximal muscle activity. There was no substantial interaction between display and desk.

Computer display height and desk design to allow forearm support are two critical design features of workstations for information technology tasks. However there is currently no 3D description of head and neck posture with different computer display heights and no direct comparison to paper based information technology tasks. There is also inconsistent evidence on the effect of forearm support on posture and no evidence on whether these features interact. This study compared the 3D head, neck and upper limb postures of 18 male and 18 female young adults whilst working with different display and desk design conditions. There was no substantial interaction between display height and desk design. Lower display heights increased head and neck flexion with more spinal asymmetry when working with paper. The curved desk, designed to provide forearm support, increased scapula elevation/protraction and shoulder flexion/abduction.

Cell surface ECTO-NOX proteins exhibit a clock-related, temperature-independent entrainable pattern of periodic (24 min) oscillations in the rate of oxidation of NAD(P)H. Aqueous solutions of copper salts also oxidize NAD(P)H with a similar temperature-independent pattern. For both, five maxima are observed, two of which are separated by 6 min and the remaining three are separated by 4.5 min. In D2O, the pattern is retained but the period length is proportionately increased to 30 min in direct relationship to the 30 h circadian day observed with D2O-grown organisms. With copper solutions, periodic changes in redox potential correlate precisely with the periodic changes in the rates of NAD(P)H oxidation. Consequently, the local environment of the Cu2+ ion in copper chloride solutions was investigated by X-ray absorption spectroscopy. Detailed extended X-ray absorption fine structure (EXAFS) analyses revealed a pattern of oscillations closely resembling those of the copper-catalyzed oxidation of NADH. With CuCl2 in D2O, a pattern with a period length of 30 min was observed. The findings suggest a regular pattern of distortion in the axial and/or equatorial oxygen atoms of the coordinated water molecules which correlate with redox potential changes sufficient to oxidize NADH. A metastable equilibrium condition in the ratio of ortho to para nuclear spin orientation of the water associated hydrogen atoms would be kinetically consistent with a 24-30 min timeframe. The temperature independence of the biological clock can thus be understood as the consequence of a physical rather than a chemical basis for the timing events.

For cancer immunotherapy the loading of dendritic cells (DCs) with whole tumor cell lysate preparations represents a simple and promising approach for presentation of tumor-associated antigens (TAAs), avoiding the disadvantages of HLA-matching and definition of TAAs. The aim of this study was to investigate whether lysate-pulsed DCs efficiently cross-prime CD8+ T cells and induce a strong T(H)1 cell response, as compared to DCs pulsed with specific peptides (FLU M1 and Melan-A/Mart-1). As a model system breast carcinoma cell lysate from either MCF-7 or MDA-MB-231 cell lines (both HLA-A*0201+) expressing the TAA MUC1 were selected. Both cell lines expressed MUC1, the epithelial mucin, which is a large molecular weight O-glycosylated protein expressed in the majority of breast, ovarian, and other epithelial malignancies and is under evaluation as a target antigen in cancer immunotherapy. We developed a simple lysate preparation method to solubilize all cell proteins without degradation. For loading of monocyte-derived dendritic cells, 100 microgmL(-1) of breast carcinoma cell lysate was used, accompanied by an adjuvant consisting of tumor necrosis factor-alpha (TNF-alpha) and prostaglandin-E2. T cells were co-cultivated with lysate or peptide pulsed DCs and were restimulated weekly. Before cultivation, and after the 3rd stimulation, tetramer frequencies for the MUC1 epitopes M1.2 and F7 as well as for the FLU M1 and Melan-A/Mart-1 epitopes were determined. After stimulation with lysate, higher frequencies for M1.2-specific T cells were observed compared with the F7 epitope. Furthermore, we found expansion factors for M1.2-specific T cells that had been stimulated with MCF-7 lysate-pulsed DCs of up to 43-fold. The analysis of typical T(H)1/T(H)2 cytokines (IFN-gamma, TNF-alpha, IL-12p70, IL-2, IL-4, IL-5, and IL-10) revealed a strong T(H)1 response. These results provide evidence for a strong T(H)1 polarization and cross-priming of MUC1-specific CD8+ T cells and demonstrate the feasibility of using lysate-pulsed dendritic cells in breast cancer immunotherapy.

In order to obtain information about the metabolism of methanol in plants, gene expression in response to methanol stimulation was analysed in leaves of Arabidopsis thaliana through the use of a 26,090 element oligonucleotide microarray. Following confirmation of data and the application of selection criteria, a total of 484 (1.9%) transcripts were shown to be regulated in response to a 10% methanol application. Samples were taken at 1, 24 and 72 h. Of the total identified transcripts, only 20 were shared between all three time points of which only two were down-regulated. Based on functional category analysis of these genes, at 1 h, the largest categories were metabolism, cell communication/signal transduction processes, defence genes associated with transcription and aspects of RNA processing; a few related to cell division and growth; but none were associated with photosynthesis. At 24 and 72 h, the number and overall proportion of regulated transcripts associated with metabolism increased further. Direct measurement of anthocyanin and flavonoid content confirmed that methanol-regulated transcripts corresponded to flavonoid pathways. Within the functional class metabolism, genes encoding detoxification proteins represented by far the most strongly regulated group. These included, cytochrome P450's, glucosyl transferases and members of the ABC transporter family. It is concluded that exposure to methanol affects the expression of hundreds of genes and that multiple detoxification and signalling pathways are activated.

Background: In response to the emergence of severe infection capable of rapid global spread, WHO will issue a pandemic alert. Such alerts are rare; however, on Feb 19, 2003, a pandemic alert was issued in response to human infections caused by an avian H5N1 influenza virus, A/Hong Kong/213/03. H5N1 had been noted once before in human beings in 1997 and killed a third (6/18) of infected people. The 2003 variant seemed to have been transmitted directly from birds to human beings and caused fatal pneumonia in one of two infected individuals. Candidate vaccines were sought, but no avirulent viruses antigenically similar to the pathogen were available, and the isolate killed embryonated chicken eggs. Since traditional strategies of vaccine production were not viable, we sought to produce a candidate reference virus using reverse genetics., Methods: We removed the polybasic aminoacids that are associated with high virulence from the haemagglutinin cleavage site of A/Hong Kong/213/03 using influenza reverse genetics techniques. A reference vaccine virus was then produced on an A/Puerto Rico/8/34 (PR8) backbone on WHO-approved Vero cells. We assessed this reference virus for pathogenicity in in-vivo and in-vitro assays., Findings: A reference vaccine virus was produced in Good Manufacturing Practice (GMP)-grade facilities in less than 4 weeks from the time of virus isolation. This virus proved to be non-pathogenic in chickens and ferrets and was shown to be stable after multiple passages in embryonated chicken eggs., Interpretation: The ability to produce a candidate reference virus in such a short period of time sets a new standard for rapid response to emerging infectious disease threats and clearly shows the usefulness of reverse genetics for influenza vaccine development. The same technologies and procedures are currently being used to create reference vaccine viruses against the 2004 H5N1 viruses circulating in Asia.

Purpose: Children with attention-deficit/hyperactivity disorder (ADHD) are at increased risk for traumatic injury than non-ADHD-children. Burn injuries in ADHD-children have not been studied. This study was conducted to determine differences in burn injuries between these groups., Methods: The charts of all children ages 5-18, admitted over a 7-year period to a single regional pediatric burn center, were reviewed. ADHD-children were compared to non-ADHD-children regarding age and gender, type and extent of burn, and burn injury outcome., Findings: There were 278 children included, 35 (13%) having ADHD. ADHD-children were more likely male but did not differ in age compared to non-ADHD-children. ADHD-children were more likely to experience a thermal rather than flame burn (83% versus 58% thermal, P < 0.01) and had more extensive burn injury (10% versus 5% median TBSA, P = 0.03). The ADHD group had a longer length of stay (11 versus 7 days, P = 0.05) and was less likely to be discharged to home (86% versus 93%, P = 0.17)., Conclusions: ADHD-children do differ from non-ADHD-children in their pattern and extent of burn injury. The impulsivity and vigilance deficits that characterize ADHD may place these children at higher risk for specific types of burn injuries.

This paper has two main objectives. Firstly, to review the feasibility and validity of the various methods that have been used to measure puberty. With an appreciation of these measurement issues, the second objective is to summarize the evidence-base as to whether the age at menarche and the age at onset of puberty has decreased in recent years. In consideration of these methodologies, all of which report different levels of reliability and validity, it is only possible at present to draw inferences about possible changes in pubertal age and onset. Moreover, the wide variations in sample size, selection of participants (usually non-random), and study design reported in the reviewed studies have compounded this situation further. The paper closes by providing a summary of the findings which will be of relevance to those intending to critically review the current evidence surrounding age of puberty, as well as those set on carrying out future research into this area., (Copyright 2002 The Association for Professionals in Services for Adolescents)

Objectives: to assess the feasibility of duplex follow up of aorto-iliac stents., Material and Methods: in addition to standard clinical and ankle/brachial pressure assessment, 75 patients undergoing aorto-iliac stenting for atherosclerotic disease were prospectively followed up with duplex ultrasound at 3 monthly interval up to 1 year and then annually. We attempted to directly visualise the stents wherever possible using standard 2D imaging, standard and power Doppler, as well as spectral Doppler. Patients had an angiogram at 12 months or sooner if a problem was identified on the duplex scan., Results: mean follow up was 14 months (2-36). Complete visualisation of the pelvic arterial tree was achieved in 94% of scans. All patients had at least one satisfactory scan. Duplex detected 30 lesions in 23 patients. Twenty-seven lesions were confirmed at angiography. There were two false positives. Overall sensitivity, specificity and accuracy were 100%, 93% and 96% for patient undergoing angiography., Conclusion: duplex ultrasound follow up is feasible in the majority of patients., (Copyright 2002 Harcourt Publishers Limited.)

Beta-lactam antibiotics are the class of drug most frequently associated with IgE-mediated allergy but the mechanisms underlying this response are poorly understood. IFN-gamma is a key cytokine in immunity with regulatory actions on monocytes, NK cells, epithelial cells, and T and B lymphocytes. IFN-gamma promotes Th1 responses and inhibits Th2- and IgE-mediated responses. In this study we show, by Western blotting, that the prototype beta-lactam benzylpenicillin (BP) conjugates to human IFN-gamma but not to IL-4. The interaction of BP with IFN-gamma inhibited the cytokine's detection by immunoassay and impaired its activity, as assessed in three different assays: upregulation of MHC molecules on monocytes plus induction of nitric oxide synthesis and expression of monocyte chemoattractant protein-1 mRNA by epithelial cells. This is the first reported example of a direct drug-cytokine interaction and suggests a mechanism by which penicillin may disrupt IFN-gamma-dependent immune responses and promote allergy., (Copyright 2001 Academic Press.)

Introduction: M cells are located in the epithelial layer covering the gut-associated lymphoid tissue and are responsible for delivery of macromolecules and microorganisms to the underlying lymphoid cells. It has been shown that the human colonic cell line Caco-2 can be converted to M cells in vitro following coculture with isolated lymphocytes from murine Peyer's patches. Studies were undertaken to evaluate and characterize the transepithelial transport of select macromolecules across these in vitro derived M cells., Methods: Caco-2 cells were converted to M cells as reported previously. The morphology of Caco-2 cells and M cells was compared by transmission electron microscopy (TEM). The transport properties of macromolecules such as horseradish peroxidase, FITC-conjugated polystyrene beads, and radiolabeled dextrans were examined. The activation of murine antigen-specific T cells following transport of the antigen ovalbumin across the M-cell barrier was assessed by measuring cytokine production., Results: M cells were shown to be irregular in shape and have fewer and shorter microvilli compared to the Caco-2 cell progenitors. These cells were still able to form tight junctions and monolayers on polycarbonate membranes. Time-course studies demonstrated that the transport of polystyrene beads and large-molecular-weight dextrans at physiological temperature across M-cell-containing monolayers was size dependent and more rapid than across Caco-2 cell monolayers. The transport of dextrans was also shown to be temperature and concentration dependent. Befitting the role of the M cell in mucosal defense, protein antigen could be delivered by these cells in order to be processed and presented to antigen-specific CD4+ T lymphocytes., Discussion: The M-cell permeability model is a functional and practical system for evaluating the transport properties of macromolecules and assessing the potential for intestinal mucosal antigen sampling to elicit immunological responses.

For pharmaceutical products, one approach developed to assure that different chromatographic systems are capable of generating valid results is the system suitability test. Typically, a system suitability test involves numerical limits for predefined chromatographic parameters such as theoretical plates, tailing factor, injector reproducibility, etc. An estimation of the ratio of signal compared to baseline is one way to measure system performance, according to a valid method, independent of the instrument. However, since this comparison relates the height of the signal to the height of the noise, it is difficult to relate to the peak area measurements that are typically used for quantification of samples. Additionally, although peak area and peak height may be highly correlated over a wide region, peak area at very low concentrations can be more sensitive to all components of noise due to peak shape. To establish a system suitability criterion, one can use the ratio of the area signal to the baseline noise for replicate injections for samples prepared at concentration equal to the limit of quantitation during the validation studies. A lower limit for this ratio can be derived using statistical tolerance intervals. This lower limit can be applied as a system suitability criterion to measure that any system is performing adequately for measuring low level components in the sample for all future use of the method.

Wild rice grain samples from various parts of the world have been found to have elevated concentrations of heavy metals, raising concern for potential effects on human health. It was hypothesized that wild rice from north-central Wisconsin could potentially have elevated concentrations of some heavy metals because of possible exposure to these elements from the atmosphere or from water and sediments. In addition, no studies of heavy metals in wild rice from Wisconsin had been performed, and a baseline study was needed for future comparisons. Wild rice plants were collected from four areas in Bayfield, Forest, Langlade, Oneida, Sawyer and Wood Counties in September, 1997 and 1998 and divided into four plant parts for elemental analyses: roots, stems, leaves and seeds. A total of 194 samples from 51 plants were analyzed across the localities, with an average of 49 samples per part depending on the element. Samples were cleaned of soil, wet digested, and analyzed by ICP for Ag, As, Cd, Cr, Cu, Hg, Mg, Pb, Se and Zn. Roots contained the highest concentrations of Ag, As, Cd, Cr, Hg, Pb, and Se. Copper was highest in both roots and seeds, while Zn was highest just in seeds. Magnesium was highest in leaves. Seed baseline ranges for the 10 elements were established using the 95% confidence intervals of the medians. Wild rice plants from northern Wisconsin had normal levels of the nutritional elements Cu, Mg and Zn in the seeds. Silver, Cd, Hg, Cr, and Se were very low in concentration or within normal limits for food plants. Arsenic and Pb, however, were elevated and could pose a problem for human health. The pathway for As, Hg and Pb to the plants could be atmospheric.

The use of host-derived PAS components by invasive bacteria is an increasingly recognized mechanism for acquisition of extracellular proteolytic activity. This overview summarizes the pertinent contributions to this field and is divided into three parts: (1) the PAS, (2) the interaction of bacteria that produce their own plasminogen activators with the host's PAS, and (3) the interaction of bacteria that do not produce their own plasminogen activators but use plasminogen activators supplied by the host. The significance of these mechanisms in relation to the invasive potentials of the various organisms is discussed.

The number of zinc enzymes for which detailed structural and mechanistic data, including high resolution crystal structures, are available is increasing rapidly. The new findings continue to support the conclusion that the majority of zinc enzymes catalyze hydrolysis or closely related transfer reactions. In a protein environment, tetrahedral or 5-coordinate Zn2+ is ideally suited to activate a coordinated water (frequently a Zn2+--OH) as a nucleophile attacking the carbonyl carbon of a peptide bond, the carbon of carbon dioxide or the phosphorus of a phosphate ester. Protein-bound Zn2+ can function catalytically by forming mixed complexes with the substrate, either by expanding its coordination sphere or by exchanging a ligand. Formation of protein-Zn2+-substrate bonds can position the substrate or polarize its electron distribution to facilitate further steps in the reaction.