Deeper Understanding of Solvent-Based Ambient Ionization Mass Spectrometry: Are Molecular Profiles Primarily Dictated by Extraction Mechanisms?

Solvent-based ambient ionization mass spectrometry (MS) techniques provide a powerful approach for direct chemical analysis and molecular profiling of biological tissues. While molecular profiling of tissues has been widely used for disease diagnosis, little is understood about how the interplay among solvent properties, matrix effects, and ion suppression can influence the detection of biological molecules. Here, we perform a systematic investigation of the extraction processes of lipids using an ambient ionization droplet microsampling platform to investigate how the physicochemical properties of the solvent systems and extraction time influence molecular extraction and detection. Direct molecular profiling and quantitative liquid chromatography-mass spectrometry (LC-MS) of discrete solvent droplets after surface sampling were investigated to provide insights into extraction and ionization mechanisms. The results of this study suggest that intermolecular interactions such as hydrogen bonding play a major role in extraction and detection of lipids using solvent-based ambient ionization techniques. In addition, extraction time was observed to impact the molecular profiles obtained, suggesting optimization of this parameter can be performed to favor detection of specific analytes.

Preoperative metabolic classification of thyroid nodules using mass spectrometry imaging of fine-needle aspiration biopsies.

Thyroid neoplasia is common and requires appropriate clinical workup with imaging and fine-needle aspiration (FNA) biopsy to evaluate for cancer. Yet, up to 20% of thyroid nodule FNA biopsies will be indeterminate in diagnosis based on cytological evaluation. Genomic approaches to characterize the malignant potential of nodules showed initial promise but have provided only modest improvement in diagnosis. Here, we describe a method using metabolic analysis by desorption electrospray ionization mass spectrometry (DESI-MS) imaging for direct analysis and diagnosis of follicular cell-derived neoplasia tissues and FNA biopsies. DESI-MS was used to analyze 178 tissue samples to determine the molecular signatures of normal, benign follicular adenoma (FTA), and malignant follicular carcinoma (FTC) and papillary carcinoma (PTC) thyroid tissues. Statistical classifiers, including benign thyroid versus PTC and benign thyroid versus FTC, were built and validated with 114,125 mass spectra, with accuracy assessed in correlation with clinical pathology. Clinical FNA smears were prospectively collected and analyzed using DESI-MS imaging, and the performance of the statistical classifiers was tested with 69 prospectively collected clinical FNA smears. High performance was achieved for both models when predicting on the FNA test set, which included 24 nodules with indeterminate preoperative cytology, with accuracies of 93% and 89%. Our results strongly suggest that DESI-MS imaging is a valuable technology for identification of malignant potential of thyroid nodules.

Wireless Measurements Using Electrical Impedance Spectroscopy to Monitor Fracture Healing.

There is an unmet need for improved, clinically relevant methods to longitudinally quantify bone healing during fracture care. Here we develop a smart bone plate to wirelessly monitor healing utilizing electrical impedance spectroscopy (EIS) to provide real-time data on tissue composition within the fracture callus. To validate our technology, we created a 1-mm rabbit tibial defect and fixed the bone with a standard veterinary plate modified with a custom-designed housing that included two impedance sensors capable of wireless transmission. Impedance magnitude and phase measurements were transmitted every 48 h for up to 10 weeks. Bone healing was assessed by X-ray, µCT, and histology. Our results indicated the sensors successfully incorporated into the fracture callus and did not impede repair. Electrical impedance, resistance, and reactance increased steadily from weeks 3 to 7-corresponding to the transition from hematoma to cartilage to bone within the fracture gap-then plateaued as the bone began to consolidate. These three electrical readings significantly correlated with traditional measurements of bone healing and successfully distinguished between union and not-healed fractures, with the strongest relationship found with impedance magnitude. These results suggest that our EIS smart bone plate can provide continuous and highly sensitive quantitative tissue measurements throughout the course of fracture healing to better guide personalized clinical care.

Calf muscle strain injuries in elite Australian Football players: A descriptive epidemiological evaluation.

BACKGROUND: Calf muscle strain injuries (CMSI) show consistent rates of prevalence and re-injury in elite Australian Football players. An epidemiological evaluation is warranted to better understand the clinical presentation and recovery of CMSI. PURPOSE: First, to describe the epidemiology of CMSI in elite Australian Football players. Second, to determine if recovery following injury is different according to: (a) injury type (index vs re-injury); (b) muscle injured (soleus vs gastrocnemius); and (c) mechanism of injury (running-related activity vs non running-related activity). STUDY DESIGN: Descriptive epidemiological. METHODS: Data retrieved from the Soft Tissue injury Registry of the Australian Football League were analyzed. Sixteen clubs submitted data on CMSI from 2014 to 2017. Data included: player characteristics, training and match history at the time of injury, MRI, and the time to reach recovery milestones. RESULTS: One hundred and eighty-four CMSI were included (149 index injuries; 35 re-injuries). Soleus injuries were most prevalent (84.6%). Soleus injuries took 25.4 ± 16.2 days to return to play, whereas gastrocnemius injuries took 19.1 ± 14.1 days (P = .097). CMSI sustained during running-related activities took approximately 12 days longer to recover than injuries sustained during non running-related activities (P = .001). Compared to index injuries, re-injuries involved older players (P = .03) and significantly more time was taken to run at >90% of maximum speed, return to full training, and return to play (P ≤ .001). Almost all of the observed re-injuries involved soleus (91.4%). CONCLUSION: Soleus injuries are more prevalent than gastrocnemius injuries in elite Australian Football players. Prognosis appears to be influenced by clinical factors, with CMSI sustained during running-related activities and re-injuries needing more time to recover.

Ultra-trace analysis of furanic compounds in transformer/rectifier oils with water extraction and high-performance liquid chromatography.

A novel approach for the determination of parts-per-billion level of 5-hydroxymethyl-2-furaldehyde, furfuryl alcohol, furfural, 2-furyl methyl ketone, and 5-methylfurfural in transformer or rectifier oils has been successfully innovated and implemented. Various extraction methods including solid-phase extraction, liquid-liquid extraction using methanol, acetonitrile, and water were studied. Water was by far the most efficient solvent for use as an extraction medium. Separation of the analytes was conducted using a 4.6 mm × 250 mm × 3.5 µm Agilent Zorbax column while detection and quantitation were conducted with a variable wavelength UV detector. Detection limits of all furans were at 1 ppb v/v with linear ranges range from 5 to 1000 ppb v/v with correlation coefficients of 0.997 or better. A relative standard deviation of at most 2.4% at 1000 ppb v/v and 7.3% at 5 ppb v/v and a recovery from 43% to 90% depending on the analyte monitored were obtained. The method was purposely designed to be environmental friendly with water as an extraction medium. Also, the method uses 80% water and 20% acetonitrile with a mere 0.2 mL/min of acetonitrile in an acetonitrile/water mixture as mobile phase. The analytical technique has been demonstrated to be highly reliable with low cost of ownership, suitable for deployment in quality control labs or in regions where available analytical resources and solvents are difficult to procure.

Large-Vessel Occlusion Is Associated with Poor Outcome in Stroke Patients Aged 80 Years or Older Who Underwent Intravenous Thrombolysis.

OBJECTIVE: We aimed to investigate the association between large-vessel occlusion (LVO) and functional outcome in elderly stroke patients treated with intravenous (IV) tissue plasminogen activator (tPA). METHODS: This was a retrospective study of acute ischemic stroke patients who received IV tPA within 4.5 hours after stroke onset between 2007 and 2013. Patients were categorized into 2 groups based on age (≥80 or < 80 years). LVO was evaluated by computed tomography angiography (CTA) before thrombolysis. Favorable outcome was defined as a modified Rankin Scale (mRS) score of 2 or lower at 3 months, or equal to the prestroke mRS score. RESULTS: Of 359 thrombolysis patients, 175 patients with CTA before a standard dose of IV tPA therapy (0.9 mg/kg body weight; maximum 90 mg) were included. Sixty-five patients were in the group aged 80 years or above with a median age of 84 (interquartile range: 82.5, 86) years. LVO was observed more often in the group with unfavorable outcome compared with the group with favorable outcome in older stroke patients (60.6% versus 21.9%, P = .002). The baseline National Institutes of Health Stroke Scale (NIHSS) score (odds ratio .864; 95% confidence interval [CI], .779-.959; P = .006) and LVO (odds ratio .233; 95% CI, .059-.930; P = .039) were independent associative factors for the unfavorable outcome in older patients treated with IV tPA after adjustment for patient characteristics. CONCLUSIONS: The baseline NIHSS score and LVO were independent predictors for functional outcome in elderly stroke patients received IV tPA.

Does large vessel occlusion affect clinical outcome in stroke with mild neurologic deficits after intravenous thrombolysis?

BACKGROUND: Large vessel occlusion (LVO) is associated with poor functional outcome in acute ischemic stroke. Given the uncertainty whether LVO has the same significance in mild and severe stroke, we compared functional outcomes after intravenous thrombolysis, based on severity and LVO. METHODS: Ischemic stroke patients were thrombolyzed in less than 4.5 hours after onset between 2007 and 2013. LVO was defined as occlusion of one of the following arteries: internal carotid, middle cerebral (M1/M2), anterior cerebral (A1), posterior cerebral (P1), basilar, or vertebral (V4) arteries on prethrombolysis computed tomography angiography. Mild stroke was defined as baseline National Institutes of Health Stroke Scale (NIHSS) score 0-6. Favorable outcome was defined as modified Rankin Scale (mRS) score 0-1 at 3 months or equal to the prestroke mRS. RESULTS: There were 175 acute stroke patients, median age 74 years (interquartile range [IQR], 64-83), median baseline NIHSS = 11 (IQR, 5-16), and 63 of 175 patients (36%) with mild stroke. LVO was associated with worse outcome in severe stroke (age-adjusted odds ratio [OR] of favorable outcome, .42; 95% confidence interval [CI], .19-.93; P = .033) and mortality (age-adjusted OR, 3.52; 95% CI, 1.08-11.48; P = .037). Although the difference in favorable outcome between mild stroke patients with and without LVO was not significant (55.6% vs. 74.1%, P = .262; age-adjusted OR of favorable outcome, .42; 95% CI, .1-1.84; P = .251), the similarity of effects across both subgroups cannot be excluded (LVO-by-stroke severity interaction test, P = .906). CONCLUSIONS: LVO is associated with worse functional outcome and mortality in severe stroke after intravenous thrombolysis. Although significant association between LVO and outcome in mild stroke was not found, there were similar effects on outcome and a larger study might well confirm a relationship.

The ABCG2 gene Q141K polymorphism contributes to an increased risk of gout: a meta-analysis of 2185 cases.

OBJECTIVES: Individual genetic association studies examining the relationship between the ABCG2 gene polymorphisms and gout have yielded inconsistent results. This study aims to evaluate the association between the ABCG2 gene variants and gout using meta-analysis. MATERIALS AND METHODS: Relevant studies were identified by searching databases extensively. The odds ratio (OR) was calculated using a random-effect or fixed-effect model. A Q statistic was used to evaluate homogeneity, and Egger's test and funnel plot were used to assess publication bias. Subgroup analyses on ethnicities and sex were also performed. RESULTS: A total of 7 studies, including 2185 gout patients and 8028 controls from 5 countries or regions, were included and identified for the current meta-analysis. It was found that the A allele or AA genotype of the ABCG2 Q141K polymorphism (rs2231142) had an increased risk of gout in the general population (A allele, p < 0.00001 and AA genotype, p < 0.00001, respectively). On the contrary, CC homozygote played a protective role against the risk of gout (p < 0.00001). Similar results were found in subgroup analyses. However, there was a significant heterogeneity among studies. CONCLUSIONS: Existing evidence indicates that the Q141K polymorphism (rs2231142, the A allele and AA genotype) is associated with an increased risk of gout.

"Driving the Ship" or Laying Buoys: Teachers' Use of Metaphor for Meaning-Making with Storylines.

Science curricula require new conceptualizations of how teachers relate to their materials. In this study of teacher learning, we analyze an experienced group of practicing Storylines teachers' use of metaphor to describe the roles and responsibilities of students and teachers in curriculum enactment. We found that every metaphor that teachers used to describe the uses of Storylines curriculum entailed a sort of wayfinding: a destination, a timeframe, a place, a journey, or the students' or teachers' respective position in that pursuit. These findings continue to indicate the usefulness of metaphor in foregrounding the central role that students play in NGSS-aligned instruction/materials, as well as the institutional forces that shape how curriculum materials get enacted inside the classroom. This study builds and contributes to current scholarship that aims to support teachers in reconceptualizing their role, relationship to students, and the institution of schooling, in the context of constructivist curricula.

EMPOWER: Enacting Materials to Promote Ownership, Engagement, and Relevance.

Fostering locally relevant and community-centered forms of science learning that develop students' critical science agency problematizes a "one-size-fits-all" model of teacher learning; teachers must examine how community needs and resources, local inequities and justice issues, and curriculum materials can converge to design novel learning opportunities for science learners. This paper presents the core commitments of EMPOWER, a cross-institutional effort that aims to support teachers' sensemaking and adaptations of curriculum materials to promote student ownership, engagement, and relevance at multiple sites across the U.S.

Advances and Emerging Medical Applications of Direct Mass Spectrometry Technologies for Tissue Analysis.

Offering superb speed, chemical specificity, and analytical sensitivity, direct mass spectrometry (MS) technologies are highly amenable for the molecular analysis of complex tissues to aid in disease characterization and help identify new diagnostic, prognostic, and predictive markers. By enabling detection of clinically actionable molecular profiles from tissues and cells, direct MS technologies have the potential to guide treatment decisions and transform sample analysis within clinical workflows. In this review, we highlight recent health-related developments and applications of direct MS technologies that exhibit tangible potential to accelerate clinical research and disease diagnosis, including oncological and neurodegenerative diseases and microbial infections. We focus primarily on applications that employ direct MS technologies for tissue analysis, including MS imaging technologies to map spatial distributions of molecules in situ as well as handheld devices for rapid in vivo and ex vivo tissue analysis.

Evaluating the Generalizability of Predictive Classifiers Built from DESI Imaging Lipid Data across Mass Spectrometry Platforms.

In this study, we evaluate the generalizability of predictive classifiers built from DESI lipid data for thyroid fine needle aspiration (FNA) biopsy analysis and classification using two high-performance mass spectrometers (time-of-flight and orbitrap) suited with different DESI imaging sources operated by different users. The molecular profiles obtained from thyroid samples with the different platforms presented similar trends, although specific differences in ion abundances were observed. When using a previously published statistical model built to discriminate thyroid cancer from benign thyroid tissues to predict on a new independent data set obtained, agreement for 24 of the 30 samples across the imaging platforms was achieved. We also tested the classifier on six clinical FNAs and obtained agreement between the predictive results and clinical diagnosis for the different conditions. Altogether, our results provide evidence that statistical classifiers generated from DESI lipid data are applicable across different high-resolution mass spectrometry platforms for thyroid FNA classification.

Reduced Hemoglobin Signal and Improved Detection of Endogenous Proteins in Blood-Rich Tissues for MALDI Mass Spectrometry Imaging.

Mass spectrometry imaging provides a powerful approach for the direct analysis and spatial visualization of molecules in tissue sections. Using matrix-assisted laser desorption/ionization mass spectrometry, intact protein imaging has been widely investigated for biomarker analysis and diagnosis in a variety of tissue types and diseases. However, blood-rich or highly vascular tissues present a challenge in molecular imaging due to the high ionization efficiency of hemoglobin, which leads to ion suppression of endogenous proteins. Here, we describe a protocol to selectively reduce hemoglobin signal in blood-rich tissues that can easily be integrated into mass spectrometry imaging workflows.

Complete genome sequence and updated annotation of Desulfovibrio alaskensis G20.

Desulfovibrio alaskensis G20 (formerly Desulfovibrio desulfuricans G20) is a Gram-negative mesophilic sulfate-reducing bacterium (SRB), known to corrode ferrous metals and to reduce toxic radionuclides and metals such as uranium and chromium to sparingly soluble and less toxic forms. We present the 3.7-Mb genome sequence to provide insights into its physiology.

Spectrum of cancer risk among US solid organ transplant recipients.

CONTEXT: Solid organ transplant recipients have elevated cancer risk due to immunosuppression and oncogenic viral infections. Because most prior research has concerned kidney recipients, large studies that include recipients of differing organs can inform cancer etiology. OBJECTIVE: To describe the overall pattern of cancer following solid organ transplantation. DESIGN, SETTING, AND PARTICIPANTS: Cohort study using linked data on solid organ transplant recipients from the US Scientific Registry of Transplant Recipients (1987-2008) and 13 state and regional cancer registries. MAIN OUTCOME MEASURES: Standardized incidence ratios (SIRs) and excess absolute risks (EARs) assessing relative and absolute cancer risk in transplant recipients compared with the general population. RESULTS: The registry linkages yielded data on 175,732 solid organ transplants (58.4% for kidney, 21.6% for liver, 10.0% for heart, and 4.0% for lung). The overall cancer risk was elevated with 10,656 cases and an incidence of 1375 per 100,000 person-years (SIR, 2.10 [95% CI, 2.06-2.14]; EAR, 719.3 [95% CI, 693.3-745.6] per 100,000 person-years). Risk was increased for 32 different malignancies, some related to known infections (eg, anal cancer, Kaposi sarcoma) and others unrelated (eg, melanoma, thyroid and lip cancers). The most common malignancies with elevated risk were non-Hodgkin lymphoma (n = 1504; incidence: 194.0 per 100,000 person-years; SIR, 7.54 [95% CI, 7.17-7.93]; EAR, 168.3 [95% CI, 158.6-178.4] per 100,000 person-years) and cancers of the lung (n = 1344; incidence: 173.4 per 100,000 person-years; SIR, 1.97 [95% CI, 1.86-2.08]; EAR, 85.3 [95% CI, 76.2-94.8] per 100,000 person-years), liver (n = 930; incidence: 120.0 per 100,000 person-years; SIR, 11.56 [95% CI, 10.83-12.33]; EAR, 109.6 [95% CI, 102.0-117.6] per 100,000 person-years), and kidney (n = 752; incidence: 97.0 per 100,000 person-years; SIR, 4.65 [95% CI, 4.32-4.99]; EAR, 76.1 [95% CI, 69.3-83.3] per 100,000 person-years). Lung cancer risk was most elevated in lung recipients (SIR, 6.13 [95% CI, 5.18-7.21]) but also increased among other recipients (kidney: SIR, 1.46 [95% CI, 1.34-1.59]; liver: SIR, 1.95 [95% CI, 1.74-2.19]; and heart: SIR, 2.67 [95% CI, 2.40-2.95]). Liver cancer risk was elevated only among liver recipients (SIR, 43.83 [95% CI, 40.90-46.91]), who manifested exceptional risk in the first 6 months (SIR, 508.97 [95% CI, 474.16-545.66]) and a 2-fold excess risk for 10 to 15 years thereafter (SIR, 2.22 [95% CI, 1.57-3.04]). Among kidney recipients, kidney cancer risk was elevated (SIR, 6.66 [95% CI, 6.12-7.23]) and bimodal in onset time. Kidney cancer risk also was increased in liver recipients (SIR, 1.80 [95% CI, 1.40-2.29]) and heart recipients (SIR, 2.90 [95% CI, 2.32-3.59]). CONCLUSION: Compared with the general population, recipients of a kidney, liver, heart, or lung transplant have an increased risk for diverse infection-related and unrelated cancers.

Return to Play and Recurrence After Calf Muscle Strain Injuries in Elite Australian Football Players.

BACKGROUND: Calf muscle strain injuries (CMSI) are prevalent in sport, but information about factors associated with time to return to play (RTP) and recurrence is limited. PURPOSE: To determine whether clinical and magnetic resonance imaging (MRI) data are associated with RTP and recurrence after CMSI. STUDY DESIGN: Case-control study; Level of evidence, 3. METHODS: Data of 149 CMSI reported to the Soft Tissue injury Registry of the Australian Football League were explored to evaluate the impact of clinical data and index injury MRI findings on RTP and recurrence. Clinical data included age, previous injury history, ethnicity, and the mechanism of injury. RESULTS: Irrespective of the anatomical location, players with CMSI with severe aponeurotic disruption (AD) took longer to RTP than players with CMSI with no AD: 31.3 ± 12.6 days vs 19.4 ± 10.8 days (mean ± SD; P = .003). A running-related mechanism of injury was associated with a longer RTP period for CMSI overall (adjusted hazard ratio [AHR], 0.59; P = .02). The presence of AD was associated with a longer RTP period for soleus injuries (AHR, 0.6; P = .025). Early recurrence (ie, ≤2 months of the index injury) was associated with older age (AHR, 1.3; P = .001) and a history of ankle injury (AHR, 3.9; P = .032). Older age (AHR, 1.1; P = .013) and a history of CMSI (AHR, 6.7; P = .002) increased the risk of recurrence within 2 seasons. The index injury MRI findings were not associated with risk of recurrence. CONCLUSION: A running-related mechanism of injury and the presence of AD on MRI were associated with a longer RTP period. Clinical rather than MRI data best indicate the risk of recurrent CMSI.

Image quality comparison between model-based iterative reconstruction and adaptive statistical iterative reconstruction chest computed tomography in cystic fibrosis patients.

INTRODUCTION: Cystic fibrosis (CF) predominantly affects young adults. Accurate radiological assessment of pulmonary disease is vital for predicting exacerbations, one of the leading causes of morbidity and mortality. We evaluated the image quality of model-based iterative reconstruction (MBIR) ultra-low-dose CT chest (ULD-CT) in CF evaluation. METHODS: We compared ULD-CT with standard adaptive statistical iterative reconstruction (ASIR) low-dose CT (LD-CT). Subjective assessment of contrast and noise were performed for each study. Background noise, signal to noise ratio (SNR) and contrast to noise ratio (CNR) were calculated and compared between the CT studies. Conspicuity of major structures was assessed. These aspects of image quality were compared to determine whether ULD-CT was superior to LD-CT in assessment of CF. RESULTS: The ULD-CT achieved median effective dose of 0.073 mSv, comparable to one standard chest radiograph and significantly lower than the median LD-CT dose of 1.22 mSv. ULD-CT had lower subjective contrast and higher subjective noise when compared to LD-CT. Objectively measured background noise was lower in ULD-CT (16.33 HU vs 38.53 HU, P < 0.0001) compared to LD-CT. ULD-CT had higher median CNR (52.65 vs 22.09, P < 0.0001) and SNR in lung (9.08 vs 7.29, P = 0.002) compared to LD-CT. ULD-CT was equal to LD-CT in identification of trachea, bronchi, pleural and pericardium. Interobserver reliability showed agreement of 80-92%. CONCLUSIONS: The image quality of ULD-CT is similar to LD-CT, at 1/16th the dose. MBIR constructed ULD-CT is an effective imaging modality for CF surveillance, with potential applications in other disease settings.

Smart bone plates can monitor fracture healing.

There are currently no standardized methods for assessing fracture healing, with physicians relying on X-rays which are only useful at later stages of repair. Using in vivo mouse fracture models, we present the first evidence that microscale instrumented implants provide a route for post-operative fracture monitoring, utilizing electrical impedance spectroscopy (EIS) to track the healing tissue with high sensitivity. In this study, we fixed mouse long bone fractures with external fixators and bone plates. EIS measurements taken across two microelectrodes within the fracture gap were able to track longitudinal differences between individual mice with good versus poor healing. We additionally present an equivalent circuit model that combines the EIS data to classify fracture repair states. Lastly, we show that EIS measurements strongly correlated with standard quantitative µCT values and that these correlations validate clinically-relevant operating frequencies for implementation of this technique. These results demonstrate that EIS can be integrated into current fracture management strategies such as bone plating, providing physicians with quantitative information about the state of fracture repair to guide clinical decision-making for patients.

US organ donation breakthrough collaborative increases organ donation.

More than 92000 Americans are on waiting lists for organ transplants, and an average of 17 of them die each day while waiting. The US Organ Donation Breakthrough Collaborative (ODBC), which began in 2003 at the request of the Secretary of the US Department of Health and Human Services, was a formal, concerted effort of the donation and transplantation community to bring about a major change to improve the organ donation system. The nationwide Collaborative was housed within a Health and Human Services agency, the Health Resources and Services Administration (HRSA) Division of Transplantation, and included participation of the organ procurement organizations (OPOs) throughout the United States and the American hospitals with the largest organ-donor potential. HRSA leaders used the Breakthrough Series Collaborative method, originally developed by the Institute for Healthcare Improvement, as the model for the intervention. Expert practitioners drawn from hospitals and OPOs that had already demonstrated their ability to achieve and sustain high organ donation rates were chosen as faculty for the collaborative and best practices were gleaned from their institutions. The number of organ donors in Collaborative hospitals increased 14.1% in the first year, a 70% greater increase than the 8.3% increase experienced by non-Collaborative hospitals. Moreover, the increased organ recovery continued into the post-Collaborative periods. Between October 2003 and September 2006, the number of total US organ donors increased 22.5%, an increase 4-fold greater than the 5.5% increase measured over the same number of years in the immediate pre-Collaborative period. The study did not involve a randomized design, but time-series analysis using statistical process control charts shows a highly significant discontinuity in the rate of increase in participating hospitals concurrent with the Collaborative program, and strongly suggests that the activities of the Collaborative were a major contributor to this increase. Given the stable nature of the historical increases over many years, the HRSA estimates that more than 4000 annual additional transplants have occurred in association and apparently as a result of these increases in organ donation.

A novel heterodimerization domain, CRM1, and 14-3-3 control subcellular localization of the MondoA-Mlx heterocomplex.

Among members of the bHLHZip family of transcriptional regulators, MondoA and Mlx have the unique property of cytoplasmic localization. We have proposed that MondoA-Mlx heterodimers accumulate in the nucleus in response to extracellular cues. Our previous work implicated heterodimerization between MondoA and Mlx and a conserved domain in the N terminus of MondoA as important determinants of MondoA-Mlx subcellular localization. MondoA and Mlx share sequence similarity in their bHLHZip domains and C termini. Here we show that for both MondoA and Mlx, this C-terminal domain has cytoplasmic localization activity that is required by the protein monomers to accumulate in the cytoplasm. This C-terminal domain is also a novel dimerization interface that functions independently of the leucine zipper to mediate heterotypic interactions between MondoA and Mlx. Dimerization between MondoA and Mlx inactivates the cytoplasmic localization activity of their C termini and is necessary for the heterocomplex to accumulate in the nucleus. MondoA-Mlx heterodimers, while poised for nuclear entry, are retained in the cytoplasm by conserved domains in the N terminus of MondoA. Mondo conserved regions (MCRs) II and III contribute to cytoplasmic localization of MondoA-Mlx by functioning as a CRM1-dependent nuclear export signal and as a novel binding site for 14-3-3 family members, respectively. We propose that the nuclear accumulation of MondoA and Mlx is a two-step process. First, heterodimerization abolishes the cytoplasmic localization activity of their C termini. Second, an extracellular signal(s) must overcome the cytoplasmic localization function imparted by CRM1 and 14-3-3 binding to the N terminus of MondoA.