Search

To test whether it is practical to use phage display coupled with proteolysis for protein design, we used this approach to convert a partially unfolded four-helix bundle protein, apocytochrome b562, to a stably folded four-helix bundle protein. Four residues expected to form a hydrophobic core were mutated. One residue was changed to Trp to provide a fluorescence probe for studying the protein''s physical properties and to partially fill the void left by the heme. The other three positions were randomly mutated. In addition, another residue in the region to be redesigned was substituted with Arg to provide a specific cutting site for protease Arg-c. This library of mutants was displayed on the surface of phage and challenged with protease Arg-c to select stably folded proteins. The consensus sequence that emerged from the selection included hydrophobic residues at only one of the three positions and non-hydrophobic residues at the other two. Nevertheless, the selected proteins were thermodynamically very stable. The structure of a selected protein was characterized using multi-dimensional NMR. All four helices were formed in the structure. Further, site-directed mutagenesis was used to change one of the two non-hydrophobic residues to a hydrophobic residue, which increased the stability of the protein, indicating that the selection result was not based solely on the protein''s global stability and that local structural characteristics may also govern the selection. This conclusion is supported by the crystal structure of another mutant that has two hydrophobic residues substituted for the two non-hydrophobic residues. These results suggest that the hydrophobic interactions in the core are not sufficient to dictate the selection and that the location of the cutting site of the protease also influences the selection of structures. [Copyright &y& Elsevier]

Introduction: We previously identified Notch2 in smooth muscle cells (SMC) in human atherosclerosis and found that signaling via Notch2 suppressed human SMC proliferation. Thus, we tested whether loss of Notch2 in SMC would alter atherosclerotic plaque progression using a mouse model., Methods: Atherogenesis was examined at the brachiocephalic artery and aortic root in a vascular SMC null (inducible smooth muscle myosin heavy chain Cre) Notch2 strain on the ApoE-/- background. We measured plaque morphology and size, as well as lipid, inflammation, and smooth muscle actin content after Western diet., Results: We generated an inducible SMC Notch2 null on the ApoE-/- background. We observed ∼90% recombination efficiency with no detectable Notch2 in the SMC. Loss of SMC Notch2 did not significantly change plaque size, lipid content, necrotic core, or medial area. However, loss of SMC Notch2 reduced the contractile SMC in brachiocephalic artery lesions and increased inflammatory content in aortic root lesions after 6 weeks of Western diet. These changes were not present with loss of SMC Notch2 after 14 weeks of Western diet., Conclusions: Our data show that loss of SMC Notch2 does not significantly reduce atherosclerotic lesion formation, although in early stages of plaque formation there are changes in SMC and inflammation., (© 2022 S. Karger AG, Basel.)

Background: Neuro-Developmental Treatment (NDT) currently embraces evidence-based concepts of motor control, motor learning and neuroplasticity. However, most research has been performed on outdated models of NDT., Objective: This case series examines the short- and long-term outcomes of a three-week intensive using contemporary NDT interventions., Methods: Six children, 2-10 years old with neurologic disorders and Gross Motor Function Classification System (GMFCS) levels I-III participated in the intervention. The three-week intensive included 60 minutes of physical, occupational and speech therapy 3-5 times weekly., Results: All children demonstrated Gross Motor Function Measure-66 gains of medium to large effect sizes. These gains were maintained or improved upon 3 months' post conclusion of the intensive intervention., Conclusions: This study supports emerging research regarding the effectiveness of intensive intervention and further study of current NDT interventions.

Background: People with advanced dementia often exhibit responsive behaviors such as apathy, depression, agitation, aggression, and psychosis. Non-pharmacological approaches (e.g., listening to music, watching television, doing arts and crafts) are now considered as a first-line strategy to manage responsive behaviors in clinical practice due to the potential risks associated with the antipsychotic medications. To date, no evaluations of immersive non-head mounted virtual reality (VR) experience as a non-pharmacologic approach for people with advanced dementia living in nursing homes have been reported., Objective: To evaluate the feasibility (acceptance and safety) of VR experience., Methods: A single site case series (nonrandomized and unblinded) with a convenience sample (N = 24; age = 85.8±8.6 years; Cognitive Performance Scale score = 3.4±0.6) measuring depression and agitation before and after the intervention. The intervention was a 30-min long research coordinator- facilitated VR experience for two weeks (10 sessions)., Results: The intervention was feasible (attrition rate = 0% ; adverse events = 0). A reduction in depression and in agitation was observed after the intervention. However, we suggest extreme caution in interpreting this result considering the study design and small sample size., Conclusion: This study provides the basis for conducting a randomized controlled trial to evaluate the effect of VR experience on responsive behaviors in nursing homes. Since our intervention uses a smart remote-controlled projector without a headset, infectious exposure can be avoided following the COVID-19 pandemic-induced physical distancing policy in care homes.

Extracellular signal-regulated kinase 1 and 2 (ERK1/2) have been implicated as important regulators of metabolic homeostasis. Here we generated a new mouse model with genetic deletion of two ERK1/2 phosphatases, dual specificity phosphatase (DUSP) 6 and 8, to further define the role of ERK1/2 in obesity development. Dusp6/8 double-null mice demonstrated elevated ERK1/2 phosphorylation in multiple tissues, without any change of phosphorylation of p38 and c-Jun N-terminal kinases (JNKs). Elevated ERK1/2 activity in Dusp6/8 double-null mice was associated with larger hearts and other organs, consistent with greater rate of cell proliferation in these mice. However, ERK1/2 activation was not sufficient to protect the mouse hearts from pathological hypertrophy and interstitial fibrosis following angiotensin II and phenylephrine stimulation. Interestingly, mice lacking DUSP6/8 were resistant to high-fat diet-induced obesity. Serum triglyceride, lipid content in the liver and visceral adipose tissues was also dramatically reduced in Dusp6/8 double-null mice. Furthermore, Dusp6/8 double-null mice had improved glucose tolerance. Mechanistically, we found out that elevated ERK1/2 activity increased the expression levels of genes involved in lipid metabolism and glucose homeostasis. Together, our data suggest that ERK1/2 play an essential role for the management of metabolic homeostasis., 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., (Published by Elsevier Inc.)

Objective: Perivascular adipose tissue (PVAT) surrounding arteries supports healthy vascular function. During obesity, PVAT loses its vasoprotective effect. We study pathological conversion of PVAT, which involves molecular changes in protein profiles and functional changes in adipocytes. Approach and Results: C57BL6/J mice were fed a 60% high-fat diet for 12 weeks or a cardioprotective 30% calorie-restricted diet for 5 weeks. Proteomic analysis identified PVAT as a molecularly distinct adipose depot, and novel markers for thermogenic adipocytes, such as GRP75 (stress-70 protein, mitochondrial), were identified. High-fat diet increased the similarity of protein signatures in PVAT and brown adipose, suggesting activation of a conserved whitening pathway. The whitening phenotype was characterized by suppression of UCP1 (uncoupling protein 1) and increased lipid deposition, leptin, and inflammation, and specifically in PVAT, elevated Notch signaling. Conversely, PVAT from calorie-restricted mice had decreased Notch signaling and less lipid. Using the Adipoq-Cre strain, we constitutively activated Notch1 signaling in adipocytes, which phenocopied the changes in PVAT caused by a high-fat diet, even on a standard diet. Preadipocytes from mouse PVAT expressed Sca1, CD140a, Notch1, and Notch2, but not CD105, showing differences compared with preadipocytes from other depots. Inhibition of Notch signaling during differentiation of PVAT-derived preadipocytes reduced lipid deposition and adipocyte marker expression., Conclusions: PVAT shares features with other adipose depots, but has a unique protein signature that is regulated by dietary stress. Increased Notch signaling in PVAT is sufficient to initiate the pathological conversion of PVAT by promoting adipogenesis and lipid accumulation and may thus prime the microenvironment for vascular disease.

End stage kidney disease affects hundreds of thousands of patients in the United States. The therapy of choice is kidney replacement, but availability of organs is limited, and alternative sources of tissue are needed. Generation of new kidney tissue in the laboratory has been made possible through pluripotent cell reprogramming and directed differentiation. In current procedures, aggregates of cells known as organoids are grown either submerged or at the air-liquid interface. These studies have demonstrated that kidney tissue can be generated from pluripotent stem cells, but they also identify limitations. The first is that perfusion of cell aggregates is limited, restricting the size to which they can be grown. The second is that aggregates lack the structural integrity required for convenient engraftment and suturing or adhesion to regions of kidney injury. In this study, we evaluated the capacity of silk to serve as a support for the growth and differentiation of kidney tissue from primary cells and from human induced pluripotent stem cells. We find that cells can differentiate to epithelia characteristic of the developing kidney on this material and that these structures are maintained following engraftment under the capsule of the adult kidney. Blood vessel investment can be promoted by the addition of vascular endothelial growth factor to the scaffold, but the proliferation of stromal cells within the graft presents a challenge, which will require some readjustment of cell growth and differentiation conditions. In summary, we find that silk can be used to support growth of stem cell derived kidney tissue., (© 2019 John Wiley & Sons, Ltd.)

Atherosclerosis is the most common cause of heart disease and stroke. The use of animal models has advanced our understanding of the molecular signaling that contributes to atherosclerosis. Further understanding of this degenerative process in humans will require human tissue. Plaque removed during endarterectomy procedures to relieve arterial obstructions is usually discarded, but can be an important source of diseased cells. Resected tissue from carotid and femoral endarterectomy procedures were compared with carotid arteries from donors with no known cardiovascular disease. Vascular smooth muscle cells (SMC) contribute to plaque formation and may determine susceptibility to rupture. Notch signaling is implicated in the progression of atherosclerosis, and plays a receptor-specific regulatory role in SMC. We defined protein localization of Notch2 and Notch3 within medial and plaque SMC using immunostaining, and compared Notch2 and Notch3 levels in total plaques with whole normal arteries using immunoblot. We successfully derived SMC populations from multiple endarterectomy specimens for molecular analysis. To better define the protein signature of diseased SMC, we utilized sequential window acquisition of all theoretical spectra (SWATH) proteomic analysis to compare normal carotid artery SMC with endarterectomy-derived SMC. Similarities in protein profile and differentiation markers validated the SMC identity of our explants. We identified a subset of differentially expressed proteins that are candidates as functional markers of diseased SMC. To understand how Notch signaling may affect diseased SMC, we performed Jagged1 stimulation of primary cultures. In populations that displayed significant growth, Jagged1 signaling through Notch2 suppressed proliferation; cultures with low growth potential were non-responsive to Jagged1. In addition, Jagged1 did not promote contractile smooth muscle actin nor have a significant effect on the mature differentiated phenotype. Thus, SMC derived from atherosclerotic lesions show distinct proteomic profiles and have altered Notch signaling in response to Jagged1 as a differentiation stimulus, compared with normal SMC.

Objective: Vascular remodeling is associated with complex molecular changes, including increased Notch2, which promotes quiescence in human smooth muscle cells. We used unbiased protein profiling to understand molecular signatures related to neointimal lesion formation in the presence or absence of Notch2 and to test the hypothesis that loss of Notch2 would increase neointimal lesion formation because of a hyperproliferative injury response., Approach and Results: Murine carotid arteries isolated at 6 or 14 days after ligation injury were analyzed by mass spectrometry using a data-independent acquisition strategy in comparison to uninjured or sham injured arteries. We used a tamoxifen-inducible, cell-specific Cre recombinase strain to delete the Notch2 gene in smooth muscle cells. Vessel morphometric analysis and immunohistochemical staining were used to characterize lesion formation, assess vascular smooth muscle cell proliferation, and validate proteomic findings. Loss of Notch2 in smooth muscle cells leads to protein profile changes in the vessel wall during remodeling but does not alter overall lesion morphology or cell proliferation. Loss of smooth muscle Notch2 also decreases the expression of enhancer of rudimentary homolog, plectin, and annexin A2 in vascular remodeling., Conclusions: We identified unique protein signatures that represent temporal changes in the vessel wall during neointimal lesion formation in the presence and absence of Notch2. Overall lesion formation was not affected with loss of smooth muscle Notch2, suggesting compensatory pathways. We also validated the regulation of known injury- or Notch-related targets identified in other vascular contexts, providing additional insight into conserved pathways involved in vascular remodeling., (© 2018 American Heart Association, Inc.)

Introduction: Health systems can be supported by collaborative networks focused on data sharing and comparative analytics to identify and rapidly disseminate promising care practices. Standardized data collection, quality assessment, and cleansing is a necessary process to facilitate meaningful analytics for operations, quality improvement, and research. We developed a framework for aligning data from health care delivery systems using the High Value Healthcare Collaborative central registry., Framework: The centralized data registry model allows for multiple layers of data quality assessment. Our framework uses an iterative approach, starting with clear specifications, maintaining ongoing dialogue with diverse stakeholders, and regular checkpoints to assess data conformance, completeness, and plausibility., Lessons Learned: We found that an iterative communication process is critical for a central registry to ensure: 1) clarity of data specifications, 2) appropriate data quality, and 3) thorough understanding of data source, purpose, and context. Engaging teams from all participating institutions and incorporating diverse stakeholders of clinicians, information technologists, data analysts, operations managers, and health services researchers in all decision making processes supports development of high quality datasets for comparative analytics across multiple institutions., Conclusion: A standard data specification and submission process alone does not guarantee aligned data for a collaborative registry. Implementing an iterative data quality improvement framework with extensive communication proved to be effective for aligning data from multiple institutions to support meaningful analytics.

Introduction: Intensive Care Unit (ICU) length of stay is a strong indicator of severity of illness and cost in the care of sepsis patients. In this case study, we examine the difference between an electronic health record (EHR) based submissions with Centers for Medicare and Medicaid Services (CMS) payment data., Methods: Member submitted EHR data contained 26,733 unique patient's records. The CMS data contained demographics, diagnosis, and revenue codes. After linking EHR data to CMS data, we found a discrepancy in ICU days from CMS claims vs. EHR data. Our hypothesis was that removing intermediate ICU LOS would result in a closer match from CMS claims with EHR data. We suspected the use of Intermediate ICU stays in our CMS ICU definition contaminated our ICU LOS data. This resulted in a review of the sepsis specification, further investigation of the data, and follow up conversations with the Member organizations., Results: Agreement between EHR and CMS data improved from 73 percent to 86 percent once the Intermediate ICU time had been removed., Discussion and Conclusions: The inclusion of Intermediate ICU in the analysis of severely ill sepsis patients from CMS data diluted the importance of using an ICU LOS for estimating the severity of illness and the cost to the healthcare system. We must ensure that clinical definitions are consistent between data sources that were built for different purposes. Additionally, we learned that engaging with clinicians, analysts, and clinical coders early in the process is required to fully understand the complexities from different sources.

This study evaluated the effectiveness of VeinViewer for peripheral vascular accessing a pediatric hematology oncology clinic. After obtaining consent, 53 patients were randomly assigned to either the VeinViewer group (n = 27) or standard methods group (n = 26). Data on number of attempts, procedural time, access complications, and patient and nurse satisfaction were collected. Patients randomized to the VeinViewer group required significantly less time to access a vein as compared with the standard methods group (P ≤ .05). Additionally, these patients rated nurses as having significantly more skill than nurses who did not use VeinViewer (P ≤ .05) and assigned significantly higher scores for "overall experience"(P ≤ .05). Responses by nurses using VeinViewer overall saw the device in a positive light., (© 2015 by Association of Pediatric Hematology/Oncology Nurses.)

Background: Pregnancy is associated with an increased sympathetic state, which can be exacerbated by gestational conditions. Research has shown that exercise during pregnancy lowers heart rate (HR) and can attenuate the symptoms of gestational conditions associated with increased sympathetic control. However, changes in maternal heart autonomic function in response to exercise have not been reported across multiple time points during pregnancy. This analysis is designed to address this gap., Objective: To determine if exercise throughout gestation improves maternal cardiac autonomic nervous system functioning, as evidenced by decreased HR and increased heart rate variability (HRV) indices., Design: Case control study., Setting: Academic medical institution., Participants: A total of 56 women with healthy, singleton, low-risk pregnancies., Methods: Participants were asked to complete 3 resting 18-minute HRV recordings at 28, 32, and 36 weeks' gestation, along with a physical activity questionnaire., Main Outcome Measures: HRV indices were calculated for time (R peak to R peak interval standard deviation and root mean squared of successive differences) and frequency (very low, low, and high frequency) domain measures. The differences between groups were compared for HRV indices at 28, 32, and 36 weeks., Results: Resting HR was significantly lower in the exercise group at 28 weeks (P < .01) compared with the control group. The exercise group had significantly (P < .05) increased measures of resting HRV time domain measures at 28, 32, and 36 weeks' gestation compared with the control group. Resting HRV power was significantly increased (P < .05) in all frequency domain measures at 32 weeks in the exercise group relative to the control group. No differences occurred in sympathovagal balance (low frequency/high frequency ratio) between groups., Conclusion: Exercise throughout pregnancy can significantly improve cardiac autonomic control. More research is needed to determine if this adaptation to exercise may reduce the risk of adverse outcomes associated with gestational conditions with poor autonomic control, such as diabetes, hypertension, pre-eclampsia, and excessive weight gain., (Copyright © 2016 American Academy of Physical Medicine and Rehabilitation. Published by Elsevier Inc. All rights reserved.)

Background: Police-community relations have catapulted onto the national stage after several high-profile instances of alleged police brutality. Blame and hostility can be barriers to positive police-community relations. Playback is a form of audience-inspired, improvisational theater designed to promote connectivity and empathy through storytelling., Objectives: We tested the feasibility and acceptability of an arts-based intervention, bringing together police officers and formerly incarcerated individuals from the same community in Memphis, Tennessee., Methods: We collected pre/post quantitative data from five police officers and five ex-offenders who took part in the intervention, as well as qualitative data to provide contextual information., Results: The project was feasible and acceptable to participants. Participants showed gains in their ability to make meaning of stressful life experiences. The officers and ex-offenders showed parallel gains in their increased positive attitudes toward the other group., Conclusions: This study demonstrates that creating contexts of safety and understanding necessary to address relational problems is both feasible and acceptable to law enforcement and ex-offenders.

The PanAfrican Acupuncture Project (PAAP) was created in 2001 and is a volunteer-based not-for-profit training organization. It encourages community empowerment and wellness through training local healthcare providers how to use simple, effective acupuncture protocols to treat the symptoms of HIV/ AIDS, malaria, tuberculosis, and other chronic conditions. A manual explains the theory of acupuncture and the techniques associated with it in clear and simple terms with specific acupuncture protocols associated with the signs and symptoms of illnesses commonly seen in Uganda. PAAP maintains contact with the trainees to provide ongoing support through: (1) three week-long meetings per year, (2) ongoing contact with the PAAP local coordinator, and (3) electronic communications including email, text messaging, and Facebook communication. A primary focus is to help integrate acupuncture into the local context and current public-health system. This is accomplished by working with local governmental and nongovernmental agencies and healthcare facilities to improve access to and effectiveness of care through collaboration with Western and traditional healthcare modalities. The integration of acupuncture and other traditional complementary and alternative medicines (TCAMs) in the international public health sector at the level of local health workers can increase access to treatment in a sustainable manner while also building the confidence and self-worth of the public health-care practitioners.

Background: Cystic fibrosis (CF) is a common genetic disease in Caucasians. Chronic pulmonary disease with progressive destruction of the pulmonary parenchyma is two of the major morbidities, but the relationship between clinical severity of CF and aortopulmonary collateral blood flow has not been assessed., Objective: The purpose of this study is to measure changes in aortopulmonary collateral blood flow by phase-contrast magnetic resonance imaging (MRI) in children with CF across the spectrum of disease severity as measured by the forced expiratory volume in one second as percent predicted value (FEV1% predicted)., Materials and Methods: Sixteen patients with CF were prospectively evaluated. Eight were classified as having mild CF lung disease (FEV1 ≥80% predicted) and eight were classified as having moderate to severe CF lung disease (FEV1 <80% predicted). Seventeen age- and gender-matched non-CF subjects without cardiac or lung disease served as controls. Phase-contrast flow was measured at the ascending aorta, main pulmonary artery and both pulmonary arteries. Aortopulmonary collateral blood flow was calculated for each subject. The relationship between collateral flow and FEV1% predicted was modeled using nonparametric regression. Group differences were assessed by analysis of variance., Results: Aortopulmonary collateral blood flow began to increase as FEV1% predicted in subjects with CF fell below 101.5% with significant further increase in the aortopulmonary collateral blood flow in the subjects with CF with moderate to severe lung disease compared to controls (0.89 vs. 0.20 L/min, P < 0.0001). Aortopulmonary collateral blood flow correlated negatively with FEV1% predicted (r=0.70, P = 0.0050) confirming its relationship to this established marker of disease severity. There was no statistically significant difference in results obtained from two independent observers., Conclusion: These preliminary findings suggest that phase-contrast MRI can be performed reliably with consistent results and without interobserver variability. While the aortopulmonary collateral blood flow is within the normal range in subjects with mild CF disease, it begins to increase even when lung function is still in the normal range. A significant increase in the aortopulmonary collateral blood flow compared to controls is measured in patients with moderate to severe CF lung disease. The studies support the notion that aortopulmonary collateral blood flow may serve as a novel and sensitive biomarker of early pulmonary disease in cystic fibrosis.

Berry syndrome comprises a rare combination of heart defects that includes aortopulmonary window, interrupted aortic arch, intact ventricular septum, and aortic origin of the right pulmonary artery. We report the case of a neonate confirmed to have Berry syndrome by transthoracic echocardiogram and computed tomography (CT). This neonate had the additional finding of an aberrant right subclavian artery arising from the descending aorta. A single-stage repair was successfully performed when the infant was 7 days of age. Genetic testing showed a 102-kb deletion within chromosome band 9p24.2; this deletion has not been previously linked to congenital heart defects. Berry syndrome can be diagnosed accurately by transthoracic echocardiogram and CT. There may be an underlying genetic etiology, and this possibility warrants further investigation.

The effect of ecdysteroid signaling on Drosophila female precopulatory behavior was investigated using two types of mutants with either globally reduced ecdysteroid availability or reduced expression of ecdysone receptors in fruitless neurons, known to control sexual behavior. While being courted by males, mutant females performed significantly less full ovipositor extrusion behavior to reject male copulation attempts. Ecdysteroid depleted females (ecdysoneless(1)) performed male-like courtship behaviors, including unilateral wing extension and song production with patterns very similar to male courtship song. These results support the hypothesis that ecdysteroids modulate female sexual behavior, perhaps acting as a regulator of sexual motivation, and as a component affecting the performance of sex specific behavior patterns., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Mutations of influenza virus increase concerns of worldwide epidemics resulting from the newly emergent strains. Current influenza vaccines are inefficient and require annual vaccinations. W805EC adjuvant is an oil-in-water emulsion composed of nanodroplets with an average diameter of approximately 400 nm. The nanoemulsion adjuvant has been used successfully to stimulate the immune response when mixed with several other antigens in animal models. In this study, W805EC nanoemulsion adjuvant activity was evaluated using nasal influenza vaccination in a murine model. Five to twenty percent W805EC adjuvant was used to inactivate influenza A/Puerto Rico/8/34/05 (H1N1). Mice immunized with the nanoemulsion adjuvanted influenza virus intranasally showed a robust specific humoral immune response as demonstrated using ELISA and HAI assays. Serum HAI titers were more than 104 following two vaccinations. Vaccinated mice were also protected against challenge with an LD80 of live influenza virus. Splenocytes from vaccinated mice were assayed for cytokine production following virus stimulation. The cytokine profile demonstrated a robust cellular immune response with enhanced Th1 and Th17 immunity that provided balanced immunity against both intracellular and extracellular forms of the virus. Additionally, the vaccine preparations showed minimal protein degradation but remained potent when stored at 4°C for up to three months. This work demonstrates that W805EC nanoemulsion adjuvant can effectively enhance the immunogenicity of influenza hemagglutinin antigen. The nanoemulsion adjuvant can result in antigen sparing and cross-protection. The potential exists for a nasally administered influenza vaccine that may require little or no refrigerated storage.

Background: Many infectious diseases that cause significant morbidity and mortality, especially in the developing world, could be preventable through vaccination. The effort to produce safe, thermally stable, and needle-free mucosal vaccines has become increasingly important for global health considerations. We have previously demonstrated that a thermally stable nanoemulsion, a mucosal adjuvant for needle-free nasal immunization, is safe and induces protective immunity with a variety of antigens, including recombinant protein. The successful use of nanoemulsion-based vaccines, however, poses numerous challenges. Among the challenges is optimization of the formulation to maintain thermal stability and potency and another is accuracy and efficiency of dispensing the vaccines to the nasal mucosa in the anterior and turbinate region of the nasal cavity or potentially to the nasopharynx-associated lymphoid tissue., Methods: We have examined the effects of different diluents [phosphate-buffered saline (PBS) and 0.9% NaCl] on the stability and potency of nanoemulsion-based vaccines. In addition, we have determined the efficiency of delivering them using commercially available nasal spray devices (Pfeiffer SAP-62602 multidose pump and the BD Hypak SCF 0.5 ml unit dose Accuspray(TM))., Results: We report the stability and potency of PBS-diluted ovalbumin-nanomeulsion mixtures for up to 8 months and NaCl-diluted mixtures up to 6 months when stored at room temperature. Significant differences in spray characteristics including droplet size, spray angle, plume width, and ovality ratios were observed between the two pumps. Further, we have demonstrated that the nanoemulsion-based vaccines are not physically or chemically altered and retain potency following actuation with nasal spray devices. Using either device, the measured spray characteristics suggest deposition of nanoemulsion-based vaccines in inductive tissues located in the anterior region of the nasal cavity., Conclusions: The results of this study suggest that nanoemulsion-based vaccines do not require specially engineered delivery devices and support their potential use as nasopharyngeal vaccine adjuvants.

Background: Hepatitis B virus infection remains an important global health concern despite the availability of safe and effective prophylactic vaccines. Limitations to these vaccines include requirement for refrigeration and three immunizations thereby restricting use in the developing world. A new nasal hepatitis B vaccine composed of recombinant hepatitis B surface antigen (HBsAg) in a novel nanoemulsion (NE) adjuvant (HBsAg-NE) could be effective with fewer administrations., Methodology and Principal Findings: Physical characterization indicated that HBsAg-NE consists of uniform lipid droplets (349+/-17 nm) associated with HBsAg through electrostatic and hydrophobic interactions. Immunogenicity of HBsAg-NE vaccine was evaluated in mice, rats and guinea pigs. Animals immunized intranasally developed robust and sustained systemic IgG, mucosal IgA and strong antigen-specific cellular immune responses. Serum IgG reached > or = 10(6) titers and was comparable to intramuscular vaccination with alum-adjuvanted vaccine (HBsAg-Alu). Normalization showed that HBsAg-NE vaccination correlates with a protective immunity equivalent or greater than 1000 IU/ml. Th1 polarized immune response was indicated by IFN-gamma and TNF-alpha cytokine production and elevated levels of IgG(2) subclass of HBsAg-specific antibodies. The vaccine retains full immunogenicity for a year at 4 degrees C, 6 months at 25 degrees C and 6 weeks at 40 degrees C. Comprehensive pre-clinical toxicology evaluation demonstrated that HBsAg-NE vaccine is safe and well tolerated in multiple animal models., Conclusions: Our results suggest that needle-free nasal immunization with HBsAg-NE could be a safe and effective hepatitis B vaccine, or provide an alternative booster administration for the parenteral hepatitis B vaccines. This vaccine induces a Th1 associated cellular immunity and also may provide therapeutic benefit to patients with chronic hepatitis B infection who lack cellular immune responses to adequately control viral replication. Long-term stability of this vaccine formulation at elevated temperatures suggests a direct advantage in the field, since potential excursions from cold chain maintenance could be tolerated without a loss in therapeutic efficacy.

General ecological methods and models that require a minimum amount of information yet are still able to inform conservation planning are particularly valuable. Nested subset analysis has been advocated as such a tool for the prediction of extinction-prone species and populations. However, such advocacy has not been without skepticism and debate, and in the majority of published examples assessing extinction vulnerability, actual extinctions are based on assumptions rather than direct evidence. Here, we empirically test the power of nested subset analysis to predict extinction-prone species, using documented Holocene insular mammal extinctions on three island archipelagos off the west coast of North America. We go on to test whether the introduction of invasive mammals promotes nestedness on islands via extinction. While all three archipelagos were significantly nested before and after the extinction events, nested subset analysis largely failed to predict extinction patterns. We also failed to detect any correlations between the degree of nestedness at the genus-level with area, isolation, or species richness and extinction risk. Biogeography tools, such as nested subset analysis, must be critically evaluated before they are prescribed widely for conservation planning. For these island archipelagos, it appears detailed natural history and taxa-specific ecology may prove critical in predicting patterns of extinction risk.

Experiments were conducted to address recent reports of precocial testis development or testicular hypertrophy induced by the anti-thyroid agent 6-N-propyl-2-thiouracil (PTU) in domestic fowl and rodents. In three experiments, PTU was administered to male turkeys during different stages of development. The PTU was given in the feed at a concentration of either 0.1 or 0.5% and effects on thyroid hormones were measured. Periods of PTU treatment were 8 to 16 or 10 to 18 wk of age (Experiment 1); 0 to 8, 0 to 16, and 8 to 16 wk of age (Experiment 2); and 8 to 16 and 20 to 35 wk of age (Experiment 3). Data were collected to determine the effect of PTU treatment on testicular growth (weight), sexual maturation (semen onset and plasma testosterone concentrations), and early adult testicular function in terms of semen quantity (semen volume and sperm concentration) and quality (sperm viability and fertilizing ability). The 0.1% PTU treatment at 8 to 16 wk of age in Experiment 1 resulted in greater testis weights and sperm production than those of untreated controls at 24 wk of age. The difference was absent by 36 wk of age. Likewise, in Experiment 2, onset of semen production was advanced by about 2 wk by the 8 to 16 wk treatment as compared to untreated controls (23.3 vs 25.9 wk), although, at 32 and 36 wk of age neither the quantity nor quality of semen were significantly affected. Also in Experiment 2, 0.1% PTU treatment from 0 to 8 or 0 to 16 wk of age delayed the onset of semen production and depressed semen volumes at 32 and 36 wk of age without affecting semen quality. In Experiment 3, the mean age at the onset of semen was not significantly affected by 0.5% PTU treatment from 8 to 16 or 20 to 35 wk of age. In addition, semen volume and quality at 32 and 36 wk of age was similar to controls. It was concluded that antithyroid treatment with PTU was dependent on dose and time of treatment for effects on both sexual development and early reproductive performance. Precocial development and associated hypertrophy of the testes occurred but was transient. Thereafter, during initial reproductive function, semen quantity and quality were normal.

From January through March of 1981, the Health Care Financing Administration (HCFA) surveyed the agencies of 49 States and the District of Columbia responsible for the administration of the Medicaid program. The purpose of the survey was to determine if the agencies had a nursing home pre-admission screening program for Medicaid patients. Twenty-eight States and the District of Columbia responded that there was a state-wide, pre-admission screening program for Medicaid patients prior to their entry into a nursing home, or that there was a program operating in a portion of the State. HCFA collected information on the scope of the programs, the agencies responsible for conducting pre-admission screening, the composition of the screening teams, and the characteristics of the client assessment instruments. Two States, Virginia and Massachusetts, provided information on program impact. This article presents the findings of the survey and explores several aspects of the Medicaid program influencing the effectiveness of pre-admission screening. It begins with an overview of the policy issues which have influenced the development of pre-admission screening and defines the core components of these programs.

The use of human monoclonal antibodies (MCA) in the detection and treatment of human cancer has been limited by the apparent scarcity of MCA to tumor cell surface antigens. Using peripheral blood lymphocytes from autologous tumor-immunized patients, we isolated 36 MCA that react to sections of colorectal carcinoma. Twenty of these human MCA appear to be directed against cell surface antigens. Two-thirds of the human MCA-producing cell lines were diploid human B-cells rather than human-mouse heterohybridomas. Direct antibody-binding assays performed with the MCA indicated that they recognized antigenic determinants preferentially expressed on tumor cells. Experiments with paired specimens of air-dried, dissociated colon tumor cells and normal colonic mucosa cells suggested that the MCA bound significantly more to the cell surfaces of tumor cells than to the surfaces of normal colonic mucosa cells. Similarly, tests with a panel of cryostat sections of paired colon tumor and normal colonic mucosa showed that MCA bound to the tumor cells and not to the normal colonic mucosa. None of the MCA bound to cells from frozen sections of normal breast, stomach, liver, skeletal muscle, or skin. Furthermore, the human MCA did not react with carcinoembryonic antigen and human erythrocyte antigens as measured by various techniques. Our data also demonstrated that these transformed B-cells and hybridomas were stable producers of human MCA. Thus, our studies show that these tumor-specific human MCA may have the specificity and stability necessary for in vivo evaluation of their use in the detection and treatment of cancer.