Search

AimTo explore whether there are racial/ethnic differences in diabetes management and outcomes among adult health center (HC) patients with type 2 diabetes.MethodsWe analyzed data from the 2014 Health Center Patient Survey, a national sample of HC patients. We examined indicators of diabetes monitoring (A1C testing, annual foot/eye doctor visits, and cholesterol checks) and care management (specialist referrals, individual treatment plan, and receipt of calls/appointments/home visits). We also examined diabetes-specific outcomes (blood glucose levels, diabetes-related emergency department [ED] visits/hospitalizations, and diabetes self-management confidence) and general outcomes (number of doctor visits, ED visits, and hospitalizations). We used multilevel logistic regression models to examine racial/ethnic disparities by the above indicators.ResultsWe found racial/ethnic parity in A1C testing, eye doctor visits, and diabetes-specific outcomes. However, Hispanics/Latinos (odds ratio [OR] 0.26), non-Hispanic African Americans (OR 0.25), and Asians (OR 0.11) were less likely to receive a cholesterol check than Whites. Non-Hispanic African Americans (OR 0.43) were less likely to have frequent doctor visits, while Hispanic/Latino patients (OR 0.45) were less likely to receive an individual treatment plan.ConclusionHCs largely provide equitable diabetes care but have room for improvement in some indicators. Tailored efforts such as culturally competent care and health education for some racial/ethnic groups may be needed to improve diabetes management and outcomes.

PURPOSE Nearly one-fifth of Americans live in rural areas and experience multiple socioeconomic and health disparities. Health Resources and Services Administration (HRSA)-funded health centers (HCs) provide comprehensive primary care in rural communities. However, no prior research has examined trends in access to care in rural HC patients. We examined the change in access to care among patients served at rural HRSA-funded HCs in the United States between 2009 and 2014. METHODS We compared patients by year to examine measures of access using multilevel generalized structural equation logistic regression models with random effects. We used the 2009 and 2014 cross-sectional Health Center Patient Surveys and identified 2,625 adult rural HC patients. Dependent variables were subjective (unmet need/delay in medical care, mental health, dental care, and prescription medications) and objective measures (preventive care and other health care utilization) in access to care. Our independent variable of interest was time, comparing access in 2009 and 2014. RESULTS Rural HC patients reported higher predicted probability of influenza vaccine receipt (37% vs 51%), and lower unmet (25% vs 14%) and delayed medical care (36% vs 18%) between 2009 and 2014. Any emergency department visits in the last year increased (32% vs 46%) and mammogram (70% vs 55%) and Pap test (83% vs 72%) screening rates decreased. CONCLUSIONS Observed increases in access to care among rural HC patients are positive developments but the challenges to access care still persist. Remote services, such as telehealth, could be cost-effective means of improving access to care among rural patients with limited provider supply.

Existing literature indicates that multimorbidity, mental health (MH) conditions, substance use disorders (SUDs), and social determinants of health are hallmarks of high-need, high-cost patients. Health Resources and Services Administration-funded health centers (HCs) provide care to nearly 30 million patients, but data on their patients' complexity and utilization patterns are limited. We identified subgroups of HC patients based on latent concepts of complexity and utilization.We used cross-sectional national data from the 2014 Health Center Patient Survey and latent class analyses to identify distinct and homogenous groups of complex high-utilizing patients aged 18 to 64 years.We included indicators of chronic conditions (CCs), MH, SUD risk, and health behavior to measure complexity. We used number of outpatient and emergency department visits in the past year to measure utilization.HC patients were separated in 9 distinct groups based on 3 complexity latent classes (MH, multiple CCs, and low risk) and 3 utilization classes (low, high, and superutilizers). Conditions associated with each subgroup differed. The highest prevalence of bipolar disorder (45%) and high SUD risk (6%) was observed among MH superutilizers, whereas the highest prevalence of cardiovascular disease (48%) and obesity (96%) was seen among CC superutilizers. Most MH superutilizer patients concurrently had MH conditions and obesity and were smokers, but most CC superutilizer patients concurrently had hypertension, obesity, and cardiovascular disease.Our examination of complexity and utilization indicated distinct HC patient populations. Managing the care of each group may require different targeted intervention approaches such as multidisciplinary care teams that include MH providers or specialists.

Policy makers are increasingly investing in programs focused on identifying and addressing the nonmedical needs of high-utilizing Medicaid beneficiaries, yet little is known about these programs’ i...

Health centers (HCs) play a crucial and integral role in addressing social determinants of health (SDOH) among vulnerable and underserved populations, yet data on SDOH assessment and subsequent actions is limited. We conducted a systematic review to understand the existing evidence of integration of SDOH into HC primary-care practices. Database searches yielded 3,516 studies, of which 41 articles met the inclusion criteria. A majority of studies showed that HCs primarily captured patient-level rather than community-level SDOH data. Studies also showed that HCs utilized SDOH in electronic health records but capabilities varied widely. A few studies indicated that HCs measured health-related outcomes of integrating SDOH data. The review highlighted that many knowledge gaps exist in the collection, use, and assessment of impact of these data on outcomes, and future research is needed to address this knowledge gap.

Frameworks for identifying and assessing social determinants of health (SDOH) are effective for developing long-term societal policies to promote health and well-being, but may be less applicable in clinical settings. The authors compared the relative contribution of a specific set of SDOH indicators with several measures of health status among patients served by health centers (HCs). The 2014 Health Center Patient Survey was used to identify a sample of HC patient adults 18 years and older that reported the HC as their usual source of care (

ObjectiveThe study objective was to examine the association between mental health staffing at health centers funded by the Health Resources and Services Administration (HRSA) and patients' receipt of mental health treatment.MethodsData were from the 2014 HRSA-funded Health Center Patient Survey and the 2013 Uniform Data System. Colocation of any mental health staff, including psychiatrists, psychologists, and other licensed staff, was examined. The outcomes of interest were whether a patient received any mental treatment and received any such treatment on site (at the health center). Analyses were conducted with multilevel generalized structural equation logistic regression models for 4,575 patients ages 18-64.ResultsPatients attending health centers with at least one mental health full-time equivalent (FTE) per 2,000 patients had a higher predicted probability of receiving mental health treatment (32%) compared with those attending centers with fewer than one such FTE (24%) or no such staffing (22%). Among patients who received this treatment, those at health centers with no staffing had a significantly lower predicted probability of receiving such treatment on site (28%), compared with patients at health centers with fewer than one such FTE (49%) and with at least one such FTE (65%). The predicted probability of receiving such treatment on site was significantly higher if there was a colocated psychiatrist versus no psychiatrist (58% versus 40%).ConclusionsColocating mental health staff at health centers increases the probability of patients' access to such treatment on site as well as from off-site providers.

Author(s): Pourat, Nadereh; Chen, Xiao; Lu, Connie; Zhou, Weihao; Hoang, Hank; Hair, Brionna; Bolton, Joshua; Sripipatana, Alek | Abstract: ObjectivesHealth Resources and Services Administration-funded health centres (HCs) are an important source of dental services for low-income and vulnerable patients in the United States. About 82% of HCs in 2018 had dental workforce, but it is unclear whether this workforce meets the oral health needs of HC patients. Thus, we first examined (a) whether dental workforce was associated with any dental visits vs none and (b) whether HC patients with any visits were more likely to have a visit at the HC vs elsewhere. We then examined (c) if need for oral health care and long-term continuity at the HC were associated with dental visits and visits at the HC.MethodsThis study used the 2014 Health Center Patient Survey, a nationally representative study of US HC patients, and the 2013 Uniform Data System, an administrative dataset of HC characteristics. We also used the 2013 Area Health Resource File to measure the contribution of local supply of dentists. We included working-age adult patients (nn=n5006) and used multilevel structural equation models with Poisson specification.ResultsLarger dental workforce at the HC was significantly associated with 1% higher likelihood (relative risk [RR]: 1.01, 1.00-1.02) of any visits and 10% higher likelihood of a visit at the HC among those with a visit (RR: 1.10, 1.06-1.14). Patient self-reported oral health need was positively associated with 157% higher likelihood of dental visits (RR: 2.57, 2.29-2.88), and 42% higher likelihood of dental visit at the HC vs elsewhere (RR: 1.42, 1.19-1.69). Long-term continuity with the HC was not significantly associated with likelihood of dental visits, but was associated with 26% higher likelihood of visits at the HC among those who had any visits (RR: 1.26, 1.02-1.56).DiscussionThe findings highlight the potential impact of increasing dental workforce at HCs to promote access; the high level of need for oral health care at HCs; and the increased effort required to promote access among newer patients who may be less familiar with the availability of oral health care at HCs. Together, these findings reinforce the importance of addressing barriers of use of oral health services among low-income and uninsured patients.

This study sought to examine racial/ethnic variations in receipt of provider recommendations on weight loss, patient adherence, perception of weight, attempts at weight loss and actual weight loss among patients with overweight/obesity status at Health Resources and Services Administration-funded health centres (HC). We used a 2014 nationally representative survey of adult HC patients with overweight/obesity status (PwOW/OB) last year and reported the HC was their usual source of care (n = 3517). We used logistic regression models to assess the interaction of race/ethnicity and having obesity in (1) provider recommendations of diet or (2) exercise, (3) patient adherence to diet or (4) exercise, (5) perceptions of weight and (6) weight loss attempts. We used a multinomial regression model to examine (7) weight loss or gain vs no change and a linear regression model to evaluate (8) percent weight change. We found Black PwOW/OB (OR = 1.65) experienced greater odds of receiving diet recommendations than Whites. We found limited racial/ethnic disparities in adherence. Black (OR = 0.41), Hispanic/Latino (OR = 0.45), and American Indian/Alaska Native (OR = 0.41) PwOW/OB had lower odds of perceiving themselves as overweight. Black (OR = 1.68) and Hispanic (OR = 1.98) PwOW/OB had a greater odds of reporting weight gain, and Asian PwOW/OB (OR = 0.42) had lower odds of reporting weight loss than Whites. Disparities in provider diet recommendations among Blacks and Hispanics indicated the importance of personalized weight management recommendations. Understanding underlying reasons for discordance between self-perception and observed weight among different groups is needed. Overall increase in weight, despite current interventions, should be addressed through targeted racially/ethnically appropriate approaches.

Policy makers are increasingly investing in programs focused on identifying and addressing the nonmedical needs of high-utilizing Medicaid beneficiaries, yet little is known about these programs' implementation. This study provides an overview of early progress in and strategies used to implement California's Whole Person Care (WPC) Pilot Program, a $3 billion Medicaid Section 1115(a) waiver demonstration project focused on improving the integrated delivery of health, behavioral health, and social services for Medicaid beneficiaries who use acute and costly services in multiple service sectors. WPC pilots reported significant progress in developing partnerships, data-sharing infrastructure, and services needed to coordinate care for identified patient populations. We also identified major barriers to WPC implementation, such as difficulty identifying and engaging eligible beneficiaries and the lack of affordable housing. Our findings offer insights to leaders and policy makers interested in testing new approaches for improving the health and well-being of medically and socially complex patients.

Background The opioid epidemic and subsequent mortality is a national concern in the U.S. The burden of this problem is disproportionately high among low-income and uninsured populations who are more likely to experience unmet need for substance use services. We assessed the impact of two Health Resources and Services Administration (HRSA) substance use disorder (SUD) service capacity grants on SUD staffing and service use in HRSA -funded health centers (HCs). Methods and findings We conducted cross-sectional analyses of the Uniform Data System (UDS) from 2010 to 2017 to assess HC (n = 1,341) trends in capacity measured by supply of SUD and medication-assisted treatment (MAT) providers, utilization of SUD and MAT services, and panel size and visit ratio measured by the number of patients seen and visits delivered by SUD and MAT providers. We merged mortality and national survey data to incorporate SUD mortality and SUD treatment services availability, respectively. From 2010 to 2015, 20% of HC organizations had any SUD staff, had an average of one full-time equivalent SUD employee, and did not report an increase in SUD patients or SUD services. SUD capacity grew significantly in 2016 (43%) and 2017 (22%). MAT capacity growth was measured only in 2016 and 2017 and grew by 29% between those years. Receipt of both supplementary grants increased the probability of any SUD capacity by 35% (95% CI: 26%, 44%) and service use, but decreased the probability of SUD visit ratio by 680 visits (95% CI: -1,013, -347), compared to not receiving grants. Conclusions The significant growth in HC specialized SUD capacity is likely due to supplemental SUD-specific HRSA grants and may vary by structure of grants. Expanding SUD capacity in HCs is an important step in increasing SUD access for low income and uninsured populations broadly and for patients of these organizations.

BackgroundIn the United States, there are nearly 1,400 Health Resources and Services Administration-funded health centers (HCs) serving low-income and underserved populations and more than 600 of these HCs are located in rural areas. Disparities in quality of medical care in urban vs. rural areas exist but data on such differences between urban and rural HCs is limited in the literature. We examined whether urban and rural HCs differed in their performance on clinical quality measures before and after controlling for patient, organizational, and contextual characteristics.Methods and findingsWe used the 2017 Uniform Data System to examine performance on clinical quality measures between urban and rural HCs (n = 1,373). We used generalized linear regression models with the logit link function and binomial distribution, controlling for confounding factors. After adjusting for potential confounders, we found on par performance between urban and rural HCs in all but one clinical quality measure. Rural HCs had lower rates of linking patients newly diagnosed with HIV to care (74% [95% CI: 69%, 80%] vs. 83% [95% CI: 80%, 86%]). We identified control variables that systematically accounted for eliminating urban vs. rural differences in performance on clinical quality measures. We also found that both urban and rural HCs had some clinical quality performance measures that were lower than available national benchmarks. Main limitations included potential discrepancy of urban or rural designation across all HC sites within a HC organization.ConclusionsFindings highlight HCs’ contributions in addressing rural disparities in quality of care and identify opportunities for improvement. Performance in both rural and urban HCs may be improved by supporting programs that increase the availability of providers, training, and provision of technical resources.

Author(s): Nguyen, Vy; Daniel, Marlon; Joskow, Renee; Lu, Connie; Chen, Xiao; Zhou, Weihao; Lin, Sue; Sripipatana, Alek; Nair, Suma; Pourat, Nadereh | Abstract: ObjectivesThis study aims to assess the impact of Health Resources and Services Administration (HRSA) investment in oral health through the HRSA FY16 Oral Health Service Expansion (OHSE) funding on workforce, access, and quality in health centers (HCs) from 2015 to 2017.MethodsAnalyses were conducted using data from the Uniform Data System from 2015 and 2017, and the 2015 Area Health Resource File. Change in indicators of oral health workforce, access, and quality of care by the receipt of OHSE funding received by HCs in 2016 were examined. Regression models for 1,345 HCs were developed to conduct a difference-in-difference analyses of the comparative change from 2015 to 2017 in the dependent variables among OHSE and non-OHSE awardees while controlling for confounders.ResultsOHSE awardees showed a significant difference in the oral health workforce with a higher mean number increase by 0.6 full time equivalent (FTE) dentists, 0.4 dental hygienists, 1.1 dental assistants, and 2.3 for other dental staff compared to non-OHSE awardees. Compared to non-OHSE awardees, OHSE awardees showed a mean increase of 712 dental patients served who received 1,402 dental visits, representing a 9-percentage point increase in the percentage of HCs that had an oral health program and a 3-percentage point increase in the ratio of dental patients to total patients.ConclusionsFunding dedicated to oral health service expansion in HCs may result in outcomes ranging from increasing workforce to reduction in access and financial barriers. Retrospective analysis demonstrated improved capacity for oral healthcare delivery attributable to HRSA support to HCs.

Enabling services address a combination of social determinants of health and barriers to access to primary care and are intended to reduce health disparities. They include care coordination; health education; transportation; and assistance with obtaining food, shelter, and benefits. Empirical evidence of enabling services' potential contribution to health outcomes is limited, which impedes their widespread dissemination. We examined how the receipt of enabling services influenced patient health care outcomes based on a nationally representative survey of patients served in 2014 at health centers funded by the Health Resources and Services Administration. We compared enabling services users and nonusers and found that enabling services were associated with 1.92 more health center visits, an 11.78-percentage-point higher probability of getting a routine checkup, a 16.34-percentage-point higher likelihood of having had a flu shot, and a 7.63-percentage-point higher probability of patient satisfaction. Our results confirm the value of systematic delivery of enabling services in reducing access barriers and improving patient satisfaction.

Some racial/ethnic minorities are more likely to have hypertension and experience increased hypertension-related morbidity and mortality compared to whites. Health Resources and Services Administration-funded health centers care for over 27 million patients, 62 percent of whom are racial/ethnic minorities. We assessed the presence of racial/ethnic disparities in (a) hypertension management and (b) hypertension outcomes among health center patients. We used data from the 2014 Health Center Patient Survey and performed multilevel logistic regression models to predict hypertension management counseling, patient adherence to counseling and medication regimen, management plan receipt, high blood pressure at last clinical visit, confidence in hypertension self-management, and hypertension-related emergency department (ED) episodes or hospitalizations in the past year. We controlled for patient characteristics including age, sex, education, nativity, health behaviors, health care access, and comorbidities. We found significantly higher odds of diet counseling (African Americans, OR: 1.87; Asian Americans, OR: 3.02, AIAN, OR: 2.01), reduced sodium intake (African American, OR: 2.42), and adherence to exercise counseling (African American, OR: 3.52; Asian Americans, OR: 2.93). We also found lower odds of taking hypertension control medication (AIAN, OR: 0.50) and higher odds of hypertension-related ED visits (African Americans, OR: 3.61, AIAN, OR: 5.31). These results highlight the success of health centers in managing hypertension by race/ethnicity but found adverse hypertension outcomes for some groups. Racial/ethnically tailored efforts might be required to manage hypertension and improve outcomes.

Aims This paper examines the association of multimorbidity, mental health (MH) conditions, and obesity severity among health center (HC) patients in the United States. Methods We used cross-sectional data from the 2014 Health Center Patient Survey (n = 5370). We examined the independent association of multimorbidity and MH conditions with obesity severity using a multinomial logistic regression model, controlling for potential confounders. Results More than half (50.4%) of HC patients had obesity, including 15% with severe obesity. In addition, 41% had multimorbidity and 49% had one of five diagnosed MH conditions. We found higher odds of having mild (OR = 4.06), moderate (OR = 3.39), and severe (OR = 4.93) obesity with the presence of both multimorbidity and MH conditions. Multimorbidity alone was also associated with higher odds of obesity, but MH conditions alone were only associated with having moderate obesity. Conclusions Findings show a higher burden of comorbidity and MH conditions given the severity of obesity among HC patients. This complex patient profile highlights the need for innovative approaches in effective care delivery and management of HC patients who are primarily low-income or uninsured. Approaches may include further integration of behavioral health and social services with primary care and the need for targeted practice guidelines for providers managing such patients.

Purpose: The Veterans Health Administration (VHA) has provided transgender transition-related care to veterans since 2011. However, little is known about the experiences of transgender veteran patients accessing transgender transition-related healthcare at Veterans Affairs (VA) clinics since the establishment of this care. The purpose of this study was to explore transgender veterans' experiences accessing and utilizing transition-related healthcare through the VA healthcare system. Methods: Eleven transgender veterans were recruited using in-person recruitment at the 2013 Southern Comfort Conference (Atlanta, GA). In-depth semistructured interviews were conducted with participants using a qualitative inquiry methodological perspective and experience-centered approach. Interviews were digitally recorded and transcribed verbatim. The transcripts were coded by two independent researchers using ATLAS.ti.© Results: Five inter-related themes were identified as key factors impacting the accessibility and quality of care transgender veterans receive through the VA: (1) long delays in receiving care; (2) needing to travel to receive care; (3) lack of patient knowledge regarding the coverage of transition-related care; (4) insensitivity, harassment, and violence among providers; and (5) a general lack of knowledge about transgender patients and care among providers. Conclusion: To our knowledge, this study is one of the first to explore the experiences of transgender veterans in accessing and utilizing transgender transition-related care at the VA after the 2011 VHA directive. Our findings suggest that although transgender healthcare coverage is available, additional patient-centered and healthcare system-level interventions are needed to improve the uptake and implementation of the VHA transgender protections and care coverage.

Shared use agreements (SUA) could increase opportunities for physical activity (PA) in under-resourced, urban areas. Despite recent investments in SUAs, the extent to which they reach communities in need and the level of community awareness and use of SUAs remains unclear. This cross-sectional study examined: 1) the distribution of SUAs in Los Angeles (LA) during the 2015–2016 academic year, 2) the characteristics of communities where SUAs were located, and 3) the extent to which community members were aware of and using available facilities. Assessment methods included: 1) abstraction of school administrative data reflecting the geographic distribution and scope of SUAs in LA, 2) collation of community-level Census and local planning data to describe demographic characteristics and per capita park acreage of communities where SUAs were located, and 3) collection of data via an Internet panel survey of LA adults (n = 371) examining awareness and use of SUA facilities. Under 3% of schools had a SUA in place during the study period. Compared to other areas of the city, areas within one mile of SUAs had more Hispanic/Latino, low-income, and lower educational status residents. Among survey respondents, 25.6% of those living within one mile of a SUA reported having access to school facilities; 48.6% of those reporting access reported using them. Although potentially targeted in high-needs areas, community members may not be aware of or utilizing SUA facilities. Additional efforts are needed to both expand access to school-based PA resources and attract community users.

The type II secretion system (T2SS) is present in many Gram-negative bacteria and is responsible for secreting a large number of folded proteins, including major virulence factors, across the outer membrane. The T2SS consists of 11-15 different proteins most of which are present in multiple copies in the assembled secretion machinery. The ATPase GspE, essential for the functioning of the T2SS, contains three domains (N1E, N2E and CTE) of which the N1E domain is associated with the cytoplasmic domain of the inner membrane protein GspL. Here we describe and analyze the structure of the GspE•cyto-GspL complex from Vibrio vulnificus in the presence of an ATP analog, AMPPNP. There are three such ∼83 kDa complexes per asymmetric unit with essentially the same structure. The N2E and CTE domains of a single V. vulnificus GspE subunit adopt a mutual orientation that has not been seen before in any of the previous GspE structures, neither in structures of related ATPases from other secretion systems. This underlines the tremendous conformational flexibility of the T2SS secretion ATPase. Cyto-GspL interacts not only with the N1E domain, but also with the CTE domain and is even in contact with AMPPNP. Moreover, the cyto-GspL domains engage in two types of mutual interactions, resulting in two essentially identical, but crystallographically independent, "cyto-GspL rods" that run throughout the crystal. Very similar rods are present in previous crystals of cyto-GspL and of the N1E•cyto-GspL complex. This arrangement, now seen four times in three entirely different crystal forms, involves contacts between highly conserved residues suggesting a role in the biogenesis or the secretion mechanism or both of the T2SS.

Summary The type II secretion system (T2SS), a multiprotein machinery spanning two membranes in Gram-negative bacteria, is responsible for the secretion of folded proteins from the periplasm across the outer membrane. The critical multidomain T2SS assembly ATPase GspE EpsE had not been structurally characterized as a hexamer. Here, four hexamers of Vibrio cholerae GspE EpsE are obtained when fused to Hcp1 as an assistant hexamer, as shown with native mass spectrometry. The enzymatic activity of the GspE EpsE -Hcp1 fusions is ∼20 times higher than that of a GspE EpsE monomer, indicating that increasing the local concentration of GspE EpsE by the fusion strategy was successful. Crystal structures of GspE EpsE -Hcp1 fusions with different linker lengths reveal regular and elongated hexamers of GspE EpsE with major differences in domain orientation within subunits, and in subunit assembly. SAXS studies on GspE EpsE -Hcp1 fusions suggest that even further variability in GspE EpsE hexamer architecture is likely.

Precise 3'-end processing of mRNA is essential for correct gene expression, yet in yeast, 3'-processing signals consist of multiple ambiguous sequence elements. Two neighboring elements upstream of the cleavage site are particularly important for the accuracy (positioning element) and efficiency (efficiency element) of 3'-processing and are recognized by the RNA-binding proteins Rna15 and Hrp1, respectively. In vivo, these interactions are strengthened by the scaffolding protein Rna14 that stabilizes their association. The NMR structure of the 34 -kDa ternary complex of the RNA recognition motif (RRM) domains of Hrp1 and Rna15 bound to this pair of RNA elements was determined by residual dipolar coupling and paramagnetic relaxation experiments. It reveals how each of the proteins binds to RNA and introduces a novel class of protein-protein contact in regions of previously unknown function. These interdomain contacts had previously been overlooked in other multi-RRM structures, although a careful analysis suggests that they may be frequently present. Mutations in the regions of these contacts disrupt 3'-end processing, suggesting that they may structurally organize the ribonucleoprotein complexes responsible for RNA processing.

Error-prone PCR was used to isolate Photinus pyralis luciferase mutants producing bright light in the red-orange region of the spectrum. All mutations were clustered in the beta5-alpha10-beta6 region of N-terminal subdomain B and appear to affect bioluminescence color by modulating the position of the Ser314-Leu319 mobile loop with respect to the putative active site. Two red variants (Q283R and S284G) and one orange mutant (S293P) contained a single substitution. Although the remaining orange variant contained two mutations, L287I mainly contributed to the color change. Emission spectra collected on whole cells at pH 7.0 revealed that while a single peak of lambdamax approximately 605 nm accounts for red light production by the Q283R and S284G variants, orange light results from the contribution of two peaks of lambdamax approximately 560 and 600 nm. All spectra underwent a red-shift when cells were assayed under acidic conditions, whereas a blue-shift was observed at pH 8.0, indicating that the internal pH of Escherichia coli is close to the external pH shortly after imposition of acid or alkaline stress. In addition, changes in assay pH led to bimodal emission spectra, lending support to the idea that bioluminescence color is determined by the relative contribution of yellow-green and red-orange peaks. The set of multicolored luciferase mutants described here may prove useful for a variety of applications including biosensing, pH monitoring, and tissue and animal imaging.

Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased tendency to aggregate. Thus, mutations in TM4 cause a range of phenotypes of dysfunctional gap junction channels that are discussed within the context of the X-ray crystallographic structure.

Size exclusion chromatography (SEC) is the most commonly used method to separate and quantify monoclonal antibody (mAb) size variants. MAb-A is an IgG1 subtype humanized monoclonal antibody recombinantly produced in Chinese hamster ovary (CHO) cells. SEC analysis of MAb-A resolved a peak, named Peak 1, which elutes between monomer and dimer peaks. MAb-A lots produced from different clones and production scales all have 0.2-0.3% of SEC Peak 1. Electron spray ionization--time of flight mass spectrometry (ESI-TOF MS), microfluidics capillary electrophoresis and sodium dodecyl sulfate-PAGE (SDS PAGE) results demonstrated that SEC Peak 1 contains two structural variants: MAb-A with one extra light chain (2H3L) and MAb-A with two extra light chains (2H4L). The C-terminal Cys of the extra light chain in Peak 1 variants is either a free thiol, capped by glutathione, cysteine, or another light chain. Both electrophoresis and LC/MS analyses of non-reduced and reduced samples suggested that the extra light chains are linked to the MAb-A light chain through disulfide bonds. Isolated SEC Peak 1 fraction had a potency of 50% relative to MAb-A reference material. The 50% potency loss may result from the reduced accessibility to the antigen-binding site caused by the extra light chain(s)' steric hindrance.

NS0 cells require exogenous cholesterol for growth. The non-glutamine synthetase (GS) cholesterol-dependent NS0 host was treated with 5-azacytidine (5azaC), a demethylation drug, and adapted to grow in cholesterol-free, chemically defined medium. Within 7 weeks, a stable, cholesterol-independent NS0 host (NS0.CF) was obtained. The new NS0.CF host, as well as the original cholesterol auxotroph host, was transfected with the same mAb expression plasmid, and the top producing clone from both hosts were compared side-by-side in the enhanced platform fed-batch cultures using chemically defined media. The NS0.CF derived clone significantly out-performed the cholesterol-dependent clone, with titer reaching 4.5 g/L versus 3.0 g/L, respectively, mainly due to higher specific productivity, while key product quality attributes remained comparable. This work demonstrated an effective and rapid approach to generate a cholesterol-independent NS0 host, and its application in recombinant protein production.

The analysis of human DNA polymorphisms is a valuable tool in genetic research and diagnostics and has become routine in clinical and forensic laboratories. It has aided in diagnosing diseases, determining paternity, identifying human remains, and attributing biological specimens to a source. However, DNA analysis for identity testing is only useful if there is a reference sample to compare with the sample in question. In cases where no obvious reference is available, it is necessary to find an alternative source. Difficulty can arise when the authenticity of the reference sample is questionable, or when the sample is taken from tissues with possible genetic variability, such as a malignant tumor (1-7). Since a known characteristic of malignant tumors are permanent alterations in the DNA molecule, DNA from these cells can display numerous anomalies including microsatellite instability (MSI), characterized by the presence of an additional allele or a size shift in an existing allele, deletions, or loss of heterozygosity (LOH) (8-13). Short tandem repeats (STRs; microsatellite DNA) commonly examined in forensic identification are not thought to be linked to visible traits or pathologies and are considered relatively stable and well distributed in the population. To that effect, some profiles obtained from nuclear and mitochondrial DNA of malignant tissues have been proven to be consistent with their normal counterparts. However, given the potential for pathological variability through genetic instability, it is necessary to evaluate its effect on the validity of the nuclear DNA analysis for identity determination and if steps can be taken to abrogate its impact. Clinical tissue samples are not usually employed in forensic casework, but can play a role in the identification of unknown bodies and paternity testing, as well as aid in resolving issues stemming from pathology mix-ups and contamination (1,2). Archival pathology specimens are carefully cataloged and widely available, given their use in diagnostics and are a potentially large source for forensic STR testing. STRs are relatively stable, but degradation may have an effect, especially when analyzing tissues which have been treated with highly aggressive chemicals (eg, formaldehyde, xylene, etc.) during typical histological processing. This research, conducted over a period of several years, analyzed and reviewed characteristics of DNA profiles obtained by means of 2 different commercially available kits, which are widely used in forensic identification. These kits both contain the widely accepted “core loci,” recommended by the Federal Bureau of Investigation for use in forensic DNA databases (http://www.promega.com/geneticidproc/eusymp2proc/17.pdf). The aim of the study was to determine STR profile differences between healthy and pathologically altered tissues from the same source using 2 widely used forensic multiplex polymerase chain reaction (PCR) tests. These genetic differences arise due to disease and can complicate the use of malignant tissues as reference samples in forensic biology. We also sought to determine if variations occur in the indicators of genetic instability (LOH and MSI) among different tumor types and to assess possible variations in genetic instability in the same tumor type. This information allows us to recommend interpretation guidelines for the use of archived potentially diseased, and thus genetically unstable, material for human identification.

Transport of folded proteins across membranes is a feat accomplished by few biomacromolecular machines. One of the machineries able to do so is the sophisticated type II secretion system (T2SS). It can translocate key virulence factors from the bacterial periplasm into the lumen of the gut of the human host. A prime example is the secretion of cholera toxin by Vibrio cholerae. The T2SS consists of ~12 different proteins, most of these present in multiple copies, organized into three subassemblies: (i) the Inner Membrane Platform; (ii) the Pseudopilus in the periplasm, which acts most likely as a piston pushing exoproteins through the outer membrane pore; (iii) the Outer Membrane Complex, allowing passage of ~100 kDa folded proteins. We have determined crystal structures from more than a dozen T2SS domains, yet, a full understanding of the architecture and mechanism of action of the T2SS remains a formidable challenge. Our approaches include the use of "assistant-multimers" to promote recalcitrant multimer formation and of nanobodies to overcome reluctant crystal formation. The Inner Membrane Platform is interacting with the secretion ATPase GspE which most likely needs to be hexameric for full activity. Full-length GspE co-crystallized with its major partner, the cytoplasmic domain of GspL, revealed a tremendous flexibility of this ATPase, and, most unexpectedly, also the organization of the same linear arrangement of cyto-GspL domains throughout three entirely different crystal forms. Two very different hexamers of GspE were elucidated by linking the GspE subunit to the subunit of Hcp1, which successfully acted as an "assistant hexamer", inducing hexamer formation by GspE. The dodecameric nature of the ~ 850 kDa GspD, the major component of the Outer Membrane Complex, evident in earlier electron microscopy studies, was observed in the dodecameric ring-like helix in crystals of its N-terminal domain. The contacts between GspD and the inner-membrane protein GspC will be discussed as well as the remarkably frequent occurrence of dimers of Inner Membrane Platform domains. How dimers are co-assembled with an ATPase hexamer with C6 symmetry and the Outer Membrane Complex dodecamer with C12 symmetry remains one of the many fascinating outstanding questions of the T2SS.

Background In 2005, we reported on the success of Comprehensive School Health (CSH) in improving diets, activity levels, and body weights. The successful program was recognized as a "best practice" and has inspired the development of the Alberta Project Promoting active Living and healthy Eating (APPLE) Schools. The project includes 10 schools, most of which are located in socioeconomically disadvantaged areas. The present study examines the effectiveness of a CSH program adopted from a "best practice" example in another setting by evaluating temporal changes in diets, activity levels and body weight. Methods In 2008 and 2010, we surveyed grade 5 students from approximately 150 randomly selected schools from the Canadian province of Alberta and students from 10 APPLE Schools. Students completed the Harvard Youth/Adolescent Food Frequency Questionnaire, questions on physical activity, and had their height and weight measured. Multilevel regression methods were used to analyze changes in diets, activity levels, and body weight between 2008 and 2010. Results In 2010 relative to 2008, students attending APPLE Schools were eating more fruits and vegetables, consuming fewer calories, were more physically active and were less likely obese. These changes contrasted changes observed among students elsewhere in the province. Conclusions These findings provide evidence on the effectiveness of CSH in improving health behaviors. They show that an example of "best practice" may lead to success in another setting. Herewith the study provides the evidence that investments for broader program implementation based on "best practice" are justified.

Connexins (Cx) assemble to form the intercellular communication channels in gap junctions (GJs). Mutations in Connexin26 (Cx26) that cause childhood pre-lingual deafness are found in human populations around the globe. Types of defect resulting from connexin mutations include loss of protein synthesis, mis-trafficking, hemichannel docking and defective channel function (for example, altered permeability or gating). We have characterized a new category of non-functional connexin mutations where substitutions in the transmembrane helix regions render the hexamer unstable (Ambrosi et al., (2010) Biophys. J. 98:1809).This work on deafness mutants focuses on the fourth transmembrane helix, M4. According to the X-ray crystallographic structure of Cx26 (Maeda et al., (2009) Nature 458:597) one face of this helix sits against the lipid bilayer. Our 6 A EM structure of the Cx26M34A mutant embedded in membrane bilayers (Oshima et al. (2011) J Mol Biol 405:724) revealed an outward expansion of the transmembrane domain relative to the X-ray crystallographic structure of the wild-type Cx26 channel in detergent. We became interested in how single site M4 amino substitutions that are distal to the channel pore, especially those that face the lipid membranes, can create non-functional channels. The 14 deafness mutants we studied in the M4 helix are not localized to a specific face of the helix and do not all cause the same type of GJ defect. Several deafness mutations in M4 cause mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P) but others (M195T, A197S, C202F, I203T, I205V, N206S) make GJs in mammalian cells. When expressed and purified from baculovirus infected Sf9 cells, C202F and N206S form hemichannels that are unstable upon detergent solubilization. Experiments are now in progress to test the functional properties of all six GJ forming mutants.