The direct role of selenocysteine in [NiFeSe] hydrogenase maturation and catalysis.

Hydrogenases are highly active enzymes for hydrogen production and oxidation. [NiFeSe] hydrogenases, in which selenocysteine is a ligand to the active site Ni, have high catalytic activity and a bias for H2 production. In contrast to [NiFe] hydrogenases, they display reduced H2 inhibition and are rapidly reactivated after contact with oxygen. Here we report an expression system for production of recombinant [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough and study of a selenocysteine-to-cysteine variant (Sec489Cys) in which, for the first time, a [NiFeSe] hydrogenase was converted to a [NiFe] type. This modification led to severely reduced Ni incorporation, revealing the direct involvement of this residue in the maturation process. The Ni-depleted protein could be partly reconstituted to generate an enzyme showing much lower activity and inactive states characteristic of [NiFe] hydrogenases. The Ni-Sec489Cys variant shows that selenium has a crucial role in protection against oxidative damage and the high catalytic activities of the [NiFeSe] hydrogenases.

The FlxABCD-HdrABC proteins correspond to a novel NADH dehydrogenase/heterodisulfide reductase widespread in anaerobic bacteria and involved in ethanol metabolism in Desulfovibrio vulgaris†Hildenborough.

Flavin-based electron bifurcation (FBEB) is an important mechanism for the energy metabolism of anaerobes. A new family of NADH dehydrogenases, the flavin oxidoreductase (FlxABCD, previously called FloxABCD), was proposed to perform FBEB in sulphate-reducing organisms coupled with heterodisulfide reductase (HdrABC). We found that the hdrABC-flxABCD gene cluster is widespread among anaerobic bacteria, pointing to a general and important role in their bioenergetics. In this work, we studied FlxABCD of Desulfovibrio vulgaris Hildenborough. The hdr-flx genes are part of the same transcriptional unit and are increased in transcription during growth in ethanol-sulfate, and to a less extent during pyruvate fermentation. Two mutant strains were generated: one where expression of the hdr-flx genes was interrupted and another lacking the flxA gene. Both strains were unable to grow with ethanol-sulfate, whereas growth was restored in a flxA-complemented strain. The mutant strains also produced very reduced amounts of ethanol compared with the wild type during pyruvate fermentation. Our results show that in D. vulgaris, the FlxABCD-HdrABC proteins are essential for NADH oxidation during growth on ethanol, probably involving a FBEB mechanism that leads to reduction of ferredoxin and the small protein DsrC, while in fermentation they operate in reverse, reducing NAD(+) for ethanol production.

New model for electron flow for sulfate reduction in Desulfovibrio alaskensis G20.

To understand the energy conversion activities of the anaerobic sulfate-reducing bacteria, it is necessary to identify the components involved in electron flow. The importance of the abundant type I tetraheme cytochrome c3 (TpIc3) as an electron carrier during sulfate respiration was questioned by the previous isolation of a null mutation in the gene encoding TpIc3, cycA, in Desulfovibrio alaskensis G20. Whereas respiratory growth of the CycA mutant with lactate and sulfate was little affected, growth with pyruvate and sulfate was significantly impaired. We have explored the phenotype of the CycA mutant through physiological tests and transcriptomic and proteomic analyses. Data reported here show that electrons from pyruvate oxidation do not reach adenylyl sulfate reductase, the enzyme catalyzing the first redox reaction during sulfate reduction, in the absence of either CycA or the type I cytochrome c3:menaquinone oxidoreductase transmembrane complex, QrcABCD. In contrast to the wild type, the CycA and QrcA mutants did not grow with H2 or formate and sulfate as the electron acceptor. Transcriptomic and proteomic analyses of the CycA mutant showed that transcripts and enzymes for the pathway from pyruvate to succinate were strongly decreased in the CycA mutant regardless of the growth mode. Neither the CycA nor the QrcA mutant grew on fumarate alone, consistent with the omics results and a redox regulation of gene expression. We conclude that TpIc3 and the Qrc complex are D. alaskensis components essential for the transfer of electrons released in the periplasm to reach the cytoplasmic adenylyl sulfate reductase and present a model that may explain the CycA phenotype through confurcation of electrons.

Thirty Years of SNAP-Ed: The Transition of the Nation's Largest Nutrition Education Program Into a Pillar of the Public Health Infrastructure.

This paper describes the 30-year evolution of Supplemental Nutrition Assistance Program-Education (SNAP-Ed) to provide evidence to support our perspective that SNAP-Ed has earned its position as a pillar of the public health infrastructure in the US. Legislatively designated as a nutrition education and obesity prevention program, its focus is the nearly 90 million Americans with limited income. This audience experiences ongoing health disparities and is disproportionately affected by public health crises. The SNAP-Ed program works to reduce nutrition-related health disparities at all levels of the Social-Ecological Model, follows a robust evaluation framework, and leverages strong partnerships between state-based practitioners, state agencies, and the US Department of Agriculture. The expansion of SNAP-Ed would enable the program to reach more Americans so that our nation can end hunger and reduce diet-related health disparities.

First Analysis of Nationwide Trends in the Use of the SNAP-Ed Evaluation Framework.

OBJECTIVE: To understand Supplemental Nutrition Assistance Program-Education (SNAP-Ed) Implementing Agencies'(SIAs) use of the SNAP-Ed Evaluation Framework (Framework), which is a tool that includes 51 indicators that SNAP-Ed programs can use to measure the success of their programs in the first 5 years after its release. METHODS: A repeated cross-sectional study design was utilized to administer electronic surveys to between 124 and 154 SIAs who received SNAP-Ed funding in fiscal years 2017, 2019, and 2021. Analyses included descriptive statistics and tests of proportions. RESULTS: Most SIAs indicated that they used the Framework to inform both data collection instruments and program planning decisions and the rates remained relatively constant over the 3 time points (> 80%). The most common specific use of the Framework across all 3 time points was to define, count, or measure the work accomplished, but this statistically decreased from 2017 (76%) to 2021 (57%) (z-score = 3.31; P < 0.001). CONCLUSIONS AND IMPLICATIONS: The results of this analysis confirmed that 5 years after its introduction, uptake and use of the Framework was high and that, as a whole, SIAs focused on priority indicators set by the US Department of Agriculture, with no notable increases in addressing and measuring longer-term, multisector, and population-wide outcomes. The systematic study of the Framework's usability over time has a broader application to other national health promotion initiatives with shared frameworks.

Adults' Reaction to Public Health Messaging: Recall, Media Type, and Behavior Change Motivation.

This paper focuses on effective messaging practices identified in data collected after 10 years of implementing a gain-framed messaging campaign encouraging healthier behaviors in middle-aged and older adults. In Study 1, we measured message recall and intended health behaviors in an intercept survey of 733 adults. Binary logistic regression indicated that women were more likely than men to report intent to change behavior. Recalling messages from billboards or fliers was associated with a lower likelihood of intended behavior change, and media type was associated with intended behavior for those who saw the message online (reducing screen time) or on television (increasing physical activity and ceasing smoking). Study 2 focused on adult generational differences in response to the campaign and types of media used to access information. Data from an intercept survey of 604 clients at agencies serving low-income adults were segmented into three age groups: under 35, ages 35-54, and ages 55+. Recall and reaction to campaign materials differed by age group, and the influence of life stage factors and health costs varied across age groups. Television and newspapers were most frequently reported by the oldest group, and social media and online news/blogs were most frequently chosen by the youngest group. Campaign response of adults older than age 35 aligned with goals of improving health behaviors. Together, these studies indicate that diffuse messaging strategies may raise overall awareness, and targeted strategies may be more influential in motivating behavior change. Influential factors and media should be differentially leveraged to target different age cohorts of adults.

Efficacy of standard- vs reduced-dose insulin for treatment of hyperkalemia: A quasi-experiment.

PURPOSE: Hyperkalemia more commonly affects patients with a glomerular filtration rate of less than 60 mL/min. Using intravenous (IV) insulin to shift potassium intracellularly may cause hypoglycemia, requiring additional treatment or longer hospitalization. Literature on insulin dosing in this context is limited, with one previous study indicating that 5 units of IV insulin might be as effective and result in less hypoglycemia than the standard dose of 10 units of IV insulin. The hyperkalemia treatment pathway at our institution was revised in May 2018 to include a reduced-dose option (5 units of insulin) for patients with end-stage renal disease. This study aimed to compare the prevalence of hypoglycemia between patients who received standard-dose vs reduced-dose IV insulin. METHODS: This single-center, retrospective, quasi-experimental study evaluated the impact of revision of the hyperkalemia treatment pathway by assessing rates of hypoglycemia during the 6 months before and after implementation of the revised pathway. The primary endpoint was prevalence of hypoglycemia, defined as a blood glucose level of less than or equal to 70 mg/dL. RESULTS: There was no statistically significant difference in the occurrence of hypoglycemia when comparing the pre- and postimplementation groups (36 [17.7%] patients vs 34 [18.7%] patients; P = 0.7924). The postimplementation group had a statistically significant lower reduction in potassium levels after treatment than the preimplementation group (mean [interquartile range], -0.9 [-1.3, -0.5] mEq/L vs -0.6 [-1.2, -0.2] mEq/L; P = 0.0095). Baseline potassium levels were similar between the groups. CONCLUSION: Administration of reduced-dose IV insulin for treatment of hyperkalemia was significantly less effective in lowering serum potassium levels and did not decrease prevalence of hypoglycemia. When accounting for potential confounders, the only variable that was associated with hypoglycemia was pretreatment glucose level.

Student Attitudes Contribute to the Effectiveness of a Genomics CURE.

The Genomics Education Partnership (GEP) engages students in a course-based undergraduate research experience (CURE). To better understand the student attributes that support success in this CURE, we asked students about their attitudes using previously published scales that measure epistemic beliefs about work and science, interest in science, and grit. We found, in general, that the attitudes students bring with them into the classroom contribute to two outcome measures, namely, learning as assessed by a pre- and postquiz and perceived self-reported benefits. While the GEP CURE produces positive outcomes overall, the students with more positive attitudes toward science, particularly with respect to epistemic beliefs, showed greater gains. The findings indicate the importance of a student's epistemic beliefs to achieving positive learning outcomes.

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.

Phase I trial of vinflunine and pemetrexed in refractory solid tumors.

BACKGROUND: Vinflunine is a novel vinca alkaloid with promising single agent clinical activity. Pemetrexed has at least additive activity with other vincas. A phase I trial was undertaken to assess the safety of vinflunine and pemetrexed in patients with refractory solid tumors. METHODS: A standard 3-patient cohort dose escalation scheme was used to determine the dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) of the vinflunine/pemetrexed combination. Pemetrexed 500 mg/m(2) was given with vinflunine 280 mg/m(2) (cohort 1), 300 mg/m(2) (cohort 2) or 320 mg/m(2) (cohort 3) on day 1 of a 21-day cycle. RESULTS: 19 patients were enrolled, median age 58 years (range 32 to 77) and had a median of 3 (range 1-6) prior therapies. DLT occurred 1 of 6 pts in cohort 1 (thrombocytopenia, hyponatremia), 2 of 10 pts in cohort 2 (febrile neutropenia, hyponatremia, hyperbilirubinema; febrile neutropenia), and 2 of 3 pts in cohort 3 (febrile neutropenia, hypokalemia; febrile neutropenia). 1 pt in cohort 2 died prior to completion of cycle 1 likely from disease progression. Most common grade 3/4 adverse events were neutropenia (7), leukopenia (5). Febrile neutropenia occurred in 4 patients (21%). No objective responses were seen. Two patients (breast and lung) had prolonged stable disease for 25 and 20 cycles respectively. CONCLUSIONS: Based on this experience we recommend vinflunine 300 mg/m(2) and pemetrexed 500 mg/m(2) in combination every 3 weeks for future study. At these doses, the combination of vinflunine and pemetrexed was tolerable in this heavily pretreated population. Hematologic toxicity, including febrile neutropenia, was prominent.

A phase I evaluation of the combination of vinflunine and erlotinib in patients with refractory solid tumors.

PURPOSE: Epidermal growth factor receptor (EGFR) inhibition may overcome chemotherapy resistance by inhibiting important anti-apoptotic signals that are constitutively activated by an overstimulated EGFR pathway. METHODS: This phase I dose escalation trial assessed the safety and efficacy of vinflunine, a novel vinca alkaloid microtubule inhibitor, with erlotinib, an EGFR tyrosine kinase inhibitor, in patients with refractory solid tumors. RESULTS: Seventeen patients were treated, 10 with continuous erlotinib, and 7 with intermittent erlotinib. At dose level 1, vinflunine 280 mg/m(2) IV day 1 and erlotinib 75 mg PO days 2-21 ("continuous erlotinib") in 21 day cycles, two of four patients experienced DLTs. At dose level -1 (vinflunine 250 mg/m(2) every 21 days and erlotinib 75 mg/day), two of six patients experienced DLTs. The study was amended to enroll to "intermittent erlotinib" dosing: vinflunine day 1 and erlotinib days 2-15 of a 21 day cycle. Two of seven experienced DLTs and the study was terminated. One patient with breast cancer had a partial response; three had stable disease ≥ 6 cycles. All were treated in the continuous erlotinib group. CONCLUSIONS: Given the marked toxicity in our patient population, the combination of vinflunine and erlotinib cannot be recommended for further study with these dosing schemas.

The SNAP-Ed Evaluation Framework: Nationwide Uptake and Implications for Nutrition Education Practice, Policy, and Research.

OBJECTIVE: Provide the first baseline census of Supplemental Nutrition Assistance Program Education (SNAP-Ed) state implementing agencies' (SIAs) intent to use and evaluate 51 indicators described in the SNAP-Ed Evaluation Framework. METHODS: A cross-sectional study design was used to administer electronic surveys to 124 SIAs who received SNAP-Ed funding in 2017. Descriptive statistics were used to analyze the results. RESULTS: Of 51 indicators, SIAs reported their intent to impact an average of 19 indicators and evaluate an average of 12. More SIAs reported the intention to impact indicators at the individual (59%) and environmental levels (48%), compared with the sectors of influence (20%) and population levels (30%) of the framework. In addition, more SIAs intended to impact and evaluate short- or medium-term indicators, compared with long-term indicators. CONCLUSIONS AND IMPLICATIONS: These findings illustrate the progress made toward aggregating metrics to measure the collective impact of SNAP-Ed.

Deletion Mutants, Archived Transposon Library, and Tagged Protein Constructs of the Model Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough.

The dissimilatory sulfate-reducing deltaproteobacterium Desulfovibrio vulgaris Hildenborough (ATCC 29579) was chosen by the research collaboration ENIGMA to explore tools and protocols for bringing this anaerobe to model status. Here, we describe a collection of genetic constructs generated by ENIGMA that are available to the research community.

Facilitating Growth through Frustration: Using Genomics Research in a Course-Based Undergraduate Research Experience.

A hallmark of the research experience is encountering difficulty and working through those challenges to achieve success. This ability is essential to being a successful scientist, but replicating such challenges in a teaching setting can be difficult. The Genomics Education Partnership (GEP) is a consortium of faculty who engage their students in a genomics Course-Based Undergraduate Research Experience (CURE). Students participate in genome annotation, generating gene models using multiple lines of experimental evidence. Our observations suggested that the students' learning experience is continuous and recursive, frequently beginning with frustration but eventually leading to success as they come up with defendable gene models. In order to explore our "formative frustration" hypothesis, we gathered data from faculty via a survey, and from students via both a general survey and a set of student focus groups. Upon analyzing these data, we found that all three datasets mentioned frustration and struggle, as well as learning and better understanding of the scientific process. Bioinformatics projects are particularly well suited to the process of iteration and refinement because iterations can be performed quickly and are inexpensive in both time and money. Based on these findings, we suggest that a dynamic of "formative frustration" is an important aspect for a successful CURE.

A molybdopterin oxidoreductase is involved in H2 oxidation in Desulfovibrio desulfuricans G20.

Three mutants deficient in hydrogen/formate uptake were obtained through screening of a transposon mutant library containing 5,760 mutants of Desulfovibrio desulfuricans G20. Mutations were in the genes encoding the type I tetraheme cytochrome c(3) (cycA), Fe hydrogenase (hydB), and molybdopterin oxidoreductase (mopB). Mutations did not decrease the ability of cells to produce H(2) or formate during growth. Complementation of the cycA and mopB mutants with a plasmid carrying the intact cycA and/or mopB gene and the putative promoter from the parental strain allowed the recovery of H(2) uptake ability, showing that these specific genes are involved in H(2) oxidation. The mop operon encodes a periplasm-facing transmembrane protein complex which may shuttle electrons from periplasmic cytochrome c(3) to the menaquinone pool. Electrons can then be used for sulfate reduction in the cytoplasm.

Development of a markerless genetic exchange system for Desulfovibrio vulgaris Hildenborough and its use in generating a strain with increased transformation efficiency.

In recent years, the genetic manipulation of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough has seen enormous progress. In spite of this progress, the current marker exchange deletion method does not allow for easy selection of multiple sequential gene deletions in a single strain because of the limited number of selectable markers available in D. vulgaris. To broaden the repertoire of genetic tools for manipulation, an in-frame, markerless deletion system has been developed. The counterselectable marker that makes this deletion system possible is the pyrimidine salvage enzyme, uracil phosphoribosyltransferase, encoded by upp. In wild-type D. vulgaris, growth was shown to be inhibited by the toxic pyrimidine analog 5-fluorouracil (5-FU), whereas a mutant bearing a deletion of the upp gene was resistant to 5-FU. When a plasmid containing the wild-type upp gene expressed constitutively from the aph(3')-II promoter (promoter for the kanamycin resistance gene in Tn5) was introduced into the upp deletion strain, sensitivity to 5-FU was restored. This observation allowed us to develop a two-step integration and excision strategy for the deletion of genes of interest. Since this in-frame deletion strategy does not retain an antibiotic cassette, multiple deletions can be generated in a single strain without the accumulation of genes conferring antibiotic resistances. We used this strategy to generate a deletion strain lacking the endonuclease (hsdR, DVU1703) of a type I restriction-modification system that we designated JW7035. The transformation efficiency of the JW7035 strain was found to be 100 to 1,000 times greater than that of the wild-type strain when stable plasmids were introduced via electroporation.

Transgender health and HIV.

Transgender persons illuminate the complex interplay of social and biological factors that determine gender identity (an individual's own sense of maleness or femaleness) and contribute to both HIV risk and HIV health. This article highlights some of the unique challenges that transgender persons face in maintaining their health--including avoiding HIV infection or living well with HIV--and discusses ways in which health-care providers can better equip themselves to provide care that meets the needs of transgender clients.

The SNAP-Ed Evaluation Framework: demonstrating the impact of a national framework for obesity prevention in low-income populations.

The United States Department of Agriculture's Supplemental Nutrition Assistance Program Education, known as SNAP-Ed, is the country's largest and most diverse community nutrition program. In 2017, nearly 140 SNAP-Ed implementing agencies (SIAs) and hundreds of contractors delivered nutrition education to almost 5 million people in nearly 60,000 low-resource sites. Millions more were impacted with social marketing campaigns and policy, systems, and environmental changes. This article introduces and describes the benefits of the newly developed SNAP-Ed Evaluation Framework (Framework) and companion Interpretive Guide to consistently measure SNAP-Ed outcomes across different settings. The Framework uses the social ecological model as its underlying theory and features 51 indicators across four levels: Individual, Environmental Supports, Sectors of Influence, and Population Results. Topline findings from the first-year Census to track Framework adoption found that most SIAs intended to impact indicators closer to the inner levels of influence: Individual (mean = 59% of SIAs; SD = 22%) and Environmental Settings (mean = 48%; SD = 23%). As yet, few SIAs targeted outcomes for long-term indicators (mean = 26%; SD = 15%), Sectors of Influence (mean = 20%; SD = 12%), or Population Results (mean = 30%; SD = 11%). An in-depth example of how one state is using the Framework is described. The SNAP-Ed Evaluation Framework offers a new suite of evaluation measures toward eliminating disparities that contribute to poor diet, physical inactivity, food insecurity and obesity. Practitioners will need technical assistance to implement the Framework, especially to measure longer-term, multi-sector and population results, and to maximize effectiveness in SNAP-Ed.

TonB/TolA amino-terminal domain modeling.

TonB and TolA proteins are energy transducers that couple the ion electrochemical gradient of the cytoplasmic membrane to support energy-dependent processes in the outer membrane of gram-negative bacteria. Energization of these proteins involves specific interactions with multiprotein cytoplasmic membrane energy harvesting complexes. The specific mechanisms by which these energy transfers occur remain unclear, but the evidence to date indicates that the amino-terminally located signal anchors of TonB and TolA play essential roles in the process. Mutant hunts have identified one motif in this region, common to both TonB and TolA, as important for energization. Because TonB and TolA each have a "preferred" energy-harvesting complex, it is clear that additional motifs, not shared between TonB and TolA, are involved in interactions with energy harvesting complexes. We have adopted a strategy of examining derivatives with multiple-residue substitutions to identify such regions. This involves the characterization of specific TonB derivatives generated by two similar approaches: the block substitutions in TonB by alanyl residues and the exchange of short regions between TonB and TolA. The methods by which these derivatives are generated are described, with an illustrative example for each.

"Keep Me Doing What I Love": A Photovoice Evaluation of the Missouri AgrAbility Project.

The Missouri AgrAbility program links the Cooperative Extension Service at a land-grant university with a nonprofit disability organization to provide practical education and assistance that promotes rural independence. This project utilized Photovoice to describe how Missouri AgrAbility clients perceived how the program impacts their lives. This article relates clients' perceptions of the impact of the AgrAbility program on their lives to the socio-ecologic model framework. Although the primary focus of AgrAbility is to help individuals who have disabilities maintain their independence, all levels of the socio-ecological model are integral in meeting the needs of program participants.