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Campylobacter jejuni, a human gastrointestinal pathogen, uses nitrate for growth under microaerophilic conditions using periplasmic nitrate reductase (Nap). The catalytic subunit, NapA, contains two prosthetic groups, an iron sulfur cluster and a molybdenum cofactor. Here we describe the cloning, expression, purification, and Michaelis-Menten kinetics (kcat of 5.91 ± 0.18 s−1 and a KM (nitrate) of 3.40 ± 0.44 μM) in solution using methyl viologen as an electron donor. The data suggest that the high affinity of NapA for nitrate could support growth of C. jejuni on nitrate in the gastrointestinal tract. Site-directed mutagenesis was used and the codon for the molybdenum coordinating cysteine residue has been exchanged for serine. The resulting variant NapA is 4-fold less active than the native enzyme confirming the importance of this residue. The properties of the C. jejuni enzyme reported here represent the first isolation and characterization of an epsilonproteobacterial NapA. Therefore, the fundamental knowledge of Nap has been expanded.

A re-investigation of the interaction with NO of the small tetraheme protein cytochrome c554 (C554) from Nitrosomonas europaea has shown that the 5-coordinate heme II of the 2-electron or 4-electron reduced protein will nitrosylate reversibly. The nitrosylation process was found to be first order in C554, first-order in NO, and second-order overall. The rate constant for NO binding to the heme was determined to be 3000 ± 140 M-1s -1, while the rate constant for dissociation was 0.034 ± 0.009 s-1; the degree of protein reduction does not appear to significantly influence the nitrosylation rate. In contrast to a previous report, [ Upadhyay, A. K., et al. (2006), J. Am. Chem. Soc. 128, 4330-4337] this study turned up no evidence of C554-catalyzed NO reduction, either with C55422- or with C55442-. Some sub-stoichiometric oxidation of the lowest potential heme IV was detected when C55444- was exposed to an excess of NO, and this could in principle be part of a process that yields N2O, though alternative explanations are equally plausible. The vacant heme II site of C554 is sterically crowded by three non-bonding hydrophobic amino acids, Thr 154, Pro 155 and Phe 156. Replacing Phe156 with a protonatable but still bulky histidine residue did not significantly alter the reactivity of the F156H mutant with NO, though the NO binding rate appeared to increase 10-fold. On the other hand, when Phe156 was replaced with the smaller but still hydrophobic alanine, the 6-coordinate low-spin hemes of the 4-electron reduced mutant oxidized over the course of several minutes after exposure to NO. Two-electron reduced F156A2– nitrosylated, but did not oxidize, upon exposure to NO. Notably, the nitrosylation rate for F156A2-and F156A4- was about 400x faster than for the wild type or for the F156H mutant, though the rate of the reverse denitrosylation process was almost the same for the three C554 variants. The midpoint potentials of C554, and of the F156A and F156H variants, were determined for all the hemes in these tetraheme proteins, using spectropotentiometric analysis. The heme II midpoint potential of F156H was profoundly altered from the wild type value, shifting about 170 mV to the negative. This is taken as evidence that the histidine ligand in the variant binds to the erstwhile vacant ferric heme II axial site, thus stabilizing the oxidized state. Consistent with this interpretation, the UV/Visible spectrum of fully oxidized F156H exhibited increased absorbance at 409 nm relative to the wild type, which suggests that the mutant protein has 4 low-spin ferrihemes, rather than three low-spin and one high-spin as seen in the wild type. Upon reduction of heme II though, the spectrum of F156H exhibited a band at 430 nm characteristic of high-spin ferrohemes, which suggests that His156 dissociates from the heme when this reduces. In contrast to the case with F156H, the midpoint potentials of hemes I and II in F156A were only slightly shifted relative to the wild type. On the other hand, the midpoint potentials of the low-potential hemes III and IV were shifted about 100 mV to the negative by mutating Phe156 to Ala, whereas mutation of Phe156 to His had minimal impact on these hemes. It appears that the substitution of bulky Phe by the small Ala significantly alters the conformation of the protein backbone, which in turn affects the environment of distant hemes enough to substantially alter their midpoint potentials. The lower heme III and IV midpoint potentials of F156A, together with the increased solvent access to the heme II vacant site in this variant, may work together in changing its reactivity to bound NO. The more strongly reducing hemes could more readily reduce bound NO, while increased solvent access could now allow protonation to accompany reduction of the bound nitrogen moiety.

Single cell profiling by genetic, proteomic and imaging methods has expanded the ability to identify programmes regulating distinct cell states. The 3-dimensional (3D) culture of cells or tissue fragments provides a system to study how such states contribute to multicellular morphogenesis. Whether cells plated into 3D cultures give rise to a singular phenotype or whether multiple biologically distinct phenotypes arise in parallel is largely unknown due to a lack of tools to detect such heterogeneity. Here we develop Traject3d (Trajectory identification in 3D), a method for identifying heterogeneous states in 3D culture and how these give rise to distinct phenotypes over time, from label-free multi-day time-lapse imaging. We use this to characterise the temporal landscape of morphological states of cancer cell lines, varying in metastatic potential and drug resistance, and use this information to identify drug combinations that inhibit such heterogeneity. Traject3d is therefore an important companion to other single-cell technologies by facilitating real-time identification via live imaging of how distinct states can lead to alternate phenotypes that occur in parallel in 3D culture. There are currently a lack of tools to detect heterogeneity in 3D cultures. Here the authors report Traject3d as a framework to identify heterogeneous states in 3D culture and to understand how these give rise to distinct phenotypes using label-free multi-day time-lapse imaging.

Acute myeloid leukemia (AML) is a typically lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually, most AML retain wild-type TP53 , encoding the pro-apoptotic tumor suppressor p53. MDM2 inhibitors (MDM2i), which activate wild-type p53, and BET inhibitors (BETi), targeting the BET-family co-activator BRD4, both show encouraging pre-clinical activity, but limited clinical activity as single agents. Here, we report enhanced toxicity of combined MDM2i and BETi towards AML cell lines, primary human blasts and mouse models, resulting from BETi’s ability to evict an unexpected repressive form of BRD4 from p53 target genes, and hence potentiate MDM2i-induced p53 activation. These results indicate that wild-type TP53 and a transcriptional repressor function of BRD4 together represent a potential broad-spectrum synthetic therapeutic vulnerability for AML. MDM2 and BET inhibitors have shown efficacy in AML treatment. Here, the authors show that the two compounds can synergize through both p53 protein stabilization and inhibition of BRD4-mediated repression of p53 target genes.

The signalling pathways underpinning cell growth and invasion use overlapping components, yet how mutually exclusive cellular responses occur is unclear. Here, we report development of 3-Dimensional culture analyses to separately quantify growth and invasion. We identify that alternate variants of IQSEC1, an ARF GTPase Exchange Factor, act as switches to promote invasion over growth by controlling phosphoinositide metabolism. All IQSEC1 variants activate ARF5- and ARF6-dependent PIP5-kinase to promote PI(3,4,5)P 3 -AKT signalling and growth. In contrast, select pro-invasive IQSEC1 variants promote PI(3,4,5)P 3 production to form invasion-driving protrusions. Inhibition of IQSEC1 attenuates invasion in vitro and metastasis in vivo. Induction of pro-invasive IQSEC1 variants and elevated IQSEC1 expression occurs in a number of tumour types and is associated with higher-grade metastatic cancer, activation of PI(3,4,5)P 3 signalling, and predicts long-term poor outcome across multiple cancers. IQSEC1-regulated phosphoinositide metabolism therefore is a switch to induce invasion over growth in response to the same external signal. Targeting IQSEC1 as the central regulator of this switch may represent a therapeutic vulnerability to stop metastasis. The signalling pathways underpinning cell growth and invasion use overlapping components, yet how mutually exclusive responses occur is unclear. Here, the authors show that alternate isoforms of the ARF GTPase exchange factor IQSEC1 direct phosphoinositide metabolism to control this switch.

This multiagency report developed by the Interagency Collaboration for Genomics for Food and Feed Safety provides an overview of the use of and transition to whole genome sequencing (WGS) technology for detection and characterization of pathogens transmitted commonly by food and for identification of their sources. We describe foodborne pathogen analysis, investigation, and harmonization efforts among the following federal agencies: National Institutes of Health; Department of Health and Human Services, Centers for Disease Control and Prevention (CDC) and U.S. Food and Drug Administration (FDA); and the U.S. Department of Agriculture, Food Safety and Inspection Service, Agricultural Research Service, and Animal and Plant Health Inspection Service. We describe single nucleotide polymorphism, core-genome, and whole genome multilocus sequence typing data analysis methods as used in the PulseNet (CDC) and GenomeTrakr (FDA) networks, underscoring the complementary nature of the results for linking genetically related foodborne pathogens during outbreak investigations while allowing flexibility to meet the specific needs of Interagency Collaboration partners. We highlight how we apply WGS to pathogen characterization (virulence and antimicrobial resistance profiles) and source attribution efforts and increase transparency by making the sequences and other data publicly available through the National Center for Biotechnology Information. We also highlight the impact of current trends in the use of culture-independent diagnostic tests for human diagnostic testing on analytical approaches related to food safety and what is next for the use of WGS in the area of food safety.

IntroductionAcute-phase serum amyloid A (A-SAA) has cytokine-like properties and is expressed at sites of inflammation. We examined whether A-SAA-induced pro-inflammatory mechanisms are mediated through Toll-like receptor 2 (TLR2) in rheumatoid arthritis (RA).MethodsThe effect of A-SAA on human embryonic kidney (HEK), TLR2 or TLR4 cells was quantified by nuclear factor (NF)-κB luciferase reporter assays. A-SAA-induced RASFC and dHMVEC function were performed in the presence of a specific neutralising anti-TLR2 mAb (OPN301) (1 μg/mL) and matched IgG isotype control Ab (1 μg/mL). Cell surface expression of intracellular adhesion molecule (ICAM)-1, chemokine expression, cell migration, invasion and angiogenesis were assessed by flow cytometry, ELISA, Matrigel invasion chambers and tube formation assays. MyD88 expression was assessed by real-time PCR and western blot.ResultsA-SAA induced TLR2 activation through induction of NF-κB (p<0.05), but failed to induce NF-κB in HEK-TLR4 cells, confirming specificity for TLR2. A-SAA-induced proliferation, invasion and migration were significantly inhibited in the presence of anti-TLR2 (all p<0.05), with no significant effect observed for tumour necrosis factor-α-induced events. Additionally, A-SAA-induced ICAM-1, interleukin-8, monocyte chemoattractant protein-1, RANTES and GRO-α expression were significantly reduced in the presence of anti-TLR2 (all p<0.05), as was A-SAA induced angiogenesis (p<0.05). Finally, A-SAA induced MyD88 signalling in RASFC and dHMVEC (p<0.05).ConclusionsA-SAA is an endogenous ligand for TLR2, inducing pro-inflammatory effects in RA. Blocking the A-SAA/TLR2 interaction may be a potential therapeutic intervention in RA.

Forming a professional identity is a process by which an individual achieves an awareness of his or her own self-concept in the context of the profession. Identity in relation to an individual's profession includes the ability to articulate one's role as a professional and professional philosophy. Professional identity has been studied extensively in other fields, but currently no professional identity scales have been validated within the athletic training profession.   To validate the Professional Identity and Values Scale (PIVS) among an athletic trainer population.   Cross-sectional study.   Web-based questionnaire.   Athletic trainers employed in National Collegiate Athletic Association Division I, II, III, or National Association of Intercollegiate Athletics colleges or universities (n = 299, 56.5% female, 43.5% male). The average age of the participants was 33.6 ± 8.3 years, and they had 10.3 ± 7.6 years of experience.   Participants were asked to complete a demographic questionnaire and the 32-item PIVS. The variables included demographics and the PIVS (Professional Orientation and Values subscale [18 items] and the Professional Development subscale [14 items]).   Exploratory factor analysis reduced the survey from 32 to 20 items and revealed 6 factors. Three factors emerged from the Professional Development subscale and emphasized professional insecurities during the early career stages, the importance of mentors during the intermediate stages, and self-confidence and awareness during the later stages of professional development. An additional 3 factors emerged from the Professional Orientation and Values subscale: (1) patient care and advocacy, (2) professional engagement and collaboration, and (3) personal wellness and values. A Cronbach α of 0.80 indicated good internal consistency.   A modified PIVS is a valid and reliable measure of professional identity among athletic trainers employed in the collegiate setting.

Recent employment data from collegiate athletic training settings have demonstrated departure trends among men and women. These trends have been hypothesized to be related to work-life balance. However, work-life balance is only 1 aspect of a myriad of factors. Due to the complex nature of the work-life interface, a multilevel examination is needed to better understand the precipitators of departure.   To quantitatively examine factors that may influence collegiate athletic trainers' (ATs') job satisfaction and career intentions via a multilevel examination of the work-life interface.   Cross-sectional study.   Web-based questionnaire.   Athletic trainers employed in National Collegiate Athletic Association Division I, II, or III or National Association of Intercollegiate Athletics colleges or universities (N = 299: 56.5% female, 43.5% male). The average age of participants was 33.6 ± 8.3 years, and their average experience was 10.3 ± 7.6 years.   Participants responded to an online questionnaire consisting of demographic questions, 9 Likert-scale surveys, and open-ended questions. Job-satisfaction Scores (JSSs) and intention-to-leave scores (ITLSs) served as the dependent variables and factors from individual, organizational, and sociocultural levels were the independent variables. Hierarchical regression analysis was run to determine the predictability of factors.   No sex differences in ITLS or JSS were found in our sample. Independent variables explained 68.5% of the variance in JSS and 28.8% of the variance in ITLS. Additions of factor levels increased the percentage of explained variance in both scores.   A combination of individual-, organizational-, and sociocultural-level factors was able to best predict JSS and ITLS among collegiate ATs.

Purpose: To examine individual and organizational level factors that may affect the job satisfaction and career intentions of athletic trainers employed in the collegiate setting. Methods: Volunteers for this qualitative study included 30 athletic trainers aged 33 ± 8 years with 10 ± 8 years of experience who were employed in the National Collegiate Athletic Association (NCAA) Division I, II, or III or National Association of Intercollegiate Athletics (NAIA) setting. Semi-structured phone interviews were digitally recorded and transcribed verbatim. Data were analyzed using a general inductive approach. The credibility of the study was maintained using peer review and multiple analyst triangulation. Results: Organizational level factors appeared to influence the participants' job satisfaction and career intentions. Although there were similarities between individual level factors among collegiate athletic trainers, these factors alone did not account for job satisfaction or career intentions. Conclusions: Continued multilevel examinations of the work–life interface are warranted to better understand issues related to retention and attrition within the athletic training profession. [Athletic Training & Sports Health Care. 2020;12(1):21–30.]