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Using the concept of methodological congruence-where the different elements of a study 'fit' together-we explore both problematic and good practice in (reflexive) thematic analysis (TA) as reported in Health Promotion International (HPI). Aligning with the importance we place on 'owning your perspectives' we situate this exploration in relation to our understanding of the variation in approaches to TA and qualitative research more broadly. This contextualization is necessary for highlighting why we judge practices to be in/congruent, and to facilitate more knowing congruence in future research. We critically reviewed a 'sample' of 31 papers published in HPI between 2010 and 2023 citing Braun and Clarke as reference for TA. We overview a range of problematic and good features of the use of TA in HPI, before focusing on two domains that seemed to present key challenges: theory and themes. Methodological incongruence can occur when postpositivist values and practices unwittingly creep into ostensibly non-positivist TA; we encourage thoughtfully and what we term 'knowing' consideration of theory, and quality practices and criteria. Methodological incongruence can also occur through mismatched conceptualizations of themes-notably, the use of 'topic summaries' as themes for reflexive TA (and fragmented thematic structures with 'thin' themes). We provide examples from the reviewed papers to demonstrate good practice in researcher reflexivity, articulation of theoretical and methodological frameworks and congruent themes. However, mindful of power dynamics, we only discuss problematic practice in general terms, to protect author anonymity. To facilitate thoughtful, quality TA-of all kinds-we provide eight pointers for researchers (and reviewers) to guide quality practice, and facilitate the use of concepts, procedures and criteria that promote knowing methodological congruence., (© The Author(s) 2024. Published by Oxford University Press.)

Bacteria are usually iron-deficient because the Fe3+ in their environment is insoluble or is incorporated into proteins. To overcome their natural iron limitation, bacteria have developed sophisticated iron transport and regulation systems. In gram-negative bacteria, these include iron carriers, such as citrate, siderophores, and heme, which when loaded with Fe3+ adsorb with high specificity and affinity to outer membrane proteins. Binding of the iron carriers to the cell surface elicits a signal that initiates transcription of iron carrier transport and synthesis genes, referred to as "cell surface signaling". Transcriptional regulation is not coupled to transport. Outer membrane proteins with signaling functions contain an additional N-terminal domain that in the periplasm makes contact with an anti-sigma factor regulatory protein that extends from the outer membrane into the cytoplasm. Binding of the iron carriers to the outer membrane receptors elicits proteolysis of the anti-sigma factor by two different proteases, Prc in the periplasm, and RseP in the cytoplasmic membrane, inactivates the anti-sigma function or results in the generation of an N-terminal peptide of ∼50 residues with pro-sigma activity yielding an active extracytoplasmic function (ECF) sigma factor. Signal recognition and signal transmission into the cytoplasm is discussed herein., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Bacteria are in constant conflict with competing bacterial and eukaryotic cells. To cope with the various challenges, bacteria developed distinct strategies, such as toxins that inhibit the growth or kill rivals of the same ecological niche. In recent years, two toxin systems have been discovered - the type VI secretion system and the contact-dependent growth inhibition (CDI) system. These systems have structural and functional similarities and share features with the long-known gram-negative bacteriocins, such as small immunity proteins that bind to and inactivate the toxins, and target sites on DNA, tRNA, rRNA, murein (peptidoglycan), or the cytoplasmic membrane. Colicins, CdiA proteins, and certain type VI toxins have a modular design with the transport functions localized in the N-terminal region and the activity functions localized in the C-terminal region. Despite these common properties, the sequences of toxins and immunity proteins of colicins, CDI systems, and type VI systems show little similarity., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Background: Many countries with shortages in health personnel are introducing task shifting in primary health care. GPs' attitudes and practices strongly affect task shifting and the expansion of the roles of physician assistants (PAs)., Objective: To assess, in a German state with shortages of health personnel, the overall willingness of GPs to delegate home visit tasks to PAs and to elicit their perceptions of barriers to and benefits of such delegation and the current practice of informal delegation., Methods: Postal self-administered anonymous survey of all practicing GPs in the rural state of Mecklenburg-Vorpommern. Main outcomes were GPs' willingness to delegate in home visit tasks to a properly trained PA, perceived barriers to and benefits of home visit delegation and current practice of informal delegation. Using multinomial logistic regression, associations were identified among outcome variables, and characteristics of the GPs and of their practices., Results: Response rate was 47%. Responders (500) were comparable to all GPs in the state (1096); 48% of practitioners are willing to delegate home visits tasks to PAs. The main barrier to delegation was the related costs of PAs' training (34%), and the main benefit that it 'saves the GP's time' (67%). The 46% of practitioners who are informally delegating home visit tasks were significantly more likely be younger [odds ratio (OR) and 95% confidence interval (CI)] [OR = 0.96 (0.93-0.99)] and female [OR = 1.70 (1.12-2.58)]., Conclusion: The increasing proportion of women in family medicine might favor task shifting in General Practice.

Background: The management of obesity with its associated morbidity and mortality is a growing problem in primary care practices. Despite numerous recommendations in response to this challenge, weight management interventions still yield poor results. This is partly due to a discrepancy between physicians' and patients' understanding of the problem and possible solutions., Objective: This study analyses patients' and physicians' visions for the future management of obesity., Methods: Qualitative in-depth semi-structured interviews were performed. Physicians and patients were asked about their individual needs, experience and views regarding the management of obesity. Fifteen GPs and 15 overweight patients participated in this study. Interviews were transcribed and submitted to qualitative content analysis., Results: The investigation reveals a high level of agreement between the two groups with regard to communication requirements for successful care. Both groups stressed the need for multimodal care concepts inside and outside of GP practices. Both also addressed the current overburdening of outpatient care structures in dealing with the management of obesity., Conclusion: Options should be developed for closer cooperation between GPs and support facilities inside and outside practices.

Objective: The increasing prevalence of obesity requires particularly primary care providers to take action. The aim of this study was to analyze general practitioners (GPs) encounters with overweight and obese patients in primary care to test the hypothesis that patients with a BMI ≥ 30 kg/m² would have longer consultations focusing on lifestyle-related issues like nutrition and physical activity than those with a BMI < 30 kg/m²., Design: Cross sectional comparison of audiotaped encounters of patients with a BMI ≥ 30 kg/m² and those with a BMI < 30 kg/m²., Setting: Twelve GP surgeries in Berlin, Germany., Participants: Fifty patients who agreed to have preventive check-up encounters audiotaped., Main Outcome Measures: Based on the Roter Interaction, Analysis: System (RIAS) we assessed duration of encounter and the prevalence of GP statements regarding cardiovascular risks, nutrition and physical activity., Results: An increased BMI was found to be a predictor for the length of encounters (P = 0.01), whereas the content of talks was mainly determined by the individual of GP and sex of the GP. Statements regarding cardiovascular risks were most frequent, followed by those regarding nutrition and physical activity. In this study the assessed physiological parameters were not associated with the specific contents of preventive encounters like nutrition or physical activity (P > 0.05)., Conclusions: Our results indicate that GPs rarely use the check-up program to conduct lifestyle consultations with obese patients. Barriers to lifestyle counseling and possible solutions are discussed with a view to promoting individualized and target management of overweight patients.

The ferric citrate transport system of Escherichia coli is the first example of a transcription initiation mechanism that starts at the cell surface. The inducer, ferric citrate, binds to an outer membrane transport protein, and without further transport elicits a signal that is transmitted across the outer membrane, the periplasm, and the cytoplasmic membrane into the cytoplasm. Signal transfer across the three subcellular compartments is mediated by the outer membrane transport protein that interacts in the periplasm with a cytoplasmic transmembrane protein. The latter is required for activation of a sigma factor which belongs to the extracytoplasmic function sigma factor family. A similar kind of transcription regulation has been demonstrated in Pseudomonas putida, P. aeruginosa, Serratia marcescens, Klebsiella pneumoniae, Aerobacter aerogenes, Bordetella pertussis, B. bronchseptica, B. avium, and Ralstonia solanacearum. The genomes of P. putida, P. aeruginosa, Nitrosomonas europaea, Bacteroides thetaiotaomicron and Caulobacter crescentus predict the existence of many more such transcriptional regulatory devices.

Background: In cases of male infertility, routine analysis for sperm characteristics is a poor predictive factor for the segmentation rate and embryo development in assisted reproductive technologies. It is assumed that epigenetic factors could have an influence on the embryo's quality. The aim of this work was to determine the relationship between sperm DNA methylation level and fertilization and pregnancy rates according to the assisted reproduction technique performed., Methods: A prospective study was undertaken. Ejaculates were obtained from men (n = 63) undergoing an assisted reproduction procedure. 5-Methylcytosine was immunostained with a polyclonal antibody and revealed by fluorescein isothiocyanate. The DNA methylation level was then quantified by flow cytometry., Results: Sixty-three conventional IVF cycles were performed, 760 oocytes were retrieved, an average of 8.1 +/- 4.8 embryos was obtained, and 2.4 embryos were transferred. Neither the fertilization rate nor the rate of good quality embryos was correlated with the DNA methylation level (r = -0.1 and r = -0.08 respectively; not significant). When sperm DNA methylation was >555 arbitrary units, the pregnancy rate was 33.3% compared with 8.3% in the lower (<555) group (P<0.05)., Conclusion: DNA methylation level in human sperm could represent a new approach to study the ability of sperm to lead to pregnancy in an assisted reproduction procedure, especially when sperm samples with normal characteristics are used.

Infection of Escherichia coli K-12 by phages T1 and phi 80 requires the FhuA outer membrane protein and the TonB protein. Mutations in the N-terminal globular domain close to the predicted channel in the beta-barrel of FhuA were created. The FhuA Delta 107-111 N104K K110D L111P mutant and the FhuA(L(109)DPNGLK(110)) insertion mutant were sensitive to phage T1, but nearly resistant to phage phi 80. FhuA Delta 107-111 N104K K110D L111P mediated phage T1 infection in a tonB mutant without formation of TonB-independent phage T1 host-range mutants. The FhuA mutants showed no altered sensitivity to phage T5. Although the phages share overlapping binding sites in FhuA, the structural alterations elicited by the mutations resulted in very different phage sensitivities. In the FhuA deletion mutant, the TonB requirement for phage T1 infection was partially bypassed.

Motivation: Animals build their bodies by altering the fates of cells. The way in which they do so is reflected in the topology of cell lineages and the fates of terminal cells. Cell lineages should, therefore, contain information about the molecular events that determined them. Here we introduce new tools for visualizing, manipulating, and extracting the information contained in cell lineages. Our tools enable us to analyze very large cell lineages, where previously analyses have only been carried out on cell lineages no larger than a few dozen cells., Results: Ales (A Lineage Evaluation System) allows the display, evaluation and comparison of cell lineages with the aim of identifying molecular and cellular events underlying development. Ales introduces a series of algorithms that locate putative developmental events. The distribution of these predicted events can then be compared to gene expression patterns or other cellular characteristics. In addition, artificial lineages can be generated, or existing lineages modified, according to a range of models, in order to test hypotheses about lineage evolution., Availability: The program can run on any operating system with a compliant Java 2 environment. Ales is free for academic use and can be downloaded from http://mbi.dkfz-heidelberg.de/mbi/research/cellsim/ales.

Background: Standard sperm characteristics are poor predictors of the outcome of IVF treatments. On the contrary, sperm genome quality has been emphasized for several years as playing a major role in early embryogenesis, thus in the success of IVF attempt., Methods: Sperm DNA fragmentation from a selected group of 104 couples undergoing assisted reproductive techniques (ART) (IVF: n = 50; and ICSI: n = 54) was measured by TUNEL assay and correlated with semen and ART outcomes., Results: A negative correlation was found between sperm characteristics and the proportion of sperm showing DNA fragmentation. For fragmentation >10%, a significant decrease of the fertilization rate was observed. No correlation was found between sperm DNA fragmentation and embryo quality. A high proportion of sperm with fragmented DNA was a pejorative factor to obtain pregnancies when ICSI was performed, but there was no relationship when conventional IVF was performed., Conclusions: The proportion of sperm with DNA fragmentation appears to be potentially useful as a predictor of ICSI outcome, whereas embryo quality based on morphological criteria, appeared unaffected by DNA fragmentation.

A particular property of Clostridium difficile strain C34 is an insertion of approximately 2 kb in the tcdA-C34 gene that does not hinder expression of a fully active TcdA-C34 molecule. Intoxication with TcdA-C34 induced an arborized appearance in eukaryotic cells (D-type cytopathic effect); intoxication with TcdB-C34 induced a spindle-like appearance of cells (S-type cytopathic effect). Inactivation of GTPases with purified toxins revealed that Rho, Rac, Cdc42, and Rap are substrates of TcdA-C34. The variant cytotoxin TcdB-C34 inactivated Rho, Rac, Cdc42, Rap, Ral, and R-Ras. Hence, this is the first 'S-type' cytotoxin which inactivates both Rho and R-Ras, and is coexpressed with a 'D-type' enterotoxin. Our results support the hypothesis that R-Ras is a key GTPase related to the S-type cytopathic effect and suggest that induction of a S-type cytopathic effect dominates induction of the D-type cytopathic effect.

The uptake of colicin U into sensitive cells is dependent on the Tol system. Like colicin A and the E-type colicins, colicin U contains the consensus pentapeptide DG(T/S)G(S/W) in the N-terminal 36 amino acids, which have been proposed to be involved in the interaction of colicin U with the Tol proteins. The role of the DGTGW motif in colicin U was studied by converting it into the TonB box motif DTMVV of colicin B. Uptake of the mutated colicin U (DTMVV) depended on TonB and ExbB and still remained TolA- and TolQ-dependent, but no longer required TolB. The Ton system could obviously replace the TolB function in the uptake of the mutated colicin U. The colicin U derivative is the first colicin whose uptake depends on the Ton and the Tol systems.

The FhuA protein of Escherichia coli K-12 transports ferrichrome and the structurally related antibiotic albomycin across the outer membrane and serves as a receptor for the phages T1, T5, and phi 80 and for colicin M. In this paper, we show that chimeric proteins consisting of the central part of FhuA and the N- and C-terminal parts of FhuE (coprogen receptor) or the N- and/or C-terminal parts of FoxA (ferrioxamine B receptor), function as ferrichrome transport proteins. Although the hybrid proteins contained the previously identified gating loop of FhuA, which is the principal binding site of the phages T5, T1, and phi 80, only the hybrid protein consisting of the N-terminal third of FoxA and the C-terminal two thirds of FhuA conferred weak phage sensitivity to cells. Apparently, the gating loop is essential, but not sufficient for wild-type levels of ferrichrome transport and for phage sensitivity. The properties of FhuA-FoxA hybrids suggest different regions of the two receptors for ferric siderophore uptake.

The multifunctional FhuA protein of Escherichia coli K-12 forms a channel that is closed by a loop, tentatively designated the 'gating loop', which is also the principal binding site for all FhuA ligands. In this report, it is shown by in vivo labeling that the two cysteines in the gating loop form a disulfide bridge, and they react weakly after reduction with biotin-maleimide, as determined by streptavidin-beta-galactosidase bound to biotin. The two cysteines close to the C-terminus of FhuA also form a disulfide bridge and react with the thiol reagents only after heat denaturation of FhuA in SDS. Replacement of the existing cysteines by serine did not alter the sensitivity of cells to the FhuA ligands tested (T5, phi 80, T1, colicin M, and albomycin) and supported growth on ferrichrome as sole iron source. The cysteines in the gating loop play no specific functional role; they are largely buried in the interior of the loop, and the disulfide bridges are not essential for maintaining the conformation of FhuA. The C-terminal cysteines are in the interior of FhuA and are also not important for the structure of FhuA. The method used allows the identification of free cysteines and disulfides in surface exposed protein regions.

In an attempt to directly approach the postulated toxic domain of Clostridium difficile's TcdB-10463, eight subclones of different size and locations in the N-terminal third of the toxin were generated. Expression of these toxin fragments was checked in Western blots and the enzymatic activity of the expressed proteins was analyzed by glucosylating Ras related small GTP-binding proteins. Two polypeptides of 875 aa (TcdBc1-3) and 557 aa (TcdBc1-H) glucosylated their targets Rho, Rac and Cdc42 with the same activity and specificity as the holotoxin. In comparison 516 aa (TcdBc1-N) and 467 aa (TcdBc1-A) protein fragments exhibited highly reduced activity, while Tcdc1 and TcdB2-3 (aa 1-243 and 244-890, respectively) were enzymatically inactive. Our results indicate that all structures involved in the catalysis are located at several different sites within the 557 aa fully active fragment. The shortest enzymatically still active protein covers aa 1-467 and obviously fulfils all minimal requirements for glucosylation. The data support the postulated three domain model of 'large clostridial cytotoxins'.

We have used six independent polymerase chain reactions (A1-A3 and B1-B3) for amplification of the entire sequence of the two toxin genes tcdA and tcdB of several Clostridium difficile strains. With this approach we have detected (1) restriction site polymorphisms which are distributed all over the genes, and (2) deletions that could be found only in tcdA. Characteristic differences between strains were mainly focused to the 5' third of tcdB (B1 fragment) and/or the 3' third of tcdA (A3 fragment). The possible use of our approach for typing of C. difficile toxin genes is discussed.

Iron in the form of ferric siderophore complexes and vitamin B12 are transported through the outer membrane of Gram-negative bacteria by a mechanism which consumes energy. There is no known energy source in the outer membrane or in the adjacent periplasmic space so that energy is provided by the electrochemical potential across the cytoplasmic membrane. Energy flows from the cytoplasmic into the outer membrane via a complex consisting of the TonB, ExbB and ExbD proteins which are anchored in the cytoplasmic membrane. It is proposed that the TonB--ExbB--ExbD complex opens--via an energized conformation of the TonB protein--channels in the outer membrane, formed by proteins which serves as highly specific binding sites for the various ferric siderophores and vitamin B12. In addition, outer membrane receptors together with the TonB--ExbB--ExbD complex are directly involved in induction of the transcription of ferric citrate and pseudobactin transport genes of Escherichia coli and Pseudomonas putida, respectively.

The current model of TonB-dependent colicin transport through the outer membrane of Escherichia coli proposes initial binding to receptor proteins, vectorial release from the receptors and uptake into the periplasm from where the colicins, according to their action, insert into the cytoplasmic membrane or enter the cytoplasm. The uptake is energy-dependent and the TonB protein interacts with the receptors as well as with the colicins. In this paper we have studied the uptake of colicins B and Ia, both pore-forming colicins, into various tonB point mutants. Colicin Ia resistance of the tonB mutant (G186D, R204H) was consistent with a defective Cir receptor-TonB interaction while colicin Ia resistance of E. coli expressing TonB of Serratia marcescens, or TonB of E. coli carrying a C-terminal fragment of the S. marcescens TonB, seemed to be caused by an impaired colicin Ia-TonB interaction. In contrast, E. coli tonB (G174R, V178I) was sensitive to colicin Ia and resistant to colicin B unless TonB, ExbB and ExbD were overproduced which resulted in colicin B sensitivity. The differential effects of tonB mutations indicate differences in the interaction of TonB with receptors and colicins.

The activity of the FhuA receptor in the outer membrane of Escherichia coli is dependent on the TonB, ExbB and ExbD proteins which are anchored to the cytoplasmic membrane. Only infection by phage T5 occurs independently of TonB, ExbB and ExbD. In this paper we describe mutated FhuA proteins which displayed either an increased or decreased FhuA activity to phage T5 when combined with mutated TonB proteins. These results suggest conformational changes in FhuA by TonB which are recognized by phage T5. Similar results were obtained with colicin M and the phages T1 and phi 80. It is proposed that the FhuA mutant proteins assume conformations which are either improved or impaired by the TonB derivatives. For the direct interaction of FhuA with TonB regions which are located outside the TonB box of FhuA and the region around residue 160 of TonB are important.

Most Serratia marcescens strains produce a new type of cytolysin (hemolysin) which is also found in other Serratia species. The hemolytic polypeptide ShlA (M(r) 162 101) is secreted across the outer membrane through the help of the ShlB protein which also involves conversion of an inactive precursor in an hemolytically active form. Both proteins are synthesized with signal sequences which are released during export across the cytoplasmic membrane. Mutants expressing inactive ShlB derivatives are impaired in activation and secretion suggesting a tight coupling between both processes. The region of ShlA for activation and secretion is confined to the N-terminal 16% of the polypeptide which contains the sequence NPNG which is also found in the Proteus hemolysin, the Bordetella pertussis filamentous hemagglutinin and two highly expressed outer membrane proteins of Haemophilus influenzae. Substitution of the first asparagine (N) residue by isoleucine converts the Serratia hemolysin into an inactive secretion incompetent form. It is concluded that this region is recognized by ShlB for activation and secretion of ShlA. The Serratia hemolysin forms defined pores in erythrocyte membranes.

The iron(III) transport system via citrate displays the characteristics of a binding protein-dependent transport mechanism through the cytoplasmic membrane. One of the five transport proteins, FecE, contains the two motifs found in ATP-binding proteins. Cloned overexpressed FecE was photoaffinity-labelled with [32P]8-azido-ATP. Labelling was inhibited by 1 mM ATP, ADP, GTP and less by CTP, demonstrating the specificity of the reaction. It is suggested that ATP is the principal energy source for iron(III) citrate transport through the cytoplasmic membrane of Escherichia coli.