Purpose The purposes of this study were to provide an outline of the existing literature on operationalization of the International Classification of Functioning, Disability and Health (ICF) within vocational rehabilitation (VR) and to explore the ICF utility within VR. Methods The process was undertaken in five stages according to a framework of scoping review. Screening and extraction of data were done by two independent reviewers, and data was summarized according to content analysis. Results Fifty papers  were included. The operationalization of the ICF was described in four different ways: In total 18 (36%) papers described use of the ICF for structuring information, 8 (15%) for linking information to ICF, 12 (24%) for analysis of results, and 12 (24%) for development of a model. In total 15 (29%) papers described VR interventions involving stakeholders, whereas 32 (62%) were reviews. Forty of the papers described all the ICF components. Conclusions The review revealed use of the ICF within the field VR in 50 papers, and in various settings. The ICF framework was most often operationalized for structuring or linking information of functioning. A majority of papers were reviews and involved researchers only, whereas different stakeholders and VR professionals were involved in the interventions. In 40 papers all the ICF components were described, and the ICF was considered a useful tool to inform the VR professionals´ assessment of functioning. However, more research within VR is needed to standardize and ease the use of the ICF.The online version of this article (10.1007/s10926-018-9788-4) contains supplementary material, which is available to authorized users. Work disability is often associated with personal suffering and loss of income, diminished productivity and increased medical and societal costs and can be addressed through vocational rehabilitation (VR) . The essThere are several definitions of VR, e.g. medical, psychological, social and occupational activities aiming to reestablish sick or injured peoples work capacity and prerequisites for returning or entering the labour market, i.e. to a job or availability for a job, 2009 [A review showed diversity in the ICF contents of the measures used in the literature, and proposed that the ICF and VR interface should be further examined . KnowingThe purposes of this review were to provide an outline of the existing literature and to explore the ICF utility within VR. The primary aim was to examine and map the operationalization of the ICF within VR. The second aim was to examine the different VR professionals´ use of the ICF. Ideally a multi-professional, multimodal approach should be used in VR .Within the WHO a number of ICF core sets have been developed in order to make the ICF more applicable for clinical practice. A third aim was to examine to what extent the components of the ICF framework, the VR core set, and other ICF core sets are used within VR. Core sets are lists of essential ICF-categories in specific health conditions and contexts to describe functioning, e.g. a comprehensive and a brief VR core set were developed and validated for interdisciplinary assessment, documentation, and communication in VR , 14.A preliminary search in PROSPERO and PubMed showed no review on the topic, and to our knowledge there are no existing systematic reviews or scoping review on how the ICF is applied within VR.The scoping review was conducted according the methodology conduced in five steps: (1) identifying the research question, (2) identifying relevant studies, (3) study selection, (4) charting the data, and (5) collating, summarizing, and reporting results –17.How is the ICF operationalized in empirical papers within VR?Who are involved and how does the ICF inform the professionals´ assessment of functioning in VR?Which of the ICF components and core sets are considered when functioning is evaluated in VR?A three-step search strategy was conducted . FirstlyInclusion criteria: ICF or International Classification of Functioning, Disability and Health mentioned in the title or abstract, ICF used in the field of VR research, peer reviewed original papers and reviews, date of publication from January 2001 to May 2016, abstract available, and study populations of working age adults. There were no limitations regarding including reviews and thereby potential overlap of individual papers included in the reviews. There were no context limitations regarding geography or culture, and papers were eligible from any healthcare setting or research setting .Exclusion criteria: papers only mentioning ICF in the abstract, background or discussion, or only mentioning ICIDH or ICIDH-2, overviews, editorials, comments, theoretical papers, text and opinion papers, theses/dissertations, books, and papers on ICF-Children and Youth.The process of study selection was reported using the PRISMA , and eliStudy characteristics were extracted from the included studies using a pilot-tested non-software template. The papers were divided in qualitative and quantitative papers according to qualifications of the review team. Two authors extracted study characteristics independently for qualitative papers (AM and RR) and quantitative papers (AM and CMS), respectively. In case of disagreement, the final decision about characteristics was resolved through discussion. The papers were divided in qualitative and quantitative papers according to the data collection method described.Study characteristics according to The Joanna Briggs Institute Reviewers’ Manual included: first author, publication year, country, setting, study type (intervention yes/no), population, aims, methods, and outcomes . InterveStructuring was considered present, when data or outcomes were categorized, or themes or information from interviews was coded according to the ICF framework. However, in case structuring was followed by other use, data was extracted according to the latter. Linking was considered present, when health information (e.g. from questionnaires or interviews) was coded to specific ICF categories, based on linking rules, e.g. linking items in a questionnaire to categories in a core set [Analysing was considered present if the paper explicitly described that data were analysed, most commonly after data or information had been structured following the ICF framework. Developing instruments or models based on the ICF framework was the last reported usage.Regarding the first research question; data was extracted according to the use of the ICF framework as described in the individual papers. Four different ways of operationalization of the ICF were the most typical descriptions used in a subset of the papers included: (a) structuring, (b) linking, (c) analysing, or (d) developing instruments or models, respectively. All the included papers´ description were categorised in these. core set , 22. AnaRegarding the second research question the description of VR professionals  involved were extracted, and a descriptive summary of their use of the ICF in order to inform the assessment of functioning was presented. Regarding the third question; the use of the ICF components  and the ICF core set(s) was extracted based on the information provided in individual papers.A descriptive summary of the charted data was done independently by two authors on all the included papers. The coded data relevant to inform the three review questions were charted from each paper included and categorized according to content analysis –25. BothIn total 1343 papers were retrieved from seven databases, of which 702 duplicates were removed; thus, 641 papers were assessed for eligibility  in Switzerland and the Netherlands; other VR settings in which the ICF was used were widespread, i.e. hospitals, rehabilitation centres, primary health care centres, and sickness certificate registration offices.A minority of papers reported interventions within VR; only seven of the qualitative papers , 61–63, In total 18 papers (36%) used the ICF as a framework for structuring of information: twelve of the qualitative papers , 72–74, In total eight papers (15%) used the ICF as a framework for linking between ICF categories and e.g. items in questionnaires: three qualitative papers , 38, 52,The analysis was performed according to the ICF framework in 12 papers (24%): five qualitative papers , 55, 63,The ICF was used as a framework for the development of an instrument or a new model for various aspects within VR in 12 papers (24%) : five qualitative papers , 65, 67;Four papers described involvement of patients and researchers , 55 or pRegarding to what extent the ICF informed professionals´ assessment of functioning; several papers reported discussions on the ICF´s applicability for VR, service delivery, and RTW support. As examples were papers reporting on potential benefits of the ICF: to structure and phrase disability evaluation in the field of social insurance , on tracOne paper concluded that a questionnaire based on the ICF proved to be a “useful framework that can be used for research but also by occupational physicians in their usual practice after specific training” . A paperA paper described how VR professionals used the ICF to guide assessment in the job placement process and used the appropriate ICF domains and categories as a template to determine what specific information needed to be obtained, and how to organize it in a systematic way. Thus, an interview format informed by the ICF structure enabled the professionals to highlight the needs for assessment information .One paper involving several health and non-health professionals concluded, that disadvantages of the ICF are the “complicated terminology, perceived subjectivity of the assessor in coding” and that ‘it is too bulky’ . AnotherExcept in 10 papers all the ICF components were described. Two papers commented on personal factors, despite the fact they are part of the ICF there are no categorizations , 35. OnlSeven papers used the ICF core set , 51, 55,The ICF was primarily used in Western VR contexts. The ICF used as a framework was the most prevalent operationalizing of ICF (18 papers), whereas linking, analysing and developing appeared in 8, 12 and 12 papers respectively. As 32 of the 50 included papers were reviews the predominant profession involved in ICF were researchers. Among the original papers no single profession stood out as particularly ICF users. In general the ICF enabled the various professions involved in VR in a structured way to obtain relevant need assessments and communicate this across professions. The majority (40) of papers described all factors, which support the bio-psycho-social approach. However, it was not clear if the ICF was suitable as an instrument for goal setting and evaluation as merely single papers mentioned these properties. Moreover, the ICF was criticised for being time consuming. Unexpectedly four papers described the ICF components body and acidity only, despite participation and environmental factors seem inseparable from VR. The VR core set was not the primary tool when functioning was evaluated within VR.Compared to the findings in another review where qualitative papers only constituted a tenth , and desThe ICF defines functioning as the interaction between an individual and that individual’s environmental and personal factors; accordingly a paper illustrated the problems of functioning in a person with low back pain by use of the framework .The ICF is seen as a useful tool for describing, comparing and contrasting information from outcome measures and clinical patient reports across diagnoses, settings, languages and countries . A revieEvaluation of functioning is relevant early in VR , and thiFurthermore, an ICF-based questionnaire regarding time to RTW, work difficulties, job satisfaction, and work relations was reportedly useful for occupational physicians assessing patients after transplant procedures . Thus, tThe present review illustrates how the ICF may support development of questionnaires , 60, likAlthough the ICF is a reasonable starting point in efforts to harmonize terminologies , the fraA review on the use of ICF in outcome measures used within VR identified that a third of categories were related to body functions . This reUnexpectedly, the review revealed a limited use of the ICF VR core sets. These include environmental factors that may prove to be useful when disability evaluation and work capacity is being assessed. However, a validation study of the comprehensive VR core set concluded, that it was insufficient from a sole physiotherapist perspective, there was a need for additional ICF categories. Although the VR core set was considered useful to clarify responsibilities and for communication in a multidisciplinary setting, it was too comprehensive for mono-disciplinary use of physiotherapist . The corOne strength was the inclusion of both qualitative and quantitative papers from multiple settings and countries. Furthermore, the scoping review format offers an overview of study findings in a field where the knowledge is still limited.The reviewers experienced difficulties in study selection, despite the method by two reviewers and how to ensure eligibility criteria is a limitation. In a scoping review the extracted data is based on information provided in individual papers without critical assessment, which is a limitation despite no scope of synthesizing evidence. Furthermore, the categories of operationalization may not be mutual exclusive.The sixth and optional stage of involving relevant stakeholders was not included but may have contributed with other VR professionals´ views .This review confirm challenges with the use of the ICF: e.g. it cannot infer causality in disability  and not Our findings revealed that the ICF has been applied in different settings and for different purposes, which has important implications for future research. In order to ensure comparability across studies and robust testing of hypotheses the use of the ICF needs to be clarified. Furthermore, how data are collected, assessed and classified based is lacking in the field of VR. Hence, research on the practical utility of the ICF across different assessment instruments is crucially needed to inform a feasible framework development in VR.Although the ICF provides a framework to evaluate contextual factors, this review finds there is a gap between the knowledge of the impact of personal factors and actual assessment within VR and more research is needed.The scoping review revealed use of the ICF within the field VR in 50 papers, and in various settings; e.g. hospitals, rehabilitation centres, primary health care centres, sickness certificate registration offices, and research departments. The operationalization of the ICF was described in four ways: for structuring information, linking of categories or content, analysis according to the ICF framework, or development of instruments or models based on the ICF.A majority of papers were reviews and involved researchers only, whereas different stakeholders and VR professionals were involved in the interventions. The components of the ICF that depict functioning and disability were largely incorporated in the VR research. This observation points to the benefit of using a common set of ICF components to inform the selection of set of measurement instruments. Such a process would lead to a single set of standardized measures looking at similar outcomes and make comparability across studies possible However, more research is needed to develop and validate instruments measuring relevant domains including personal factors and to standardize and ease the VR professionals´ use of the ICF.Below is the link to the electronic supplementary material.Supplementary material 1 (DOCX 50 KB)


Plasmodium chabaudi is coordinated with host circadian rhythms. When this coordination is disrupted, parasites suffer a 50% reduction in both asexual stages and sexual stage gametocytes over the acute phase of infection. Reduced gametocyte density may not simply follow from a loss of asexuals because investment into gametocytes (“conversion rate”) is a plastic trait; furthermore, the densities of both asexuals and gametocytes are highly dynamic during infection. Hence, the reasons for the reduction of gametocytes in infections that are out-of-synch with host circadian rhythms remain unclear. Here, two explanations are tested: first, whether out-of-synch parasites reduce their conversion rate to prioritize asexual replication via reproductive restraint; second, whether out-of-synch gametocytes experience elevated clearance by the host’s circadian immune responses.The intraerythrocytic development cycle (IDC) of the rodent malaria P. chabaudi that were in-synch or 12 h out-of-synch with host circadian rhythms. Second, three new experiments examined whether the inflammatory cytokine TNF varies in its gametocytocidal efficacy according to host time-of-day and gametocyte age.First, conversion rate data were analysed from a previous experiment comparing infections of There was no evidence that parasites reduce conversion or that their gametocytes become more vulnerable to TNF when out-of-synch with host circadian rhythms.The factors causing the reduction of gametocytes in out-of-synch infections remain mysterious. Candidates for future investigation include alternative rhythmic factors involved in innate immune responses and the rhythmicity in essential resources required for gametocyte development. Explaining why it matters for gametocytes to be synchronized to host circadian rhythms might suggest novel approaches to blocking transmission. Plasmodium) parasite is synchronous, rhythmic development during asexual replication cycles within host red blood cells. For Plasmodium chabaudi, each intraerythrocytic development cycle (IDC) spans 24 h, at the end of which mature parasites burst to release their merozoite progeny. Each merozoite is committed to either asexual replication or to differentiating into a sexual stage (gametocyte) for transmission to mosquitoes. Like the human malaria Plasmodium falciparum, the asexual development of the rodent malaria P. chabaudi progresses through sequential stages within the IDC in synchrony with each other, transitioning between IDC stages at particular times-of-day [A hallmark of many species of malaria  rather than in the early morning , parasites suffer a 50% reduction of both asexually replicating stages and gametocytes across the acute phase . AsexualP. chabaudi and P. falciparum in response to treatment with low doses of anti-malarial drugs and within-host competition [The “parasite strategy” scenario stems from a body of work revealing that malaria parasites adjust their conversion rate in response to changes in the within-host environment in ways that maximise their fitness , 11–14. petition , 15–18. P. chabaudi gametocytes from the blood is the pro-inflammatory cytokine tumour necrosis factor (TNF) [P. chabaudi-infected mice increases (on average by 44%), regardless of parasite clone and asexual parasite density [P. falciparum via a calcium-cAMP downstream signalling, with PCNA1 (proliferating‐cell nuclear antigen-1) as a possible target [P. chabaudi infected mice, rhythmicity in TNF is also linked to the time-of-day that schizogony occurs [Alternatively , the host’s circadian immune responses could be more effective at removing out-of-synch gametocytes from circulation. This “immune killing” hypothesis requires that: (i) the appearance of a gametocytocidal immune factor in the blood follows a circadian rhythm set by the host’s circadian clock; (ii) the vulnerability of gametocytes varies throughout their development, such that during in-synch infections, gametocytes are at a less vulnerable age when the immune factor appears or peaks, and so, a more vulnerable age coincides with the immune factor in out-of-synch infections; and (iii) the gametocytocidal factor is part of the innate immune response because the costs of being out-of-synch occur in the first few days of infection when primarily innate responses are active. The only gametocytocidal factor reported to rapidly clear or (TNF) . When th density . This in density . Asexuale target . Whethere target . HoweverP. chabaudi. First, conversion was estimated for a previously collected dataset in which parasites were either in-synch or out-of-synch with host rhythms. Conversion rates were estimated using a method for statistical inference which follows each time-series of within-host infection dynamics, including the densities of asexual parasites, RBCs, and the starting gametocyte density for each infection [Here, both the “parasite strategy” (conversion rate modulation) and “immune killing” hypotheses were investigated using nfection , 22. NexP. chabaudi strain AJ ring stage parasites (1 × 106 parasitized RBCs) were harvested from donor mice kept in standard (12-h light: 12-h dark) or reversed (12-h dark: 12-h light) lighting schedules and used to infect recipient mice  in the same lighting schedule as their donor mice, or into mice kept under the opposite (reversed) lighting schedule. Thus, parasites in recipient hosts kept in the same lighting schedule as the donor host remained in synchrony or “in-synch” with the host circadian rhythm and parasites moved between lighting schedules became 12-h “out-of-synch”. This produced four groups of infections: two in-synch and two out-of-synch. Data were collected for days 0–7 post-treatment; blood samples to quantify gametocyte densities (10 μL) were taken every day and total parasite densities (5 μL) were taken on days 1, 3, 5, and 7. DNA and RNA were extracted as described in Schneider et al. [A previously published data set  was usedr et al. . AsexualUnlike previous methods which made unrealistic assumptions about infections , the method used here more realistically infers conversion in dynamic infections . The metThree experiments were conducted to test: (i) whether changes in the concentration of TNF vary by time-of-day of injection and dose ; (ii) whether gametocyte vulnerability to injected TNF varies according to host time-of-day; (iii) whether gametocyte age mediates vulnerability to injected TNF.P. chabaudi clone ER parasitized red blood cells (1 × 106) via intraperitoneal (IP) injection.All mice were 6- to 8-week-old male and female WT C57/BL6 mice . Mice were provided water containing 0.05% para-aminobenzoic acid  and food ad libitum and kept under a standard 12-h light: 12-h dark schedule (except in experiment iii). All experimental mice that were infected received ring stage Uninfected mice received either 30 μg/kg or 60 μg/kg TNF in 100 μL PBS carrier at either lights on  or lights off  via IP injection. ZT refers to “Zeitgeber Time” which is defined as the number of hours elapsed since lights on. Two hours post-treatment with TNF, blood was obtained from all mice individually by cardiac puncture and centrifuged to collect plasma for a murine TNF-specific quantitative enzyme-linked immunosorbent assay .To coincide with peak gametocyte density in control infections , 24 micePer1/2 null mice , generated by David Weaver ) were used. These clock mutant have an impaired TTFL clock and exhibit arrhythmic behaviour when placed in constant conditions such as constant darkness [Both host-time-of day and gametocyte age co-vary in experiment ii and it is possible these factors oppose the effects of TNF on gametocyte density. Therefore, to focus on gametocyte age without the confounding effect of host rhythms, C57/BL6 darkness , 26. To Under a 12-h light: 12-h dark schedule, a new cohort of gametocytes is produced at each ~ ZT17 , 27, traPlasmodium chabaudi gametocytes reach maturity between 24 and 36 h after RBC invasion and become identifiable on blood smears between 18 and 24 h old by their morphology and senesce rapidly post-maturation [P. chabaudi, then infections were run until day 7 PI and ring stages were collected to infect the experimental, arrhythmic Per1/2(−/−) mice. Parasites begin to lose synchrony in Per1/2(−/−) mice after five replication cycles [−1 phenylhydrazine dissolved in 100 μL PBS  [Donor mice were allowed to entrain to their schedules for 1 week prior to infection with n cycles , and so,anaemia)  4 days p10-transformed. Gametocyte densities for “testing the immune killing hypothesis” parts ii and iii were square root-transformed . Five outliers were eliminated in “testing the immune killing hypothesis” part ii due to poor fit, and data were scaled in “testing the immune killing hypothesis” parts ii and iii to have a mean of 0 and a standard deviation of 1, post square root-transformation. Linear models were used to analyse conversion rate estimates (testing the conversion hypothesis) and the effects of host time-of-day on TNF concentration (testing the immune killing hypothesis part (i). Linear mixed-effect models were used to analyse gametocyte density (testing the immune killing hypothesis parts (ii) and (iii), using mouse ID as a random effect. To avoid overfitting due to small sample sizes, “Akaike information criterion-corrected” (AICc) values were calculated for each model, and a change in 2 AICc (ΔAICc = 2) was chosen to select the most parsimonious model. Only models directly reflecting the hypotheses under test were fitted.All analyses were performed using R v. 3.5.1 . To meet assumptions of homogeneity and variance, conversion rate estimates were logNo evidence was found to support the hypothesis that parasites out-of-synch with host rhythms reduce conversion Table ; Fig. 2.Dose (P < 0.0001) and host time-of-day (P < 0.001) contributed substantially to TNF concentration 2 HPI Fig.  but therNo evidence was found to support the interaction between host time-of-day (i.e. injection time) and TNF on gametocyte density Fig. . Indeed,w = 0.409; Table w = 0.267; Table The most parsimonious model included only gametocyte age  and also the full dataset of all infections  [The conversion results suggest that either parasites do not adjust their conversion rate when out-of-synch with host rhythms, or that the methods utilised here were unable detect change. Conversion is a key determinant of transmission potential  and a phenotypically plastic trait. Conversion is reduced by ication)  so parasication)  is not sication) —particulication)  to reliaication) . WhetherThat TNF rhythms and the age of gametocytes had no significant effect on gametocyte density was unexpected. Schizogony  causes an elevation of TNF that may be capable of sterilizing gametocytes . The conTo address the potentially confounding effects of schizogony-induced TNF, future work could aim to block TNF (following ) in circUnderstanding consequences of being out-of-synch with host circadian rhythms is important for unravelling the evolutionary drivers of rhythmic development in malaria parasites. Knowledge of the benefits, or costs, to parasites of being in-synch with host rhythms will allow them to be harnessed for the development of novel anti-malarial treatments; for example, if gametocytes show time-of-day-specific vulnerabilities, drugs could take advantage of rhythmic weaknesses. Further, understanding how malaria parasites respond to temporal variation in the within-host environment is increasingly important as some mosquito populations are reported to have shifted the timing of blood-foraging rhythms in response to the widespread use of long-lasting insecticide-treated bed nets , 34. SinAdditional file 1. Additional table and figure.


Background:Mycobacterium tuberculosis (TB) is one of the most important causes of human mortality. Approximately one-third of the world’s population is infected with TB and 5–10% of them develop the active form of the disease. Cytokines play a major role in the host defense process against Mycobacterium infections. Among these cytokines, tumor necrosis factor alpha (TNF-α) has a prominent role in the defense of and pathological responses to tuberculosis.Materials and methods: A case-control study was carried out from May 2016 to June 2017. 45 patients with diagnosis of tuberculosis (smear and positive culture) were included as case group and 45 healthy subjects as control group. The serum levels of TNF-α were determined with the enzyme-linked immunosorbent assay (ELISA) method.Results: The concentration of TNF-α in patients with TB was significantly higher than in the control group (P<0.05). However, the difference was only significant in the age groups 20–30 and 50–60 years; in the age groups 30–40, 40–50 and 50–70 years, the difference was not significant, although certain trends were apparent.Discussion and conclusion: Since the level of serum TNF-α is higher in patients with pulmonary tuberculosis than in individuals without it, the measurement of TNF-α levels can be useful as a probable marker for the diagnosis of tuberculosis. Mycobacterium tuberculosis (TB) is one of the most important causes of mortality in humans. According to the World Health Organization report in 2017, tuberculosis is responsible for 1.7 million deaths every year . It. ItMycobery year , 7]. T-. T-Mycobery year . Tumor Nery year . TNF-α bery year . Awareneery year . The purA case-control study was conducted from June 2016 to July 2017. The case group consisted of 45 patients with a definite tuberculosis diagnosis (with smear and positive culture), and 45 healthy subjects were selected as the control group.TNF-α was measured using the enzyme-linked immunosorbent assay (ELISA) .ANOVA, Tukey’s test, and Bonferroni correction were employed, as was the Shapiro-Wilk test (P≤0.05) due to the low sample size.The age of patients ranged from 20 to 60 years, with a mean age of 41±1.5 years. The study population included 52 (68.8%) men and 38 (42.2%) women. The mean serum level of TNF-α in the patient group were 10.6 pg/ml with the lowest and highest rates being 1.2 and 186.2 pg/ml, respectively. The mean serum level of TNF-α in the control group was 8.26 pg/ml, with the lowest and highest rates being 1.2 and 240 pg/ml, respectively Figure 1 . Data were evaluated in Prism 6 GraphPad software, using one-way ANOVA and Tukey’s tests. A comparison of serum levels of TNF-α with different age groups is shown in Figure 3 Mycobacterium tuberculosis. The results of this study showed that TNF-α production in patients with Mycobacterium tuberculosis infection was greater than serum levels of TNF-α in the control group. Cytokines play a crucial role in the host defense process against Mycobacterium infections . I. IMycobafections . In thisfections  studied fections , 17], [, [Mycobafections . The resfections  studied fections , serum lfections , the serpatients . The difThe findings of this study show that cytokines can be used as a possible marker for the diagnosis of tuberculosis. Measuring the level of cytokines can be used to supplement the diagnostic tests for Mycobacterium. More studies are needed to confirm these findings.The authors declare that they have no competing interests.


Sturnus vulgaris) flocks are under attack by a raptor, such as a peregrine falcon , they show a great diversity of patterns of collective escape. The corresponding structural complexity concerns rapid variation in density and shape of the flock over time. Here, we present a first step towards unravelling this complexity. We apply a time series analysis to video footage of 182 sequences of hunting by falcons on flocks of thousands of starlings close to two urban roosts during winter. We distinguish several types of collective escape by determining the position and movement of individuals relative to each other (which determines darkness and shape of the flock over time) as well as relative to the predator, namely ‘flash expansion’, ‘blackening’, ‘wave event’, ‘vacuole’, ‘cordon’ and ‘split’. We show that the specific type of collective escape depends on the collective pattern that precedes it and on the level of threat posed by the raptor. A wave event was most likely to occur when the predator attacked at medium speed. Flash expansion occurred more frequently when the predator approached the flock at faster rather than slower speed and attacked from above rather than from the side or below. Flash expansion was often followed by split, but in many cases, the flock showed resilience by remaining intact. During a hunting sequence, the frequencies of different patterns of collective escape increased when the frequency of attack by the raptor was higher. Despite their complexity, we show that patterns of collective escape depend on the predatory threat, which resembles findings in fish.Collective behaviour of animals has been a main focus of recent research, yet few empirical studies deal with this issue in the context of predation, a major driver of social complexity in many animal species. When starling  depend on the preceding pattern and on details of attack. A higher frequency of attack during a hunting sequence resulted in a higher frequency of collective escape events. Flash expansion happened most often when the predator attacks at greater speed. A wave event was most likely when the raptor attacks at medium (rather than high or low) speed. These results provide a first quantitative approach to social complexity in collective avoidance of a predator.The online version of this article (10.1007/s00265-018-2609-0) contains supplementary material, which is available to authorized users. Predator-prey relationships are the result of an evolutionary arms race in which the prey adopts strategies to reduce the risk of being captured  and patterns of collective escape of schools of minnows  in response to a raptor . Termini is situated in the city centre, and the roost there was used by up to 20,000 starlings. This site was under a relatively low predatory pressure and experienced regular hunts of only two peregrine falcons. The other roosting site (Eur) is located in the southern part of Rome, and was used by about 60,000 starlings and five peregrine falcons during the study period , data were collected on flocks of European starlings at two urban roosting sites (Termini and Eur) in Rome, Italy. These roosting locations are spaced about 10 km from each other and are both regularly visited by peregrine falcons  and MiniDV digital tapes were used. Two operators (CC and FZ) stood at a fixed location in front of the roost. In total, these recordings comprise 16 h of footage. Aerial manoeuvres of starling flocks, occurring as collective responses to the falcon, were recorded whenever possible.All video materials were digitalized in MP4. Sequences in which the falcons were hunting a flock were selected. We defined a hunting sequence from when the falcon starts to pursue the flock until it either catches a starling or withdraws from the flock  and location relative to the flock (below/side/above). For this classification, we only included attacks that occurred primarily in the two-dimensional plane . We distinguished between attacks that followed each other fast (referred to as repetitive attacks) from attacks that were isolated in time (referred to as isolated attacks) based on an inter-attack time of 5 s. The threshold of 5 s came from the distribution of inter-attack times Fig. . We measFor wave events that showed two or more pulses, we analysed the duration of the interval between pulses. The frames showing the wave event were analysed using ImageJ  higher than 2 were considered as significantly worse than the best model.The time it took for flock dilution to occur after an attack was 15.0 ± 2.4 s (mean ± SEM). Using a chi-squared test, we tested whether certain collective escape responses occurred more often than expected by a random distribution of events before or after an attack within a hunting sequence. The null hypothesis was that there is no relationship between the occurrence of attacks and collective escape patterns within a hunting sequence; thus, the distribution of collective escape patterns through time is unaffected by the timing of the attacks, which would make them random. For all statistics, we applied a Bonferroni correction. General linear models were used to examine correlations among behaviours of the flock and the falcon. In one of these models, the log-transformed frequency at which collective escape responses occurred within a hunting sequence was the response variable and the candidate explanatory variables were attack frequency, collective escape type, roosting site and the success of the falcon, using a Gaussian error distribution. Other models were used to test the effects of attack strategy, including explanatory variables such as attack speed and location, and roosting site on the probability of each of the specific collective responses occurring within or after 5 s of an attack. For these models, we used a logistic regression. Selection of the best model was based on the Akaike information criterion (AIC) scores, where models with a Δt test for independent samples  and vacuole the least common (N = 5)  = 365.14, p < 0.001  = 52.849, p = 0.004 , sometimes from the side (N = 36) and seldom from below (N = 18). It mostly attacked at medium speed (N = 144), in 16 cases at low speed and in 15 cases at high speed. Fifty-five of 175 attacks occurred within 5 s before or after another attack.Hunting sequences with a higher frequency of attack were associated with a higher frequency of blackening, wave events and flash expansion Fig. , Table 3The probability of a flash expansion occurring immediately after an attack (within 5 s) was highest when attacks were done at high speed rather than medium or low speed and when they came from above rather than from the side or below Fig. . Wave evn = 54). Wave events lasted 3.5 ± 0.23 s (mean ± SEM) and comprised 2.88 ± 0.19 pulses . The time between pulses was on average 0.86 ± 0.44 s when estimated with ImageJ , an individual-level analysis was not possible. Thus, the mechanisms underlying these escape responses cannot be determined conclusively in starling flocks with our data. In fish schools, blackening was shown to result from compacting  are clearly distinct from one another and could not be confused. Our observations were conservative, in the sense that we may have missed some of the collective patterns if they were hidden to the observer by overlapping flocks in between the observer and the collective event. Note that our observations were continuous, and we selected only footage where the flock and the falcon were clearly visible (omitting unclear footage).Another potential limitation of our study is that falcons experience patterns of collective escape differently from an observer because of their closer spatial location relative to the flock. Indeed, future work focusing on the viewpoint of the raptor (e.g. with the use of drones) is of great importance to gain information on how collective escape patterns are perceived by the falcon.In sum, we have shown that the pattern of collective escape in starling flocks depends on the specifics of the hunting behaviour of the falcon and on the preceding pattern of collective escape. Such complex and dynamic patterns of collective escape emerge from the interplay between the antipredatory actions of thousands of prey and the hunting behaviour of the predator. These dynamics show similarity with collective patterns of escape in fish, suggesting that despite being complex, general rules may underlie these systems.ESM 1ESM 2ESM 3(DOCX 1203 kb)


Additional measurements comprised key circulating BV‐regulating hormones such as pro‐atrial natriuretic peptide (proANP), copeptin, aldosterone and erythropoietin (EPO), as well as central hemodynamics and arterial stiffness via carotid–femoral pulse wave velocity (PWV). Carotid–femoral PWV was increased (+20%) in HFpEF patients versus control individuals. With respect to hematological variables, plasma volume (PV) did not differ between groups, whereas BV was decreased (−14%) in HFpEF patients. In consonance with the hypovolemic status, Hbmass was reduced (−27%) in HFpEF patients, despite they presented more than a twofold elevation of circulating EPO (+119%). Plasma concentrations of BV‐regulating hormones, including proANP (+106%), copeptin (+99%), and aldosterone (+62%), were substantially augmented in HFpEF patients. HFpEF patients may present with hypovolemia and markedly reduced Hbmass, underpinned by a generalized overactivation of endocrine systems regulating fluid homeostasis and erythropoiesis. These findings provide a novel perspective on the pathophysiological basis of the HFpEF condition.A fundamental tenet of heart failure (HF) pathophysiology hinges on a propensity for fluid retention leading to blood volume (BV) expansion and hemodilution. Whether this can be applied to heart failure patients with preserved ejection fraction (HFpEF) remains uncertain. The present study sought to determine BV status and key hormones regulating fluid homeostasis and erythropoiesis in HFpEF patients. BV and hemoglobin mass (Hb HFpEF patients may present with hypovolemia and markedly reduced hemoglobin mass, underpinned by a generalized overactivation of endocrine systems regulating fluid homeostasis and erythropoiesis. These findings provide a novel perspective on the pathophysiological basis of the HFpEF condition. Prior to the start of the experiments, informed oral and written consents were obtained from all participants.−1), relevant structural/functional cardiac alterations, and no history of iron deficiency (ID) anemia. Healthy age‐ and sex‐matched individuals were recruited from the community and excluded if they had any chronic medical illness, were taking daily prescription medications, had current medical symptoms, or were performing aerobic exercise on a regular basis given its potential effect on BVs and Hbmass (Montero and Flammer Twenty stable HFpEF patients (left ventricular ejection fraction (LVEF) = 55.7 ± 4.7%, 25% females) were recruited from the HF outpatient clinic of the University Hospital of Zurich. Inclusion criteria comprised: signs and symptoms of HF . Duplicates of blood samples were centrifuged and stored at −80°C for further examination.All measurements were accomplished within a period spanning from 8:30 mass comprised the classic carbon monoxide (CO) rebreathing technique integrated in a fully automated system with a very low typical error of measurement  , and hematocrit (Hct) with an hemoximeter . Subsequently, individuals breathed 100% O2 for 4 min to flush the nitrogen from the airways. After closing the O2 input, a bolus of 1.5 mL kg−1 of 99.997% chemically pure CO  was administrated into the breathing circuit. Individuals rebreathed this gas mixture for 10 min. Then, an additional 2‐mL blood sample was obtained and analyzed in duplicate as aforementioned. The change in %HbCO is used to calculate Hbmass, taking into account the amount of CO that remains in the rebreathing circuit at the end of the procedure. Total red blood cell volume (RBCV), plasma volume (PV), and BV were determined from Hbmass, Hb, and Hct .Blood samples (5 mL) from the antecubital vein were collected anaerobically in heparinized glass syringes. Serum creatinine was assessed via the kinetic Jaffe reaction . Estimated glomerular filtration rate (eGFR) was calculated using the CKD‐EPI Creatinine Equation (2009) according to the guidelines from the National Kidney Foundation  aspect ratio, a surrogate of central venous pressure (CVP), was determined at the level of the cricoid cartilage using the method described by Keller et al.  In briefCarotid–femoral pulse wave velocity (PWV) was assessed with arterial tonometry  according to established guidelines .where Statistical analysis was performed using SPSS 22.0 . Data were tested for normal distribution with the Kolmogorov–Smirnov test and for homogeneity of variances with Levene's test.t‐test, and Mann–Whitney U‐test were used to compare dichotomous, parametric, and nonparametric data, respectively, between HFpEF patients and control individuals. Data are reported as mean ± SD unless otherwise stated. A two‐tailed P‐value less than 0.05 was considered significant.Fisher's exact test, independent P = 0.003) and NT‐proBNP (P < 0.001) while eGFR was mildly reduced (P = 0.002) compared with control individuals. Venous Hb concentration (P = 0.002) and Hct  were also reduced in HFpEF patients. No difference was detected for venous sO2  between groups. The most prevalent comorbidities in HFpEF patients were hypertension (80%), coronary artery disease (55%), and diabetes mellitus (25%). With respect to the medication, beta‐blockers (70%), loop diuretics (60%), and angiotensin‐converting enzyme inhibitors/angiotensin II‐receptor blockers (60%) were common and similarly prevalent in HFpEF patients. Control individuals were healthy and did not take medications.Baseline characteristics of HFpEF patients and control individuals are reported in Table P = 0.034). Aortic Aix@75 was similar between HFpEF patients and control individuals. Likewise, carotid distensibility did not differ between groups. In contrast, carotid–femoral PWV was increased in HFpEF patients (P = 0.047).Table −1, P = 0.324), whereas RBCV  and BV  were diminished in HFpEF patients. Similar results were observed for BVs expressed in absolute units   and absolute units .Blood volumes in HFpEF patients and control individuals are displayed in Figure −1, P < 0.001), copeptin , and aldosterone  concentrations were substantially augmented in HFpEF patients. Moreover, HFpEF patients presented more than twofold enhancement of EPO concentration , which resulted in similar O/P EPO ratio  between groups. Albumin levels did not differ between HFpEF patients and control individuals .Figure mass, and endocrine regulation in stable HFpEF patients and healthy age‐ and sex‐matched control individuals. The main findings evidence that HFpEF patients may present with: (1) preserved PV, (2) marked reductions in RBCV and Hbmass, resulting in hypovolemia, and (3) augmented levels of BV‐regulating hormones including copeptin, aldosterone, and EPO, denoting the activation of negative feedback mechanisms elicited by hypovolemia.The present study assessed intravascular volumes, HbFluid retention represents a primary clinical manifestation of decompensation in HF patients , a hallmark of exercise tolerance routinely used to ascertain cardiac transplant eligibility in patients with advanced HF  and hemodynamic  variables determining convective oxygen delivery (Montero et al. This investigation reveals another intriguing hematological characteristic of stable HFpEF patients. While the high prevalence of anemia, determined from Hb concentration cutoff values, is well established in HF populations (Brucks et al. mass in HFpEF patients. BV is subjected to endocrine regulation via natriuretic peptides, vasopressin, and the RAAS (Gauer and Henry The question arises as to which mechanisms underlie hypovolemia and reduced Hbmass might be exacerbated in later stages of the HFpEF condition. Moreover, the narrow range of diuretic doses in our cohort limits the possibility to assess the specific impact of diuretics on PV and hemodynamics (Montero and Flammer mass in HFpEF patients. In addition, HFpEF patients may present ID, a prevalent comorbidity associated with severe diastolic dysfunction and decreased exercise capacity (Bekfani et al. There are some limitations to this study that require comment. The recruitment of stable HFpEF patients with preserved functional capacity precludes the generalization of findings to patients with advanced disease, albeit the demographics and clinical characteristics of the HFpEF group closely matched those of systematic HF reviews (Montero et al. mass, in parallel to a generalized overactivation of hormones regulating fluid homeostasis and erythropoiesis. These data provide key pathophysiological evidence distinguishing a plausibly prevalent phenotype of HFpEF. The implication of these findings for identifying effective therapies in this population remains to be substantiated in future studies.In stable HFpEF patients, intravascular volume status is characterized by hypovolemia and reduced HbNone declared.Figure S1. Potential mechanisms linking hypovolemia with impaired cardiac function.Click here for additional data file.


Indy (I’m not dead yet) gene encodes a plasma membrane transporter of Krebs’ cycle intermediates with highest affinity for citrate. Indy is the fly homolog of a mammalian mIndy (SLC13A5), which has the same physiological function. Reduced expression of the Indy gene extends longevity in fruit flies and worms. Genetic and pharmacological INDY reduction affects metabolism in flies, worms, mice, rats and non-human primates by affecting the levels of cytoplasmic citrate. In flies, INDY is predominantly expressed in the midgut, fat body and oenocytes, all tissues with a key role in metabolism. Our first goal was to examine our working hypothesis that INDY reduction in the midgut regulates citrate levels leading to metabolic changes that preserve intestinal stem cell (ISCs) homeostasis and slows aging by modifying Insulin/Insulin-like signaling (IIS). ISC homeostasis is vital for midgut homeostasis and contributes to health and longevity. We found that reduction of Indy preserves ISC homeostasis and intestinal integrity. The IIS is a key nutrient sensing pathway, which regulates growth, metabolism and longevity. Indy reduction is associated with decreased IIS activity. Our second goal was to examine the role of IIS in Indy mediated changes in ISC homeostasis and health. We found that at least some of INDY’s beneficial effects on fly health are mediated by the IIS.


The present study aimed to compare variations in quantified tumor necrosis factor-alpha ; those with T2D (n=37); those with POD2B/T2D (n=40); and healthy subjects (n=39). Glycosylated hemoglobin (HbA1c) and blood glucose (BG) levels were quantified in each subject. Data were collected on body mass index (BMI), loss of insertion (LI), and probe depth (PD). Gingival crevicular fluid samples were collected from the most acutely affected periodontal pocket and gingival sulcus in each subject, and TNF-α was quantified by multiplex analysis. Levels of the cytokine TNF-α levels, LI, PD, BMI, BG, and HbA1c by group. Differences (p<0.001) were found for LI, PD, BG, and HbA1c. A Spearman test was used to calculate possible correlations between TNF-α levels and LI or PD identified a weak but significant negative correlation of TNF-α with LI , and a moderately positive correlation of LI with PD . Kruskal Wallis tests was used to identify differences in TNF-α levels and the presence of POD2B, POD2B/T2D, or T2D, suggesting the absence of any direct relationship between progression of these diseases and TNF-α levels. However, a correlation was present between low TNF-α concentrations and greater LI. No variation was found between TNF- Diabetes mellitus is a public health problem in many countries. In medical terms, it encompasses a group of metabolic disorders with heterogeneous clinical and genetic characteristics, which manifest as abnormally high blood glucose levels. These disorders have a profound impact on health in affected individuals, causing high morbidity and mortality rates, and constitute an economic burden on health systems , 2. The e.g., diabetes mellitus, HIV), which can depress host defenses, and the environment  [Periodontitis (POD) is the most common form of periodontal disease. It is more prevalent in adults but can occur in children. Although multifactorial in origin, three main factors are involved in POD appearance and evolution: accumulation of plaque and calculus; level of bacterial virulence; and cellular immune response . Evoluti stress) .Both T2D and POD influence oral cavity health. Diabetes is a risk factor for gingivitis and POD when related to formation of a more persistent inflammatory infiltrate. An inverse influence may also exist in that POD could be a risk factor for diabetic decompensation . A complα) is a proinflammatory mediator considered to be a soluble mediator released from immunocompetent cells. It exercises a wide range of proinflammatory and immunomodulatory effects in different cell populations, such as stimulating prostaglandin synthesis, promoting tumors in a variety of cancers, producing proteases, and activating osteoclastic function and therefore bone resorption. Its myriad functions suggest that TNF-α plays an important role in mediating the immune-inflammatory responses initiated by infection or other types of damage [α is also vital for maintaining chronic inflammation, angiogenesis, tissue remodeling, tumor growth, and metastasis; TNF-α blockers are therefore effective in treating a variety of acute and chronic inflammatory conditions [Recent studies have been focused on the role of cytokines in periodontal diseases in diabetic patients. Cytokines are a group of cell regulators vital in the production and activation of effector cells that initiate and regulate different immune and inflammatory responses –16. Tumof damage . During nditions .α production occurs in response to stimuli from cell types such as macrophages, neutrophils, keratinocytes, adipocytes, fibroblasts, and NK, T and B cells. High serum levels of this cytokine have been detected in patients with POD, suggesting it may be contributing to pathogenesis. Its activation also stimulates bone resorption by induction in osteoclast progenitor proliferation, and production of extracellular matrix metalloproteinases, cytokines, collagenase, and prostaglandins [An important proinflammatory cytokine and immune response modulator, TNF-glandins –22.α levels in patients with POD and/or T2D and to identify any relationships between this cytokine and these diseases.The aim of this study was to compare the variations in quantified TNF-A cross-sectional study was done and approved by the Institutional Bioethics Committee. Subjects were selected from the Admission Dental Clinic, Faculty of Dentistry, Autonomous University of Yucatan (UADY); after explanation of the procedure, those choosing to participate signed an informed consent.Presence of T2D was identified based on the 2019 American Diabetes Association parameters : glycosy g for 5 minutes and at 4°C, until reaching a final elution volume of 80 μL. Of this volume, 40 μL were tested with Luminex platform  and analyzed with a MILLIPLEX analyst software . Results were expressed per mL of elution to measure TNF-α levels in the total amount (pg) and concentration volume according to the formula TNF-α (pg)/volume (μL).According to specific classification for periodontal and peri-implant diseases and conditions, patients included were targeted with stage 2, grade B POD . Periodoα, loss of insertion , probe depth (PD), body mass index (BMI), blood glucose (BG), and glycosylated hemoglobin (HbA1c) by group. A Spearman test was used to evaluate the possible existence of a correlation between TNF-α count and LI or PD .Kruskal-Wallis test was applied to identify any differences in the data for TNF-Analysis of the BMI, BG, and HbA1c data showed 31.87% of the total sample to exhibit glycemic levels outside normal levels . In the α concentration were found between groups. A Spearman test identified a weak but significant negative correlation between TNF-α levels and LI , and a moderate but significant positive correlation between LI and PD .Analysis of periodontal condition identified differences between groups for LI and PD (p<0.001), with the highest values in both cases being in POD2B and POD2B/T2D . No diffThere are multiple conditions that can affect the periodontal health. Locally, factors such as dental malposition, poorly adjusted restorations, maxillofacial fractures, and uncontrolled use of chlorhexidine-based products have been reported , 26. Likα is an important proinflammatory cytokine and modulator of the immune response. It is produced in response to stimuli from cell types such as macrophages, neutrophils, keratinocytes, adipocytes, fibroblasts, and NK, T and B cells. High serum TNF-α levels have been detected in patients with POD since it contributes to disease pathogenesis. This cytokine stimulates bone resorption by inducing osteoclast progenitor proliferation, as well as production of chemokine, extracellular matrix metalloproteinases, cytokines, collagenase, and prostaglandins. Cytokine concentration in GCF has been linked to degree of glycemic control in diabetic patients [TNF-patients –22.γ, CCL5, TNF-α, prostaglandins, and metalloproteinases. These mediators alter the activity of leukocytes and osteoblasts-osteoclasts and promote the tissue remodeling process both locally and systemically. The proinflammatory cytokine TNF-α regulates production of collagenase, prostaglandin E2, molecular adhesion cells, and factors related to bone resorption. Secreted mainly by monocytes and macrophages, elevated TNF-α levels have been observed in chronic gingival inflammation processes and in GCF in patients with POD [Studies have shown that diabetic patients with periodontal infection have a higher risk of losing control of their glycemic condition; and this deteriorates over the long term compared to diabetic patients who do not suffer POD –32. In twith POD , 33, 34.α concentration were observed in the present study. This is similar to the lack of differences in TNF-α concentration in the GCF between patients with POD2B, aggressive POD, or systemically healthy patients reported in a Turkish population [α concentration were observed when comparing active and inactive sites in 56 patients in a Chilean population diagnosed with moderate to advanced chronic POD [α concentration was noted between POD2B/T2D and POD2B patients [No intergroup differences in TNF-pulation . Howeveronic POD , 37. Thepatients , 39.α concentration in patients with POD2B/T2D or POD2B have also been described in a Portuguese population, but with no differences between groups [α levels and gingival tissues in patients with POD [α expression in POD2B/T2D patients because hyperglycemia can overregulate levels of TNF-α, and other cytokines such as GM-CSF and IL-6, in both healthy and periodontal affected tissues [Increases in TNF-n groups , 41, likwith POD , 31. The tissues , 43. Thi tissues .α levels in patients with or without POD2B, and neither did they observe decreases in TNF-α levels after nonsurgical periodontal treatment [α concentrations between sites with mucositis and peri-implantitis in both GCF and saliva [α concentration between data from the baseline condition, at 14 days and at 6 months [Reis et al. found no differences in TNF-reatment . In anotd saliva . In a co6 months .α concentration and T2D when comparing healthy and infected sites in patients with and without T2D [α can be attributed to an increase in RAGE or expression of TLR4, which directly affect the response of epithelial cells and their antagonists inducing the proinflammatory cytokine response, as well as an increase in cell surface receiving capacity [Duarte et al. found significant differences between TNF-hout T2D , 47. Thicapacity . These rcapacity .α concentrations have been reported after nonsurgical periodontal treatment, with higher levels in healthy subjects than in unhealthy patients [α levels were observed in healthy subjects than in the POD2B, POD2B/T2D, and T2D groups. A possible explanation for these reduced inflammatory protein levels in patients with these chronic diseases is that host immune response may be diminished.Increases in TNF-patients . These rα concentrations may be due to several factors including periodontal disease severity, subject age, sample type, population type, and technique details such as storage temperature and pretest storage time [Interstudy variation in TNF-age time .α concentrations, possibly explaining the contrasts between different studies mentioned previously.GCF has been widely used as a diagnostic tool for various periodontal diseases. Available evidence indicates that GCF can influence the progression of periodontal diseases when combined with systemic diseases, suggesting that local changes in GCF can be reflected as systemic inflammation through direct expression of circulating inflammatory mediators . The preα cytokine; however, low TNF-α concentrations are directly correlated with greater insertion loss and probe depth.The present results indicate that good control of BG in patients with POD2B/T2D can directly influence expression of the TNF-


Escherichia coli, DnaA molecules construct functional oligomers at the origin oriC, enabling localized unwinding of oriC and stable binding of DnaB helicases via multiple domain I molecules of oriC-bound DnaA. DnaA-bound DnaB helicases are then loaded onto the unwound region of oriC for construction of a pair of replisomes for bidirectional replication. However, mechanisms of DnaB loading to the unwound oriC remain largely elusive. In this study, we determined that His136 of DnaA domain III has an important role in loading of DnaB helicases onto the unwound oriC. DnaA H136A mutant protein was impaired in replication initiation in vivo, and in DnaB loading to the unwound oriC in vitro, whereas the protein fully sustained activities for oriC unwinding and DnaA domain I-dependent stable binding between DnaA and DnaB. Functional and structural analyses supported the idea that transient weak interactions between DnaB helicase and DnaA His136 within specific protomers of DnaA oligomers direct DnaB to a region in close proximity to single stranded DNA at unwound oriC bound to DnaA domain III of the DnaA oligomer. The aromatic moiety of His136 is basically conserved at corresponding residues of eubacterial DnaA orthologs, implying that the guidance function of DnaB is common to all eubacterial species.Chromosomal replication initiation requires dynamic mechanisms in higher-order nucleoprotein complexes that are constructed at the origin of replication. In  Escherichia coli occur at the unique replication origin, oriC, which has a sophisticated structure that directs unwinding of duplex DNA and loading of replicative helicases . In these steps, the initiator DnaA molecules construct specific oligomers with the aid of DiaA (a DnaA-binding protein) and appropriately located DnaA-binding sequences (DnaA boxes) in the oriC DnaA-oligomerization region (DOR) . The left-half DOR adjoins the oriC duplex unwinding element (DUE) and includes a specific binding site (IBS) for IHF (Integration host factor), a DNA-bending protein. The DnaA subcomplex including the left-half DOR and IHF causes localized unwinding of DNA in the DUE . The unwound single-stranded (ss) DUE is stabilized by binding to the DnaA oligomer, which is a prerequisite for DnaB loading  . ATP-bou loading .oriC . DnaB is a replicative helicase with a homohexamer structure and ring  configuration : a deletion analysis of oriC suggests that each DnaA subcomplex constructed on the left- and right-half subregions of DOR binds a single DnaB helicase (or DnaBC complex)  . Also, dcomplex) . The twoh strand .Aquifex aeolicus DnaC  and competes with the DnaBC complex in DnaA binding . Domain I contains a specific site for binding to DnaB and DiaA . A previous study assessed specific inhibition with monoclonal anti-DnaA antibodies, and the results suggested that the DnaA Pro111–Gln148 region includes a site for DnaB interaction .In addition to domain I, DnaA domains II–III are thought to contain a second site for DnaB binding, which is important for DnaB loading on eraction . Anothereraction . Deletioon  has been described previously (The DOR dsDNA fragment (ΔDUE) and 28-mer T-rich ssDUE strand have been described previously . M13KEW1eviously . pBSoriCeviously ; the ampeviously . M13-A-seviously .Buffer G contained 20 mM HEPES-KOH (pH 7.6), 5 mM magnesium acetate, 1 mM EDTA, 10% glycerol, 0.1% Triton X-100, and 4 mM dithiothreitol (DTT). Buffer F contained 20 mM Tris–HCl (pH 7.5), 8 mM DTT, 10 mM magnesium acetate, 125 mM potassium glutamate, 3 mM ATP, and 0.5 mg/mL bovine serum albumin (BSA). Buffer ABC contained 20 mM Tris–HCl (pH 7.5), 0.1 mg/mL BSA, 8 mM DTT, 8 mM magnesium acetate, 0.01% Brij-58, and 125 mM potassium glutamate.E. coli strain KA450 [ΔoriC1071::Tn10 rnhA199(Am) dnaA17(Am)] from pKA234 or pH136A (a derivative of pKA234 encoding DnaA H136A), and purified as previously described (Wild-type DnaA and DnaA H136A proteins were overproduced in escribed .Flow cytometry was performed as described previously . BrieflyoriC replication (except for DnaA) was prepared from E. coli strain WM433 (dnaA204) , and WM433 crude extract (200 μg), as described  . Reactioescribed .oriC plasmid  and HU protein (16 ng), followed by brief incubation with P1 nuclease. DNA was purified and incubated with restriction enzyme BseRI, followed by analysis by agarose-gel electrophoresis, with ethidium bromide staining.The assay was performed essentially as described previously . BrieflyoriC dsDNA fragment, DOR(ΔDUE) (5 nM) were incubated on ice for 5 min, followed by incubation for 5 min at 30°C with 32P-labeled 28-mer T-rich ssDUE strand (2.5 nM) in the presence of λphage DNA (25 ng). The resultant DNA complexes were analyzed by 4% polyacrylamide-gel electrophoresis at 4°C.The assay was performed as described previously . BrieflyThe assay was performed as previously described . BrieflyoriC , including the entire oriC and flanking regions , washed twice with buffer G (12.5 μL) containing 75 mM KCl, and dissolved in SDS sample buffer. Proteins were quantified using silver staining and quantitative protein standards, as we previously performed  and pBSoriC or pBSoriCΔDUE (1.6 nM) were incubated for 15 min at 30°C in buffer F (10 μL) containing 40 nM IHF, 200 nM His-DnaB K236A, and the native (non-tagged) DnaC (200 nM). After addition of Co2+-conjugated magnetic beads  and incubation for 15 min at 4°C, the beads and bound materials were collected by magnetic pull-down and washed in buffer F containing 100 mM NaCl and excluding BSA. His-DnaB-bound plasmid DNA was eluted in standard SDS sample buffer, and analyzed by 1% agarose-gel electrophoresis and ethidium bromide staining.The assay was performed under similar conditions to the Form I∗, 26 ng β-clamp subunit, 1 mM ATP, 0.25 mM each of GTP, CTP, and UTP, and 0.1 mM each of dNTP and [α-32P]dATP. DNA polymerase III∗ is a subcomplex of DNA polymerase III holoenzyme lacking the β-clamp subunit. Reactions were stopped by addition of 1 mL 10% trichloroacetic acid, and the amounts of synthesized DNA were measured by liquid scintillation.The assay was performed as described previously . BrieflydnaA-coding region downstream of the arabinose-inducible PBAD promoter -sensitive dnaA46 host strain (KA413). Even in the absence of the inducer arabinose, introduction of pKA234, but not the vector pING1, enabled the KA413 cells to grow at 42°C, presumably because of leaky expression of the pKA234 dnaA gene (Table 1). In similar experiments that we previously performed, DnaA amounts in cells bearing pKA234 was 1.3- to 2.8-fold higher than those in cells bearing pING1  sensitive growth (Table 1).A putative DnaB interaction site has been speculated to reside in a DnaA domain III N-terminal region spanning Lys135–Gln148. We conducted an alanine-scanning analysis on all the amino acid residues in this region except for Gly144, which lacks a side chain. Plasmid pKA234 contains the wild-type promoter . Derivatng pING1 . Unlike Table 1). DnaA F141A also resulted in growth inhibition at 42°C, suggesting that substitution of the hydrophobic aromatic side chain of phenylalanine resulted in DnaA structural changes that indirectly inhibited the function of the N-linker motif residues, such as Val142.Val142 is a component of the N-linker motif , a conserved sequence in AAA+ family proteins that is thought to interact with the adenine moiety of ATP . DnaA V1oriC repressing overinitiation (Table 1). When cells were incubated at 42°C, a temperature at which replication initiation is stimulated even in wild-type cells, a severe inhibitory effect of DnaA E143A for cell growth by overinitiation might be suppressed. Based on these ideas, we excluded DnaA F141A and V142A for further analyses in this study.We previously indicated that Glu143 within the N-linker motif is important for stable binding of ATP and ADP, and DnaA E143A causes moderate overinitiation of replication at 30°C in the absence of wild-type DnaA, presumably because of rapid exchange of bound ADP to ATP . When coitiation . These pin vivo of DnaA H136A, we used flow cytometry to determine the chromosomal replication modes of dnaA46 cells containing derivatives of pKA234. Cells were cultured for exponential growth at 30°C and shifted to 42°C for inactivation of the intrinsic DnaA46. Further incubation with rifampicin and cefalexin enabled run-out replication of the chromosomes. At 30°C, distinct DNA peaks were seen for each strain . In dnaA46 cells with the pING vector, the two-chromosome peak predominated, with minor peaks for one, three, four, and five chromosomes, indicating asynchronous initiations (dnaA (pKA234) into dnaA46 cells stimulated initiation and resulted in a predominant four-chromosome peak. DnaA F46A protein is impaired in stable DnaB binding and DnaB loading at oriC  gave a similar profile to that of dnaA46 cells with the pING vector, whereas moderate stimulation was detected with dnaA H136A (pH136A). This stimulation could be a consequence of mixture of DnaA46 and DnaA H136A proteins (see below). At 42°C, the profiles with pING and dnaA H136A were fundamentally similar with respect to inhibition of initiation, whereas moderate stimulation occurred with dnaA F46A, as previously observed , suggesting preservation of the overall protein structure of domain III. By contrast, when replication initiation was assessed with an oriC plasmid and a replicative-protein extract, even the ATP form of DnaA H136A was inactive, unlike that of wild-type DnaA .DnaA H136A protein was overproduced and purified, and shown to have high-affinity binding of ATP and ADP at levels similar to wild-type DnaA .Functions of DnaA His136 were assessed with reconstituted systems for oriC DOR, and DnaA. In this assay, ATP-DnaA molecules can construct homo-oligomers on the DOR and bind ssDUE with high affinity, producing DOR-DnaA-ssDUE ternary complexes  . DnaA-ssDUE binding was dependent on DOR as we previously demonstrated. Faint signals in the gel wells were irregular aggregates of DnaA which involved 32P-ssDUE. Those were unstable and slowly degraded during electrophoresis, resulting in faint smeared bands  using ssDUE, omplexes . Constru Arg245) . Here, ted bands . These r∗ assay to determine DnaB loading onto the unwound strands of oriC (via indirect interactions with DnaC and DnaA) and its subsequent helicase action on DNA strands. Loading on ssDUE activates DnaB helicase, expanding the ssDNA region and introducing positive supercoils. Activation of gyrase then produces highly negatively supercoiled oriC plasmid (Form I∗), and this topoisomeric form is distinguished from Form I by gel electrophoresis . Compared with wild-type DnaA, DnaA H136A, and the negative control DnaA F46A (which is inactive in primary DnaB binding) only had low levels of Form I∗ production, even in the presence of excessive amounts of DnaBC proteins . The left-half oriC is a minimal region for DUE unwinding and DnaB loading , consistent with the idea that DnaB loading reactions per se are inhibited also with the full-length oriC, but not with the idea that DnaBC complexes binds simultaneously to left- and right-half DnaA–oriC subcomplexes, causing abortive interactions with each other, and inhibiting DnaB loading on ssDUE. Taken together, the results suggest that the DnaA His136 residue has a predominant role in the process of productive loading of DnaB to unwound oriC.Form I loading . Compare∗ assay. In these experiments, complexes including both the wild-type DnaA and H136A (or F46A) proteins should be constructed on the same oriC molecule. Thus, if a subgroup of DnaA protomers is not used for DnaB loading, the partial inclusion of DnaA H136A (or F46A) which is fully active in DUE unwinding, might retain DUE unwinding and DnaB loading. DnaA F46A, although inactive for DnaB binding by itself, can help achieve optimal Form I∗ formation when wild-type DnaA is provided suboptimally , as previously demonstrated  of DnaA molecules assembled on oriC would require Phe46 for DnaB loading .Here, activity of mixtures of the wild-type DnaA and DnaA H136A or DnaA F46A proteins were analyzed using Form Instrated , suggest loading . Here, w∗ assay, but did not produce substantial amounts of Form I∗ . Only weak Form I∗ formation due to the residual activity of DnaA H136A was observed. These results suggest that, at least in a subgroup of DnaA molecules assembled on oriC, both the His136 and Phe46 residues must be present in the same DnaA protomer for DnaB loading.DnaA H136A and DnaA F46A proteins were mixed at various ratios and assessed by Form IoriC, resulting in largely reduced binding of DnaB compared with wild-type DnaA, although DUE unwinding activity is sustained , indicating that DnaA H136A is competent for DnaA assembly, which is consistent with its activities in DUE unwinding and ssDUE binding. In the presence of DnaA H136A and DnaB, oriC pull-down of DnaB was similar to that in the presence of wild-type DnaA and DnaB, indicating stable binding of DnaB helicase by DnaA H136A . Also, even when DnaC was coincubated, DnaA H136A stably bound DnaB at a level similar to wild-type DnaA . As previously shown , suggesting that stable complexes with DnaB hexameric rings  encircling the ssDNA of oriC are necessary for oriC recovery. A low level of recovery of oriC plasmid in the absence of DnaC presumably represented direct binding of DnaB to DnaA oligomers on oriC involving DnaA Phe46. These complexes were likely to have been largely dissociated by the use of wash buffer. In agreement with this idea, the use of wild-type DnaA and mutant oriC with DUE deletion (ΔDUE), which is inactive for unwinding, only resulted in a low level of recovery of oriC plasmid. Moreover, the use of DnaA F46A resulted in no observable recovery of oriC plasmid . These results were consistent with DnaA Phe46-dependent binding between DnaB and DnaA oligomers constructed on oriC being responsible for the basal recovery level, with considerable enhancement of recovery resulting from DnaB loading on ssDNA.Loading of DnaB was further examined by a novel pull-down assay using His-tagged DnaB, DnaC, DnaA, IHF, and oriC in this assay in the presence of DnaB and DnaC resulted in moderate inhibition in oriC recovery, compared to the level seen with wild-type DnaA . A low level of recovery (similar to that with wild-type DnaA) was seen with DnaA H136A in the absence of DnaC or in the presence of ΔDUE. These results further support the idea that DnaA H136 residue is specifically important for functional DnaB loading to the unwound site of oriC .The inclusion of DnaA H136A and wild-type Figure 7). DnaA binding to the hairpin structure enables recruitment of DnaBC complex and DnaB loading to the SSB-coated ssDNA, followed by primer and DNA synthesis by DnaG primase and DNA polymerase III holoenzyme. Whereas DnaA F46A was essentially inactive in this system, DnaA H136A was fully active relative to wild-type DnaA , indicating that DnaA H136A promoted DnaB loading to ssDNA in this simple system. Notably, unlike the more complex oriC, only 2–4 DnaA molecules bind to this M13 hairpin region, with its single DnaA box (oriC and A-site ssDNA (see section “Discussion”).DnaA–DnaB interactions were also studied using the simplified reconstituted ABC primosome system, which uses M13-phage-derived ssDNA with a hairpin structure containing the DnaA box R1 sequence (A-site ssDNA) . Even in the hyperthermophile A. aeolicus DnaA ortholog, the corresponding residue is tyrosine . Thus, the aromatic moiety at the position of E. coli DnaA His136 appears to have an evolutionarily conserved role in the loading of DnaB helicases at oriC.Among bacterial DnaA orthologs, the position corresponding to tyrosine . In γ prE. coli, this step depends on dynamic interactions between the initiator DnaA and DnaB replicative helicase. Stable binding between the two depends on a site including DnaA Glu21 and Phe46 of domain I. This site is suggested to interact with a specific site of DnaB, which resides in a region including the N-terminus and its franking region of DnaB CTD. In addition, the DnaA region Lys135–Gln148 within domain III has been implicated as having weak physical contact with DnaB NTD. However, the biological importance of this interaction has not previously been determined. Here, we used alanine scanning of these DnaA residues to highlight the role of His136 in replication initiation in vivo. The plasmid complementation test and flow cytometry analysis demonstrated that the dnaA H136A allele has only low in vivo initiation activity. In-depth biochemical characterization of DnaA H136A revealed that His136 is indispensable for DnaB loading at oriC, but that DUE unwinding, ssDUE binding, and assembly on oriC do not depend on this residue. Intriguingly, DnaA H136A was fully active in an oriC-independent DnaB loading system for ssDNA replication . These observations suggest that DnaA His136 residue has a distinct role in DnaB loading at oriC. Because DnaA H136A-oriC complexes maintain the primary contact between DnaA Phe46 and DnaB, we conclude that a secondary, weak contact via His136 might allow DnaA-directed DnaB loading on the ssDUE region. The conservation of His136 suggests that its role is conserved in eubacterial DnaA orthologs.Loading of replicative helicases on unwound origin DNA region is a crucial step in replication initiation of chromosomes. In oriC relies on stable formation of DnaA oligomers on oriC. DnaA Arg281 residue indirectly facilitates DnaB binding and loading by contributing to stable binding of DnaA molecules to oriC .Loading of DnaB at  to oriC . By cont of DnaA , the locin vitro and in vivo studies as well as the present data are in support of the ssDUE-recruitment mechanism in which DnaA oligomers constructed on DOR stably bind ssDUE using DnaA domain III H/B-motifs .Our previous Figure 5). We previously reported that the DnaA domain I Phe46 exhibits high-affinity binding to DnaB when DnaA oligomers are constructed on oriC , inhibition by DnaA H136A was moderate compared to the severe inhibition of Form I∗ production . Given that DnaA Phe46-dependent DnaB interaction is intact even in DnaA H136A, a possible explanation to this is that interaction of DnaB with DnaA H136A at oriC results in abortive loading complexes of DnaB that are not competent for helicase activity. Alternatively, loading orientation of DnaB helicases on ssDUE or DUE strand-specific loading of DnaB might be compromised by the DnaA H136A mutation, resulting in abortive complexes.In the oriC by a combination of DnaA domain I F46A and domain III H136A are substantially inactive in DnaB loading , suggesting that both Phe46 and His136 must be present in identical DnaA protomers for helicase loading at oriC. Although it has previously been suggested that a weak contact between DnaB and a DnaA region consisting of residues 111–148 (which includes His136) may precede stable binding involving DnaA domain I .Two distinct DnaB-binding modes of DnaA seem to be required for the loading of DnaB onto ssDUE. Notably, heterologous complexes formed at domain I , our resdomain I  in whichFigure 10). Because the T-rich strand of the ssDUE directly binds to the H/B motifs of DnaA domain III . Notably, in our high-definition structural model of DnaA–oriC complexes, DnaA His136 residues are exposed on the surface of the DnaA protomers, suggesting that they are accessible to DnaB without physically obstructing ssDUE binding to the H/B-motifs .We now suggest a model in which the secondary, weak contact via His136 facilitates accessibility of DnaB to the ssDUE , which might enable formation of multiple contact points with a single DnaB homohexamer. By contrast, in the ABC primosome system, only a few DnaA protomers are involved in a region bearing only a single DnaA box  even if each individual contact only has weak affinity . A similDnaA box , suggestWhereas DnaA His136 was dispensable in the ABC primosome system, DnaA domain III is important. Specifically, two residues (Lys234 and Arg285) exposed on the protein surface that are important for DnaA–DnaA interaction stimulate ssDNA replication in the ABC primosome system . BindingoriC for duplex unwinding and helicase loading. This study reveals the essential function for DnaA domain III His136 in the loading of DnaB replicative helicases on the ssDUE. Further analyses are required to dissect the DnaA protomers responsible for the DnaB interaction during DnaB loading on ssDUE and the DnaA-interacting sites on DnaB as well as to uncover the dynamic mechanisms of DnaA–DnaB complexes underlying the loading orientation of DnaB on ssDUE and DUE strand-specific loading of DnaB.Dynamic protein complexes are constructed and change structurally at YS, MN, SO, and TK conceived the experiments and wrote the paper. YS, MN, YA, and CH performed the experiments. All authors analyzed the data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.