Assessing the Effectiveness of a Multicomponent Intervention on Hand Hygiene and Well-Being in Primary Health Care Centers and Schools Lacking Functional Water Supply in Protracted Conflict Settings: Protocol for a Cluster Randomized Controlled Trial.

BACKGROUND: Hand hygiene is crucial in health care centers and schools to avoid disease transmission. Currently, little is known about hand hygiene in such facilities in protracted conflict settings. OBJECTIVE: This protocol aims to assess the effectiveness of a multicomponent hand hygiene intervention on handwashing behavior, underlying behavioral factors, and the well-being of health care workers and students. Moreover, we report our methodology and statistical analysis plan transparently. METHODS: This is a cluster randomized controlled trial with 2 parallel arms taking place in 4 countries for 1 year. In Burkina Faso and Mali, we worked in 24 primary health care centers per country, whereas in Nigeria and Palestine, we focused on 26 primary schools per country. Facilities were eligible if they were not connected to a functioning water source but were deemed accessible to the implementation partners. Moreover, health care centers were eligible if they had a maternity ward and ≥5 employees, and schools if they had ≤7000 students studying in grades 5 to 7. We used covariate-constrained randomization to assign intervention facilities that received a hardware, management and monitoring support, and behavior change. Control facilities will receive the same or improved intervention after endline data collection. To evaluate the intervention, at baseline and endline, we used a self-reported survey, structured handwashing observations, and hand-rinse samples. At follow-up, hand-rinse samples were dropped. Starting from the intervention implementation, we collected longitudinal data on hygiene-related health conditions and absenteeism. We also collected qualitative data with focus group discussions and interviews. Data were analyzed descriptively and with random effect regression models with the random effect at a cluster level. The primary outcome for health centers is the handwashing rate, defined as the number of times health care workers performed good handwashing practice with soap or alcohol-based handrub at one of the World Health Organization 5 moments for hand hygiene, divided by the number of moments for hand hygiene that presented themselves during the patient interaction within an hour of observation. For schools, the primary outcome is the number of students who washed their hands before eating. RESULTS: The baseline data collection across all countries lasted from February to June 2023. We collected data from 135 and 174 health care workers in Burkina Faso and Mali, respectively. In Nigeria, we collected data from 1300 students and in Palestine from 1127 students. The endline data collection began in February 2024. CONCLUSIONS: This is one of the first studies investigating hand hygiene in primary health care centers and schools in protracted conflict settings. With our strong study design, we expect to support local policy makers and humanitarian organizations in developing sustainable agendas for hygiene promotion. TRIAL REGISTRATION: ClinicalTrials.gov NCT05946980 (Burkina Faso and Mali); https://www.clinicaltrials.gov/study/NCT05946980 and NCT05964478 (Nigeria and Palestine); https://www.clinicaltrials.gov/study/NCT05964478. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/52959.

Applying the photovoice method with adolescents in mining areas in rural Mozambique: critical reflections and lessons learned.

There is a recognised need for innovative methods to elicit the perspective of adolescents on public health issues, particularly when addressing sensitive topics such as the impact of mining projects on their health. Participatory approaches such as "photovoice" allow for deep engagement of vulnerable and marginalised populations, including adolescents. However, few existing studies have used the photovoice method to reflect on issues related to the environment and its impact on public health. To date, no studies have been found that have used photovoice to gain insight into adolescents' perspectives in mining areas. In this paper, we discuss the application of the photovoice method to understand adolescents' perceptions about the impact of mining on their health and well-being in rural areas in Mozambique. The study was conducted in northern and central Mozambique. Photovoice was successfully integrated into eight focus group discussions with adolescent girls and boys aged 15 to 17 years. Several lessons for guiding future research were learned. First, it provided an understanding of the perceived impacts of mining on their health and well-being. Second, photovoice promoted active engagement and interest in the study by the adolescents. Finally, compared to its ability to capture perceptions of physical and environmental aspects affecting adolescents' well-being, the method was less straightforward in revealing their concerns regarding social, relational and community aspects that are less tangible. Programs can make use of photovoice to address health issues without setting adolescents' views and priorities aside, allowing them to influence health decisions on issues that are meaningful to them. Future studies should explore strategies to minimise the role of the power dynamics that affect the engagement and contribution of adolescents in advocating for necessary and meaningful changes. Additionally, it is important to investigate how health programs and policies can help to reduce the impact of existing inequalities.

Components of local adaptation and divergence in pollination efficacy in a coevolving species interaction.

Selection leading to adaptation to interactions may generate rapid evolutionary feedbacks and drive diversification of species interactions. The challenge is to understand how the many traits of interacting species combine to shape local adaptation in ways directly or indirectly resulting in diversification. We used the well-studied interactions between Lithophragma plants (Saxifragaceae) and Greya moths (Prodoxidae) to evaluate how plants and moths together contributed to local divergence in pollination efficacy. Specifically, we studied L. bolanderi and its two specialized Greya moth pollinators in two contrasting environments in the Sierra Nevada in California. Both moths pollinate L. bolanderi during nectaring, one of them-G. politella-also while ovipositing through the floral corolla into the ovary. First, field surveys of floral visitors and the presence of G. politella eggs and larvae in developing capsules showed that one population was visited only by G. politella and few other pollinators, whereas the other was visited by both Greya species and other pollinators. Second, L. bolanderi in these two natural populations differed in several floral traits putatively important for pollination efficacy. Third, laboratory experiments with greenhouse-grown plants and field-collected moths showed that L. bolanderi was more efficiently pollinated by local compared to nonlocal nectaring moths of both species. Pollination efficacy of ovipositing G. politella was also higher for local moths for the L. bolanderi population, which relies more heavily on this species in nature. Finally, time-lapse photography in the laboratory showed that G. politella from different populations differed in oviposition behavior, suggesting the potential for local adaptation also among Greya populations. Collectively, our results are a rare example of components of local adaptation contributing to divergence in pollination efficacy in a coevolving interaction and, thus, provide insights into how geographic mosaics of coevolution may lead to coevolutionary diversification in species interactions.

Digest: Recreating ancestral trait variation to understand adaptation.

Two highly correlated anther traits affect pollination efficacy in a wild radish population. Does the strength and type of selection on these traits differ through male and female fitness with increased ancestral trait variation? Waterman et al. (2023) found stabilizing selection on one trait and disruptive selection on the other trait, with no difference between male and female fitness. Such quantifications of selection in populations with the increased variation that reflects ancestral trait variation provide insights into processes of trait adaptation.

Measuring, comparing and interpreting phenotypic selection on floral scent.

Natural selection on floral scent composition is a key element of the hypothesis that pollinators and other floral visitors drive scent evolution. The measure of such selection is complicated by the high-dimensional nature of floral scent data and uncertainty about the cognitive processes involved in scent-mediated communication. We use dimension reduction through reduced-rank regression to jointly estimate a scent composite trait under selection and the strength of selection acting on this trait. To assess and compare variation in selection on scent across species, time and space, we reanalyse 22 datasets on six species from four previous studies. The results agreed qualitatively with previous analyses in terms of identifying populations and scent compounds subject to stronger selection but also allowed us to evaluate and compare the strength of selection on scent across studies. Doing so revealed that selection on floral scent was highly variable, and overall about as common and as strong as selection on other phenotypic traits involved in pollinator attraction or pollen transfer. These results are consistent with an important role of floral scent in pollinator attraction. Our approach should be useful for further studies of plant-animal communication and for studies of selection on other high-dimensional phenotypes. In particular, our approach will be useful for studies of pollinator-mediated selection on complex scent blends comprising many volatiles, and when no prior information on the physiological responses of pollinators to scent compounds is available.

Cemented versus screw-retained posterior implant-supported single crowns: A 24-month randomized controlled clinical trial.

OBJECTIVES: To compare the incidence of biological and technical complications of cemented and screw-retained monolithic lithium-disilicate implant-supported posterior single crowns. MATERIAL AND METHODS: Forty-one subjects with a total of 56 implants received randomly allocated 28 cemented and 28 screw-retained crowns. In the screw-retained group, monolithic lithium-disilicate restorations were luted to titanium bases extraorally. In the cemented group, monolithic lithium-disilicate crowns were cemented on individualized titanium abutments intraorally. All restorations were examined according to modified FDI criteria within 2 weeks of inserting the crowns (baseline) and after 12 (n = 46) and 24 (n = 43) months. Bone loss was evaluated by standardized radiographs at baseline and 12 months. RESULTS: After 12 months, the incidence of mucositis (positive bleeding on probing) was 14.2% (screw-retained) and 17.9% (cement-retained). The gingival and plaque index and a mean marginal bone loss between 0.03-0.15 mm showed no significant difference between the groups. In the cemented group, cement residues were detected at baseline at two restorations (6.9%) by radiographic examination. A complete digital workflow was realized in most cases (85.7%). At 24 months, no restoration had failed, and no chipping of the ceramic had occurred. In the screw-retained group, screw loosening occurred in one implant. In both groups, there was obvious deterioration in the quality of 32% of the occlusal and of 18% of the proximal contact points. CONCLUSIONS: The type of retention mode of monolithic implant-retained lithium-disilicate posterior crowns had no influence on the biological and technical complication rate.

Alfred Gysi (1865-1957) - More than a Pioneer of Dental Prosthetics and Articulation.

Alfred Gysi was one of the most influential dental scientists of the last century. He is primarily considered a pioneer of modern articulation and occlusion theory, and several of his technical developments still bear his name. But what milieu did this dentist come from, what characterized his academic career and what research contributions did he make beyond the theory of articulation? How was he perceived by contemporary experts ­ as a researcher and as a colleague ­ and how can his scientific contributions be classified from today's perspective? These are precisely the questions that this article examines. The study is based on contemporary primary sources and on Gysi's own publications. In addition, a systematic re-analysis of the available secondary literature on the life and work of Alfred Gysi and on the history of the Zurich Dental Institute was carried out. The analysis shows that Gysi had a strong influence on contemporary dental prosthodontics and contributed considerably to its scientific character, especially due to the development of articulators and face bows, which received worldwide attention. But Gysi's scientific influence was by no means limited to prosthetics. Rather, he also provided important studies on caries and the dental pulp. Moreover, he published fundamental work on dental histopathology and microphotography. The current image of Gysi therefore needs to be revised. In view of the thematic breadth of his scientific contributions, Gysi is not only the most important prosthodontist of his time, but also one of the most versatile and innovative representatives in the history of Swiss dentistry.

Introduction to a Landscape Analysis of Multisectoral Approaches for Prevention and Control of Infectious and Vector-Borne Diseases.

The Swiss Development Cooperation, Canada's International Development Research Centre, the Swiss Tropical Public Health Institute, and the UNICEF/United Nations Development Programme (UNDP)/World Bank/World Health Organization (WHO) Special Programme for Research and Training in Tropical Diseases (TDR) collaborated on a project to review, understand and promote the use of multisectoral approaches (MSAs) in the prevention and control of vector-borne diseases (VBDs). The objectives of the project were to support a landscape analysis of how MSAs have been used in the prevention and control of VBDs; to develop a theoretical framework for guiding the implementation of interventions; and to test the recommendations in real-life conditions. To realize these objectives, the project supported several activities, including commissioning a series of scientific reviews on MSAs in 5 thematic areas, sharing the key findings of these reviews in workshops and events, and developing a guidance framework for the implementation of MSAs. These activities have produced the theoretical framework that will be tested in real-life conditions through the support of case studies. The collaboration on implementing multisectoral activities against VBDs will continue among TDR, the Swiss Tropical Public Health Institute, and new partners such as the WHO Water Sanitation and Hygiene Group, UNDP, and UN-Habitat, in order to face the challenges identified and propose solutions tailored to specific contexts. The prevention and control of VBDs require strong and adapted MSAs with the full participation of all relevant sectors.

Digitalization in dentistry: ethical challenges and implications.

OBJECTIVE: Scientific papers and books on digital dentistry are in vogue. In most cases, these publications focus clearly on the - undoubted - potentials and possibilities offered by digitalization. The fact that digital dentistry necessarily entails risks and ethical challenges, by contrast, is rarely discussed. This paper aims to complement the discourse on digitalization in dentistry by analyzing precisely these challenges. METHOD AND MATERIALS: The study is based on an analysis of international publications and specialist writings on digitalization and its applications in the fields of dentistry and medicine, as well as on the analysis of specific contributions from the disciplines of medical ethics and medical law, and from the public media. RESULTS: The paper identifies and discusses eight core challenges: (1) big data ("digital double" and falsification in dentistry), (2) the dental practitioner-patient relationship, (3) digital literacy, (4) the assumption of responsibility in complex systems, (5) accompanying changes in the dental professions, (6) cost trap and risks of overtreatment in dentistry, (7) consumption spiral and ecologic footprint, and (8) clinical evidence in dental treatments. In addition, a catalog of criteria for assessing the effects of digitalization in dentistry is developed. CONCLUSION: It is crucial to closely monitor both the potentials and the challenges posed by digitalization in dentistry. Ultimately, it is only those problems that are identified as such that can be resolved and only those technologies that are accepted by dentists, patients, and society that will prevail in the long term.

Preventing lung pathology and mortality in rabbit Staphylococcus aureus pneumonia models with cytotoxin-neutralizing monoclonal IgGs penetrating the epithelial lining fluid.

Staphylococcus aureus pneumonia is associated with high mortality irrespective of antibiotic susceptibility. Both MRSA and MSSA strains produce powerful cytotoxins: alpha-hemolysin(Hla) and up to five leukocidins - LukSF-PV, HlgAB, HlgCB, LukED and LukGH (LukAB) - to evade host innate defense mechanisms. Neutralizing cytotoxins has been shown to provide survival benefit in rabbit S. aureus pneumonia models. We studied the mechanisms of protection of ASN100, a combination of two human monoclonal antibodies (mAbs), ASN-1 and ASN-2, that together neutralize Hla and the five leukocidins, in rabbit MRSA and MSSA pneumonia models. Upon prophylactic passive immunization, ASN100 displayed dose-dependent increase in survival and was fully protective against all S. aureus strains tested at 5 or 20 mg/kg doses. Macroscopic and microscopic lung pathology, edema rate, and bacterial burden were evaluated 12 hours post infection and reduced by ASN100. Pharmacokinetic analysis of ASN100 in bronchoalveolar-lavage fluid from uninfected animals detected efficient penetration to lung epithelial lining fluid reaching peak levels between 24 and 48 hours post dosing that were comparable to the mAb concentration measured in serum. These data confirm that the ASN100 mAbs neutralize the powerful cytotoxins of S. aureus in the lung and prevent damage to the mucosal barrier and innate immune cells.

Assessing the function of pneumococcal neuraminidases NanA, NanB and NanC in in vitro and in vivo lung infection models using monoclonal antibodies.

Streptococcus pneumoniae isolates express up to three neuraminidases (sialidases), NanA, NanB and NanC, all of which cleave the terminal sialic acid of glycan-structures that decorate host cell surfaces. Most research has focused on the role of NanA with limited investigations evaluating the roles of all three neuraminidases in host-pathogen interactions. We generated two highly potent monoclonal antibodies (mAbs), one that blocks the enzymatic activity of NanA and one cross-neutralizing NanB and NanC. Total neuraminidase activity of clinical S. pneumoniae isolates could be inhibited by this mAb combination in enzymatic assays. To detect desialylation of cell surfaces by pneumococcal neuraminidases, primary human tracheal/bronchial mucocilial epithelial tissues were infected with S. pneumoniae and stained with peanut lectin. Simultaneous targeting of the neuraminidases was required to prevent desialylation, suggesting that inhibition of NanA alone is not sufficient to preserve terminal lung glycans. Importantly, we also found that all three neuraminidases increased the interaction of S. pneumoniae with human airway epithelial cells. Lectin-staining of lung tissues of mice pre-treated with mAbs before intranasal challenge with S. pneumoniae confirmed that both anti-NanA and anti-NanBC mAbs were required to effectively block desialylation of the respiratory epithelium in vivo. Despite this, no effect on survival, reduction in pulmonary bacterial load, or significant changes in cytokine responses were observed. This suggests that neuraminidases have no pivotal role in this murine pneumonia model that is induced by high bacterial challenge inocula and does not progress from colonization as it happens in the human host.

Ethical dilemmas of dental implantology: Ready for aftercare?

Dental implantology is one of the most attractive and dynamic fields of work within clinical dentistry. It provides many patients with new and improved opportunities for stable (implant-supported) rehabilitation and also offers dentists a field that is interesting in both technical and economic terms. Adverse clinical outcomes are, however, often the subject of discussion. These relate both to prosthetic failure and to biologic complications and side effects, namely persistent infections surrounding implants. A further point of discussion relates to the question as to how far the triumphant march of implantology is accompanied by a reduction in the willingness to retain teeth. It thus appears all the more important to ask some questions regarding the normative overall conditions of responsible implantologic care on the basis of the present state of research and currently available studies. In line with this, this paper deals with both existing preconceptions and ethically relevant parameters of responsible implantology. These function simultaneously as concrete clinical-ethical guidelines for dentists practicing implantology.

Disarming Staphylococcus aureus from destroying human cells by simultaneously neutralizing six cytotoxins with two human monoclonal antibodies.

Pathogenesis of Staphylococcus aureus is increasingly recognized to be driven by powerful toxins. Staphylococcus aureus employs up to six pore-forming toxins to subvert the human host defense and to promote bacterial invasion: alpha-hemolysin that disrupts epithelial and endothelial barriers and five leukocidins that lyse phagocytes involved in bacterial clearance. Previously, we described two human monoclonal antibodies (mAbs), ASN-1 that neutralizes alpha-hemolysin and four leukocidins (LukSF-PV, LukED, HlgAB, HlgCB), and ASN-2 that inactivates the 5th leukocidin, LukGH. In this study we tested the individual and combined effects of ASN-1 and ASN-2 in multiple in vitro models employing relevant human target cells. We found that diverse S. aureus isolates with different genetic backgrounds (based on MLST- and spa-typing) and antibiotic sensitivity (both MRSA and MSSA) displayed greatly different cytotoxin expression patterns influenced by the type of growth medium used. Both mAbs were required to fully prevent the lysis of human neutrophils exposed to the mixture of recombinant cytotoxins or native toxins present in the culture supernatants of S. aureus isolates. Flow cytometry confirmed the protective effects of ASN-1 + ASN-2 (known as ASN100) on granulocytes, monocytes, NK-cells and T-lymphocytes. ASN-1 alone preserved the integrity of a 3D-primary culture of human tracheal/bronchial mucociliary epithelial tissue infected with S. aureus. We conclude that simultaneous inhibition of alpha-hemolysin and five leukocidins by ASN100 blocks cytolytic activity of S. aureus towards human target cells in vitro.

Selective sensitization of human neutrophils to LukGH mediated cytotoxicity by Staphylococcus aureus and IL-8.

OBJECTIVES: Staphylococcus aureus produces up to five bi-component leukocidins - LukSF-PV, gamma-hemolysins AB and CB, LukGH (LukAB) and LukED - to evade innate immunity by lysing phagocytic cells. Species specificity of these leukocidins limits the relevance of animal models, therefore we assessed their individual contribution using human neutrophils. METHODS: Human polymorphonuclear leukocytes (PMNs) were activated with stimuli relevant during bacterial infections and sensitivity to recombinant leukocidins was measured in cell-viability assays. Leukocidin receptor expression was quantified by flow cytometry. RESULTS: We observed greatly variable sensitivities of different PMN preparations towards LukGH. Activation of PMNs by lipopolysaccharide (LPS) or S. aureus culture supernatant (CS) lacking all leukocidins resulted in higher surface expression of CD11b, the LukGH receptor, and greatly enhanced the sensitivity towards LukGH, eliminating the variability observed with unstimulated cells. In contrast, CS induced a decrease in sensitivity of PMNs to the other four leukocidins and reduced surface staining for their cognate receptors (CXCR1, CXCR2, C5aR, C5L2). Delta-toxin and peptidoglycan mimicked the effect of CS. Moreover, IL-8, an important cytokine in neutrophil activation, also selectively increased LukGH sensitivity. Deletion of lukGH, but not other leukocidin genes, prevented PMN killing upon infection with USA300 CA-MRSA. CONCLUSION: Inflammatory signals enhance the susceptibility of human PMNs to lysis by LukGH rendering this toxin dominant among the S. aureus leukocidins in vitro.

Context matters: The importance of dimerization-induced conformation of the LukGH leukocidin of Staphylococcus aureus for the generation of neutralizing antibodies.

LukGH (LukAB) is a potent leukocidin of Staphylococcus aureus that lyses human phagocytic cells and is thought to contribute to immune evasion. Unlike the other bi-component leukocidins of S. aureus, LukGH forms a heterodimer before binding to its receptor, CD11b expressed on professional phagocytic cells, and displays significant sequence variation. We employed a high diversity human IgG1 library presented on yeast cells to discover monoclonal antibodies (mAbs) neutralizing the cytolytic activity of LukGH. Recombinant LukG and LukH monomers or a LukGH dimer were used as capture antigens in the library selections. We found that mAbs identified with LukG or LukH as bait had no or very low toxin neutralization potency. In contrast, LukGH dimer-selected antibodies proved to be highly potent, and several mAbs were able to neutralize even the most divergent LukGH variants. Based on biolayer interferometry and mesoscale discovery, the high affinity antibody binding site on the LukGH complex was absent on the individual monomers, suggesting that it was generated upon formation of the LukG-LukH dimer. X-ray crystallography analysis of the complex between the LukGH dimer and the antigen-binding fragment of a very potent mAb (PDB code 5K59) indicated that the epitope is located in the predicted cell binding region (rim domain) of LukGH. The corresponding IgG inhibited the binding of LukGH dimer to target cells. Our data suggest that knowledge of the native conformation of target molecules is essential to generate high affinity and functional mAbs.

Why Do Floral Perfumes Become Different? Region-Specific Selection on Floral Scent in a Terrestrial Orchid.

Geographically structured phenotypic selection can lead to adaptive divergence. However, in flowering plants, such divergent selection has rarely been shown, and selection on floral signals is generally little understood. In this study, we measured phenotypic selection on display size, floral color, and floral scent in four lowland and four mountain populations of the nectar-rewarding terrestrial orchid Gymnadenia odoratissima in two years. We also quantified population differences in these traits and pollinator community composition. Our results show positive selection on display size and positive, negative, or absence of selection on different scent compounds and floral color. Selection on the main scent compounds was consistently stronger in the lowlands than in the mountains, and lowland plants emitted higher amounts of most of these compounds. Pollinator community composition also differed between regions, suggesting different pollinators select for differences in floral volatiles. Overall, our study is the first to document consistent regional differences in selection on floral scent, suggesting this pattern of selection is one of the evolutionary forces contributing to regional divergence in floral chemical signaling.

'I know it has worked for millions of years': the role of the 'natural' in parental reasoning against child immunization in a qualitative study in Switzerland.

BACKGROUND: Despite efforts of international and national health authorities, immunization coverage and timeliness of vaccination against dangerous childhood diseases have been adversely affected by parental hesitation to vaccinate their children in high-income countries. Literature shows that social and political processes and shifts in conceptual structures, such as emerging views linked to health and 'natural' lifestyles, have shaped parents' immunization decisions. This paper investigates how Swiss parents argued along the lines of a natural development of the child to explain their critical attitudes towards immunization against measles and other childhood diseases. METHODS: A total of 32 semi-structured interviews were conducted with parents of children between 0 and 16 years of age who decided not to fully immunize their children. The interviews were analyzed using qualitative content analysis and an interpretative approach. RESULTS: Parents built their arguments against immunization on a strong faith in the strength of the naturally acquired immune system. Childhood diseases were not perceived as a threat but as part of the natural way to reinforce the body and to acquire a "natural" and thus strong immunity. Parents understood immunization as an artificial intrusion into the natural development of the immune system and feared overloading the still immature immune system of their young children and infants through current vaccination schemes. CONCLUSIONS: In the context of emerging trends towards natural lifestyles and ideas of holistic health in Switzerland and Europe, where many well-informed parents express concerns towards vaccinating their children, public vaccination strategies require reconsideration. Public immunization schedules need to acknowledge parents' wish for more flexibility and demand for an individualized patient-centered approach to immunization.

Are tetraploids more successful? Floral signals, reproductive success and floral isolation in mixed-ploidy populations of a terrestrial orchid.

BACKGROUND AND AIMS: Polyploidization, the doubling of chromosome sets, is common in angiosperms and has a range of evolutionary consequences. Newly formed polyploid lineages are reproductively isolated from their diploid progenitors due to triploid sterility, but also prone to extinction because compatible mating partners are rare. Models have suggested that assortative mating and increased reproductive fitness play a key role in the successful establishment and persistence of polyploids. However, little is known about these factors in natural mixed-ploidy populations. This study investigated floral traits that can affect pollinator attraction and efficiency, as well as reproductive success in diploid and tetraploid Gymnadenia conopsea (Orchidaceae) plants in two natural, mixed-ploidy populations. METHODS: Ploidy levels were determined using flow cytometry, and flowering phenology and herbivory were also assessed. Reproductive success was determined by counting fruits and viable seeds of marked plants. Pollinator-mediated floral isolation was measured using experimental arrays, with pollen flow tracked by means of staining pollinia with histological dye. KEY RESULTS: Tetraploids had larger floral displays and different floral scent bouquets than diploids, but cytotypes differed only slightly in floral colour. Significant floral isolation was found between the two cytotypes. Flowering phenology of the two cytotypes greatly overlapped, and herbivory did not differ between cytotypes or was lower in tetraploids. In addition, tetraploids had higher reproductive success compared with diploids. CONCLUSIONS: The results suggest that floral isolation and increased reproductive success of polyploids may help to explain their successful persistence in mixed-ploidy populations. These factors might even initiate transformation of populations from pure diploid to pure tetraploid.

Structure-function analysis of heterodimer formation, oligomerization, and receptor binding of the Staphylococcus aureus bi-component toxin LukGH.

The bi-component leukocidins of Staphylococcus aureus are important virulence factors that lyse human phagocytic cells and contribute to immune evasion. The γ-hemolysins (HlgAB and HlgCB) and Panton-Valentine leukocidin (PVL or LukSF) were shown to assemble from soluble subunits into membrane-bound oligomers on the surface of target cells, creating barrel-like pore structures that lead to cell lysis. LukGH is the most distantly related member of this toxin family, sharing only 30-40% amino acid sequence identity with the others. We observed that, unlike other leukocidin subunits, recombinant LukH and LukG had low solubility and were unable to bind to target cells, unless both components were present. Using biolayer interferometry and intrinsic tryptophan fluorescence we detected binding of LukH to LukG in solution with an affinity in the low nanomolar range and dynamic light scattering measurements confirmed formation of a heterodimer. We elucidated the structure of LukGH by x-ray crystallography at 2.8-Šresolution. This revealed an octameric structure that strongly resembles that reported for HlgAB, but with important structural differences. Structure guided mutagenesis studies demonstrated that three salt bridges, not found in other bi-component leukocidins, are essential for dimer formation in solution and receptor binding. We detected weak binding of LukH, but not LukG, to the cellular receptor CD11b by biolayer interferometry, suggesting that in common with other members of this toxin family, the S-component has the primary contact role with the receptor. These new insights provide the basis for novel strategies to counteract this powerful toxin and Staphylococcus aureus pathogenesis.

Five birds, one stone: neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody.

Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis.