Correction: An in vitro method for inducing titan cells reveals novel features of yeast-to-titan switching in the human fungal pathogen Cryptococcus gattii.

[This corrects the article DOI: 10.1371/journal.ppat.1010321.].

An in vitro method for inducing titan cells reveals novel features of yeast-to-titan switching in the human fungal pathogen Cryptococcus gattii.

Cryptococcosis is a potentially lethal fungal infection of humans caused by organisms within the Cryptococcus neoformans/gattii species complex. Whilst C. neoformans is a relatively common pathogen of immunocompromised individuals, C. gattii is capable of acting as a primary pathogen of immunocompetent individuals. Within the host, both species undergo morphogenesis to form titan cells: exceptionally large cells that are critical for disease establishment. To date, the induction, defining attributes, and underlying mechanism of titanisation have been mainly characterized in C. neoformans. Here, we report the serendipitous discovery of a simple and robust protocol for in vitro induction of titan cells in C. gattii. Using this in vitro approach, we reveal a remarkably high capacity for titanisation within C. gattii, especially in strains associated with the Pacific Northwest Outbreak, and characterise strain-specific differences within the clade. In particular, this approach demonstrates for the first time that cell size changes, DNA amplification, and budding are not always synchronous during titanisation. Interestingly, however, exhibition of these cell cycle phenotypes was correlated with genes associated with cell cycle progression including CDC11, CLN1, BUB2, and MCM6. Finally, our findings reveal exogenous p-Aminobenzoic acid to be a key inducer of titanisation in this organism. Consequently, this approach offers significant opportunities for future exploration of the underlying mechanism of titanisation in this genus.

Indigenous birth support worker (IBSW) program evaluation: a qualitative analysis of program workers and clients' perspectives.

BACKGROUND: The Indigenous Birth Support Worker (IBSW) Program provides Indigenous women with respectful, culturally safe, and trauma-informed care and supports women and families during labor and delivery. Located in the Jim Pattison Children's Hospital (JPCH) Maternal Care Centre in Saskatoon, Saskatchewan, Canada, the program served 1023 clients between December 2019 and January 2021. METHODS: The study objective was to explore the perspectives of the IBSWs and program clients one year post-implementation. The research plan was developed in collaboration with the IBSW program director and manager, IBSWs, and partners from the First Nation and Métis Health departments within the health region. A focus group with four IBSWs and individual interviews with ten clients who received services were conducted using a qualitative research design. RESULTS: Thematic analysis revealed that clients greatly appreciated and respected the IBSWs' cultural support and their compassionate, nonjudgmental, and safe care. IBSWs emphasized the importance of culturally safe and client-centered treatment, more effective pain management solutions, and that relationships with Elders and community healthcare personnel should be built and strengthened to improve pregnancy and postnatal care delivery. IBSWs desire to work with community healthcare providers to provide prenatal care and build relationships before delivery. IBSWs advocated for collaborative cooperation with community healthcare professionals and rural healthcare teams to enable a smooth care flow to and from communities. CONCLUSION: The Indigenous Birth Support Worker (IBSW) Program provides safe and client-centred care to Indigenous women during pregnancy, labour, and postpartum, consistent with the six principles proposed by BC perinatal services. IBSWs advocate for and assist Indigenous women in obtaining quality healthcare, provide traditional and cultural support, and positively affect mental health. However, the evaluation has revealed that healthcare provider insensitivity towards Indigenous clients persists. There is a need for greater role clarity and collaboration with healthcare practitioners to ensure evidence-based healthcare of the highest standard. This requires a commitment to addressing systemic issues and implementing broader calls to action and justice proposed by the Truth and Reconciliation Commission Calls to Action, the Missing and Murdered Indigenous Women and Girls Calls for Justice, and the United Nations Declaration on the Rights of Indigenous Peoples. The IBSW program offers vital support to Indigenous women during childbirth, but it must be viewed in the context of ongoing colonialism and the need for reconciliation and decolonization, requiring genuine collaboration with Indigenous peoples.

Emulsifiers in ultra-processed foods in the UK food supply.

OBJECTIVE: Ultra-processed foods (UPF), including those containing food additive emulsifiers, have received research attention due to evidence implicating them in the pathogenesis of certain diseases. The aims of this research were to develop a large-scale, brand-level database of UPF in the UK food supply and to characterise the occurrence and co-occurrence of food additive emulsifiers. DESIGN: A database was compiled sampling all products from the food categories contributing to energy intake from UPF in the UK from the National Diet and Nutrition Survey (2008-2014). Every food in these categories were identified from online supermarket provision from the 'big four' supermarkets that dominate the market share in the UK, comprising Tesco, Sainsbury's, Asda and Morrisons. SETTING: Major supermarkets in the UK. RESULTS: A total of 32 719 food products in the UK supermarket food supply were returned in searches. Of these, 12 844 products were eligible and manually reviewed for the presence of emulsifiers. Emulsifiers were present in 6642 (51·7 %) food products. Emulsifiers were contained in 95·0 % of 'Pastries, buns and cakes', 81·9 % of 'Milk-based drinks', 81·0 % of 'Industrial desserts' and 77·5 % of 'Confectionary'. Fifty-one per cent of all emulsifier-containing foods contained multiple emulsifiers. Across emulsifier-containing foods, there were a median of two emulsifiers (IQR 2) per product. The five most common emulsifiers were lecithin (23·4 % of all products), mono- and diglycerides of fatty acids (14·5 %), diphosphates (11·6 %), and xanthan gum and pectin (8·0 %). CONCLUSIONS: Findings from this study are the first to demonstrate the widespread occurrence and co-occurrence of emulsifiers in UPF in the UK food supply.

Viral infection triggers interferon-induced expulsion of live Cryptococcus neoformans by macrophages.

Cryptococcus neoformans is an opportunistic human pathogen, which causes serious disease in immunocompromised hosts. Infection with this pathogen is particularly relevant in HIV+ patients, where it leads to around 200,000 deaths per annum. A key feature of cryptococcal pathogenesis is the ability of the fungus to survive and replicate within the phagosome of macrophages, as well as its ability to be expelled from host cells via a novel non-lytic mechanism known as vomocytosis. Here we show that cryptococcal vomocytosis from macrophages is strongly enhanced by viral coinfection, without altering phagocytosis or intracellular proliferation of the fungus. This effect occurs with distinct, unrelated human viral pathogens and is recapitulated when macrophages are stimulated with the anti-viral cytokines interferon alpha or beta (IFNα or IFNß). Importantly, the effect is abrogated when type-I interferon signalling is blocked, thus underscoring the importance of type-I interferons in this phenomenon. Lastly, our data help resolve previous, contradictory animal studies on the impact of type I interferons on cryptococcal pathogenesis and suggest that secondary viral stimuli may alter patterns of cryptococcal dissemination in the host.

A group program for overweight and obese women with urinary incontinence (ATHENA): an implementation-effectiveness hybrid type 3 study.

INTRODUCTION AND HYPOTHESIS: Supervised pelvic floor muscle training (PFMT), weight loss and exercise are recommended for overweight/obese women with urinary incontinence (UI). This study aimed to implement and evaluate the feasibility, acceptability and effectiveness of a 12-week group exercise and healthy eating program (ATHENA) for overweight/obese women with UI. METHODS: This study, using an implementation-effectiveness hybrid type 3 design, was conducted within a Women's Health Physiotherapy outpatient service at an Australian tertiary public hospital. Intervention feasibility and acceptability were assessed through process evaluation of implementation, while clinical effectiveness was assessed via pre-/post-clinical and quality of life surveys. Process data were analyzed using descriptive statistics and effectiveness data were compared pre-/post-intervention using inferential statistics. RESULTS: Of 156 eligible patients, 37 (24%) agreed to participate; 29 (78%) completed the ATHENA program. Median (IQR) age and body mass index were 53 (47-65) years and 30.8 (29.1-34.8) kg/m2 respectively. ATHENA was feasible to implement, with all components delivered as intended and high participant satisfaction. Ninety-seven percent of participants reported improved UI symptoms (global rating of change) and significant improvements in overall pelvic floor dysfunction and quality of life utility scores (p = 0.001). While weight did not change, significant improvements were found in body-food choice congruence (intuitive eating scale-2; p < 0.01). CONCLUSIONS: The ATHENA intervention was feasible, acceptable and clinically effective for overweight and obese women with urinary incontinence at a tertiary public hospital in Australia. Further research into longer term outcomes and the cost effectiveness of this group intervention is recommended. TRIAL REGISTRATION: N/A. Ethics approval, HREC/2018/QGC/46582, date of registration 14/11/2018.

Whole almond consumption is associated with better diet quality and cardiovascular disease risk factors in the UK adult population: National Diet and Nutrition Survey (NDNS) 2008-2017.

PURPOSE:  This work aimed to estimate whole almond consumption in a nationally representative UK survey population and examine associations with diet quality and cardiovascular disease (CVD) risk. METHODS:  Four-day food record data from the National Diet and Nutrition Survey (NDNS) 2008-2017 (n = 6802, age ≥ 19 year) were analyzed to investigate associations between whole almond consumption and diet quality, measured by the modified Mediterranean Diet Score (MDS) and modified Healthy Diet Score (HDS), and CVD risk markers, using survey-adjusted multivariable linear regression. RESULTS:  Whole almond consumption was reported in 7.6% of the population. Median intake in whole almond consumers was 5.0 g/day (IQR 9.3). Consumers had higher diet quality scores relative to non-consumers; higher intakes of protein, total fat, monounsaturated, n-3 and n-6 polyunsaturated fats, fiber, folate, vitamin C, vitamin E, potassium, magnesium, phosphorus, and iron; and lower intakes of trans-fatty acids, total carbohydrate, sugar, and sodium. BMI and WC were lower in whole almond consumers compared to non-consumers: 25.5 kg/m2 (95% CI 24.9, 26.2) vs 26.3 kg/m2 (25.9, 26.7), and 88.0 cm (86.2, 89.8) vs 90.1 cm (89.1, 91.2), respectively. However, there were no dose-related fully adjusted significant associations between increasing almond intake (g per 1000 kcal energy intake) and lower CVD risk markers. CONCLUSIONS:  Almond intake is low in the UK population, but consumption was associated with better dietary quality and lower CVD risk factors. Habitual consumption of whole almonds should be encouraged as part of a healthy diet.

Evaluation of the enhanced upper limb therapy programme within the Robot-Assisted Training for the Upper Limb after Stroke trial: descriptive analysis of intervention fidelity, goal selection and goal achievement.

OBJECTIVE: To report the fidelity of the enhanced upper limb therapy programme within the Robot-Assisted Training for the Upper Limb after stroke (RATULS) randomized controlled trial, the types of goals selected and the proportion of goals achieved. DESIGN: Descriptive analysis of data on fidelity, goal selection and achievement from an intervention group within a randomized controlled trial. SETTING: Out-patient stroke rehabilitation within four UK NHS centres. SUBJECTS: 259 participants with moderate-severe upper limb activity limitation (Action Research Arm Test 0-39) between one week and five years post first stroke. INTERVENTION: The enhanced upper limb therapy programme aimed to provide 36 one-hour sessions, including 45 minutes of face-to-face therapy focusing on personal goals, over 12 weeks. RESULTS: 7877/9324 (84%) sessions were attended; a median of 34 [IQR 29-36] per participant. A median of 127 [IQR 70-190] repetitions were achieved per participant per session attended. Based upon the Canadian Occupational Performance Measure, goal categories were: self-care 1449/2664 (54%); productivity 374/2664 (14%); leisure 180/2664 (7%) and 'other' 661/2664 (25%). For the 2051/2664 goals for which data were available, 1287 (51%) were achieved, ranging between 27% by participants more than 12 months post stroke with baseline Action Research Arm Test scores 0-7, and 88% by those less than three months after stroke with scores 8-19. CONCLUSIONS: Intervention fidelity was high. Goals relating to self-care were most commonly selected. The proportion of goals achieved varied, depending on time post stroke and baseline arm activity limitation.

The Cryptococcus neoformans Titan cell is an inducible and regulated morphotype underlying pathogenesis.

Fungal cells change shape in response to environmental stimuli, and these morphogenic transitions drive pathogenesis and niche adaptation. For example, dimorphic fungi switch between yeast and hyphae in response to changing temperature. The basidiomycete Cryptococcus neoformans undergoes an unusual morphogenetic transition in the host lung from haploid yeast to large, highly polyploid cells termed Titan cells. Titan cells influence fungal interaction with host cells, including through increased drug resistance, altered cell size, and altered Pathogen Associated Molecular Pattern exposure. Despite the important role these cells play in pathogenesis, understanding the environmental stimuli that drive the morphological transition, and the molecular mechanisms underlying their unique biology, has been hampered by the lack of a reproducible in vitro induction system. Here we demonstrate reproducible in vitro Titan cell induction in response to environmental stimuli consistent with the host lung. In vitro Titan cells exhibit all the properties of in vivo generated Titan cells, the current gold standard, including altered capsule, cell wall, size, high mother cell ploidy, and aneuploid progeny. We identify the bacterial peptidoglycan subunit Muramyl Dipeptide as a serum compound associated with shift in cell size and ploidy, and demonstrate the capacity of bronchial lavage fluid and bacterial co-culture to induce Titanisation. Additionally, we demonstrate the capacity of our assay to identify established (cAMP/PKA) and previously undescribed (USV101) regulators of Titanisation in vitro. Finally, we investigate the Titanisation capacity of clinical isolates and their impact on disease outcome. Together, these findings provide new insight into the environmental stimuli and molecular mechanisms underlying the yeast-to-Titan transition and establish an essential in vitro model for the future characterization of this important morphotype.

Characterizing the Mechanisms of Nonopsonic Uptake of Cryptococci by Macrophages.

The pathogenic fungus Cryptococcus enters the human host via inhalation into the lung and is able to reside in a niche environment that is serum- (opsonin) limiting. Little is known about the mechanism by which nonopsonic phagocytosis occurs via phagocytes in such situations. Using a combination of soluble inhibitors of phagocytic receptors and macrophages derived from knockout mice and human volunteers, we show that uptake of nonopsonized Cryptococcus neoformans and C. gattii via the mannose receptor is dependent on macrophage activation by cytokines. However, although uptake of C. neoformans is via both dectin-1 and dectin-2, C. gattii uptake occurs largely via dectin-1. Interestingly, dectin inhibitors also blocked phagocytosis of unopsonized Cryptococci in wax moth (Galleria mellonella) larvae and partially protected the larvae from infection by both fungi, supporting a key role for host phagocytes in augmenting early disease establishment. Finally, we demonstrated that internalization of nonopsonized Cryptococci is not accompanied by the nuclear translocation of NF-κB or its concomitant production of proinflammatory cytokines such as TNF-α. Thus, nonopsonized Cryptococci are recognized by mammalian phagocytes in a manner that minimizes proinflammatory cytokine production and potentially facilitates fungal pathogenesis.

Tree nut snack consumption is associated with better diet quality and CVD risk in the UK adult population: National Diet and Nutrition Survey (NDNS) 2008-2014.

OBJECTIVES: To examine associations of tree nut snack (TNS) consumption with diet quality and cardiovascular disease (CVD) risk in UK adults from National Diet and Nutrition Survey (NDNS) 2008-2014. DESIGN: Cross-sectional analysis using data from 4-d food diaries, blood samples and physical measurements for CVD risk markers. To estimate diet quality, modified Mediterranean Diet Score (MDS) and modified Healthy Diet Score (HDS) were applied. Associations of TNS consumption with diet quality and markers of CVD risk were investigated using survey-adjusted multivariable linear regression adjusted for sex, age, ethnicity, socio-economic and smoking status, region of residency and total energy and alcohol intake. SETTING: UK free-living population. SUBJECTS: 4738 adults (≥19 years). RESULTS: TNS consumers had higher modified MDS and HDS relative to non-consumers. TNS consumers also had lower BMI, WC, SBP and DBP and higher HDL compared to non-consumers, although a dose-related fully adjusted significant association between increasing nut intake (g per 4184 kJ/1000 kcal energy intake) and lower marker of CVD risk was only observed for SBP. TNS consumption was also associated with higher intake of total fat, mono-, n-3 and n-6 polyunsaturated fatty acids, fibre, vitamin A, thiamin, folate, vitamin C, vitamin E, potassium, magnesium, phosphorus, selenium and iron; and lower intake of saturated fatty acids, trans fatty acids, total carbohydrate, starch, free sugar, sodium and chloride. CONCLUSIONS: TNS consumers report better dietary quality and consumption was associated with lower CVD risk factors. Encouraging replacement of less healthy snacks with TNS should be encouraged as part of general dietary guidelines.

The fungal pathogen Cryptococcus neoformans manipulates macrophage phagosome maturation.

Phagocytosis by cells of the innate immune system, such as macrophages, and the subsequent successful maturation of the phagosome, is key for the clearance of pathogens. The fungal pathogen Cryptococcus neoformans is known to overcome killing by host phagocytes and both replicate within these cells and also escape via a non-lytic process termed vomocytosis. Here we demonstrate that, during intracellular growth, cryptococci modify phagolysosome maturation. Live cryptococci, but not heat-killed pathogens or inert targets, induce the premature removal of the early phagosome markers Rab5 and Rab11. In addition, significant acidification of the phagosome, calcium flux and protease activity is hindered, thus rendering the phagosome permissive for cryptococcal proliferation. Interestingly, several attenuated cryptococcal mutants retain this ability to subvert phagosomal maturation, suggesting that hitherto unidentified pathogen mechanisms regulate this process.

Opportunistic adolescent health assessment in the child protection unit.

AIM: Adolescent health assessments are recommended to identify health-risk behaviours. Adolescents who experience maltreatment are more likely to engage in such behaviours. This study (i) describes the frequency of health-risk behaviours amongst adolescents attending a hospital-based child protection unit (CPU) and (ii) determines whether use of a health assessment questionnaire increases the identification of these behaviours. METHODS: A retrospective audit was performed of case notes of adolescents (aged ≥ 12 years) presenting to the CPU over 5 years (2007-2011). Data regarding health-risk behaviours were extracted. In 2012, following the introduction of a standardised HEADSS-based four-page questionnaire, health-risk data were collected prospectively over 18 months. The proportion of subjects reporting health-risk behaviours, before and after questionnaire introduction, was analysed. RESULTS: Two hundred fifty-eight subjects, median age 13 (range 12-18) years, 78% female, were included in the pre-questionnaire period; and 85 subjects, median age 14 (range 12-17) years, 86% female, were included following introduction of the questionnaire. Questionnaire use was associated with an increase in the frequency of health-risk behaviours identified in the following domains: Education (odds ratio 4.48 [confidence interval 2.56-7.96] P < 0.001), Activities (16.18 [6.70-42.74] P < 0.001), Drugs/alcohol (4.00 [2.23-7.16] P < 0.001) and Suicidality (8.27 [4.59-14.92] P < 0.001). Participants reported higher rates of health-risk behaviours than the national population. CONCLUSION: Adolescents attending a hospital-based CPU report high rates of health-risk behaviours. A standardised questionnaire results in increased identification of such behaviours.

An assessment of dental caries among young Aboriginal children in New South Wales, Australia: a cross-sectional study.

BACKGROUND: Limited research has been undertaken in Australia to assess the dental status of pre-school Aboriginal children. This cross-sectional study records the number of decayed, missing and filled teeth (dmft) and surfaces (dmfs) of pre-school Aboriginal children living in different locations in New South Wales (NSW), Australia. METHODS: A convenience sample of young children from seven Aboriginal communities in rural, remote and metropolitan areas of NSW, was recruited. One calibrated examiner recorded the dmft/s of children with written parental consent. RESULTS: 196 children were invited to participate and 173 children aged two to five years were examined, a response rate of 88.3%. Forty percent (n = 69) of the children were diagnosed with dental caries with a mean of 2.1 (SD = 3.6). The dmft scores were significantly higher in remote locations when compared to rural (p = <0.0001) and metropolitan areas (p = 0.0155). Children 4-5 years old living in remote NSW had a mean dmft of 3.5 and mean dmfs of 8.0 compared with children living in rural areas who had a dmft and dmfs of 1.5 and 4.2 respectively. Untreated dental caries was the primary contributor to the scores, and children who had previously received dental treatment still had active carious lesions. CONCLUSION: There was a high prevalence of untreated dental caries among the Aboriginal children, particularly for those in remote locations.

Noninvasive biomarkers of necrotizing enterocolitis.

Necrotizing enterocolitis is an acute inflammatory disease, which primarily affects preterm infants, and is a leading cause of morbidity and mortality in the neonatal intensive care unit. Unfortunately, necrotizing enterocolitis can be difficult to distinguish from other diseases and clinical conditions especially during the early course of the disease. This diagnostic uncertainty is particularly relevant to clinical evaluation and medical management and potentially leads to unnecessary and extended periods of cessation of enteral feedings and prolonged courses of parenteral nutrition and antibiotics. Biomarkers are molecular indicators of a disease process, diagnosis, prognosis and can be used to monitor the effects of disease management. Historically, there has been a paucity of reliable and robust biomarkers for necrotizing enterocolitis. However, several studies have recently identified promising biomarkers. Noninvasive samples for biomarker measurement are preferred and may have certain advantages in the preterm infant. In this review article, we focus on recent exciting and promising discoveries in noninvasive biomarkers for necrotizing enterocolitis.

Misdiagnosing New Hires Is Costly.

Winning the war for talent will propel profitable practices ahead of the competitive flow. Today there are psychometric tools that can support busy leaders who are tired of a range of disappointments, from mis-hires to underperforming employees. This article discusses several of these assessment tools, addressing when and where they should be used. Additionally, it shows how by adopting the three Ds--Diagnosis, Determine, and Decide--you can stop losing money, stop the employee aggravation, and reduce your stress.

Taking a Pulse on Your Practice.

Each medical practice, like a living organism, occasionally requires reading of its vital signs. As with human beings, one of those vital signs is the pulse. For your medical practice, just like your patients, there are numerous places from which to take that reading. This article reviews seven key pulses that provide insight into what is happening within the workplace culture of your practice.

Developing leaflets to give dental health advice to Aboriginal families with young children.

OBJECTIVES: Dental caries (decay) is a serious problem for young Aboriginal children, causing pain and stress. Treatment often involves extraction of teeth under a general anaesthetic. However, dental caries can be prevented by reducing the frequency of sugar consumption and brushing teeth twice a day with fluoride toothpaste. Such straightforward advice could be given to families by Aboriginal Health Workers who are trusted by their communities and have an existing advisory role. This paper reports on the development of dental health advice leaflets for use in Aboriginal communities. METHODS: An Aboriginal reference panel was recruited to comment on dental health advice leaflets prepared by an Aboriginal graphic designer. The panel was asked to consider the design, cultural appropriateness and practicality of the leaflets. Comments were collected through email and face-to-face discussions, which were collated and the leaflets altered accordingly. RESULTS: The advice from the panel resulted in greater use of pictures. For example large green ticks and red crosses highlighted healthy and unhealthy behaviours, respectively. The tooth brushing leaflet was amended to emphasise the safe storage of toothpaste in order to keep it out of reach of young children. The panel stated that all leaflets should incorporate the Aboriginal flag, and proposed that fridge magnets might be beneficial as all family members would benefit from seeing the messages every day. CONCLUSION: The consultation process refined dental advice leaflets to reflect the views of an Aboriginal Reference Panel, in terms of design, cultural competence and practicality.

Extended family support for parents faced with life-support decisions for extremely premature infants.

PURPOSE: To outline parents' descriptions of extended family involvement and support surrounding decision making for their extremely preterm infant. DESIGN: Collective case study design in a prospective, descriptive, longitudinal research. Seventy-five digitally recorded interviews were done with parents before and after the birth. SAMPLE: Fifty-four parents (40 mothers, 14 fathers). MAIN OUTCOME VARIABLE: Categories of family involvement and support in the parents' decision making. RESULTS: Most parents did not seek advice from family members for life-support decisions made prenatally. Instead, parents made the decision as a couple with their physician without seeking family input. Family members provided certain types of support: emotional support, advice and information, prayer, and instrumental help such as child care. Most parents described at least one way their family supported them. For postnatal and end-of-life decisions, parents were more likely to seek advice from extended family in addition to the other forms of support.

Mechanisms of microbial escape from phagocyte killing.

Phagocytosis and phagosome maturation are crucial processes in biology. Phagocytosis and the subsequent digestion of phagocytosed particles occur across a huge diversity of eukaryotes and can be achieved by many different cells within one organism. In parallel, diverse groups of pathogens have evolved mechanisms to avoid killing by phagocytic cells. The present review discusses a key innate immune cell, the macrophage, and highlights the myriad mechanisms microbes have established to escape phagocytic killing.