In vitro activity of Western Australian honeys and Manuka honey against clinically important yeasts.

With the steady rise in antifungal resistance amongst clinically important yeasts, antifungal drug discovery remains of the utmost importance. To determine the potential of some honeys as alternative antifungal agents, we quantified the antifungal activity of 12 Western Australian honey samples, two Manuka honey samples and an artificial honey against 10 yeast isolates including clinical and reference strains. Results showed that the tested honeys varied in activity, and yeasts species also differed in susceptibility, with minimum inhibitory concentrations (MICs) determined by broth microdilution ranging from 8% to >44% w/v honey. Honeys with the highest overall activity were derived from Blackbutt (Eucalyptus patens), Jarrah (E. marginata), and Karri (E. diversicolor). The optical density of each MIC microtitre plate was determined after incubation and showed that at relatively low concentrations of honey the growth of all yeasts was enhanced compared to the untreated control, whereas at and above approximately 12% w/v, honeys exerted a dose-dependent growth inhibitory effect, the extent of which varied by honey type. Time-kill studies with 64% w/v honey showed that all eight of the natural honeys tested had greater fungicidal activity than the comparator artificial honey. Our findings suggest that the specific nectar-derived phytochemicals present within each honey play an important role in antifungal activity, and support the notion that activity is due to a combination of factors including osmotic activity, hydrogen peroxide and phytochemical compounds. These data indicate that honey is worthy of further investigation as a potential therapeutic agent for superficial yeast infections.

See us, hear us! children, adolescents and families in Saskatchewan coping with mental health during the COVID-19 pandemic.

PURPOSE: To examine the lived experiences of children and adolescents coping with mental health issues and seeking mental health services in Saskatchewan during the COVID-19 pandemic. METHODS: In our descriptive phenomenological qualitative study, we interviewed forty-six individuals, including children aged 8-15 and their parents. Thematic analysis was applied to interpret the interview data. RESULTS: Our analysis identified three key themes: pyscho-behavioural impact, academic impact, and social impact. The pandemic adversely affected children due to factors like changes in behaviours such as increased screen time and decreased physical activity, limited access to mental health services, and disruptions to schooling and social interactions. Coping mechanisms varied, ranging from the utilization of available mental health supports and services to individual and family-based strategies. Disparities in timely access to mental health services were evident, with financially stable families accessing private services, while others struggled, particularly in rural areas. Families demonstrated resilience through parental efforts to seek balance and prioritize safety amidst COVID-19 challenges. CONCLUSIONS: Social connectedness served as a crucial buffer against pandemic-induced stress. Children faced difficulty in accessing timely mental health services and supports. Echoing participant experiences, our findings emphasize the urgency of targeted interventions and policy adjustments to address existing gaps in mental health service accessibility and availability.

Functional outcomes of diets in multiple sclerosis (FOOD for MS): Protocol for a parallel arm randomized feeding trial for low glycemic load and calorie restriction.

BACKGROUND: Pilot trials indicate that both a low glycemic load (GL) diet and calorie restriction (CR) can be implemented successfully in people with multiple sclerosis (pMS) and may improve MS symptoms and physical function, but large randomized clinical trials (RCTs) have not yet been conducted. The purpose of this study is to test these interventions alone and in combination to determine their efficacy for improving clinical and patient reported outcomes (PROs) in pMS. METHODS: This 32-week, two-arm, RCT at two centers will randomly assign 100 adults with relapsing-remitting or secondary progressive MS to a low GL diet (n = 50) or a standard GL diet (n = 50). Both diet groups will complete two study phases: a eucaloric phase (16 weeks) and a CR phase (16 weeks). Groceries for the study meal plans will be delivered to participants' homes weekly. The primary outcome is physical function, measured by timed 25-ft walk test. Secondary outcomes are pain, fatigue, mood, and anxiety. DISCUSSION: This will be the most rigorous intervention trial to date of a low GL diet and CR in adults with MS, and among the first to assess the impact of intentional weight loss on MS symptoms. Results will provide valuable insight for recommending dietary change, weight loss, or both to adults with MS. These non-drug interventions pose few risks and have potential to yield significant improvements in MS symptoms. TRIAL REGISTRATION ID: NCT05327322.

Bioactivities and Phenolic Profiles of Honeys Derived from Plants of the Goldfields, Esperance and Wheatbelt Regions of Western Australia.

The aim of this study was to examine a collection of 79 honeys derived from plants endemic to several Western Australian unique bioregions for bioactivity and physicochemical characteristics. For physicochemical analyses, total phenolic content, high performance thin layer chromatography (HPTLC) fingerprints, pH, Brix, colour and hydrogen peroxide generation were examined. Brix (82.6±1.3) and pH (4.34±0.24) values were within expected ranges, whereas hydrogen peroxide levels determined using an o-dianisidine/horseradish peroxidase assay were relatively low, ranging from 0-244 µM. Antibacterial activity determined by the broth microdilution assay showed that Moort (Eucalyptus platypus) and Yate (Eucalyptus occidentalis) honeys had the highest overall activity with mean minimum inhibitory concentrations of 24.8 % and 25.1 % (w/v) honey, respectively. Yate honey also had the highest overall antioxidant activity (4.38±0.58 mmol Fe2+ /kg of honey), followed by Mallee honeys from various eucalypts, as determined by FRAP (Ferric reducing antioxidant power) and DPPH⋅ (2,2-Diphenyl-1-picrylhydrazyl) assays. This study identified new sources of honeys with potentially useful therapeutic properties from bioregions within Western Australia.

Protocol to visualize glycan-mediated binding between virus and human blood mononuclear cells using thin-section transmission electron microscopy.

Glycan-glycan interactions between viral particles and host cells may lengthen the dwell time of the virus on the cell surface to facilitate cellular receptor engagement. Here, we present a protocol for visualizing glycan-mediated binding between virus or virus-like-particles (VLPs) and human peripheral blood mononuclear cells using transmission electron microscopy (TEM). We describe steps for virus and VLP production, isolation of human peripheral blood mononuclear cells, and sample preparation. We then detail procedures for thin-section TEM. For complete details on the use and execution of this protocol, please refer to Spillings et al.1.

Changes in antibacterial activity, colour, and hydrogen peroxide content of Western Australian Jarrah and Marri honeys after storage at different temperatures over time.

AIMS: This study aimed to evaluate the effects of storage and different temperatures on the antibacterial activity and physicochemical characteristics of several types of honey. METHODS AND RESULTS: Honeys stored for 16 weeks at 37 and 45°C showed significant declines in antibacterial activity determined by minimum inhibitory concentrations, the loss of hydrogen peroxide, decreases in honey pH, and increases in honey colour, with changes most pronounced at 45°C. In contrast, honeys stored for 16 weeks at ambient (∼22°C) and cold (4, -20, and -80°C) temperatures showed only minor changes. In a second set of 12 honeys stored for 16-32 months at ambient temperature and then 4°C, honeys showed minor changes in antibacterial activity, increases in colour, and decreases in pH. For a third set of 17 honeys stored for five years at ambient temperature, the honeys showed almost complete loss of hydrogen peroxide and were all significantly darker in colour, but showed varied changes in antibacterial activity. CONCLUSIONS: Heat was detrimental to the antibacterial activity of honeys, as was long-term storage at ambient temperatures for some honeys but not others.

Mental Health and Health-Related Quality of Life of Children and Youth during the First Year of the COVID-19 Pandemic: Results from a Cross-Sectional Survey in Saskatchewan, Canada.

For children and youth, the COVID-19 pandemic surfaced at a critical time in their development. Children have experienced extended disruptions to routines including in-person schooling, physical activities, and social interactions-things that bring meaning and structure to their daily lives. We estimated the prevalence of anxiety and depression symptoms of children and youth and their experiences of health-related quality of life (HRQoL), during the first year of the pandemic, and identified factors related to these outcomes. Further, we examined these effects among ethnocultural minority families. We conducted an online survey (March-July 2021) with 510 children and youth aged 8-18 years and their parents/caregivers. The sample was representative of the targeted population. We modelled the relationship between anxiety, depression (measured using the Revised Child Anxiety and Depression Scale), HRQoL (measured using KIDSCREEN-10), and sociodemographic, behavioural, and COVID-19-contributing factors using binary logistic regression. A priori-selected moderating effects of sociodemographic characteristics and self-identified ethnocultural minority groups on the outcomes were tested. The point-in-time prevalence of medium-to-high anxiety symptoms and depression symptoms was 10.19% and 9.26%, respectively. Almost half (49.15%) reported low-to-moderate HRQoL. Children reporting medium-to-high anxiety symptoms, depression symptoms, and low-to-moderate HRQoL were more likely to be aged 8-11 years, 16-18 years, ethnocultural minority participants, living in rural/urban areas, having good/fair MH before COVID-19, experiencing household conflicts, having less physical activity, and having ≥3 h of recreational screen time. Those who had more people living at home and ≥8 h of sleep reported low anxiety and depression symptoms. Ethnocultural minority 16-18-year-olds were more likely to report low-to-moderate HRQoL, compared to 12-15-year-olds. Additionally, 8-11-year-olds, 16-18-year-olds with immigrant parents, and 16-18-year-olds with Canadian-born parents were more likely to report low-moderate HRQoL, compared to 12-15-year-olds. Children and youth MH and HRQoL were impacted during the pandemic. Adverse MH outcomes were evident among ethnocultural minority families. Our results reveal the need to prioritize children's MH and to build equity-driven, targeted interventions.

Hydrocephalus in Nfix-/- Mice Is Underpinned by Changes in Ependymal Cell Physiology.

Nuclear factor one X (NFIX) is a transcription factor required for normal ependymal development. Constitutive loss of Nfix in mice (Nfix-/-) is associated with hydrocephalus and sloughing of the dorsal ependyma within the lateral ventricles. Previous studies have implicated NFIX in the transcriptional regulation of genes encoding for factors essential to ependymal development. However, the cellular and molecular mechanisms underpinning hydrocephalus in Nfix-/- mice are unknown. To investigate the role of NFIX in hydrocephalus, we examined ependymal cells in brains from postnatal Nfix-/- and control (Nfix+/+) mice using a combination of confocal and electron microscopy. This revealed that the ependymal cells in Nfix-/- mice exhibited abnormal cilia structure and disrupted localisation of adhesion proteins. Furthermore, we modelled ependymal cell adhesion using epithelial cell culture and revealed changes in extracellular matrix and adherens junction gene expression following knockdown of NFIX. Finally, the ablation of Nfix from ependymal cells in the adult brain using a conditional approach culminated in enlarged ventricles, sloughing of ependymal cells from the lateral ventricles and abnormal localisation of adhesion proteins, which are phenotypes observed during development. Collectively, these data demonstrate a pivotal role for NFIX in the regulation of cell adhesion within ependymal cells of the lateral ventricles.

Antibacterial interactions between two monofloral honeys and several topical antiseptics, including essential oils.

BACKGROUND: Honey has broad spectrum antibacterial activity against clinically important organisms and may be suitable for treating superficial bacterial infections. However, very little data are available describing potential interactions between honey and other topically applied agents such as antiseptics or essential oils. METHODS: Interactions between pairs of antibacterial agents were investigated by performing checkerboard assays and determining the fractional inhibitory concentration indices (FICIs). Interactions between the two monofloral honeys marri (from Corymbia calophylla) and manuka, and the antiseptic agents benzalkonium chloride, chlorhexidine digluconate, silver (I) nitrate, tea tree oil, and Eucalyptus polybractea oil were investigated against Staphylococcus aureus ATCC® 43300 and Pseudomonas aeruginosa ATCC® 27853. RESULTS: Additive or indifferent interactions (FICI 0.5-2) were observed for all combinations against both organisms tested, with the exception of chlorhexidine and honey. Chlorhexidine and marri honey showed an antagonistic relationship against S. aureus (median FICI 2.00, range 1.25-4.83). Similarly, chlorhexidine and manuka honey showed antagonism against S. aureus (median FICI 2.33, range 2.00-2.67). CONCLUSIONS: With the exception of chlorhexidine, these data indicate that honey does not interfere with the antimicrobial activity of the tested agents, and that honey may be suitable for combination therapy with other topically applied antibacterial agents for treating superficial bacterial infections.

In vitro antibacterial activity of Western Australian honeys, and manuka honey, against bacteria implicated in impetigo.

Impetigo is a contagious skin disease caused by Staphylococcus aureus and Streptococcus pyogenes. Without treatment, impetigo may be recurrent, develop into severe disease, or have serious, life-threatening sequelae. Standard treatment consists of topical or systemic antibiotic therapy (depending on severity), however, due to antibiotic resistance some therapies are increasingly ineffective. In this study we evaluated the potential for honey as an alternative treatment for impetigo. A broth microdilution assay in 96-well microtitre trays was used to determine the minimum inhibitory concentrations (MICs) of six monofloral honeys (jarrah, marri, red bell, banksia, wandoo, and manuka), a multifloral honey and artificial honey against S. aureus (n = 10), S. pyogenes (n = 10), and coagulase-negative staphylococci (CoNS) (n = 10). The optical density (OD) of all microtitre tray wells was also determined before and after assay incubation to analyse whether sub-MIC growth inhibition occurred. Jarrah, marri, red bell, banksia, and manuka honeys were highly effective at inhibiting S. aureus and CoNS, with MIC50 values ranging from 4 to 8% w/v honey. S. pyogenes was also inhibited by these same honeys, albeit at higher concentrations (8-29% w/v). Wandoo and multifloral honeys had the least antibacterial activity with MICs of >30% (w/v) for all isolates. However, OD data indicated that sub-MIC concentrations of honey were still partially restricting bacterial growth. Our pre-clinical data indicate that honey may be a potential therapeutic agent for the routine treatment of mild impetigo, and we suggest that clinical trials would be appropriate to further investigate this.

Correlation of the antibacterial activity of commercial manuka and Leptospermum honeys from Australia and New Zealand with methylglyoxal content and other physicochemical characteristics.

Variation in the antibacterial potency of manuka honey has been reported in several published studies. However, many of these studies examine only a few honey samples, or test activity against only a few bacterial isolates. To address this deficit, a collection of 29 manuka/Leptospermum honeys was obtained, comprising commercial manuka honeys from Australia and New Zealand and several Western Australian Leptospermum honeys obtained directly from beekeepers. The antibacterial activity of honeys was quantified using several methods, including the broth microdilution method to determine minimum inhibitory concentrations (MICs) against four species of test bacteria, the phenol equivalence method, determination of antibacterial activity values from optical density, and time kill assays. Several physicochemical parameters or components were also quantified, including methylglyoxal (MGO), dihydroxyacetone (DHA), hydroxymethylfurfural (HMF) and total phenolics content as well as pH, colour and refractive index. Total antioxidant activity was also determined using the DPPH* (2,2-diphenyl-1-picrylhydrazyl) and FRAP (ferric reducing-antioxidant power) assays. Levels of MGO quantified in each honey were compared to the levels stated on the product labels, which revealed mostly minor differences. Antibacterial activity studies showed that MICs varied between different honey samples and between bacterial species. Correlation of the MGO content of honey with antibacterial activity showed differing relationships for each test organism, with Pseudomonas aeruginosa showing no relationship, Staphylococcus aureus showing a moderate relationship and both Enterococcus faecalis and Escherichia coli showing strong positive correlations. The association between MGO content and antibacterial activity was further investigated by adding known concentrations of MGO to a multifloral honey and quantifying activity, and by also conducting checkerboard assays. These investigations showed that interactions were largely additive in nature, and that synergistic interactions between MGO and the honey matrix did not occur.

An investigation of the suitability of melissopalynology to authenticate Jarrah honey.

This study reports on the analysis of eleven Jarrah (Eucalyptus marginata) honeys, of which nearly half (n = 5) were re-classified as Blackbutt (E. patens) honey on the grounds of the predominant flower pollen identified by melissopalynology. Based on a comprehensive analysis of the honeys' physico- and phytochemical characteristics and antioxidant activity data, taking into account pH, electrical conductivity, refractive index and Brix values as well as moisture content, individual fructose and glucose content and derived fructose to glucose ratio alongside total phenolic content and antioxidant activity determined by the DPPH assay, no statistically significant difference was found amongst the eleven honeys classified by pollen analysis into two honey groups, 'Jarrah' or 'Blackbutt'. This study therefore draws into question the value of melissopalynology as an analysis tool to authenticate Jarrah honey.

Quantification of Angiogenesis in Laser Choroidal Neovascularization.

Murine laser-induced laser choroidal neovascularization is a widely used and robust model of wet (exudative) age-related macular degeneration (wAMD). wAMD is one of the leading causes of blindness in the Western world. In brief, a focused laser beam is used to penetrate Bruch's membrane, which separates the choriocapillaris (well-vascularized choroid layer) from the pigmented layers of the retina. Damage to the integrity of this membrane during diabetes leads to fluid accumulation and vascular invasion into the subretinal layers resulting in a progressive worsening of vision. Here we describe a 14-day model using untreated C57/Bl6 mice, but it is equally applicable to incorporation into transgenic studies and therapeutic agent development (such as eye drops), injection of therapeutic agents (including antibodies), and for longer time course studies. In vivo functional analysis or lesioned choroids can be studied with further immunohistochemical staining for further analyses.

Simple Gene Knockdown in Endothelial Cells Using Short Interfering RNA Oligonucleotides.

Cell transfection using short interfering RNAs (siRNAs) is a widely used technique to perform loss of function studies by "knocking down" genes of interest. Oftentimes, primary cells can be difficult to transfect, but here we provide a simple and robust method using cultured endothelial cells and routine transfection reagents. Knockdown studies can be used to complement overexpression studies and validate biochemical pathway analysis, as well as functional assays. The enclosed protocol will compliment other in vitro assays detailed in this edition.

Feasibility and acceptability of time-restricted eating in a group of adults with multiple sclerosis.

Introduction: Intermittent fasting (IF) has become a popular dietary pattern for adults with multiple sclerosis (MS), and initial studies in animal models and human trials indicate promising results for improving symptoms and slowing disease progression. Most studies published to date have focused on alternate day fasting or fasting mimicking diets including a 5:2 pattern, in which participants greatly restrict calorie intake on two non-consecutive days and eat regularly on other days; however, time restricted eating (TRE) may be equally effective for improving symptoms and may lead to better long term adherence due to its focus only on the time of day in which calories are consumed with no restriction on number of calories or types of food consumed. Methods: The purpose of this pilot study was to determine the feasibility and acceptability of a TRE intervention in adults with relapsing remitting MS (RRMS). Participants (n = 12) were instructed to eat all food within an 8-h window every day and fast the remaining 16 h for 8 weeks. Results: The eating pattern was determined to be feasible based on retention rates (n = 11; 92%) and acceptable based on participant feedback. Discussion: Exploratory results of changes in cognition, pain, and fatigue, indicate that further study of TRE in this population is warranted. Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT04389970; NCT04389970.

COVID-19 vaccine hesitancy and refusal and associated factors in an adult population in Saskatchewan, Canada: Evidence from predictive modelling.

BACKGROUND: A high population level of vaccination is required to control the COVID-19 pandemic, but not all Canadians are convinced of the value and safety of vaccination. Understanding more about these individuals can aid in developing strategies to increase their acceptance of a COVID-19 vaccine. The objectives of this study were to describe COVID-19 vaccine acceptance, hesitancy and refusal rates and associated factors in Saskatchewan, Canada. METHODS: This is a cross-sequential study that consisted of pooled responses from weighted samples of 9,252 Saskatchewan adults (≥18 years) across nine rounds of data collection between May 4, 2020 and April 3, 2021. The outcome variable was vaccine intention: vaccine acceptance, hesitancy, and refusal. The independent variables were layered into socio-demographic factors, risk of exposure to coronavirus, mitigating behaviours, and perceptions of COVID-19. Data were analyzed using multinomial logistic regression and a classification and regression tree. RESULTS: Seventy-six percent of the respondents indicated that they had been or were willing to be vaccinated, 13% had not yet decided, and the remaining 11% said they would not be vaccinated. Factors that increased the likelihood of vaccine refusal and hesitancy were lower education level, financial instability, Indigenous status, and not being concerned about spreading the coronavirus. Perceiving COVID-19 to be more of a threat to one's community and believing that one had a higher risk of illness or death from COVID-19 decreased the likelihood of both vaccine refusal and hesitancy. Women and newcomers to Canada were more likely to be unsure about getting vaccinated. Respondents who did not plan to be vaccinated were less likely to wear face masks and practice physical distancing. CONCLUSION: While many Canadians have voluntarily and eagerly become vaccinated already, reaching sufficient coverage of the population is likely to require targeted efforts to convince those who are resistant or unsure. Identifying and overcoming any barriers to vaccination that exist within the socio-demographic groups we found were least likely to be vaccinated is a crucial component.

Early Life Nociception is Influenced by Peripheral Growth Hormone Signaling.

A number of cellular systems work in concert to modulate nociceptive processing in the periphery, but the mechanisms that regulate neonatal nociception may be distinct compared with adults. Our previous work indicated a relationship between neonatal hypersensitivity and growth hormone (GH) signaling. Here, we explored the peripheral mechanisms by which GH modulated neonatal nociception under normal and injury conditions (incision) in male and female mice. We found that GH receptor (GHr) signaling in primary afferents maintains a tonic inhibition of peripheral hypersensitivity. After injury, a macrophage dependent displacement of injury-site GH was found to modulate neuronal transcription at least in part via serum response factor (SRF) regulation. A single GH injection into the injured hindpaw muscle effectively restored available GH signaling to neurons and prevented acute pain-like behaviors, primary afferent sensitization, and neuronal gene expression changes. GH treatment also inhibited long-term somatosensory changes observed after repeated peripheral insult. Results may indicate a novel mechanism of neonatal nociception.SIGNIFICANCE STATEMENT Although it is noted that mechanisms of pain development in early life are unique compared with adults, little research focuses on neonatal-specific peripheral mechanisms of nociception. This gap is evident in the lack of specialized care for infants following an injury including surgeries. This report evaluates how distinct cellular systems in the periphery including the endocrine, immune and nervous systems work together to modulate neonatal-specific nociception. We uncovered a novel mechanism by which muscle injury induces a macrophage-dependent sequestration of peripheral growth hormone (GH) that effectively removes its normal tonic inhibition of neonatal nociceptors to promote acute pain-like behaviors. Results indicate a possible new strategy for treatment of neonatal postsurgical pain.

Development and validation of a new microplate assay that utilises optical density to quantify the antibacterial activity of honeys including Jarrah, Marri and Manuka.

The phenol equivalence assay is the current industry-adopted test used to quantify the antibacterial activity of honeys in Australia and New Zealand. Activity is measured based on the diffusion of honey through agar and resulting zone of growth inhibition. Due to differences in the aqueous solubilities of antibacterial compounds found in honeys, this method may not be optimal for quantifying activity. Therefore, a new method was developed based on the existing broth microdilution assay that is widely used for determining minimum inhibitory concentrations (MICs). It utilises the four organisms Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853, and an optical density endpoint to quantify bacterial growth. Decreases in bacterial growth in the presence of honey, relative to the positive growth control, are then used to derive a single value to represent the overall antibacterial activity of each honey. Antibacterial activity was quantified for a total of 77 honeys using the new method, the phenol equivalence assay and the standard broth microdilution assay. This included 69 honeys with undisclosed floral sources and the comparators Manuka, Jarrah (Eucalyptus marginata), Marri (Corymbia calophylla), artificial and multifloral honey. For the 69 honey samples, phenol equivalence values ranged from 0-48.5 with a mean of 34 (% w/v phenol). Mean MICs, determined as the average of the MICs obtained for each of the four organisms for each honey ranged from 7-24% (w/v honey). Using the new assay, values for the 69 honeys ranged from 368 to 669 activity units, with a mean of 596. These new antibacterial activity values correlated closely with mean MICs (R2 = 0.949) whereas the relationship with phenol equivalence values was weaker (R2 = 0.649). Limit of detection, limit of quantitation, measuring interval, limit of reporting, sensitivity, selectivity, repeatability, reproducibility, and ruggedness were also investigated and showed that the new assay was both robust and reproducible.