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.

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.

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.

Single-cell visualization indicates direct role of sponge host in uptake of dissolved organic matter.

Marine sponges are set to become more abundant in many near-future oligotrophic environments, where they play crucial roles in nutrient cycling. Of high importance is their mass turnover of dissolved organic matter (DOM), a heterogeneous mixture that constitutes the largest fraction of organic matter in the ocean and is recycled primarily by bacterial mediation. Little is known, however, about the mechanism that enables sponges to incorporate large quantities of DOM in their nutrition, unlike most other invertebrates. Here, we examine the cellular capacity for direct processing of DOM, and the fate of the processed matter, inside a dinoflagellate-hosting bioeroding sponge that is prominent on Indo-Pacific coral reefs. Integrating transmission electron microscopy with nanoscale secondary ion mass spectrometry, we track 15N- and 13C-enriched DOM over time at the individual cell level of an intact sponge holobiont. We show initial high enrichment in the filter-feeding cells of the sponge, providing visual evidence of their capacity to process DOM through pinocytosis without mediation of resident bacteria. Subsequent enrichment of the endosymbiotic dinoflagellates also suggests sharing of host nitrogenous wastes. Our results shed light on the physiological mechanism behind the ecologically important ability of sponges to cycle DOM via the recently described sponge loop.

Soil Organic Carbon Stabilization: Mapping Carbon Speciation from Intact Microaggregates.

The clearing of land for agricultural production depletes soil organic carbon (OC) reservoirs, yet despite their importance, the mechanisms by which C is stabilized in soils remain unclear. Using synchrotron-based infrared microspectroscopy, we have for the first time obtained in situ, laterally resolved data regarding the speciation of C within sections taken from intact free microaggregates from two contrasting soils (Vertisol and Oxisol, 0-20 cm depth) impacted upon by long-term (up to 79 y) agricultural production. There was no apparent gradient in the C concentration from the aggregate surface to the interior for any of the three forms of C examined (aliphatic C, aromatic C, and polysaccharide C). Rather, organo-mineral interactions were of critical importance in influencing overall C stability, particularly for aliphatic C, supporting the hypothesis that microaggregates form through organo-mineral interactions. However, long-term cropping substantially decreased the magnitude of the organo-mineral interactions for all three forms of C. Thus, although organo-mineral interactions are important for OC stability, C forms associated with the mineral phases are not entirely resistant to degradation. These results provide important insights into the underlying mechanisms by which microaggregates form and the factors influencing the persistence of OC in soils.

Muscle IL1ß Drives Ischemic Myalgia via ASIC3-Mediated Sensory Neuron Sensitization.

UNLABELLED: Musculoskeletal pain is a significantly common clinical complaint. Although it is known that muscles are quite sensitive to alterations in blood flow/oxygenation and a number of muscle pain disorders are based in problems of peripheral perfusion, the mechanisms by which ischemic-like conditions generate myalgia remain unclear. We found, using a multidisciplinary experimental approach, that ischemia and reperfusion injury (I/R) in male Swiss Webster mice altered ongoing and evoked pain-related behaviors in addition to activity levels through enhanced muscle interleukin-1 beta (IL1ß)/IL1 receptor signaling to group III/IV muscle afferents. Peripheral sensitization depended on acid-sensing ion channels (ASICs) because treatment of sensory afferents in vitro with IL1ß-upregulated ASIC3 in single cells, and nerve-specific knock-down of ASIC3 recapitulated the results of inhibiting the enhanced IL1ß/IL1r1 signaling after I/R, which was also found to regulate afferent sensitization and pain-related behaviors. This suggests that targeting muscle IL1ß signaling may be a potential analgesic therapy for ischemic myalgia. SIGNIFICANCE STATEMENT: Here, we have described a novel pathway whereby increased inflammation within the muscle tissue during ischemia/reperfusion injury sensitizes group III and IV muscle afferents via upregulation of acid-sensing ion channel 3 (ASIC3), leading not only to alterations in mechanical and chemical responsiveness in individual afferents, but also to pain-related behavioral changes. Furthermore, these I/R-induced changes can be prevented using an afferent-specific siRNA knock-down strategy targeting either ASIC3 or the upstream mediator of its expression, interleukin 1 receptor 1. Therefore, this knowledge may contribute to the development of alternative therapeutics for muscle pain and may be especially relevant to pain caused by issues of peripheral circulation, which is commonly observed in disorders such as complex regional pain syndrome, sickle cell anemia, or fibromyalgia.

Screen detection of ductal carcinoma in situ and subsequent incidence of invasive interval breast cancers: a retrospective population-based study.

BACKGROUND: The value of screen detection and treatment of ductal carcinoma in situ (DCIS) is a matter of controversy. At present, the extent to which the diagnosis and treatment of DCIS could prevent the occurrence of invasive breast cancer in the future is not clear. We sought to estimate the association between detection of DCIS at screening and invasive interval cancers subsequent to the relevant screen. METHODS: We obtained aggregate data for screen-detected cancers from 84 local screening units within 11 regional Quality Assurance Reference Centres in England, Wales, and Northern Ireland from the National Health Service Breast Screening Programme. Data for DCIS diagnoses were obtained for women aged 50-64 years who were invited to and attended mammographic breast screening from April 1, 2003, to March 31, 2007 (4 screening years). Patient-level data for interval cancer arising in the 36 months after each of these were analysed by Poisson regression with invasive interval cancer screen detection rate as the outcome variable; DCIS detection frequencies were fitted first as a continuous and then as a categorical variable. We repeated this analysis after adjustment with both small size and high-grade invasive screen-detected cancers. FINDINGS: We analysed data for 5,243,658 women and on interval cancers occurring in the 36 months after the relevant screen. The average frequency of DCIS detected at screening was 1·60 per 1000 women screened (median 1·50 [unit range 0·54-3·56] [corrected to] per 1000 women). There was a significant negative association of screen-detected DCIS cases with the rate of invasive interval cancers (Poisson regression coefficient -0·084 [95% CI -0·13 to -0·03]; p=0·002). 90% of units had a DCIS detection frequency within the range of 1·00 to 2·22 per 1000 women; in these units, for every three screen-detected cases of DCIS, there was one fewer invasive interval cancer in the next 3 years. This association remained after adjustment for numbers of small screen-detected invasive cancers and for numbers of grade 3 invasive screen-detected cancers. INTERPRETATION: The association between screen-detected DCIS and subsequent invasive interval cancers suggests that detection and treatment of DCIS is worthwhile in prevention of future invasive disease. FUNDING: UK Department of Health Policy Research Programme and NHS Cancer Screening Programmes.

Identification of the primary lesion of toxic aluminum in plant roots.

Despite the rhizotoxicity of aluminum (Al) being identified over 100 years ago, there is still no consensus regarding the mechanisms whereby root elongation rate is initially reduced in the approximately 40% of arable soils worldwide that are acidic. We used high-resolution kinematic analyses, molecular biology, rheology, and advanced imaging techniques to examine soybean (Glycine max) roots exposed to Al. Using this multidisciplinary approach, we have conclusively shown that the primary lesion of Al is apoplastic. In particular, it was found that 75 µm Al reduced root growth after only 5 min (or 30 min at 30 µm Al), with Al being toxic by binding to the walls of outer cells, which directly inhibited their loosening in the elongation zone. An alteration in the biosynthesis and distribution of ethylene and auxin was a second, slower effect, causing both a transient decrease in the rate of cell elongation after 1.5 h but also a longer term gradual reduction in the length of the elongation zone. These findings show the importance of focusing on traits related to cell wall composition as well as mechanisms involved in wall loosening to overcome the deleterious effects of soluble Al.

Evaluation of the effect of socio-economic status on performance in a Multiple Mini Interview for admission to medical school.

INTRODUCTION: Widening participation in Medicine is a key policy priority as it helps promote a diverse and representative workforce and improves patient care. The selection process employed can influence the socio-economic composition of the student cohort and this study therefore evaluated whether Multiple Mini Interview (MMI) performance was influenced by school type or area-level Higher Education (HE) participation rates. METHODS: MMI performance for all UK applicants was recorded and consent to link performance with socio-economic data was requested using an applicant questionnaire. Station-level and total MMI scores, and offer rates were compared between applicants from non-selective, non-fee-paying schools and from selective and/or fee-paying schools; and between applicants from each quintile of area-level HE participation. RESULTS: 793 applicants were included in the analysis. MMI performance and offer rates were slightly higher for applicants from non-selective, non-fee-paying schools and/or from lower HE participation quintiles, but the effects were small and not statistically significant. CONCLUSIONS: The MMI did not favour applicants from selective and/or fee-paying schools, or from areas with high HE participation rates. Work to evaluate whether the relationship between MMI and medical school performance is the same for all sub-groups of students is now required.

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.

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.

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.

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.

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.

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.

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.