Mushrooms: a food-based solution to vitamin D deficiency to include in dietary guidelines.

Vitamin D deficiency and insufficiency is a public health issue, with low dietary vitamin D intakes a contributing factor. Rates of vitamin D deficiency are 31% in Australia, and up to 72% in some regions globally. While supplementation is often prescribed as an alternative to additional sun exposure, complementary approaches including food-based solutions are needed. Yet, food-centric dietary guidelines are not always adequate for meeting vitamin D needs. Edible mushrooms such as Agaricus bisporus can produce over 100% of vitamin D recommendations (10 µg/day, Institute of Medicine) per 75 g serve (18 µg) on exposure to UV-light, with the vitamin D2 produced showing good stability during cooking and processing. However, mushrooms are overlooked as a vitamin D source in dietary guidelines. Our dietary modelling shows that four serves/week of UV-exposed button mushrooms can support most Australian adults in meeting vitamin D recommendations, and UV-exposed mushrooms have been found to increase vitamin D status in deficient individuals. While recent evidence suggests some differences between vitamin D2 and vitamin D3 in physiological activities, vitamin D2 from mushrooms can be part of a larger solution to increasing dietary vitamin D intakes, as well as an important focus for public health policy. Mushrooms exposed to UV represent an important tool in the strategic toolkit for addressing vitamin D deficiency in Australia and globally. Health authorities lead the recognition and promotion of mushrooms as a natural, vegan, safe, and sustainable vitamin D food source.

Health effects of drinking 100% juice: an umbrella review of systematic reviews with meta-analyses.

CONTEXT: Low fruit and vegetable intakes are major modifiable determinants of disease. One hundred percent juice may facilitate intake and deliver essential nutrients and bioactive compounds. However, the position of 100% juice in healthy eating guidelines remains controversial due to its lower dietary fiber and higher free-sugar contents compared with whole fruits and vegetables. OBJECTIVE: To conduct an umbrella review of systematic literature reviews with meta-analyses (MAs) to summarize the health benefits of drinking 100% fruit and/or vegetable juice. DATA SOURCES: Four databases (Medline, The Cochrane Library, EMBASE, and CINAHL) were systematically searched for MAs of 100% juice and any health outcomes. DATA ANALYSIS: Screening, quality, risk of bias, and content overlap tools were applied, and extracted data were narratively synthesized. No eligible studies for vegetable juice were found. Fifteen systematic literature reviews (51 primary MAs, 6 dose-response, and 87 subanalyses; 50-1200 mL/day; hours to years of duration) were included. Ten MAs (19.6%) reported health benefits (4 for blood pressure, 2 for vascular function, 3 for inflammation, 1 for stroke mortality), 3 MAs (5.9%) reported adverse risks (1 each for cardiovascular disease mortality, prostate cancer, type 2 diabetes risk), while most (74.5%) reported no effect (blood lipids, body composition, liver function, metabolic health, cancers, and inflammation). Risks were limited to cohort studies and benefits were found in both cohort and intervention studies. CONCLUSION: The findings collate evidence showing some potential health benefits associated with 100% juice consumption, with fewer potential risks. The balance of evidence does not support the exclusion of 100% juice from food-based guides to healthy eating, although caution may be warranted in certain groups or individuals, and the body of evidence is not yet conclusive. SYSTEMATIC REVIEW REGISTRATION: PROSPERO registration no. CRD42022380588.

Priority nutrients to address malnutrition and diet-related diseases in Australia and New Zealand.

Background: The double burden of malnutrition and diet-related disease has been attributed to diets high in ultra-processed and discretionary foods, with increased sugars, saturated fats, and sodium, and insufficient dietary fibre. There is a limited understanding of the role of other macronutrients and micronutrients. Objective: Determine the highest priority nutrients to address both malnutrition and diet-related disease in Australia and New Zealand, for each demographic group and the total population. Methods: A novel four-step methodological approach was undertaken to identify: 1. Demographic (age-sex) groups; 2. Health priorities; 3. Potential nutrients based on inadequacy, increased requirements, and health priority association; and 4. Priority nutrients. Nutrient intake data was obtained from the most recent Australian and New Zealand nutrition surveys. Health priorities were based on national statistical data and expert consultation. High-level scientific literature (systematic reviews) was scoped for associations with health priorities and the suitability of recommended intakes. A quantitative scoring matrix was developed and used to determine the highest priority nutrients, with scoring over three domains: extent of inadequacy; consensus for increased requirements; and degree of association with health priorities. Results: Nutritional inadequacies were common, with 22 of 31 essential nutrients consumed below recommended levels. Nine priority nutrients were identified across the demographic groups, with each demographic group characterised by a specific subset of these. Six nutrients were highest priority within the total population: vitamin D, calcium, omega-3 fatty acids, magnesium, folate, dietary fibre. Conclusion: The extent of nutritional inadequacies in Australia and New Zealand is high, both within each demographic group and the entire population, relative to both recommended intakes and key health outcomes. The methodology can be applied to other countries and globally. Findings make a significant contribution to understanding the nutrients to prioritise in future-proofing the health of the Australian and New Zealand populations. Guidelines and policies can target priority nutrients to address the malnutrition and diet-related disease double burden.

Should We 'Eat a Rainbow'? An Umbrella Review of the Health Effects of Colorful Bioactive Pigments in Fruits and Vegetables.

Health promotion campaigns have advocated for individuals to 'eat a rainbow' of fruits and vegetables (FV). However, the literature has only focused on individual color pigments or individual health outcomes. This umbrella review synthesized the evidence on the health effects of a variety of color-associated bioactive pigments found in FV (carotenoids, flavonoids, betalains and chlorophylls), compared to placebo or low intakes. A systematic search of PubMed, EMBASE, CINAHL and CENTRAL was conducted on 20 October 2021, without date limits. Meta-analyzed outcomes were evaluated for certainty via the GRADE system. Risk of bias was assessed using the Centre for Evidence-Based Medicine critical appraisal tools. A total of 86 studies were included, 449 meta-analyzed health outcomes, and data from over 37 million participants were identified. A total of 42% of health outcomes were improved by color-associated pigments (91% GRADE rating very low to low). Unique health effects were identified: n = 6 red, n = 10 orange, n = 3 yellow, n = 6 pale yellow, n = 3 white, n = 8 purple/blue and n = 1 green. Health outcomes associated with multiple color pigments were body weight, lipid profile, inflammation, cardiovascular disease, mortality, type 2 diabetes and cancer. Findings show that color-associated FV variety may confer additional benefits to population health beyond total FV intake.

Methodology for the Review and Update of Nutrient Criteria Underpinning Front-of-Pack Labeling: Application to the Glycemic Index Symbol.

Background: Nutrient criteria underlying front-of-pack food labeling programs can play an important role in improving dietary intakes. Currently, no methodology for the development or update of nutrient criteria has been published, nor the methods used by food regulatory bodies. The scientific publication of methodology outlining the development and update of nutrient criteria underpinning front-of-pack food labeling programs highlighting healthier food choices is needed. Objective: To develop and provide a globally applicable and transparent methodology for researchers to follow when reviewing existing or developing new nutrient criteria for front-of-pack labeling. Methods: The Nutrient Criteria Methodology involved five phases: Phase I, the development of guiding principles; Phase II, collection of information for subsequent phases, including a pre-scope of the literature and selection of food composition database(s) for modeling; Phase III, literature review of all possible nutrients relevant to the nutrient criteria; Phase IV, database modeling to set quantitative limits for each selected nutrient; Phase V, assessment of the criteria against an established nutritional quality assessment tool. As an example, the methodology was applied to the update of the GI Symbol Product Eligibility and Nutrient Criteria (PENC). Results: A comprehensive and replicable methodology, based on best practice protocols and ensuring both scientific credibility and practicality of use by industry, was developed. Application of the five phases of the methodology to the GI Symbol PENC highlighted the ability of the methodology to uncover nutritional measures currently missing in many nutrient criteria for front-of-pack food labeling programs and other national food labeling systems, such as glycemic load and the unsaturated to saturated fat ratio. Foods achieving the PENC had a higher Health Star Rating than foods not achieving the PENC. Conclusion: Our Nutrient Criteria Methodology can be applied to the development and update of global nutrient criteria underpinning front-of-pack food labeling programs. Further research into the implementation of additional nutritional measures found to be important for human health is recommended, with the goal of the prevention of diet-related disease.

Nutrient Dense, Low-Cost Foods Can Improve the Affordability and Quality of the New Zealand Diet-A Substitution Modeling Study.

The high prevalence of non-communicable disease in New Zealand (NZ) is driven in part by unhealthy diet selections, with food costs contributing to an increased risk for vulnerable population groups. This study aimed to: (i) identify the nutrient density-to-cost ratio of NZ foods; (ii) model the impact of substituting foods with a lower nutrient density-to-cost ratio with those with a higher nutrient density-to-cost ratio on diet quality and affordability in representative NZ population samples for low and medium socioeconomic status (SES) households by ethnicity; and (iii) evaluate food processing level. Foods were categorized, coded for processing level and discretionary status, analyzed for nutrient density and cost, and ranked by nutrient density-to-cost ratio. The top quartile of nutrient dense, low-cost foods were 56% unprocessed (vegetables, fruit, porridge, pasta, rice, nuts/seeds), 31% ultra-processed (vegetable dishes, fortified bread, breakfast cereals unfortified <15 g sugars/100 g and fortified 15-30 g sugars/100 g), 6% processed (fruit juice), and 6% culinary processed (oils). Using substitution modeling, diet quality improved by 59% and 71% for adults and children, respectively, and affordability increased by 20-24%, depending on ethnicity and SES. The NZ diet can be made healthier and more affordable when nutritious, low-cost foods are selected. Processing levels in the healthier, modeled diet suggest that some non-discretionary ultra-processed foods may provide a valuable source of low-cost nutrition for food insecure populations.

Diet and Economic Modelling to Improve the Quality and Affordability of the Australian Diet for Low and Medium Socioeconomic Households.

Food costs are a barrier to healthier diet selections, particularly for low socioeconomic households who regularly choose processed foods containing refined grains, added sugars, and added fats. In this study, the objectives were to: (i) identify the nutrient density-to-cost ratio of Australian foods; (ii) model the impact of substituting foods with lower nutrient density-to-cost ratio with those with the highest nutrient density-to-cost ratio for diet quality and affordability in low and medium socioeconomic households; and (iii) evaluate food processing levels. Foods were categorized, coded for processing level, analysed for nutrient density and cost, and ranked by nutrient density-to-cost ratio. The top quartile of nutrient dense, low-cost foods included 54% unprocessed (vegetables and reduced fat dairy), 33% ultra-processed (fortified wholegrain bread and breakfast cereals <20 g sugars/100 g), and 13% processed (fruit juice and canned legumes). Using substitution modelling, diet quality improved by 52% for adults and 71% for children across all households, while diet affordability improved by 25% and 27% for low and medium socioeconomic households, respectively. The results indicate that the quality and affordability of the Australian diet can be improved when nutritious, low-cost foods are selected. Processing levels in the healthier modelled diets suggest that some ultra-processed foods may provide a beneficial source of nutrition when consumed within national food group recommendations.

Effect of SunGold Kiwifruit and Vitamin C Consumption on Ameliorating Exercise-Induced Stress Response in Women.

INTRODUCTION: Markers of oxidative and psychological stress are elevated during high-intensity exercise. Additionally, when energy intake does not match expenditure, women who actively participate in sports and exercise are at risk of developing menstrual dysfunction, infertility, and osteoporosis. Vitamin C is known to reduce exercise-induced stress. Here, this study investigates the efficacy of consuming vitamin C from SunGold kiwifruit compared to in isolation, in ameliorating exercise-induced stress in recreationally active women. METHODS AND RESULTS: Ten eumenorrheic women are recruited in this crossover study and attended three exercise and one rest trial. In the exercise trials, participants consumed 300 mg vitamin C from kiwifruit or drink, or have a placebo drink, followed by 30-min exercise on a cycle ergometer at 60% power. During rest visit, participants sat quietly and consumed a placebo drink. Salivary uric acid (oxidative stress) and cortisol (psychological stress) are measured before and immediately after exercise for 2 h. Both vitamin C and kiwifruit reduced exercise-induced uric acid, immediately after exercise. Vitamin C drink continued to decrease uric acid for a further 30 min and slightly attenuated exercise-induced cortisol. CONCLUSIONS: Consuming liquid vitamin C prior to high-intensity cycling appears more effective than eating kiwifruit, in ameliorating exercise-induced stress in recreationally active women of reproductive age.

The impact of Hayward green kiwifruit on dietary protein digestion and protein metabolism.

PURPOSE: The purpose of the study was to determine if an actinidin protease aids gastric digestion and the protein anabolic response to dietary protein. METHODS: Hayward green kiwifruit (containing an actinidin protease) and Hort 16A gold kiwifruit (devoid of actinidin protease) were given in conjunction with a beef meal to healthy older subjects. Twelve healthy older males (N = 6) and females (N = 6) were studied with a randomized, double-blinded, crossover design to assess muscle and whole-body protein metabolism before and after ingestion of kiwifruit and 100 g of ground beef. Subjects consumed 2 of each variety of kiwifruit daily for 14 d prior to each metabolic study, and again during each study with beef intake. RESULTS: Hayward green kiwifruit consumption with beef resulted in a more rapid increase in peripheral plasma essential amino acid concentrations. There were significant time by kiwifruit intake interactions for plasma concentrations of EAAs, branched chain amino acids (BCAAs), and leucine (P < 0.01). However, there was no difference in the total amount of EAAs absorbed. As a result, there were no differences between kiwifruit in any of the measured parameters of protein kinetics. CONCLUSION: Consumption of Hayward green kiwifruit, with a beef meal facilitates protein digestion and absorption of the constituent amino acids as compared to Hort 16A gold kiwifruit. CLINICAL TRIAL: NCT04356573, April 21, 2020 "retrospectively registered".

Quantifying the contribution of dietary protein to whole body protein kinetics: examination of the intrinsically labeled proteins method.

Intrinsically labeled dietary proteins have been used to trace various aspects of digestion and absorption, including quantifying the contribution of dietary protein to observed postprandial amino acid and protein kinetics in human subjects. Quantification of the rate of appearance in peripheral blood of an unlabeled (tracee) amino acid originating from an intrinsically labeled protein (exogenous Ra) requires the assumption that there is no dilution of the isotope enrichment of the protein-bound amino acid in the gastrointestinal tract or across the splanchnic bed. It must also be assumed that the effective volume of distribution into which the tracer and tracee appear can be reasonably estimated by a single value and that any recycling of the tracer is minimal and thus does not affect calculated rates. We have assessed these assumptions quantitatively using values from published studies. We conclude that the use of intrinsically labeled proteins as currently described to quantify exogenous Ra systematically underestimates the true value. When used with the tracer-determined rates of amino acid kinetics, underestimation of exogenous Ra from the intrinsically labeled protein method likely translates to incorrect conclusions regarding protein breakdown, including the effect of a protein meal and the anabolic impact of the speed of digestion and absorption of amino acids. Estimation of exogenous Ra from the bioavailability of ingested protein has some advantages as compared with the intrinsically labeled protein method. We therefore conclude that the bioavailability method for estimating exogenous Ra is preferable to the intrinsically labeled protein method.

Endogenous Amino Acid Losses from the Gastrointestinal Tract of the Adult Human-A Quantitative Model.

Background: The loss of endogenous (nondietary) amino acids (AAs) from the gastrointestinal tract (GIT) is an important component underlying the adult human dietary requirement for protein and essential AAs (EAAs). Although data with regard to endogenous AA losses to the end of the small intestine have been published, to our knowledge there are no direct measures of colonic endogenous AA losses. Objective: The objective was to derive quantitative estimates for daily endogenous protein and EAAs lost from the colon of the adult human. Methods: A factorial model was developed for the prediction of endogenous AA losses across the adult human GIT. Estimates of AAs entering the upper GIT lumen were combined with relative protein synthesis rates in the colon to predict colonic AA losses. The AA composition of human colonic endogenous protein was calculated by estimating the relative contributions of epithelial cell protein and mucin protein on the basis of published data for cell shedding in the pig small intestine, small intestinal protein synthesis rates in pigs and humans, and human upper and lower GIT surface areas. Colonic AA losses were summed with empirical estimates of ileal AA losses in humans to estimate total daily GIT endogenous AA losses. Results: Colonic AA loss was estimated to total 3.5 g/d in the adult male human, comprising 33% of total GIT endogenous AA loss (10.2 g/d). GIT essential AA losses accounted for 25-97% of the current recommended daily AA requirement for adult humans. For threonine, colonic losses were 54% of total GIT threonine losses, which were 97% of the current recommended daily threonine requirement. Conclusions: Colonic endogenous AA losses represent a significant fraction of total GIT endogenous AA losses. The requirement of the GIT for EAAs to replace AAs lost via the gut lumen comprises a substantial proportion of the Recommended Daily Intake of AAs.

Factors contributing to the selection of dietary protein food sources.

Protein is the only dietary macronutrient required for life. As such, it is reasonable to consider dietary protein as the centerpiece of a healthy eating pattern. To do so requires consideration of what type of protein should be eaten. Account should be taken of the quality of the protein, the density of the protein in the protein food source, and the non-protein components of protein food source. The quality of protein can be quantified based on the amount and profile of essential amino acids (EAAs), as well as the true ileal digestibility of the EAAs in the protein. The density of protein in a food source can be quantified on the basis of the amount of total calories ingested to achieve intake of the daily requirement of all EAAs. Non-protein components of protein food sources can be considered in terms of the amount and nature of fat, carbohydrate and fiber, as well as the content of micronutrients. Potential beneficial effects of high-quality protein food sources should be balanced against any possible adverse effects. When all of these factors are considered we conclude that animal-based protein foods (e.g., eggs, dairy, meat, fish, poultry) occupy an important place in a healthy eating pattern.

Salivary diagnostic markers in males and females during rest and exercise.

BACKGROUND: Saliva is a useful diagnostic tool for analysis in sports, exercise and nutrition research, as collection is easy and non-invasive and it contains a large number of analytes affected by a range of physiological and pathological stressors and conditions. This study examined key salivary electrolytes and stress and immune markers in males and females at rest and during exercise. METHODS: Unstimulated whole saliva from 20 healthy, recreationally active participants (8 males and 12 females) was analysed for flow rate, osmolality, sodium (Na+), potassium (K+), chloride (Cl-), secretory immunoglobulin A (SIgA), α-amylase activity and cortisol during both rest and moderate intensity (70% peak power) cycling exercise in a randomised crossover design. Each trial lasted 60 min and sampling was carried out at 15 and 45 min after the start of the trial. Saliva was collected using the gold-standard drool method; participants were required to provide at least 1 mL sample over 2 or 3-min period. RESULTS: Females showed a greater response to steady-state exercise stress than males, with significant increases in osmolality (P < 0.001), α-amylase activity (P = 0.001) and secretion rate (P = 0.023) and SIgA secretion rate (P = 0.023), with trends for an increase in K+ (P = 0.053) and decrease in Cl- (P = 0.067). There were no differences between rest and exercise for any salivary analytes in males. In addition, females showed a trend for higher levels of cortisol than males at both rest (P = 0.099) and exercise (P = 0.070), as well as a higher heart rate (P < 0.001) and greater ratings of perceived exertion (P < 0.001) during the exercise trial. The coordination of the two stress response pathways (α-amylase vs cortisol) was positive in males (r = 0.799; P = 0.017) yet negative in females (r = -0.475; P = 0.036). CONCLUSIONS: Males and females show a markedly different response to steady-state exercise stress as measured in unstimulated whole saliva.

Dietary thiols in exercise: oxidative stress defence, exercise performance, and adaptation.

Endurance athletes are susceptible to cellular damage initiated by excessive levels of aerobic exercise-produced reactive oxygen species (ROS). Whilst ROS can contribute to the onset of fatigue, there is increasing evidence that they play a crucial role in exercise adaptations. The use of antioxidant supplements such as vitamin C and E in athletes is common; however, their ability to enhance performance and facilitate recovery is controversial, with many studies suggesting a blunting of training adaptations with supplementation. The up-regulation of endogenous antioxidant systems brought about by exercise training allows for greater tolerance to subsequent ROS, thus, athletes may benefit from increasing these systems through dietary thiol donors. Recent work has shown supplementation with a cysteine donor (N-acetylcysteine; NAC) improves antioxidant capacity by augmenting glutathione levels and reducing markers of oxidative stress, as well as ergogenic potential through association with delayed fatigue in numerous experimental models. However, the use of this, and other thiol donors may have adverse physiological effects. A recent discovery for the use of a thiol donor food source, keratin, to potentially enhance endogenous antioxidants may have important implications for endurance athletes hoping to enhance performance and recovery without blunting training adaptations.

Caffeine ingestion enhances perceptual responses during intermittent exercise in female team-game players.

We examined the influence of caffeine supplementation on cognitive performance and perceptual responses in female team-game players taking low-dose monophasic oral contraceptives of the same hormonal composition. Ten females (24 ± 4 years; 59.7 ± 3.5 kg body mass; 2-6 training sessions per week) took part in a randomised, double-blind, placebo-controlled crossover-design trial. A 90-min intermittent treadmill-running protocol was completed 60 min following ingestion of a capsule containing either 6 mg â€¢ kg(-1) anhydrous caffeine or artificial sweetener (placebo). Perceptual responses (ratings of perceived exertion (RPE), feeling scale (FS), felt arousal scale (FAS)), mood (profile of mood states (POMS)) and cognitive performance (Stroop test, choice reaction time (CRT)) were completed before, during and after the exercise protocol, as well as after ~12 h post exercise. Caffeine ingestion significantly enhanced the ratings of pleasure (P = 0.008) and arousal (P = 0.002) during the exercise protocol, as well as increased vigour (POMS; P = 0.007), while there was a tendency for reduced fatigue (POMS; P = 0.068). Caffeine ingestion showed a tendency to decrease RPE (P = 0.068) and improve reaction times in the Stroop (P = 0.072) and CRT (P = 0.087) tests. Caffeine supplementation showed a positive effect on perceptual parameters by increasing vigour and a tendency to decrease fatigue during intermittent running activity in female games players taking low-dose monophasic oral contraceptive steroids (OCS).

The C313Y Piedmontese mutation decreases myostatin covalent dimerisation and stability.

BACKGROUND: Myostatin is a key negative regulator of muscle growth and development, whose activity has important implications for the treatment of muscle wastage disorders. Piedmontese cattle display a double-muscled phenotype associated with the expression of C313Y mutant myostatin. In vivo, C313Y myostatin is proteolytically processed, exported and circulated extracellularly but fails to correctly regulate muscle growth. The C313Y mutation removes the C313-containing disulphide bond, an integral part of the characteristic TGF-ß cystine-knot structural motif. RESULTS: Here we present in vitro analysis of the structure and stability of the C313Y myostatin protein that reveals significantly decreased covalent dimerisation for C313Y myostatin accompanied by a loss of structural stability compared to wild type. The C313Y myostatin growth factor, processed from full length precursor protein, fails to inhibit C2C12 myoblast proliferation in contrast to wild type myostatin. Although structural modeling shows the substitution of tyrosine causes structural perturbation, biochemical analysis of additional disulphide mutants, C313A and C374A, indicates that an intact cystine-knot motif is a major determinant in myostatin growth factor stability and covalent dimerisation. CONCLUSIONS: This research shows that the cystine-knot structure is important for myostatin dimerisation and stability, and that disruption of this structural motif perturbs myostatin signaling.

Cytotoxic aggregation and amyloid formation by the myostatin precursor protein.

Myostatin, a negative regulator of muscle growth, has been implicated in sporadic inclusion body myositis (sIBM). sIBM is the most common age-related muscle-wastage disease with a pathogenesis similar to that of amyloid disorders such as Alzheimer's and Parkinson's diseases. Myostatin precursor protein (MstnPP) has been shown to associate with large molecular weight filamentous inclusions containing the Alzheimer's amyloid beta peptide in sIBM tissue, and MstnPP is upregulated following ER stress. The mechanism for how MstnPP contributes to disease pathogenesis is unknown. Here, we show for the first time that MstnPP is capable of forming amyloid fibrils in vitro. When MstnPP-containing Escherichia coli inclusion bodies are refolded and purified, a proportion of MstnPP spontaneously misfolds into amyloid-like aggregates as characterised by electron microscopy and binding of the amyloid-specific dye thioflavin T. When subjected to a slightly acidic pH and elevated temperature, the aggregates form straight and unbranched amyloid fibrils 15 nm in diameter and also exhibit higher order amyloid structures. Circular dichroism spectroscopy reveals that the amyloid fibrils are dominated by beta-sheet and that their formation occurs via a conformational change that occurs at a physiologically relevant temperature. Importantly, MstnPP aggregates and protofibrils have a negative effect on the viability of myoblasts. These novel results show that the myostatin precursor protein is capable of forming amyloid structures in vitro with implications for a role in sIBM pathogenesis.