Endoplasmic reticulum stress and associated apoptosis are linked with the pathogenesis of white striping in broiler breast muscles.

White striping (WS) that appears as white stripes parallel to the muscle fibrils is an emerging growth-related abnormality of broiler breast meat. The pathomechanism of this defect has not been fully understood despite intensive studies over the past decade. In the present study, endoplasmic reticulum (ER) stress and its associated apoptotic pathways were investigated to elucidate the potential role of these pathways in the development of WS. To this end, a total of 60 Pectoralis major (Pm) muscle samples were collected from 55-d-old Ross 308 male broiler chickens according to the severity of gross WS lesions (normal, mild, and severe). Histopathological and molecular analyses were conducted to evaluate the lesions and genes involved in the ER stress and related apoptosis. All the Pm samples, both with and without macroscopic WS lesions, showed varying degrees of myodegenerative lesions. Molecular analysis revealed that the transcript abundances of many components related to protein kinase R-like ER kinase (PERK) and inositol-requiring enzyme type 1 (IRE-1) signals of the ER stress response were significantly greater in severely WS-affected breast tissues compared to their mildly affected and normal counterparts. Similarly, the transcript abundances of apoptotic markers related to both signaling pathways were significantly greater in severe WS lesions than those of mildly affected and normal Pm tissues. Besides these, a significant increase in caspase-3 transcript abundance was seen in severe WS lesions in comparison with mild WS and normal breast muscles. Findings of this study suggest that ER stress response and its related apoptotic pathways are possibly activated in the breast muscle of broiler chickens with severe WS lesions. Based on these findings, it is speculated that ER stress-mediated apoptosis occupies a central role in the progression of WS in broiler chickens.

Association of computed tomography-derived pectoralis muscle area and density with disease severity and respiratory symptoms in patients with chronic obstructive pulmonary disease: A case-control study.

RATIONALE AND OBJECTIVES: Computed tomography (CT) is commonly used and offers an additional viewpoint for evaluating extrapulmonary symptoms, disease severity, and muscle atrophy. This study assessed whether the pectoralis muscle area (PMA) and pectoralis muscle density (PMD) are lower in patients with chronic obstructive pulmonary disease (COPD) than in healthy controls and elucidated their relationships with these variables. MATERIALS AND METHODS: The participants were enrolled in the hospital outpatient clinic between October 2023 and May 2024. Information was obtained from questionnaires, lung function, and CT imaging findings. On full-inspiratory CT, the PMA and PMD were measured at the aortic arch level using predetermined attenuation ranges of -29 and 150 Hounsfield units. We observed lower PMA and PMD and evaluated their associations with lung function, respiratory symptoms, and CT imaging findings in patients with COPD. RESULTS: Overall, 120 participants were enrolled at baseline (60 healthy controls and 60 patients with COPD). PMA and PMD were lower with progressive airflow limitation severity in those with COPD. The degree of emphysema and air trapping, as well as lung function, were correlated with PMA and PMD (P < 0.05), although not with the COPD Assessment Test or modified Medical Research Council scores (P > 0.05). CONCLUSION: Participants with COPD had smaller PMA and PMD. These measurements were correlated with the severity of airflow limitation, lung function, emphysema, and air trapping, suggesting that these features of the pectoralis muscle obtained from CT are helpful in assessments of patients with COPD.

RNA-sequencing revisited data shed new light on wooden breast myopathy.

Wooden Breast (WB) abnormality represents one of the major challenges that the poultry industry has faced in the last 10 years. Despite the enormous progress in understanding the mechanisms underlying WB, the precise initial causes remain to be clarified. In this scenario, the present research is intended to characterize the gene expression profiles of broiler Pectoralis major muscles affected by WB, comparing them to the unaffected counterpart, to provide new insights into the biological mechanisms underlying this defect and potentially identifying novel genes likely involved in its occurrence. To this purpose, data obtained in a previous study through the RNA-sequencing technology have been used to identify differentially expressed genes (DEGs) between 6 affected and 5 unaffected broilers' breast muscles, by using the newest reference genome assembly for Gallus gallus (GRCg7b). Also, to deeply investigate molecular and biological pathways involved in the WB progression, pathways analyses have been performed. The results achieved through the differential gene expression analysis mainly evidenced the downregulation of glycogen metabolic processes, gluconeogenesis, and tricarboxylic acid cycle in WB muscles, thus corroborating the evidence of a dysregulated energy metabolism characterizing breasts affected by this abnormality. Also, genes related to hypertrophic muscle growth have been identified as differentially expressed (e.g., WFIKKN1). Together with that, a downregulation of genes involved in mitochondrial biogenesis and functionality has been detected. Among them, PPARGC1A and PPARGC1B chicken genes are particularly noteworthy. These genes not only have essential roles in regulating mitochondrial biogenesis but also play pivotal roles in maintaining glucose and energy homeostasis. In view of that, their downregulation in WB-affected muscle may be considered as potentially related to both the mitochondrial dysfunction and altered glucose metabolism in WB muscles, and their key involvement in the molecular alterations characterizing this muscular abnormality might be hypothesized.

Review: Myopathies in broilers: supply chain approach to provide solutions to challenges related to raising fast growing birds.

This review is a summary of a Poultry Science Association symposium addressing myopathies in broilers' breast meat, focusing on the interactions between genetics, nutrition, husbandry, and meat processing. The Pectoralis major myopathies (woody breast [WB]; white striping [WS]; spaghetti meat [SM]) and Pectoralis minor ("feathering") are described, followed by discussing their prevalence, potential causes, current and future ways to mitigate, as well as detection methods (in live birds and meat) as well as ways to utilize affected meat. Overall, breast myopathies remain an important focus across the poultry industry and whilst a lot of data and knowledge has been gathered, it is clear that there is still a lot to understand. As there are multiple factors impacting the occurrence of breast myopathies, their reduction relies on a holistic approach. Ongoing balanced breeding strategies by poultry breeders is targeting the longer-term genetic component but comprehending the significant influence from nongenetic factors (short-term solutions such as nutrition) remains a key area of opportunity. Consequently, understanding the physiology and biological needs of the muscle through the life of the bird is critical to reduce the myopathies (e.g., minimizing oxidative stress) and gain more insight into their etiology.

Research Note: Relationships between texture and water property measurements in raw intact broiler breast fillets with the wooden breast condition.

Relationships between texture measurements and meat water properties were investigated in raw intact broiler breast fillets with the wooden breast (WB) condition. Texture measurements included subjective WB scores and blunt Meullenet-Owens Razor Shear (BMORS). Water properties were determined with low-field nuclear magnetic resonance (LF-NMR). Spearman correlation was used to estimate relationships between WB scores and water properties, while Pearson correlation was used for relationships between BMORS force and water properties. LF-NMR measurements exhibited 3 water components: protein-associated or hydration water T2b, intra-myofibrillar water or immobilized water T21, and extra-myofibrillar water or free water T22 in chicken breast meat. Significant and strong Spearman correlations were found between the WB scores and T21 time constant, the abundance (normalized areas) of T22, and the proportion of T21 and T22 (rs > 0.60, P < 0.001). Strong Pearson correlations (r = 0.72) were noted only between the T21 time constant and BMORS force. These results demonstrate that water may contribute to the specific texture characteristics measured with subjective WB scoring (palpable hardness and rigidity) and BMORS (hardness and share force) in raw broiler breast fillets with the WB condition.

Research Note: Prospects for early detection of breast muscle myopathies by automated image analysis.

White Striping (WS), Wooden Breast (WB), and Spaghetti Meat (SM) are documented breast muscle myopathies (BMM) affecting broiler chickens' product quality, profitability and welfare. This study evaluated the efficacy of our newly developed deep learning-based automated image analysis tool for early detection of morphometric parameters related to BMM in broiler chickens. Male chicks were utilized, and muscle samples were collected on d 14 of rearing. Histological procedures, including microscopic scoring, blood vessel count, and collagen quantification, were conducted. A previous study demonstrated our automated image analysis as a reliable tool for evaluating myofiber size, conforming with manual histological measurements. A threshold for BMM detection was established by normalizing and consolidating myofiber diameter and area into a unified metric based on automated measurements, also termed as "relative myofiber size value." Results show that severe myopathy broilers consistently exhibited lower relative myofiber size values, effectively detecting myopathy severity. Our study, aimed as proof of concept, underscores the potential of our automated image analysis tool as an early detection method for BMM.

Relationships of quality indices with wooden breast myopathy severity in chicken breast meat under refrigerated storage.

1. This study investigated the relationships of quality indices with the severity of wooden breast (WB) myopathy in chicken breast meat under refrigerated storage. The physicochemical properties, water-holding capacity (WHC), microbial quality and fatty acid profiles of normal chicken breast meat samples (NOR samples, n = 63), moderate WB (MWB, n = 63) myopathy and severe WB (SWB, n = 63) myopathy (MWB and SWB samples, respectively) were evaluated immediately after sampling and after 4 and 8 d of refrigerated storage at 4°C.2. Total collagen, fat, saturated and monounsaturated fatty acid contents, redness and pH of the SWB and MWB samples were higher than the NOR samples. The SWB samples that were stored for 8 d had poor WHC, total viable counts (TVC) of higher than 7.0log colony-forming units, total volatile basic nitrogen (TVB-N) content of greater than 15 mg/100 g and a thiobarbituric acid - reactive substance level of higher than 1 mg/kg malondialdehyde.3. No significant difference was observed in the TVB-N content and TVC of the MWB and NOR samples during storage. Polyunsaturated fatty acid content was lower in the SWB and MWB samples than in the NOR samples. The SWB samples were tougher than the MWB and NOR samples after 8 d of refrigeration.4. In conclusion, the quality of chicken breast meat with SWB myopathy degraded considerably over time; thus, such meat should not be subjected to extended refrigeration for storage.

Spatial transcriptomics reveals alterations in perivascular macrophage lipid metabolism in the onset of Wooden Breast myopathy in broiler chickens.

This study aims to use spatial transcriptomics to characterize the cell-type-specific expression profile associated with the microscopic features observed in Wooden Breast myopathy. 1 cm3 muscle sample was dissected from the cranial part of the right pectoralis major muscle from three randomly sampled broiler chickens at 23 days post-hatch and processed with Visium Spatial Gene Expression kits (10X Genomics), followed by high-resolution imaging and sequencing on the Illumina Nextseq 2000 system. WB classification was based on histopathologic features identified. Sequence reads were aligned to the chicken reference genome (Galgal6) and mapped to histological images. Unsupervised K-means clustering and Seurat integrative analysis differentiated histologic features and their specific gene expression pattern, including lipid laden macrophages (LLM), unaffected myofibers, myositis and vasculature. In particular, LLM exhibited reprogramming of lipid metabolism with up-regulated lipid transporters and genes in peroxisome proliferator-activated receptors pathway, possibly through P. Moreover, overexpression of fatty acid binding protein 5 could enhance fatty acid uptake in adjacent veins. In myositis regions, increased expression of cathepsins may play a role in muscle homeostasis and repair by mediating lysosomal activity and apoptosis. A better knowledge of different cell-type interactions at early stages of WB is essential in developing a comprehensive understanding.

Pectoralis Muscle Dosimetry and Posttreatment Rehabilitation Utilization for Patients With Early-Stage Breast Cancer.

PURPOSE: Up to 50% of women treated for localized breast cancer will experience some degree of arm or shoulder morbidity. Although radiation is thought to contribute to this morbidity, the mechanism remains unclear. Prior studies have shown biologic and radiographic changes in the pectoralis muscles after radiation. This study thus aimed to investigate the relationship between radiation to the pectoralis muscles and referrals for rehabilitation services posttreatment for arm and shoulder morbidity. METHODS AND MATERIALS: A retrospective 1:1 matched case-control study was conducted for patients with breast cancer who were and were not referred for breast or shoulder rehabilitation services between 2014 and 2019 at a single academic institution. Patients were included if they had a lumpectomy and adjuvant radiation. Patients who underwent an axillary lymph node dissection were excluded. Cohorts were matched based on age, axillary surgery, and use of radiation boost. Muscle doses were converted to equivalent dose in 2 Gy fractions assuming an α:ß ratio of 2.5 and were compared between the 2 groups. RESULTS: In our cohort of 50 patients of a median age 60 years (interquartile range, 53-68 years), 36 patients (72%) underwent a sentinel lymph node biopsy in addition to a lumpectomy. Although pectoralis muscle doses were generally higher in those receiving rehabilitation services, this was not statistically significant. Pectoralis major V20-40 Gy reached borderline significance, as did pectoralis major mean dose (17.69 vs 20.89 Gy; P = .06). CONCLUSIONS: In this limited cohort of patients, we could not definitively conclude a relationship between pectoralis muscle doses and use of rehabilitation services. Given the borderline significant findings, this should be further investigated in a larger cohort.

Acute muscle loss and early effects of COVID-19 on skeletal muscle in adult patients: A retrospective cohort study.

OBJECTIVES: It is known that COVID-19 has multisystemic effects. However, its early effects on muscle tissue have not been clearly elucidated. The aim of this study is to investigate early changes in the pectoral muscle in patients with COVID-19 infection. MATERIALS AND METHODS: The pectoral muscle areas (PMA) and pectoral muscle index (PMI) of 139 patients diagnosed with COVID-19 were measured from chest CTs taken at the time of the first diagnosis and within 6 months after the diagnosis. The effect of the infection on the muscle area was investigated by evaluating whether there was a change between the two measurements. Lung involvement of the infection in the first CT was scored with the CT severity score (CT-SS). In addition, the effects of patients' clinics, CT-SS, length of hospital stay, and intubation history on changes in the muscle area were investigated. RESULTS: When the PMA and PMI values were compared, there was a statistically significant decrease in the values in the control CT group compared to the first diagnosis CT group. The difference was found higher in intubated patients. CT-SS was associated with a decrease in PMI. CONCLUSION: COVID-19 is one of the causes of acute sarcopenia. Pectoralis muscle is part of the skeletal muscle, and there may be a decrease in the muscle area in the early period of the disease.

Mitochondrial dysfunction and calcium dyshomeostasis in the pectoralis major muscle of broiler chickens with wooden breast myopathy.

The incidence of wooden breast (WB) meat of commercial broiler chicken has been increasing in recent years. Histological examination found that the occurrence of WB myopathy was accompanied by the pectoralis major (PM) muscle damage. So far, the potential mechanisms are not fully understood. This study aimed to explore the underlying mechanism of the damage of WB-affected PM muscle caused by changes in mitochondrial function, mitochondrial redox status and Ca2+ homeostasis. A total of 80 market-age Arbor Acres male broiler chickens were sampled and categorized into control (CON) and WB groups based on the evaluation of myopathic lesions. PM muscle samples were collected (n = 8 in each group) for histopathological evaluation and biochemical analyses. Ultrastructural examination and histopathological changes suggested the occurrence of PM muscle damage in broiler chickens with WB myopathy. The WB group showed an increased level of reactive oxygen species and enhanced antioxidant capacities in mitochondria of PM muscle. These changes were related to impaired mitochondria morphology and mitochondrial dysfunction. In addition, abnormal expressions of Ca2+ channels led to substantial Ca2+ loss in SR and cytoplasmic Ca2+ overload, as well as Ca2+ accumulation in mitochondria, resulting in Ca2+ dyshomeostasis in PM muscle of broiler chickens with WB myopathy. Combined, these findings indicate that WB myopathy is related to mitochondrial dysfunction, mitochondrial redox status imbalance and Ca2+ dyshomeostasis, leading to WB-affected PM muscle damage.

Low muscle mass in lung cancer is associated with an inflammatory and immunosuppressive tumor microenvironment.

BACKGROUND: Computed tomographies (CT) are useful for identifying muscle loss in non-small lung cancer (NSCLC) cachectic patients. However, we lack consensus on the best cutoff point for pectoralis muscle loss. We aimed to characterize NSCLC patients based on muscularity, clinical data, and the transcriptional profile from the tumor microenvironment to build a cachexia classification model. METHODS: We used machine learning to generate a muscle loss prediction model, and the tumor's cellular and transcriptional profile was characterized in patients with low muscularity. First, we measured the pectoralis muscle area (PMA) of 211 treatment-naive NSCLC patients using CT available in The Cancer Imaging Archive. The cutoffs were established using machine learning algorithms (CART and Cutoff Finder) on PMA, clinical, and survival data. We evaluated the prediction model in a validation set (36 NSCLC). Tumor RNA-Seq (GSE103584) was used to profile the transcriptome and cellular composition based on digital cytometry. RESULTS: CART demonstrated that a lower PMA was associated with a high risk of death (HR = 1.99). Cutoff Finder selected PMA cutoffs separating low-muscularity (LM) patients based on the risk of death (P-value = 0.003; discovery set). The cutoff presented 84% of success in classifying low muscle mass. The high risk of LM patients was also found in the validation set. Tumor RNA-Seq revealed 90 upregulated secretory genes in LM that potentially interact with muscle cell receptors. The LM upregulated genes enriched inflammatory biological processes. Digital cytometry revealed that LM patients presented high proportions of cytotoxic and exhausted CD8+ T cells. CONCLUSIONS: Our prediction model identified cutoffs that distinguished patients with lower PMA and survival with an inflammatory and immunosuppressive TME enriched with inflammatory factors and CD8+ T cells.

Periosteal reaction-like lesions at the cranial aspect of the humeral diaphysis have a high prevalence in older, large breed dogs and may represent entheses of the superficial pectoral muscles.

Authors have commonly observed lamellar periosteal new bone formation at the cranial aspect of the humeral diaphysis in mediolateral radiographs of the humerus for large breed dogs with no evidence of pain or lameness. The aim of this retrospective, analytical study was to investigate the appearance and prevalence of "humeral periosteal reaction-like lesions" (HPRLL) in dogs and identify any predispositions. Mediolateral radiographs of humeri were evaluated and the presence and extent of "humeral periosteal reaction-like lesions" at the cranial aspect of the humerus were recorded. Macroscopic and histological examination of the humeri were performed for one dog with HPRLL. A total of 2877 mediolateral radiographs of 1727 dogs were included and focal or extended periosteal reaction-like lesions were found in 643 humeri of 387 dogs. Body weight ≥ 30 kg and age ≥ 7 years had a statistically significant, positive effect (P < 0.001) on the presence of HPRLL. German Shepherd dogs and Rottweilers were overrepresented in the group with HPRLL (P < 0.01). At the level of the HPRLL, the enthesis of the superficial pectoral muscles (M. pectoralis descendens and M. pectoralis transversus) to the Crista tuberculi majoris and Crista humeri were macroscopically and histologically identified. The authors propose that higher mechanical loads to the enthesis in large breed dogs may lead to physiological, age-related remodeling processes of the muscular attachment. The finding should not be confused with a pathological condition such as bone neoplasia.

Characterization of dorsal recumbency syndrome associated with woody breast in broiler flocks from Ontario, Canada.

A dorsal recumbency syndrome (DRS) has been recently described in market-age broiler chickens. Affected broilers fall onto their backs, and are unable to right themselves, and eventually die of cardiopulmonary insufficiency. These broilers are referred to as turtle chickens. A previous report and anecdotal evidence suggest that breast myopathies, such as woody breast (WB), may be associated with DRS due to impaired contractility of the pectoral muscles. In this study, we aimed to provide additional evidence to document DRS in broilers, and its possible association with breast myopathies. A total of 64 broilers (Ross 708), 33 DRS-affected and 31 controls, were culled between 42 and 48 d of age from 3 different commercial farms over 4 visits. All broilers underwent postmortem analysis; breast muscles were scored grossly and/or histologically to determine the presence and severity of myopathies, and sera were used to determine the level of aspartate aminotransferase (AST) and creatine kinase (CK). A gross diagnosis of WB was moderately associated with DRS broilers, and DRS broilers displayed a greater microscopic severity of lesions (P < 0.001) in the Pectoralis major, as typically observed with WB. Levels of AST and CK were greater (P < 0.001) in the sera of DRS-affected compared to control broilers, consistent with muscular damage. The frequency of cardiac changes, such as mild hydropericardium and right ventricular dilation, or severity of microscopic pulmonary lesions, such as edema, were not significantly different between the 2 groups. The odds of DRS increased with the histology score of the P. major (OR = 1.37, 95% CI 1.02-1.85). The data presented in this study support an association between DRS and muscular damage of the P. major, suggesting that WB may predispose broilers to DRS. DRS might be a cause of broiler death, and this syndrome could be responsible for significant financial loss to the farmers and to the whole poultry industry.

Evaluating the impact of organic chromium with flax seed in broiler diets: effects on production performance, breast muscle pathology, and meat quality aspects.

The study investigated the impact of organic chromium (Cr) and flax seed supplementation on live performance, carcass yield, muscle lipid profile, histopathological aspects, and meat quality parameters in broilers. Ninety (n = 90), day-old Cobb chicks were fed a corn-soybean meal-based diet containing 0% flax seed (Control), 10% flax seed (Diet 1) and Diet 1 + 0.05% organic Cr (Diet 2). The experiment was a completely randomized design and chicks were placed in 6 pens with 5 chicks per each pen. Pen was the experimental unit for production performance and bird collected from each pen was considered as experimental unit for all other analysis. On d 43, 45, and 48, one bird per pen were euthanized. A one-way ANOVA was performed with diet as the main factor and significance was set at P < 0.05. Significant differences between each treatment were analyzed by GLM Lean Square Method and Tukey's Honestly Significant Difference test. Weight gain and feed:gain was determined at d (1-11), (12-21), and (22-42). For all response variables, the effects among dietary treatments were compared using ANOVA separately using SAS 9.4. P -values were considered significant at ≤0.05. At d 22, Diet 1 and Diet 2 birds had lower BW and feed:gain than Control (P < 0.05). At d 42, Diet 2 birds were higher in BW with improved feed:gain when compared to Diet 1 (P < 0.05) but were not different from Control (P > 0.05). The overall weight gain was higher in Diet 2 and Control compared to Diet 1 (P < 0.05) and overall feed:gain was the highest in Control than the experimental diets (P < 0.05). Histopathological changes in breast muscle including floccular/vacuolar degeneration, fibrosis, lipidosis, interstitial inflammation, and muscle lysis were less pronounced in Diet 1 compared to Diet 2 (P < 0.05). Breast muscle total fat and cholesterol was lower in Diet 1 compared to Control (P < 0.05). Diet 1 and Diet 2 increased (>2-5 fold) total and long chain (≥20C) n-3 fatty acids (FA) in the breast muscle (P < 0.05) compared to Control. Lipid peroxidation products measured as thiobarbituric acid reactive substances were lower in the breast muscle of Diet 1 and Diet 2 compared to Control (P < 0.05). Phospholipid n-3 FA molecular species in phosphatidylcholine (PC) 36:5, 38:6, and phosphatidylethanolamine (PE) 36:5 were higher in breast muscle of Diet 1 than Control (P < 0.05) and was not different from Diet 2 (P > 0.05). A decrease in n-6 FA species (36:4 and 38:4) was observed in PC and PE of Diet 1and Diet 2 compared to Control (P < 0.05). Drip loss values were reduced in Diet 1 and Diet 2 versus Control (P < 0.05). As consumer demand for n-3 FA-rich poultry products are on the rise, Cr may serve as a feed supplement that could be used in broilers fed flax seed-containing diets for enriching edible tissues with n-3 FA, while enhancing production performance.

Intermittent dilution of dietary digestible lysine lowers the incidence of white striping by suppressing the growth, lipid synthesis, and muscle damage in broiler chickens.

BACKGROUND: White striping (WS) is a myopathy of breast muscle (Pectoralis major) that affects the quality and consumer acceptance of breast fillets of broiler chickens. Previous studies have shown that intermittent dilution of dietary nutrients suppresses the development of WS on the breast muscle of broiler chickens. However, the mechanism by which these interventions reduce the occurrence of WS remains inconclusive. In this study, we adopted intermittent reduction of dietary digestible lysine (dLys) density or metabolizable energy (ME) and amino acid (AA) density using chemical and fatty acid composition of breast fillets, and blood metabolites to understand the mechanism while histopathology and immunohistochemistry of breast muscles were used for confirmation. RESULTS: Occurrence of WS was lower in broiler chickens fed 85% dLys diets in comparison with other groups. Crude protein and ether extract in breast meat of 85% dLys groups were greater (P < 0.001) and lower (P = 0.010), respectively. Serum concentrations of lipid metabolites and enzymes were lower in broiler chickens fed 85% dLys diets than control group (P < 0.05). Feeding 85% dLys diets had low degree of myodegeneration and necrosis, inflammation, lipid deposition, infiltration of T-lymphocyte (CD3+) and macrophages (Iba-1+), and low expression of heat-shock protein 70 (HSP70) than other groups (P < 0.001). CONCLUSION: Dilution of dietary dLys to 85% of the required quantities reduces the development of WS in broiler chickens by slowing the growth, lipid synthesis, and muscle damage confirmed by lower extent of histopathological lesions. © 2022 Society of Chemical Industry.