Role of Heat-Shock Proteins in the Determination of Postmortem Metabolism and Meat Quality Development of DFD Meat.

This research explored the potential role of various heat-shock proteins (HSPs) in the determination of postmortem metabolism and the development of meat quality of normal, atypical DFD, and typical DFD beef. Beef longissimus thoracis muscle samples were classified into normal, atypical DFD, and typical DFD beef. The HSP27, HSP70, and HSP90 levels, meat quality parameters, and glycolytic metabolites were tested. The results showed that color coordinates (L*, a*, and b*), glycogen, and lactate contents were lower, whereas water-holding capacity was higher in the typical DFD beef than in the normal and atypical DFD beef (p < 0.05). The expression of HSP27 on day 1 was higher in atypical DFD beef. However, expressions of HSP70 on days 1 and 3 were higher in typical DFD, while the expression of HSP90 on day 1 was higher in atypical and typical DFD compared to the normal beef (p < 0.05). Interestingly, the expression of HSP27 was positively correlated with shear force readings. HSP70 and HSP90 presented a direct correlation with pH and water-holding capacity and an indirect correlation with a* and b*, glycogen and lactate contents (p < 0.05). The study concluded that the heat-shock proteins could influence the formation of DFD beef possibly by regulating the development of postmortem metabolism and meat quality traits.

In-depth exploration of the high and normal pH beef proteome: First insights emphasizing the dynamic protein changes in Longissimus thoracis muscle from pasture-finished Nellore bulls over different postmortem times.

This study aimed to evaluate for the first time the temporal dynamic changes in early postmortem proteome of normal and high ultimate pH (pHu) beef samples from the same cattle using a shotgun proteomics approach. Ten selected carcasses classified as normal (pHu < 5.8; n = 5) or high (pHu ≥ 6.2; n = 5) pHu beef from pasture-finished Nellore (Bos taurus indicus) bulls were sampled from Longissimus thoracis muscle at 30 min, 9 h and 44 h postmortem for proteome comparison. The temporal proteomics profiling quantified 863 proteins, from which 251 were differentially abundant (DAPs) between high and normal pHu at 30 min (n = 33), 9 h (n = 181) and 44 h (n = 37). Among the myriad interconnected pathways regulating pH decline during postmortem metabolism, this study revealed the pivotal role of energy metabolism, cellular response to stress, oxidoreductase activity and muscle system process pathways throughout the early postmortem. Twenty-three proteins overlap among postmortem times and may be suggested as candidate biomarkers to the dark-cutting condition development. The study further evidenced for the first time the central role of ribosomal proteins and histones in the first minutes after animal bleeding. Moreover, this study revealed the disparity in the mechanisms underpinning the development of dark-cutting beef condition among postmortem times, emphasizing multiple dynamic changes in the muscle proteome. Therefore, this study revealed important insights regarding the temporal dynamic changes that occur in early postmortem of high and normal muscle pHu beef, proposing specific pathways to determine the biological mechanisms behind dark-cutting determination.

Non-invasive meat quality assessment: Exploring the potential of ocular infrared thermography to predict ultimate pH in Nellore beef cattle.

This study investigated the use of infrared thermography (IRT) to identify the dark, firm, and dry (DFD) phenomenon in Brazilian beef, which is a significant concern for the industry because of its inferior quality and reduced shelf life. This study examined 113 Nellore bulls and analyzed their minimum and maximum ocular temperatures using IRT. The results highlight the efficacy of thermal images (IRTmax) as a significant predictor, with R2 values ranging from 0.84 to 0.88 for calibration models. The inclusion of parameters such as glucose and lactate further enhanced prediction accuracy. The models also revealed that the combination of features, such as lightness (L*), redness (a*), and yellowness (b*), contributed to the precise prediction of pHu, with an R2 of 0.88. In model validation, RMSEP ranged from 0.104 to 0.158, indicating good generalization capability. The RPD, ranging from 1.7 to 2.6, suggests satisfactory quantitative prediction. The statistical significance of all models, evidenced by P-values <0.001, strengthens the reliability of the results. In conclusion, the models support the use of IRT as a tool for identifying pHu alterations in carcasses. When combined with blood parameters, they may exhibit even greater efficiency in predicting pHu in Nelore cattle carcasses, highlighting the potential applicability of these methods in the beef industry.

Exploring the miRNAs Profile in Dark-Cutting Beef.

In an animal production system, different stressors may cause the depletion of muscle glycogen stores, resulting in an elevated pH at 24 h post mortem (pH24), which leads to cell metabolism alterations that affect the conversion of muscle into meat, causing meat quality defects, such as dark-cutting beef, also known as dark, firm, and dry (DFD) beef. This process may involve the alteration of small non-coding RNAs (miRNAs), which play critical regulatory roles in cellular processes. Here, we determined whether differential miRNA expression in the Longissimus thoracis et lumborum muscle from the Asturiana de los Valles breed at 24 h post mortem could serve as an early indicator of beef quality defects. Following total RNA extraction, complete miRNAome sequencing revealed 12 miRNAs that were significantly upregulated (p < 0.001) in DFD beef compared to the levels in CONTROL beef. These miRNAs are mainly involved in the cellular responses to redox imbalances and apoptosis. Among these, four miRNAs known to be related to oxidative stress (bta-miR-1246, bta-miR-2332, bta-miR-23b-5p, and bta-miR-2411-3p) were validated via quantitative RT-PCR. Some of their target proteins were also analyzed using Western blotting. High 70 kDa heat shock protein and low Caspase-9 expressions (p < 0.01) were found in DFD beef, suggesting the downregulation of apoptosis. These results suggest the importance of miRNAs in regulating stress in muscle cells during early post mortem, as differences in the abundance of some of these miRNAs are still observed at 24 h post mortem. These changes lead to an inadequate conversion of muscle into meat, resulting in meats with quality defects.

A Monocular Variable Magnifications 3D Laparoscope System Using Double Liquid Lenses.

During minimal invasive surgery (MIS), the laparoscope only provides a single viewpoint to the surgeon, leaving a lack of 3D perception. Many works have been proposed to obtain depth and 3D reconstruction by designing a new optical structure or by depending on the camera pose and image sequences. Most of these works modify the structure of the conventional laparoscopes and cannot provide 3D reconstruction of different magnification views. In this study, we propose a laparoscopic system based on double liquid lenses, which provide doctors with variable magnification rates, near observation, and real-time monocular 3D reconstruction. Our system composes of an optical structure that can obtain auto magnification change and autofocus without any physically moving element, and a deep learning network based on the Depth from Defocus (DFD) method, trained to suit inconsistent camera intrinsic situations and estimate depth from images of different focal lengths. The optical structure is portable and can be mounted on conventional laparoscopes. The depth estimation network estimates depth in real-time from monocular images of different focal lengths and magnification rates. Experiments show that our system provides a 0.68-1.44x zoom rate and can estimate depth from different magnification rates at 6fps. Monocular 3D reconstruction reaches at least 6mm accuracy. The system also provides a clear view even under 1mm close working distance. Ex-vivo experiments and implementation on clinical images prove that our system provides doctors with a magnified clear view of the lesion, as well as quick monocular depth perception during laparoscopy, which help surgeons get better detection and size diagnosis of the abdomen during laparoscope surgeries.

Sensory quality of beef with different ultimate pH values - A Brazilian perspective.

This study evaluated Brazilian consumer perceptions of beef with different pH values at 48 h post-mortem (pHu) through sensory analysis. A total of 138 consumers evaluated raw and grilled steaks. The steaks were divided according to their pHu (normal pH < 5.8, atypical darkness, firmness, and dryness [DFD] > 5.8 pH < 6, and typical DFD pH ≥ 6). There was no difference in the visual evaluation of raw steaks or purchase intention. Evaluation of the grilled steaks showed that consumers preferred typical DFD steaks in terms of tenderness, and there was a tendency for the same behavior in terms of juiciness. No differences were observed in other evaluated parameters. Cluster analysis identified three consumer segments for visual evaluation, indicating a preference for steaks with higher pHu in terms of freshness. Additionally, four segments were identified for evaluation, with some groups expressing a preference for higher pHu meat in terms of freshness appearance, tenderness liking, and overall liking. The developed regression models for overall liking and purchase intention exhibited favorable adjustment indices, with r2 values of 0.86 and 0.57, respectively, for raw steaks and 0.90 for grilled steaks for in overall liking. Regression models indicated a strong influence of color and freshness appearance, in addition to tenderness liking and juiciness liking, on the overall liking scores of consumers. These results indicate that Brazilian consumers do not dislike dark cutting and, despite differentiating their greater tenderness, do not show a preference between the different pHu values.

Socioeconomic Deprivation as a Predictor of Lower Limb Amputation in Diabetic Foot Disease.

Objective The prevalence of diabetes and its complications are on the rise worldwide. This is particularly prevalent in low- and middle-income countries. The Index of Multiple Deprivation (IMD) is a measure of relative social deprivation. This index classifies England into small subsets called lower layer super output areas (LSOAs) and then ranks these LSOAs into deciles from the most deprived to the least deprived area. We used this to analyse the relationship between deprivation and outcomes in diabetic foot disease (DFD). Methods We evaluated patients admitted to our multidisciplinary unit with DFD over a five-year period. Their postal codes were used to classify these patients into IMD deciles with decile 1 representing the 10% of most deprived LSOAs and decile 10 the least deprived areas in the country. We used this classification to analyse the relationship between deprivation and its influence on surgical outcomes and lower limb amputation. We specifically compared those falling in the top five against the bottom five deciles. Results Our cohort consisted of 70 patients with diabetes who had surgery on their diabetic foot. Of this cohort, 33 (47%) of these underwent amputation. The majority of these procedures were minor amputations conducted on the forefoot (75.6%) and there were no below knee amputations. Of those requiring an amputation, 27 patients (81.8%) had an IMD decile of 5 or below, and only six (18.2%) patients had above 5. Conclusion Our study shows that the likelihood of amputations related to diabetic foot disease is inversely proportional to the index of multiple deprivation.

Role of the endoplasmic reticulum in the search for early biomarkers of meat quality.

Defects in meat quality such as dark, firm and dry (DFD) beef have been related to high levels of oxidative stress that produce cellular alterations that may affect to the process of meat quality acquisition. Despite the important role of endoplasmic reticulum (ER) in the cellular response to oxidative stress, its function in the muscle-to-meat conversion process has not yet been studied. In this study, differences in muscular antioxidant defense and the unfolded protein response (UPR) of the ER in CONTROL (normal pH24) and dark, firm, and dry (DFD, pH24 ≥ 6.2) beef at 24 h post-mortem were analyzed to understand the changes in the muscle-to-meat conversion process related to meat quality defects. DFD meat showed poor quality, lower antioxidant activity (P < 0.05) and higher UPR activation (P < 0.05), which indicates higher oxidative stress what could partly explain the occurrence of meat quality defects. Therefore, the biomarkers of these cellular processes (IRE1α, ATF6α, and p-eIF2α) are putative biomarkers of meat quality.

Behaviour of Escherichia coli O157:H7 and Listeria monocytogenes in Normal and DFD Beef of an Autochthonous Portuguese Breed.

This study was carried out to identify the behaviour of Escherichia coli O157:H7 and of Listeria monocytogenes inoculated in Maronesa breed beef with different ultimate pH (pHu) (Normal and DFD), and stored at two different temperatures (4 and 9 °C), during 28 days post mortem (pm). The main objective was to illustrate the problematic feature of dealing with beef showing high pHu and stored at mild abusive temperatures (9 °C). Beef steaks (ms. longissimus dorsi) were inoculated with low levels (2-3 log CFU/g) of those both pathogens and packed in air, vacuum and three gaseous mixtures with decreasing O2 and increasing CO2 concentrations (MAP70/20, MAP50/40 and MAP30/60). At 4 °C, the growth of E. coli O157:H7 presented the same pattern on Normal and DFD meat. On the contrary, the growth of L. monocytogenes was higher in DFD meat, revealing the effect of the pHu and its psychotropic character. At abusive temperatures, both pathogens grew, achieving high levels in DFD meat. In these cases, the MAP with the highest CO2 concentration (60%) was revealed to be more effective against the development of E. coli O157:H7, therefore, not exceeding levels of 5 log CFU/g at the end of storage, while in L. monocytogenes, it reaches 8 log CFU/g under the same conditions.

Effect of postmortem pH on the physical, biochemical, and sensory characteristics of bovine longissimus thoracis et lumborum muscle.

From a large feeding trial study consisting of 299 bulls and steers, 15 carcasses exhibited stress-related syndromes manifested by atypical color and pH which were then selected for subsequent analysis. Samples of longissimus thoracis et lumborum muscle with postmortem pH in the range of 5.5-6.9 were subjected to a 14-day aging period at 2°C. Sensory panel tenderness, connective tissue, juiciness, and flavor intensity of high pH (6.4-6.9) meat were significantly different (p < .05) from samples of intermediate pH (6.0-6.1) as well as normal pH (5.5). Muscles at pH 6.0-6.1 were the toughest samples. This was confirmed by Warner-Bratzler shear force (WBSF), residual force, and myofibril fragmentation index. Palatability attributes of normal pH (5.5) samples were significantly different (p < .05) from dark-cutting beef in terms of tenderness and flavor and at the high pH extreme. The increase in WBSF at pH 6.0-6.1, lack of extensive degradation of muscle proteins, and the decreased sarcomere length resulted in tougher meat than low or high pH muscles. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis of meat at the high pH extreme (6.7-6.9) revealed that the breakdown of troponin-T to 30 kD was complete while at intermediate pH (6.0-6.1) was incomplete. In addition, the appearance of a 'doublet' on high-molecular-weight resolution gels may also account for the greater tenderness experienced by sensory panelists.

Effect of the addition of chokeberry leaf extract on the physicochemical and sensory properties of burgers from dark cutting veal.

The occurrence of the DFD defect shortens microbiological stability and reduces consumer acceptance of meat. The effect of a chokeberry leaf extract (ChLE) addition at concentrations of 0.01 %, 0.05 % and 0.1 % on the sensory and physicochemical properties of burgers prepared from DFD meat during refrigerated storage under vacuum for 10 days was assessed. The pH, water activity, texture, colour, degree of lipid oxidation, and content of α-tocopherol were measured. The extract at concentrations of 0.05 % and 0.1 % significantly influenced lower levels of lipid oxidation and higher content of α-tocopherol on each tested period. Moreover, these concentrations had a beneficial effect on the instrumental and sensory evaluation of texture parameters and on overall quality. It was concluded that the addition of 0.1 % extract was the most beneficial over the entire 10-day storage period. In summary, use of ChLE may reduce the loss of raw meat caused by DFD defect.

Wild boar captured in a large corral-style trap or hunted: preliminary comparison of meat quality traits.

The management and numerical control of wild boars mainly depend on hunting practices, even if other alternative strategies such as the use of traps and cages can be adopted. There is little information available on the quality of captured wild boar meat. The aim of this study was to evaluate the meat quality of wild boars captured with a large corral-style trap compared to still hunting and collective hunting methods. Longissimus dorsi samples were collected from 60 wild boars, 20 of which were obtained by trapping, 20 by still hunting, and 20 by collective hunting. The animals considered were 32 males and 28 females, weighing between 42 and 68 kg. Muscle pH has been recorded at 1, 24, and 48 hours post-mortem. Furthermore, after 24 hours, color, drip loss, cooking loss, and Warner-Bratzler shear force were also evaluated. Trapping with large enclosures such as corral-style traps, if properly managed, does not seem to adversely affect the quality traits of wild boar meat, which were found to be like those obtained by the still hunting method.

Changes in postmortem metabolites profile of atypical and typical DFD beef.

This study explored the changes in postmortem metabolites of atypical and typical DFD beef using UHPLC-Q-TOF/MS. The longissimus thoracis muscles were categorized into normal, atypical DFD and typical DFD groups. The L⁎, a⁎, b⁎, glycogen and lactic acid contents were lower (P < 0.05) in typical DFD than normal beef. A total of 240 compounds were significantly different among groups. Levels of glycerol-3-phosphate, d-ribose-5-phosphate, d-glucose-6-phosphate, d-fructose-6-phosphate, alpha-d-glucose-1-phosphate and dihydroxyacetone phosphate were higher in atypical DFD than those in the normal and typical DFD beef and the contents of 9 amino acids were elevated in typical DFD. KEGG analysis showed that metabolomic changes were primarily related with ATP-binding cassette transporters, metabolism of purine, central carbon, glycine, serine and threonine, biosynthesis of aminoacyl-tRNA, digestion and absorption of proteins, metabolism of amino sugar and nucleotide sugar, pyrimidine, bile secretion and galactose. In conclusion, development of atypical and typical DFD meat results in differences at level of postmortem metabolite concentrations of different metabolic pathways.

Material selection in design for deconstruction using Kano model, fuzzy-AHP and TOPSIS methodology.

During last decade, design for deconstruction (DfD) has attracted the attention of researchers and project managers as an environmentally friendly alternative to the conventional demolition of buildings. Yet, the intensity of raw materials consumption, waste generation and greenhouse gas (GhG) emission in the construction industry proves that current methods of selecting building components have failed to make the deconstruction effectively feasible. Specifically, in the material selection process, most research studies concentrate on assessing environmental and economic aspects while in selecting material for DfD various factors must be considered. To overcome this gap, this study aims to propose a DfD-based material selection model which enables designers to choose materials that make the recyclability and reusability of building components feasible. To this end, the Kano model is first applied to categorize selection criteria identified via a questionnaire. After extracting the weights of criteria by using Fuzzy-Analytical Hierarchy Process, a Technique for Order Preference by Similarity to the Ideal Solution-based multi-criteria decision-making framework is proposed for choosing the best possible alternatives. Based on the research results, the framework enables designers to find decent materials in terms of DfD requirements. A numerical example is also provided to examine the proposed framework for selecting the most appropriate materials for walls.

Sarcoplasmic and myofibrillar phosphoproteins profile of beef M. longissimus thoracis with different pHu at different days postmortem.

BACKGROUND: The abnormal ultimate pH (pHu ) in postmortem muscles affect the meat quality and results in substantial economic losses. Dark, firm, and dry (DFD) meat linked with the higher postmortem pHu values and exhibited many quality issues such as dark color, tough texture and shorter shelf life. This research aimed to investigate the effect of protein phosphorylation on variations in beef pHu in order to explore the possible mechanisms underlying DFD meat formation. RESULTS: Glycogen and lactate contents were higher, while L* and a* were lower in high pHu beef. Shear force was higher in intermediate pHu group. Global phosphorylation of sarcoplasmic proteins was higher in low pHu samples on day 1 and of myofibrillar proteins was higher in intermediate pHu meat on days 1 and 2 postmortem. Sarcoplasmic protein bands with different phosphorylation levels were identified as containing some glycometabolism and stress response proteins and phosphorylated myofibrillar protein bands were identified sarcomeric and metabolic proteins. CONCLUSIONS: Phosphorylation of multiple proteins of glycolytic pathway and contractile machinery may play critical roles in development of DFD beef. © 2021 Society of Chemical Industry.

Use of Near-Infrared Spectroscopy to Discriminate DFD Beef and Predict Meat Quality Traits in Autochthonous Breeds.

The potential of near-infrared reflectance spectroscopy (NIRS) to discriminate Normal and DFD (dark, firm, and dry) beef and predict quality traits in 129 Longissimus thoracis (LT) samples from three Spanish purebreeds, Asturiana de los Valles (AV; n = 50), Rubia Gallega (RG; n = 37), and Retinta (RE; n = 42) was assessed. The results obtained by partial least squares-discriminant analysis (PLS-DA) indicated successful discrimination between Normal and DFD samples of meat from AV and RG (with sensitivity over 93% for both and specificity of 100 and 72%, respectively), while RE and total sample sets showed poorer results. Soft independent modelling of class analogies (SIMCA) showed 100% sensitivity for DFD meat in total, AV, RG, and RE sample sets and over 90% specificity for AV, RG, and RE, while it was very low for the total sample set (19.8%). NIRS quantitative models by partial least squares regression (PLSR) allowed reliable prediction of color parameters (CIE L*, a*, b*, hue, chroma). Results from qualitative and quantitative assays are interesting in terms of early decision making in the meat production chain to avoid economic losses and food waste.

PlGF Reduction Compromises Angiogenesis in Diabetic Foot Disease Through Macrophages.

Diabetic foot disease (DFD) is a common and serious complication for diabetes and is characterized with impaired angiogenesis. In addition to the well-defined role of vascular endothelial growth factor (VEGF) -A and its defect in the pathogenesis of DFD, another VEGF family member, placental growth factor (PlGF), was also recently found to alter expression pattern in the DFD patients with undetermined mechanisms. This question was thus addressed in the current study. We detected attenuated PlGF upregulation in a mouse DFD model. In addition, the major cell types at the wound to express the unique PlGF receptor, VEGF receptor 1 (VEGFR1), were macrophages and endothelial cells. To assess how PlGF regulates DFD-associated angiogenesis, we injected recombinant PlGF and depleted VEGF1R specifically in macrophages by local injection of an adeno-associated virus (AAV) carrying siRNA for VEGFR1 under a macrophage-specific CD68 promoter. We found that the angiogenesis and recovery of the DFD were both improved by PlGF injection. The PlGF-induced improvement in angiogenesis and the recovery of skin injury were largely attenuated by macrophage-specific depletion of VEGF1R, likely resulting from reduced macrophage number and reduced M2 polarization. Together, our data suggest that reduced PlGF compromises angiogenesis in DFD at least partially through macrophages.

Evaluation of colorimetric pH strips for wild animal meat using image analysis.

Colorimetric pH strips (MColorpHast™) are very convenient to use, but the decision of pH is based on an individual's color perception and is, therefore, subjective. We developed a pH calculation program for the image of coloring strips on CIE1976 (L*a*b* color space), which slightly underestimated human judgment as the color of pH darker. This image analysis and three individuals' judgments were used for evaluating the strip's features for various qualities of meat from wild animals, and the results were compared with the assessments based on potentiometric pH. In both methods, dipping the strips in distilled water just before use improved the regression coefficient compared with that mentioned in the manual. The image analysis showed higher correlation than human judgments but slightly underestimate pH by a maximum of 0.13 unit from the regression line of the potentiometric pH. In addition, the image analysis revealed meat pigment changed pH higher on the color scale in the lower meat pH region. The strips must be used according to the manual, but dipping is effective when the meat surface is dry, and keeping the strips from touching the meat drip is important in lower pH region because the pigment affects the color of pH.

Dark-cutting beef: A brief review and an integromics meta-analysis at the proteome level to decipher the underlying pathways.

Comprehensive characterization of the post-mortem muscle proteome defines a fundamental goal in meat proteomics. During the last decade, proteomics tools have been applied in the field of foodomics to help decipher factors underpinning meat quality variations and to enlighten us, through data-driven methods, on the underlying mechanisms leading to meat quality defects such as dark-cutting meat known also as dark, firm and dry (DFD) meat. In cattle, several proteomics studies have focused on the extent to which changes in the post-mortem muscle proteome relate to dark-cutting beef development. The present data-mining study firstly reviews proteomics studies which investigated dark-cutting beef, and secondly, gathers the protein biomarkers that differ between dark-cutting versus beef with normal-pH in a unique repertoire. A list of 130 proteins from eight eligible studies was curated and mined through bioinformatics for Gene Ontology annotations, molecular pathways enrichments, secretome analysis and biological pathways comparisons to normal beef color from a previous meta-analysis. The major biological pathways underpinning dark-cutting beef at the proteome level have been described and deeply discussed in this integromics study.

Impact of Extraction Method on the Detection of Quality Biomarkers in Normal vs. DFD Meat.

The objective of this work was to demonstrate how the extraction method affects the reliability of biomarker detection and how this detection depends on the biomarker location within the cell compartment. Different extraction methods were used to study the sarcoplasmic and myofibrillar fractions of the Longissimus thoracis et lumborum muscle of young bulls of the Asturiana de los Valles breed in two quality grades, standard (Control) or dark, firm, and dry (DFD) meat. Protein extractability and the expression of some of the main meat quality biomarkers-oxidative status (lipoperoxidation (LPO) and catalase activity (CAT)), proteome (SDS-PAGE electrophoretic pattern), and cell stress protein (Hsp70)-were analyzed. In the sarcoplasmic fraction, buffers containing Triton X-100 showed significantly higher protein extractability, LPO, and higher intensity of high-molecular-weight protein bands, whereas the TES buffer was more sensitive to distinguishing differences in the protein pattern between the Control and DFD meat. In the myofibrillar fraction, samples extracted with the lysis buffer showed significantly higher protein extractability, whereas samples extracted with the non-denaturing buffer showed higher results for LPO, CAT, and Hsp70, and higher-intensity bands in the electrophoretic pattern. These findings highlight the need for the careful selection of the extraction method used to analyze the different biomarkers considering their cellular location to adapt the extractive process.