Granule Morphology Research Articles

Insulin granule morphology and crinosome formation in mice lacking the pancreatic β cell-specific phogrin (PTPRN2) gene.

We recently reported that phogrin, also known as IA-2β or PTPRN2, forms a complex with the insulin receptor in pancreatic β cells upon glucose stimulation and stabilizes insulin receptor substrate 2. In β cells of systemic phogrin gene knockout (IA-2β-/-) mice, impaired glucose-induced insulin secretion, decreased insulin granule density, and an increase in the number and size of lysosomes have been reported. Since phogrin is expressed not only in β cells but also in various neuroendocrine cells, the precise impact of phogrin expressed in β cells on these cells remains unclear. In this study, we performed a comprehensive analysis of morphological changes in RIP-Cre+/-Phogrinflox/flox (βKO) mice with β cell-specific phogrin gene knockout. Compared to control RIP-Cre+/- Phogrin+/+ (Ctrl) mice, aged βKO mice exhibited a decreased density of insulin granules, which can be categorized into three subtypes. While no differences were observed in the density and size of lysosomes and crinosomes, organelles involved in insulin granule reduction, significant alterations in the regions of lysosomes responding positively to carbohydrate labeling were evident in young βKO mice. These alterations differed from those in Ctrl mice and continued to change with age. These electron microscopic findings suggest that phogrin expression in pancreatic β cells plays a role in insulin granule homeostasis and crinophagy during aging, potentially through insulin autocrine signaling and other mechanisms.

Caenorhabditis elegans germ granules are present in distinct configurations and assemble in a hierarchical manner.

RNA silencing pathways are complex, highly conserved, and perform crucial regulatory roles. In Caenorhabditis elegans germlines, RNA surveillance occurs through a series of perinuclear germ granule compartments - P granules, Z granules, SIMR foci, and Mutator foci - multiple of which form via phase separation. Although the functions of individual germ granule proteins have been extensively studied, the relationships between germ granule compartments (collectively, 'nuage') are less understood. We find that key germ granule proteins assemble into separate but adjacent condensates, and that boundaries between germ granule compartments re-establish after perturbation. We discover a toroidal P granule morphology, which encircles the other germ granule compartments in a consistent exterior-to-interior spatial organization, providing broad implications for the trajectory of an RNA as it exits the nucleus. Moreover, we quantify the stoichiometric relationships between germ granule compartments and RNA to reveal discrete populations of nuage that assemble in a hierarchical manner and differentially associate with RNAi-targeted transcripts, possibly suggesting functional differences between nuage configurations. Our work creates a more accurate model of C. elegans nuage and informs the conceptualization of RNA silencing through the germ granule compartments.

Starch phosphorylation regulates starch granule morphological homogeneity in Arabidopsis thaliana.

Starch granule morphological homogeneity presents a gap in starch research. Transitory starch granules in wild-type plants are discoid, regardless of species. Notably, while the shape of starch granules can differ among mutants, it typically remains homogeneous within a genotype. We found an Arabidopsis thaliana mutant, dpe2sex4, lacking both the cytosolic disproportionating enzyme 2 (DPE2) and glucan phosphatase SEX4, showing an unprecedented bimodal starch granule diameter distribution when grown under a light/dark rhythm. dpe2sex4 contained 2 types of starch granules: large granules and small granules. In contrast to the double starch initiation in wheat (Triticum aestivum) endosperm, where A-type granules are initiated first and B-type granules are initiated later, dpe2sex4 small and large granules developed simultaneously in the same chloroplast. Compared with the large granules, the small granules had more branched amylopectin and less surface starch-phosphate, thus having a more compact structure that may hinder starch synthesis. During plant aging, the small granules barely grew. In in vitro experiments, fewer glucosyl residues were incorporated in small granules. Under continuous light, dpe2sex4 starch granules were morphologically homogeneous. Omitting the dark phase after a 2-wk light/dark cycle by moving plants into continuous light also reduced morphological variance between these 2 types of granules. These data shed light on the impact of starch phosphorylation on starch granule morphology homogeneity.

Synergistic modification of ultrasound and bamboo leaf flavonoid on the rheological properties, multi-scale structure, and in vitro digestibility of pea starch.

In this study, the effect of ultrasonic treatment (UT), bamboo leaf flavonoid (BLF), ultrasonic treatment prior to bamboo leaf flavonoid (UT-BLF), and bamboo leaf flavonoid prior to the ultrasonic treatment (BLF-UT) on the rheological properties, multi-scale structure, and digestibility of pea starch (PS) were investigated. The morphology and crystal structure of starch granules were destroyed by UT, thereby promoting starch retrogradation and digestion. The binding between BLF and starch through hydrophobic interactions and hydrogen bonds inhibited the interaction between starch molecular chains and impaired their double helix structure, thus effectively retarding starch retrogradation. The anti-digestibility of starch was enhanced after synergistic treatment. Compared with single treatment, synergistic treatment increased the ordered structure and gelatinization enthalpy of starch. In comparison with the UT-BLF group, the viscoelastic and thermal stability of BLF-UT group were improved with the increase in ordered structure. This study could provide valuable information for PS modification.

Factors Affecting the Morphology of Granular Sludge in Phosphorus-Accumulating Organism (PAO) and Denitrifying PAO (DPAO) Sequencing Batch Reactors

This study aimed to investigate the influencing factors and characteristics of granule morphology through approximately 500 d of long-term monitoring of two types of anaerobic–aerobic phosphorus-accumulating organism (PAO) and anaerobic–anoxic denitrifying PAO (DPAO) sequencing batch reactors (SBRs). The results show that granules were present in the DPAO SBR and PAO SBR after 200 d and 250 d of operation, respectively. The average diameters of the granules were 2.2 ± 0.7 mm in the DPAO SBR and 0.4 ± 0.3 mm in the PAO SBR, respectively. The DPAO granular sludge contained rod-shaped microorganisms, whereas the PAO granular sludge contained cocci-type microorganisms. A precipitated core consisting of hydroxyapatite was found in the DPAO granules. A comparative analysis conducted under various operating conditions revealed that the availability and type of the electron acceptors (EAs) may have a significant impact on granulation. This observation suggests that the presence and diversity of EAs are crucial factors for the development of different granule sizes and morphologies.

Dithiothreitol Affects the Fertilization Response in Immature and Maturing Starfish Oocytes.

Immature starfish oocytes isolated from the ovary are susceptible to polyspermy due to the structural organization of the vitelline layer covering the oocyte plasma membrane, as well as the distribution and biochemical properties of the actin cytoskeleton of the oocyte cortex. After the resumption of the meiotic cycle of the oocyte triggered by the hormone 1-methyladenine, the maturing oocyte reaches fertilizable conditions to be stimulated by only one sperm with a normal Ca2+ response and cortical reaction. This cytoplasmic ripening of the oocyte, resulting in normal fertilization and development, is due to the remodeling of the cortical actin cytoskeleton and germinal vesicle breakdown (GVBD). Since disulfide-reducing agents such as dithiothreitol (DTT) are known to induce the maturation and GVBD of oocytes in many species of starfish, we analyzed the pattern of the fertilization response displayed by Astropecten aranciacus oocytes pre-exposed to DTT with or without 1-MA stimulation. Short treatment of A. aranciacus immature oocytes with DTT reduced the rate of polyspermic fertilization and altered the sperm-induced Ca2+ response by changing the morphology of microvilli, cortical granules, and biochemical properties of the cortical F-actin. At variance with 1-MA, the DTT treatment of immature starfish oocytes for 70 min did not induce GVBD. On the other hand, the DTT treatment caused an alteration in microvilli morphology and a drastic depolymerization of the cortical F-actin, which impaired the sperm-induced Ca2+ response at fertilization and the subsequent embryonic development.

Neural networks allow the automatic verification of the type of flour, analysing the starch granule morphology, to ensure the protected geographical indication ‘Galician Bread’

Quality control of flour is essential to control the quality of bread produced from it. We propose a control method based on the morphological characteristics of the granules of starch. The automation of the identification, segmentation and determination of the average size of the granules of starch of each of the cereals that make up a flour, from microscopy images, is an essential procedure for producers who want to produce bread under the protected geographical indication (PGI) ‘Galician Bread’. This identification and counting procedure, if performed manually, is a tedious activity for a trained expert, and is very time consuming. Thus, automating this task would streamline the process, in addition to saving a great deal of time. This paper addresses this problem by using deep learning approaches (Mask R–CNN) to predict the type of the granule of starch and its size for the first time. The trained models are then evaluated with the same raw microscopy images of these granules observed under polarized light, as has been previouly used for manual identification and counting. A dataset comprising 1308 2564 × 1924-pixel images is analysed. The images contain 17000 labelled granules of starch for two types of wheat: commercial wheat flour from ‘Castilla’ (type 0) and the Galician autochthonous flour ‘Caaveiro’ (type 1). The number of samples is approximately the same for each class. Instance segmentation with Mask R–CNN (Model II) achieved valid results for unseen images, with a categorical global accuracy of about 88.6% and with a discrepancy with respect to the areas of the granules as estimated by a human expert of less than 4%. The performance achieved by Mask R–CNN produces a strong correlation between the results of an expert and the results of the network, confirming the practical validity of our proposal.

Carbohydrate Profiling of Different Accessions of Three Underutilized Legumes: A Potential Source of Valuable Nutraceuticals and Pharmaceuticals

AbstractCarbohydrates are significant components of legumes, and their profiling can provide information about their nutritional value and potential health benefits. This study explores the proximate composition and carbohydrate profiles of accessions of three underutilized legumes, namely, Vigna radiata, Vigna mungo, and Macrotyloma uniflorum. The total starch and soluble sugar are determined. Starch granule morphology and diameter are determined using scanning electron microscopy and chain length distribution of amylopectin is analyzed using Capillary Electrophoresis. All accessions of the legumes vary both in their proximate composition and carbohydrate components. Accessions of M. uniflorum (PI‐658594‐01‐SD) have the highest carbohydrate component (7.6%) while V. radiata (TVR‐42) has the least (4.2%). Macrotyloma uniflorum accensions (PI‐658594‐01‐SD, and PI‐180437‐01‐SD) have the highest starch and sucrose contents, while V. mungo accensions (TVM‐13, and TVM‐11) have the lowest. The glucose and fructose contents are lower than sucrose in all the accessions of the legumes. Macrotyloma uniflorum accensions exhibit larger granules while accessions of Vigna species have the smallest granules. All species accessions exhibit similar amylopectin chain length distribution profiles although accessions of V. mungo slightly differ in their proportion of long and short glucan chains. The three species of underutilized legumes exhibit unique characteristics which make them suitable for consumption and may be exploited as a source of nutraceuticals and pharmaceuticals.

Effects of explosion puffing on the native structural organization and oil adsorption properties of starch

The effects of explosion puffing (EP) on the native structural organization (i.e., thermal properties, crystalline structure, short-range order, granule morphology and powder properties) and oil adsorption properties of puffed starch (PS) were investigated. The results showed that EP treatment could decrease the melting enthalpy of starch double helices and increase the V-type crystallinity. The highest V-type crystallinity (24.7 %) was obtained when the puffing pressure was 0.4 MPa and the starch:ethanol:water ratio was 1:2:1 (w/w). By controlling the puffing conditions, EP treatment can alter the morphology, and increase the particle size, flowability and specific surface area of PS. The high amorphous proportion and porous sheet structure of PS resulted in the highest oil adsorption capacity when the starch:ethanol:water ratio was 1:1:1 (w/w). Pearson correlation analysis showed that oil adsorption capacity was significantly and positively correlated with the 1022/995 cm−1 value and V-type crystallinity, but negatively correlated with bulk density and angle of repose. Furthermore, oil retention capacity was strongly dependent on V-type crystallinity. These findings demonstrated that EP is an innovative technology with the potential to enhance the V-type crystallinity and adsorption performance of starch.

Effects of Continuous and Cycled Annealing on the Physicochemical Properties and Digestibility of Water Caltrop Starch.

The effects of treatment time of continuous annealing (ANN) and cycle numbers of cycled ANN on the structural, physicochemical, and digestive properties of water caltrop starch were studied under 70% moisture at 65 °C. It was found that continuous and cycled ANN have no significant effects on the morphology of starch granules. However, the relative crystallinity and content of resistant starch increased pronouncedly, possibly due to crystalline perfection, which also led to the rise in gelatinization temperature and the narrowed gelatinization temperature range of starch. The treatment time in continuous ANN generally showed a pronounced effect on the rheological properties of water caltrop starch. During pasting, the breakdown viscosity and setback viscosity of all treatment decreased, implying that ANN modified starch was less susceptible to the condition in heating and continuous shearing, and less likely to cause short-term retrogradation. In contrast, peak viscosity decreased with increasing treatment time of continuous ANN, indicating crystalline perfection restricted the swelling of starch granules and viscosity development during pasting process, which was consistent with the results of steady and dynamic rheological evaluation. All ANN-modified samples showed pseudoplastic behavior with weak gel viscoelastic characteristic. Under a total annealing time of 96 h, the pasting and rheological properties of water caltrop starch were essentially less affected by annealing cycle numbers. However, multistage ANN showed stronger resistance to enzyme hydrolysis.

A comparative study on digestion and SEM based topological behaviour of six native starches hydrolysed using porcine pancreatic enzymes

The variation in structure and function of any biomolecule plays a vital role as far as the digestibility, absorption and other metabolic regulations are concerned. The amorphous to crystalline arrangement of any starchy granule reflects the polymorphic type and so-forth determines the digestion pattern as rapid, slow or resistant. The granule morphology in its native form and its ultrastructural arrangement are also crucial factors in determining the digestion behaviour of any native starch granule. The present study depicted that the digestion profile of any starchy granule is not only dictated by the compositional variation of amylose to amylopectin ratio but also the thickness of the granule layer. Although the native starch of banana and potato resisted digestion by porcine pancreatic enzymes due to the presence of thick granule layer, but the thin granule layer of taro, cassava, rice and maize starch were not able to resist the action of hydrolytic enzymes.

Process optimization for the development of traditionally roasted chickpea flour for meal replacement beverages

Chickpeas are gaining importance as a sustainable, plant-based protein source. The present study was conducted to optimize the conditions for roasting chickpea seeds that can be used in the development of products having characteristics similar to those made by the traditional approach. The temperature range was selected as 140–220 °C and the time was 1–5 min. The effect of roasting parameters on puffing index, bulk density, water absorption capacity, browning index, color difference, TPC, antioxidant activity, and overall acceptability was analyzed. Based on the criteria set, optimum roasting conditions were selected using numerical optimization. The optimum solution was found at 181.64 °C and a time of 3.17 min, with a desirability of 0.871. Roasting significantly affected chickpea flour's physical, proximate, functional, microstructural, thermal, and pasting properties. TPC and antioxidant activity increased, whereas TFC decreased after roasting. The morphology of starch granules was highly modified, and their thermal properties also varied considerably.

Metagenomic Analysis of a Continuous-Flow Aerobic Granulation System for Wastewater Treatment.

Aerobic granulation is an emerging process in wastewater treatment that has the potential to accelerate sedimentation of the microbial biomass during secondary treatment. Aerobic granulation has been difficult to achieve in the continuous flow reactors (CFRs) used in modern wastewater treatment plants. Recent research has demonstrated that the alternation of nutrient-abundant (feast) and nutrient-limiting (famine) conditions is able to promote aerobic granulation in a CFR. In this study, we conducted a metagenomic analysis with the objective of characterizing the bacterial composition of the granular biomass developed in three simulated plug flow reactors (PFRs) with different feast-to-famine ratios. Phylogenetic analyses revealed a clear distinction between the bacterial composition of aerobic granules in the pilot simulated PFRs as compared with conventional activated sludge. Larger and denser granules, showing improved sedimentation properties, were observed in the PFR with the longest famine time and were characterized by a greater proportion of bacteria producing abundant extracellular polymeric substances (EPS). Functional metagenomic analysis based on KEGG pathways indicated that the large and dense aerobic granules in the PFR with the longest famine time showed increased functionalities related to secretion systems and quorum sensing, which are characteristics of bacteria in biofilms and aerobic granules. This study contributes to a further understanding of the relationship between aerobic granule morphology and the bacterial composition of the granular biomass.

Influence of drying and granulation process conditions on the characteristics of micronutrient chelates granules

Abstract Fluidized-bed spray granulation (FBSG) enables manufacturing particles with desired characteristics, including particle size distribution (PSD), density, or dust content. This study investigated the effect of selected factors on the granules obtained in a continuous FBSG of chelated fertilizers for foliar applications. The effect of surfactant addition to the solution sprayed into the bed and perturbations of operating parameters on PSD and granules morphology was studied. The experiments were supplemented with calculations based on a population balance equation (PBE). It was shown that granules manufactured with the tenside addition are more regular in shape, and thus less prone to mechanical wear. It was demonstrated that increasing rotational mill speed does contribute to a slight increase in the amount of dust, but in the long term, it does not disturb the regular agglomeration process. The computational results confirm that, despite the complexity of the process, its description with PBE is feasible.

The physicochemical and structural properties and in vitro digestibility of pea starch isolated from flour ground by milling and air classification.

The fine, coarse and parent starches were isolated from pea flour by milling and air-classification. Their structural, thermal, physicochemical properties and in vitro digestibility were investigated. Particle Size Distribution showed the fine starch with the smallest unimodal distribution (18.33 and 19.02μm) displayed higher degree of short-range molecular order and lower number of double helix structure. Scanning Electron Microscopy showed the morphology of the coarse starch granules as uniform in size and lacking protein particles on its smooth surface. Differential Scanning Calorimetry revealed the coarse starch had higher enthalpy changes while Rapid Visco Analysis showed higher peak, trough, and breakdown viscosities for the fine starch. In vitro digestibility featured the fine starch containing lower fast digesting starch contents, but with higher resistant starch content, indicating its resistance to enzymatic hydrolysis. The results could provide theoretical support for application of pea starch in functional foods and the manufacture of emerging starch products.

Physicochemical and Functional Properties of Banana Starch and Its Alternative Returns

The current study’s goal is to isolate the starch from different varieties of banana i.e. Local Banana (LB) and BARI Banana2 (BB2)and characterize them in order to explore their physical and chemical compositions, amylose content, resistant starch content, functional properties, granule morphology and crystalline pattern. Different techniques were used for characterization viz. X-ray diffractometery (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Spectroscopy (SEM) etc. The starch extracted from banana showed significant difference in the amylose content and the value was found 28.26%and 25.93%respectively for local banana starch (LBS) and bari banana 2 starch (BB2S). This study also revealed that selected banana varieties can be a potential source for the production of resistant starch. The resistant starch (RS) content of the banana was found 46.93% and 45.78% respectively for LBS and BB2S. Using de-branching and autoclaving technique in the starch, there was a significant increase in RS content. Paste clarity was decreased gradually with time for all the starch samples. Water absorption capacity (WAC) and oil absorption capacity (OAC) of LBS and BB2Swere found in the ranged of 165.8-170.7% and 132.6-168.1% respectively. XRD showed both LB and BB2 starches had B-type crystal pattern. The d-spacing of banana starches were ranged from 3.86-5.88Á. SEM data revealed that banana starches possessed the regular elongated round ridges with smooth surface in the range of 4.3-12.4 µm. The information obtained from this research indicates that both banana starches can be used in different areas; especially in food, pharmaceutical and cosmetics industries as an edible coating, thickening agent, fat replacer, etc. Also, banana starch containing functional food can be recommended for diabetic patients.

Enhancing Starch-Based Packaging Materials: Optimization of Plasticizers and Process Parameters.

In order to actively promote green production and address these concerns, there is an urgent need for new packaging materials to replace traditional plastic products. Starch-based packaging materials, composed of starch, fiber, and plasticizers, offer a degradable and environmentally friendly alternative. However, there are challenges related to the high crystallinity and poor compatibility between thermoplastic starch and fibers, resulting in decreased mechanical properties. To address these challenges, a novel approach combining plasticizer optimization and response surface method (RSM) optimization has been proposed to enhance the mechanical properties of starch-based packaging materials. This method leverages the advantages of composite plasticizers and process parameters. Scanning electron microscopy and X-ray crystallography results demonstrate that the composite plasticizer effectively disrupts the hydrogen bonding and granule morphology of starch, leading to a significant reduction in crystallinity. Fourier transform infrared spectroscopy results show that an addition of glycerol and D-fructose to the starch can form new hydrogen bonds between them, resulting in an enhanced plasticizing effect. The optimal process parameters are determined using the RSM, resulting in a forming temperature of 198 °C, a forming time of 5.4 min, and an AC content of 0.84 g. Compared with the non-optimized values, the tensile strength increases by 12.2% and the rebound rate increases by 8.1%.

Morphology and structure characterization of hollow granule mold powder for continuous casting

Characteristic parameters of granule morphology (granule diameter and sphericity) and structure (pore size, cross-sectional structure, and porosity) of mold powders were qualitatively and quantitatively analyzed. The results indicated that the granules had a rough surface, good sphericity, and a hollow structure; the internal pores had different diameters, location distributions, and irregular shapes; and the raw material powder particles were interwoven to form a cross-linked network. Energy-dispersive X-ray spectroscopy of the surface and cross-section of the mold powder granules revealed the internal structure and elemental distribution pattern, migration mechanism of the binder, and formation mechanism of the solid crust. The sphericity s values of the mold powder granules were mostly of class B (0.90–0.75 mm), accounting for 72.39%. The s values of more than 90% of granules exceeded 0.8. The porosity values, such as H and ε, increased as the internal pore diameter increased, reaching 0.92 and 0.96, respectively. The H and ε values of more than 90% of the granules were mainly concentrated in the 0.4–0.8 range, and the relationship between H and ε values as well as the variation in granule diameter and pore diameter were mainly consistent.

The Effect of Dual-Modification by Heat-Moisture Treatment and Octenylsuccinylation on Physicochemical and Pasting Properties of Arrowroot Starch.

Starch is widely applied in various industrial sectors, including the food industry. Starch is used as a thickener, stabilizer, or emulsifier. However, arrowroot starch generally has weaknesses, such as unstable under heating and acidic conditions, which are generally applied to processing in the food industry. Modifications were applied to improve the characteristics of native arrowroot starch. In this study, arrowroot starch was modified by heat-moisture treatment (HMT), octenylsuccinylation (OSA), and dual modification between OSA and HMT in a different sequence--namely, HMT followed by OSA, and OSA followed by HMT. This study aims to determine the effect of different modification methods on the physicochemical and functional properties of native arrowroot starch. The result shows that both single HMT and dual modification caused damage to native starch granules, such as the formation of cracks and roughness. For single OSA treatment, especially, there is no significant change in granule morphology after modification. All modification treatments did not change the crystalline type of starch but reduced the RC of native starch. Both single HMT and dual modifications (HMT-OSA, OSA-HMT) increased pasting temperature and setback, but, conversely, decreased the peak and the breakdown viscosity of native starch, whereas single OSA had the opposite trend compared with the other modifications. HMT played a greater role in increasing the thermal stability and the retrogradation ability of arrowroot starch. Both single modifications (HMT and OSA) increased the hardness and gumminess of native starch, and the opposite was true for the dual modifications. HMT had a greater effect on color characteristics, where the lightness and whiteness index of native arrowroot starch decreased. Single OSA modification increased swelling volume higher than dual modification. Both single HMT and dual modifications increased water absorption capacity and decreased the oil absorption capacity of native arrowroot starch.

ESEM-EDX Mineralization and Morphological Analysis of Human Retrieved Maxillary Sinus Bone Graft Biopsies before Loading.

This study aimed to analyze the morphology of bone graft granules, the presence of granule demineralization, and bone morphology in retrieved human maxillary sinus bone graft biopsies. Healthy patients underwent sinus bone augmentation using lateral access. Two different dimensions of the antrostomy were performed, a 4 mm or 8 mm height. After 6 months, all sites received one implant using a flap technique, crestal positioning, and submerged healing. Implant biopsies were retrieved after 3 months and were histologically processed. The ESEM analysis was performed on the entire portion of the peri-implant bone (up to 750 µm from the implant thread). Three different regions of interest (ROIs) were selected: the coronal, middle, and apical portions of the implant. In these areas, EDX was performed, and calcium (Ca), phosphate (P), nitrogen (N), and their atomic ratios (Ca/P, Ca/N, and P/N) were calculated. Different bone tissue electron-dense areas were detected through grayscale intensity quantification of ESEM images with different organic (N) or inorganic (Ca,P) compositions. A total of 16 biopsies from 16 healthy patients were analyzed. Bone graft granules were mostly detected in the apical ROI. New bone tissue bridges were detected in the apical and middle ROI. These structures, with lower Ca/N and P/N ratios, were connected and enveloped the bone graft granules. Cortical ROI revealed the most mineralized bone tissue. Conclusions: After 9 months, bone graft resorption was only partially completed and new bone tissue appeared less mineralized in the middle and apical ROI than in the coronal ROI.