Targeting dysregulated phago-/auto-lysosomes in Sertoli cells to ameliorate late-onset hypogonadism.

Age-related changes in testicular function can impact health and well-being. The mechanisms underlying age-related testicular dysfunction, such as late-onset hypogonadism (LOH), remain incompletely understood. Using single-cell RNA sequencing on human testes with LOH, we delineated Sertoli cells (SCs) as pivotal metabolic coordinators within the testicular microenvironment. In particular, lysosomal acidity probing revealed compromised degradative capacity in aged SCs, hindering autophagy and phagocytic flux. Consequently, SCs accumulated metabolites, including cholesterol, and have increased inflammatory gene expression; thus, we termed these cells as phago-/auto-lysosomal deregulated SCs. Exposure to a high-fat diet-induced phago-/auto-lysosomal dysregulated-like SCs, recapitulating LOH features in mice. Notably, efferent ductular injection and systemic TRPML1 agonist administration restored lysosomal function, normalizing testosterone deficiency and associated abnormalities in high-fat diet-induced LOH mice. Our findings underscore the central role of SCs in testis aging, presenting a promising therapeutic avenue for LOH.

Rbm46 inhibits reactive oxygen species in mouse embryonic stem cells through modulating BNIP3-mediated mitophagy.

Embryonic stem cells (ESCs) exhibit a metabolic preference for glycolysis over oxidative phosphorylation to meet their substantial adenosine triphosphate (ATP) demands during self-renewal. This metabolic choice inherently maintains low mitochondrial activity and minimal reactive oxygen species (ROS) generation. Nonetheless, the intricate molecular mechanisms governing the restraint of ROS production and the mitigation of cellular damage remain incompletely elucidated. In this study, we reveal the pivotal role of RNA-binding motif protein 46 (RBM46) in ESCs, acting as a direct post transcriptional regulator of ROS levels by modulating BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3) mRNA expression. Rbm46 knockout lead to diminished mitochondrial autophagy, culminating in elevated ROS within ESCs, disrupting the delicate balance required for healthy self-renewal. These findings provide insights into a novel mechanism governing ROS regulation in ESCs.

Bioaccessibility of lead and cadmium in soils around typical lead-acid power plants and their effect on gut microorganisms.

Potentially toxic elements (Pb and Cd) contamination of soil can adversely affect human health. Moreover, these metal ions interact with the gut microbiota after entering the human digestive system. Based on the physiologically based extraction test and the simulator of human intestinal microbial ecosystem, the bioaccessibility of Pb and Cd in soils contaminated with lead-acid power plants was assessed. The gastric stage exhibited the greatest average bioaccessibility of lead and cadmium (63.39% and 57.22%), followed by the small intestinal stage (6.86% and 36.29%); due to gut microorganisms, the bioaccessibility of lead and cadmium was further reduced in the colon stage (1.86% and 4.22%). Furthermore, to investigate soil contamination's effects on gut microbes, 16S rRNA high-throughput sequencing was used to identify the gut microbial species after the colon period. Due to Pb and Cd exposure, the relative abundance of Firmicutes and unidentified_Bacteria decreased, while the relative abundance of Proteobacteria, Synergistota, and Bacteroidota increased. The relationship between environmental factors and the number of microbial species in the gut was also examined using Spearman correlation analysis. Pb and Cd exposure has been found to affect the composition and structure of the gut microbiota.

Evaluating the Metabolic Basis of α-Gal A mRNA Therapy for Fabry Disease.

mRNA injection-based protein supplementation has emerged as a feasible treatment for Fabry disease. However, whether the introduction of LNP-encapsulated mRNA results in the alteration of metabolomics in an in vivo system remains largely unknown. In the present study, α-galactosidase A (α-Gal A) mRNA was generated and injected into the Fabry disease mouse model. The α-Gal A protein was successfully expressed. The level of globotriaosylsphingosine (Lyso-Gb3), a biomarker for Fabry disease, as well as pro-inflammatory cytokines such as nuclear factor kappa-B (NF-κB), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α), were greatly decreased compared to the untreated control, indicating the therapeutic outcome of the mRNA drug. Metabolomics analysis found that the level of 20 metabolites was significantly altered in the plasma of mRNA-injected mice. These compounds are primarily enriched in the arachidonic acid metabolism, alanine, aspartate and glutamate metabolism, and glycolysis/gluconeogenesis pathways. Arachidonic acid and 5-hydroxyeicosatetraenoic acid (5-HETE), both of which are important components in the eicosanoid pathway and related to inflammation response, were significantly increased in the injected mice, possibly due to the presence of lipid nanoparticles. Moreover, mRNA can effectively alter the level of metabolites in the amino acid and energy metabolic pathways that are commonly found to be suppressed in Fabry disease. Taken together, the present study demonstrated that in addition to supplementing the deficient α-Gal A protein, the mRNA-based therapeutic agent can also affect levels of metabolites that may help in the recovery of metabolic homeostasis in the full body system.

Microbial removal of uranyl from aqueous solution by Leifsonia sp. in the presence of different forms of iron oxides.

Immobilization of uranyl by indigenous microorganisms has been proposed as an economic and clean in-situ approach for removal of uranium, but the potential mechanisms of the process and the stability of precipitated uranium in the presence of widespread Fe(III) (hydr)oxides remain elusive. The potential of iron to serve as a reductant and/or an oxidant of uranium indicates that bioemediation strategies which mainly rely on the reduction of highly soluble U(VI) to poorly soluble U(IV) minerals to retard uranium transport in groundwater may be enhanced or hindered under different environmental conditions. This study purposes to determine the effect of ubiquitous Fe(III) (hydr)oxides (two-line ferrihydrite, hematite and goethite) on the removal of U(VI) by Leifsonia sp. isolated from an acidic tailings pond in China. The removal mechanism was elucidated via SEM-EDS, XPS and Mössbauer. The results show that the removal of U(VI) was retarded by Fe(III) (hydr)oxides when the initial concentration of U(VI) was 10 mg/L, pH was 6, temperature was 25 °C. Particularly, the retardatory effect of hematite on U(VI) removal was blindingly obvious. Also, it is worth noting that the U(VI) in the precipitate slow-released in the Fe(III) (hydrodr) oxide treatment groups, accompanied by an increase in Fe(II) concentration. SEM-EDS results demonstrated that the ferrihydrite converted to goethite may be the reason for U(VI) release in the process of 15 days culture. Mössbauer spectra fitting results further imply that the metastable iron oxides were transformed into stable Fe3O4 state. XPS measurements results showed that uranium product is most likely a mixture of Iron-U(IV) and Iron-U(VI), which indicated that the hexavalent uranium was converted into tetravalent uranium. These observations imply that the stability of the uranium in groundwater may be impacted on the prevailing environmental conditions, especially the solid-phase Fe(III) (hydr)oxide in groundwater or sediment.

Hydration and mechanical properties of arabinoxylan, (1,3;1,4)-ß-glucan, and cellulose multilayer films simulating the cell wall of wheat endosperm.

The cell walls of wheat endosperm, which play a pivotal role in seed germination, exhibit a laminated structure primarily composed of polysaccharides. In this study, composite multilayer films were prepared using arabinoxylan (AX), (1,3;1,4)-ß-D-glucan (MLG), and cellulose nanofibers (CNFs), and the effect of polymer blend structure on cell wall hydration and mechanical properties was investigated. Atomic force microscopy and X-ray diffraction indicated that the network structure of MLG/CNF exhibits a higher degree of continuity and uniformity compared to that of AX/CNF. Mechanically, the extensive linkages between MLG and CNFs chains enhance the mechanical properties of the films. Moreover, water diffusion experiments and TD-NMR analysis revealed that water molecules diffuse faster in the network structure formed by AX. We propose a structural model of the endosperm cell wall, in which the CNFs polymer blend coated with MLG serves as the framework, and the AX network fills the gaps between them, providing diffusion channels for water molecules.

Study on arsenic speciation, bioaccessibility, and gut microbiota in realgar-containing medicines by DGT technique and artificial gastrointestinal extraction (PBET) combine with simulated human intestinal microbial ecosystem (SHIME).

It is well-known that several Chinese patent medicines use realgar as a specific component. People are more aware of the health dangers associated with realgar since it includes arsenic. Previous research overstated the arsenic toxicity of realgar-containing Chinese prescription medications because little thought was given to the influence of arsenic bioaccessibility by gut microbiota. In light of this, this study examined the total content, bioaccessibility and speciation of targeted medications while also examining intestinal epithelial transit utilizing the diffusive gradients in thin-films (DGT). All samples contained arsenic, and the bioaccessibilities of the colon, intestine and gastric regions ranged from 0.19% to 1.73%, 0.25-1.88% and 0.21-1.70% respectively. The range of DGT-bioaccessibility is 0.01-0.0018%. Three steps of analysis were conducted on inorganic As(III) and As(V). In health risk assessment, the ADDs and HQs of DGT-bioaccessibility were below the threshold levels when compared to computing average daily intake dose (ADD) and hazard quotient (HQ) by bioaccessibility of gastric, intestinal and colon. Additionally, Proteobacteria and Firmicutes were discovered to be the two predominant kinds of gut microbes in this study. Under arsenic exposure, the abundance of Christensenellaceae, Desulfovibrionaceae and Akkermansiaceae increased, but the quantity of Rikenellaceae decreased. These findings revealed that alterations in gut microbiota had an impact on host metabolism.

Dual-Hand Motion Capture by Using Biological Inspiration for Bionic Bimanual Robot Teleoperation.

Bionic bimanual robot teleoperation can transfer the grasping and manipulation skills of human dual hands to the bionic bimanual robots to realize natural and flexible manipulation. The motion capture of dual hands plays an important role in the teleoperation. The motion information of dual hands can be captured through the hand detection, localization, and pose estimation and mapped to the bionic bimanual robots to realize the teleoperation. However, although the motion capture technology has achieved great achievements in recent years, visual dual-hand motion capture is still a great challenge. So, this work proposed a dual-hand detection method and a 3-dimensional (3D) hand pose estimation method based on body and hand biological inspiration to achieve convenient and accurate monocular dual-hand motion capture and bionic bimanual robot teleoperation. First, a dual-hand detection method based on body structure constraints is proposed, which uses a parallel structure to combine hand and body relationship features. Second, a 3D hand pose estimation method with bone-constraint loss from single RGB images is proposed. Then, a bionic bimanual robot teleoperation method is designed by using the proposed hand detection and pose estimation methods. Experiment results on public hand datasets show that the performances of the proposed hand detection and 3D hand pose estimation outperform state-of-the-art methods. Experiment results on a bionic bimanual robot teleoperation platform shows the effectiveness of the proposed teleoperation method.

Contrasting effects of a novel biochar-microalgae complex on arsenic and mercury removal.

Biochar and algae were commonly used as environmental-friendly adsorbents to treat wastewater contaminated with heavy metals. In the study, we used a biochar-microalgae complex of Coconut shell activated carbon (Csac) and Chlorella to evaluate and compare the adsorption ability of arsenic and mercury. The adsorption kinetic study showed that the adsorption efficiency of the biochar-microalgae complex for mercury was better remarkably than arsenic (about 74.84% higher in initial 1 min and 71.62% higher at adsorption equilibrium), which could be interpreted as the complex had excellent adsorption capacity for mercury. The new biochar-microalgae complex adsorbed up to 46.8 µg·g-1 of mercury at 100 µg·L-1 concentration. FTIR and XPS indicated that the surface of biochar-microalgae complex adsorbent had abundant oxygen-containing functional groups that could provide active sites during the adsorption process, i.e., -COOH, -OH and C-O-C et al. Compared with arsenic, the adsorption peaks of mercury moved or changed significantly, suggesting that the complex strongly adsorbed mercury and the main adsorption mechanisms were the ion exchange and complexation between functional groups and mercury ion. What must be emphasized was arsenic mainly existed as negative ions (AsO2-, AsO23-) in water, which was the reason for the weak adsorption capacity of the biochar-microalgae complex for arsenic. In short, the adsorption efficiency and performance of the biochar-microalgae complex was significantly higher than that of arsenic (p < 0.01), and the adsorption of mercury by biochar-microalgae was chemisorption based on the single molecular layer theory.

[Chloroplast genomic characterization and phylogenetic analysis of Castanopsis hystrix].

The structure and size of the chloroplast genome of Castanopsis hystrix was determined by Illumina HiSeq 2500 sequencing platform to understand the difference between C. hystrix and the chloroplast genome of the same genus, and the evolutionary position of C. hystrix in the genus, so as to facilitate species identification, genetic diversity analysis and resource conservation of the genus. Bioinformatics analysis was used to perform sequence assembly, annotation and characteristic analysis. R, Python, MISA, CodonW and MEGA 6 bioinformatics software were used to analyze the genome structure and number, codon bias, sequence repeats, simple sequence repeat (SSR) loci and phylogeny. The genome size of C. hystrix chloroplast was 153 754 bp, showing tetrad structure. A total of 130 genes were identified, including 85 coding genes, 37 tRNA genes and 8 rRNA genes. According to codon bias analysis, the average number of effective codons was 55.5, indicating that the codons were highly random and low in bias. Forty-five repeats and 111 SSR loci were detected by SSR and long repeat fragment analysis. Compared with the related species, chloroplast genome sequences were highly conserved, especially the protein coding sequences. Phylogenetic analysis showed that C. hystrix is closely related to the Hainanese cone. In summary, we obtained the basic information and phylogenetic position of the chloroplast genome of red cone, which will provide a preliminary basis for species identification, genetic diversity of natural populations and functional genomics research of C. hystrix.

Advances in air pollution control for key industries in China during the 13th five-year plan.

Industrial development is an essential foundation of the national economy, but the industry is also the largest source of air pollution, of which power plants, iron and steel, building materials, and other industries emit large amounts of pollutants. Therefore, the Chinese government has promulgated a series of stringent emission regulations, and it is against this backdrop that research into air pollution control technologies for key industrial sectors is in full swing. In particular, during the 13th Five-Year Plan, breakthroughs have been made in pollution control technology for key industrial sectors. A multi-pollutant treatment technology system of desulfurization, denitrification, and dust collection, which applies to key industries such as power plants, steel, and building materials, has been developed. High-performance materials for the treatment of different pollutants, such as denitrification catalysts and desulfurization absorbers, were developed. At the same time, multi-pollutant synergistic removal technologies for flue gas in various industries have also become a hot research topic, with important breakthroughs in the synergistic removal of NOx, SOx, and Hg. Due to the increasingly stringent emission standards and regulations in China, there is still a need to work on the development of multi-pollutant synergistic technologies and further research and development of synergistic abatement technologies for CO2 to meet the requirements of ultra-low emissions in industrial sectors.

SGLT2 inhibitor improves the prognosis of patients with coronary heart disease and prevents in-stent restenosis.

Coronary heart disease is a narrowing or obstruction of the vascular cavity caused by atherosclerosis of the coronary arteries, which leads to myocardial ischemia and hypoxia. At present, percutaneous coronary intervention (PCI) is an effective treatment for coronary atherosclerotic heart disease. Restenosis is the main limiting factor of the long-term success of PCI, and it is also a difficult problem in the field of intervention. Sodium-glucose cotransporter 2 (SGLT2) inhibitor is a new oral glucose-lowering agent used in the treatment of diabetes in recent years. Recent studies have shown that SGLT2 inhibitors can effectively improve the prognosis of patients after PCI and reduce the occurrence of restenosis. This review provides an overview of the clinical studies and mechanisms of SGLT2 inhibitors in the prevention of restenosis, providing a new option for improving the clinical prognosis of patients after PCI.

Development and students' evaluation of a blended online and offline pedagogy for physical education theory curriculum in China during the COVID-19 pandemic.

The outbreak and continuation of the COVID-19 pandemic has challenged the implementation of physical education theory (PET) curriculums among global colleges and universities. This study aimed to describe the design and students' evaluation of a blended "Sports Multimedia Courseware Design" course among Chinese university students during the COVID-19 pandemic. Using information communication technologies, a 4-month blended course was developed, which consisted of 36 credits (18-credit online self-learning + 18-credit offline group-learning). A total of 1300 Chinese university students who majored in physical education, completed the blended course from Mar to Jun 2020, among which 238 (69.75% males; 21 ± 1.2 years) were randomly recruited to evaluate the course in terms of three aspects: (1) online self-learning, (2) offline group-learning, and (3) overall learning outcomes. A descriptive analysis was conducted using the IBM SPSS 27.0. Students' overall positive evaluation supported a successful development and implementation of the blended course. Over 90% of students fulfilled the learning tasks and satisfied with the online learning resources. About 83% of students indicated high levels of autonomous motivation and engagement in online self-learning. Approximately 88% of students showed positive attitudes to the offline group-learning content, while the participation rate (60%) was relatively lower than of the online self-learning. Over 50% of the students indicated self-improvements in diverse aspects after attending the blended course. Blended online and offline pedagogy shows apparent promise in delivering the PET course among Chinese university students during the COVID-19 pandemic. Further application and comprehensive evaluation are warranted in the future.

Size Changeable Nanomedicines Assembled by Noncovalent Interactions of Responsive Small Molecules for Enhancing Tumor Therapy.

The size of nanocarriers strongly affects their performance in biological systems, especially the capacity to overcome various barriers before delivering the payloads to destinations. However, the optimum size varies at different delivery stages in cancer therapy due to the complicated tumor microenvironment. Relatively large particles are favored for long-term circulation in vivo, while smaller particles contribute to deep penetration into tumor tissues. This dilemma in the size of particles stimulates the development of stimuli-responsive size-shrinking nanocarriers. Herein, we report a facile strategy to construct a tumor-triggered tannic acid (TA) nanoassembly with improved drug delivery efficiency. Cystamine (CA), a small molecule with a disulfide bond, is thus used to mediate TA assembling via cooperative noncovalent interactions, which endows the nanoassembly with intrinsic pH/GSH dual-responsiveness. The obtained TA nanoassemblies were systematically investigated. DOX encapsulated nanoassembly labeled TCFD NP shows high drug loading efficiency, pH/GSH-responsiveness and significant size shrinkage from 122 to 10 nm with simultaneous drug release. The in vitro and in vivo experimental results demonstrate the excellent biocompatibility, sufficient intracellular delivery, enhanced tumor retention/penetration, and superior anticancer efficacy of the small-molecule-mediated nanoassembly. This noncovalent strategy provides a simple method to fabricate a tumor-triggered size-changeable delivery platform to overcome biological barriers.

A pH-responsive hyaluronic acid hydrogel for regulating the inflammation and remodeling of the ECM in diabetic wounds.

Diabetes is a universal disease in the world. In the wounds of diabetic individuals, chronic inflammation and an inefficient fibrogenic process hinder the formation and deposition of the ECM, which delays the process of wound healing. To reconstruct the ECM of a diabetic patient's wound, in this work, we designed a pH-responsive "Double H-bonds" (hydrogen bond and hydrazone bond) hyaluronic acid-collagen hydrogel. This hydrogel can be self-gelled quickly in neutral and alkaline environments. But the weakly acidic inflammatory environment of diabetic wounds may accelerate the degradation of the hydrogel and the release of metformin. The in vitro results showed that the hydrogel can enhance the adhesion and infiltration of fibroblasts while inhibiting the growth of macrophages. Meanwhile, metformin could be released and polarize macrophages from M1 to M2, thereby accelerating the migration of fibroblasts and the production of collagen in a high glucose environment. The in vivo results proved that this hydrogel could remodel the ECM in diabetic mice wounds.

Study on the mechanism of biochar loaded typical microalgae Chlorella removal of cadmium.

Coconut shell activated carbon (Csac) is one of the most widely used materials to remove cadmium (Cd) from contaminated water. A large diversity of microorganisms exists in various aquatic systems and may aid Cd removal by Csac. In this study, we explored the reactions of Csac with microalgae (Chlorella) in Cd-containing media. The results of scanning electron microscope (SEM) imaging, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), superconducting pulse-Fourier transform nuclear magnetic resonance (pulse-FT NMR) and X-ray photoelectron spectroscopy (XPS) indicated that Chlorella could adhere in the micropores of Csac formed Csac@Chlorella composite adsorbent loading Chlorella. Furthermore, the composite adsorbent surface had abundant functional groups such -COOH, -OH and C-O-C, which served as active sites during the adsorption process. Compared with Csac, Csac@Chlorella had an enhanced Cd adsorption capacity evidently. The results showed that pH 8, 0.2 g Csac, OD680 of 0.1 for Chlorella were optimal conditions for maximum Cd adsorption capacity within one hour contact time. Furthermore, the Cd adsorption process was well described by the pseudo-second-order and Langmuir adsorption isotherm models. The models revealed that the adsorption process was mainly based on chemical adsorption of a single molecular layer, accompanied by electrostatic attraction, complexation and intracellular adsorption, amongst other parameters. Collectively, the findings illustrate that the microalgae (Chlorella)-Csac-Cd interaction is complex and will thus have immense interest to a broad range of biological, environmental, and geoscience communities.

Polydopamine Nanosheets Doped Injectable Hydrogel with Nitric Oxide Release and Photothermal Effects for Bacterial Ablation and Wound Healing.

The development of wound dressings with combined antibacterial activities and pro-healing functions has always been an intractable medical task for treating bacterial wound infection. Herein, a novel injectable hybrid hydrogel dressing is developed, which is doped with nitric oxide (NO) donor (N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine, BNN6) loaded two-dimensional polydopamine nanosheets (PDA NS). The hydrogel matrix is in situ formed through dynamic Schiff base crosslinking between hydrazide-modified γ-polyglutamic acid (γ-PGA-ADH) and aldehyde-terminated Pluronic F127 (F127-CHO). Under 808 nm irradiation, the embedded PDA NS exhibits outstanding photothermal transform properties (56.1%) and on-demand NO release. The combination of photothermal and NO gas therapy with a synergistic antibacterial effect works on both Escherichia coli and Staphylococcus aureus in vitro. Furthermore, a full-thickness skin defect model also demonstrates that the hybrid hydrogel shows outstanding antibacterial properties and effectively accelerates the wound healing process. Overall, this study provides a facile and promising method for the fabrication of PDA NS based multifunctional hydrogel dressing for the application of infectious skin wound healing.

The complete chloroplast genome sequence of Bambusa albolineata (bambusodae).

Bambusa albolineata (local name: Hua Zhu) is found in Zhejiang, Jiangxi, Fujian, Taiwan, and Guangdong provinces of China, and is often cultivated on low hills, flatlands, and along streams and rivers. Due to its long internodes and flexible material, it is used as timber wood in China. In the current research, the complete chloroplast (CP) genome of B. albolineata was sequenced and reported for the first time. The complete CP genome sequence was 139,326 bp, including a large single-copy (LSC) region of 82,862 bp, a small single-copy (SSC) region of 12,870 bp, and a pair of invert repeats (IR) regions of 21,798 bp. Besides, the plastid genome consisted of 129 genes; having 82 protein-coding genes, 39 tRNA genes, and eight rRNA genes. The overall GC content of the genome was 44.2%. The phylogenetic analysis based on the complete chloroplast genome indicates that B. albolineata is strongly related to B. flexuosa and B. boniopsis.

Data assimilation of flow-acoustic resonance.

A data assimilation (DA) strategy was developed for accurate prediction of the flow-acoustic resonant fields within a channel-branch system. The challenges of numerical simulation of such internal aeroacoustic systems are primarily associated with determination of the transfer loss between the acoustic waves and the shear layer vortices. Thus, a data-assimilated momentum loss model that comprises a viscous loss item and an inertial loss item was established and embedded into the Navier-Stokes equations. During the DA, the acoustic pressure pulsations measured from a dynamic pressure array served as the observational data, the ensemble Kalman filter served as the optimization algorithm, and a three-dimensional transient computational fluid dynamics method comprising an explicit algebraic Reynolds stress model (EARSM) served as the predictive model system. EARSM was used because its ability to predict internal flow-acoustic resonances was superior to that of other eddy viscosity models and Reynolds stress models. The data-assimilated flow-acoustic resonant fields were then comprehensively validated in terms of their acoustic fields, time-averaged flow fields, and phase-dependent flow fields. The time-averaged flow fields were obtained from planar particle-image velocimetry (PIV) measurements, and the phase-dependent flow fields were obtained from field programmable gate array-based phase-locking PIV measurements. The results demonstrate that the use of DA afforded an optimal simulation that efficiently decreased the numerical errors in the frequencies and amplitudes of the acoustic pressure pulsations, thereby achieving better agreement between time-averaged flow distributions and fluctuations. In addition, the data-assimilated numerical simulation completely reproduced the spatiotemporal evolution of the shear layer vortices, that is, their formation, developing, transport, and collapsing regions.