Intercellular Distance Research Articles

Long-term remission of Hailey-Hailey disease by Er:YAG ablative laser therapy.

Hailey-Hailey disease (HHD) is a rare genetic therapy-resistant blistering disease with great disease burden. Treatment is currently focused on symptomatic relief. Er:YAG ablative laser therapy is a therapeutic modality with promising results, though evidence is currently scarce. To analyse the effect of Er:YAG ablative laser therapy on clinical remission of erosive plaques in HHD and on patient's quality of life (QoL). In this observational study eight patients were included and 77 erosive plaques were treated once only by Er:YAG laser and assessed for clinical remission. QoL was evaluated by obtaining Skindex-29 and DLQI questionnaires before laser therapy, 6 weeks and 3 years after laser therapy. Skin biopsies were taken to evaluate the depth of laser ablation. The intercellular distance between keratinocytes, the number of desmosomes and intermediate filament distribution were studied by electron microscopy before and after laser therapy and in clinically uninvolved skin and were compared to subjects without HHD. One single Er:YAG laser ablation to mid-dermis resulted in complete remission of 97.4% of HHD plaques (75/77) after median 38 months (range 7-63 months) and significantly improved QoL. Laser therapy restored the number of desmosomes, decreased intercellular distance and diminished perinuclear retraction of keratin filaments to a level comparable to the patient's clinically uninvolved skin. After laser ablation, the skin showed significantly fewer ultrastructural aberrations compared to the patient's clinically uninvolved skin and rather resembled the skin of healthy control individuals. One single Er:YAG laser treatment resulted in long-term remission of HHD and significantly improved QoL. Our findings support a greater role for ablative laser surgery in the management of this recalcitrant disorder.

Histological differences related to autophagy in the minor salivary gland between primary and secondary types of Sjögren’s syndrome

Some forms of Sjögren’s syndrome (SS) follow a clinical course accompanied by systemic symptoms caused by lymphocyte infiltration and proliferation in the liver, kidneys, and other organs. To better understand the clinical outcomes of SS, here we used minor salivary gland tissues from patients and examine their molecular, biological, and pathological characteristics. A retrospective study was performed, combining clinical data and formalin-fixed paraffin-embedded (FFPE) samples from female patients over 60 years of age who underwent biopsies at Okayama University Hospital. We employed direct digital RNA counting with nCounter® and multiplex immunofluorescence analysis with a PhenoCycler™ on the labial gland biopsies. We compared FFPE samples from SS patients who presented with other connective tissue diseases (secondary SS) with those from stable SS patients with symptoms restricted to the exocrine glands (primary SS). Secondary SS tissues showed enhanced epithelial damage and lymphocytic infiltration accompanied by elevated expression of autophagy marker genes in the immune cells of the labial glands. The close intercellular distance between helper T cells and B cells positive for autophagy-associated molecules suggests accelerated autophagy in these lymphocytes and potential B cell activation by helper T cells. These findings indicate that examination of FFPE samples from labial gland biopsies can be an effective tool for evaluating molecular histological differences between secondary and primary SS through multiplexed analysis of gene expression and tissue imaging.

Design of a Membrane-Anchored DNAzyme-Based Molecular Machine for Enhanced Cancer Therapy by Customized Cascade Regulation.

Synthetic DNAzyme-based structures enable dynamic cell regulation. However, engineering an effective and targeted DNAzyme-based structure to perform customizable multistep regulation remains largely unexplored. Herein, we designed a membrane-anchored DNAzyme-based molecular machine to implement dynamic inter- and intracellular cascade regulation, which realizes efficient T-cell/cancer cell interactions and subsequent receptor mediated cancer cell uptake. Using CD8+ T-cells and HeLa cancer cells as a proof of concept, we demonstrate that the designed DNAzyme-based molecular machine enables customized cascade regulation including (1) specific recognition between T-cells and cancer cells, (2) specific response and fluorescence sensing upon extracellular stimuli, and (3) cascade regulation including intercellular distance shortening, cell-cell communication, and intracellular delivery of anticancer drugs. Together, this work provides a promising pathway for customized cascade cell regulation based on a DNAzyme-based molecular machine, which enables enhanced cancer therapy by combining T-cell immunotherapy and chemotherapy.

The investigation of retinoic acid on spermatogenetic cell types of rats

Background/Aim: Vitamin A is crucial for male fertility and the progression of the spermatogenetic process. Retinoic acid (RA), a metabolite of vitamin A, binds to three nuclear receptors, functioning similarly to a hormone by activating them. Our study aimed to investigate the effects of exogenous RA on spermatogenetic cell types, assessing them histochemically, immunohistochemically, and ultrastructurally. Methods: We used three groups: a control group and experimental groups treated with 40 mg/kg RA and 80 mg/kg RA. Each group contained eight adult Sprague-Dawley rats. RA, dissolved in corn oil, was administered to the experimental groups via gavage for 3 weeks. After 3 weeks, testes from the sacrificed animals were evaluated using light and electron microscopy. The sections were stained histochemically with hematoxylin-eosin (H&E) and periodic acid Schiff (PAS). Seminiferous tubules in the rats were staged using PAS staining. The cellular localization of the RARα receptor in the seminiferous tubules was identified after immunohistochemical analysis. Results: Immunoreactivity was qualitatively observed and graded from no staining to strong. In the immunohistochemical analysis, the experimental groups, particularly in stages VI, VII, VIII, and XIV, showed a significant difference in immunoreactivity compared to the control group. This difference was particularly evident in stage VI spermatogonia – the stage at which the first meiosis begins. A morphologically observed reduction in the seminiferous tubules, likely due to the loss of germ cells, was statistically significant in terms of the average diameter of the seminiferous tubules in the 80 mg/kg experimental group compared to both the control and the 40 mg/kg experimental group (P<0.001). Electron microscopic examination revealed an increase in intercellular distance, especially between basal compartment cells, in both experimental groups. Additionally, compared to the control group, both experimental groups showed an increase in the number of lipid-like granules on the membrane, particularly in the cytoplasm of spermatogonia and Sertoli cells. Conclusion: Based on our observations, this study suggests that exogenous RA can impact the overall histology of the testis. Moreover, it may play a significant role in the meiosis process by influencing the internal dynamics of spermatogenetic cell types.

Information Transmission via Molecular Communication in Astrobiological Environments.

The ubiquity of information transmission via molecular communication between cells is comprehensively documented on Earth; this phenomenon might even have played a vital role in the origin(s) and early evolution of life. Motivated by these considerations, a simple model for molecular communication entailing the diffusion of signaling molecules from transmitter to receiver is elucidated. The channel capacity C (maximal rate of information transmission) and an optimistic heuristic estimate of the actual information transmission rate are derived for this communication system; the two quantities, especially the latter, are demonstrated to be broadly consistent with laboratory experiments and more sophisticated theoretical models. The channel capacity exhibits a potentially weak dependence on environmental parameters, whereas the actual information transmission rate may scale with the intercellular distance d as ∝ d-4 and could vary substantially across settings. These two variables are roughly calculated for diverse astrobiological environments, ranging from Earth's upper oceans (C ∼ 3.1 × 103 bits/s; ∼ 4.7 × 10-2 bits/s) and deep sea hydrothermal vents (C ∼ 4.2 × 103 bits/s; ∼ 1.2 × 10-1 bits/s) to the hydrocarbon lakes and seas of Titan (C ∼ 3.8 × 103 bits/s; ∼ 2.6 × 10-1 bits/s).

Unique characteristics of the tumor immune microenvironment in young patients with metastatic colorectal cancer.

Metastatic colorectal cancer (mCRC) remains a common and highly morbid disease, with a recent increase in incidence in patients younger than 50 years. There is an acute need to better understand differences in tumor biology, molecular characteristics, and other age-related differences in the tumor microenvironment (TME). 111 patients undergoing curative-intent resection of colorectal liver metastases were stratified by age into those <50 years or >65 years old, and tumors were subjected to multiplex fluorescent immunohistochemistry (mfIHC) to characterize immune infiltration and cellular engagement. There was no difference in infiltration or proportion of immune cells based upon age, but the younger cohort had a higher proportion of programmed death-ligand 1 (PD-L1)+ expressing antigen presenting cells (APCs) and demonstrated decreased intercellular distance and increased cellular engagement between tumor cells (TCs) and cytotoxic T lymphocytes (CTLs), and between TCs and APCs. These trends were independent of microsatellite instability in tumors. Age-related differences in PD-L1 expression and cellular engagement in the tumor microenvironment of patients with mCRC, findings which were unrelated to microsatellite status, suggest a more active immune microenvironment in younger patients that may offer an opportunity for therapeutic intervention with immune based therapy.

Quantitative biochemical phenotypic heterogeneity of senescent macrophage at a single cell level by Synchrotron Radiation Fourier Transform Infrared Microspectroscopy

Macrophage senescence plays an important role in pathophysiological process of age-related diseases such as atherosclerosis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, and lung cancer. After macrophage senescence, the biochemical phenotypes related to biological functions showed great heterogeneity. However, the biochemical phenotype and phenotypic heterogeneity of senescent macrophage has not been fully understood. Exploring the phenotype of biochemical substances in senescent macrophage will be helpful for understanding the function of senescent macrophage and finding out the potential mechanism between immune macrophage senescence and age-related diseases. In this study, we employed SR-FTIR microspectroscopy to detect the biochemical phenotype and phenotypic heterogeneity of single macrophage. The whole infrared spectra of senescent macrophages shifted, indicating biochemical substance changes within senescent macrophages. PCA and intercellular Euclidean distance statistical analysis based on specific spectra regions revealed dynamic changes of lipids and proteins during macrophage senescence. This proved that SR-FTIR microspectroscopy is an effective tool to detect the single cell biochemical phenotype transformation and phenotypic heterogeneity during macrophage senescence. It is of great significance to provide an evaluation method or clue for the study of cellular functions related to intracellular biochemical substances.Graphical abstract

Intercellular mechanical signalling in a 3D nonlinear fibrous network model

The extracellular matrix (ECM) consists of a network of polymeric fibres in which cells are embedded and interact with each other. The nonlinear elasticity of the ECM aids in directing the transmission of cell-induced mechanical loads over long distances, thereby facilitating mechanical communication between cells that are not in direct contact. Previous computational models have established the role of compression-buckling of the ECM fibres in increasing the propagation range of mechanical loads and fibre remodelling, however, most of them are two-dimensional (2D). Here, we generate a more realistic, 3D finite-element simulation of two cells contracting within the ECM by means of two contractile spheres embedded within a network of beam elements. We explore the effects of the mechanical behaviour of the fibres (linear-elastic, compression-bucklable), network connectivity (3.7, 8, 12) and intercellular distance (1–5 cell radii) on the structural remodelling and mechanical loads occurring on the cell surface and within the intercellular region of the ECM. When cells were embedded in a matrix of bucklable rather than linear-elastic fibres, contraction-induced loads were more directed towards the neighbouring cell, and the fibre rearrangement in the intercellular ECM was more eminent up to an intercellular distance of ∼2.5–5 cell radii. Our 3D discrete model highlights the role of the ECM nonlinear elasticity in the efficient transfer of mechanical signals between cells, a mechanism governing numerous multicellular processes such as tissue morphogenesis, wound healing, angiogenesis and cancer metastasis. The findings reemphasise the importance of using nonlinear elastic gels in in vitro cellular models.

Study on the preservation effects of the amputated forelimb by machine perfusion at physiological temperature

PurposeIschemia and hypoxia are the main factors limiting limb replantation and transplantation. Static cold storage (SCS), a common preservation method for tissues and organs, can only prolong limb ischemia time to 4 – 6 h. The normothermic machine perfusion (NMP) is a promising method for the preservation of tissues and organs, which can extend the preservation time in vitro by providing continuous oxygen and nutrients. This study aimed to evaluate the difference in the efficacy of the 2 limb preservation methods. MethodsThe 6 forelimbs from beagle dogs were divided into 2 groups. In the SCS group (n = 3), the limbs were preserved in a sterile refrigerator at 4 °C for 24 h, and in the NMP group (n = 3), the perfusate prepared with autologous blood was used for the oxygenated machine perfusion at physiological temperature for 24 h, and the solution was changed every 6 h. The effects of limb storage were evaluated by weight gain, perfusate biochemical analysis, enzyme-linked immunosorbent assay, and histological analysis. All statistical analyses and graphs were performed using GraphPad Prism 9.0 one-way or two-way analysis of variance. The p value of less than 0.05 was considered to indicate statistical significance. ResultsIn the NMP group, the weight gained percentage was 11.72% ± 4.06%; the hypoxia-inducible factor-1α contents showed no significant changes; the shape of muscle fibers was normal; the gap between muscle fibers slightly increased, showing the intercellular distance of (30.19 ± 2.83) μm; and the vascular α-smooth muscle actin (α-SMA) contents were lower than those in the normal blood vessels. The creatine kinase level in the perfusate of the NMP group increased from the beginning of perfusion, decreased after each perfusate change, and remained stable at the end of perfusion showing a peak level of 4097.6 U/L. The lactate dehydrogenase level of the NMP group increased near the end of perfusion and reached the peak level of 374.4 U/L. In the SCS group, the percentage of weight gain was 0.18% ± 0.10%, and the contents of hypoxia-inducible factor-1α increased gradually and reached the maximum level of (164.85 ± 20.75) pg/mL at the end of the experiment. The muscle fibers lost their normal shape and the gap between muscle fibers increased, showing an intercellular distance of (41.66 ± 5.38) μm. The contents of vascular α-SMA were much lower in the SCS group as compared to normal blood vessels. ConclusionsNMP caused lesser muscle damage and contained more vascular α-SMA as compared to SCS. This study demonstrated that NMP of the amputated limb with perfusate solution based on autologous blood could maintain the physiological activities of the limb for at least 24 h.

Abstract 1787: Design of bispecific adapters for universal CAR T cell therapies of solid tumors

Abstract Recent success in the use of chimeric antigen receptor (CAR) T cell therapies to treat hematologic cancers has encouraged the exploration of CAR T cell technologies to treat solid tumors. For establishment of a productive immunologic synapse between the CAR T cell and cancer cell, multiple CARs on the T cell surface must engage multiple tumor antigens on the cancer cell surface. Whereas these binding interactions are mediated in classical CAR T cells by direct recognition of the tumor antigen by the scFv on the CAR, in the case of universal CAR T cells this recognition must be created by a bispecific adapter that can bridge between the tumor antigen and the CAR. While a diversity of bispecific adapters have been explored in the literature, the bispecific adapter used here is comprised of a low molecular weight ligand that binds avidly to the desired tumor antigen, the yellow dye fluorescein (FL) that binds tightly to an anti-fluorescein scFv on the CAR, and a polyethylene glycol (PEG) spacer that spans between the two. The length of this PEG spacer appears to be critical to ensure that 1) the bispecific adapter is long enough to mediate engagement of the universal CAR T cell with the cancer cell, and 2) the resulting intercellular distance is optimal for activating the necessary co-receptor interactions.In our study, we have utilized protein structure data and molecular modeling to estimate the optimal adapter spacer lengths for bridging between our CAR T cell and two important target cells, namely PSMA positive prostate cancer cells and fibroblast activation protein (FAP) expressing cancer associated fibroblasts. We then synthesized the desired bispecific adapters with a variety lengths of PEG spacer and evaluated their effects in mediating the cytotoxicity of our universal anti-FL CAR T cells both in vitro and in vivo. Our results reveal that the optimal spacer lengths in the bispecific adapters for targeting PSMA (DUPA-FL) and FAP (FAPL-FL) are different and depend on the locations of the adapter binding pockets on their respective protein targets, as predicted by molecular modeling. Specifically, a shorter PEG spacer is required for maximal cytoxicity when employing the PSMA-targeted adapter, whereas a much longer PEG spacer is needed to achieve maximal cell killing with the FAP-targeted adapter. In addition, spacer length can also be adjusted to avoid overactivation-induced T cell exhaustion. Taken together, our studies demonstrate that instead of optimizing the hinge length of a CAR for each antigen epitope, our bispecific adapter/universal CAR platform provides a highly flexible strategy for optimizing the intermembrane distance between the universal CAR T cell and cancer cell to achieve maximal target cell killing with minimal CAR T cell exhaustion. Citation Format: Suilan Zheng, Bo Huang, Yashapal Singh, Haiyan Chu, Suresh K. Bowroju, Md Sazzadul Bari, Madduri Srinivasarao, Sudarsan R. Kasireddy, Ramesh Mukkamala, Laurie Beitz, Byoung Ryu, Andrew M. Scharenberg, Michael Jensen, Philip S. Low. Design of bispecific adapters for universal CAR T cell therapies of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1787.

Combination Cancer Treatment: Using Engineered DNAzyme Molecular Machines for Dynamic Inter‐ and Intracellular Regulation

AbstractDespite the promise of combination cancer therapy, it remains challenging to develop targeted strategies that are nontoxic to normal cells. Here we report a combination therapeutic strategy based on engineered DNAzyme molecular machines that can promote cancer apoptosis via dynamic inter‐ and intracellular regulation. To achieve external regulation of T‐cell/cancer cell interactions, we designed a DNAzyme‐based molecular machine with an aptamer and an i‐motif, as the MUC‐1‐selective aptamer allows the specific recognition of cancer cells. The i‐motif is folded under the tumor acidic microenvironment, shortening the intercellular distance. As a result, T‐cells are released by metal ion activated DNAzyme cleavage. To achieve internal regulation of mitochondria, we delivered another DNAzyme‐based molecular machine with mitochondria‐targeted peptides into cancer cells to induce mitochondria aggregation. Our strategy achieved an enhanced killing effect in zinc deficient cancer cells.

Combination Cancer Treatment: Using Engineered DNAzyme Molecular Machines for Dynamic Inter- and Intracellular Regulation.

Despite the promise of combination cancer therapy, it remains challenging to develop targeted strategies that are nontoxic to normal cells. Here we report a combination therapeutic strategy based on engineered DNAzyme molecular machines that can promote cancer apoptosis via dynamic inter- and intracellular regulation. To achieve external regulation of T-cell/cancer cell interactions, we designed a DNAzyme-based molecular machine with an aptamer and an i-motif, as the MUC-1-selective aptamer allows the specific recognition of cancer cells. The i-motif is folded under the tumor acidic microenvironment, shortening the intercellular distance. As a result, T-cells are released by metal ion activated DNAzyme cleavage. To achieve internal regulation of mitochondria, we delivered another DNAzyme-based molecular machine with mitochondria-targeted peptides into cancer cells to induce mitochondria aggregation. Our strategy achieved an enhanced killing effect in zinc deficient cancer cells.

Expansion of fibroblast cell sheets using a modified MEEK micrografting technique for wound healing applications

Cell sheet engineering, a scaffold-free approach to fabricate functional tissue constructs from several cell monolayers, has shown promise in tissue regeneration and wound healing. Unfortunately, these cell sheets are often too small to provide sufficient wound area coverage. In this study, we describe a process to enlarge cell sheets using MEEK micrografting, a technique extensively used to expand skin autografts for large burn treatments. Human dermal fibroblast cell sheets were placed on MEEK’s prefolded gauze without any use of adhesive, cut along the premarked lines and stretched out at various expansion ratios (1:3, 1:6 and 1:9), resulting in regular distribution of many square islands of fibroblasts at a much larger surface area. The cellular processes essential for wound healing, including reattachment, proliferation, and migration, of the fibroblasts on expanded MEEK gauze were superior to those on nylon dressing which served as a control. The optimal expansion ratio with the highest migration rate was 1:6, possibly due to the activation of chemical signals caused by mechanical stretching and an effective intercellular communication distance. Therefore, the combination of cell sheet engineering with the MEEK micrografting technique could provide high quality cells with a large coverage area, which would be particularly beneficial in wound care applications.

Adaptive tracking algorithm for trajectory analysis of cells and layer-by-layer assessment of motility dynamics

Tracking biological objects such as cells or subcellular components imaged with time-lapse microscopy enables us to understand the molecular principles about the dynamics of cell behaviors. However, automatic object detection, segmentation and extracting trajectories remain as a rate-limiting step due to intrinsic challenges of video processing. This paper presents an adaptive tracking algorithm (Adtari) that automatically finds the optimum search radius and cell linkages to determine trajectories in consecutive frames. A critical assumption in most tracking studies is that displacement remains unchanged throughout the movie and cells in a few frames are usually analyzed to determine its magnitude. Tracking errors and inaccurate association of cells may occur if the user does not correctly evaluate the value or prior knowledge is not present on cell movement. The key novelty of our method is that minimum intercellular distance and maximum displacement of cells between frames are dynamically computed and used to determine the threshold distance. Since the space between cells is highly variable in a given frame, our software recursively alters the magnitude to determine all plausible matches in the trajectory analysis. Our method therefore eliminates a major preprocessing step where a constant distance was used to determine the neighbor cells in tracking methods. Cells having multiple overlaps and splitting events were further evaluated by using the shape attributes including perimeter, area, ellipticity and distance. The features were applied to determine the closest matches by minimizing the difference in their magnitudes. Finally, reporting section of our software were used to generate instant maps by overlaying cell features and trajectories. Adtari was validated by using videos with variable signal-to-noise, contrast ratio and cell density. We compared the adaptive tracking with constant distance and other methods to evaluate performance and its efficiency. Our algorithm yields reduced mismatch ratio, increased ratio of whole cell track, higher frame tracking efficiency and allows layer-by-layer assessment of motility to characterize single-cells. Adaptive tracking provides a reliable, accurate, time efficient and user-friendly open source software that is well suited for analysis of 2D fluorescence microscopy video datasets.

Thermal optimization of intercellular distance in lithium-ion batteries and numerical analysis of the original honeycomb metal integrated battery pack

In Lithium-ion batteries, it is necessary to keep the system temperature within an acceptable range to extend the working life. A thermal management system is also required to maintain temperature uniformity. In this study, first, the steady and transient thermal behavior of 18650 Lithium-ion batteries was investigated, and the results were validated. After this, a module design consisting of 14 battery cells was made and the distance intercellular in this module was optimized. A thermal analysis study of the battery pack was carried out for the most suitable layout. In the study, the differences among modules with cell connection distances of 20, 25, 30, 35 and 40 mm were compared in terms of heat transfer and mounting. To calculate heat transfer data, a Computational Fluid Dynamics based program, FLOEFD was used. According to the results obtained from the analyses, it was determined that the distances between cell centers should be 20 mm, or 25 mm in terms of heat transfer and mounting. Additionally, the design of a metal insert with a honeycomb shape inside the plastic enclosure of the module containing 14 battery cells was examined. It was found that the designed metal insert package had a significant effect on lowering the temperature of the batteries. While the open module maximum temperature was 41.89 °C, the module maximum temperature in the package dropped to 37.39 °C. In addition, effective results were obtained for equal aging. The cell temperature differences were found to be 0.24 °C, 0.17 °C, and 0.59 °C for Aluminum, Copper and Steel, respectively in metal integrated battery pack analyzes.

RBIO-06. MECHANISM OF ACTION AND ASSOCIATED EFFECTS OF TUMOR TREATING FIELDS (TTFIELDS) ON LIVING CELLS USING SIMULATIONS

Abstract Following FDA approval, TTFields treatment has become a commonly used modality for treating patients with Glioblastoma (GBM) and Mesothelioma. From the early 2000’s, extensive research has been performed in in-vitro systems for studying the effects of TTFields on living cells. These studies have shown that multiple cellular functions are affected by TTFields. However, the physical mechanism by which the fields exert effects on cells are not well understood. We propose an analytical model for predicting the geometric and electrical parameters enabling amplification of the electric field in the living cells. This amplification favors the emergence of local heating, dielectrophoretic (DEP) force, or electrostatic pressure at TTFields frequencies. This model is supported by simulations of cells in different configurations. Computational studies were performed with Comsol Multiphysics software. Cell models were constituted of cytoplasm, membrane and extracellular matrix. A field of 1V/cm was generated at different frequencies between 10kHz and 1GHz. Maximal field amplification of X20 of the applied field (@200 kHz) was observed in a model of confluent cells with 5nm intercellular distance. Such field amplification could create electrostatic pressure on the membrane potentially leading to its deformation and to stress on the cytoskeleton. Analytical calculations show the field gradient could result in DEP forces of ~10pN on the membrane. Such force could potentially disrupt the membrane or junctions. Results show that a 10nm pore in membrane would lead to a 450 times amplification in the pore’s vicinity, potentially resulting in forces of between 0.1pN and 100pN on intracellular structures. Those forces are sufficient for disrupting microtubules. Specific Absorption Rates of up to 106 W/kg were observed in the vicinity of the pore, suggesting that strong thermal effects may also explain the effect of TTFields on cells. Our generic analytical model predicts the conditions for field amplification at TTFields frequencies.

Duodenal Mucosal Barrier in Functional Dyspepsia

In addition to gastric sensorimotor dysfunctions, functional dyspepsia (FD) is also variably associated with duodenal micro-inflammation and epithelial barrier dysfunction, the pathogenesis and clinical significance of which are unknown. Our hypothesis was that miRNAs and/or inflammation degrade epithelial barrier proteins, resulting in increased duodenal mucosal permeability in FD. We compared the duodenal mucosal gene expression and miRNAs, invivo permeability (lactulose-mannitol excretion between 0 and 60 and 60 and 120 minutes after saccharide ingestion), exvivo assessments (transmucosal resistance, fluorescein isothiocyanate [FITC]-dextran flux, and basal ion transport), and duodenal histology (light and electron microscopy) in 40 patients with FD and 24 controls. Compared with controls, the mRNA expression of several barrier proteins (zonula occludens-1, occludin, claudin-12, and E-cadherin) was modestly reduced (ie, a fold change of 0.8-0.85) in FD with increased expression of several miRNAs (eg, miR-142-3p and miR-144-3-p), which suppress these genes. The urinary lactulose excretion and the lactulose:mannitol ratio between 60 and 120 minutes were greater in FD than in controls (P < .05). The FITC-dextran flux, which reflects paracellular permeability, was inversely correlated (r= -0.32, P= .03) with transmucosal resistance and directly correlated (r= 0.4, P= .02) with lactulose:mannitol ratio. Other parameters (mucosal eosinophils, intraepithelial lymphocytes, and mast cells, transmucosal resistance, FITC-dextran flux, average intercellular distance, and proportion of dilated junctions) were not significantly different between groups. In FD, there is a modest reduction in the expression of several duodenal epithelial barrier proteins, which may be secondary to up-regulation of regulatory miRNAs, and increased small intestinal permeability measured invivo.

Assessing Different Temporal Scales of Calcium Dynamics in Networks of Beta Cell Populations.

Beta cells within the pancreatic islets of Langerhans respond to stimulation with coherent oscillations of membrane potential and intracellular calcium concentration that presumably drive the pulsatile exocytosis of insulin. Their rhythmic activity is multimodal, resulting from networked feedback interactions of various oscillatory subsystems, such as the glycolytic, mitochondrial, and electrical/calcium components. How these oscillatory modules interact and affect the collective cellular activity, which is a prerequisite for proper hormone release, is incompletely understood. In the present work, we combined advanced confocal Ca2+ imaging in fresh mouse pancreas tissue slices with time series analysis and network science approaches to unveil the glucose-dependent characteristics of different oscillatory components on both the intra- and inter-cellular level. Our results reveal an interrelationship between the metabolically driven low-frequency component and the electrically driven high-frequency component, with the latter exhibiting the highest bursting rates around the peaks of the slow component and the lowest around the nadirs. Moreover, the activity, as well as the average synchronicity of the fast component, considerably increased with increasing stimulatory glucose concentration, whereas the stimulation level did not affect any of these parameters in the slow component domain. Remarkably, in both dynamical components, the average correlation decreased similarly with intercellular distance, which implies that intercellular communication affects the synchronicity of both types of oscillations. To explore the intra-islet synchronization patterns in more detail, we constructed functional connectivity maps. The subsequent comparison of network characteristics of different oscillatory components showed more locally clustered and segregated networks of fast oscillatory activity, while the slow oscillations were more global, resulting in several long-range connections and a more cohesive structure. Besides the structural differences, we found a relatively weak relationship between the fast and slow network layer, which suggests that different synchronization mechanisms shape the collective cellular activity in islets, a finding which has to be kept in mind in future studies employing different oscillations for constructing networks.

Experience in Manufacturing and Mastering Roll Forming Mills for the Production of Profiled Flooring

The purpose of the study is to develop and master the designs of rolling mills for the production of profile flooring. In recent years, the volume of the profile flooring production has been increasing. This leads to an increase in the number and expansion of types of structures of rolling mills. An urgent problem in the production of rolling mills is not only ensuring the high quality of the products, but also choosing the optimal design of the rolling mill from the point of view of reliability, rigidity and manufacturability. The designs of rolling mills are considered: with stands without a frame (housingless stands); with frames representing steel plates, with openings for the installation of bearing supports of working rolls; with frames made of stand; with free-standing frames for each cage made of steel plates. Also, different types of roller drive (using gears and a chain drive), adjustment methods are considered. The advantages and disadvantages of each design are presented. Based on the experience of implementing (mastering) mill designs in relation to the height of the profile of the resulting sheet, it can be noted that for corrugated profiles with a corrugation height of no more than 16 mm, a rolling mill with an intercellular distance of 200...300 mm and a bed representing steel plates, with openings for installing bearing supports of working rolls; for profiles with a corrugation height of more than 16 mm, rolling mills with an intercellular distance of more than 350 mm and beds of stands with guides are optimal. It is rational to drive only the lower rolls in order to reduce the cost of the drive and improve the speed mode. In mills with a bed representing steel plates, with openings and an intercellular distance of up to 300 mm, it is advisable to use a drive of working shafts using gears. And as for rolling mills with an intercellular distance of more than 350 mm and in which the frames are made of stands with guides, it is desirable to equip them with a chain drive of rotation of the working rolls.

Regulation of Chondrocyte Differentiation by Changing Intercellular Distances Using Type II Collagen Microfibers.

Osteoarthritis is a common degenerative disease that mainly occurs in older age groups, and the search for an effective cure remains a major global challenge. The technology of constructing 3D in vitro cartilage tissue with zonal differentiated structures for use as alternative implants for treating osteoarthritis has attracted researchers' attention. For this challenge, it is important for understanding the relationship between chondrocyte differentiation and the amount of extracellular matrix by modulating intercellular distance. This study investigates the interplay between chondrocyte differentiation and intercellular distance. Type II collagen microfibers (CMF II) were used as a distance regulator by varying their amounts. The results indicated that the secretion of cartilage-specific glycosaminoglycan after 2 weeks of differentiation from the chondrogenic cells, ATDC5, was decreased with an increased intercellular distance. Also, the shortest intercellular distance, being ATDC5 cells without CMF II, presented an upregulated gene expression profile of cartilage markers. The groups with CMF II-mediated intracellular distances, however, did not show the upregulation. The elastic modulus of the 3D samples increased depending on the amount of CMF II, relating to the differentiation preventing property of the CMF II. These findings suggest the promising potential of this approach for the modulation of chondrocyte differentiation.