Integrating single-nucleus RNA sequencing and spatial transcriptomics to elucidate a specialized subpopulation of astrocytes, microglia and vascular cells in brains of mouse model of lipopolysaccharide-induced sepsis-associated encephalopathy.

BACKGROUND: Understanding the mechanism behind sepsis-associated encephalopathy (SAE) remains a formidable task. This study endeavors to shed light on the complex cellular and molecular alterations that occur in the brains of a mouse model with SAE, ultimately unraveling the underlying mechanisms of this condition. METHODS: We established a murine model using intraperitoneal injection of lipopolysaccharide (LPS) in wild type and Anxa1-/- mice and collected brain tissues for analysis at 0-hour, 12-hour, 24-hour, and 72-hour post-injection. Utilizing advanced techniques such as single-nucleus RNA sequencing (snRNA-seq) and Stereo-seq, we conducted a comprehensive characterization of the cellular responses and molecular patterns within the brain. RESULTS: Our study uncovered notable temporal differences in the response to LPS challenge between Anxa1-/- (annexin A1 knockout) and wild type mice, specifically at the 12-hour and 24-hour time points following injection. We observed a significant increase in the proportion of Astro-2 and Micro-2 cells in these mice. These cells exhibited a colocalization pattern with the vascular subtype Vas-1, forming a distinct region known as V1A2M2, where Astro-2 and Micro-2 cells surrounded Vas-1. Moreover, through further analysis, we discovered significant upregulation of ligands and receptors such as Timp1-Cd63, Timp1-Itgb1, Timp1-Lrp1, as well as Ccl2-Ackr1 and Cxcl2-Ackr1 within this region. In addition, we observed a notable increase in the expression of Cd14-Itgb1, Cd14-Tlr2, and Cd14-C3ar1 in regions enriched with Micro-2 cells. Additionally, Cxcl10-Sdc4 showed broad upregulation in brain regions containing both Micro-2 and Astro-2 cells. Notably, upon LPS challenge, there was an observed increase in Anxa1 expression in the mouse brain. Furthermore, our study revealed a noteworthy increase in mortality rates following Anxa1 knockdown. However, we did not observe substantial differences in the types, numbers, or distribution of other brain cells between Anxa1-/- and wildtype mice over time. Nevertheless, when comparing the 24-hour post LPS injection time point, we observed a significant decrease in the proportion and distribution of Micro-2 and Astro-2 cells in the vicinity of blood vessels in Anxa1-/- mice. Additionally, we noted reduced expression levels of several ligand-receptor pairs including Cd14-Tlr2, Cd14-C3ar1, Cd14-Itgb1, Cxcl10-Sdc4, Ccl2-Ackr1, and Cxcl2-Ackr1. CONCLUSIONS: By combining snRNA-seq and Stereo-seq techniques, our study successfully identified a distinctive cellular colocalization, referred to as a special pathological niche, comprising Astro-2, Micro-2, and Vas-1 cells. Furthermore, we observed an upregulation of ligand-receptor pairs within this niche. These findings suggest a potential association between this cellular arrangement and the underlying mechanisms contributing to SAE or the increased mortality observed in Anxa1 knockdown mice.

Changes in the tissue elements of the gastric mucosa interacting with different strains of Helicobacter pylori, taking into consideration the patient's genotype.

The present study aimed to investigate the peculiarities of adaptation of tissue elements of the gastric mucosa during interaction with Helicobacter pylori, as determined by genetic characteristics of the bacterium and the host. Venous blood and biopsy samples of the mucosa of the antrum and body of the stomach from young patients (18 to 25 years old) were examined. The condition of the gastric mucosa was assessed using stained histological preparations. Venous blood was collected from the patients to ascertain the polymorphisms of the IL-lß and IL-IRN genes. The most pronounced changes were observed in the parameters of reparative regeneration of epithelial differentiation during colonization of the gastric mucosa by H. pylori strains carrying the CagA(+) and BabA2(+) genes. These included an increase in proliferation and apoptosis rates and alterations in epithelial differentiation markers characterized by elevated production of Shh and MUC5AC, as well as a reduction in the production of the protective mucin MUC6 by isthmus gland cells. The presence of the vacAs1 and vacAs2 genes of H. pylori results in a high level of apoptosis in epithelial cells without accelerating proliferation. It was found that after eradication, patients with preserved cellular infiltrates in their gastric mucosa plates were carriers of mainly the IL-1ß*T/IL-1RN*2R haplotypes after 12 months.

Morphology of human sinoatrial node and its surrounding right atrial muscle in the global obesity pandemic-does fat matter?

Introduction: The sinus node (SN) is the main pacemaker site of the heart, located in the upper right atrium at the junction of the superior vena cava and right atrium. The precise morphology of the SN in the human heart remains relatively unclear especially the SN microscopical anatomy in the hearts of aged and obese individuals. In this study, the histology of the SN with surrounding right atrial (RA) muscle was analyzed from young non-obese, aged non-obese, aged obese and young obese individuals. The impacts of aging and obesity on fibrosis, apoptosis and cellular hypertrophy were investigated in the SN and RA. Moreover, the impact of obesity on P wave morphology in ECG was also analyzed to determine the speed and conduction of the impulse generated by the SN. Methods: Human SN/RA specimens were dissected from 23 post-mortem hearts (preserved in 4% formaldehyde solution), under Polish local ethical rules. The SN/RA tissue blocks were embedded in paraffin and histologically stained with Masson's Trichrome. High and low-magnification images were taken, and analysis was done for appropriate statistical tests on Prism (GraphPad, USA). 12-lead ECGs from 14 patients under Polish local ethical rules were obtained. The P wave morphologies from lead II, lead III and lead aVF were analyzed. Results: Compared to the surrounding RA, the SN in all four groups has significantly more connective tissue (P ≤ 0.05) (young non-obese individuals, aged non-obese individuals, aged obese individuals and young obese individuals) and significantly smaller nodal cells (P ≤ 0.05) (young non-obese individuals, aged non-obese individuals, aged obese individuals, young obese individuals). In aging, overall, there was a significant increase in fibrosis, apoptosis, and cellular hypertrophy in the SN (P ≤ 0.05) and RA (P ≤ 0.05). Obesity did not further exacerbate fibrosis but caused a further increase in cellular hypertrophy (SN P ≤ 0.05, RA P ≤ 0.05), especially in young obese individuals. However, there was more infiltrating fat within the SN and RA bundles in obesity. Compared to the young non-obese individuals, the young obese individuals showed decreased P wave amplitude and P wave slope in aVF lead. Discussion: Aging and obesity are two risk factors for extensive fibrosis and cellular hypertrophy in SN and RA. Obesity exacerbates the morphological alterations, especially hypertrophy of nodal and atrial myocytes. These morphological alterations might lead to functional alterations and eventually cause cardiovascular diseases, such as SN dysfunction, atrial fibrillation, bradycardia, and heart failure.

Porcine lungs perfused with three different flows using the 8-h open-atrium cellular ex vivo lung perfusion technique.

The number of lung transplantations is limited due to the shortage of donor lungs fulfilling the standard criteria. The ex vivo lung perfusion (EVLP) technique provides the ability of re-evaluating and potentially improving and treating marginal donor lungs. Accordingly, the technique has emerged as an essential tool to increase the much-needed donor lung pool. One of the major EVLP protocols, the Lund protocol, characterized by high pulmonary artery flow (100% of cardiac output [CO]), an open atrium, and a cellular perfusate, has demonstrated encouraging short-EVLP duration results. However, the potential of the longer EVLP duration of the protocol is yet to be investigated, a duration which is considered necessary to rescue more marginal donor lungs in future. This study aimed to achieve stable 8-h EVLP using an open-atrium cellular model with three different pulmonary artery flows in addition to determining the most optimal flow in terms of best lung performance, including lung electrolytes and least lung edema formation, perfusate and tissue inflammation, and histopathological changes, using the porcine model. EVLP was performed using a flow of either 40% (n = 6), 80% (n = 6), or 100% (n = 6) of CO. No flow rate demonstrated stable 8-h EVLP. Stable 2-h EVLP was observed in all three groups. Insignificant deterioration was observed in dynamic compliance, peak airway pressure, and oxygenation between the groups. Pulmonary vascular resistance increased significantly in the 40% group (p < .05). Electrolytes demonstrated an insignificant worsening trend with longer EVLP. Interleukin-8 (IL-8) in perfusate and tissue, wet-to-dry weight ratio, and histopathological changes after EVLP were insignificantly time dependent between the groups. This study demonstrated that stable 8-h EVLP was not feasible in an open-atrium cellular model regardless of the flow of 40%, 80%, or 100% of CO. No flow was superior in terms of lung performance, lung electrolytes changes, least lung edema formation, minimal IL-8 expression in perfusate and tissue, and histopathological changes.

Metabolic Responses to Redox Stress in Vascular Cells.

Significance: Redox stress underlies numerous vascular disease mechanisms. Metabolic adaptability is essential for vascular cells to preserve energy and redox homeostasis. Recent Advances: Single-cell technologies and multiomic studies demonstrate significant metabolic heterogeneity among vascular cells in health and disease. Increasing evidence shows that reductive or oxidative stress can induce metabolic reprogramming of vascular cells. A recent example is intracellular L-2-hydroxyglutarate accumulation in response to hypoxic reductive stress, which attenuates the glucose flux through glycolysis and mitochondrial respiration in pulmonary vascular cells and provides protection against further reductive stress. Critical Issues: Regulation of cellular redox homeostasis is highly compartmentalized and complex. Vascular cells rely on multiple metabolic pathways, but the precise connectivity among these pathways and their regulatory mechanisms is only partially defined. There is also a critical need to understand better the cross-regulatory mechanisms between the redox system and metabolic pathways as perturbations in either systems or their cross talk can be detrimental. Future Directions: Future studies are needed to define further how multiple metabolic pathways are wired in vascular cells individually and as a network of closely intertwined processes given that a perturbation in one metabolic compartment often affects others. There also needs to be a comprehensive understanding of how different types of redox perturbations are sensed by and regulate different cellular metabolic pathways with specific attention to subcellular compartmentalization. Lastly, integration of dynamic changes occurring in multiple metabolic pathways and their cross talk with the redox system is an important goal in this multiomics era.

Enhancing neovascularization post-myocardial infarction through injectable hydrogel functionalized with endothelial-derived EVs.

Over the past three decades, cell therapy development has fallen short of expectations, with many cellular sources demonstrating a "Janus effect" and raising safety concerns. Extracellular vesicles (EVs), supported by advanced technologies, present a promising avenue in regenerative medicine, offering benefits such as immune tolerance and avoidance of negative aspects associated with cell transplants. Our previous research showcased enhanced and organized subcutaneous vascularization using three-dimensional bioprinted patches containing HUVEC-derived EVs in immunodeficient animal models. In this context, stress conditions on the cells of origin further boosted the EVs' neoangiogenic potential. Since neovascularization is the first regenerative target requiring restoration, the present study aims to complement our previous work by employing an injectable gelatin methacrylate (GelMA) hydrogel functionalized with HUVEC-derived EVs in a pathological condition of acute myocardial infarction. This bioactive hydrogel resulted in reduced fibrosis, improved contractility, and promoted angiogenesis, showing promise in countering tissue deterioration and addressing vascular deficits. Moreover, the molecular characterization of EVs through miRNome and proteomic analyses further supports their potential as bio-additives for hydrogel functionalization. This cell-free approach mitigates immune rejection and oncogenic risks, offering innovative therapeutic advantages.

Nanoparticle-plant interactions: Physico-chemical characteristics, application strategies, and transmission electron microscopy-based ultrastructural insights, with a focus on stereological research.

Ensuring global food security is pressing among challenges like population growth, climate change, soil degradation, and diminishing resources. Meeting the rising food demand while reducing agriculture's environmental impact requires innovative solutions. Nanotechnology, with its potential to revolutionize agriculture, offers novel approaches to these challenges. However, potential risks and regulatory aspects of nanoparticle (NP) utilization in agriculture must be considered to maximize their benefits for human health and the environment. Understanding NP-plant cell interactions is crucial for assessing risks of NP exposure and developing strategies to control NP uptake by treated plants. Insights into NP uptake mechanisms, distribution patterns, subcellular accumulation, and induced alterations in cellular architecture can be effectively drawn using transmission electron microscopy (TEM). TEM allows direct visualization of NPs within plant tissues/cells and their influence on organelles and subcellular structures at high resolution. Moreover, integrating TEM with stereological principles, which has not been previously utilized in NP-plant cell interaction assessments, provides a novel and quantitative framework to assess these interactions. Design-based stereology enhances TEM capability by enabling precise and unbiased quantification of three-dimensional structures from two-dimensional images. This combined approach offers comprehensive data on NP distribution, accumulation, and effects on cellular morphology, providing deeper insights into NP impact on plant physiology and health. This report highlights the efficient use of TEM, enhanced by stereology, in investigating diverse NP-plant tissue/cell interactions. This methodology facilitates detailed visualization of NPs and offers robust quantitative analysis, advancing our understanding of NP behavior in plant systems and their potential implications for agricultural sustainability.

Atypical cellular neurothekeoma of the oral mucosa: A rare case report and literature review.

Cellular neurothekeoma (CN) is a benign dermal neoplasm that most often affects the head and neck region and rarely occurs in the oral mucosa. We report a rare case of CN with atypical features on the floor of the mouth and summarize the reported cases of oral CN in English-language literature. A 62-year-old woman presented with a 6-month history of a painless mass on the floor of the mouth. Histopathological analysis of the excised specimen revealed a proliferation of neoplastic cells with oval to spindle morphology arranged in a vaguely nested and multinodular architecture separated by scarce hyaline collagen within a predominantly myxoid-rich stroma. The tumor cells were positive for NSE, and CD63 (NKI/C3), and negative for S100 protein, CD34, and SMA. Thus, the final diagnosis was CN. In addition, we summarized all clinicopathological data on oral CNs reported in the English-language literature. Nineteen cases were reviewed. Among them, only one case affected the floor of the mouth of a young girl, in contrast to the present case that occurred in an elderly woman. CN is particularly rare in this location and may be a diagnostic challenge for oral pathologists due to its rarity and morphological similarity with other lesions.

In situ visualization of the cellular uptake and sub-cellular distribution of mussel oligosaccharides.

Unlike chemosynthetic drugs designed for specific molecular and disease targets, active small-molecule natural products typically have a wide range of bioactivities and multiple targets, necessitating extensive screening and development. To address this issue, we propose a strategy for the direct in situ microdynamic examination of potential drug candidates to rapidly identify their effects and mechanisms of action. As a proof-of-concept, we investigated the behavior of mussel oligosaccharide (MOS-1) by tracking the subcellular dynamics of fluorescently labeled MOS-1 in cultured cells. We recorded the entire dynamic process of the localization of fluorescein isothiocyanate (FITC)-MOS-1 to the lysosomes and visualized the distribution of the drug within the cell. Remarkably, lysosomes containing FITC-MOS-1 actively recruited lipid droplets, leading to fusion events and increased cellular lipid consumption. These drug behaviors confirmed MOS-1 is a candidate for the treatment of lipid-related diseases. Furthermore, in a high-fat HepG2 cell model and in high-fat diet-fed apolipoprotein E (ApoE) -/- mice, MOS-1 significantly promoted triglyceride degradation, reduced lipid droplet accumulation, lowered serum triglyceride levels, and mitigated liver damage and steatosis. Overall, our work supports the prioritization of in situ visual monitoring of drug location and distribution in subcellular compartments during the drug development phase, as this methodology contributes to the rapid identification of drug indications. Collectively, this methodology is significant for the screening and development of selective small-molecule drugs, and is expected to expedite the identification of candidate molecules with medicinal effects.

Projection neurons from medial entorhinal cortex to basolateral amygdala are critical for the retrieval of morphine withdrawal memory.

The medial entorhinal cortex (MEC) is crucial for contextual memory, yet its role in context-induced retrieval of morphine withdrawal memory remains unclear. This study investigated the role of the MEC and its projection neurons from MEC layer 5 to the basolateral amygdala (BLA) (MEC-BLA neurons) in context-induced retrieval of morphine withdrawal memory. Results show that context activates the MEC in morphine withdrawal mice, and the inactivation of the MEC inhibits context-induced retrieval of morphine withdrawal memory. At neural circuits, context activates MEC-BLA neurons in morphine withdrawal mice, and the inactivation of MEC-BLA neurons inhibits context-induced retrieval of morphine withdrawal memory. But MEC-BLA neurons are not activated by conditioning of context and morphine withdrawal, and the inhibition of MEC-BLA neurons do not influence the coupling of context and morphine withdrawal memory. These results suggest that MEC-BLA neurons are critical for the retrieval, but not for the formation, of morphine withdrawal memory.

Bone marrow-derived myeloid cells transiently colonize the brain during postnatal development and interact with glutamatergic synapses.

Although the roles of embryonic yolk sac-derived, resident microglia in neurodevelopment were extensively studied, the possible involvement of bone marrow-derived cells remains elusive. In this work, we used a fate-mapping strategy to selectively label bone marrow-derived cells and their progeny in the brain (FLT3+IBA1+). FLT3+IBA1+ cells were confirmed to be transiently present in the healthy brain during early postnatal development. FLT3+IBA1+ cells have a distinct morphology index at postnatal day(P)0, P7, and P14 compared with neighboring microglia. FLT3+IBA1+ cells also express the microglial markers P2RY12 and TMEM119 and interact with VGLUT1 synapses at P14. Scanning electron microscopy indeed showed that FLT3+ cells contact and engulf pre-synaptic elements. Our findings suggest FLT3+IBA1+ cells might assist microglia in their physiological functions in the developing brain including synaptic pruning which is performed using their purinergic sensors. Our findings stimulate further investigation on the involvement of peripheral macrophages during homeostatic and pathological development.

Integrating serum pharmacochemistry and network pharmacology to explore potential compounds and mechanisms of Alpiniae oxyphyllae fructus in the treatment of cellular senescence in diabetic kidney disease.

Background: Diabetic kidney disease (DKD), one of the microvascular complications in patients with diabetes mellitus, is a common cause of end-stage renal disease. Cellular senescence is believed to be an essential participant in the pathogenesis of DKD. Although there is evidence that Alpiniae oxyphyllae fructus (AOF) can ameliorate DKD progression and organismal senescence, its ability to ameliorate renal cellular senescence in DKD as well as active components and molecular mechanisms remain to be explored. Purpose: This study aimed to investigate the role of AOF in the treatment of cellular senescence in DKD and to explore its active components and potential molecular mechanisms. Methods: The pharmacological efficacy of AOF in ameliorating cellular senescence in DKD was assessed by establishing DKD mouse models and HK-2 cells under high glucose stress. UHPLC-QTOF-MS was used to screen the active compounds in AOF, which were used in conjunction with network pharmacology to predict the molecular mechanism of AOF in the treatment of cellular senescence in DKD. Results: In vivo experiments showed that AOF reduced GLU, mAlb, Scr, BUN, MDA, SOD levels, and ameliorated renal pathological damage and renal cell senescence in DKD mice. In vitro experiments showed that AOF-containing serum improved the decline in HK-2 cell viability and alleviated cellular senescence under high glucose intervention. The results of the UHPLC-QTOF-MS screened 26 active compounds of AOF. The network pharmacological analyses revealed that Cubebin, 2',6'-dihydroxy-4'-methoxydihydrochalcone, Chalcone base + 3O,1Prenyl, Batatasin IV, and Lucidenolactone were the five core compounds and TP53, SRC, STAT3, PIK3CA, and AKT1 are the five core targets of AOF in the treatment of DKD. Molecular docking simulation results showed that the five core compounds had good binding ability to the five core targets. Western blot validated the network pharmacological prediction results and showed that AOF and AOF-containing serum down-regulate the expression of TP53, and phosphorylation of SRC, STAT3, PIK3CA, and AKT. Conclusion: Our study shows that AOF may delay the development of cellular senescence in DKD by down-regulating the levels of TP53, and phosphorylation of SRC, STAT3, PIK3CA, and AKT.

Shufeng Jiedu capsule alleviates influenza A (H1N1) virus induced acute lung injury by regulating the lung inflammatory microenvironment.

Background: Death caused by respiratory tract infection is one of the leading causes of death in the world today. Shufeng Jiedu Capsule (SFJDC) is a traditional Chinese medicine that has been widely used clinically for coronavirus disease 2019 (COVID-19), H1N1 influenza virus pneumonia and other diseases. Its pharmacological effect is to inhibit inflammation and improve the body's ability to clear viruses. However, the mechanism of SFJDC in the treatment of viral pneumonia, especially its effect on the inflammatory-immune microenvironment of lung tissue remains unclear. Methods: Mice with H1N1 influenza virus pneumonia were used as a model to verify the efficacy of SFJDC through death protection, lung index, viral load, and HE staining of lung tissue. The levels of inflammatory cytokines and chemokines in lung tissue were investigated by multi-analyte immunoassay. The number and proportion of cells in peripheral blood were detected by blood routine. The percentage of infiltrating immune cells in lung tissue was detected by flow cytometry and immunofluorescence. Results: SFJDC (2.2 g/kg·d-1 and 1.1 g/kg·d-1) increased survival rate (P＜0.01, P＜0.05), prolonged the survival period of mice, and alleviated the histopathological damage in lung (P＜0.01). SFJDC (2.2 g/kg·d-1, 1.1 g/kg·d-1 and 0.055 g/kg·d-1) increased body weight(P＜0.01, P＜0.05), improved activity status, reduced the lung index (P＜0.01, P＜0.05) and viral load (P＜0.01). SFJDC (2.2 g/kg·d-1 and 1.1 g/kg·d-1) reduced interleukin-1ß (IL-1ß), interleukin-18(IL-18), tumour necrosis factor α (TNF-α), monocyte chemoattractant protein (MCP), chemokine (C-X-C motif) ligand 1 (CXCL1) (P＜0.01, P＜0.05), and SFJDC (2.2 g/kg·d-1) increased IL-10 levels (P＜0.05) to regulate inflammation. SFJDC (2.2 g/kg·d-1) increased the percentages of CD4+ T cells (P＜0.01), CD8+ T cells (P＜0.05), and B cells(P＜0.05), and decreased F4/80+ macrophages (P＜0.05). Conclusion: Our findings indicated that SFJDC could inhibit inflammation and lung injury while maintaining the function of the adaptive immune response mediated by T and B cells, and promote the clearance of the virus, thereby treating influenza A (H1N1) virus-induced pneumonia.

Enzymatic control of intermolecular interactions for generating synthetic nanoarchitectures in cellular environment.

Nanoarchitectonics, as a technology to arrange nano-sized structural units such as molecules in a desired configuration, requires nano-organization, which usually relies on intermolecular interactions. This review briefly introduces the development of using enzymatic reactions to control intermolecular interactions for generating artificial nanoarchitectures in a cellular environment. We begin the discussion with the early examples and uniqueness of enzymatically controlled self-assembly. Then, we describe examples of generating intracellular nanostructures and their relevant applications. Subsequently, we discuss cases of forming nanostructures on the cell surface via enzymatic reactions. Following that, we highlight the use of enzymatic reactions for creating intercellular nanostructures. Finally, we provide a summary and outlook on the promises and future direction of this strategy. Our aim is to give an updated introduction to the use of enzymatic reaction in regulating intermolecular interactions, a phenomenon ubiquitous in biology but relatively less explored by chemists and materials scientists. Our goal is to stimulate new developments in this simple and versatile approach for addressing societal needs.

Enzymatic reactions in cells create precise nanoarchitectures, offering insights into cell biology through controllable nanoarchitectonics, as shown by numerous examples in this review.

Immune interactions and regulation with CD39+ extracellular vesicles from platelet concentrates.

Introduction: CD39 plays an important role in the immunoregulation and inhibition of effector cells. It is expressed on immune cells, including Tregs, and on extracellular vesicles (EVs) budding from the plasma membrane. Platelet transfusion may induce alloimmunization against HLA-I antigens, leading to refractoriness to platelet transfusion with severe consequences for patients. Tregs may play a key role in determining whether alloimmunization occurs in patients with hematologic disorders. We hypothesized that CD39+ EVs might play an immunoregulatory role, particularly in the context of platelet transfusions in patients with hematologic disorders. Such alloimmunization leads to the production of alloantibodies and is sensitive to the regulatory action of CD39. Methods: We characterized CD39+ EVs in platelet concentrates by flow cytometry. The absolute numbers and cellular origins of CD39+ EVs were evaluated. We also performed functional tests to evaluate interactions with immune cells and their functions. Results: We found that CD39+ EVs from platelet concentrates had an inhibitory phenotype that could be transferred to the immune cells with which they interacted: CD4+ and CD8+ T lymphocytes (TLs), dendritic cells, monocytes, and B lymphocytes (BLs). Moreover, the concentration of CD39+ EVs in platelet concentrates varied and was very high in 10% of concentrates. The number of these EVs present was determinant for EV-cell interactions. Finally, functional interactions were observed with BLs, CD4+ TLs and CD39+ EVs for immunoglobulin production and lymphoproliferation, with potential implications for the immunological management of patients.

Gap junctions fine-tune ganglion cell signals to equalize response kinetics within a given electrically coupled array.

Retinal ganglion cells (RGCs) summate inputs and forward a spike train code to the brain in the form of either maintained spiking (sustained) or a quickly decaying brief spike burst (transient). We report diverse response transience values across the RGC population and, contrary to the conventional transient/sustained scheme, responses with intermediary characteristics are the most abundant. Pharmacological tests showed that besides GABAergic inhibition, gap junction (GJ)-mediated excitation also plays a pivotal role in shaping response transience and thus visual coding. More precisely GJs connecting RGCs to nearby amacrine and RGCs play a defining role in the process. These GJs equalize kinetic features, including the response transience of transient OFF alpha (tOFFα) RGCs across a coupled array. We propose that GJs in other coupled neuron ensembles in the brain are also critical in the harmonization of response kinetics to enhance the population code and suit a corresponding task.

A Flow Cytometry-Based Method for Assessing CAR Cell Binding Kinetics Using Stable CAR Jurkat Cells.

Chimeric antigen receptors (CARs) are synthetic fusion proteins that can reprogram immune cells to target specific antigens. CAR-expressing T cells have emerged as an effective treatment method for hematological cancers; despite this success, the mechanisms and structural properties that govern CAR responses are not fully understood. Here, we provide a simple assay to assess cellular avidity using a standard flow cytometer. This assay measures the interaction kinetics of CAR-expressing T cells and targets antigen-expressing target cells. By co-culturing stably transfected CAR Jurkat cells with target positive and negative cells for short periods of time in a varying effector-target gradient, we were able to observe the formation of CAR-target cell doublets, providing a readout of actively bound cells. When using the optimized protocol reported here, we observed unique cellular binding curves that varied between CAR constructs with differing antigen binding domains. The cellular binding kinetics of unique CARs remained consistent, were dependent on specific target antigen expression, and required active biological signaling. While existing literature is not clear at this time whether higher or lower CAR cell binding is beneficial to CAR therapeutic activity, the application of this simplified protocol for assessing CAR binding could lead to a better understanding of the proximal signaling events that regulate CAR functionality. Key features • Determines CAR receptor cellular interaction kinetics using a Jurkat cell model. • Can be used for a wide variety of CAR target antigens, including both hematological and solid tumor targets. • Experiments can be performed in under two hours with no staining using a standard flow cytometer. • Requires stable CAR Jurkat cells and target cells with stable fluorescent marker expression for optimal results.

Trypanosoma cruzi Vps34 colocalizes with Beclin1 and plays a role in parasite invasion of the host cell by modulating the expression of a sub-group of trans-sialidases.

Trypanosoma cruzi, the etiological agent of Chagas' disease, can infect both phagocytic and non-phagocytic cells. T. cruzi gp82 and gp90 are cell surface proteins belonging to Group II trans-sialidases known to be involved in host cell binding and invasion. Phosphatidylinositol kinases (PIK) are lipid kinases that phosphorylate phospholipids in their substrates or in themselves, regulating important cellular functions such as metabolism, cell cycle and survival. Vps34, a class III PIK, regulates autophagy, trimeric G-protein signaling, and the mTOR (mammalian Target of Rapamycin) nutrient-sensing pathway. The mammalian autophagy gene Beclin1 interacts to Vps34 forming Beclin 1-Vps34 complexes involved in autophagy and protein sorting. In T. cruzi epimastigotes, (a non-infective replicative form), TcVps34 has been related to morphological and functional changes associated to vesicular trafficking, osmoregulation and receptor-mediated endocytosis. We aimed to characterize the role of TcVps34 during invasion of HeLa cells by metacyclic (MT) forms. MTs overexpressing TcVps34 showed lower invasion rates compared to controls, whilst exhibiting a significant decrease in gp82 expression in the parasite surface. In addition, we showed that T. cruzi Beclin (TcBeclin1) colocalizes with TcVps34 in epimastigotes, thus suggesting the formation of complexes that may play conserved cellular roles already described for other eukaryotes.

The Role of Dislocation Type in the Thermal Stability of Cellular Structures in Additively Manufactured Austenitic Stainless Steel.

The ultrafine cellular structure promotes the extraordinary mechanical performance of metals manufactured by laser powder-bed-fusion (L-PBF). An in-depth understanding of the mechanisms governing the thermal stability of such structures is crucial for designing reliable L-PBF components for high-temperature applications. Here, characterizations and 3D discrete dislocation dynamics simulations are performed to comprehensively understand the evolution of cellular structures in 316L stainless steel during annealing. The dominance of screw-type dislocation dipoles in the dislocation cells is reported. However, the majority of dislocations in sub-grain boundaries (SGBs) are geometrically necessary dislocations (GNDs) with varying types. The disparity in dislocation types can be attributed to the variation in local stacking fault energy (SFE) arising from chemical heterogeneity. The presence of screw-type dislocations facilitates the unpinning of dislocations from dislocation cells/SGBs, resulting in a high dislocation mobility. In contrast, the migration of SGBs with dominating edge-type GNDs requires collaborative motion of dislocations, leading to a sluggish migration rate and an enhanced thermal stability. This work emphasizes the significant role of dislocation type in the thermal stability of cellular structures. Furthermore, it sheds light on how to locally tune dislocation structures with desired dislocation types by adjusting local chemistry-dependent SFE and heat treatment.