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In binocular animals that exhibit stereoscopic visual responses, the axons of retinal ganglion cells (RGCs) connect to brain areas bilaterally by forming a commissure called the optic chiasm (OC). Ventral anterior homeobox 1 (Vax1) contributes to the formation of the OC, acting endogenously in optic pathway cells and exogenously in growing RGC axons. Here, we generated Vax1AA/AA mice expressing the Vax1AA mutant, which is incapable of intercellular transfer. We found that RGC axons cannot take up Vax1AA protein from the Vax1AA/AA mouse optic stalk (OS) and grow slowly to arrive at the hypothalamus at a late stage. The RGC axons of Vax1AA/AA mice connect exclusively to ipsilateral brain areas after failing to access the midline, resulting in reduced visual acuity and abnormal oculomotor responses. Overall, our study provides physiological evidence for the necessity of intercellular transfer of Vax1 and the importance of the bilateral RGC axon projection in proper visuomotor responses.

The spatiotemporal organization of intracellular features is fundamental to understanding the intercommunication of neurons in the brain. Neuronal communication is regulated by receptor activation, which is influenced by signaling messenger availability and internal calcium (Ca2+) concentrations. An excess or deficiency of cytoplasmic Ca2+ can lead to changes in secondary messenger diffusion, synaptic plasticity, gene expression, and even cause cell apoptosis. Existing models of intracellular calcium movement have used image-based recording methods to approximate a uniform distribution within the cytosolic space, but it is still unclear whether or how somatic features shape calcium mobility. A methodic analysis of the relationship between somatic arrangement and the Ca2+ signaling pathway does not currently exist. In this study, we used fine-scale electron tomogram reconstructions of cerebellar Purkinje neurons to create a precise model of the interior of the cell. We established structural motifs of endoplasmic reticulum (ER) and noticed a heterogeneous arrangement of organelles within the soma. In addition, we used fluorescent immunohistochemistry to analyze localization patterns of signaling proteins and observed how two receptors that mediate Ca2+ efflux from internal ER stores: inositol 1,4,5-trisphosphate receptor 1 (IP3R1) and ryanodine receptor 1 (RyR1) have distinct angular and radial domains. Overall, we found that ER serves as a diffusion barrier and can cause nonuniform diffusion of Ca2+. This thesis demonstrates how the localization organelles and calcium modulating proteins significantly contribute to intracellular communication and provides an architectural reference for emerging research on somatic neuronal signaling.

Calcineurin (CaN) is a unique calcium (Ca2+) and calmodulin (CaM)-dependent serine/threonine phosphatase that becomes activated in the presence of increased intracellular Ca2+ level. CaN then functions to dephosphorylate target substrates including various transcription factors, receptors, and channels. Once activated, the CaN signaling pathway participates in the development of multiple organs as well as the onset and progression of various diseases via regulation of different cellular processes. Here, we review current literature regarding the structural and functional properties of CaN, highlighting its crucial role in the development and pathogenesis of immune system disorders, neurodegenerative diseases, kidney disease, cardiomyopathy and cancer.

Abstract: Skeletal muscles are considered the largest reservoirs of the protein pool in the body and are critical for the maintenances of body homeostasis. Skeletal muscle atrophy is supported by various physiopathological conditions that lead to loss of muscle mass and contractile capacity of the skeletal muscle. Lysosomal mediated autophagy and ubiquitin-proteasomal system (UPS) concede the major intracellular systems of muscle protein degradation that result in the loss of mass and strength. Both systems recognize ubiquitination as a signal of degradation through different mechanisms, a sign of dynamic interplay between systems. Hence, growing shreds of evidence suggest the interdependency of autophagy and UPS in the progression of skeletal muscle atrophy under various pathological conditions. Therefore, understanding the molecular dynamics and associated factors responsible for their interdependency is necessary for the new therapeutic insights to counteract muscle loss. Based on current literature, the present review summarizes the factors that interplay between autophagy and UPS in favor of enhanced proteolysis of skeletal muscle and how they affect the anabolic signaling pathways under various conditions of skeletal muscle atrophy.

The cytosolic antibody receptor and RING E3 ligase TRIM21 targets intracellular, antibody-coated immune complexes for degradation and activates the immune system. Here we review how TRIM21 degrades diverse targets and how this activity can be exploited in molecular biology and for the development of new therapeutics. In addition, we compare what is known about TRIM21's mechanism to other TRIM proteins and RING E3 ligases.

Antibodies mediate the majority of their effects in the extracellular domain, or in intracellular compartments isolated from the cytosol. Under a growing list of circumstances, however, antibodies are found to gain access to the cytoplasm. Cytosolic immune complexes are bound by the atypical antibody receptor TRIM21, which mediates the rapid degradation of the immune complexes at the proteasome. These discoveries have informed the development of TRIM-Away, a technique to selectively deplete proteins using delivery of antibodies into cells. A range of related approaches that elicit selective protein degradation using intracellular constructs linking antibody fragments to degradative effector functions have also been developed. These methods hold promise for inducing the degradation of proteins as both research tools and as a novel therapeutic approach. Protein aggregates are a pathophysiological feature of neurodegenerative diseases and are considered to have a causal role in pathology. Immunotherapy is emerging as a promising route towards their selective targeting, and a role of antibodies in the cytosol has been demonstrated in cell-based assays. This review will explore the mechanisms by which therapeutic antibodies engage and eliminate intracellularly aggregated proteins. We will discuss how future developments in intracellular antibody technology may enhance the therapeutic potential of such antibody-derived therapies.

When the brain is in a pathological state, the content of lipid droplets (LDs), the lipid storage organelles, is increased, particularly in glial cells, but rarely in neurons. The biology and mechanisms leading to LD accumulation in astrocytes, glial cells with key homeostatic functions, are poorly understood. We imaged fluorescently labeled LDs by microscopy in isolated and brain tissue rat astrocytes and in glia‐like cells in Drosophila brain to determine the (sub)cellular localization, mobility, and content of LDs under various stress conditions characteristic for brain pathologies. LDs exhibited confined mobility proximal to mitochondria and endoplasmic reticulum that was attenuated by metabolic stress and by increased intracellular Ca2+, likely to enhance the LD–organelle interaction imaged by electron microscopy. When de novo biogenesis of LDs was attenuated by inhibition of DGAT1 and DGAT2 enzymes, the astrocyte cell number was reduced by ~40%, suggesting that in astrocytes LD turnover is important for cell survival and/or proliferative cycle. Exposure to noradrenaline, a brain stress response system neuromodulator, and metabolic and hypoxic stress strongly facilitated LD accumulation in astrocytes. The observed response of stressed astrocytes may be viewed as a support for energy provision, but also to be neuroprotective against the stress‐induced lipotoxicity., Main points Astroglial lipid droplets are dynamic organelles with confined mobility.Lipid droplet turnover is important for astrocyte survival/proliferation.Stressed astrocytes accumulate lipid droplets likely to provide energy and reduce lipotoxicity.

Failure of embryo implantation has been introduced as an important limiting parameter in early assisted reproduction and pregnancy. The embryo-maternal interactions, endometrial receptivity, and detections of implantation consist of the embryo viability. For regulating the implantation, multiple molecules may be consistent; however, their specific regulatory mechanisms still stand unclear. MicroRNAs (miRNAs) have attracted a lot of attention due to their important effect on human embryo implantation. MicroRNA (miRNA), which acts as the transcriptional regulator of gene expression, is consisted of embryo implantation. Recent studies indicated that miRNAs not only act inside the cells but also can be secreted by cells into the extracellular environment via multiple packaging forms, facilitating intercellular communication and providing indicative information related to various conditions. The detection of extracellular miRNAs provided new information in cases of implantation studies. For embryo-maternal communication, MiRNAs offered novel approaches. In addition, in assisted reproduction, for embryo choice and prediction of endometrial receptivity, they can act as non-invasive biomarkers and can enhance the accuracy in the process of reducing the mechanical damage for the tissue.

Macrophages (MQ) are major constituents of chronically inflamed periapical tissues in apical periodontitis. This study aimed to investigate the immunomodulatory effect of engineered bioactive chitosan-based nanoparticles (CSnp) antibiofilm medication on MQ cocultured with periodontal ligament fibroblasts (PdLF). Cells viability, spreading, PdLF migration, and intracellular CSnp uptake were characterized. Tandem Mass Tag-based proteomics was applied to analyze MQ global protein expression profiles after interaction with Enterococcus faecalis biofilm, CSnp-treated biofilm, and CSnp. Secreted inflammatory mediators were analyzed. Following bioinformatics analyses, candidate proteins were validated via targeted proteomics. CSnp maintained cells viability, increased MQ spreading, and PdLF migration (p 1.5-folds, p 1.5-folds, p, Graphical abstract This study elucidated the immunomodulatory effect of engineered bioactive chitosan-based nanoparticles (CSnp) as antibiofilm medication on macrophage (MQ) co-cultured with periodontal ligament fibroblasts (PdLF) via proteomic and cytokine analyses. CSnp were internalized via different endocytic pathways. Cells interaction with CSnp resulted in increased MQ spreading and PdLF migration. MQ inflammatory response to CSnp-treated biofilm was compared to untreated biofilm. Quantitative proteomics of MQ was done using tandem mass tag (TMT)-mass spectrometry (MS). Bioinformatics analysis of global proteomic data was followed by validation of candidate proteins via parallel reaction monitoring (PRM)-targeted MS. CSnp upregulated proteins having immunoregulatory and antioxidant activities. MQ stimulated with CSnp-treated biofilm produced higher anti-inflammatory but lower pro-inflammatory mediators. Engineered CSnp has the potential to regulate biofilm-mediated inflammation to promote periapical healing in the treatment of apical periodontitis (Schematic created with BioRender.com).Image 1., Highlights • CSnp internalized via macropinocytosis, clathrin-mediated endocytosis, and phagocytosis. • Enterococcus faecalis biofilm altered macrophage proteomic profile. • Macrophage proteome upon CSnp-treated biofilm interaction was distinct from biofilm. • CSnp upregulated proteins with immunoregulatory and antioxidant activities. • CSnp reduced proinflammatory but increased anti-inflammatory mediators.

Background Lidocaine is a local anesthetic that wildly used in surgical treatment and postoperative medical care for lung cancers. We hypothesized that lidocaine at clinical plasma concentration can inhibit CXCL12/CXCR4 axis-regulated cytoskeletal remodeling thereby reduce the migration of Non-small-cell lung cancers (NSCLC) cells. Methods We determined the effect of lidocaine at clinical plasma concentration on CXCL12-induced cell viability, apoptosis, cell death, monolayer cell wound healing rate, individual cell migration indicators, expression of CXCR4, CD44, and ICAM-1, intracellular Ca2+ level, and filamentous actin level alteration of NSCLC cells A549 and CXCR4-knocked down A549 cells using CCK-8, Bcl-2 ELISA, Cell death ELISA, wound healing assay, chemotaxis assay, western blotting, QPCR, Fura-2-based intracellular Ca2+ assay, and Fluorescein Phalloidin staining respectively. Results Lidocaine did not affect cell viability, apoptosis, and cell death but inhibited CXCL12-induced migration, intracellular Ca2+ releasing, and filamentous actin increase. Lidocaine decreased expression of CXCR4, increased CD44, but had no effect on ICAM-1. CXCL12 induced the increase of CD44 and ICAM-1 but did not affect CD44 in the presence of lidocaine. The knockdown of CXCR4 eliminated all the effects of lidocaine. The overexpression of CXCR4 promoted migration but the migration was inhibited by lidocaine. Conclusion Lidocaine at clinical plasma concentrations inhibited CXCL12-induced CXCR4 activation, thereby reduced the intracellular Ca2+-dependent cytoskeleton remodeling, resulting in slower migration of A549 cells.

Commensal intestinal protozoa, unlike their pathogenic relatives, are neglected members of the mammalian microbiome. These microbes have a significant impact on the host’s intestinal immune homeostasis, typically by elevating anti-microbial host defense. Tritrichomonas musculis (T. mu), a protozoan gut commensal, strengthens the intestinal host defense against enteric Salmonella infections through Asc- and Il1r1-dependent Th1 and Th17 cell activation. However, the underlying inflammasomes mediating this effect remain unknown. Here, we report that colonization with T. mu results in an increase in luminal extracellular ATP, elevated levels of IL-1β, and increased numbers of IL-18 receptor-expressing Th1 and Th17 cells in the colon. Mice deficient in either Nlrp1b or Nlrp3 failed to display these protozoan-driven immune changes and lost resistance to enteric Salmonella infections even in the presence of T. mu. These findings demonstrate that T. mu-mediated host protection requires sensors of extra and intracellular ATP to confer resistance to enteric Salmonella infections.KEY POINTSIntestinal colonization with the commensal Tritrichomonas musculis elevates luminal ATP levelsNLRP1B and NLRP3 activation is required for Tritrichomonas musculis-driven Th cell response.

The study aimed to determine the sterilization effect of a combination of high-pressure thermal sterilization (HPTS) and e-polylysine (PL) on Bacillus subtilis spores. The spores were treated with HPTS (550 MPa at 25°C, 65°C and 75°C) combining with e-PL at 0.1% and 0.3%. The results showed that HPTS and e-PL synergistically inactivated the spores. The increased temperature and e-PL concentration decreased the number of surviving spores, with the maximal inactivation of the spores in the treatment of 550 MPa, 75°C combining with 0.3% e-PL. The increases in the temperature and e-PL concentration significantly increased the release of the intracellular components in the spore suspension, with the maximal value for the spores treated with 550 MPa, 75°C and 0.3% e-PL. The maximal fluidity and permeability of the cell inner membrane were observed in the treatment of 550 MPa, 75°C combining with 0.3% e-PL. Changes in membrane lipids were detected from 3000 to 2800 cm -1 by Fourier transform infrared spectroscopy. The results may provide new insights into the mechanism by which HPTS and e-PL synergistically sterilize Bacillus subtilis spores.

Major histocompatibility complex class I (MHC-I), a key element of the acquired immune system, plays essential roles in activating CD8+ T cells by recognizing intracellular antigens derived from pa...

Molecular targeted therapy has become an emerging promising strategy in cancer treatment, and screening the agents targeting at cancer cell specific targets is very desirable for cancer treatment. Our previous study firstly found that a secretory peroxidase of class III derived from foxtail millet bran (FMBP) exhibited excellent targeting anti-colorectal cancer (CRC) activity in vivo and in vitro, whereas its underlying target remains unclear. The highlight of present study focuses on the finding that cell surface glucose-regulated protein 78 (csGRP78) abnormally located on CRC is positively correlated with the anti-CRC effects of FMBP, indicating it serves as a potential target of FMBP against CRC. Further, we demonstrated that the combination of FMBP with the nucleotide binding domain (NBD) of csGRP78 interfered with the downstream activation of signal transducer and activator of transcription 3 (STAT3) in CRC cells, thus promoting the intracellular accumulation of reactive oxygen species (ROS) and cell grown inhibition. These phenomena were further confirmed in nude mice tumor model. Collectively, our study highlights csGRP78 acts as an underlying target of FMBP against CRC, uncovering the clinical potential of FMBP as a targeted agent for CRC in the future.

In recent years, the strategy of inhibiting the interactions of p53 with murine double minute 2 (MDM2) and murine double minute X (MDMX) has been proved to be a promising approach for tumor therapy. However, the poor proteolytical stability and low intracellular delivery efficiency of peptide inhibitors limit their clinical application. Here, we designed and synthesized the bicyclic stapled peptides based on p53 by combining all-hydrocarbon stapling and lactam stapling strategies. We demonstrated that bicyclic stapled peptide p53-16 significantly improved α-helicity and proteolytic stability. Especially, p53-16 showed nanomolar binding affinity for MDM2 and MDMX. In addition, p53-16 could penetrate the cell membrane, and selectively inhibited the activity of tumor cells via activating p53 pathway in vitro. Our data suggest that p53-16 is a potential dual inhibitor of MDM2 and MDMX interactions. The bicyclic stapling strategy is a promising drug design strategy for protein–protein interactions inhibitors.

Ciprofloxacin (CIP) is an antibiotic used to treat infections caused by bacteria. In this experiment, key enzymes and intracellular metabolites of Lactobacillus plantarum DNZ-4 was researched under CIP stress. The results showed that the activities of hexokinase, pyruvate kinase, β-galactosidase and Na+, K+-ATPase after 1/2 minimum bacteriostatic concentration (MIC) CIP treatment were significantly decreased (P

Fusarium moniliforme (F. moniliforme) and its secondary metabolite fumonisin pose a severe threat to food safety, and searching for effective antimicrobial agents is a focus of current research. In this study, the secondary structure of Sub3 was analyzed by circular dichroism, meanwhile, the inhibition rate of Sub3 against F. moniliforme infection of maize was studied. To explore the possible inhibition mechanisms, morphological and structural changes of spores treated with Sub3 at 0, 1/2MIC and MIC were observed by scanning electron microscopy and transmission electron microscopy; the cell wall integrity, membrane integrity, reactive oxygen species, mitochondrial membrane potential, ATP synthase activity, redox reactions, and the nuclear damage of F. moniliforme were also investigated. The results showed that Sub3 was mostly in the state of random in deionized water, while mainly showed the β-sheet structure in the hydrophobic environment of 50% TFE solution, indicating that Sub3 might generate partial structure deformation when acting on the cell membrane; and its minimum inhibitory concentration (MIC) on F. moniliforme spores was 0.2 g/L. Under the 1/2MIC and MIC, the inhibition rates of Sub3 against F. moniliforme infected maize were 34.3% and 75.6%, respectively. The results of inhibition mechanisms revealed that the defective pathogenicity of F. moniliforme caused by Sub3 was attributed to damages on both the cell wall and the cell membrane, which might upset balance of intracellular redox system and mitochondrial energy metabolism and trigger nucleus damage, ultimately leading to cell death. The results provided direct evidence for inhibition of F. moniliforme infection of maize by Sub3, and useful knowledge applicable for food preservation.

Large amounts of ATP are produced in mitochondria especially in the brain and heart, where energy consumption is high compared with other organs. Thus, a decrease in ATP production in such organs could be a cause of many diseases such as neurodegenerative diseases and heart disease. Based on thus assumption, increasing intracellular ATP production in such organs could be a therapeutic strategy. In this study, we report on the delivery of vitamin B1, a coenzyme that activates the tricarboxylic acid (TCA) cycle, to the inside of mitochondria. Since the TCA cycle is responsible for ATP production, we hypothesized delivering vitamin B1 to mitochondria would enhance ATP production. To accomplish this, we used a mitochondrial targeted liposome a “MITO-Porter” as the carrier. Using SH-SY5Y cells, a model neuroblast, cellular uptake and intracellular localization were analyzed using flow cytometry and confocal laser scanning microscopy. The optimized MITO-Porter containing encapsulated vitamin B1 (MITO-Porter (VB1)) was efficiently accumulated in mitochondria of SH-SY5Y cells. Further studies confirmed that the level of ATP production after the MITO-Porter (VB1) treatment was significantly increased as compared to a control group that was treated with naked vitamin B1. This study provides the potential for an innovative therapeutic strategy in which the TCA cycle is activated, thus enhancing ATP production. Relative ATP ratio (%) = IS/IU × 100, where IS and IU represent the intracellular ATP amounts for the treated and untreated cells with samples, respectively.

Capsid allosteric modulators (CAMs) inhibit the encapsidation of hepatitis B virus (HBV) pregenomic RNA (pgRNA), which contains a pathogen-associated molecular pattern motif. However, the effect of CAMs on the innate immune response of HBV-infected hepatocytes remains unclear, and we examined this effect in this study. Administration of a CAM compound, BAY41-4109 (BAY41), to HBV-infected primary human hepatocytes (PHHs) did not change the total cytoplasmic pgRNA levels but significantly reduced intracapsid pgRNA levels, suggesting that BAY41 increased extracapsid pgRNA levels in the cytoplasm. BAY41 alone did not change the intracellular interferon (IFN)-stimulated gene (ISG) expression levels. However, BAY41 enhanced antiviral ISG induction by IFN-α in HBV-infected PHHs but did not change ISG induction by IFN-α in uninfected PHHs. Compared with BAY41 or IFN-α alone, coadministration of BAY41 and IFN-α significantly suppressed extracellular HBV-DNA levels. HBV-infected human liver-chimeric mice were treated with vehicle, BAY41, pegylated IFN-α (pegIFN-α), or BAY41 and pegIFN-α together. Compared with the vehicle control, pegIFN-α highly up-regulated intrahepatic ISG expression levels, but BAY41 alone did not change these levels. The combination of BAY41 and pegIFN-α further enhanced intrahepatic antiviral ISG expression, which was up-regulated by pegIFNα. The serum HBV-DNA levels in mice treated with the combination of BAY41 and pegIFN-α were the lowest observed in all the groups. Conclusion: CAMs enhance the host IFN response when combined with exogenous IFN-α, likely due to increased cytoplasmic extracapsid pgRNA.

Nanoparticles (NPs) that can activate macrophages infected with the tuberculosis causative pathogen Mycobacterium tuberculosis, could be an effective host directed therapy for the disease. In this study, curdlan was conjugated to poly(lactic-co-glycolic acid) (PLGA) to produce immunotherapeutic NPs. Various physicochemical characterizations were used to evaluate the curdlan-PLGA copolymer and the NPs. Molecular dynamics and simulation studies were used to characterize the interaction between curdlan, on the polymer and on NPs, with the Dectin-1 macrophage receptor. NPs with varying curdlan densities were evaluated for their effects on the production of pro- and anti-inflammatory cytokines in M. tuberculosis infected RAW264.7 macrophages. The killing efficacy of the NPs against intracellular M. tuberculosis was assessed. Physicochemical characterization of the curdlan-PLGA copolymer and NPs indicated successful formation of curdlan-PLGA copolymer and NPs of varying curdlan density (0 - 8% w/w) had sizes between 330 – 453 nm. Modelling studies showed curdlan to have a strong affinity for Dectin-1. Cytotoxicity assays showed the NPs to be non-toxic over 72 h. The proinflammatory cytokine TNF-α was found to be significantly upregulated by the NPs. The NPs reduced intracellular M. tuberculosis burden over 72 h. These NPs are a promising host directed therapy for intracellular eradication of M. tuberculosis.

The fungal cell wall and membrane are the principal targets of antifungals. Herein, we report that myricetin exerts antifungal activity against Candida albicans by damaging the cell wall integrity and notably enhancing the membrane permeability. In the presence of sorbitol, an osmotic protectant, the minimum inhibitory concentration (MIC) of myricetin against C. albicans increased from 20 to 40 and 80 ?g/ml in 24 and 72 h, respectively, demonstrating that myricetin disturbs the cell wall integrity of C. albicans. Fluorescence microscopic images showed the presence of propidium iodide-stained C. albicans cells, indicating the myricetin-induced initial damage of the cell membrane. The effects of myricetin on the membrane permeability of C. albicans cells were assessed using crystal violet-uptake and intracellular material-leakage assays. The percentage uptakes of crystal violet for myricetin-treated C. albicans cells at 1×, 2×, and 4× the MIC of myricetin were 36.5, 60.6, and 79.4%, respectively, while those for DMSO-treated C. albicans cells were 28.2, 28.9, and 29.7%, respectively. Additionally, myricetin-treated C. albicans cells showed notable DNA and protein leakage, compared with the DMSO-treated controls. Furthermore, treatment of C. albicans cells with 1× the MIC of myricetin showed a 17.2 and 28.0% reduction in the binding of the lipophilic probes diphenylhexatriene and Nile red, respectively, indicating that myricetin alters the lipid components or order in the C. albicans cell membrane, leading to increased membrane permeability. Therefore, these data will provide insights into the pharmacological worth of myricetin as a prospective antifungal for treating C. albicans infections.

Background: Amyloid-β (Aβ), which derives from the amyloid-β protein precursor (AβPP), forms plaques and serves as a fluid biomarker in Alzheimer’s disease (AD). How Aβ forms from AβPP is known, but questions relating to AβPP and Aβ biology remain unanswered. AD patients show mitochondrial dysfunction, and an Aβ/AβPP mitochondria relationship exists. Objective: We considered how mitochondrial biology may impact AβPP and Aβ biology. Methods: SH-SY5Y cells were transfected with AβPP constructs. After treatment with FCCP (uncoupler), Oligomycin (ATP synthase inhibitor), or starvation Aβ levels were measured. β-secretase (BACE1) expression was measured. Mitochondrial localized full-length AβPP was also measured. All parameters listed were measured in ρ0 cells on an SH-SY5Y background. iPSC derived neurons were also used to verify key results. Results: We showed that mitochondrial depolarization routes AβPP to, while hyperpolarization routes AβPP away from, the organelle. Mitochondrial AβPP and cell Aβ secretion inversely correlate, as cells with more mitochondrial AβPP secrete less Aβ, and cells with less mitochondrial AβPP secrete more Aβ. An inverse relationship between secreted/extracellular Aβ and intracellular Aβ was observed. Conclusion: Our findings indicate mitochondrial function alters AβPP localization and suggest enhanced mitochondrial activity promotes Aβ secretion while depressed mitochondrial activity minimizes Aβ secretion. Our data complement other studies that indicate a mitochondrial, AβPP, and Aβ nexus, and could help explain why cerebrospinal fluid Aβ is lower in those with AD. Our data further suggest Aβ secretion could serve as a biomarker of cell or tissue mitochondrial function.

Epigenetic modifications, such as histone or DNA modifications are key regulators of gene transcription and changes are often associated with maladaptive processes underlying cardiovascular disease. Epigenetic regulators therefore likely play a crucial role in cardiomyocyte homeostasis and facilitate the cellular adaption to various internal and external stimuli, responding to different intercellular and extracellular cues. Class IIa histone deacetylases are a class of epigenetic regulators that possess a myriad of post-transcriptional modification sites that modulate their activity in response to oxidative stress, altered catecholamine signalling or changes in the cellular metabolism. This review summaries the known reversible, post-translational modifications (PTMs) of class IIa histone deacetylases (HDACs) that ultimately drive transcriptional changes in homeostasis and disease. We also highlight the idea of a crosstalk of various PTMs on class IIa HDACs potentially leading to compensatory or synergistic effects on the class IIa HDAC-regulated cell behavior.

Despite recent progress recognizing the importance of aryl hydrocarbon receptor (Ahr)-dependent signaling in suppressing colon tumorigenesis, its role in regulating colonic crypt homeostasis remains unclear. To assess the effects of Ahr on intestinal epithelial cell heterogeneity and functional phenotypes, we utilized single-cell transcriptomics and advanced analytic strategies to generate a high-quality atlas for colonic intestinal crypts from wild-type and intestinal-specific Ahr knockout mice. Here we observed the promotive effects of Ahr deletion on Foxm1-regulated genes in crypt-associated canonical epithelial cell types and subtypes of goblet cells and deep crypt–secretory cells. We also show that intestinal Ahr deletion elevated single-cell entropy (a measure of differentiation potency or cell stemness) and RNA velocity length (a measure of the rate of cell differentiation) in noncycling and cycling Lgr5+ stem cells. In general, intercellular signaling cross-talk via soluble and membrane-bound factors was perturbed in Ahr-null colonocytes. Taken together, our single-cell RNA sequencing analyses provide new evidence of the molecular function of Ahr in modulating putative stem cell driver genes, cell potency lineage decisions, and cell–cell communication in vivo. Prevention Relevance: Our mouse single-cell RNA sequencing analyses provide new evidence of the molecular function of Ahr in modulating colonic stemness and cell–cell communication in vivo. From a cancer prevention perspective, Ahr should be considered a therapeutic target to recalibrate remodeling of the intestinal stem cell niche.

Autophagy is a highly conserved process of cellular self-digestion that involves the formation of autophagosomes for the delivery of intracellular components and dysfunctional organelles to lysosomes. This process is induced by different signals including starvation, mitochondrial dysfunction, and DNA damage. The molecular link between autophagy and DNA damage is not well understood yet. Importantly, tumor cells utilize the mechanism of autophagy to cope with genotoxic anti-cancer drug therapy. Another mechanism of drug resistance is provided to cancer cells via the execution of the EMT program. One of the critical transcription factors of EMT is Zeb1. Here we demonstrate that Zeb1 is involved in the regulation of autophagy in several breast cancer cell models. On the molecular level, Zeb1 likely facilitates autophagy through the regulation of autophagic genes, resulting in increased LC3-II levels, augmented staining with Lysotracker, and increased resistance to several genotoxic drugs. The attenuation of Zeb1 expression in TNBC cells led to the opposite effect. Consequently, we propose that Zeb1 augments the resistance of breast cancer cells to genotoxic drugs, at least partially, via autophagy. Collectively, we have uncovered a novel function of Zeb1 in the regulation of autophagy in breast cancer cells.

Cyclin M (CNNM) and its prokaryotic ortholog CorC belong to a family of proteins that function as Mg2+-extruding transporters by stimulating Na+/Mg2+ exchange, and thereby control intracellular Mg2+ levels. The Mg2+-extruding function of CNNM is inhibited by the direct binding of an oncogenic protein, phosphatase of regenerating liver (PRL), and this inhibition is responsible for the PRL-driven malignant progression of cancers. Studies with mouse strains deficient for the CNNM gene family revealed the importance of CNNM4 and CNNM2 in maintaining organismal Mg2+ homeostasis by participating in intestinal Mg2+ absorption and renal reabsorption, respectively. Moreover, CNNM proteins are involved in various diseases, and gene mutations in CNNM2 and CNNM4 cause dominant familial hypomagnesemia and Jalili syndrome, respectively. Genome wide association studies have also revealed the importance of CNNM2 in multiple major diseases, such as hypertension and schizophrenia. Collectively, the molecular and biological characterizations of CNNM/CorC show that they are an intriguing therapeutic target; the current status of drug development targeting these proteins is also discussed.

‘Candidatus Liberibacter asiaticus’ (CLas), the devastating pathogen related to Huanglongbing (HLB), is a phloem-limited, fastidious, insect-borne bacterium. Rapid spread of HLB disease relies on CLas-efficient propagation in the vector, the Asian citrus psyllid Diaphorina citri, in a circulative manner. Understanding the intracellular lifecycle of CLas in psyllid midgut, the major organ for CLas transmission, is fundamental to improving current management strategies. Using a microscopic approach within CLas-infected insect midgut, we observed the entry of CLas into gut cells inside vesicles, termed Liberibacter-containing vacuoles (LCVs), by endocytosis. Endocytosis is followed by the formation of endoplasmic reticulum-related and replication permissive vacuoles (rLCVs). Additionally, we observed the formation of double membrane autophagosome-like structure, termed autophagy-related vacuole (aLCV). Vesicles containing CLas egress from aLCV and fuse with the cell membrane. Immunolocalization studies showed that CLas uses endocytosis- and exocytosis-like mechanisms that mediates bacterial invasion and egress. Upregulation of autophagy-related genes indicated subversion of host autophagy by CLas in psyllid vector to promote infection. These results indicate that CLas interacts with host cellular machineries to undergo a multistage intracellular cycle through endocytic, secretory, autophagic, and exocytic pathways via complex machineries. Potential tactics for HLB control can be made depending on further investigations on the knowledge of the molecular mechanisms of CLas intracellular cycle.

Extracellular vesicles, including exosomes, are released by all cells, including those of the nervous system. Capable of delivering lipid, protein and nucleic acids to both nearby and distal cells, exosomes have been hypothesized to play a role in progression of many diseases of the nervous system. To date, most analyses on the role of these vesicles in the healthy and diseased state have relied on studying vesicles from in vitro sources, such as conditioned cell culture media, or body fluids. Here we have taken a critical approach to the enrichment and characterization of exosomes from human frontal cortex. This method maintains the integrity of the vesicles and their cargo, and comprehensive proteomic and genomic characterization confirms the legitimacy of the resulting extracellular vesicles as endosome-derived exosomes. This method will enable neuroscientists to acquire more detailed information about exosomes in the brain and explore the role(s) this form of intercellular communication and unique source of lipid, protein and RNA has in healthy brain function and pathogenic conditions. Furthermore, this method may have important utility in the isolation of exosomes from other tissues.

Intracellular bacteria use multiple strategies to enter and persist in host cells. In doing so they must survive a plethora of host defense strategies that straddle innate and adaptive immune mechanisms. Intriguingly, it is the combination of intracellular infection and deployment of host immunity that define some of the most notorious bacterial-borne diseases known to humans, namely typhoid, tuberculosis and leprosy. Despite this, improvements in our understanding of the molecular processes involved on the side of both bacteria and host are new concepts in microbiology, immunology, molecular biology and ultimately, clinical medicine. New therapeutic approaches emanating from these insights should aid us in defying the continuing threat of increasing antibiotic resistance that is rendering many long-relied upon drugs ineffective.

Twenty years of research have led to the model, acknowledged by the 2013 Nobel Prize in Physiology and Medicine, that soluble N -ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate the fusion of intracellular membranes. SNAREs are also able to recruit other molecules playing essential roles in the spatial and temporal regulation of vesicular traffic, and they now even appear as multitask proteins in intracellular vesicle biogenesis, transport and fusion. Here we focus on recent findings at the molecular level which suggest how SNAREs operate as membrane fusion nanomachines that are finely tuned to allow fusion to occur at the right time and place, and how they are integrated into complex molecular networks of interaction.

Sorting nexins (SNXs) are a family of proteins that regulate cellular cargo sorting and trafficking, maintain intracellular protein homeostasis, and participate in intracellular signaling. SNXs are also important in the regulation of blood pressure via several mechanisms. Aberrant expression and dysfunction of SNXs participate in the dysregulation of blood pressure. Genetic studies show a correlation between SNX gene variants and the response to antihypertensive drugs. In this review, we summarize the progress in SNX-mediated regulation of blood pressure, discuss the potential role of SNXs in the pathophysiology and treatment of hypertension, and propose novel strategies for the medical therapy of hypertension.

In this study, the intracellular β-galactosidases of Bacillus subtilis 4NK and Bacillus paralicheniformis 5NK isolated from Bingöl Binkap hot spring was partially purified and characterized. As a result of purification, the yield of the enzyme for B. subtilis 4NK was 85.2% and the purification fold was 2.8. The yield for B. paralicheniformis 5NK was 76.8% and the purification fold was 2.0. The optimum temperature of the enzyme was determined as 45 oC for B. subtilis 4NK and 55 oC for B. paralicheniformis 5NK and the optimum pH was 6.0 for both. In addition, in the thermal stability experiments even at the end of 120 min both enzymes were stable at 50 oC. It was determined that the partially purified enzyme activity increased in the presence of iodoacetamide and phenylmethylsulfonylfluoride for B. subtilis 4NK, dithiothreitol, N-ethylenemaleimide and phenylmethylsulfonylfluoride for B. paralicheniformis 5NK. The metals were found to activate the enzyme at low concentrations of Co2+, Cd2+ and Mn2+ for B. subtilis 4NK, Cu2+ and Cd2+ were found to inhibit the enzyme at high rates for B. paralicheniformis 5NK. Km and Vmax values for 4NK and 5NK, respectively; 23.80 mM, 1.978 μmol/min and 5.61 mM, 1.869 μmol/min.

Secreted acid phosphatases (APases) dephosphorylate extracellular organic phosphate compounds to supply inorganic phosphate (Pi) to maintain cellular functions. Here, we show that APases are necessary to maintain a normal replicative lifespan in Saccharomyces cerevisiae. Deletion of all four APase genes shortened the lifespan in yeast strains on synthetic media (irrespective of the concentrations of Pi in the media), but it did not affect the intracellular ortho- and polyphosphate levels. Deletion of IPK1, which encodes inositol-pentakisphosphate 2-kinase, restored the lifespan in APase-null mutants, and IPK1 overexpression shortened the lifespan in wild-type strains. Overexpression of inositol hexakisphosphate (IP6 ) and heptakisphosphate kinases, KCS1 and VIP1, recovered the lifespan in APase-null mutants. Thus, yeast APases modulate the replicative lifespan, probably through dephosphorylation of intracellular IP6 .

Targeting intracellular components for lysosomal degradation by autophagy not only maintains cellular homeostasis but also counteracts the effects of external stimuli, including invading pathogens. Among various kinds of pathogens, viruses have been extensively shown to induce autophagy to benefit viral growth in infected cells and to modulate host defense responses, such as innate antiviral immunity. Recently, numerous lines of evidence have implied that virus-induced autophagy triggers multilayer mechanisms to regulate the innate antiviral response of host cells, thus promoting a balance in virus-host cell interactions. In this review, the detailed mechanisms underlying autophagy and the innate antiviral immune response are first described. Then, I summarize the current information regarding the diverse functional role(s) of autophagy in the control of antiviral defenses against different types of viral infections. Moreover, the physiological significance of autophagy-regulated antiviral responses on the viral life cycle and the potential autophagy alterations induced by virus-associated antiviral signaling is further discussed.

SUMMARYPlants use electrical and chemical signals for systemic communication. Herbivory, for instance, appears to trigger local apoplasmic glutamate accumulation, systemic electrical signals and calcium waves that travel to report insect damage to neighboring leaves and initiate defense. To monitor extra- and intracellular glutamate concentrations in plants, we generated Arabidopsis lines expressing genetically encoded fluorescent glutamate sensors. In contrast to cytosolically localized sensors, extracellularly displayed variants inhibited plant growth and proper development. Phenotypic analyses of high-affinity display sensor lines revealed that root meristem development, particularly the quiescent center (QC), number of lateral roots, vegetative growth and floral architecture were impacted. Notably, the severity of the phenotypes was positively correlated with the affinity of the display sensors, intimating that their ability to sequester glutamate at the surface of the plasma membrane was responsible for the defects. Root growth defects were suppressed by supplementing culture media with low levels of glutamate. Together, the data indicate that sequestration of glutamate at the cell surface either disrupts the supply of glutamate to meristematic cells and/or impairs localized glutamatergic signaling important for developmental processes.SIGNIFICANCE STATEMENTGlutamate is an important signaling molecule in animals and plants. The affinity-dependent effects of surface-displayed glutamate sensors on plant development intimate that glutamate levels in the vicinity of the plasma membrane play important roles in signaling in plants.

Haematopoietic stem/progenitor cell (HSPC) integrates intracellular signal network from growth factors (GFs) and utilizes its proliferation feature to generate high yields of transplantable cells upon ex vivo culture. However, the molecular basis for HSPC activation and proliferation is not completely understood. The goal of this study was to investigate proliferation regulator in the downstream of GFs and develop HSPC expansion strategy. Microarray and Ingenuity Pathway Analysis were performed to evaluate differentially expressed genes in cytokine-induced CD34+ cells after ex vivo culture. We identified that MEK1 was a potential HSPC proliferation regulator, which represented indispensable roles and MEK1 silence attenuated the proliferation of HSPC. Notably, 500 nM MEK1 agonist, PAF C-16, increased the numbers of phenotypic HSPC and induced cell cycling of HSPC. The PAF C-16 expanded HSPC demonstrated comparative clonal formation ability and secondary expansion capacity compared to the vehicle control. Our results provide insights into regulating the balance between proliferation and commitment of HSPC by targeting the HSPC proliferation-controlling network. This study demonstrates that MEK1 critically regulates HSPC proliferation and cell production in the ex vivo condition for transplantation.

The intracellular pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) exploits host macrophage as a crucial survival and replicative niche. To minimize host immune response stimulated by flagellin, the expression of flagellar genes is downregulated during S. Typhimurium growth within host macrophages. However, the underlying mechanisms are largely unknown. In this study, we show that STM14_1285 (named AsiR), a putative RpiR-family transcriptional regulator, which is downregulated within macrophages as previously reported and also confirmed here, positively regulates the expression of flagellar genes by directly binding to the promoter of flhDC. By generating an asiR mutant strain and a strain that persistently expresses asiR gene within macrophages, we confirmed that the downregulation of asiR contributes positively to S. Typhimurium replication in macrophages and systemic infection in mice, which could be attributed to decreased flagellar gene expression and therefore reduced flagellin-stimulated secretion of pro-inflammatory cytokines IL-1β and TNF-α. Furthermore, the acidic pH in macrophages is identified as a signal for the downregulation of asiR and therefore flagellar genes. Collectively, our results reveal a novel acidic pH signal-mediated regulatory pathway that is utilized by S. Typhimurium to promote intracellular replication and systemic pathogenesis by repressing flagellar gene expression.

Extracellular vesicles (EVs) are vehicles of intercellular communication that are released from various cell types under physiological and pathological conditions, with differing effects on the body. Under physiological conditions, EVs mediate cell-to-cell and intertissue communication and participate in maintaining homeostasis. Certain EV types have emerged as biological therapeutic agents in various fields, such as cell-free regenerative medicine, drug delivery and immunotherapy. However, the low yield of EVs is a bottleneck in the large-scale implementation of these therapies. Conversely, more EVs in the microenvironment in other circumstances, such as tumor metastasis, viral particle transmission, and the propagation of neurodegenerative disease, can exacerbate the situation, and the inhibition of EV secretion may delay the progression of these diseases. Therefore, the promotion and inhibition of EV release is a new and promising field because of its great research potential and wide application prospects. We first review the methods and therapeutic opportunities for the regulation of EV release based on the mechanism of EV biogenesis and consider the side effects and challenges.

The accurate definition of an epitranscriptome is endangered by artefacts resulting from RNA degradation after cell death, a ubiquitous yet little investigated process. By tracing RNA marker modifications through tissue preparation protocols, we identified a major blind spot from daily lab routine, that has massive impact on modification analysis in small RNAs. In particular, m6,6A and Am as co-varying rRNA marker modifications, appeared in small RNA fractions following rRNA degradation in vitro and in cellulo. Analysing mouse tissue at different time points post mortem, we tracked the progress of intracellular RNA degradation after cell death, and found it reflected in RNA modification patterns. Differences were dramatic between liver, where RNA degradation commenced immediately after death, and brain, yielding essentially undamaged RNA. RNA integrity correlated with low amounts of co-varying rRNA markers. Thus validated RNA preparations featured differentially modified tRNA populations whose information content allowed a distinction even among the related brain tissues cortex, cerebellum and hippocampus. Inversely, advanced cell death correlated with high rRNA marker content, and correspondingly little with the naïve state of living tissue. Therefore, unless RNA and tissue preparations are executed with utmost care, interpretation of modification patterns in tRNA and small RNA are prone to artefacts.

The potential clinical applications of the powerful in vitro-transcribed (IVT)-mRNAs, to restore defective protein functions, strongly depend on their successful intracellular delivery and transient translation through the development of safe and efficient delivery platforms. In this study, an innovative (international patent-pending) methodology was developed, combining the IVT-mRNAs with the protein transduction domain (PTD) technology, as an efficient delivery platform. Based on the PTD technology, which enables the intracellular delivery of various cargoes intracellularly, successful conjugation of a PTD to the IVT-mRNAs was achieved and evaluated by band-shift assay and NMR spectroscopy. In addition, the PTD-IVT-mRNAs were applied and evaluated in two protein-disease models, including the mitochondrial disorder fatal infantile cardioencephalomyopathy and cytochrome c oxidase (COX) deficiency (attributed to SCO2 gene mutations) and β-thalassemia. The PTD-IVT-mRNA of SCO2 was successfully transduced and translated to the corresponding Sco2 protein inside the primary fibroblasts of a SCO2/COX-deficient patient, whereas the PTD-IVT-mRNA of β-globin was transduced and translated in bone marrow cells, derived from three β-thalassemic patients. The transducibility and the structural stability of the PDT-IVT-mRNAs, in both cases, were confirmed at the RNA and protein levels. We propose that our novel delivery platform could be clinically applicable as a protein therapy for metabolic/genetic disorders., Graphical abstract, The corresponding author and colleagues successfully developed a universal delivery platform of therapeutic in vitro-transcribed (IVT)-mRNAs conjugated to a selected PTD through an innovative patent-pending methodology. PTD-IVT-mRNAs exhibited significant stability, intracellular transduction, and protein expression efficiency and were applied in patients’ cells of two different monogenic/metabolic disorders, revealing powerful future perspectives.

In this study, we obtained four alternative splicing transcripts of intracellular copper/zinc superoxide dismutase 1 (icCuZnSOD1) in Oxya chinensis. OcicCuZnSOD1a has all common characteristics of CuZnSOD family and is a canonical CuZnSOD. OcicCuZnSOD1b is missing a Zn binding site. OcicCuZnSOD1c lacks Zn ion and is a Cu-only SOD. OcicCuZnSOD1d is missing a CuZnSOD conserved sequence and lacks the E-loop, a conserved disulfide bond, and an active site arginine. OcicCuZnSOD1a was the most heat-resistant and OcicCuZnSOD1c was the most unstable at high temperatures above 55 °C. They were stable at a wide pH range, especially in alkaline conditions. The four variants expressed at the throughout developmental stages and had various tissue expression patterns. OcicCuZnSOD1a and OcicCuZnSOD1d were significantly induced by 8.79 mM CuCl2 and OcicCuZnSOD1b was significantly up-regulated by 14.67 mM CuCl2. OcicCuZnSOD1a was significantly inhibited by 19.13 mM ZnSO4 while OcicCuZnSOD1d were significantly induced by 22.61 mM ZnSO4. Disc diffusion assay showed that the four isoforms of OcicCuZnSOD1 made the killing zones smaller surrounding the CdCl2-soaked filter discs. However, the reduction ratios of OcicCuZnSOD1a were the highest. These results implied that the four transcripts played roles in defense against CdCl2-induced oxidative stress while OcicCuZnSOD1a had stronger antioxidant capacity.

Elevated blood pressure caused by excessive salt intake is common and associated with cardiovascular diseases in most countries. However, the composition and responses of vascular cells in the progression of hypertension have not been systematically described. We performed single-cell RNA sequencing on the aortic arch from C57BL/6J mice fed a chow/high-salt diet. We identified 19 distinct cell populations representing 12 lineages, including smooth muscle cells (SMCs), fibroblasts, endothelial cells (ECs), B cells, and T cells. During the progression of hypertension, the proportion of three SMC subpopulations, two EC subpopulations, and T cells increased. In two EC clusters, the expression of reactive oxygen species-related enzymes, collagen and contractility genes was upregulated. Gene set enrichment analysis showed that three SMC subsets underwent endothelial-to-mesenchymal transition. We also constructed intercellular networks and found more frequent cell communication among aortic cells in hypertension and that some signaling pathways were activated during hypertension. Finally, joint public genome-wide association study data and our single-cell RNA-sequencing data showed the expression of hypertension susceptibility genes in ECs, SMCs, and fibroblasts and revealed 21 genes involved in the initiation and development of high-salt-induced hypertension. In conclusion, our data illustrate the transcriptional landscape of vascular cells in the aorta associated with hypertension and reveal dramatic changes in cell composition and intercellular communication during the progression of hypertension., High blood pressure: how salt affects cells in the aorta Salt-induced high blood pressure involves dramatic changes in cell composition and communication in blood vessels, a genetic analysis reveals. Over 99% of the world’s adult population has an excessive salt intake, which is associated with hypertension and numerous cardiovascular diseases. Xin Ma and co-workers at Jiangnan University in Wuxi, China, performed single-cell RNA sequencing on aortas from mice that were fed a high-salt diet, and combined their results with genome-wide association studies. They revealed that as hypertension progresses the proportion of smooth muscle cells, cells lining blood vessels, and T cells increases, while several signaling pathways are enriched and new pathways become activated. They also identified 21 associated genes, including Nox4, which promotes collagen synthesis and leads to arterial stiffness. This new understanding provides potential targets for prevention and treatment of hypertension.

Calcium ion (Ca(2+)) signaling is one of the most frequently employed mechanisms of signal transduction by eukaryotic cells, and starts with either Ca(2+) release from intracellular stores or Ca(2+) entry through the plasma membrane. In intracellular protist parasites Ca(2+) signaling initiates a sequence of events that may facilitate their invasion of host cells, respond to environmental changes within the host, or regulate the function of their intracellular organelles. In this review we examine recent findings in Ca(2+) signaling in two groups of intracellular protist parasites that have been studied in more detail, the apicomplexan and the trypanosomatid parasites.

Citrobacter rodentium, a natural mouse pathogen which colonises the colon of immuno-competent mice, provides a robust model for interrogating host-pathogen-microbiota interactions in vivo. This model has been key to providing new insights into local host responses to enteric infection, including changes in intestinal epithelial cell immunometabolism and mucosal immunity. C. rodentium injects 31 bacterial effectors into epithelial cells via a type III secretion system (T3SS). Recently, these effectors were shown to be able to form multiple intracellular subnetworks which can withstand significant contractions whilst maintaining virulence. Here we highlight recent advances in understanding gut mucosal responses to infection and effector biology, as well as potential uses for artificial intelligence (AI) in understanding infectious disease and speculate on the role of T3SS effector networks in host adaption.

Metal–organic framework (MOF) nanoparticles are promising carriers for the internal delivery of therapeutics to the cells; however, the effects of their shape on cellular uptake have not yet been clearly evaluated. Therefore, we synthesized biocompatible Fe-based MOF nanoparticles of two distinct shapes—rods and octahedrons (NH2-MIL-88B(Fe)-ROD and NH2-MIL-88B(Fe)-OCTA, respectively)—and evaluated their cellular uptake kinetics and mechanisms using three different cell lines (HeLa, MDA-MB-231, and L929). Although both nanoparticles possessed the same constituent metal, organic linkers, and thus, crystalline structures, a more rapid uptake was observed with NH2-MIL-88B(Fe)-OCTA, whereas NH2-MIL-88B(Fe)-ROD were endocytosed in a more sustained manner. This could be ascribed to the large number of sharp edges available in NH2-MIL-88B(Fe)-OCTA particles; hence, there were more points of expedited entry into the cells via a clathrin-mediated pathway. The findings herein provide important information for the application of the MOF nanoparticles as intracellular drug delivery carriers, especially when they can be prepared in different shapes with the same composition.

Ferroptosis is a non-apoptotic regulated cell death caused by iron accumulation and subsequent lipid peroxidation. Currently, the therapeutic role of ferroptosis on cancer is gaining increasing interest. Baicalin an active component in Scutellaria baicalensis Georgi with anticancer potential various cancer types; however, the effects of baicalein on bladder cancer and the underlying molecular mechanisms remain largely unknown. In the study, we investigated the effect of baicalin on bladder cancer cells 5637 and KU-19-19. As a result, we show baicalin exerted its anticancer activity by inducing apoptosis and cell death in bladder cancer cells. Subsequently, we for the first time demonstrate baicalin-induced ferroptotic cell death in vitro and in vivo, accompanied by reactive oxygen species (ROS) accumulation and intracellular chelate iron enrichment. The ferroptosis inhibitor deferoxamine but not necrostatin-1, chloroquine (CQ), N-acetyl-l-cysteine, l-glutathione reduced, or carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK) rescued baicalin-induced cell death, indicating ferroptosis contributed to baicalin-induced cell death. Mechanistically, we show that ferritin heavy chain 1 (FTH1) was a key determinant for baicalin-induced ferroptosis. Overexpression of FTH1 abrogated the anticancer effects of baicalin in both 5637 and KU19-19 cells. Taken together, our data for the first time suggest that the natural product baicalin exerts its anticancer activity by inducing FTH1-dependent ferroptosis, which will hopefully provide a prospective compound for bladder cancer treatment., Graphical abstract Baicalin exerts its anticancer activity in bladder cancer by inducing ferritin heavy chain 1 (FTH1)-dependent ferroptosis, which will hopefully provide great therapeutic potential for bladder cancer treatment.Image 1

Cyanobacteria can utilize CO2 or even N2 to produce a variety of high value-added products efficiently. Plastoquinone (PQ) is an important electron carrier in both of the photosynthetic and respiratory electron transport chain. Although the content of PQ, as well as their redox state, have an important effect on physiology and metabolism, there are relatively few studies on the synthesis of PQ and its related metabolic regulation mechanism in photosynthetic microorganisms. In this study, the strategies of overexpression of Geranyl diphosphate: 4-hydroxybenzoate geranyltransferase (lepgt) and addition of 4-hydroxybenzoate (4-HB) as the quinone ring precursor were adopted to regulate the biosynthesis of PQ in Synechocystis PCC 6803. Combined with the analysis the photosystem activity, respiration rate and metabolic components, we found the changes of intracellular PQ reprogrammed the metabolism of Synechocystis PCC 6803. The results showed that the overexpression of lepgt reduced PQ content dramatically, by 22.18%. Interestingly, both of the photosynthesis and respiration rate were enhanced. In addition, the intracellular lipid and protein contents were significantly increased. Whereas, the addition of low concentrations of 4-HB enhanced the biosynthesis of PQ, and the intracellular PQ contents were increased by 14.76%–70.86% in different conditions. Addition of 4-HB can regulate the photosystem efficiency and respiration and reprogram the metabolism of Synechocystis PCC 6803 efficiently. In a word, regulating the PQ biosynthesis provided a novel idea for promoting the reprogramming the physiology and metabolism of Synechocystis.

Protozoa of the genus Leishmania are intracellular parasites that cause human leishmaniasis, a disease spread mostly in the tropics and subtropics. Leishmania cycle between the midgut of female sand flies and phagolysosome of mammalian macrophages. During their life cycle they constantly encounter changing nutritional environments. To monitor the external concentration of essential nutrients, the invading parasites employ sensors that report on the availability of these nutrients; but to-date only a few sensing pathways have been identified in Leishmania. This review focuses on the Arginine Deprivation Response, which both extracellular and intracellular Leishmania utilize to monitor environmental arginine and adjust their arginine transporter (AAP3) levels accordingly.

© The Author(s) 2021., Temperature fluctuation is one of the most frequent threats to which organisms are exposed in nature. The activation of gene expression programs that trigger the transcription of heat stress-protective genes is the main cellular response to resist high temperatures. In addition, reversible accumulation and compartmentalization of thermosensitive proteins in high-order molecular assemblies are emerging as critical mechanisms to ensure cellular protection upon heat stress. Here, we summarize representative examples of membrane-less intracellular bodies formed upon heat stress in yeasts and human cells and highlight how protein aggregation can be turned into a cytoprotective mechanism., This work was supported by the Ministerio de Ciencia, Innovación y Universidades (Grant: PGC2018-099849-B-I00 to R.R. Daga) and Junta de Andalucía-FEDER-UPO (grant: UPO-1264663 to S.S.P.).

Objective. To explore the effects of silicone gel nanoparticles modified with octacalcium phosphate on the surface (silica/OCP) polymer drugs on the proliferation of osteoblasts and autophagy. Method. Silica/OCP was prepared in vitro, and the quality of the sample preparation was tested through characterization experiments. The osteoblast cell line (hFOB1.19) was treated with silica/OCP, autophagy inhibitor (3-methyladenine (3-MA)), and silica/OCP+3-MA, respectively. The proliferation of hFOB1.19 cells was detected through the methylthiazolyldiphenyl-tetrazolium bromide (MTT) kit. Flow cytometry was used to detect the cell apoptosis. The change in protein beclin1 and P62 expression in hFOB1.19 cells was observed in Western blot. An ROS detection kit was used to detect the content of reactive oxygen species in hFOB1.19 cells. Results. Silica/OCP was a sphere with a particle size of 50 nm to 130 nm and had an OCP phase in electron projection microscopy and X-ray diffraction techniques. The results indicated that OCP successfully modified silica and the material was successfully prepared. An MTT kit and flow cytometry test showed that the cell viability of the cells treated with silica/OCP increased significantly ( P < 0.05 ), and the intracellular apoptosis phenomenon was significantly decreased ( P < 0.05 ) compared to the control group. Moreover, the inhibition of cell viability and promotion of apoptosis caused by the autophagy inhibitor 3-MA can be rescued. Western blotting demonstrated that the protein level of beclin1 in osteoblasts reached the highest after six hours of treatment with silica/OCP, and the protein level of p62, the substrate protein of autophagy, reached the lowest. At the same time, treatment of cells with the autophagy inhibitor 3-MA and silica/OCP+3-MA found that the protein levels of beclin1 and p62 in the silica/OCP+3-MA group were adjusted back compared to the 3-MA group. After adding the autophagy inhibitor, the reactive oxygen content in the cell was significantly increased ( P < 0.05 ) in the silica/OCP group. In the presence of intracellular reactive oxygen inhibitors catalase and silica/OCP, the cell viability of osteoblasts was significantly lower than that of the silica/OCP group but significantly higher than that of the silica/OCP+3-MA group. The apoptosis level of the silica/OCP+catalase group was also significantly lower than that of the silica/OCP+3-MA group ( P < 0.05 ) but was significantly higher than that of the silica/OCP group ( P < 0.05 ). Conclusion. Silica/OCP nanoparticles can upregulate the level of autophagy in osteoblasts and promote the proliferation of osteoblasts.