Cells — Open Access Journal of Cell Biology

The anaerobic pathogen Clostridium difficile is of growing significance for the health care system due to its increasing incidence and mortality. As C. difficile infection is both supported and treated by antibiotics, a deeper knowledge on how antimicrobial agents affect the physiology of this important pathogen may help to understand and prevent the development and spreading of antibiotic resistant strains. As the proteomic response of a cell to stress aims at counteracting the harmful effects of this stress, it can be expected that the pattern of a pathogen’s responses to antibiotic treatment will be dependent on the antibiotic mechanism of action. Hence, every antibiotic treatment is expected to result in a specific proteomic signature characterizing its mode of action. In the study presented here, the proteomic response of C. difficile 630∆erm to vancomycin, metronidazole, and fidaxomicin stress was investigated on the level of protein abundance and protein synthesis based on 2D PAGE. The quantification of 425 proteins of C. difficile allowed the deduction of proteomic signatures specific for each drug treatment. Indeed, these proteomic signatures indicate very specific cellular responses to each antibiotic with only little overlap of the responses. Whereas signature proteins for vancomycin stress fulfil various cellular functions, the proteomic signature of metronidazole stress is characterized by alterations of proteins involved in protein biosynthesis and protein degradation as well as in DNA replication, recombination, and repair. In contrast, proteins differentially expressed after fidaxomicin treatment can be assigned to amino acid biosynthesis, transcription, cell motility, and the cell envelope functions. Notably, the data provided by this study hint also at so far unknown antibiotic detoxification mechanisms. Full article

Treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) improves the overall survival of patients with EGFR-mutated non-small-cell lung cancer (NSCLC). First-generation EGFR-TKIs (e.g., gefitinib and erlotinib) or second-generation EGFR-TKIs (e.g., afatinib and dacomitinib) are effective for the treatment of EGFR-mutated NSCLC, especially in patients with EGFR exon 19 deletions or an exon 21 L858R mutation. However, almost all cases experience disease recurrence after 1 to 2 years due to acquired resistance. The EGFR T790M mutation in exon 20 is the most frequent alteration associated with the development of acquired resistance. Osimertinib—a third-generation EGFR-TKI—targets the T790M mutation and has demonstrated high efficacy against EGFR-mutated lung cancer. However, the development of acquired resistance to third-generation EGFR-TKI, involving the cysteine residue at codon 797 mutation, has been observed. Other mechanisms of acquired resistance include the activation of alternative pathways or downstream targets and histological transformation (i.e., epithelial–mesenchymal transition or conversion to small-cell lung cancer). Furthermore, the development of primary resistance through overexpression of the hepatocyte growth factor and suppression of Bcl-2-like protein 11 expression may lead to problems. In this report, we review these mechanisms and discuss therapeutic strategies to overcome resistance to EGFR-TKIs. Full article

Ephexin4 is a guanine nucleotide-exchange factor (GEF) for RhoG and is involved in various RhoG-related cellular processes such as phagocytosis of apoptotic cells and migration of cancer cells. Ephexin4 forms an oligomer via an intermolecular interaction, and its GEF activity is increased in the presence of Elmo, an Ephexin4-interacting protein. However, it is uncertain if and how Ephexin4 is autoinhibited. Here, using an Ephexin4 mutant that abrogated the intermolecular interaction, we report that this interaction impeded binding of RhoG to Ephexin4 and thus inhibited RhoG activation. Mutation of the glutamate residue at position 295, which is a highly conserved residue located in the region of Ephexin4 required for the intermolecular interaction, to alanine (Ephexin4^E295A) disrupted the intermolecular interaction and increased binding of RhoG, resulting in augmented RhoG activation. In addition, phagocytosis of apoptotic cells and formation of membrane ruffles were increased more by expression of Ephexin4^E295A than by expression of wild-type Ephexin4. Taken together, our data suggest that Ephexin4 is autoinhibited through its intermolecular interaction, which impedes binding of RhoG. Full article

Aquaporin 1 (AQP1) and aquaporin 4 (AQP4) have been identified in the eye as playing an essential role in the formation of the aqueous humor along with the Na⁺/K⁺ ATPase pump. Different authors have described the relationship between blood pressure, aqueous humor production, and intraocular pressure with different conclusions, with some authors supporting a positive correlation between blood pressure and intraocular pressure while others disagree. The aim of this work was to study the effect of high blood pressure on the proteins involved in the production of aqueous humor in the ciliary body (CB) and iris. For this purpose, we used the eyes of spontaneously hypertensive rats (SHR) and their control Wistar-Kyoto rats (WKY). Immunofluorescence was performed in different eye structures to analyze the effects of hypertension in the expression of AQP1, AQP4, and the Na⁺/K⁺ ATPase α1 and α2 subunits. The results showed an increase in AQP1 and Na⁺/K⁺ ATPase α1 and a decrease in AQP4 and Na⁺/K⁺ ATPase α2 in the CB of SHR, while an increase in AQP4 and no significant differences in AQP1 were found in the iris. Therefore, systemic hypertension produced changes in the proteins implicated in the movement of water in the CB and iris that could influence the production rate of aqueous humor, which would be affected depending on the duration of systemic hypertension. Full article

Aquaporins (AQPs) function as tetrameric structures in which each monomer has its own permeable pathway. The combination of structural biology, molecular dynamics simulations, and experimental approaches has contributed to improve our knowledge of how protein conformational changes can challenge its transport capacity, rapidly altering the membrane permeability. This review is focused on evidence that highlights the functional relationship between the monomers and the tetramer. In this sense, we address AQP permeation capacity as well as regulatory mechanisms that affect the monomer, the tetramer, or tetramers combined in complex structures. We therefore explore: (i) water permeation and recent evidence on ion permeation, including the permeation pathway controversy—each monomer versus the central pore of the tetramer—and (ii) regulatory mechanisms that cannot be attributed to independent monomers. In particular, we discuss channel gating and AQPs that sense membrane tension. For the latter we propose a possible mechanism that includes the monomer (slight changes of pore shape, the number of possible H-bonds between water molecules and pore-lining residues) and the tetramer (interactions among monomers and a positive cooperative effect). Full article

The Hedgehog (Hh) pathway is a signaling cascade that plays a crucial role in many fundamental processes, including embryonic development and tissue homeostasis. Moreover, emerging evidence has suggested that aberrant activation of Hh is associated with neoplastic transformations, malignant tumors, and drug resistance of a multitude of cancers. At the molecular level, it has been shown that Hh signaling drives the progression of cancers by regulating cancer cell proliferation, malignancy, metastasis, and the expansion of cancer stem cells (CSCs). Thus, a comprehensive understanding of Hh signaling during tumorigenesis and development of chemoresistance is necessary in order to identify potential therapeutic strategies to target various human cancers and their relapse. In this review, we discuss the molecular basis of the Hh signaling pathway and its abnormal activation in several types of human cancers. We also highlight the clinical development of Hh signaling inhibitors for cancer therapy as well as CSC-targeted therapy. Full article

The aquaglyceroporin AQP7, a family member of aquaporin membrane channels, facilitates the permeation of water and glycerol through cell membranes and is crucial for body lipid and energy homeostasis. Regulation of glycerol permeability via AQP7 is considered a promising therapeutic strategy towards fat-related metabolic complications. Here, we used a yeast aqy-null strain for heterologous expression and functional analysis of human AQP7 and investigated its regulation by pH. Using a combination of in vitro and in silico approaches, we found that AQP7 changes from fully permeable to virtually closed at acidic pH, and that Tyr135 and His165 facing the extracellular environment are crucial residues for channel permeability. Moreover, instead of reducing the pore size, the protonation of key residues changes AQP7’s protein surface electrostatic charges, which, in turn, may decrease glycerol’s binding affinity to the pore, resulting in decreased permeability. In addition, since some pH-sensitive residues are located at the monomer-monomer interface, decreased permeability may result from cooperativity between AQP7’s monomers. Considering the importance of glycerol permeation via AQP7 in multiple pathophysiological conditions, this mechanism of hAQP7 pH-regulation may help the design of selective modulators targeting aquaglyceroporin-related disorders. Full article

The comprehension of dental pathogenesis and disorders derived from eruption failure requires a deep understanding of the molecular mechanisms underlying normal tooth eruption. As intense remodelling is needed during tooth eruption, we hypothesize that matrix metalloproteinase-1 (MMP-1) and acid phosphatase (ACP) play a role in the eruptive pathway degradation. We evaluated MMP-1-immunoexpression and the collagen content in the lamina propria at different eruptive phases. Immunohistochemistry and ultrastructural cytochemistry for detection of ACP were also performed. In the maxillary sections containing first molars of 9-, 11-, 13-, and 16-day-old rats, the birefringent collagen of eruptive pathway was quantified. MMP-1 and ACP-2 immunohistochemical reactions were performed and the number of MMP-1-immunolabelled cells was computed. Data were analyzed by one-way ANOVA and Tukey post-test (p ≤ 0.05). ACP cytochemistry was evaluated in specimens incubated in sodium β-glycerophosphate. In the eruptive pathway of 13- and 16-day-old rats, the number of MMP-1-immunolabelled cells increased concomitantly to reduction of collagen in the lamina propria. Enhanced ACP-2-immunolabelling was observed in the lamina propria of 13- and 16-day-old rats. Fibroblasts and macrophages showed lysosomes and vacuoles containing fragmented material reactive to ACP. MMP-1 degrades extracellular matrix, including collagen fibers, being responsible for the reduction in the collagen content during tooth eruption. The enhanced ACP activity at the mucosal penetration stage indicates that this enzyme plays a role in the degradation of remnant material, which is engulfed by macrophages and fibroblasts of the eruptive pathway. Therefore, enzymatic failure in the eruptive pathway may disturbs tooth eruption. Full article

H3K9me3, H3K27me3, and H4K20me1 are epigenetic markers associated with chromatin condensation and transcriptional repression. Previously, we found that migration of melanoma cells is associated with and dependent on global chromatin condensation that includes a global increase in these markers. Taken together with more recent reports by others suggests it is a general signature of migrating cells. Here, to learn about the function of these markers in migrating cells, we mapped them by ChIP-seq analysis. This analysis revealed that induction of migration leads to expansion of these markers along the genome and to an increased overlapping between them. Significantly, induction of migration led to a higher increase in H3K9me3 and H4K20me1 signals at repetitive elements than at protein-coding genes, while an opposite pattern was found for H3K27me3. Transcriptome analysis revealed 182 altered genes following induction of migration, of which 33% are dependent on H3K27me3 for these changes. H3K27me3 was also required to prevent changes in the expression of 501 other genes upon induction of migration. Taken together, our results suggest that heterochromatinization in migrating cells is global and not restricted to specific genomic loci and that H3K27me3 is a key component in executing a migration-specific transcriptional plan. Full article

In neutrophils, intracellular Ca²⁺ levels are regulated by several transporters and pathways, namely SERCA [sarco(endo)plasmic reticulum Ca²⁺-ATPase], SOCE (store-operated calcium entry), and ROCE (receptor-operated calcium entry). However, the exact mechanisms involved in the communication among these transporters are still unclear. In the present study, thapsigargin, an irreversible inhibitor of SERCA, and ML-9, a broadly used SOCE inhibitor, were applied in human neutrophils to better understand their effects on Ca²⁺ pathways in these important cells of the immune system. The thapsigargin and ML-9 effects in the intracellular free Ca²⁺ flux were evaluated in freshly isolated human neutrophils, using a microplate reader for monitoring fluorimetric kinetic readings. The obtained results corroborate the general thapsigargin-induced intracellular pattern of Ca²⁺ fluctuation, but it was also observed a much more extended effect in time and a clear sustained increase of Ca²⁺ levels due to its influx by SOCE. Moreover, it was obvious that ML-9 enhanced the thapsigargin-induced emptying of the internal stores. Indeed, ML-9 does not have this effect by itself, which indicates that, in neutrophils, thapsigargin does not act only on the influx by SOCE, but also by other Ca²⁺ pathways, that, in the future, should be further explored. Full article

Estrogens acting through the classic estrogen receptors (ERs) and the G protein estrogen receptor (GPER) regulate the expression of diverse miRNAs, small sequences of non-coding RNA involved in several pathophysiological conditions, including breast cancer. In order to provide novel insights on miRNAs regulation by estrogens in breast tumor, we evaluated the expression of 754 miRNAs by TaqMan Array in ER-negative and GPER-positive SkBr3 breast cancer cells and cancer-associated fibroblasts (CAFs) upon 17β-estradiol (E2) treatment. Various miRNAs were regulated by E2 in a peculiar manner in SkBr3 cancer cells and CAFs, while miR-338-3p displayed a similar regulation in both cell types. By METABRIC database analysis we ascertained that miR-338-3p positively correlates with overall survival in breast cancer patients, according to previous studies showing that miR-338-3p may suppress the growth and invasion of different cancer cells. Well-fitting with these data, a miR-338-3p mimic sequence decreased and a miR-338-3p inhibitor sequence rescued the expression of genes and the proliferative effects induced by E2 through GPER in SkBr3 cancer cells and CAFs. Altogether, our results provide novel evidence on the molecular mechanisms by which E2 may regulate miR-338-3p toward breast cancer progression. Full article

The centrosome is not only the largest and most sophisticated protein complex within a eukaryotic cell, in the light of evolution, it is also one of its most ancient organelles. This special issue of “Cells” features representatives of three main, structurally divergent centrosome types, i.e., centriole-containing centrosomes, yeast spindle pole bodies (SPBs), and amoebozoan nucleus-associated bodies (NABs). Here, I discuss their evolution and their key-functions in microtubule organization, mitosis, and cytokinesis. Furthermore, I provide a brief history of centrosome research and highlight recently emerged topics, such as the role of centrioles in ciliogenesis, the relationship of centrosomes and centriolar satellites, the integration of centrosomal structures into the nuclear envelope and the involvement of centrosomal components in non-centrosomal microtubule organization. Full article

Current treatments for Parkinson’s disease (PD) only alleviate symptoms doing little to inhibit the onset and progression of the disease, thus we must research the mechanism of Parkinson’s. Rotenone is a known inducer of parkinsonian conditions in rats; we use rotenone to induce parkinsonian cellular conditions in Dictyostelium discoideum. In our model we primarily focus on mitochondrial dynamics. We found that rotenone disrupts the actin and microtubule cytoskeleton but mitochondrial morphology remains intact. Rotenone stimulates mitochondrial velocity while inhibiting mitochondrial fusion, increases reactive oxygen species (ROS) but has no effect on ATP levels. Antioxidants have been shown to decrease some PD symptoms thus we added ascorbic acid to our rotenone treated cells. Ascorbic acid administration suggests that rotenone effects may be specific to the disruption of the cytoskeleton rather than the increase in ROS. Our results imply that D. discoideum may be a valid cellular PD model and that the rotenone induced velocity increase and loss of fusion could prevent mitochondria from effectively providing energy and other mitochondrial products in high demand areas. The combination of these defects in mitochondrial dynamics and increased ROS could result in degeneration of neurons in PD. Full article

Chagas disease, caused by Trypanosoma cruzi, is a potentially life-threatening tropical disease endemic to Latin American countries that affects approximately 8 million people. In the chronic phase of the disease, individuals are classified as belonging to the indeterminate clinical form or to the cardiac and/or digestive forms when clinical symptoms are apparent. The relationship between monocytes and lymphocytes may be an important point to help clarify the complexity that surrounds the clinical symptoms of the chronic phase of Chagas disease. The co-stimulatory signals are essential to determining the magnitude of T cell response to the antigen. The signals are known to determine the regulation of subsequent adaptive immune response. However, little is known about the expression and function of these molecules in Chagas disease. Therefore, this review aims to discuss the possible role of main pathways of co-stimulatory molecule-receptor interactions in this pathology that could be crucial to understand the disease dynamics. Full article

It is well known that fibroblast growth factor receptor 2 (FGFR2) interacts with its ligand of fibroblast growth factor (FGF) therefore exerting biological functions on cell proliferation and differentiation. In this study, we first reported that the FGFR2 gene could generate a circular RNA of circFGFR2, which regulates skeletal muscle development by sponging miRNA. In our previous study of circular RNA sequencing, we found that circFGFR2, generated by exon 3–6 of FGFR2 gene, differentially expressed during chicken embryo skeletal muscle development. The purpose of this study was to reveal the real mechanism of how circFGFR2 affects skeletal muscle development in chicken. In this study, cell proliferation was analyzed by both flow cytometry analysis of the cell cycle and 5-ethynyl-2′-deoxyuridine (EdU) assays. Cell differentiation was determined by analysis of the expression of the differentiation marker gene and Myosin heavy chain (MyHC) immunofluorescence. The results of flow cytometry analysis of the cell cycle and EdU assays showed that, overexpression of circFGFR2 accelerated the proliferation of myoblast and QM-7 cells, whereas knockdown of circFGFR2 with siRNA reduced the proliferation of both cells. Meanwhile, overexpression of circFGFR2 accelerated the expression of myogenic differentiation 1 (MYOD), myogenin (MYOG) and the formation of myotubes, and knockdown of circFGFR2 showed contrary effects in myoblasts. Results of luciferase reporter assay and biotin-coupled miRNA pull down assay further showed that circFGFR2 could directly target two binding sites of miR-133a-5p and one binding site of miR-29b-1-5p, and further inhibited the expression and activity of these two miRNAs. In addition, we demonstrated that both miR-133a-5p and miR-29b-1-5p inhibited myoblast proliferation and differentiation, while circFGFR2 could eliminate the inhibition effects of the two miRNAs as indicated by rescue experiments. Altogether, our data revealed that a novel circular RNA of circFGFR2 could promote skeletal muscle proliferation and differentiation by sponging miR-133a-5p and miR-29b-1-5p. Full article

Protein secretion in general depends on signal sequence (also named leader sequence), a hydrophobic segment located at or close to the NH2-terminus of a secretory or membrane protein. This sequence guides the entry of nascent polypeptides into the lumen or membranes of the endoplasmic reticulum (ER) for folding, assembly, and export. However, evidence accumulated in recent years has suggested the existence of a collection of unconventional protein secretion (UPS) mechanisms that are independent of the canonical vesicular trafficking route between the ER and the plasma membrane (PM). These UPS mechanisms export soluble proteins bearing no signal sequence. The list of UPS cargos is rapidly expanding, along with the implicated biological functions, but molecular mechanisms accountable for the secretion of leaderless proteins are still poorly defined. This review summarizes our current understanding of UPS mechanisms with an emphasis on the emerging role of endo-lysosomes in this process. Full article

The development in digital screen technology has exponentially increased in the last decades, and many of today’s electronic devices use light-emitting diode (LED) technology producing very strong blue light (BL) waves. Long-term exposure at LED-BL seems to have an implication in the dehydration of the epidermis, in the alterations of shape and number of the keratinocytes, and in the aging of the skin. Aquaporins (AQPs) are water membrane channels that permeate both water and glycerol and play an important role in the hydration of epidermis, as well as in proliferation and differentiation of keratinocytes. Thus, we have hypothesized that AQPs could be involved in the aging of the skin exposed to LED-BL. Therefore, we have examined the expression of AQPs in human keratinocytes exposed to LED-BL at dose of 45 J/cm², used as an in vitro model to produce the general features of photo aging of the skin. The aim was to verify if LED-BL induces changes of the basal levels of AQPs. The keratinocytes exposure to LED-BL produced an increase of reactive oxygen species (ROS), an activation of 8-hydroxy-2’-deoxyguanosine (8-OHdG), an alteration of proliferating cell nuclear antigen (PCNA), and a down-regulation of AQP1, 3 and 9. These findings are preliminary evidences that may be used as starting points for further investigations about the mechanistic involvement of AQP1, 3, and 9 in LED-BL-induced skin aging. Full article

Background: Advanced cirrhosis is related to alterations in immunity. We aimed to evaluate the levels of peripheral CD4⁺ T cells (Tregs) and plasma cytokine in patients coinfected with human immunodeficiency virus and hepatitis C virus (HIV/HCV) according to liver fibrosis stages [evaluated as liver stiffness measure (LSM)] and their linear relationship. Methods: We performed a cross-sectional study on 238 HIV/HCV-coinfected patients (119 had <12.5 kPa, 73 had 12.5–25 kPa, and 46 had >25 kPa). Peripheral T-cell subsets were phenotyped by flow cytometry, plasma biomarkers were assessed by multiplex immunoassays, and LSM was assessed by transient elastography. Results: We found HIV/HCV-coinfected patients had higher values of CD4⁺ Tregs (p < 0.001), memory Tregs (p ≤ 0.001), and plasma cytokine levels [IFN-γ (p ≤ 0.05) and IL-10 (p ≤ 0.01)] compared with healthy donors and HIV-monoinfected patients. In the multivariate analysis, higher LSM values were associated with reduced levels of IL-10 (adjusted arithmetic mean ratio (aAMR) = 0.83; p = 0.019), IL-2 (aAMR = 0.78; p = 0.017), TNF-α (aAMR = 0.67; p < 0.001), and IL-17A (aAMR = 0.75; p = 0.006). When we focus on HIV/HCV-coinfected patients analyzed by LSM strata, patients with ≥25 kPa had lower values of IL-2 (aAMR = 0.66; p = 0.021), TNF-α (aAMR = 0.565; p = 0.003), and IL-17A (aAMR = 0.58; p = 0.003) than patients with <12.5 kPa. Conclusion: HIV/HCV-coinfected patients showed an immunosuppressive profile compared to healthy controls and HIV-monoinfected patients. Additionally, HIV/HCV-coinfected patients with advanced cirrhosis (LSM ≥ 25 kPa) had the lowest plasma values of cytokines related to Th1 (IL-2 and TNF-α) and Th17 (IL-17A) response. Full article

Water supply is essential for maintaining normal physiological function during the rapid growth of bamboo. Aquaporins (AQPs) play crucial roles in water transport for plant growth and development. Although 26 PeAQPs in bamboo have been reported, the aquaporin-led mechanism of maintaining diurnal water balance in bamboo shoots remains unclear. In this study, a total of 63 PeAQPs were identified, based on the updated genome of moso bamboo (Phyllostachys edulis), including 22 PePIPs, 20 PeTIPs, 17 PeNIPs, and 4 PeSIPs. All of the PeAQPs were differently expressed in 26 different tissues of moso bamboo, based on RNA sequencing (RNA-seq) data. The root pressure in shoots showed circadian rhythm changes, with positive values at night and negative values in the daytime. The quantitative real-time PCR (qRT-PCR) result showed that 25 PeAQPs were detected in the base part of the shoots, and most of them demonstrated diurnal rhythm changes. The expression levels of some PeAQPs were significantly correlated with the root pressure. Of the 86 sugar transport genes, 33 had positive co-expression relationships with 27 PeAQPs. Two root pressure-correlated PeAQPs, PeTIP4;1 and PeTIP4;2, were confirmed to be highly expressed in the parenchyma and epidermal cells of bamboo culm, and in the epidermis, pith, and primary xylem of bamboo roots by in situ hybridization. The authors’ findings provide new insights and a possible aquaporin-led mechanism for bamboo fast growth. Full article