Cells — Open Access Journal of Cell Biology

A combinatorial approach integrating stem cells and capable of exploiting available cues is likely needed to regenerate lost neural tissues and ultimately restore neurologic functions. This study investigates the effects of the subcutaneous maturation of adult-derived neural stem cell (aNSCs) seeded into biomaterial constructs on aNSC differentiation and ultimate regional neuronal identity as a first step toward a future spinal cord injury treatment. To achieve this, we encapsulated rat aNSCs in chitosan-based hydrogels functionalized with immobilized azide-tagged interferon-γ inside a chitosan conduit. Then, we implanted these constructs in the subcutaneous tissues in the backs of rats in the cervical, thoracic, and lumbar regions for 4, 6, and 8 weeks. After harvesting the scaffolds, we analyzed cell differentiation qualitatively using immunohistochemical analysis and quantitatively using RT-qPCR. Results revealed that the hydrogels supported aNSC survival and differentiation up to 4 weeks in the subcutaneous environment as marked by the expression of several neurogenesis markers. Most interesting, the aNSCs expressed region-specific Hox genes corresponding to their region of implantation. This study lays the groundwork for further translational work to recapitulate the potentially undiscovered patterning cues in the subcutaneous tissue and provide support for the conceptual premise that our bioengineering approach can form caudalized region-specific neuroepithelium. Full article

The mitochondrial proteome contains proteins from two different genetic systems. Proteins are either synthesized in the cytosol and imported into the different compartments of the organelle or directly produced in the mitochondrial matrix. To ensure proteostasis, proteins are monitored by the mitochondrial quality control system, which will degrade non-native polypeptides. Defective mitochondrial membrane proteins are degraded by membrane-bound AAA-proteases. These proteases are regulated by factors promoting protein turnover or preventing their degradation. Here we determined genetic interactions between the mitoribosome receptors Mrx15 and Mba1 with the quality control system. We show that simultaneous absence of Mrx15 and the regulators of the i-AAA protease Mgr1 and Mgr3 provokes respiratory deficiency. Surprisingly, mutants lacking Mrx15 were more tolerant against proteotoxic stress. Furthermore, yeast cells became hypersensitive against proteotoxic stress upon deletion of MBA1. Contrary to Mrx15, Mba1 cooperates with the regulators of the m-AAA and i-AAA proteases. Taken together, these results suggest that membrane protein insertion and mitochondrial AAA-proteases are functionally coupled, possibly reflecting an early quality control step during mitochondrial protein synthesis. Full article

Sustained endoplasmic reticular stress (ERS) is implicated in aggressive metastasis of cancer cells and increased tumor cell proliferation. Cancer cells activate the unfolded protein response (UPR), which aids in cellular survival and adaptation to harsh conditions. Inhibition of apoptosis, in contrast, is a mechanism adopted by cancer cells with the help of the inhibitor of an apoptosis (IAP) class of proteins such as Survivin to evade cell death and gain a proliferative advantage. In this study, we aimed to reveal the interrelation between ERS and Survivin. We initially verified the expression of Survivin in Winnie (a mouse model of chronic ERS) colon tissues by using immunohistochemistry (IHC) and immunofluorescence (IF) in comparison with wild type Blk6 mice. Additionally, we isolated the goblet cells and determined the expression of Survivin by IF and protein validation. Tunicamycin was utilized at a concentration of 10 µg/mL to induce ERS in the LS174T cell line and the gene expression of the ERS markers was measured. This was followed by determination of inflammatory cytokines. Inhibition of ERS was carried out by 4Phenyl Butyric acid (4PBA) at a concentration of 10 mM to assess whether there was a reciprocation effect. The downstream cell death assays including caspase 3/7, Annexin V, and poly(ADP-ribose) polymerase (PARP) cleavage were evaluated in the presence of ERS and absence of ERS, which was followed by a proliferative assay (EdU click) with and without ERS. Correspondingly, we inhibited Survivin by YM155 at a concentration of 100 nM and observed the succeeding ERS markers and inflammatory markers. We also verified the caspase 3/7 assay. Our results demonstrate that ERS inhibition not only significantly reduced the UPR genes (Grp78, ATF6, PERKandXBP1) along with Survivin but also downregulated the inflammatory markers such as IL8, IL4, and IL6, which suggests a positive correlation between ERS and the inhibition of apoptosis. Furthermore, we provided evidence that ERS inhibition promoted apoptosis in LS174T cells and shortened the proliferation rate. Moreover, Survivin inhibition by YM155 led to a comparable effect as that of ERS inhibition, which includes attenuation of ERS genes and inflammatory markers as well as the promotion of programmed cell death via the caspase 3/7 pathway. Together, our results propose the interrelation between ERS and inhibition of apoptosis assigning a molecular and therapeutic target for cancer treatment. Full article

LMNA linked-Emery-Dreifuss muscular dystrophy (EDMD2) is a rare disease characterized by muscle weakness, muscle wasting, and cardiomyopathy with conduction defects. The mutated protein lamin A/C binds several nuclear envelope components including the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex and the inner nuclear membrane protein Samp1 (Spindle Associated Membrane Protein 1). Considering that Samp1 is upregulated during muscle cell differentiation and it is involved in nuclear movement, we hypothesized that it could be part of the protein platform formed by LINC proteins and prelamin A at the myotube nuclear envelope and, as previously demonstrated for those proteins, could be affected in EDMD2. Our results show that Samp1 is uniformly distributed at the nuclear periphery of normal human myotubes and committed myoblasts, but its anchorage at the nuclear poles is related to the presence of farnesylated prelamin A and it is disrupted by the loss of prelamin A farnesylation. Moreover, Samp1 is absent from the nuclear poles in EDMD2 myotubes, which shows that LMNA mutations associated with muscular dystrophy, due to reduced prelamin A levels in muscle cell nuclei, impair Samp1 anchorage. Conversely, SUN1 pathogenetic mutations do not alter Samp1 localization in myotubes, which suggests that Samp1 lies upstream of SUN1 in nuclear envelope protein complexes. The hypothesis that Samp1 is part of the protein platform that regulates microtubule nucleation from the myotube nuclear envelope in concert with pericentrin and LINC components warrants future investigation. As a whole, our data identify Samp1 as a new contributor to EDMD2 pathogenesis and our data are relevant to the understanding of nuclear clustering occurring in laminopathic muscle. Full article

Telomeres (TLs) protect chromosome ends from chromosomal fusion and degradation, thus conferring genomic stability, and play crucial roles in cellular aging and disease. Recent studies have found a correlation between environmental, physiological and even mental stresses on TL dynamics in humans. However, the causal relationship between stress and TL length and the molecular mechanisms underlying that relationship are far from being understood. This study describes the effect of moderate concentrations of ethanol, equivalent to social drinking, on human TL dynamics and partially elucidates the mechanism mediating this effect. The exposure of Immortalized human foreskin fibroblast, primary human foreskin fibroblast and human hepatocellular carcinoma cells to 25 mM ethanol for one week moderately shortened telomeres in all cells. Similar TL shortening was obtained following cells’ exposure to 25 µM acetaldehyde (AcH) and to a much lower extent after exposure to 4-methylpyrazolean, an inhibitor of alcoholdehydrogenase, suggesting that AcH plays a key role in ethanol-dependent telomere shortening. Telomerase activity was not involved in this effect. TRF2 and several TRF2 binding proteins increased their binding to TLs after ethanol treatment, implying their involvement in this effect. The methylation status of several sub-telomeric regions increased in response to EtOH exposure. Gene expression profiling showed distinct patterns in cells treated with EtOH and in cells recovered from EtOH. In addition to cellular ageing, the described telomere shortening may contribute to the carcinogenic potential of acute alcohol consumption; both are associated with the shortening of TLs and provide new insights regarding the moderate consumption of alcohol referred to as “social drinking.” Full article

Most cytotoxic agents have limited efficacy for solid cancers. Cell-cycle phase analysis at the single-cell level in solid tumors has shown that the majority of cancer cells in tumors is not cycling and is therefore resistant to cytotoxic chemotherapy. Intravital cell-cycle imaging within tumors demonstrated the cell-cycle position and distribution of cancer cells within a tumor, and cell-cycle dynamics during chemotherapy. Understanding cell-cycle dynamics within tumors should provide important insights into novel treatment strategies. Full article

Extracellular vesicles (EVs) are membrane enclosed micro- and nano-sized vesicles that are secreted from almost every species, ranging from prokaryotes to eukaryotes, and from almost every cell type studied so far. EVs contain repertoire of bioactive molecules such as proteins (including enzymes and transcriptional factors), lipids, carbohydrates and nucleic acids including DNA, coding and non-coding RNAs. The secreted EVs are taken up by neighboring cells where they release their content in recipient cells, or can sail through body fluids to reach distant organs. Since EVs transport bioactive cargo between cells, they have emerged as novel mediators of extra- and intercellular activities in local microenvironment and inter-organ communications distantly. Herein, we review the activities of EV-associated matrix-remodeling enzymes such as matrix metalloproteinases, heparanases, hyaluronidases, aggrecanases, and their regulators such as extracellular matrix metalloproteinase inducers and tissue inhibitors of metalloproteinases as novel means of matrix remodeling in physiological and pathological conditions. We discuss how such EVs act as novel mediators of extracellular matrix degradation to prepare a permissive environment for various pathological conditions such as cancer, cardiovascular diseases, arthritis and metabolic diseases. Additionally, the roles of EV-mediated matrix remodeling in tissue repair and their potential applications as organ therapies have been reviewed. Collectively, this knowledge could benefit the development of new approaches for tissue engineering. Full article

Receptor tyrosine kinases (RTKs) regulate cellular processes by converting signals from the extracellular environment to the cytoplasm and nucleus. Tyro3, Axl, and Mer (TAM) receptors form an RTK family that plays an intricate role in tissue maintenance, phagocytosis, and inflammation as well as cell proliferation, survival, migration, and development. Defects in TAM signaling are associated with numerous autoimmune diseases and different types of cancers. Here, we review the structure of TAM receptors, their ligands, and their biological functions. We discuss the role of TAM receptors and soluble circulating TAM receptors in the autoimmune diseases systemic lupus erythematosus (SLE) and multiple sclerosis (MS). Lastly, we discuss the effect of TAM receptor deregulation in cancer and explore the therapeutic potential of TAM receptors in the treatment of diseases. Full article

Hepatitis C virus (HCV) represents a challenging global health threat to ~200 million infected individuals. Clinical data suggest that only ~10–15% of acutely HCV-infected individuals will achieve spontaneous viral clearance despite exuberant virus-specific immune responses, which is largely attributed to difficulties in recognizing the pathognomonic symptoms during the initial stages of exposure to the virus. Given the paucity of a suitable small animal model, it is also equally challenging to study the early phases of viral establishment. Further, the host factors contributing to HCV chronicity in a vast majority of acutely HCV-infected individuals largely remain unexplored. The last few years have witnessed a surge in studies showing that HCV adopts myriad mechanisms to disconcert virus-specific immune responses in the host to establish persistence, which includes, but is not limited to viral escape mutations, viral growth at privileged sites, and antagonism. Here we discuss a few hitherto poorly explained mechanisms employed by HCV that are believed to lead to chronicity in infected individuals. A better understanding of these mechanisms would aid the design of improved therapeutic targets against viral establishment in susceptible individuals. Full article

Overexpression of human epidermal growth factor receptor type 3 (HER3) is associated with tumour cell resistance to HER-targeted therapies. Monoclonal antibodies (mAbs) targeting HER3 are currently being investigated for treatment of various types of cancers. Cumulative evidence suggests that affibody molecules may be appropriate alternatives to mAbs. We previously reported a fusion construct (3A3) containing two HER3-targeting affibody molecules flanking an engineered albumin-binding domain (ABD₀₃₅) included for the extension of half-life in circulation. The 3A3 fusion protein (19.7 kDa) was shown to delay tumour growth in mice bearing HER3-expressing xenografts and was equipotent to the mAb seribantumab. Here, we have designed and explored a series of novel formats of anti-HER3 affibody molecules fused to the ABD in different orientations. All constructs inhibited heregulin-induced phosphorylation in HER3-expressing BxPC-3 and DU-145 cell lines. Biodistribution studies demonstrated extended the half-life of all ABD-fused constructs, although at different levels. The capacity of our ABD-fused proteins to accumulate in HER3-expressing tumours was demonstrated in nude mice bearing BxPC-3 xenografts. Formats where the ABD was located on the C-terminus of affibody binding domains (3A, 33A, and 3A3) provided the best tumour targeting properties in vivo. Further development of these promising candidates for treatment of HER3-overexpressing tumours is therefore justified. Full article

Mitochondria are dynamic, semi-autonomous organelles that execute numerous life-sustaining tasks in eukaryotic cells. Functioning of mitochondria depends on the adequate action of versatile proteinaceous machineries. Fine-tuning of mitochondrial activity in response to cellular needs involves continuous remodeling of organellar proteome. This process not only includes modulation of various biogenetic pathways, but also the removal of superfluous proteins by adenosine triphosphate (ATP)-driven proteolytic machineries. Accordingly, all mitochondrial sub-compartments are under persistent surveillance of ATP-dependent proteases. Particularly important are highly conserved two inner mitochondrial membrane-bound metalloproteases known as m-AAA and i-AAA (ATPases associated with diverse cellular activities), whose mis-functioning may lead to impaired organellar function and consequently to development of severe diseases. Herein, we discuss the current knowledge of yeast, mammalian, and plant AAA proteases and their implications in mitochondrial function and homeostasis maintenance. Full article

Multiple tissues and systems in the organism undergo modifications during aging due to an accumulation of damaged proteins, lipids, and genetic material. To counteract this process, the cells are equipped with specific mechanisms, such as autophagy and senescence. Particularly, the immune system undergoes a process called immunosenescence, giving rise to a chronic inflammatory status of the organism, with a decreased ability to counteract antigens. The obvious result of this process is a reduced defence capacity. Currently, there is evidence that some pathogens are able to accelerate the immunosenescence process for their own benefit. Although to date numerous reports show the autophagy–senescence relationship, or the connection between pathogens with autophagy or senescence, the link between the three actors remains unexplored. In this review, we have summarized current knowledge about important issues related to aging, senescence, and autophagy. Full article

Rheumatoid arthritis (RA) is a chronic, autoimmune, systemic, inflammatory disorder that affects synovial joints, both small and large joints, in a symmetric pattern. This disorder usually does not directly cause death but significantly reduces the quality of life and life expectancy of patients if left untreated. There is no cure for RA but, patients are usually on long-term disease modifying anti-rheumatic drugs (DMARDs) to suppress the joint inflammation, to minimize joint damage, to preserve joint function, and to keep the disease in remission. RA is strongly associated with various immune cells and each of the cell type contributes differently to the disease pathogenesis. Several types of immunomodulatory molecules mainly cytokines secreted from immune cells mediate pathogenesis of RA, hence complicating the disease treatment and management. There are various treatments for RA depending on the severity of the disease and more importantly, the patient’s response towards the given drugs. Early diagnosis of RA and treatment with (DMARDs) are known to significantly improve the treatment outcome of patients. Sensitive biomarkers are crucial in early detection of disease as well as to monitor the disease activity and progress. This review aims to discuss the pathogenic role of various immune cells and immunological molecules in RA. This review also highlights the importance of understanding the immune cells in treating RA and in exploring novel biomarkers. Full article

MYC-translocated T-lineage acute lymphoblastic leukemia (T-ALL) is a rare subgroup of T-ALL associated with CDKN2A/B deletions, PTEN inactivation, and absence of NOTCH1 or FBXW7 mutations. This subtype of T-ALL has been associated with induction failure and aggressive disease. Identification of drug targets and mechanistic insights for this disease are still limited. Here, we established a human NOTCH1-independent MYC-translocated T-ALL cell line that maintains the genetic and phenotypic characteristics of the parental leukemic clone at diagnosis. The University of Padua T-cell acute lymphoblastic leukemia 13 (UP-ALL13) cell line has all the main features of the above described MYC-translocated T-ALL. Interestingly, UP-ALL13 was found to harbor a heterozygous R882H DNMT3A mutation typically found in myeloid leukemia. Chromatin immunoprecipitation coupled with high-throughput sequencing for histone H3 lysine 27 (H3K27) acetylation revealed numerous putative super-enhancers near key transcription factors, including MYC, MYB, and LEF1. Marked cytotoxicity was found following bromodomain-containing protein 4 (BRD4) inhibition with AZD5153, suggesting a strict dependency of this particular subtype of T-ALL on the activity of super-enhancers. Altogether, this cell line may be a useful model system for dissecting the signaling pathways implicated in NOTCH1-independent T-ALL and for the screening of targeted anti-leukemia agents specific for this T-ALL subgroup. Full article

Inflammatory granulocytes are characterized by an oxidative burst, which may promote oxidative stress and lipid modification both in affected tissues and on a systemic level. On the other hand, redox signaling involving lipid peroxidation products acting as second messengers of free radicals play important yet not fully understood roles in the pathophysiology of inflammation and various stress-associated disorders. Therefore, the aim of this study was to evaluate the onset of oxidative stress and alterations of enzyme-dependent lipid metabolism resulting from redox imbalance in granulocytes and plasma obtained from patients with psoriasis vulgaris or psoriatic arthritis in comparison to the healthy subjects. The results obtained revealed enhanced activity of pro-oxidant enzymes nicotinamide adenine dinucleotide phosphate (NADPH) and xanthine oxidases in granulocytes with a decrease of enzymatic and non-enzymatic antioxidants in the plasma of psoriatic patients. The nuclear factor erythroid 2–related factor 2 (Nrf2) and its regulators were increased in both forms of psoriasis while heme oxygenase 1 levels were increased only in psoriasis vulgaris. The redox imbalance was associated with decreased levels of phospholipids and of free polyunsaturated fatty acids but with enhanced activity of enzymes involved in lipid metabolism (phospholipase A2, acetylhydrolase PAF, cyclooxygenases 1 and 2) and increased lipid peroxidation products 4-hydroxynonenal, isoprostanes, and neuroprostanes. Increased endocannabinoids and G protein-coupled receptor 55 were observed in both forms of the disease while expression of the cannabinoid type 1 receptor (CB1) was increased only in patients with psoriatic arthritis, which is opposite to the cannabinoid type 2 receptor. This receptor was increased only in psoriasis vulgaris. Changes in protein expression promoted the apoptosis of granulocytes by increased caspases mainly in psoriasis vulgaris. This study indicates that inhibition of the Nrf2 pathway in psoriatic arthritis promotes a redox imbalance. In addition, increased expression of CB1 receptors leads to increased oxidative stress, lipid modifications, and inflammation, which, in turn, may promote the progression of psoriasis into the advanced, arthritic form of the disease. Full article

The extracellular matrix (ECM) in the tumor microenvironment modulates the cancer cell phenotype, especially in pancreatic ductal adenocarcinoma (PDAC), a tumor characterized by an intense desmoplastic reaction. Because the epithelial-to-mesenchymal transition (EMT), a process that provides cancer cells with a metastatic phenotype, plays an important role in PDAC progression, the authors aimed to explore in vitro the interactions between human PDAC cells and ECM components of the PDAC microenvironment, focusing on the expression of EMT markers and matrix metalloproteinases (MMPs) that are able to digest the basement membrane during tumor invasion. EMT markers and the invasive potential of HPAF-II, HPAC, and PL45 cells grown on different ECM substrates (fibronectin, laminin, and collagen) were analyzed. While N-cadherin, αSMA, and type I collagen were not significantly affected by ECM components, the E-cadherin/β-catenin complex was highly expressed in all the experimental conditions, and E-cadherin was upregulated by collagen in PL45 cells. Cell migration was unaffected by fibronectin and delayed by laminin. In contrast, collagen significantly stimulated cell migration and the secretion of MMPs. This study’s results showed that ECM components impacted cell migration and invasive potential differently. Collagen exerted a more evident effect, providing new insights into the understanding of the intricate interplay between ECM molecules and cancer cells, in order to find novel therapeutic targets for PDAC treatment. Full article

Nucleoli are emerging as key sensors of cellular stress and regulators of the downstream consequences on proliferation, metabolism, senescence, and apoptosis. NF-κB signalling is activated in response to a similar plethora of stresses, which leads to modulation of cell growth and death programs. While nucleolar and NF-κB pathways are distinct, it is increasingly apparent that they converge at multiple levels. Exposure of cells to certain insults causes a specific type of nucleolar stress that is characterised by degradation of the PolI complex component, TIF-IA, and increased nucleolar size. Recent studies have shown that this atypical nucleolar stress lies upstream of cytosolic IκB degradation and NF-κB nuclear translocation. Under these stress conditions, the RelA component of NF-κB accumulates within functionally altered nucleoli to trigger a nucleophosmin dependent, apoptotic pathway. In this review, we will discuss these points of crosstalk and their relevance to anti-tumour mechanism of aspirin and small molecule CDK4 inhibitors. We will also briefly the discuss how crosstalk between nucleoli and NF-κB signalling may be more broadly relevant to the regulation of cellular homeostasis and how it may be exploited for therapeutic purpose. Full article

Hydrogen peroxide (H₂O₂) is an important metabolite involved in most of the redox metabolism reactions and processes of the cells. H₂O₂ is recognized as one of the main molecules in the sensing, modulation and signaling of redox metabolism, and it is acting as a second messenger together with hydrogen sulfide (H₂S) and nitric oxide (NO). These second messengers activate in turn a cascade of downstream proteins via specific oxidations leading to a metabolic response of the cell. This metabolic response can determine proliferation, survival or death of the cell depending on which downstream pathways (homeostatic, pathological, or protective) have been activated. The cells have several sources of H₂O₂ and cellular systems strictly control its concentration in different subcellular compartments. This review summarizes research on the role played by H₂O₂ in signaling pathways of eukaryotic cells and how this signaling leads to homeostatic or pathological responses. Full article

Patient derived anti-CD19 chimeric antigen receptor-T (CAR-T) cells are a powerful tool in achieving a complete remission in a range of B-cell malignancies, most notably B-acute lymphoblastic leukaemia (B-ALL) and diffuse large B-cell lymphoma (DLBCL). However, there are limitations, including inability to manufacture CAR-T cells from the patient’s own T cells, disease progression and death prior to return of engineered cells. T cell dysfunction is known to occur in cancer patients, and several groups have recently described differences in CAR-T cells generated from chronic lymphocytic leukaemia (CLL) patients compared with those from a healthy donor. This is thought to contribute to the low response rate in this disease group. Healthy donor, gene-edited CAR-T cells which do not require human leucocyte antigen (HLA) matching have the potential to provide an ‘off the shelf’ product, overcoming the manufacturing difficulties of producing CAR-T cells for each individual patient. They may also provide a more functional, potent product for malignancies such as CLL, where T cell dysfunction is common and frequently cannot be fully reversed during the manufacturing process. Here we review the potential benefits and obstacles for healthy donor, allogeneic CAR-T cells. Full article