Lower Cell Proliferation Rate Research Articles

The Deubiquitinase CYLD Acts As an Oncogene in a Cellular Model of Chronic Lymphocytic Leukemia

The cylindromatosis protein (CYLD) is a functional deubiquitinase that regulates critical signaling pathways, e.g. NF-κB and Wnt, thus modulating several cellular functions. CYLD acts as a tumor suppressor gene in solid tumors, and is also involved in the pathogenesis of hematological malignancies, including B cell lymphomas, in as yet unclear ways. In chronic lymphocytic leukemia (CLL), preliminary evidence suggests that reduced expression of CYLD correlates with a worse clinical prognosis, which appears to be in line with its postulated role as a tumor suppressor. Here we sought to gain insight into the function of CYLD in CLL through genetic engineering, molecular characterization and bioenergetic profiling. To this end, we used CRISPR/Cas9 technology and a CYLD-targeting or an unrelated gRNA to generate stable CYLD-knockout ( CYLDko) and control ( CYLDwt) MEC1 cells. Phenotypic characterization of CYLDkoversus CYLDwt MEC1 cells by flow cytometry showed (i) significantly reduced viability, assessed by Annexin V (fold difference, FD=1.2, p<0.05); (ii) lower cell proliferation rate, assessed by Ki67 expression (FD=1.4, p<0.05); (iii) increased apoptosis, determined by measuring active caspase 3 expression levels (FD=6.1, p<0.01); and, (iv) reduced expression of CD86 (FD=4.3, p<0.001) and CD40 (FD=4.1, p=0.05). Western blotting analysis of CYLDko versus CYLDwtMEC1 cells revealed down-regulation of the NF-κB pathway evidenced by diminished expression of ΙΚΚβ (FD=2.4, p<0.01), phospho-ΙκΒα (FD=3.4, p<0.01) and phospho-p105 (FD=2.5, p=0.2); and, the Wnt pathway evidenced by reduced β-catenin levels (FD=2, p<0.05). Transcriptome profiling by RNA-seq gave concordant results, in documenting increased apoptosis and decreased NF-κΒ signaling in CYLDkoversus CYLDwtMEC1 cells. The former also showed downregulation of calcium, BcR and PI3K/AKT/mTOR signaling pathways and, in contrast, upregulation of the glutathione and KEAP1-NFE2L2 pathways, which contribute to antioxidant defense and nutrient metabolism, as well as, regulation of redox balance and cellular metabolism, respectively. To evaluate the impact of CYLD deletion on CLL bioenergetics, we assessed ATP production rate, as a marker of active cellular metabolism, using the Seahorse XF Real-Time ATP Rate assay. CYLDkoMEC1 cells exhibited impaired ATP production reflected in decreased OCR (oxygen consumption rate) and ECAR (extracellular acidification rate), that are proportional to OXPHOS (oxidative phosphorylation) and glycolysis, respectively. Regarding the metabolic phenotype, CYLDwtMEC1 cells displayed a shift towards mitochondrial OXPHOS, whereas, in CYLDkoMEC1 cells ATP production was mostly based on glycolysis. High-performance liquid chromatography was performed using culture supernatants obtained at sequential time points to quantify glucose uptake and lactate secretion rates. The analysis revealed that CYLDwt MEC1 cells primarily directed glucose carbon toward biomass biosynthetic pathways, as reflected in both higher cell number and proliferation rate achieved, while CYLD deletion redirected carbon flux enhancing lactate formation. In both cases, once glucose was consumed (day 4), the secreted lactate was re-used, yet this was more pronounced in CYLDko MEC1 cells. Finally, we explored whether CYLD knockout might impact MEC1 sensitivity to targeted agents i.e. the BTK inhibitor ibrutinib and the BCL2 inhibitor venetoclax. We found that CYLDkoMEC1 cells presented increased apoptosis compared to their CYLDwtcounterparts when cultured in the presence of either drug. Moreover, treatment with ibrutinib or venetoclax led to reduced ATP production rates in both CYLDkoand CYLDwtMEC1 cells, albeit the reduction was more pronounced in the former (FD=4.5 for ibrutinib; FD=10 for venetoclax compared to the respective CYLDwt MEC1 treated cells). Taken together, we demonstrate for the first time that CYLD can also act as an oncogene, at least in the context of CLL and in particular in the MEC1 cell line model of CLL, since its elimination leads to (i) lower proliferation and increased apoptosis rates coupled with diminished signaling capacity; (ii) metabolic rewiring toward enhanced lactate formation; and, (iii) augmented sensitivity to CLL therapeutic agents. It remains to be elucidated under which condition this could also occur in the patients with CLL or other B lymphoproliferative disorders.

Study on biological activity and p38 mitogen-activated protein kinases of magnetic nanoparticles on hepatoma cells

This study assessed effects of magnetic nanoparticles (MNPs) on biological activity of hepatoma cells. HepG2 cells were cultured with MNPs, then morphological changes, cell proliferation, number of cell clones, apoptosis, and expressions of p38MAPK and p53 were analyzed. Hepatoma cells in the group grew fusiform and were connected with each other, while cell density and growth connection in the group B decreased, while cell number in group C decreased with changed cell morphology changed, while the number of cells in group D was least. The number of round suspension cells, volume, and nucleus increased, and apoptotic bodies gradually appeared. Compared with group A, groups B, C and D had lower cell proliferation rate, clone number and higher apoptosis rate (P < 0.05). The expressions of p38 MAPK and p53 in groups B, C and D was decreased (P < 0.05). The expression of p38MAPK and p53 in other groups was different (P < 0.05). MNPs changed cell morphology, reduced proliferation and clonal ability of HepG2 cells, and accelerated apoptosis, which is related to activation of p38MAPK and p53 expression.

Exploring a new family of designer copper(II) complexes of anthracene-appended polyfunctional organic assembly displaying potential anticancer activity via cytochrome c mediated mitochondrial apoptotic pathway

The present article describes the systematic study on design and synthesis, physicochemical properties and spectroscopic features, and potential anticancer activities of a family of novel copper(II)-based designer metal complexes [Cu2(acdp)(μ-Cl)(H2O)2] (1), [Cu2(acdp)(μ-NO3)(H2O)2] (2) and [Cu2(acdp)(μ-O2CCF3)(H2O)2] (3) of anthracene-appended polyfunctional organic assembly, H3acdp (H3acdp = N,N′-bis[anthracene-2-ylmethyl]-N,N′-bis[carboxymethyl]-1,3-diaminopropan-2-ol). Synthesis of 1–3 was accomplished under facile experimental conditions, preserving their overall integrity in solution. The incorporation of polycyclic anthracene skeleton within the backbone of organic assembly increases lipophilicity of resulting complexes, thereby dictating the degree of cellular uptake with improved biological activity. Complexes 1–3 were characterized by elemental analysis, molar conductance, FTIR, UV–Vis absorption/fluorescence emission titration spectroscopy, PXRD and TGA/DTA studies, including DFT calculations. The cellular cytotoxicity of 1–3 when studied in HepG2 cancer cell line showed substantial cytotoxic effects, whereas no such cytotoxicity was observed when exposed to normal L6 skeletal muscle cell line. Thereafter, the signaling factors involved in the process of cytotoxicity in HepG2 cancer cells were investigated. Alteration of cytochrome c and Bcl-2 protein expression levels along with modulation of mitochondrial membrane potential (MMP) in the presence of 1–3, strongly suggested the possibility of activating mitochondria-mediated apoptotic pathway involved in halting the cancer cell propagation. However, when a comparative assessment on their bio-efficacies was made, 1 showed higher cytotoxicity, nuclear condensation, DNA binding and damage, ROS generation and lower rate of cell proliferation compared to 2 and 3 in HepG2 cell line, indicating that the anticancer activity of 1 is significantly higher than that of 2 and 3.

Bone Marrow Mesenchymal Stem Cells (BMSC) Exosome-Derived mR-512-5p Inhibits the Proliferation and Apoptosis of Glioblastoma Cells

This study discusses the mechanism of miR-512-5p derived from BMSC in restraining the proliferation and prompting apoptosis of GBM. BMSC exosome was obtained through ultra-centrifugation and assessed by TEM. The positive presentation of CD63 and HSP70 was detected with Western Blot. The GBM cell line LN229 was divided into WM set, NC set, and ZR set followed by analysis of cell proliferation by MTT method, invasive ability by Tranwell chamber, apoptotic rate by FCM and the expression of JAG1 and notch2 by Western Blot. miR-512-5p level in LN229 cells was significantly lower than U87MG and SHG44 cells. There was positive expression of CD63 and HSP70 in exosome. LN229 cell proliferation was restrained by the drug. ZR set had lower cell proliferation rate and invasive quantity and higher apoptotic rate than WM set and NC set. The protein expressions of JAG1 and notch2 in ZR set was reduced compared with WM set and NC set (P <0.05) without difference between NC set and WM set (P >0.05). In conclusion, GBM cell proliferation could be restrained and apoptosis could be increased by miR-512-5p derived from BMSC through targeting JAG1. It could provide a brand-new therapeutic strategy for the treatment on GBM.

Hyperosmolar expansion medium improves nucleus pulposus cell phenotype.

BackgroundRepopulating the degenerated intervertebral disc (IVD) with tissue‐specific nucleus pulposus cells (NPCs) has already been shown to promote regeneration in various species. Yet the applicability of NPCs as cell‐based therapy has been hampered by the low cell numbers that can be extracted from donor IVDs and their potentially limited regenerative capacity due to their degenerated phenotype. To optimize the expansion conditions, we investigated the effects of increasing culture medium osmolarity during expansion on the phenotype of dog NPCs and their ability to produce a healthy extracellular matrix (ECM) in a 3D culture model.MethodsDog NPCs were expanded in expansion medium with a standard osmolarity of 300 mOsm/L or adjusted to 400 or 500 mOsm/L in both normoxic and hypoxic conditions. Following expansion, NPCs were cultured in a 3D culture model in chondrogenic culture medium with a standard osmolarity. Read‐out parameters included cell proliferaton rate, morphology, phenotype and healthy ECM production.ResultsIncreasing the expansion medium osmolarity from 300 to 500 mOsm/L resulted in NPCs with a more rounded morphology and a lower cell proliferation rate accompanied by the expression of several healthy NPC and progenitor markers at gene (KRT18, ACAN, COL2, CD73, CD90) and protein (ACAN, PAX1, CD24, TEK, CD73) level. The NPCs expanded at 500 mOsm/L were able to retain most of their phenotypic markers and produce healthy ECM during 3D culture independent of the oxygen level used during expansion.ConclusionsAltogether, our findings show that increasing medium osmolarity during expansion results in an NPC population with improved phenotype, which could enhance the potential of cell‐based therapies for IVD regeneration.

Proliferation of osteoblast precursor cells on the surface of TiO2 nanowires anodically grown on a β-type biomedical titanium alloy

Studies have shown that anodically grown TiO2 nanotubes (TNTs) exhibit excellent biocompatibility. However, TiO2 nanowires (TNWs) have received less attention. The objective of this study was to investigate the proliferation of osteoblast precursor cells on the surfaces of TNWs grown by electrochemical anodization of a Ti-35Nb-7Zr-5Ta (TNZT) alloy. TNT and flat TNZT surfaces were used as control samples. MC3T3-E1 cells were cultured on the surfaces of the samples for up to 5 days, and cell viability and proliferation were investigated using fluorescence microscopy, colorimetric assay, and scanning electron microscopy. The results showed lower cell proliferation rates on the TNW surface compared to control samples without significant differences in cell survival among experimental conditions. Contact angles measurements showed a good level of hydrophilicity for the TNWs, however, their relatively thin diameter and their high density may have affected cell proliferation. Although more research is necessary to understand all the parameters affecting biocompatibility, these TiO2 nanostructures may represent promising tools for the treatment of bone defects and regeneration of bone tissue, among other applications.

The effects of CD148 Q276P/R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling

BackgroundCD148 is a transmembrane protein tyrosine phosphatase that is expressed in multiple cell types. Previous studies have shown that CD148 dephosphorylates growth factor receptors and their signaling molecules, including EGFR and ERK1/2, and negatively regulates cancer cell growth. Furthermore, research of clinical patients has shown that highly linked CD148 gene polymorphisms, Gln276Pro (Q276P) and Arg326Gln (R326Q), are associated with an increased risk of several types of cancer. However, the biological effects of these missense mutations have not been studied.AimWe aimed to determine the biological effects of CD148 Q276P/R326Q mutations in cancer cell proliferation and growth factor signaling, with emphasis on EGFR signaling.MethodsCD148 forms, wild‐type (WT) or Q276P/R326Q, were retrovirally introduced into A431D epidermoid carcinoma cells that lacks CD148 expression. The stable cells that express comparable levels of CD148 were sorted by flow cytometry. A431D cells infected with empty retrovirus was used as a control. CD148 localization, cell proliferation rate, EGFR signaling, and the response to thrombospondin‐1 (TSP1), a CD148 ligand, were assessed by immunostaining, cell proliferation assay, enzyme‐linked immunosorbent assay, and Western blotting.ResultsBoth CD148 forms (WT, Q276P/R326Q) were distributed to cell surface and all three cell lines expressed same level of EGFR. Compared to control cells, the A431D cells that express CD148 forms showed significantly lower cell proliferation rates. EGF‐induced EGFR and ERK1/2 phosphorylation as well as cell proliferation were also significantly reduced in these cells. Furthermore, TSP1 inhibited cell proliferation in CD148 (WT, Q276P/R326Q)‐expressing A431D cells, while it showed no effects in control cells. However, significant differences were not observed between CD148 WT and Q276P/R326Q cells.ConclusionOur data demonstrates that Q276P/R326Q mutations do not have major effects on TSP1‐CD148 interaction as well as on CD148's cellular localization and activity to inhibit EGFR signaling and cell proliferation.

Muscone suppresses gastric cancer via regulation of miRNA-145.

This study aims to determine the effects and mechanism of action of muscone on the biological activity of the gastric cancer cell lines SGC‐7901 and MGC‐803 (proliferation, apoptosis, invasion, and migration) in vitro. An optimal muscone concentration was determined using MTT and cell apoptosis tests. The SGC‐7901 and MGC‐803 cells were divided into five groups: normal control, muscone, miRNA, muscone + miRNA, and muscone + miRNA inhibitor. Cell proliferation rate, apoptosis rate, cell cycle phase distribution, number of invading cells, and wound healing rate were compared among the five groups using MTT, flow cytometry, transwell, and wound healing assays. Relative expression levels of the proteins PI3K, AKT, P21, c‐Myc, MMP‐2, and MMP‐9 were measured by Western blot. Compared with the control group, the groups treated with muscone and miRNA showed significantly lower cell proliferation rate, number of invading cells, and wound healing rate (p < .05 for all), but significantly higher rates of cell apoptosis rate and numbers of cells in the G1 phase (p < .05 for all). These groups also showed significantly lower expression of the proteins PI3K, AKT, c‐Myc, MMP‐2, and MMP‐9 but significantly increased expression of the protein P21 (p < .05). Transfecting muscone‐treated SGC‐7901 and MGC‐803 cells with miRNA‐145 inhibitor resulted in a significant recovery of biological activity (p < .05). Muscone suppresses the biological activity of SGC‐7901 and MGC‐803 gastric cancer cells in vitro via regulation of miRNA‐145.

Uniaxial Cyclic Stretching Promotes Chromatin Accessibility of Gene Loci Associated With Mesenchymal Stem Cells Morphogenesis and Osteogenesis.

It has been previously demonstrated that uniaxial cyclic stretching (UCS) induces differentiation of mesenchymal stem cells (MSCs) into osteoblasts in vitro. It is also known that interactions between cells and external forces occur at various aspects including cell–matrix, cytoskeleton, nucleus membrane, and chromatin. However, changes in chromatin landscape during this process are still not clear. The present study was aimed to determine changes of chromatin accessibility under cyclic stretch. The influence of cyclic stretching on the morphology, proliferation, and differentiation of hMSCs was characterized. Changes of open chromatin sites were determined by assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq). Our results showed that UCS induced cell reorientation and actin stress fibers realignment, and in turn caused nuclear reorientation and deformation. Compared with unstrained group, the expression of osteogenic and chondrogenic marker genes were the highest in group of 1 Hz + 8% strain; this condition also led to lower cell proliferation rate. Furthermore, there were 2022 gene loci with upregulated chromatin accessibility in 1 Hz + 8% groups based on the analysis of chromatin accessibility. These genes are associated with regulation of cell morphogenesis, cell–substrate adhesion, and ossification. Signaling pathways involved in osteogenic differentiation were found in up-regulated GO biological processes. These findings demonstrated that UCS increased the openness of gene loci associated with regulation of cell morphogenesis and osteogenesis as well as the corresponding transcription activities. Moreover, the findings also connect the changes in chromatin accessibility with cell reorientation, nuclear reorientation, and deformation. Our study may provide reference for directed differentiation of stem cells induced by mechanical microenvironments.

Modulation of cell cycle progression and chromatin dynamic as tolerance mechanisms to salinity and drought stress in maize.

Salinity and drought are the major abiotic stresses that disturb several aspects of maize plants growth at the cellular level, one of these aspects is cell cycle machinery. In our study, we dissected the molecular alterations and downstream effectors of salinity and drought stress on cell cycle regulation and chromatin remodeling. Effects of salinity and drought stress were determined on maize seedlings using 200 mM NaCl (induced salinity stress), and 250 mM mannitol (induced drought stress) treatments, then cell cycle progression and chromatin remodeling dynamics were investigated. Seedlings displayed severe growth defects, including inhibition of root growth. Interestingly, stress treatments induced cell cycle arrest in S-phase with extensive depletion of cyclins B1 and A1. Further investigation of gene expression profiles of cell cycle regulators showed the downregulation of the CDKA, CDKB, CYCA, and CYCB. These results reveal the direct link between salinity and drought stress and cell cycle deregulation leading to a low cell proliferation rate. Moreover, abiotic stress alters chromatin remodeling dynamic in a way that directs the cell cycle arrest. We observed low DNA methylation patterns accompanied by dynamic histone modifications that favor chromatin decondensation. Also, the high expression of DNA topoisomerase 2, 6 family was detected as consequence of DNA damage. In conclusion, in response to salinity and drought stress, maize seedlings exhibit modulation of cell cycle progression, resulting in the cell cycle arrest through chromatin remodeling.

Systematic Review-The Potential Implications of Different Platelet-Rich Plasma (PRP) Concentrations in Regenerative Medicine for Tissue Repair.

The number of studies evaluating platelet-rich plasma (PRP) concentration has substantially grown in the last fifteen years. A systematic review on this field has been realized by evaluating in the identified studies the in vitro PRP concentration—also analyzing the platelet amount—and the in vivo PRP effects in tissue regeneration compared to any control. The protocol has been developed in agreement with the Preferred Reporting for Items for Systematic Reviews and Meta-Analyses-Protocols (PRISMA-P) guidelines. Multistep research of the PubMed, MEDLINE, Embase, PreMEDLINE, Ebase, CINAHL, PsycINFO, Clinicaltrials.gov, Scopus database and Cochrane databases has permitted to identify articles on different concentrations of PRP in vitro and related in vivo impact for tissue repair. Of the 965 articles initially identified, 30 articles focusing on PRP concentration have been selected and, consequently, only 15 articles have been analyzed. In total, 40% (n = 6) of the studies were related to the fixed PRP Concentration Group used a fixed PRP concentration and altered the platelet concentration by adding the different volumes of the PRP (lysate) to the culture. This technique led to a substantial decrease in nutrition available at higher concentrations. Sixty percent (n = 9) of the studies were related to the fixed PRP Volume Group that used a fixed PRP-to-media ratio (Vol/Vol) throughout the experiment and altered the concentration within the PRP volume. For both groups, when the volume of medium (nutrition) decreases, a lower rate of cell proliferation is observed. A PRP concentration of 1.0 × 106 plt/μL, appears to be optimal thanks to the constant and plentiful capillary nutrition supply and rapid diffusion of growth factors that happen in vivo and it also respects the blood decree-law. The PRP/media ratio should provide a sufficient nutrition supply to prevent cellular starvation, that is, PRP ≤ 10% (Vol/Vol) and thus best mimic the conditions in vivo.

Heterozygous mutation of the splicing factor Sf3b4 affects development of the axial skeleton and forebrain in mouse.

Splicing factor 3B subunit 4 (SF3B4) is a causative gene of an acrofacial dysostosis, Nager syndrome. Although in vitro analyses of SF3B4 have proposed multiple noncanonical functions unrelated to splicing, less information is available based on in vivo studies using model animals. We performed expression and functional analyses of Sf3b4 in mice. The mouse Sf3b4 transcripts were found from two-cell stage, and were ubiquitously present during embryogenesis with high expression levels in several tissues such as forming craniofacial bones and brain. In contrast, expression of a pseudogene-like sequence of mouse Sf3b4 (Sf3b4_ps) found by in silico survey was not detected up to embryonic day 10. We generated a Sf3b4 knockout mouse using CRISPR-Cas9 system. The homozygous mutant mouse of Sf3b4 was embryonic lethal. The heterozygous mutant of Sf3b4 mouse (Sf3b4+/- ) exhibited smaller body size compared to the wild-type from postnatal to adult period, as well as homeotic posteriorization of the vertebral morphology and flattened calvaria. The flattened calvaria appears to be attributable to mild microcephaly due to a lower cell proliferation rate in the forebrain. Our study suggests that Sf3b4 controls anterior-posterior patterning of the axial skeleton and guarantees cell proliferation for forebrain development in mice.

Fibroblast growth factor-2 signaling modulates matrix reorganization and cell cycle turnover rate in the regenerating tail of Hemidactylus flaviviridis

Lizards restore their lost tail by the recruitment of multipotent cells which are selectively differentiated into varied cell types so as to sculpt a new tail. The precise coordination of the events involved in this complex process requires crosstalk between many signaling molecules and differential regulation of several mediators that facilitate the achievements of various milestones of regeneration. Fibroblast growth factor-2 is one such signaling molecule which activates a number of intracellular signaling pathways. Herein, the regulatory role of FGF2 during tail regeneration in Hemidactylus flaviviridis was investigated. Upon inhibition of FGFR using SU5402, the FGF2 levels were found to be significantly reduced at both transcript and protein level. Further, the compromised levels of the gelatinases, namely MMP2 and MMP9 in the tail tissues of treated lizards indicate that FGF2 regulates the activity of these enzymes perhaps to facilitate the recruitment of multipotent mesenchymal cells (blastema). The in vivo 5BrdU incorporation assay showed a lower cell proliferation rate in FGF2 signal inhibited animals during all the proliferative stages of regeneration studied. This observation was substantiated by decreased levels of PCNA in treated group. Moreover, from the combined results of Caspase-3 localization and its expression levels in the regenerates of control and SU5402 treated lizards it can be deduced that FGF2 signal regulates apoptosis as well during early stages of regeneration. Overall, the current study indicates beyond doubt that FGF2 signaling plays a pivotal role in orchestrating the matrix reorganization and cell cycle turnover during lizard tail regeneration.

Negative Regulation of mTOR Signaling Restricts Cell Proliferation in the Floor Plate.

The neural tube is composed of a number of neural progenitors and postmitotic neurons distributed in a quantitatively and spatially precise manner. The floor plate, located in the ventral-most region of the neural tube, has a lot of unique characteristics, including a low cell proliferation rate. The mechanisms by which this region-specific proliferation rate is regulated remain elusive. Here we show that the activity of the mTOR signaling pathway, which regulates the proliferation of the neural progenitor cells, is significantly lower in the floor plate than in other domains of the embryonic neural tube. We identified the forkhead-type transcription factor FoxA2 as a negative regulator of mTOR signaling in the floor plate, and showed that FoxA2 transcriptionally induces the expression of the E3 ubiquitin ligase RNF152, which together with its substrate RagA, regulates cell proliferation via the mTOR pathway. Silencing of RNF152 led to the aberrant upregulation of the mTOR signal and aberrant cell division in the floor plate. Taken together, the present findings suggest that floor plate cell number is controlled by the negative regulation of mTOR signaling through the activity of FoxA2 and its downstream effector RNF152.

Polyphenols obtained from Didymosphenia geminata (Lyngbye) Schmith altered the viability and proliferation of salmonids cells lines SHK-1 and CHSE-214

Didymosphenia geminata (Lyngbye) Schmidt, also referred to as Didymo, is an invasive diatom that forms nuisance mats. Since it was first reported in our country in approximately 2010, Didymo has expanded and colonized different rivers in the Zona Austral region of Chile. Its biology and effects on ecosystems are still being studied because Didymo is an invasive algal mat that forms in a range of systems from oligotrophic austral rivers to more subtropical systems. We aimed to evaluate the viability of two salmonid cell lines, CHSE-214 and SHK-1 (somatic and embryonic cell lines, respectively), in dilutions of river water alone and in river water contaminated with Didymo or polyphenols extracted from Didymo under controlled conditions. We developed an artificial river system (2 aquariums/replicate) from five different rivers from the central area (Bio-Bio) and Patagonia area (Futaleufú) of Chile to maintain Didymo in the benthic phase. The Didymo populations were maintained for six months in the water from the rivers, after which samples were obtained. Following the extraction of polyphenols from the Didymo samples maintained in the artificial rivers, toxicity assays (10 assays) were performed to determine cell viability. Our results indicated that the CHSE-214 cells were highly sensitive to increasing concentrations of Didymo extracts. We observed a 50% reduction in cell viability after 24 h of exposure to a 0.01 V/V dilution, and this treatment further reduced the proliferative capacity by 70% after 120 h. The SHK-1 cells were less responsive, showing only a 20% decrease in viability at 24 h and a lower cell proliferation rate (45%) after 120 h, which remained higher than that of the CHSE-214 cells. We conclude that certain cell types are sensitive to Didymo in rivers, suggesting that there are chronic effects on several aquatic species following exposure to these diatom substances. These effects should be further studied using this laboratory model to understand the full impact of Didymo on river ecosystems.

Telomeres in neurological disorders.

Ever since their discovery, the telomeres and the telomerase have been topics of intensive research, first as a mechanism of cellular aging and later as an indicator of health and diseases in humans. By protecting the chromosome ends, the telomeres play a vital role in preserving the information in our genome. Telomeres shorten with age and the rate of telomere erosion provides insight into the proliferation history of cells. The pace of telomere attrition is known to increase at the onset of several pathological conditions. Telomere shortening has been emerging as a potential contributor in the pathogenesis of several neurological disorders including autism spectrum disorders (ASD), schizophrenia, Alzheimer's disease (AD), Parkinson's disease (PD) and depression. The rate of telomere attrition in the brain is slower than that of other tissues owing to the low rate of cell proliferation in brain. Telomere maintenance is crucial for the functioning of stem cells in brain. Taking together the studies on telomere attrition in various neurological disorders, an association between telomere shortening and disease status has been demonstrated in schizophrenia, AD and depression, in spite of a few negative reports. But, studies in ASD and PD have failed to produce conclusive results. The cause-effect relationship between TL and neurological disorders is yet to be elucidated. The factors responsible for telomere erosion, which have also been implicated in the pathogenesis of neurological disorders, need to be explored in detail. Telomerase activation is now being considered as a potential therapeutic strategy for neurological disorders.

Leiomyoma of the sigmoid mesocolon associated to hemorrhagic infarct and high count of mast cells

Leiomyoma is a soft-tissue benign tumor and its occurrence in the mesocolon is extremely rare. We present a particular case of a leiomyoma of the sigmoid mesocolon of a 58-year-old man, associated to a hemorrhagic infarction treated surgically and without recurrences after 1 year of follow-up. The macroscopic, histopathologic, and immunohistochemical features are presented and discussed. The high mast cell count associated with a low rate of cell proliferation is a sign of the benign biological behavior in this entity and may be helpful hallmarks for the differential diagnosis with other gastroenterological neoplasia.

Osteosarcoma of the Mandible

Osteosarcoma is a malignant tumor that rarely affects the jaws and has a slight predilection for men. This study presents a case of osteosarcoma in a woman, involving the mandible body and causing tooth resorption. A 36-year-old woman presented with a swelling in the alveolar ridge in the buccal surface of tooth #36. This was initially asymptomatic but later presented paresthesia of the inferior alveolar nerve. Radiographic examinations showed a mixed lesion with poorly defined limits, and root reabsorption of tooth #36. Provisional diagnoses were ossifying fibroma and osteosarcoma. Biopsy was performed, and microscopic analysis detected malignant neoplastic cells exhibiting pleomorphism and hyperchromatism permeating to an extracellular matrix with irregular and atypical calcification foci. Malignant neoplastic cells were negative for AE1/AE3, CD99, desmin, and EMA. Some chondrocyte-like cells were positive for S-100; Ki-67 analysis indicated a low rate of cell proliferation. The lesion was diagnosed as osteosarcoma. The patient was sent for treatment to the oncology center. Although uncommonly found in the jaws, osteosarcoma should be considered in the differential diagnosis of aggressive and rapidly evolving lesions.

Acquired resistance to BRAFi reverses senescence-like phenotype in mutant BRAF melanoma.

Targeting MAPK pathway in mutant BRAF melanoma with the specific BRAF inhibitor vemurafenib showed robust initial responses in the majority of patients followed by relapses due to acquired resistance to the drug. In V600EBRAF melanoma cell lines, senescence-associated β-galactosidase activity is often encountered in a constitutive manner or induced after MAPK inhibition. However, the link between the senescence-like phenotype and the resistance to BRAF inhibition is not fully understood yet. Our data validate a senescence-like phenotype (low cell proliferation, high cell volume, and high β-Gal activity) in mutant BRAF cells. Vemurafenib increased β-Gal activity in 4 out of 5 sensitive lines and in 2 out of 5 lines with intrinsic resistance to the drug. Interestingly, the 3 lines with acquired resistance to vemurafenib became depending on the drug for proliferation. In absence of drug, these lines showed a lower cell proliferation rate together with a substantial increase of β-Gal activity both in vitro and in vivo. In all settings, the senescence-like phenotype was significantly associated with an inhibition of pRB and cyclin D1, explaining the inhibition of cell proliferation. In conclusion, β-Gal activity is increased by V600EBRAF inhibition in the majority of sensitive and intrinsically resistant melanoma cells. Acquired resistance to vemurafenib is associated with a dependence to the drug for cell proliferation and tumor growth, and, in this case, drug removal stimulate β-Gal activity suggesting that the senescence-like phenotype could contribute to the acquired resistance to BRAF inhibition.

Arabidopsis NUCLEOSTEMIN-LIKE 1 (NSN1) regulates cell cycling potentially by cooperating with nucleosome assembly protein AtNAP1;1

BackgroundIn mammals, nucleostemin (NS), a nucleolar GTPase, is involved in stem cell proliferation, embryogenesis and ribosome biogenesis. Arabidopsis NUCLEOSTEMIN-LIKE 1 (NSN1) has previously been shown to be essential for plant growth and development. However, the role of NSN1 in cell proliferation is largely unknown.ResultsUsing nsn1, a loss-of-function mutant of Arabidopsis NSN1, we investigated the function of NSN1 in plant cell proliferation and cell cycle regulation. Morphologically, nsn1 exhibited developmental defects in both leaves and roots, producing severely reduced vegetative organs with a much smaller number of cells than those in the wild type. Dynamic analysis of leaf and root growth revealed a lower cell proliferation rate and slower cell division in nsn1. Consistently, the transcriptional levels of key cell cycle genes, including those regulating the transition of G1-S and G2-M, were reduced drastically in nsn1. The introduction of CYCLIN B1::GUS into nsn1 resulted in confined expression of GUS in both the leaf primordia and root meristem, indicating that cell proliferation was hampered by the mutation of NSN1. Upon subjection to treatment with bleomycin and methyl methanesulfonate (MMS), nsn1 plants exhibited hypersensitivity to the genotoxic agents. In the nucleus, NSN1 interacted with nucleosome assembly protein1 (AtNAP1;1), a highly conserved histone chaperone functioning in cell proliferation. Notably, the N-terminal conserved domains of Arabidopsis NSN1 were critical for the physical interaction.ConclusionsAs a conserved homolog of mammalian nucleostemin, Arabidopsis NSN1 plays pivotal roles in embryogenesis and ribosome biogenesis. In this study, NSN1 was found to function as a positive regulator in cell cycle progression. The interaction between NSN1 and histone chaperone AtNAP1;1, and the high resemblance in sensitivity to genotoxics between nsn1 and atnap1;1 imply the indispensability of the two nuclear proteins for cell cycle regulation. This work provides an insight into the delicate control of cell proliferation through the cooperation of a GTP-binding protein with a nucleosome assembly/disassembly protein in Arabidopsis.