Increase In Doubling Time Research Articles

Primary fibroblast co-culture stimulates growth and metabolism in Sdhb-impaired mouse pheochromocytoma MTT cells.

Pheochromocytomas and paragangliomas (PGLs) due to mutations of succinate dehydrogenase (SDH) B, a subunit of the SDH complex with a role in the Krebs cycle and the respiratory chain, tend to be larger at diagnosis and more prone to metastatic disease than other tumors. This presentation contrasts with the behavior of some cell line models of SDHB impairment, which show reduced growth compared to wild type. We hypothesize that reduced growth of SDHB-impaired monolayer culture models might reflect lack of support from sources within the tumor microenvironment. The present study therefore investigates how the microenvironment, modeled here by fibroblast co-culture, modulates cell metabolism, growth and invasion in an Sdhb-impaired mouse pheochromocytoma cell line. We employed two different constructs of short hairpin RNA to knockdown Sdhb and compared growth in a monolayer with and without fibroblast co-culture. Sdhb-silenced cells showed functional impairment of SDH with elevated succinate to fumarate ratio and decreased oxidative capacity. Cell growth was delayed with an increase in doubling time of 2h or 20h. Clonogenic cell survival and viability, on the other hand, were either unchanged or increased compared to control. In standard monolayer culture, no differences in pro-metastatic features were present. Co-culture with primary mouse fibroblast reversed the difference of proliferation between control and Sdhb knockdown but was unable to significantly influence invasiveness under these culture conditions. Metabolic studies identified that lactate secreted by fibroblasts was taken up preferentially by Sdhb-silenced cells. In summary, the present study identified a potential role for the tumor microenvironment in influencing phenotypic features of SDHB-mutated PGLs, providing a basis for the use of therapies targeted towards the tumor microenvironment.

Characterization of Human Fetal Cartilage Progenitor Cells During Long-Term Expansion in a Xeno-Free Medium.

Stem cell therapy requires a serum-free and/or chemically-defined medium for commercialization, but it is difficult to find one that supports long-term expansion of cells without compromising their stemness, particularly for novel stem cells. In this study, we tested the efficiency of StemPro® MSC SFM Xeno Free (SFM-XF), a serum-free medium, for the long-term expansion of human fetal cartilage-derived progenitor cells (hFCPCs) from three donors in comparison to that of the conventional α-Modified Eagle's Medium (α-MEM) supplemented with 10% fetal bovine serum (FBS). We found that SFM-XF supported the expansion of hFCPCs for up to 28-30 passages without significant changes in the doubling time, while α-MEM with 10% FBS showed a rapid increase in doubling time at 10-18 passages depending on the donor. Senescence of hFCPCs was not observed until passage 10 in both media but was induced in approximately 15 and 25% of cells at passage 20 in SFM-XF and α-MEM with 10% FBS, respectively. The colony forming ability of hFCPCs in SFX-XF was also comparable to that in α-MEM with 10% FBS. hFCPCs expressed pluripotency genes like Oct-4, Sox-2, Nanog, SCF, and SSEA4 at passage 20 and 31 in SFM-XF; however, this was not observed when cells were cultured in α-MEM with 10% FBS. The ability of hFCPCs to differentiate into three mesodermal lineages decreased gradually in both media but it was less significant in SFM-XF. Finally we found no chromosomal abnormality after long-term culture of hFCPCs until passage 17 by karyotype analysis. These results suggest that SFM-XF supports the long-term expansion of hFCPCs without significant phenotypic and chromosomal changes. This study have also shown that hFCPCs can be mass-produced in vitro, proving their commercial value as a novel source for developing cell therapies.

Lack of acquired resistance in HER2-positive breast cancer cells after long-term HER2 siRNA nanoparticle treatment.

Intrinsic and acquired resistance to current HER2 targeted therapies remains a challenge in clinics. We have developed a therapeutic HER2 siRNA delivered using mesoporous silica nanoparticles modified with polymers and conjugated with HER2 targeting antibodies. Our previous studies have shown that our HER2 siRNA nanoparticles could overcome intrinsic and acquired resistance to trastuzumab and lapatinib in HER2-positive breast cancers. In this study, we investigated the effect of long-term (7 months) treatment using our therapeutic HER2 siRNA. Even after the removal of HER2 siRNA, the long-term treated cells grew much slower (67% increase in doubling time) than cells that have not received any treatment. The treated cells did not undergo epithelial-mesenchymal transition or showed enrichment of tumor initiating cells. Unlike trastuzumab and lapatinib, which induced resistance in BT474 cells after 6 months of treatment, HER2 siRNA did not induce resistance to HER2 siRNA, trastuzumab, or lapatinib. HER2 ablation with HER2 siRNA prevented reactivation of HER2 signaling that was observed in cells resistant to lapatinib. Altogether, our results indicate that a HER2 siRNA based therapeutic provides a more durable inhibition of HER2 signaling in vitro and can potentially be more effective than the existing therapeutic monoclonal antibodies and small molecule inhibitors.

FK506‐binding protein 51 plays a role in pulmonary endothelial cell phenotype switching

It is well‐established that endothelial cells exhibit heterogeneity. Indeed, in the pulmonary vasculature, pulmonary artery endothelial cells (PAECs) and pulmonary microvascular endothelial cells (PMVECs) exhibit a many differences, including differences in proliferation rate and growth inhibition. PMVECs have a greater proliferative potential than PAECS and do not exhibit contact inhibition of growth in culture. Additionally, PMVECs express higher levels of FK506‐binding protein 51 (FKBP51) than PAECs, a trait that confers resistance to the store‐operated calcium current, Isoc. Here, we asked whether FKBP51 affects endothelial cell phenotype. Pulmonary arterial hypertension is marked by endothelial dysfunction and vascular remodeling, and it has been suggested that phenotype switching via endothelial‐to‐mesenchymal transition or resident stem cells may contribute to disease progression. We investigated the role of FKBP51 in phenotype switching using an FKPB51‐overexpressing PAEC cell line. FKBP51‐overexpressing PAECs showed a two‐fold increase in doubling time compared to wild type PAECs. PAECs grow in islands, exhibiting a cobblestone appearance and contact‐dependent growth inhibition. On the other hand, FKBP51‐overexpressing PAECs have a PMVEC‐like phenotype, wherein cells take on a spindle‐like morphology when sub‐confluent and are not growth inhibited at confluence. In addition, FKBP51‐overexpressing PAECs show increased smooth muscle actin, an indicator of endothelial cell dedifferentiation, as compared to wild‐type PAECs. Taken together, these data suggest that FKBP51 may play an important role in phenotype switching in PAECs.Support or Funding InformationAmerican Heart Association #17AIREA33680155This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evidence that DNA polymerase \u03b4 contributes to initiating leading strand DNA replication in Saccharomyces cerevisiae

To investigate nuclear DNA replication enzymology in vivo, we have studied Saccharomyces cerevisiae strains containing a pol2-16 mutation that inactivates the catalytic activities of DNA polymerase ε (Pol ε). Although pol2-16 mutants survive, they present very tiny spore colonies, increased doubling time, larger than normal cells, aberrant nuclei, and rapid acquisition of suppressor mutations. These phenotypes reveal a severe growth defect that is distinct from that of strains that lack only Pol ε proofreading (pol2-4), consistent with the idea that Pol ε is the major leading-strand polymerase used for unstressed DNA replication. Ribonucleotides are incorporated into the pol2-16 genome in patterns consistent with leading-strand replication by Pol δ when Pol ε is absent. More importantly, ribonucleotide distributions at replication origins suggest that in strains encoding all three replicases, Pol δ contributes to initiation of leading-strand replication. We describe two possible models.

Ion-crosslinked wood-derived nanocellulose hydrogels with tunable antibacterial properties: Candidate materials for advanced wound care applications

Development of advanced dressings with antimicrobial properties for the treatment of infected wounds is an important approach in the fight against evolution of antibiotic resistant bacterial strains. Herein, the effects of ion-crosslinked nanocellulose hydrogels on bacteria commonly found in infected wounds were investigated in vitro. By using divalent calcium or copper ions as crosslinking agents, different antibacterial properties against the bacterial strains Staphylococcus epidermidis and Pseudomonas aeruginosa were obtained. Calcium crosslinked hydrogels were found to retard S. epidermidis growth (up to 266% increase in lag time, 36% increase in doubling time) and inhibited P. aeruginosa biofilm formation, while copper crosslinked hydrogels prevented S. epidermidis growth and were bacteriostatic towards P. aeruginosa (49% increase in lag time, 78% increase in doubling time). The wound dressing candidates furthermore displayed barrier properties towards both S. epidermidis and P. aeruginosa, hence making them interesting for further development of advanced wound dressings with tunable antibacterial properties.

Mitofusin 1 and optic atrophy 1 shift metabolism to mitochondrial respiration during aging

SummaryReplicative and chronological lifespan are two different modes of cellular aging. Chronological lifespan is defined as the duration during which quiescent normal cells retain their capacity to re‐enter the proliferative cycle. This study investigated whether changes in metabolism occur during aging of quiescent normal human fibroblasts (NHFs) and the mechanisms that regulate these changes. Bioenergetics measurements were taken in quiescent NHFs from younger (newborn, 3‐day, 5‐month, and 1‐year) and older (58‐, 61‐, 63‐, 68‐, and 70‐year) healthy donors as well as NHFs from the same individual at different ages (29, 36, and 46 years). Results show significant changes in cellular metabolism during aging of quiescent NHFs: Old NHFs exhibit a significant decrease in glycolytic flux and lactate levels, and increase in oxygen consumption rate (OCR) and ATP levels compared to young NHFs. Results from the Seahorse XF Cell Mito Stress Test show that old NHFs with a lower Bioenergetic Health Index (BHI) are more prone to oxidative stress compared to young NHFs with a higher BHI. The increase in OCR in old NHFs is associated with a shift in mitochondrial dynamics more toward fusion. Genetic knockdown of mitofusin 1 (MFN1) and optic atrophy 1 (OPA1) in old NHFs decreased OCR and shifted metabolism more toward glycolysis. Downregulation of MFN1 and OPA1 also suppressed the radiation‐induced increase in doubling time of NHFs. In summary, results show that a metabolic shift from glycolysis in young to mitochondrial respiration in old NHFs occurs during chronological lifespan, and MFN1 and OPA1 regulate this process.

Abstract 1080: Targeting the PI3K-Akt and NF-κB pathways as a combination therapy in blocking prostate cancer progression

Abstract Akt/protein kinase B and transcription factor NF-κB are important signaling molecules and key survival factors involved in the control of cell proliferation, apoptosis and oncogenesis. Although Akt and NF-κB are components of separate signaling pathways, Akt can induce cell survival signal through NF-κB activation. Previous studies from our laboratory have shown constitutive activation of NF-κB/p65 and PI3K-Akt in clinical prostate cancer specimens and in autochthonous mouse model of prostate cancer. In this study we investigated if these two signaling pathways converge and whether dual targeting is effective in blocking disease progression. Using tissue samples obtained during transurethral prostatic resection and paraffin-embedded sections of benign and malignant prostate tissue, we first analyzed nuclear levels of NF-κB/p65, native/activated Akt levels, and evaluated co-localization of these two proteins. Compared to benign tissue, cancer specimens exhibited constitutive Akt and NF-κB/p65 activation which was more pronounced in high-grade cancer (Gleason grade 7-10). Immunohistochemical analyses further confirmed a progressive increase in the activated form of Akt and NF-κB/p65 in the nucleus of cancer tissues compared to benign specimens, and exhibited co-localization of these proteins in a subset of aggressive cancer. Utilizing androgen-responsive prostate cancer LNCaP cells, with increased Akt activity, and androgen-refractory PC-3 cells harboring high levels of Akt and NF-κB. Individual treatment of cells with Akt Inhibitor VIII (0.075-1.8µM) and NF-κB inhibitor Parthenolide (0.32-60µM) for 24-72 h demonstrated partial suppressive effect in cell growth. Strikingly, concurrent blocking of Akt and NF-κB/p65 at 1:10 molar ratio resulted in potentiated toxicity with marked inhibition in proliferation and induction of cell cycle arrest. Interruption of Akt activation using dominant-negative approach resulted in significant inhibition in Akt-phosphorylation (Ser473) and induction of apoptosis in LNCaP cells, whereas ectopic expression of NF-κB/p65 increased doubling time and invasiveness in these cells, which was abrogated by combinational treatment. Furthermore, combination treatment decreased the expression of p-IKK (Ser176/180), p-NF-κB/p65 (Ser536), and p-Akt (Ser473) and downstream targets viz. cyclin D1, Bcl-2, VEGF and MMP9 in both cell lines. In vivo administration of Inhibitor VIII and Parthenolide at 1:10 ratio to PC-3 tumor xenograft for 8 weeks resulted in marked decrease in tumor volume and reduced expression of target genes, compared to individual treatments. These results suggest that nuclear co-localization of Akt and NF-κB/p65 may be developed as prognostic biomarker and concurrent targeting of these molecules by combination of pharmacological inhibitors is an effective approach and is more efficacious than use of single agent blocking prostate cancer progression. Citation Format: Eswar Shankar, Rajnee Kanwal, Aditi Goel, Xiaoping Yang, Sanjeev Shukla, Gregory T. MacLennan, Pingfu Fu, Anant Madabhushi, Parameswaran Ramakrishnan, Sanjay Gupta. Targeting the PI3K-Akt and NF-κB pathways as a combination therapy in blocking prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1080. doi:10.1158/1538-7445.AM2017-1080

Cytostatic Effects of Methanolic Extracts of Amsonia orientalis Decne. on MCF-7 and DU145 Cancer Cell Lines

Amsonia orientalis is an ornamental plant with anti-cancer properties. To characterize the anti-cancer potential of the species, methanolic extracts were prepared from leaf and stem tissues of field-grown individuals. Also, a mixture of stems, leaves and roots of in vitro grown plantlets (SLR) were extracted with methanol and used. Effects of these extracts on the cancer cell lines MCF-7 (breast) and DU145 (prostate) were investigated. After incubation for 48 h, the leaf extract gave the lowest IC50 values (197.8 µM for MCF-7 and 300.9 µM for DU145). The SLR extract also gave the similar results, but with higher IC50 values. The stem extract had the weakest effect on cancer-cell-specific cytotoxicity. The extracts did not have a significant effect on DU145, compared to fibroblasts. The leaf extract increased the number of active-Caspase-3 positive cells in MCF-7 culture slightly, but the main effect of all extracts was on the cell proliferation rather than apoptosis. Real-time cellular analysis of MCF-7 demonstrated a 1.8-fold increase of doubling time for the leaf extract. The expression of proliferation-related gene MCM2 was found to be decreased in MCF-7 cells treated with the leaf extract, while its expression increased in fibroblasts after the same treatment. In conclusion, the extracts affected the MCF-7 cells at moderate concentrations. This study suggests that the leaf methanolic extracts might be a good source of compounds with cytostatical properties.

Inhibition of androgen receptor by decoy molecules delays progression to castration-recurrent prostate cancer.

Androgen receptor (AR) is a member of the steroid receptor family and a therapeutic target for all stages of prostate cancer. AR is activated by ligand binding within its C-terminus ligand-binding domain (LBD). Here we show that overexpression of the AR NTD to generate decoy molecules inhibited both the growth and progression of prostate cancer in castrated hosts. Specifically, it was shown that lentivirus delivery of decoys delayed hormonal progression in castrated hosts as indicated by increased doubling time of tumor volume, prolonged time to achieve pre-castrate levels of serum prostate-specific antigen (PSA) and PSA nadir. These clinical parameters are indicative of delayed hormonal progression and improved therapeutic response and prognosis. Decoys reduced the expression of androgen-regulated genes that correlated with reduced in situ interaction of the AR with androgen response elements. Decoys did not reduce levels of AR protein or prevent nuclear localization of the AR. Nor did decoys interact directly with the AR. Thus decoys did not inhibit AR transactivation by a dominant negative mechanism. This work provides evidence that the AR NTD plays an important role in the hormonal progression of prostate cancer and supports the development of AR antagonists that target the AR NTD.

Growth Rate of Paragangliomas Related to Germline Mutations of the SDHx Genes

The purpose was to determine the growth rate of succinate dehydrogenase subunit (SDHx) gene-related paragangliomas based on computed tomography (CT) measurements. Twenty-seven patients with SDHx mutations who underwent subsequent CT examinations were enrolled in the study. Tumors were classified as head and neck (HNP), thoracic, or abdominal/pelvic paragangliomas (PGLs). The percentage volume increase and volume doubling time were estimated. We analyzed 56 PGLs (21 with SDHD, 6 with SDHB mutations) in 27 patients (16 men, 11 women; mean age 37.7 years). The estimated median of the follow-up was 23 months. Twenty-two (39.3%) PGLs were located in the abdomen, 8 (14.3%) in the thorax, and 26 (46.4%) in the head and neck region. The median volume growth rate was estimated at 10.4% per year (interquartile range [IQR]: -1.3; 36.3). The volume doubling time was estimated as 7.01 (2.24;+∞) years. By tumor site, the estimated medians of the annual volume growth rates were 13.6% (IQR:0.8 -30.4) for HNP, -6.06% (IQR: -1.79;47.32) for thoracic PGLs, and 10.5% (IQR: -2.2;44.6) for abdominal PGLs. The volume doubling time was 5.44 years (2.61; 87.0) for HNP, 11.8 years (1.79;+∞) for thoracic PGLs, and 6.94 years (1,88;+∞) for abdominal PGLs. There was no significant difference in the volume growth rate according to tumor location or initial size (P>.7 and P = .07, respectively) or gene mutation type (SDHB vs. SDHD, P>.8). PGLs related to SDHx mutations are slowly growing tumors. There were no correlations between tumor location, growth rate or initial size over a 23-month follow-up period. CT = computed tomography HNP = head and neck paraganglioma IQR = interquartile range PGL = paraganglioma PPGL = pheochromocytoma and paraganglioma SDH = succinate dehydrogenase.

Modeling Williams syndrome with induced pluripotent stem cells.

The development of induced pluripotent stem cells (iPSCs) like never before has opened novel opportunity to study diseases in relevant cell types. In our recent study, Williams syndrome (WS), a rare genetic neurodevelopmental disorder, that is caused by hemizygous deletion of 25-28 genes on chromosome 7, is of interest because of its unique cognitive and social profiles. Little is known about haploinsufficiency effect of those deleted genes on molecular and cellular phenotypes at the neural level due to the lack of relevant human cellular model. Using the cellular reprogramming approach, we reported that WS iPSC-derived neural progenitor cells (NPCs) has increased apoptosis and therefore increased doubling time, which could be rescued by complementation of frizzled 9, one of the genes typically deleted in WS. Moreover, WS iPSC-derived CTIP2-positive pyramidal neurons exhibit morphologic alterations including longer total dendrites and increasing dendritic spine number. In addition, WS iPSC-derived neurons show an increase in calcium transient frequency and synchronized activity likely due to increased number of dendritic spines and synapses. Our work integrated cross-level data from genetics to behavior of WS individuals and revealed altered cellular phenotypes in WS human NPCs and neurons that could be validated in other model systems such as magnetic resonance imaging (MRI) in live subjects and postmortem brain tissues.

Relative resistance of sorghum varieties as revealed by variation in biological fitness parameters of the sugarcane aphid, Melanaphis sacchari (Zehntner) (Hemiptera: Aphididae)

A study was conducted using life table parameters to assess the relative resistance in eleven botswana sorghum varieties all local, namely ‘town’, ‘PMK 132’, ‘PL 174’, ‘PsA 158’, ‘PsA 161’, ‘PMK 110’, ‘PL 177’ ‘PMK 109’, 'sephala’, ‘PMK 108’ and ‘PMK 129’ against sugarcane aphid Melanaphis sacchari (Zehntner). biological fitness of the aphid was determined using population and life table parameters: developmental rate, fecundity and survival, longevity/lifespan, intrinsic rate increase (rm), net reproduction rate (Ro), generation time (T), doubling time (DT) as well as finite rate of increase (λ). Life table and population parameters of the aphids varied significantly among varieties. A significant reduction in fecundity and survival rate was observed in ‘town’, ‘PL 174’ and ‘PMK 132’. In addition aphid life table analysis showed significantly lower net reproduction rate (Ro), intrinsic rate of increase (rm), finite rate of increase (λ) and prolonged generation time (T) and doubling time (DT) on ‘town’, ‘PL 174’ and ‘PMK 132’compared to others. the negative impact of ‘town’, ‘PL 174’ and ‘PMK 132’ on biological fitness of M. sacchari suggest antibiosis mechanism of resistance in these varieties.

Antibacterial Resistance of Bacteria in Biofilms

A biofilm is a slimy layer composed of bacteria embedded in a self produced hydrated polymer mixture consisting of polysaccharide, protein and DNA. These sessile communities and their inherent resistance to antimicrobial agents, disinfectant chemicals, components of body's defense system causes chronic bacterial infections. The mechanisms of resistance in biofilms are different from now familiar plasmids, tansoposons and mutations that confer innate resistance to individual bacterial cells. Eg: s taphylococcal infections related to foreign bodies is due to bio film formation. Pseudomonas aeruginosa lung infection in cystic fibrosis patients is caused by biofilm growing mucoid strain. Nutrients and oxygen that exist in the biofilms are the gradients that are associated with decreased bacterial metabolic activity and increased doubling times of cells. These gradients are responsible for some of tolerance of antibiotics. Early aggressive antibiotic prophylaxis and chronic suppresive therapy can be given to prevent bio film formation.

Is Low Non-Lethal Concentration of Methylmercury Really Safe? A Report on Genotoxicity with Delayed Cell Proliferation.

Human exposure to relatively low levels of methylmercury is worrying, especially in terms of its genotoxicity. It is currently unknown as to whether exposure to low levels of mercury (below established limits) is safe. Genotoxicity was already shown in lymphocytes, but studies with cells of the CNS (as the main target organ) are scarce. Moreover, disturbances in the cell cycle and cellular proliferation have previously been observed in neuronal cells, but no data are presently available for glial cells. Interestingly, cells of glial origin accumulate higher concentrations of methylmercury than those of neuronal origin. Thus, the aim of this work was to analyze the possible genotoxicity and alterations in the cell cycle and cell proliferation of a glioma cell line (C6) exposed to a low, non-lethal and non-apoptotic methylmercury concentration. Biochemical (mitochondrial activity) and morphological (integrity of the membrane) assessments confirmed the absence of cell death after exposure to 3 μM methylmercury for 24 hours. Even without promoting cell death, this treatment significantly increased genotoxicity markers (DNA fragmentation, micronuclei, nucleoplasmic bridges and nuclear buds). Changes in the cell cycle profile (increased mitotic index and cell populations in the S and G2/M phases) were observed, suggesting arrest of the cycle. This delay in the cycle was followed, 24 hours after methylmercury withdrawal, by a decrease number of viable cells, reduced cellular confluence and increased doubling time of the culture. Our work demonstrates that exposure to a low sublethal concentration of MeHg considered relatively safe according to current limits promotes genotoxicity and disturbances in the proliferation of cells of glial origin with sustained consequences after methylmercury withdrawal. This fact becomes especially important, since this cellular type accumulates more methylmercury than neurons and displays a vital role protecting the CNS, especially in chronic intoxication with this heavy metal.

A human neurodevelopmental model for Williams syndrome.

SummaryWilliams syndrome (WS) is a genetic neurodevelopmental disorder characterized by an uncommon hypersociability and a mosaic of retained and compromised linguistic and cognitive abilities. Nearly all clinically diagnosed individuals with WS lack precisely the same set of genes, with breakpoints in chromosome band 7q11.231–5. The contribution of specific genes to the neuroanatomical and functional alterations, leading to behavioral pathologies in humans, remains largely unexplored. Here, we investigate neural progenitor cells (NPCs) and cortical neurons derived from WS and typically developing (TD) induced pluripotent stem cells (iPSCs). WS NPCs have an increased doubling time and apoptosis compared to TD NPCs. Using an atypical WS subject6, 7, we narrowed this cellular phenotype to a single gene candidate, FZD9. At the neuronal stage, WS-derived layers V/VI cortical neurons were characterized by longer total dendrites, increased numbers of spines and synapses, aberrant calcium oscillation and altered network connectivity. Morphometric alterations observed in WS neurons were validated after Golgi staining of postmortem layers V/VI cortical neurons. This human iPSC model8 fills in the current knowledge gap in WS cellular biology and could lead to further insights into the molecular mechanism underlying the disorder and the human social brain.

Observational and mechanistic links between C-reactive protein and blood pressure in elderly women

It is hypothesized that chronic systemic inflammation contributes to the age-related decline in cardiovascular function. The aim of the present study was to combine an assessment of the relationship between the serum level of C-reactive protein (CRP) and systolic and diastolic blood pressure in 108 elderly women (65 and 70 years) with an in-vitro exploration of the effects of CRP on the proliferative and angiogenic potential of endothelial cells exposed to serum in elderly women. Based on the median CRP level in our population, LowCRP (CRP<1.3mg/L) and HighCRP (>1.3mg/L) groups were identified. Body mass index, waist circumference, systolic blood pressure (SBP) and diastolic blood pressure (DBP) were significantly higher in the HighCRP group than in the LowCRP group (p<0.05). The influence of CRP on SBP and DBP remained significant after adjustments for BMI and use of antihypertensive medication (p<0.05). When adjusting for waist circumference the observed influence of CRP on SPB was attenuated (p=0.062). We next evaluated the ability to form capillary tubes (angiogenesis assay) and the proliferation rate of endothelial cells exposed to the sera of elderly women. Increased serum CRP levels were associated with an increased doubling time of endothelial cells (R2=0.39; p<0.05) and decreased capillary tube length (R2=0.30; p<0.05), indicating a reduction in the proliferation rate of endothelial cells and angiogenic potential. In conclusion, chronic inflammation influences blood pressure in elderly women and compromises endothelial cell function, thus contributing to the age-related decline in vascular health.

Microalgal sensitivity varies between a diuron-resistant strain and two wild strains when exposed to diuron and irgarol, alone and in mixtures

A wild strain of Chaetoceros calcitrans and wild and diuron-resistant strains of Tetraselmis suecica, were exposed to the PSII inhibitor herbicides diuron and irgarol, individually and in mixtures. The effects of three concentrations of diuron and irgarol and four binary mixtures were evaluated on doubling time, relative reactive oxygen species and lipid content by flow cytometry, and on photosynthetic efficiency by pulse amplitude modulated fluorescence.In both wild strains, significant effects were observed for each molecule at the highest concentration tested: at irgarol 0.5 μg L−1, C. calcitrans was shown to be more sensitive than T. suecica (+52% and +19% in doubling time, respectively), whereas at diuron 5 μg L−1, T. suecica was more affected (+125% in doubling time) than C. calcitrans (+21%). Overall, irgarol had a higher toxicity at a lower concentration than diuron (no effect at diuron 0.5 μg L−1) for both wild strains. The strongest mixture (irgarol 0.5 μg L−1 + diuron 5 μg L−1) increased doubling time by 356% for T. suecica, thus showing amplified effects when the two compounds were mixed.Sequencing of the diuron-resistant strain demonstrated a single mutation in the psbA gene coding sequence. Although resistance of this strain to diuron was confirmed with no effect at the highest diuron concentration, no resistance to irgarol was shown. In addition, the mutant strain exposed to the strongest mixture showed a 3.5-fold increase in doubling time compared with irgarol alone, thereby supporting the hypothesis of a biochemical interaction between these two compounds.

ZD7288, a blocker of the HCN channel family, increases doubling time of mouse embryonic stem cells and modulates differentiation outcomes in a context-dependent manner

Pluripotent stem cells are the starting cell type of choice for the development of many cell-based regenerative therapies due to their rapid and unlimited proliferation and broad differentiation potential. The unique pluripotent cell cycle underlies both these properties. Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) family channels have previously been reported to modulate mouse embryonic stem cell (ESC) proliferation and here we characterize the effects of HCN inhibitor ZD7288 on ESC proliferation and stem cell identity. The doubling time of cells treated with the HCN blocker increased by ~30 % due to longer G1 and S phases, resulting in a nearly twofold reduction in ESC numbers after 4 day serum-free culture. Slower progression through S phase was not accompanied by H2AX phosphorylation or cell stalling at transition points, although EdU incorporation in treated cells was reduced. Despite the drastic cell cycle perturbations, the pluripotent status of the cells was not compromised by treatment. Cultures treated with the HCN blocker in maintenance conditions maintained pluripotency marker expression on both RNA and protein level, although we observed a reversible effect on morphology and colony formation frequency. Addition of ZD7288 in differentiating media improved FBS-driven differentiation, but not directed differentiation to neuroectoderm, further indicating that altered cell cycle structure does not necessarily compromise pluripotency and drive ESCs to differentiation. The categorically different outcomes of ZD7288 use during differentiation indicate that cell culture context can be determinative for effects of ion-modulatory molecules and underscores the need for exploring their action in serum-free conditions demanded by potential clinical use.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-1678-7) contains supplementary material, which is available to authorized users.

Overexpression of Cyclin E and its Low Molecular Weight Isoforms Cooperate with Loss of p53 in Promoting Oncogenic Properties of MCF-7 Breast Cancer Cells.

Cyclin E, a key coordinator of the G1 to S transition in the cell cycle, may be deregulated in several malignancies, including breast cancer. The most significant aberration in cyclin E is its elastase mediated proteolytic cleavage into tumor specific low molecular weight isoforms (LMW-Es). LMW-Es are biochemically hyperactive and biologically drive tumorigenesis in transgenic mouse models. Additionally, expression of LMW-Es has been correlated with poor survival in breast cancer cases. Here we determine whether expression of LMW-Es in a breast cancer cell line that is naturally devoid of these deregulated forms would alter their progression through each phase of the cell cycle. The results revealed that LMW-Es expression resulted in an increased doubling time, concomitant with a predominant increase in the population in the S phase of the cell cycle. Moreover, downregulation of p53 in LMW-Es cells resulted in additional shortening of the doubling time and enrichment of cells in the S and G2/M phases of the cell cycle. Furthermore, expression of LMW-Es sensitized cells to β-estradiol (E2) mediated growth and changed expression patterns of estrogen receptor and Bcl-2. Intriguingly, expression of LMW-Es could surpass anti-apoptotic effects raised by p53 upregulation. Taken together these studies suggest that overexpression of LMW-Es in collaboration with p53 loss results in altered growth properties of MCF-7 cells, enhancing the oncogenic activity of these ER positive breast cancer cells.