Number Of Living Cells Research Articles

SHED's Response to Various Pulpotomy Materials: Cytotoxicity and Gene Expression Analysis.

To examine the cytotoxicity and genetic expression of SHEDs cultured in eluates of various calcium silicate based pulpotomy materials. MTT assay, flow cytometry, alizarin red staining and scratch assay was used to assess the cellular viability, apoptosis, calcium matrix deposits and cell migration respectively. The gene expression of ALP, OCN and BMP -2, were measured with rtPCR. One way ANNOVA and Bonferroni post test was used for statistical analysis. MTT assay analysis reported that all the test specimen had no cytotoxic effects. The highest number of live cells [ % ] was found in RetroMTA. The highest percentage of cell migration was observed in SHEDs cultured in EndoCem Zr. The mean absorbance for calcium matrix deposition was higher or similar in all test specimens, when compared to control groups. The expression of BMP -2 and OCN were significantly higher in cells exposed to RetroMTA and NeoMTA respectively after 24 hrs of incubation. After 72 hrs of incubation the mRNA expression of ALP was significantly higher in MTA. SHEDs cultured in eluates of various calcium silicate based cements exhibited cytocompatibility and maintained odontogenic like phenotype differentiation in SHEDs.

IMMU-07. REPROGRAMMING OF MICROGLIA/MACROPHAGES IN GLIOBLASTOMA IMPROVES ANTI-TUMOR T CELL RESPONSES

Abstract Glioblastoma (GBM) still remains incurable. In search for new treatment modalities immunotherapy might be attractive but highly depends on a sufficient infiltration and function of effector T cells in an immunosuppressive microenvironment. In this study, we analyzed if blocking or reprogramming M2polarized glioma-associated microglia/macrophages (GAMs) could improve spontaneous effector T cell responses and thus enhance the effectiveness of immunotherapy. This was tested by sorting patient-derived CD11b+ cells from GBM tissues and treating these GAMs with small molecule inhibitors (SMI) targeting the colony stimulating factor1 receptor (CSF1R). Especially CD11b+ cells treated with the SMI GW2580 presented with a reduced expression of the M2 marker CD163 (p < 0.01) and an increased expression of HLA-DR (p < 0.05). Conditioned media of SMI-treated GAMs also contained significantly higher levels of nitrite (p < 0.001) and a reduced concentration of the immunosuppressive cytokine IL-6 (p < 0.05). Moreover, gene expression profiles of GW2580-treated GAMs showed a shift from an immunosuppressive towards a pro-inflammatory phenotype. Most importantly, the addition of conditioned media of GW2580-treated GAMs to a co-culture of autologous tumor and T cells significantly reduced the number of live tumor cells as compared to the use of conditioned media of untreated GAMs (p < 0.05). Interestingly, in some cases the ability of T cells to transmigrate through a dense barrier of autologous tumor-derived endothelial cells could also be increased. In summary, we showed that CSF-1R blockade with the SMI GW2580 can reprogram GAM phenotype and thereby improve T cell activation. This strongly suggests further studies on the use of GW2580 in combination with immunotherapeutic approaches for the treatment of GBM.

Graphene Oxide Nanoparticels Interaction with Jurkat Cell Line in Cell-IQ System.

In recent years, materials based on graphene oxide (GO) have been actively studied for their use in biomedicine. The aim of our study was to investigate the increase in cell mass and viability of Jurkat tumor line T cells during 24 h of contact with GO nanoparticles in the Cell-IQ system of intravital observation. We used nanoparticles of different sizes coated with linear or branched polyethylene glycol (PEG) at concentrations of 5 and 25 μg/mL. It was shown for the first time that direct contact with GO nanoparticles reduced the growth in cell mass at the visualization points by more than twofold, regardless of nanoparticle size and concentration. Moreover, the number of live cells in the culture decreased by 5-9% after 24 h of monitoring. Thus, PEG-coated GO nanoparticles were found to suppress the proliferation and viability of Jurkat cell line T lymphocytes.

Development of Simvastatin Loaded Electrospun Zein Nanofiber Membranes for Bone Repair.

The present study aims to prepare and And Characterization of zein nanofibers with simvastatin-loaded in it by electrospinning method using a polymeric matrix prepared from zein solution containing simvastatin antibiotics. The physical, cellular properties and drug release kinetics of the fibers were studied. Fiber formation was investigated by implementing a scanning electron microscope (SEM). The SEM images show a uniform network bead-free fiber structure created from the 1.5% simvastatin/zeinsolution. The viscosity of the 1.5% simvastatin/zein mixture increased to 0.402 P due to the presence of drug in zein solution. Also, the presence of the indicative peaks associated with simvastatin in the Fourier transform infrared (FTIR) spectra of the samples containing the drug, verifies the existence of the drug on the polymeric base of zein/polyethylene oxide (PEO). Also, around 53.887% of the loaded drug was released in the first 12 h of the release kinetics assay and the results of the MTT assay shows reduction in the number of live cells in the test sample. In conclusion, zein nanofibers containing simvastatin drug were found to be potential biodegradable polymers that can find application in bone tissue repair.

Calcium Peroxide-Containing Polydimethylsiloxane-Based Microwells for Inhibiting Cell Death in Spheroids through Improved Oxygen Supply

Multicellular spheroids are expected to be used for in vivo-like tissue models and cell transplantation. Microwell devices are useful for the fabrication of multicellular spheroids to improve productivity and regulate their size. However, the high cell density in microwell devices leads to accelerated cell death. In this study, we developed O2-generating microwells by incorporating calcium peroxide (CaO2) into polydimethylsiloxane (PDMS)-based microwells. The CaO2-containing PDMS was shown to generate O2 for 3 d. Then, CaO2-containing PDMS was used to fabricate O2-generating microwells using a micro-molding technique. When human hepatocellular carcinoma (HepG2) spheroids were prepared using the conventional microwells, the O2 concentration in the culture medium reduced to approx. 67% of the cell-free level. In contrast, the O2-generating microwells maintained O2 at constant levels. The HepG2 spheroids prepared using the O2-generating microwells had a larger number of live cells than those prepared using the conventional microwells. In addition, the O2-generating microwells rescued hypoxia in the HepG2 spheroids and increased cell viability. Lastly, the O2-generating microwells were also useful for the preparation of multicellular spheroids of other cell types (i.e., MIN6, B16-BL6, and adipose-derived stem cells) with high cell viability. These results showed that the O2-generating microwells are useful for preparing multicellular spheroids with high cell viability.

Development and characterization of matrix-derived microcarriers from decellularized tissues using electrospraying techniques.

Stirred bioreactor systems integrating microcarriers represent a promising approach for therapeutic cell manufacturing. While a variety of microcarriers are commercially available, current options do not integrate the tissue-specific composition of the extracellular matrix (ECM), which can play critical roles in directing cell function. The current study sought to generate microcarriers comprised exclusively of ECM from multiple tissue sources. More specifically, porcine decellularized dermis, porcine decellularized myocardium, and human decellularized adipose tissue were digested with α-amylase to obtain ECM suspensions that could be electrosprayed into liquid nitrogen to generate 3D microcarriers that were stable over a range of ECM concentrations without the need for chemical crosslinking or other additives. Characterization studies confirmed that all three microcarrier types had similar soft and compliant mechanical properties and were of a similar size range, but that their composition varied depending on the native tissue source. In vivo testing in immunocompetent mice revealed that the microcarriers integrated into the host tissues, supporting the infiltration of host cells including macrophages and endothelial cells at 2 weeks post-implantation. In vitro cell culture studies validated that the novel microcarriers supported the attachment of tissue-specific stromal cell populations under dynamic culture conditions within spinner flasks, with a significant increase in live cell numbers observed over 1week on the dermal- and adipose-derived microcarriers. Overall, the findings demonstrate the versatility of the electrospraying methods and support the further development of the microcarriers as cell culture and delivery platforms.

Effectiveness of D,L-2-hydroxyisocaproic acid (HICA) and alpha-mangostin against endodontopathogenic microorganisms in a multispecies bacterial-fungal biofilm in an ex vivo tooth model.

To develop a defined multispecies root canal biofilm model ex vivo, and to perform viable compositional analysis following D,L-2-hydroxyisocaproic acid (HICA), alpha-mangostin, Calcicur® , and Odontopaste® exposure. Time-kill assays were conducted in vitro using HICA, alpha-mangostin, Calcicur® , Odontopaste® , and saline solution on the planktonic cultures of C.albicans, E.faecalis, L.rhamnosus, and S.gordonii. Human root dentine blocks were prepared (n=100) ex vivo, and multispecies suspensions containing each of 1.5×108 CFU/mL C.albicans, E.faecalis, L.rhamnosus, and S.gordonii in brain heart infusion (BHI) were incubated within the root canals for 21days. Canals (n=20/group) were then exposed to medicaments for 7days. Samples taken from the inner (first 0.1mm) and deeper (second 0.1mm) dentine by drilling with Ash Steel Burs No. 5 and No. 6, and residual roots were cultured in broth for 24h. Cell growth was detected by spectrophotometry and confirmed by culture on agar. The other set of inner dentine, deeper dentine, and residual root samples were sonicated, and then exposed with 50μM PMA before DNA was extracted using the QIAamp DNA mini kit. Real-time quantitative PCR was performed to determine the biofilm composition as well as the number of live and total cells remaining in the biofilm following each treatment. The OD data were analysed with Kruskal-Wallis and Friedman with Wilcoxon signed-rank test between and within groups, respectively, agar culture and qPCR data with Pearson chi-square with Mann-Whitney and Cochran with McNemar tests, respectively (p<.0001). Time-kill assays revealed that HICA and Calcicur® killed all planktonic organisms within 24h, whilst alpha-mangostin killed the organisms within 72h. However, Odontopaste® was a slow-killing agent: 10 cells of planktonic organisms survived after exposure to the agent for 7days. The ex vivo tooth model demonstrated that HICA and alpha-mangostin significantly inhibited the cell growth in all sampling depths (p<.0001). All species-specific data revealed the effectiveness of each medicament on the biofilm composition. D,L-2-hydroxyisocaproic acid and alpha-mangostin had antimicrobial activity against multispecies bacterial-fungal biofilms.

Impact of Zinc Oxide Nanoparticles on the Composition of Gut Microbiota in Healthy and Autism Spectrum Disorder Children.

Autism spectrum disorder (ASD) seriously affects children’s health, while the gut microbiome has been widely hypothesized to be involved in the regulation of ASD behavior. This study investigated and compared the number, diversity, and population structure of gut microbiota between healthy and ASD children and their susceptibility to zinc oxide nanoparticles (ZnONPs) based on the measurement of live cell number, living/dead bacterial staining test, flow cytometry observation and bacterial community analysis using 16S rRNA gene amplicon sequencing. The result of this present study revealed that ASD children not only significantly reduced the live cell number and the community diversity of gut bacteria, but also changed the gut bacterial community composition compared to the healthy children. In addition, this result revealed that ZnONPs significantly reduced the number of live bacterial cells in the gut of healthy children, but not in that of ASD children. In contrast, ZnONPs generally increased the gut bacterial community diversity in both ASD and healthy children, while a greater increase was found in ASD children than that of healthy children. Furthermore, this study successfully isolated and identified some representative nanoparticle-resistant bacteria based on the color, shape, and edge of colony as well as the 16S rDNA sequence analysis. The community of nanoparticle-resistant bacteria differed in between healthy and ASD children. Indeed, the representative strains 6-1, 6-2, 6-3 and 6-4 from healthy children were identified as Bacillus anthracis, Escherichia coli, Bacillus cereus and Escherichia coli with sequence similarity of 97.86%, 99.86%, 99.03% and 99.65%, respectively, while the representative strains 8-1, 8-2 and 8-3 from ASD children were identified as Bacillus cereus, with sequence similarities of 99.58%, 99.72% and 99.72%, respectively. Overall, this study demonstrated that ZnONPs caused a change in number, diversity, and species composition of gut bacteria, but differed in healthy and ASD children.

Does combination of estradiol and sesame oil improve the oocyte quality, embryo development and expressions of Zp3, E-cad, and Ctnnb1 genes in mice? An experimental study.

BackgroundAging may reduce oocyte maturation, embryo quality, and fertility potential.ObjectiveTo compare the effect of estradiol (E2) and sesame oil on oocyte and embryo quality between young and old mice.Materials and MethodsSixty old and young female mice were divided in to two groups (30 mice/group, grouped by age). Each group was divided into three subgroups of mice treated with sesame oil, E2 + sesame oil, and normal saline as control group. After ovulation induction, some oocytes were considered for in vitro fertilization and the rest were assessed for morphological status. After obtaining the two-cell embryos, the embryos were collected to determine the expression of zona pellucida (ZP) glycoprotein 3, E-cadherin, and β-catenin genes and some of them followed until the blastocysts stage to evaluate the viability.Results The findings showed that the mean ZP and perivitelline space thickness increased in the old mice that received the E2 + sesame oil treatment. The number of 2-cell embryos, blastocysts, and live cells were significantly higher in the old group treated with sesame oil respectively (p = 0.018, 0.002, and 0.0001, respectively). The normal ZP shape and refractile body numbers increased in the old mice that were treated with sesame oil, respectively. The E-cadherin gene was downregulated in the treatment groups compared to the controls.ConclusionSesame oil showed a better response in the old mice, because aging is associated with an increased rate of reactive oxygen species, causing deficiencies in both oocyte and embryo qualities.

Abstract MP231: Long Non-coding Rna Camirt Plays A Sentinel Role In Aging-related Heart Failure Via Interaction With Phb2 To Modulate Mitophagy Signaling In The Heart

Background: Mitochondrial dysfunction is an important risk factor for heart failure in elderly people. Mitophagy, a physiological process that controls the removal of damaged mitochondria, is compromised in aging or failing hearts. In this study, we examined the physiological role of a cardiac-specific lncRNA Camirt that can potentially modulate mitophagy in the heart. Methods and Results: RNA-seq analysis and RT-PCR reveal a lncRNA is highly expressed in both mouse and human hearts, with undetectable levels in other vital organs. Furthermore, Real time qPCR was used to examine the expression of lncRNA in different animal models and in human hearts, which results showed that the expression of this lncRNA is decreased in aging mouse and human hearts, and failing mouse hearts induced by isoproterenol and doxorubicin. RNA pull-down and RNA-binding protein immunoprecipitation assays identify prohibitin-2 (Phb2), a known mitophagy receptor, as a binding partner for this lncRNA. Thus, we name this novel lncRNA as a cardiac-specific mitophagy-associated RNA transcript (Camirt). Camirt conditional (flox) knockout mice were created via CRISPR /Cas9 technology, and subjected to the longitudinal echocardiographic and survival studies. Mice with cardiac specific deletion of Camirt (Camirt-cKO) display progressive heart failure and die within 12 month after birth. RNA sequencing and gene ontology analysis revealed that genes involved in mitophagy signaling were significantly altered in the Camirt-cKO hearts compared with the littermate wild type mice. Transmission electron microscopy were used to examine the mitochondrial morphology in mouse hearts, and reveal excessive accumulation of mitolysosomes in cardiomyocytes derived from the Camirt-cKO mice. In vitro study with Annexin-V/PI staining showed an increased number of live cells and decreased number of apoptotic cells in cultured neonatal cardiomyocytes with overexpression of Camirt following oxidative stress induced by treatment with H2O2. Increased autophagy (or mitophagy) activity was observed in HL-1 cells with stable overexpression of Camirt and in the presence of chloroquine (an inhibitor for the lysosome degradation). While reduced Camirt expression via shRNA knock-down leads to compromised mitophagy activity in HL-1 cells. Further biochemical studies support the function of Camirt/Phb2 in maintenance of mitochondria function and mitophagy signaling under stress conditions. Conclusion: Overall, our results suggested that Camirt plays a sentinel role in aging-related heart failure via interaction with Phb2 to modulate mitophagy signaling in the heart. Future studies will focus on elucidating the in vivo role and mechanisms of Camirt in modulation of mitophagy under natural aging or stress-induced pathologic conditions using the loss- or gain-of-function of Camirt mouse models.

Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope

Base editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Cas-deaminase fusion proteins critically depends on the availability of a protospacer adjacent motif (PAM) at the target locus and is limited to a window within the CRISPR-Cas R-loop, where single-stranded DNA (ssDNA) is accessible to the deaminase. Here, we reason that the Cas9-HNH nuclease domain sterically constrains ssDNA accessibility and demonstrate that omission of this domain expands the editing window. By exchanging the HNH nuclease domain with a monomeric or heterodimeric adenosine deaminase, we furthermore engineer adenine base editor variants (HNHx-ABEs) with PAM-proximally shifted editing windows. This work expands the targeting scope of base editors and provides base editor variants that are substantially smaller. It moreover informs of potential future directions in Cas9 protein engineering, where the HNH domain could be replaced by other enzymes that act on ssDNA.

Biosynthesis and Characterization of Zinc Oxide Nanoparticles and Their Impact on the Composition of Gut Microbiota in Healthy and Attention-Deficit Hyperactivity Disorder Children.

Attention-deficit hyperactivity disorder (ADHD) seriously affects children’s health, and the gut microbiome has been widely hypothesized to play a role in the regulation of ADHD behavior. The present study aims to the biosynthesize of zinc oxide nanoparticles (ZnONPs) by using Acinetobacter johnsonii strain RTN1, followed by their characterization through state-of-the-art material characterization techniques, viz., UV–vis spectroscopy, Fourier transform infrared spectroscopy, and transmission and scanning electron microscopic analyses with energy dispersive spectrometry. Moreover, we investigated and compared the population composition of gut microbiota and their susceptibility to biogenic ZnONPs between healthy and ADHD children based on the traditional plate method and 16S rRNA amplicon sequence analysis. The antibacterial effect of ZnONPs against gut bacteria was also determined by measurement of live cell number, living/dead bacterial staining test, and flow cytometry observation. The present study revealed that the number of live gut bacteria in healthy children was more than 10-fold higher than that in ADHD children; however, the community structure of gut bacteria has changed, while greater diversity was found in gut bacteria from ADHD children. In addition, we found that the number of live gut bacteria in healthy and ADHD children was reduced by ZnONPs, which shows an increased and reduced effect in composition of gut bacteria from healthy and ADHD children, respectively. It was also noted that the main mechanism of ZnONPs may be to inhibit the growth of gut bacteria rather than to kill them, while the nanoparticle-resistant strains in healthy children is also different from that in ADHD children. Some representative bacteria, in particular nanoparticle-resistant bacteria, were successfully isolated and identified. Overall, this study revealed the potential correlation of ADHD with gut bacteria and provided a new possibility to prevent ADHD by the combination of nanoparticle and its resistant bacteria.

Terahertz circular dichroism sensing of living cancer cells based on microstructure sensor

Terahertz (THz) waves have the advantages of being noninvasive and nonionizing because of their low radiation energy, so they have potential applications in the biomedical field, but thus far, those have been limited by the strong absorption in water and low detection sensitivity. Herein, we propose a reflective THz time-domain circular dichroism (CD) sensing system and a silicon subwavelength grating as the microstructure sensor to generate and detect the THz chiral polarization states, to realize quantitative detection of living cell numbers and qualitative identification of cell kinds in a liquid environment. Three kinds of hepatoma cell proliferation and inhibition with different concentrations of aspirin were measured by this sensing method, and the experimental results show that the sensitivities for CD resonance intensity and frequency shift can reach 3.44 dB mL/106 cells and 5.88 GHz mL/106 cells, respectively, and the minimum detection concentration is in the order of 104 cells/mL for THz detection in a liquid environment for the first time. This new THz sensing system and sensing method are expected to become a broadband, label-free, noncontact, real-time detection technology that can be used for quantitative detection and qualitative identification of cells or other active biochemical materials.

MiR-181c regulates MCL1 and cell survival in GATA2 deficient cells.

GATA2 is a transcription factor critical for hematopoiesis. Germline mutations in GATA binding protein 2 (GATA2) led to haploinsufficiency, severe cytopenias of multiple cell lineages, susceptibility to infections and strong propensity to develop myelodysplastic syndrome, and acute myeloid leukemia. Mechanisms of progressive cytopenias remain unclear. MicroRNA (miRNA) represents a unique mechanism of post-transcriptional gene regulation. In this study, miRNA profiles were evaluated and eight miRNAs were found to be differentially expressed (≥2-fold, P ≤ 0.05) in patient-derived cell lines (N=13) in comparison to controls (N=10). miR-9, miR-181a-2-3p, miR-181c, miR-181c-3p, miR-486-3p, and miR-582 showed increased expression, whereas miR-223 and miR-424-3p showed decreased expression. Cell death assays indicated that miR-181c potently induces cell death in lymphoid (Ly-8 and SP-53) and myeloid (HL-60) cell lines. miR-181c was predicted to target myeloid cell leukemia (MCL)1, which was confirmed by transfection assays, resulting in significantly reduced MCL1 mRNA and decreased live cell numbers. Bone marrow analysis of 34 GATA2 patients showed significantly decreased cellularity, CD34-positive cells, monocytes, dendritic cells, NK cells, B cells, and B cell precursors in comparison to healthy controls (N=29; P<0.001 for each), which was accompanied by decreased levels of MCL1 (P<0.05). GATA2 expression led to significant repression of miR-181c expression in transfection experiments. Conversely, knockdown of GATA2 led to increased miR-181c expression. These findings indicate that miR-181c expression is increased and MCL1 levels decreased in GATA2 deficiency cells, and that GATA2 represses miR-181c transcription. Increased miR-181c may contribute to elevated cell death and cytopenia in GATA2 deficiency potentially through down-regulation of MCL1.

An experimental-mathematical approach to predict tumor cell growth as a function of glucose availability in breast cancer cell lines.

We present the development and validation of a mathematical model that predicts how glucose dynamics influence metabolism and therefore tumor cell growth. Glucose, the starting material for glycolysis, has a fundamental influence on tumor cell growth. We employed time-resolved microscopy to track the temporal change of the number of live and dead tumor cells under different initial glucose concentrations and seeding densities. We then constructed a family of mathematical models (where cell death was accounted for differently in each member of the family) to describe overall tumor cell growth in response to the initial glucose and confluence conditions. The Akaikie Information Criteria was then employed to identify the most parsimonious model. The selected model was then trained on 75% of the data to calibrate the system and identify trends in model parameters as a function of initial glucose concentration and confluence. The calibrated parameters were applied to the remaining 25% of the data to predict the temporal dynamics given the known initial glucose concentration and confluence, and tested against the corresponding experimental measurements. With the selected model, we achieved an accuracy (defined as the fraction of measured data that fell within the 95% confidence intervals of the predicted growth curves) of 77.2 ± 6.3% and 87.2 ± 5.1% for live BT-474 and MDA-MB-231 cells, respectively.

The Effect of the Addition of Chokeberry Fiber on the Quality of Sheep's Milk Fermented by Lactobacillus rhamnosus and Lactobacillus acidophilus.

The global market for sheep's milk and its products is increasing due to higher demand for cheese and traditional dairy products, and as a novelty, sheep's milk is an ingredient in infant formulas and nutraceuticals. The aim of this study was to determine the properties of fermented sheep's milk, which combines probiotic benefits with increased dietary fiber content. The influence of the applied dose of chokeberry fiber on the growth of living cells of Lactobacillus acidophilus and Lactobacillus rhamnosus in fermented sheep's milk was also evaluated. Sheep milk with the addition of 0% (control sample), 1.5%, and 3.0% chokeberry fiber was fermented by two different probiotic monocultures L. acidophilus and L. rhamnosus. In the fermented milk, pH value, syneresis (%), color, and texture were determined. Furthermore, the microbiological analysis and an organoleptic evaluation were performed. With the increasing dose of chokeberry fiber, the pH value decreased already before the fermentation process. After fermentation, the milk's pH values with fiber were still lower than those in the control sample. Presumably, more acidic metabolites were produced by L. acidophilus, causing a more intense reduction of the pH value than L. rhamnosus both in control milk and in milk with the addition of fiber. The addition of chokeberry fiber affected the stimulation of the growth of both types of bacteria. In the milk sample without fiber addition, a more significant number of viable cells were counted for L. acidophilus by 0.5 log CFU g−1 more than the milk fermented with L. rhamnosus. Furthermore, in milk fermented by L. acidophilus with 1.5% chokeberry fiber (LA1), the number of viable bacterial cells was higher than that in milk fermented by L. rhamnosus with the same addition of fiber (LR1). However, in sample LA3, the number of viable bacterial cells was lower than that in sample LR3. Tested fermented sheep milk met the Recommendation of the International Dairy Federation's requirements regarding the number of live bacterial cells for dairy probiotics. The addition of chokeberry fiber increased syneresis in each fermented milk group, regardless of the bacteria used for fermentation. Moreover, the use of fiber caused a significant reduction in brightness L∗, an increase of red color, and a decrease of yellow color. Milk fermented with L. acidophilus was characterized by a harder gel, compared to their analogues fermented with L. rhamnosus. The addition of fiber intensified the sour taste and the taste of the additive in both types of fermented sheep milk.

Effect of reciprocating instrumentation on chlorhexidine substantivity on human dentin: chemical analysis followed by confocal laser microscopy

The present research analyzed the reciprocating instrumentation associated to chlorhexidine (CHX) substantivity as its correlation with E. faecalis viability in ex vivo root canals. Eighty extracted single-rooted human teeth were used, being 40 to high-performance liquid chromatography (HPLC) and 40 to confocal laser scanning microscopy (CLSM). In both, teeth were decoronated and the cervical third was prepared. In the CLSM analysis, the root canals were inoculated with E. faecalis for 14 days. Samples were divided into 4 groups (n=10) according to instrumentation technique: no instrumentation and irrigation with distilled water (control); manual instrumentation (K-File); rotary instrumentation (ProTaper Next); and reciprocating instrumentation (Reciproc R25). Two percent chlorhexidine was applied as irrigating substance in experimental groups. Longitudinal grooves resulted in 2 halves root and 20 proof bodies in each group. Samples were divided by chance in two groups (n=10) and the outcomes were evaluated after two days and one week. The retained chlorhexidine and live cells after instrumentation techniques in each evaluation time was measured by HPLC and CLSM, respectively. Specific analysis was applied for experimental tests (p≤0.05). Both rotary as well as reciprocating techniques significantly reduced the amount of chlorhexidine on dentin in all observation periods (p&lt;0.05). After evaluation times, all experimental groups presented lower live cells compared to control, but without statistically difference. Intragroup comparisons in times of evaluation showed no differences in instrumentation techniques, in chlorhexidine retention and number of live cells (p&gt;0.05). Reciprocating instrumentation does not interfere on chlorhexidine substantivity.

Inhibition of Translation Initiation and Heat Shock Factor 1 (HSF1): A Novel Strategy Targeting FLT3 Double Mutant and Non-FLT3 Mutant AML Progenitor/Stem Cells

The transcription factor Heat shock factor 1 (HSF1) is a key sensor of proteotoxic stress and plays a major role in cancer biology. In various tumors, downregulation of HSF1 inhibits growth and induces cell death, and its activation was reported to be an adverse prognostic factor in breast and lung cancers. We have reported that inhibition of translation represses DNA-binding of HSF1 in cancers; large scale drug screening identified the eIF4a antagonist rohinitib (RHT) as a potent inhibitor of the HSF1 activation state (Santagata et al, Science 2013). Review of public databases provided us with additional rationale for targeting HSF1 and eIF4a in acute myeloid leukemia (AML): 1) mRNA levels of HSPA8, a primary HSF1 target, are correlated with poor prognosis in AML; 2) eIF4a mRNA levels are highest in AML among 12 cancer types; and 3) Gene set enrichment analysis using microarray dataset of functionally-defined leukemia stem cells (LSCs) and non-LSCs (Nature, Ng et al), based on intra-patient comparisons, reveals that a gene set for translation initiation is highly enriched in LSCs (NES 2.73, FDR = 0.000, p= 0.000).RHT induced marked apoptogenic effects in seven human AML cell lines in culture at low nanomolar concentrations (LD50s; 9.5 to 99.5 nM, IC50s; 4.7 to 8.8 nM, based on AnnexinV/PI-positivity at 72hr). RHT inhibited HSF1 transcriptional activity, as shown by 70% reduction in HSPA8 mRNA levels, and 50% reduction in a luciferase reporter driven by the HSPA1A or HSPA6 promoters. We then tested primary samples from 17 AML patients and bone marrow (BM) samples from eight healthy donors. RHT potently induced apoptosis in AML cells, while relatively sparing normal BM cells (%apoptosis: 65.4% vs 6.67%, P < 0.0001). Importantly, a similarly significant difference in sensitivity was also observed between AML and normal stem/progenitor cell populations (CD45+CD34+CD38-). We further found that the activity of RHT was significantly higher in FLT3-ITD than in FLT3-wt AMLs. Comparing isogenic versions of the cell line Ba/F3, RHT more potently killed FLT3-ITD cells than wild-type (wt) cells (LD50s; 65.3 vs 20.1 nM). Immunoblot analysis showed higher activation-associated phospho-HSF1 (Serine 326) in FLT3-ITD Ba/F3 than in FLT3-wt cells, and growth inhibition by HSF1 knockdown was FLT3-ITD-dependent in those lines, indicating greater dependence of FLT3-ITD cells on HSF1. In an AML xenograft model with GFP-luciferase labeled MOLM-13 cells, RHT significantly reduced tumor burden as measured by bioluminescence imaging and reduction in GFP+ leukemic cells peripheral blood (PB) and BM by day 16. Median survival of the treatment group was significantly longer (24 vs 18 days, p < 0.0001).We also found that RHT was synergistic when combined with FLT3 inhibitors of Type I (sorafenib, quizartinib, midostaurin) or Type II (gilteritinib, and E-6201). Immunoblot analysis showed that oncogenic FLT3 activation is associated with increased phosphorylation of HSF1 (S326), suggesting that decreased phosphorylation of the S326 site associated with FLT3 inhibition and diminished DNA-binding by HSF1 driven by eIF4a inhibition combine to promote synergistic lethality. In support of this, HSF1 knockdown sensitized MOLM-13 and FLT3-ITD+ Ba/F3 cells to FLT3 inhibitors. Importantly, similar combinatorial synergistic effects with type II FLT3 inhibitors were observed in cells with dual mutations of FLT3-ITD and FLT3-D835Y/H. This observation has significant clinical relevance because the development of secondary point mutations is a major resistance mechanism against FLT3 inhibitors.Finally, to test the hypothesis that eIF4a inhibition impairs LSC functions, we treated primary patient-derived xenograft AML PDX cells with RHT ex vivo, then injected identical numbers of live cells into recipient NSG mice. Subsequent leukemia engraftment was delayed by one week vs. controls, and human CD45+ cells in PB and BM at 4 weeks post transplantation were significantly reduced (n = 9, 3.30% vs 0.26% in PB, P < 0.0001). This reduced engraftment capacity of RHT-treated PDX AML-LSCs indicates a toxic effect of eIF4a inhibition on AML-LSCs.Collectively, these preclinical findings strongly support the further development of eIF4a inhibitors for the treatment of AML, especially for high-risk FLT3-mutated AML, with additional potential as LSC-targeted therapy. DisclosuresNo relevant conflicts of interest to declare.

Identification of Target Pathways of Acquired IMiD and Proteasome Inhibitor Resistance in Multiple Myeloma

Immunomodulatory drugs (IMiDs) such as lenalidomide and pomalidomide, and proteasome inhibitors (PIs) such bortezomib and carfilzomib are critical components of anti-multiple myeloma (MM) therapy. Despite the initial effectiveness of these therapies, MM patients all eventually develop refractory disease. Therefore, new agents and new combinations are needed to bolster the current armamentarium and to meet the individual needs of MM patients. We developed a non-biased method to identify upregulated pathways in IMiD and PI-resistant myeloma cell lines using a combination of activity-based protein profiling (ABPP) and a high-throughput protein kinase inhibitor (PKI) viability screen. Target validation was then performed using ex vivo functional PKI screens of MM patient specimens. The MM cell lines MM1.S, KAS6, 8226, and U266 along with their drug resistant counterparts MM1.S-R10R, KAS6-R10R, 8226-B25, and U266-PR, were grown in mono-culture for 24h and lysates were enriched for ATP binding proteins by affinity purification versus a chemical probe (ActiveX, Thermo). Tryptic peptides were measured using discovery proteomics (nano-UPLC and QExactive Plus mass spectrometer). Using this method, 59, 87, 85, and 35 kinases out of a total of 660, 1153, 715, and 688 proteins were preferentially enriched by 2-fold change from MM1.S-R10R, KAS6-R10R, 8226-B25, and U266-PR myeloma cells compared to parental cell lines, respectively. As proof of concept, 11 proteins dysregulated in PI-resistant lines are critical for proteasome function, including core subunit (20S) and regulatory subunit (19S) proteins. Twenty-four kinases were common among IMiD or PI-resistant cell line pairs and eight kinases were common to all four resistant cell lines, representing pathways with increased activity in acquired resistance to these two classes of therapeutic agents: CDK1, PLK1, ILK, DNAPK, Syk, MKK7, NEK2, and MARK3. These eight kinases were chosen for target validation, along with 21 others identified by pathway analysis using the Kyoto Encyclopedia of Genes and Genomes database to identify signaling networks. A label-free, high throughput assay was used to measure the viability of MM cells grown in a collagen matrix with bone marrow stroma cells in 384-well plates to simulate the tumor microenvironment by capturing brightfield images every 30 minutes for 96h using a motorized microscope equipped with an incubation chamber. Digital image analysis software measures live cell numbers by detecting membrane motion and generates viability curves as a function of drug concentration and exposure time (Silva et al . Cancer Research 2017). This functional screen confirmed known MM survival networks, validated three kinases/PKIs and highlighted novel targetable pathways. Two PKIs, dinaciclib (CDK1) and volasertib (PLK1) consistently achieved LD50s in the low-nanomolar range in all eight cell lines in accordance with the proteomic data. To provide an additional level of screening, the same PKI panel was tested by viability screening of CD138-MACS-selected cells from MM patient specimens. Several PKIs showed significant activity in primary MM specimens, with dinaciclib active in 100% (n=22), volasertib in 100% (n=5) and the PLK1i, BI2536, in 87% (n=38) of patient samples examined. Among these, dinaciclib showed the most activity in patient specimens with an average 96h LD50 of 56 nM. Future work will use molecular strategies, including RNAi and inducible expression systems, to explore dysregulated pathways in IMiD and PI resistant cell lines and refractory or relapsed patient specimens. As an additional strategy, we have processed over 220 relapsed/refractory and newly diagnosed MM patients that have RNAseq data obtained from the same biopsy through the Total Cancer Care/M2Gen/ORIEN network at Moffitt Cancer Center, over two-thirds of these were tested for sensitivity to PIs and over half were tested for sensitivity to IMiDs. Patients will be stratified into cohorts based on ex vivo drug sensitivity to mine gene expression data with the goal of discovering tumor vulnerabilities that arise during therapy. Our three-tiered pharmaco-proteomic screen identified kinases critical to MM survival in the context of acquired drug resistance and represents a unique workflow to find tumor vulnerabilities that arise during therapy. DisclosuresSiqueira Silva:AbbVie: Research Funding. Shain:Celgene: Consultancy, Honoraria, Speakers Bureau; AbbVie: Research Funding; Amgen: Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau.

Investigation of the Antioxidant and Antitumour Properties of Some Cyclobutane Ring Containing 2,5-Thiophene Diacyl Compounds

In this research, the effect of thiophene derivatives on the (MDA) concentration which is an indicator of lipid peroxidation, antioxidant vitamins A, E and in vitro antioxidant activity and antitumor activity, were investigated in cell culture media. In the comparison done among groups, it was observed that MDA and vitamin A concentrations were statistically (P&lt;0.05) changed. Looking at the measured parameters during the experiment, the number of live cells, the compounds is an effective antitumor activity in the L 1210 cell types. The oxidative stress parameters are used for all compounds.