Large Fraction Of Cells Research Articles

Targeting Homologous Recombination Increases Cytotoxicity While Reducing Genomic Instability Induced By Melphalan in Myeloma

Currently the greatest problem in successfully treating cancer is the genomic adaptability of the tumor cells leading to their continual survival and progression including development of resistance to treatment. Using genome sequencing, we have previously demonstrated that multiple myeloma (MM) cells derived from patients display heterogeneity of mutational spectrum and diverse patterns of clonal evolution, including linear evolution, differential clonal response, or branching evolution. This clonal evolution could be driven by pressures from inherent or acquired genomic and epigenomic changes, microenvironment and/or therapy itself, leading to both clonal selection and formation of new clones. A fundamental problem is that most chemotherapeutics agents are DNA damaging agents which kill cancer cells by increasing damage to their DNA. Although such a treatment can kill a large fraction of cancer cells, it is also likely to increase genomic instability in surviving cancer cells, possibly resulting in emergence of drug-resistant clones and subsequent relapse. A similar effect may emerge in normal cells of body.The purpose of this study was to investigate the impact of a chemotherapeutic agent melphalan on genome stability in the presence or absence of inhibitors of homologous recombination (HR) . MM cells were cultured with or without melphalan, RI-1 (which inhibits HR through inhibition of RAD51), nilotinib (which inhibits HR through inhibition of ABL-kinase) and combination of melphalan with these HR inhibitors. The cells were evaluated for viability using CellTiter-Glo luminescent c ell viability assay and apoptosis (using flow cytometry) at different time points. Cells were also evaluated for HR activity using a plasmid based assay as well as DNA breaks which were assessed from levels of gH2AX (detected by Western blotting and immunofluorescence). To investigate the impact of these treatments on genomic instability, dead cells were removed and live cells were examined for micronuclei, a marker of genomic instability. Treatment of MM cells with HR inhibitors was associated with both inhibition of HR activity and near complete loss of gH2AX signal indicating strong inhibition of spontaneous DNA breaks, whereas treatment with melphalan caused an increase in both the HR activity (by ~ 2.4-fold) and levels of gH2AX (by > 3-fold). Interestingly, although both HR inhibitors could completely reverse melphalan-induced HR, the levels of gH2AX were in fact increased ~ ≥ 2-fold. The increased levels of gH2AX by melphalan and combination treatments were also associated with a proportionate increase in cell death and apoptosis in these cells. These data indicate that inhibition of HR-mediated repair increases cytotoxicity of melphalan by increasing DNA breaks. Although the melphalan treatment increased the genomic instability (as assessed from micronuclei) in MM cells by ~80%, both the HR inhibitors could completely reverse melphalan-induced genomic instability.Taken together these data suggest that dysregulated HR in MM cells not only contributes to increased DNA breaks but is also involved in recombinational repair/utilization of DNA damaging agent-induced breaks. Thus providing a mechanism for both the genomic adaptability as well as survival against genotoxic insults. Inhibitors of HR, therefore, have potential to inhibit/reduce intrinsic as well as chemotherapy-induced genomic instability in myeloma cells as well as can increase in the efficacy of chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of Human Uracil DNA Glycosylase Sensitizes a Large Fraction of Colorectal Cancer Cells to 5-Fluorodeoxyuridine and Raltitrexed but Not Fluorouracil.

Previous short-hairpin RNA knockdown studies have established that depletion of human uracil DNA glycosylase (hUNG) sensitizes some cell lines to 5-fluorodeoxyuridine (FdU). Here, we selectively inhibit the catalytic activity of hUNG by lentiviral transduction of uracil DNA glycosylase inhibitor protein into a large panel of cancer cell lines under control of a doxycycline-inducible promoter. This induced inhibition strategy better assesses the therapeutic potential of small-molecule targeting of hUNG. In total, 6 of 11 colorectal lines showed 6- to 70-fold increases in FdU potency upon hUNG inhibition ("responsive"). This hUNG-dependent response was not observed with fluorouracil (FU), indicating that FU does not operate through the same DNA repair mechanism as FdU in vitro. Potency of the thymidylate synthase inhibitor raltitrexed (RTX), which elevates deoxyuridine triphosphate levels, was only incrementally enhanced upon hUNG inhibition (<40%), suggesting that responsiveness is associated with incorporation and persistence of FdU in DNA rather than deoxyuridine. The importance of FU/A and FU/G lesions in the toxicity of FdU is supported by the observation that dT supplementation completely rescued the toxic effects of U/A lesions resulting from RTX, but dT only increased the IC50 for FdU, which forms both FU/A and FU/G mismatches. Contrary to previous reports, cellular responsiveness to hUNG inhibition did not correlate with p53 status or thymine DNA glycosylase expression. A model is suggested in which the persistence of FU/A and FU/G base pairs in the absence of hUNG activity elicits an apoptotic DNA damage response in both responsive and nonresponsive colorectal lines. SIGNIFICANCE STATEMENT: The pyrimidine base 5-fluorouracil is a mainstay chemotherapeutic for treatment of advanced colorectal cancer. Here, this study shows that its deoxynucleoside form, 5-fluorodeoxyuridine (FdU), operates by a distinct DNA incorporation mechanism that is strongly potentiated by inhibition of the DNA repair enzyme human uracil DNA glycosylase. The hUNG-dependent mechanism was present in over 50% of colorectal cell lines tested, suggesting that a significant fraction of human cancers may be sensitized to FdU in the presence of a small-molecule hUNG inhibitor.

Resistance of Hypoxic Cells to Ionizing Radiation Is Mediated in Part via Hypoxia-Induced Quiescence.

Double strand breaks (DSBs) are highly toxic to a cell, a property that is exploited in radiation therapy. A critical component for the damage induction is cellular oxygen, making hypoxic tumor areas refractory to the efficacy of radiation treatment. During a fractionated radiation regimen, these hypoxic areas can be re-oxygenated. Nonetheless, hypoxia still constitutes a negative prognostic factor for the patient’s outcome. We hypothesized that this might be attributed to specific hypoxia-induced cellular traits that are maintained upon reoxygenation. Here, we show that reoxygenation of hypoxic non-transformed RPE-1 cells fully restored induction of DSBs but the cells remain radioresistant as a consequence of hypoxia-induced quiescence. With the use of the cell cycle indicators (FUCCI), cell cycle-specific radiation sensitivity, the cell cycle phase duration with live cell imaging, and single cell tracing were assessed. We observed that RPE-1 cells experience a longer G1 phase under hypoxia and retain a large fraction of cells that are non-cycling. Expression of HPV oncoprotein E7 prevents hypoxia-induced quiescence and abolishes the radioprotective effect. In line with this, HPV-negative cancer cell lines retain radioresistance, while HPV-positive cancer cell lines are radiosensitized upon reoxygenation. Quiescence induction in hypoxia and its HPV-driven prevention was observed in 3D multicellular spheroids. Collectively, we identify a new hypoxia-dependent radioprotective phenotype due to hypoxia-induced quiescence that accounts for a global decrease in radiosensitivity that can be retained upon reoxygenation and is absent in cells expressing oncoprotein E7.

A Robust Method for Generating, Quantifying, and Testing Large Numbers of Escherichia coli Persisters.

Bacteria can exhibit phenotypes that render them tolerant against antibiotics. However, often only a few cells of a bacterial population show the so-called persister phenotype, which makes it difficult to study this health-threatening phenotype. We recently found that certain abrupt nutrient shifts generate Escherichia coli populations that consist almost entirely of antibiotic-tolerant cells. These nearly homogeneous persister cell populations enable assessment with population-averaging experimental methods, such as high-throughput methods. In this chapter, we provide a detailed protocol for generating a large fraction of tolerant cells using the nutrient-switch approach. Furthermore, we describe how to determine the fraction of cells that enter the tolerant state upon a sudden nutrient shift and we provide a new way to assess antibiotic tolerance using flow cytometry. We envision that these methods will facilitate research into the important and exciting phenotype of bacterial persister cells.

Somatostatin expressing GABAergic interneurons in the medial entorhinal cortex preferentially inhibit layerIII-V pyramidal cells

GABA released from heterogeneous types of interneurons acts in a complex spatio-temporal manner on postsynaptic targets in the networks. In addition to GABA, a large fraction of GABAergic cells also express neuromodulator peptides. Somatostatin (SOM) containing interneurons, in particular, have been recognized as key players in several brain circuits, however, the action of SOM and its downstream network effects remain largely unknown. Here, we used optogenetics, electrophysiologic, anatomical and behavioral experiments to reveal that the dendrite-targeting, SOM+ GABAergic interneurons demonstrate a unique layer-specific action in the medial entorhinal cortex (MEC) both in terms of GABAergic and SOM-related properties. We show that GABAergic and somatostatinergic neurotransmission originating from SOM+ local interneurons preferentially inhibit layerIII-V pyramidal cells, known to be involved in memory formation. We propose that this dendritic GABA–SOM dual inhibitory network motif within the MEC serves to selectively modulate working-memory formation without affecting the retrieval of already learned spatial navigation tasks.

KLF4 (Kruppel-Like Factor 4)-Dependent Perivascular Plasticity Contributes to Adipose Tissue inflammation.

Smooth muscle cells and pericytes display remarkable plasticity during injury and disease progression. Here, we tested the hypothesis that perivascular cells give rise to Klf4-dependent macrophage-like cells that augment adipose tissue (AT) inflammation and metabolic dysfunction associated with diet-induced obesity (DIO). Approach and Results: Using Myh11-CreERT2 eYFP (enhanced yellow fluorescent protein) mice and flow cytometry of the stromovascular fraction of epididymal AT, we observed a large fraction of smooth muscle cells and pericytes lineage traced eYFP+ cells expressing macrophage markers. Subsequent single-cell RNA sequencing, however, showed that the majority of these cells had no detectable eYFP transcript. Further exploration revealed that intraperitoneal injection of tamoxifen in peanut oil, used for generating conditional knockout or reporter mice in thousands of previous studies, resulted in large increase in the autofluorescence and false identification of macrophages within epididymal AT as being eYFP+; and unintended proinflammatory consequences. Using newly generated Myh11-DreERT2tdTomato mice given oral tamoxifen, we virtually eliminated the problem with autofluorescence and identified 8 perivascular cell dominated clusters, half of which were altered upon DIO. Given that perivascular cell KLF4 (kruppel-like factor 4) can have beneficial or detrimental effects, we tested its role in obesity-associated AT inflammation. While smooth muscle cells and pericytes-specific Klf4 knockout (smooth muscle cells and pericytes Klf4Δ/Δ) mice were not protected from DIO, they displayed improved glucose tolerance upon DIO, and showed marked decreases in proinflammatory macrophages and increases in LYVE1+ lymphatic endothelial cells in the epididymal AT. Perivascular cells within the AT microvasculature dynamically respond to DIO and modulate tissue inflammation and metabolism in a KLF4-dependent manner.

Low-dose DNA demethylating therapy induces reprogramming of diverse cancer-related pathways at the single-cell level

BackgroundEpigenetic reprogramming using DNA demethylating drugs is a promising approach for cancer therapy, but its efficacy is highly dependent on the dosing regimen. Low-dose treatment for a prolonged period shows a remarkable therapeutic efficacy, despite its small demethylating effect. Here, we aimed to explore the mechanisms of how such low-dose treatment shows this remarkable efficacy by focusing on epigenetic reprograming at the single-cell level.MethodsExpression profiles in HCT116 cells treated with decitabine (DAC) were analyzed by single-cell RNA-sequencing (scRNA-seq). Functional consequences and DNA demethylation at the single-cell level were analyzed using cloned HCT116 cells after DAC treatment.ResultsscRNA-seq revealed that DAC-treated cells had highly diverse expression profiles at the single-cell level, and tumor-suppressor genes, endogenous retroviruses, and interferon-stimulated genes were upregulated in random fractions of cells. DNA methylation analysis of cloned HCT116 cells revealed that, while only partial reduction of DNA methylation levels was observed in bulk cells, complete demethylation of specific cancer-related genes, such as cell cycle regulation, WNT pathway, p53 pathway, and TGF-β pathway, was observed, depending upon clones. Functionally, a clone with complete demethylation of CDKN2A (p16) had a larger fraction of cells with tetraploid than parental cells, indicating induction of cellular senescence due to normalization of cell cycle regulation.ConclusionsEpigenetic reprogramming of specific cancer-related pathways at the single-cell level is likely to underlie the remarkable efficacy of low-dose DNA demethylating therapy.

Model-based optimization of combination protocols for irradiation-insensitive cancers

Alternations in the p53 regulatory network may render cancer cells resistant to the radiation-induced apoptosis. In this theoretical study we search for the best protocols combining targeted therapy with radiation to treat cancers with wild-type p53, but having downregulated expression of PTEN or overexpression of Wip1 resulting in resistance to radiation monotherapy. Instead of using the maximum tolerated dose paradigm, we exploit stochastic computational model of the p53 regulatory network to calculate apoptotic fractions for both normal and cancer cells. We consider combination protocols, with irradiations repeated every 12, 18, 24, or 36 h to find that timing between Mdm2 inhibitor delivery and irradiation significantly influences the apoptotic cell fractions. We assume that uptake of the inhibitor is higher by cancer than by normal cells and that cancer cells receive higher irradiation doses from intersecting beams. These two assumptions were found necessary for the existence of protocols inducing massive apoptosis in cancer cells without killing large fraction of normal cells neighboring tumor. The best found protocols have irradiations repeated every 24 or 36 h with two inhibitor doses per irradiation cycle, and allow to induce apoptosis in more than 95% of cancer cells, killing less than 10% of normal cells.

Structural Control of Boronic Acid Ligands Enhances Intratumoral Targeting of Sialic Acid To Eradicate Cancer Stem-like Cells.

Aberrant sialylation of cancer cells is emerging as an attractive method for generating effective antitumor strategies. However, as sialic acid (SA) is also present in healthy tissues, systems targeting SA in tumors must be strategically designed to be specifically activated in an intratumoral environment while avoiding systemic interaction. Phenylboronic acid (PBA) and its derivatives have shown potential for developing such smart ligands based on its triggered binding to SA at intratumoral pH. Because the affinity of PBAs against SA can be structurally controlled, the approach may further offer the possibility to enhance tumor targeting by molecularly engineering PBAs. Thus, to demonstrate that the modification of the chemical structure of PBAs can promote tumor targeting, we compared nanomedicines installed with the standard PBA or 5-boronopicolinic acid (5-BPA), which shows an exceptionally high binding affinity to SA in acidic pH. Platinum anticancer drugs were loaded into these nanomedicines and evaluated against orthotopic head and neck tumors, featuring a large fraction of SA-rich cancer stem-like cells (CSCs) that are resistant to platinum drugs. The 5-BPA ligands increased intracellular drug delivery of nanomedicines at intratumoral pH (pH 6.5) and enhanced the accumulation of nanomedicines in tumors to efficaciously eliminate the malignant CSCs, suppress tumor growth, and prolong mice survival. These findings indicate the potential of engineered PBA ligands for developing effective strategies targeting SA in tumors.

Colon Cancer Heterogeneity: Welcome to the RiboZone.

In this issue of Cell Stem Cell, Morral etal. (2020) shed new light on the hierarchical organization of cells within colorectal cancer. They show that tumor cells at the apex of this hierarchy reside in particular tumor zones and possess high protein synthesis activity. Interference with their biosynthetic activity results in an irreversible growth arrest of CRC.

Dissimilar tumor cell populations in ascitic fluid of ovarian cancer patients

Ovarian cancer is one of the most aggressive and hard-to-treat cancers. About 75% of ovarian cancer cases are detected at later stages of the disease. Ascitic fluid is promising biological material to get information about the tumor nature in ovarian cancer. Peritoneal dissemination is one of the most unfavorable factors of malignant tumor progression. However, prognostic factors associated with malignant ascites are not well understood. The aim of the study was to evaluate various tumor cell populations in ascitic fluid of ovarian cancer patients by laser multicolor flow cytometry using a molecular panel of EpCam, CD45, CD44, CD24, CD133, and N-cadherin markers. The prospective study included 16 patients aged 36 to 76 years with newly diagnosed FIGO stage Ic–IV ovarian cancer, who were admitted for treatment to the Cancer Research Institute of Tomsk National Research Medical Center. The study material included EDTAstabilized ascitic fluid sampled during laparoscopy. Various populations of ascitic tumor cells (with stemness features, with epithelial mesenchymal transition (EMT) features, without stemness and EMT features, with a combination of these features, as well as atypical / hybrid cell populations) were identified by multicolor flow cytometry on a BDFACSCanto apparatus (USA) using fluorochrome-labeled EpCam, CD45, CD44, CD24, CD133, and N-cadherin monoclonal antibodies and the BD FACSDiva software. The study revealed twelve populations of Epcam-positive cells in ascitic fluid of ovarian cancer patients. The cell composition of ascitic fluid in ovarian cancer patients is represented by a heterogeneous population. A large fraction of ascitic tumor cells are atypical / hybrid tumor cells with stemness features as well as Epcam+CD45–CD44+CD24+CD133+/-cancer stem cells, both with and without EMT features.

Nwd1 Regulates Neuronal Differentiation and Migration through Purinosome Formation in the Developing Cerebral Cortex.

SummaryEngagement of neural stem/progenitor cells (NSPCs) into proper neuronal differentiation requires the spatiotemporally regulated generation of metabolites. Purines are essential building blocks for many signaling molecules. Enzymes that catalyze de novo purine synthesis are assembled as a huge multienzyme complex called “purinosome.” However, there is no evidence of the formation or physiological function of the purinosome in the brain. Here, we showed that a signal transduction ATPases with numerous domains (STAND) protein, NACHT and WD repeat domain-containing 1 (Nwd1), interacted with Paics, a purine-synthesizing enzyme, to regulate purinosome assembly in NSPCs. Altered Nwd1 expression affected purinosome formation and induced the mitotic exit and premature differentiation of NSPCs, repressing neuronal migration and periventricular heterotopia. Overexpression/knockdown of Paics or Fgams, other purinosome enzymes, in the developing brain resulted in a phenocopy of Nwd1 defects. These findings indicate that strict regulation of purinosome assembly/disassembly is crucial for maintaining NSPCs and corticogenesis.

Melanopsin Driven Light Responses Across a Large Fraction of Retinal Ganglion Cells in a Dystrophic Retina.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and project to central targets, allowing them to contribute to both image-forming and non-image forming vision. Recent studies have highlighted chemical and electrical synapses between ipRGCs and neurons of the inner retina, suggesting a potential influence from the melanopsin-born signal to affect visual processing at an early stage of the visual pathway. We investigated melanopsin responses in ganglion cell layer (GCL) neurons of both intact and dystrophic mouse retinas using 256 channel multi-electrode array (MEA) recordings. A wide 200 μm inter-electrode spacing enabled a pan-retinal visualization of melanopsin’s influence upon GCL activity. Upon initial stimulation of dystrophic retinas with a long, bright light pulse, over 37% of units responded with an increase in firing (a far greater fraction than can be expected from the anatomically characterized number of ipRGCs). This relatively widespread response dissipated with repeated stimulation even at a quite long inter-stimulus interval (ISI; 120 s), to leave a smaller fraction of responsive units (<10%; more in tune with the predicted number of ipRGCs). Visually intact retinas appeared to lack such widespread melanopsin responses indicating that it is a feature of dystrophy. Taken together, our data reveal the potential for anomalously widespread melanopsin responses in advanced retinal degeneration. These could be used to probe the functional reorganization of retinal circuits in degeneration and should be taken into account when using retinally degenerate mice as a model of disease.

Loss-Function Learning for Digital Tissue Deconvolution.

The gene expression profile of a tissue averages the expression profiles of all cells in this tissue. Digital tissue deconvolution addresses the following inverse problem: given the expression profile y of a tissue, what is the cellular composition c of that tissue? If X is a matrix whose columns are reference profiles of individual cell types, the composition c can be computed by minimizing for a given loss function . Current methods use predefined all-purpose loss functions. They successfully quantify the dominating cells of a tissue, while often falling short in detecting small cell populations. In this study we use training data to learn the loss function along with the composition c . This allows us to adapt to application-specific requirements such as focusing on small cell populations or distinguishing phenotypically similar cell populations. Our method quantifies large cell fractions as accurately as existing methods and significantly improves the detection of small cell populations and the distinction of similar cell types.

Characterization of Bulk Rigidity of Rigid Red Blood Cell Populations in Sickle Cell Patients Using a Parameterization Model of Ektacytometry

Introduction: One of the critical characteristics of sickle-cell disease (SCD) is the increased rigidity of the red blood cell (RBC) membrane. This decreased flexibility of hemoglobin S (HbS)-rich RBCs contributes to reduced lifespan of the cell and is known to be a primary cause of vaso-occlusion, leading to painful crisis episodes and other complications in SCD. Although often overlooked as a factor in disease severity, increased RBC rigidity has been recently shown to reduce the vascular wall adhesion of white blood cells (WBCs) drastically - hinting at a larger role for RBC rigidity in patient well-being beyond instigating vasocclusion. The rigidity and composition of RBCs in SCD patients varies highly, i.e., there can be a large fraction of HbS cells with moderate stiffness or a small fraction of extremely rigid HbS cells. Ektacytometry is a primary technique used for measuring RBC deformability in SCD via laser diffraction viscometry, but is limited in that it renders an average measurement of deformability for the entire RBC population, i.e., across both stiff and healthy RBCs. This average measurement of deformability may underestimate the rigidity of HbS-rich (%S fraction) or sickle RBC population in the blood. Here, we present an approach to estimate rigidity of HbS cells in SCD via a method that implements artificially rigidified RBCs mixed into whole blood at different ratios. Methods: We develop an approach to estimate rigidity of HbS cells in SCD via a method that implements artificially rigidified RBCs. RBCs from healthy (non-SCD) are incubated in solutions with varying concentrations of tert-butyl hydroperoxide to stiffen the cell membrane. These artificially rigidified RBCs are then mixed into whole blood at different ratios/fractions. Four parent concentrations of TBHP are chosen as base RBC rigidities: 1.0, 0.9, 0.75, and 0.5 mM TBHP. After adequate washing, stiffened RBCs are mixed with healthy RBCs in whole blood in increments of 10% rigid RBC fractions up to a total of 100% while holding the hematocrit constant at ~40%. All samples are then measured independently using a LoRRca MaxSis Ektacytometer (Mechatronics Instruments BV, Zwaag, The Netherlands). We then parameterize these ektacytometry deformability measurements to develop a numerical model that predicts the bulk rigidity of the rigid RBC population in SCD blood samples. We measured the EI for fifteen different SCD patient blood samples, details including patient SCD genotype, age, gender, current medical intervention/therapies, %S fraction, and measured maximum bulk EI. Results: With the predicted maximum EI of the rigid RBC population in patient blood samples, we plotted deformability curves using techniques described by Baskurt et al. We utilized a simplified Skreestra-Bronkhorst model for plotting predicted deformability curves implementing critical parameters such as maximum EI, shear where half the maximum EI is achieved, and the range of shear stress in the standard ektacytometry deformability measurement. The predicted bulk SCD whole blood deformability curves are overlaid with the healthy blood curve, experimental bulk SCD whole blood standard measurement, and with the predicted curve of the sickle RBC population for patients 2, 6, and 7 in Figure 1a, 1b, and 1c. These plots show that the precision of the parameterized deformability curves compared to a standard healthy curve and actual measurement of the patient sample. Specifically, in Figure 1a and 1b shows there is large differences in the elongation curve of the sickle population in comparison to the bulk measurements, yielding a maximum EIs that are ~2 times smaller than the bulk measurement. Conversely, in Figure 1c we see there is not much difference in the elongation curve of the sickle population compared to the actual raw standard measurement. Conclusions: Overall, this work presents an innovative method for a more thorough examination of ektacytometry deformability measurements of SCD patients. An understanding of bulk stiffness of the HbS population is crucial as it would allow for more accurate characterization of the impact of RBC rigidity on SCD patient crisis and general wellbeing. More detailed information on the rigidity of the HbS RBC population in SCD has the potential to be utilized to understand phenomena in hemodynamics and connection between RBC rigidity and occurrences of painful crisis in SCD patients. Disclosures No relevant conflicts of interest to declare.

MORPHOLOGICAL AND MORPHOMETRICAL FEATURES IN DUNALIELLA SALINA (CHLAMYDOMONADALES, DUNALIELLACEAE) DURING THE TWO-PHASE CULTIVATION MODE

The paper presents studies of morphological and morphometrical characteristics of green halophilic carotenogenic microalga Dunaliella salina (Dunal) Teodoresco, 1905, from the south-west region of Crimean Peninsula. D. salina was cultivated in two-phase mode under conditions of natural illumination at the premises of A.O. Kovalevsky Institute of Biology of the Southern Seas of RAS (IBSS), Sevastopol, Russia. The maximum D. salina cell density was 1.69·106 cell/ml in the “green” phase and 0.84·106 cell/ml in the “red” growth phase. The maximum productivity by cell number reached 0.15 ∙ 106 cell/(ml·day)in the “green” phase while it was lower by 73% in the “red” phase (0.04∙106 cell/(ml·day). Along with the maximum productivity, linear growth stage in the first phase was characterized by a maximum fraction of small (up to 500 μm3 in volume) cells (about 15-29%) and a decrease in cell volume by 40-45% as compared with initial value. The mean of D. salina cell volume in the “red” phase was 30% higher than in the “green” phase. At the same time, the large cell fraction in the “red” phase was consistently high (15-35%). The patterns of change in morphological and morphometrical cell parameters were in accordance to stage and conditions of growth. Thus, cell elongation was noted in the stage of linear growth, while under unfavorable conditions at growth-declining stage cells became more round-shaped, with orange and tile-red coloration and granulation of cell content. It was shown that morphological and morphometric cell parameters can serve as additional criteria for assessment of physiological condition in D. salina culture. The experiment demonstrated the prospects for two-stage D. salina cultivation in Crimea.

Establishing a local bank and comparing efficacies of third party viral-specific cytotoxic T cells (VST) and cytokine induced killer cells (VSCIK)

Background & Aim Reactivation of dormant viruses is not uncommon after allogeneic haematopoietic stem cell transplants (allo-HSCT), which invariably carry risk of opportunistic infections. Although antiviral drugs are effective against a few viruses, drug therapy is limited by refractory strains and toxicities, while matched donor-derived VSTs have been successfully used to treat allo-HSCT patients with viral reactivation and there is promising data that minimally matched 3rd party banked VSTs are also effective and safe. Hence, we seek to establish a compact but effective bank of multi virus-specific cytotoxic cells for antiviral therapy not only for allo-HSCT patients but other immune compromised patients and patients with virus-associated malignancies more common in Asia. Methods, Results & Conclusion Previous studies have identified the common HLA types in Singaporeans: 96% of Singaporeans carry one or more of the common 6 HLA*A, 6 HLA*B and 5 HLA*C alleles. We are recruiting donors with these HLA types and screening their cells for reactivity against CMV, HHV6, AdV, EBV and BKV. Donor cells that express CD137 upon stimulation with commercially available virus-specific peptides will be further cultured with viral peptides, antibodies and cytokines to produce vsCIKs (with anti-CD137, IFNγ, OKT3 and IL2) or VSTs (with IL4 and IL7). Both cell types have, in clinical studies, been shown to be effective in modulating viral infections and elicit minimal or no graft-vs-host disease (GvHD). VsCIKs also have the advantage of retaining non-HLA restricted anti-tumour effects. As expected, both protocols produce a very small subset of naive T cells, which predicts low GvHD potential. Interestingly, vsCIKs consistently kill more target cells than VSTs, although a larger fraction of cells in VST cultures degranulate upon peptide stimulation. This incongruity warrants a more detailed study of the cytokines/enzymes released by the vsCIKs and VSTs, and the mechanisms involved. So far, no donor cells have shown reactivity against EBV or BKV peptides. This is despite a high percentage of EBV carriers among Singaporeans and the successful use of the same peptides in published reports. We are unsure if this is due to the unique repertoire of HLA types or the prevalent EBV strains in the local population. We have successfully generated CMV, HHV6 and AdV-specific vsCIKs and VSTs. However, further screening of viral peptides and mechanistic studies will be required to determine a suitable cell type for the proposed bank.

Cortical circuit alterations precede motor impairments in Huntington\u2019s disease mice

Huntington’s disease (HD) is a devastating hereditary movement disorder, characterized by degeneration of neurons in the striatum and cortex. Studies in human patients and mouse HD models suggest that disturbances of neuronal function in the neocortex play an important role in disease onset and progression. However, the precise nature and time course of cortical alterations in HD have remained elusive. Here, we use chronic in vivo two-photon calcium imaging to longitudinally monitor the activity of identified single neurons in layer 2/3 of the primary motor cortex in awake, behaving R6/2 transgenic HD mice and wildtype littermates. R6/2 mice show age-dependent changes in cortical network function, with an increase in activity that affects a large fraction of cells and occurs rather abruptly within one week, preceeding the onset of motor defects. Furthermore, quantitative proteomics demonstrate a pronounced downregulation of synaptic proteins in the cortex, and histological analyses in R6/2 mice and human HD autopsy cases reveal a reduction in perisomatic inhibitory synaptic contacts on layer 2/3 pyramidal cells. Taken together, our study provides a time-resolved description of cortical network dysfunction in behaving HD mice and points to disturbed excitation/inhibition balance as an important pathomechanism in HD.

Growth-inhibition of cell lines derived from B cell lymphomas through antagonism of serotonin receptor signaling

A majority of lymphomas are derived from B cells and novel treatments are required to treat refractory disease. Neurotransmitters such as serotonin and dopamine influence activation of B cells and the effects of a selective serotonin 1A receptor (5HT1A) antagonist on growth of a number of B cell-derived lymphoma cell lines were investigated. We confirmed the expression of 5HT1A in human lymphoma tissue and in several well-defined experimental cell lines. We discovered that the pharmacological inhibition of 5HT1A led to the reduced proliferation of B cell-derived lymphoma cell lines together with DNA damage, ROS-independent caspase activation and apoptosis in a large fraction of cells. Residual live cells were found ‘locked’ in a non-proliferative state in which a selective transcriptional and translational shutdown of genes important for cell proliferation and metabolism occurred (e.g., AKT, GSK-3β, cMYC and p53). Strikingly, inhibition of 5HT1A regulated mitochondrial activity through a rapid reduction of mitochondrial membrane potential and reducing dehydrogenase activity. Collectively, our data suggest 5HT1A antagonism as a novel adjuvant to established cancer treatment regimens to further inhibit lymphoma growth.

Taurine Is a Major Carbon and Energy Source for Marine Prokaryotes in the North Atlantic Ocean off the Iberian Peninsula

Taurine, an amino acid-like compound, acts as an osmostress protectant in many marine metazoans and algae and is released via various processes into the oceanic dissolved organic matter pool. Taurine transporters are widespread among members of the marine prokaryotic community, tentatively indicating that taurine might be an important substrate for prokaryotes in the ocean. In this study, we determined prokaryotic taurine assimilation and respiration throughout the water column along two transects in the North Atlantic off the Iberian Peninsula. Taurine assimilation efficiency decreased from the epipelagic waters from 55 ± 14% to 27 ± 20% in the bathypelagic layers (means of both transects). Members of the ubiquitous alphaproteobacterial SAR11 clade accounted for a large fraction of cells taking up taurine, especially in surface waters. Archaea (Thaumarchaeota + Euryarchaeota) were also able to take up taurine in the upper water column, but to a lower extent than Bacteria. The contribution of taurine assimilation to the heterotrophic prokaryotic carbon biomass production ranged from 21% in the epipelagic layer to 16% in the bathypelagic layer. Hence, we conclude that dissolved free taurine is a significant carbon and energy source for prokaryotes throughout the oceanic water column being utilized with similar efficiencies as dissolved free amino acids.