Nucleolar c‐Myc recruitment by a Vibrio T3SS effector promotes host cell proliferation and bacterial virulence

Pathogen type 3 secretion systems (T3SS) manipulate host cell pathways by directly delivering effector proteins into host cells. In Vibrio parahaemolyticus, the leading cause of bacterial seafood‐borne diarrheal disease, we showed that a T3SS effector, VgpA, localizes to the host cell nucleolus where it binds Epstein–Barr virus nuclear antigen 1‐binding protein 2 (EBP2). An amino acid substitution in VgpA (VgpA^L10A) did not alter its translocation to the nucleus but abolished the effector’s capacity to interact with EBP2. VgpA‐EBP2 interaction led to the re‐localization of c‐Myc to the nucleolus and increased cellular rRNA expression and proliferation of cultured cells. The VgpA‐EBP2 interaction elevated EBP2’s affinity for c‐Myc and prolonged the oncoprotein’s half‐life. Studies in infant rabbits demonstrated that VgpA is translocated into intestinal epithelial cells, where it interacts with EBP2 and leads to nucleolar re‐localization of c‐Myc. Moreover, the in vivo VgpA‐EBP2 interaction during infection led to proliferation of intestinal cells and heightened V. parahaemolyticus’ colonization and virulence. These observations suggest that direct effector stimulation of a c‐Myc controlled host cell growth program can contribute to pathogenesis.     

The relationship between cell division and elongation during development of the nectar-yielding petal spur in Centranthus ruber (Valerianaceae)

Background and Aims Floral spurs are hollow, tubular outgrowths that typically conceal nectar. By their involvement in specialized pollinator interactions, spurs have ecological and evolutionary significance, often leading to speciation. Despite their importance and diversity in shape and size among angiosperm taxa, detailed investigations of the mechanism of spur development have been conducted only recently.Methods Initiation and growth of the nectar-yielding petal spur of Centranthus ruber ‘Snowcloud’ was investigated throughout seven stages, based on bud size and developmental events. The determination of the frequency of cell division, quantified for the first time in spurs, was conducted by confocal microscopy following 4'',6-diamidino-2-phenylindole (DAPI) staining of mitotic figures. Moreover, using scanning electron microscospy of the outer petal spur surface unobstructed by trichomes, morphometry of epidermal cells was determined throughout development in order to understand the ontogeny of this elongate, hollow tube.Key Results Spur growth from the corolla base initially included diffuse cell divisions identified among epidermal cells as the spur progressed through its early stages. However, cell divisions clearly diminished before a petal spur attained 30 % of its final length of 4·5 mm. Thereafter until anthesis, elongation of individual cells was primarily responsible for the spur’s own extension. Consequently, a prolonged period of anisotropy, wherein epidermal cells elongated almost uniformly in all regions along the petal spur’s longitudinal axis, contributed principally to the spur’s mature length.Conclusions This research demonstrates that anisotropic growth of epidermal cells – in the same orientation as spur elongation – chiefly explains petal spur extension in C. ruber. Representing the inaugural investigation of the cellular basis for spur ontogeny within the Euasterids II clade, this study complements the patterns in Aquilegia species (order Ranunculales, Eudicots) and Linaria vulgaris (order Lamiales, Euasterids I), thereby suggesting the existence of a common underlying mechanism for petal spur ontogeny in disparate dicot lineages.     

Identifying rare and common disease associated variants in genomic data using Parkinson’s disease as a model

Background

Genome-wide association studies have been successful in identifying common genetic variants for human diseases. However, much of the heritable variation associated with diseases such as Parkinson’s disease remains unknown suggesting that many more risk loci are yet to be identified. Rare variants have become important in disease association studies for explaining missing heritability. Methods for detecting this type of association require prior knowledge on candidate genes and combining variants within the region. These methods may suffer from power loss in situations with many neutral variants or causal variants with opposite effects.

Results

We propose a method capable of scanning genetic variants to identify the region most likely harbouring disease gene with rare and/or common causal variants. Our method assigns a score at each individual variant based on our scoring system. It uses aggregate scores to identify the region with disease association. We evaluate performance by simulation based on 1000 Genomes sequencing data and compare with three commonly used methods. We use a Parkinson’s disease case–control dataset as a model to demonstrate the application of our method.Our method has better power than CMC and WSS and similar power to SKAT-O with well-controlled type I error under simulation based on 1000 Genomes sequencing data. In real data analysis, we confirm the association of α-synuclein gene (SNCA) with Parkinson’s disease (p = 0.005). We further identify association with hyaluronan synthase 2 (HAS2, p = 0.028) and kringle containing transmembrane protein 1 (KREMEN1, p = 0.006). KREMEN1 is associated with Wnt signalling pathway which has been shown to play an important role for neurodegeneration in Parkinson’s disease.

Conclusions

Our method is time efficient and less sensitive to inclusion of neutral variants and direction effect of causal variants. It can narrow down a genomic region or a chromosome to a disease associated region. Using Parkinson’s disease as a model, our method not only confirms association for a known gene but also identifies two genes previously found by other studies. In spite of many existing methods, we conclude that our method serves as an efficient alternative for exploring genomic data containing both rare and common variants.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0088-9) contains supplementary material, which is available to authorized users.     

Ganglioside-Dependent Neural Stem Cell Proliferation in Alzheimer’s Disease Model Mice

The aggregation and formation of amyloid plaques by amyloid β-peptides (Aβs) is believed to be one of the pathological hallmarks of Alzheimer’s disease (AD). Intriguingly, Aβs have also been shown to possess proliferative effects on neural stem cells (NSCs). Many essential cellular processes in NSCs, such as fate determination and proliferation, are heavily influenced by cell surface glycoconjugates, including gangliosides. It has recently been shown that Aβ1-42 alters several key glycosyltransferases and glycosidases. To further define the effects of Aβs and to clarify the potential mechanisms of action of those peptides on NSCs, NSCs were cultured from embryonic brains of the double-transgenic mouse model of AD [B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J] coexpressing mutants of amyloid precursor protein (APPswe) and presenilin1 (PSEN1dE9). We found that Aβs not only promoted cell proliferation but also altered expression of several key glycogenes for glycoconjugate metabolism, such as sialyltransferases II and III (ST-II & -III) in AD NSCs. In addition, we found upregulation of epidermal growth factor receptor and Notch1 intracellular domain. Moreover, the increased expression of ST-II and -III coincided with the elevated levels of c-series gangliosides (A2B5+ antigens) in AD NSCs. Further, we revealed that epidermal growth factor signaling and gangliosides are necessary components on Aβ-stimulated NSC proliferation. Our present study has thus provided a novel mechanism for the upregulation of c-series ganglioside expression and increases in several NSC markers to account for the proliferative effect of Aβs on NSCs in AD mouse brain. These observations support the potential beneficial effects of Aβs and gangliosides in promoting neurogenesis in AD brain.     

Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell

Delineation of a cell’s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused throughout tissues made of diverse cell types, such as enteroendocrine cells of the intestinal epithelium. These gastrointestinal sensors of food and bacteria have been difficult to study because they are dispersed among other epithelial cells at a ratio of 1:1,000. Recently, transgenic reporter mice have been generated to identify enteroendocrine cells by means of fluorescence. One of those is the peptide YY-GFP mouse. Using this mouse, we developed a method to correlate confocal and serial block-face scanning electron microscopy. We named the method cocem3D and applied it to identify a specific enteroendocrine cell in tissue and unveil the cell’s ultrastructure in 3D. The resolution of cocem3D is sufficient to identify organelles as small as secretory vesicles and to distinguish cell membranes for volume rendering. Cocem3D can be easily adapted to study the 3D ultrastructure of other specific cell types in their native tissue.     

Comparison of 2.5D and 3D Quantification of Femoral Head Coverage in Normal Control Subjects and Patients with Hip Dysplasia

Hip dysplasia is characterized by insufficient femoral head coverage (FHC). Quantification of FHC is of importance as the underlying goal of the surgery to treat hip dysplasia is to restore a normal acetabular morphology and thereby to improve FHC. Unlike a pure 2D X-ray radiograph-based measurement method or a pure 3D CT-based measurement method, previously we presented a 2.5D method to quantify FHC from a single anteriorposterior (AP) pelvic radiograph. In this study, we first quantified and compared 3D FHC between a normal control group and a patient group using a CT-based measurement method. Taking the CT-based 3D measurements of FHC as the gold standard, we further quantified the bias, precision and correlation between the 2.5D measurements and the 3D measurements on both the control group and the patient group. Based on digitally reconstructed radiographs (DRRs), we investigated the influence of the pelvic tilt on the 2.5D measurements of FHC. The intraclass correlation coefficients (ICCs) for absolute agreement was used to quantify interobserver reliability and intraobserver reproducibility of the 2.5D measurement technique. The Pearson correlation coefficient, r, was used to determine the strength of the linear association between the 2.5D and the 3D measurements. Student’s t-test was used to determine whether the differences between different measurements were statistically significant. Our experimental results demonstrated that both the interobserver reliability and the intraobserver reproducibility of the 2.5D measurement technique were very good (ICCs > 0.8). Regression analysis indicated that the correlation was very strong between the 2.5D and the 3D measurements (r = 0.89, p < 0.001). Student’s t-test showed that there were no statistically significant differences between the 2.5D and the 3D measurements of FHC on the patient group (p > 0.05). The results of this study provided convincing evidence demonstrating the validity of the 2.5D measurements of FHC from a single AP pelvic radiograph and proved that it could serve as a surrogate for 3D CT-based measurements. Thus it may be possible to use this method to avoid a CT scan for the purpose of estimating 3D FHC in diagnosis and post-operative treatment evaluation of patients with hip dysplasia.     

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel’s Cartilage

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. Patterning of the Meckel''s cartilage, a first pharyngeal arch derivative, involves the migration of cranial neural crest (CNC) cells and the progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described CNC migration during lower jaw morphogenesis, but the details of how the chondrocytes achieve organization in the growth and extension of Meckel’s cartilage remains unclear. The sox10 restricted and chemically induced Cre recombinase-mediated recombination generates permutations of distinct fluorescent proteins (RFP, YFP and CFP), thereby creating a multi-spectral labeling of progenitor cells and their progeny, reflecting distinct clonal populations. Using confocal time-lapse photography, it is possible to observe the chondrocytes behavior during the development of the zebrafish Meckel’s cartilage.Multispectral cell labeling enables scientists to demonstrate extension of the Meckel’s chondrocytes. During extension phase of the Meckel’s cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single-cell layered row. Failure of this organized intercalating process to mediate cell extension provides the cellular mechanistic explanation for hypoplastic mandible that we observe in mandibular malformations.     

Intercellular adhesion in butyrate-treated HeLa S3 cultures : Flow cytometric analysis

The application of DNA flow cytometry (FCM) for analysis of sodium butyrate-induced intercellular adhesion in human carcinoma (HeLa S3) cell cultures is described. To prepare cell suspensions for FCM, the monolayers of cells were treated with medium containing 10% serum, 0.2% non-ionic detergent Triton X-100 and 1 μg/ml DNA fluorochrome 4,6′-diamidino-2-phenylindole (DAPI). Total numbers of single cells, and aggregates containing two, three, four or more cells, were determined from DNA histograms. In cultures treated with 5 mM butyrate for 16 h, more than 80% of the cells were aggregated. Intercellular adhesion began to appear 8 h after addition of butyrate, was maximal at 16–24 h and stable in the presence of butyrate, but disappeared 24 h after its removal. Treatment with EDTA (0.2%) dissociated only 50%, whereas trypsin (0.1%) separated all cell aggregates into single cells. Actinomycin D (actD) (0.5 μg/ml) prevented cell adhesion while blocking of cells in S phase with 250 μM 5-fluorouracil or 10 μM methotrexate did not interfere with aggregation. The number of cell aggregates estimated from DNA histograms of butyrate-treated HeLa S3 cultures was the same after staining with DAPI in the presence of Triton X-100 or after vital staining with Hoechst 33342. The DNA content was used as a marker to estimate the cellular composition of aggregates in mixed cultures of HeLa S3 cells and human fibroblasts (U cells). Intercellular adhesion in these cultures was seen only between HeLa S3 cells, indicating specificity of butyrate-induced cell aggregation. FCM provides fast automatic measurement of cell aggregate formation, estimates frequency of aggregates containing different cell numbers, shows participation of cells at different cycle phases in aggregates, and allows the detection of homotypic from heterotypic cell aggregates if the interacting cells have different DNA ploidy.     

Single cell-derived spheroids capture the self-renewing subpopulations of metastatic ovarian cancer

High Grade Serous Ovarian cancer (HGSOC) is a major unmet need in oncology, due to its precocious dissemination and the lack of meaningful human models for the investigation of disease pathogenesis in a patient-specific manner. To overcome this roadblock, we present a new method to isolate and grow single cells directly from patients’ metastatic ascites, establishing the conditions for propagating them as 3D cultures that we refer to as single cell-derived metastatic ovarian cancer spheroids (sMOCS). By single cell RNA sequencing (scRNAseq) we define the cellular composition of metastatic ascites and trace its propagation in 2D and 3D culture paradigms, finding that sMOCS retain and amplify key subpopulations from the original patients’ samples and recapitulate features of the original metastasis that do not emerge from classical 2D culture, including retention of individual patients’ specificities. By enabling the enrichment of uniquely informative cell subpopulations from HGSOC metastasis and the clonal interrogation of their diversity at the functional and molecular level, this method provides a powerful instrument for precision oncology in ovarian cancer.Subject terms: Cancer models, Cancer stem cells, Experimental models of disease     

Phosphatidylinositol 3-Kinase/AKT Pathway Inhibition by Doxazosin Promotes Glioblastoma Cells Death,Upregulation of p53 and Triggers Low Neurotoxicity

Glioblastoma is the most frequent and malignant brain tumor. Treatment includes chemotherapy with temozolomide concomitant with surgical resection and/or irradiation. However, a number of cases are resistant to temozolomide, as well as the human glioblastoma cell line U138-MG. We investigated doxazosin’s (an antihypertensive drug) activity against glioblastoma cells (C6 and U138-MG) and its neurotoxicity on primary astrocytes and organoptypic hippocampal cultures. For this study, the following methods were used: citotoxicity assays, flow cytometry, western-blotting and confocal microscopy. We showed that doxazosin induces cell death on C6 and U138-MG cells. We observed that doxazosin’s effects on the PI3K/Akt pathway were similar as LY294002 (PI3K specific inhibitor). In glioblastoma cells treated with doxasozin, Akt levels were greatly reduced. Upon examination of activities of proteins downstream of Akt we observed upregulation of GSK-3β and p53. This led to cell proliferation inhibition, cell death induction via caspase-3 activation and cell cycle arrest at G0/G1 phase in glioblastoma cells. We used in this study Lapatinib, a tyrosine kinase inhibitor, as a comparison with doxazosin because they present similar chemical structure. We also tested the neurocitotoxicity of doxazosin in primary astrocytes and organotypic cultures and observed that doxazosin induced cell death on a small percentage of non-tumor cells. Aggressiveness of glioblastoma tumors and dismal prognosis require development of new treatment agents. This includes less toxic drugs, more selective towards tumor cells, causing less damage to the patient. Therefore, our results confirm the potential of doxazosin as an attractive therapeutic antiglioma agent.     

Polycystin‐1 regulates cell proliferation and migration through AKT/mTORC2 pathway in a human craniosynostosis cell model

Craniosynostosis is the premature fusion of skull sutures and has a severe pathological impact on childrens’ life. Mechanical forces are capable of triggering biological responses in bone cells and regulate osteoblastogenesis in cranial sutures, leading to premature closure. The mechanosensitive proteins polycystin‐1 (PC1) and polycystin‐2 (PC2) have been documented to play an important role in craniofacial proliferation and development. Herein, we investigated the contribution of PC1 to the pathogenesis of non‐syndromic craniosynostosis and the associated molecular mechanisms. Protein expression of PC1 and PC2 was detected in bone fragments derived from craniosynostosis patients via immunohistochemistry. To explore the modulatory role of PC1 in primary cranial suture cells, we further abrogated the function of PC1 extracellular mechanosensing domain using a specific anti‐PC1 IgPKD1 antibody. Effect of IgPKD1 treatment was evaluated with cell proliferation and migration assays. Activation of PI3K/AKT/mTOR pathway components was further detected via Western blot in primary cranial suture cells following IgPKD1 treatment. PC1 and PC2 are expressed in human tissues of craniosynostosis. PC1 functional inhibition resulted in elevated proliferation and migration of primary cranial suture cells. PC1 inhibition also induced activation of AKT, exhibiting elevated phospho (p)‐AKT (Ser473) levels, but not 4EBP1 or p70S6K activation. Our findings indicate that PC1 may act as a mechanosensing molecule in cranial sutures by modulating osteoblastic cell proliferation and migration through the PC1/AKT/mTORC2 cascade with a potential impact on the development of non‐syndromic craniosynostosis.     

Distinct Biology of Kaposi’s Sarcoma-Associated Herpesvirus from Primary Lesions and Body Cavity Lymphomas

The DNA sequence for Kaposi’s sarcoma-associated herpesvirus was originally detected in Kaposi’s sarcoma biopsy specimens. Since its discovery, it has been possible to detect virus in cell lines established from AIDS-associated body cavity-based B-cell lymphoma and to propagate virus from primary Kaposi’s sarcoma lesions in a human renal embryonic cell line, 293. In this study, we analyzed the infectivity of Kaposi’s sarcoma-associated herpesvirus produced from these two sources. Viral isolates from cultured cutaneous primary KS cells was transmitted to an Epstein-Barr virus-negative Burkitt’s B-lymphoma cell line, Louckes, and compared to virus induced from a body cavity-based B-cell lymphoma cell line. While propagation of body cavity-based B-cell lymphoma-derived virus was not observed in 293 cell cultures, infection with viral isolates obtained from primary Kaposi’s sarcoma lesions induced injury in 293 cells typical of herpesvirus infection and was associated with apoptotic cell death. Interestingly, transient overexpression of the Kaposi’s sarcoma-associated herpesvirus v-Bcl-2 homolog delayed the process of apoptosis and prolonged the survival of infected 293 cells. In contrast, the broad-spectrum caspase inhibitors Z-VAD-fmk and Z-DEVD-fmk failed to protect infected cell cultures, suggesting that Kaposi’s sarcoma-associated herpesvirus-induced apoptosis occurs through a Bcl-2-dependent pathway. Kaposi’s sarcoma-associated herpesvirus isolates from primary Kaposi’s sarcoma lesions and body cavity-based lymphomas therefore may differ and are likely to have distinct contributions to the pathophysiology of Kaposi’s sarcoma.     

Erianin induces triple-negative breast cancer cells apoptosis by activating PI3K/Akt pathway

Background: Triple-negative breast cancer (TNBC) is a refractory subtype of breast cancer, 25–30% of which have dysregulation in the PI3K/AKT pathway. The present study investigated the anticancer effect of erianin on TNBC cell line and its underlying mechanism.Methods: After treatment with erianin, MTT assay was employed to determine the MDA-MB-231 and EFM-192A cell proliferation, the nucleus morphological changes were observed by DAPI staining. The cell cycle and apoptotic proportion were detected by flow cytometry. Western blot was performed to determine the cell cycle and apoptosis-related protein expression and PI3K pathways. Finally, the antiproliferative activity of erianin was further confirmed by adding or not adding PI3K agonists SC79.Results: Erianin inhibited the proliferation of MDA-MB-231 and EFM-192A cells in a dose-dependent manner, the IC₅₀ were 70.96 and 78.58 nM, respectively. Erianin could cause cell cycle arrest at the G₂/M phase, and the expressions of p21 and p27 were up-regulated, while the expressions of CDK1 and Cyclin B1 were down-regulated. Erianin also induced apoptosis via the mitochondrial pathway, with the up-regulation of the expression of Cyto C, PARP, Bax, active form of Caspase-3, and Caspase-9. Furthermore, p-PI3K and p-Akt expression were down-regulated by erianin. After co-incubation with SC79, the cell inhibition rate of erianin was decreased, which further confirmed that the attenuated PI3K/Akt pathway was relevant to the pro-apoptotic effect of erianin.Conclusions: Erianin can inhibit the proliferation of TNBC cells and induce cell cycle arrest and apoptosis, which may ascribe to the abolish the activation of the PI3K/Akt pathway.     

Adaptive Geometric Tessellation for 3D Reconstruction of Anisotropically Developing Cells in Multilayer Tissues from Sparse Volumetric Microscopy Images

The need for quantification of cell growth patterns in a multilayer, multi-cellular tissue necessitates the development of a 3D reconstruction technique that can estimate 3D shapes and sizes of individual cells from Confocal Microscopy (CLSM) image slices. However, the current methods of 3D reconstruction using CLSM imaging require large number of image slices per cell. But, in case of Live Cell Imaging of an actively developing tissue, large depth resolution is not feasible in order to avoid damage to cells from prolonged exposure to laser radiation. In the present work, we have proposed an anisotropic Voronoi tessellation based 3D reconstruction framework for a tightly packed multilayer tissue with extreme z-sparsity (2–4 slices/cell) and wide range of cell shapes and sizes. The proposed method, named as the ‘Adaptive Quadratic Voronoi Tessellation’ (AQVT), is capable of handling both the sparsity problem and the non-uniformity in cell shapes by estimating the tessellation parameters for each cell from the sparse data-points on its boundaries. We have tested the proposed 3D reconstruction method on time-lapse CLSM image stacks of the Arabidopsis Shoot Apical Meristem (SAM) and have shown that the AQVT based reconstruction method can correctly estimate the 3D shapes of a large number of SAM cells.     

SH-SY5Y and LUHMES cells display differential sensitivity to MPP+, tunicamycin,and epoxomicin in 2D and 3D cell culture

SH-SY5Y and LUHMES cell lines are widely used as model systems for studying neurotoxicity. Most of the existing data regarding the sensitivity of these cell lines to neurotoxicants have been recorded from cells growing as two-dimensional (2D) cultures on the surface of glass or plastic. With the emergence of 3D culture platforms designed to better represent native tissue, there is a growing need to compare the toxicology of neurons grown in 3D environments to those grown in 2D to better understand the impact that culture environment has on toxicant sensitivity. Here, a simple 3D culture method was used to assess the impact of growth environment on the sensitivity of SH-SY5Y cells and LUHMES cells to MPP+, tunicamycin, and epoxomicin, three neurotoxicants that have been previously used to generate experimental models for studying Parkinson's disease pathogenesis. SH-SY5Y cell viability following treatment with these three toxicants was significantly lower in 2D cultures as compared to 3D cultures. On the contrary, LUHMES cells did not show significant differences between growth conditions for any of the toxicants examined. However, LUHMES cells were more sensitive to MPP+, tunicamycin, and epoxomicin than SH-SY5Y cells. Thus, both the choice of cell line and the choice of growth environment must be considered when interpreting in vitro neurotoxicity data.     

Manually and automatically produced pellet cultures of human primary chondrocytes: A comparative analysis

Cartilage defects are often associated with restriction of the locomotor system. New methods are required to investigate cartilage tissue and for the repair of cartilage tissue. 3D cultures are promising due to better simulation of in vivo conditions. The aim of this study was to provide a model system for studying cartilage tissue. We solved this problem by automated production of pellet cultures of human primary chondrocytes in media with and without antibiotics using the Biomek® Cell Workstation and consequent automated bioscreening with a high‐throughput screening system, and compared with the regular manual processes. The Biomek® Cell Workstation allows the cultivation of different cell types (suspensions cells and adherent cells) and 3D cell cultures (pellet cultures, alginate beads and spheroid cultures). The proliferation was analyzed by DNA quantification and compared with the EZ4U proliferation assay as a new tool for pellet cultures. The toxicity was evaluated by the detection of ubiquitous adenylate kinases. The proliferation increased from day 14 until day 35 and was associated with a decrease in the cytotoxicity. The comparative analysis showed similar results for manual and automated processes. We concluded that the manual methods can be replaced by automated processes (pellet manufacturing and screening), which would allow large‐scale procedures to support studies on cartilage regeneration.     

Halofuginone Synergistically Enhances Anti-Proliferation of Rapamycin in T Cells and Reduces Cytotoxicity of Cyclosporine in Cultured Renal Tubular Epithelial Cells

Both rapamycin (RAPA) and cyclosporin A (CsA) are commonly used for immunosuppression, however their adverse side effects limit their application. Thus, it is of interest to develop novel means to enhance or preserve the immunosuppressive activity of RAPA or CsA while reducing their toxicity. Halofuginone (HF) has been recently tested as a potential immunosuppressant. This study investigated the interaction of HF with RAPA or with CsA in cell cultures. Cell proliferation in cultures was determined using methylthiazol tetrazolium assay, and cell apoptosis assessed by flow cytometric analysis and Western blot. The drug-drug interaction was determined according to Loewe’s equation or Bliss independence. Here, we showed that addition of HF to anti-CD 3 antibody-stimulated splenocyte cultures induced synergistic suppression of T cell proliferation in the presence of RAPA, indicated by an interaction index (γ) value of < 1.0 between HF and RAPA, but not in those with CsA. The synergistic interaction of RAPA with HF in the suppression of T cell proliferation was also seen in a mixed lymphocyte reaction and Jurkat T cell growth, and was positively correlated with an increase in cell apoptosis, but not with proline depletion. In cultured kidney tubular epithelial cells, HF attenuated the cytotoxicity of CsA. In conclusion, these data indicate that HF synergistically enhances anti-T cell proliferation of RAPA and reduces the nephrotoxicity of CsA in vitro, suggesting the potential use of HF for enhancing anti-T cell proliferation of RAPA and reducing CsA-mediated nephrotoxicity.     

Insights into the regulation of neuronal viability by nucleophosmin/B23

The vastness of the neuronal network that constitutes the human brain proves challenging when trying to understand its complexity. Furthermore, due to the senescent state they enter into upon maturation, neurons lack the ability to regenerate in the face of insult, injury or death. Consequently, their excessive death can be detrimental to the proper functioning of the brain. Therefore, elucidating the mechanisms regulating neuronal survival is, while challenging, of great importance as the incidence of neurological disease is becoming more prevalent in today’s society. Nucleophosmin/B23 (NPM) is an abundant and ubiquitously expressed protein that regulates vital cellular processes such as ribosome biogenesis, cell proliferation and genomic stability. As a result, it is necessary for proper embryonic development, but has also been implicated in many cancers. While highly studied in the context of proliferative cells, there is a lack of understanding NPM’s role in post-mitotic neurons. By exploring its role in healthy neurons as well as its function in the regulation of cell death and neurodegeneration, there can be a better understanding of how these diseases initiate and progress. Owing to what is thus far known about its function in the cell, NPM could be an attractive therapeutic target in the treatment of neurodegenerative diseases.     

Differential Growth Response of Colony-Forming α- and γ-Proteobacteria in Dilution Culture and Nutrient Addition Experiments from Lake Kinneret (Israel), the Eastern Mediterranean Sea, and the Gulf of Eilat

Even though it is widely accepted that bacterioplankton growth in lakes and marine ecosystems is determined by the trophic status of the systems, knowledge of the relationship between nutrient concentrations and growth of particular bacterial species is almost nonexistent. To address this question, we performed a series of culture experiments with water from Lake Kinneret (Israel), the eastern Mediterranean Sea, and the Gulf of Eilat (northern Red Sea). In the initial water samples, the proportion of CFU was typically <0.002% of the 4′,6′-diamidino-2-phenylindole (DAPI) counts. During incubation until the early stationary phase, the proportion of CFU increased to 20% of the DAPI counts and to 2 to 15% of the DAPI counts in unenriched lake water and seawater dilution cultures, respectively. Sequencing of the 16S ribosomal DNA of colony-forming bacteria in these cultures consistently revealed an abundance of α-proteobacteria, but notable phylogenetic differences were found at the genus level. Marine dilution cultures were dominated by bacteria in the Roseobacter clade, while lake dilution cultures were dominated by bacteria affiliated with the genera Sphingomonas and Caulobacter. In nutrient (glucose, ammonium, phosphate) addition experiments the CFU comprised 20 to 83% of the newly grown cells. In these incubation experiments fast-growing γ-proteobacteria dominated; in the marine experiments primarily different Vibrio and Alteromonas species appeared, while in the lake water experiments species of the genera Shewanella, Aeromonas, and Rheinheimera grew. These results suggest that major, but different, γ-proteobacterial genera in both freshwater and marine environments have a preference for elevated concentrations of nutrients and easily assimilated organic carbon sources but are selectively outcompeted by α-proteobacteria in the presence of low nutrient concentrations.