Dishevelled is a NEK2 kinase substrate controlling dynamics of centrosomal linker proteins.

Dishevelled (DVL) is a key scaffolding protein and a branching point in Wnt signaling pathways. Here, we present conclusive evidence that DVL regulates the centrosomal cycle. We demonstrate that DVL dishevelled and axin (DIX) domain, but not DIX domain-mediated multimerization, is essential for DVL's centrosomal localization. DVL accumulates during the cell cycle and associates with NIMA-related kinase 2 (NEK2), which is able to phosphorylate DVL at a multitude of residues, as detected by a set of novel phospho-specific antibodies. This creates interfaces for efficient binding to CDK5 regulatory subunit-associated protein 2 (CDK5RAP2) and centrosomal Nek2-associated protein 1 (C-NAP1), two proteins of the centrosomal linker. Displacement of DVL from the centrosome and its release into the cytoplasm on NEK2 phosphorylation is coupled to the removal of linker proteins, an event necessary for centrosomal separation and proper formation of the mitotic spindle. Lack of DVL prevents NEK2-controlled dissolution of loose centrosomal linker and subsequent centrosomal separation. Increased DVL levels, in contrast, sequester centrosomal NEK2 and mimic monopolar spindle defects induced by a dominant negative version of this kinase. Our study thus uncovers molecular crosstalk between centrosome and Wnt signaling.

The Effect of Uncoated SPIONs on hiPSC-Differentiated Endothelial Cells.

BACKGROUND: Endothelial progenitor cells (EPCs) were indicated in vascular repair, angiogenesis of ischemic organs, and inhibition of formation of initial hyperplasia. Differentiation of endothelial cells (ECs) from human induced pluripotent stem cells (hiPSC)-derived endothelial cells (hiPSC-ECs) provides an unlimited supply for clinical application. Furthermore, magnetic cell labelling offers an effective way of targeting and visualization of hiPSC-ECs and is the next step towards in vivo studies. METHODS: ECs were differentiated from hiPSCs and labelled with uncoated superparamagnetic iron-oxide nanoparticles (uSPIONs). uSPION uptake was compared between hiPSC-ECs and mature ECs isolated from patients by software analysis of microscopy pictures after Prussian blue cell staining. The acute and long-term cytotoxic effects of uSPIONs were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) and Annexin assay. RESULTS: We showed, for the first time, uptake of uncoated SPIONs (uSPIONs) by hiPSC-ECs. In comparison with mature ECs of identical genetic background hiPSC-ECs showed lower uSPION uptake. However, all the studied endothelial cells were effectively labelled and showed magnetic properties even with low labelling concentration of uSPIONs. uSPIONs prepared by microwave plasma synthesis did not show any cytotoxicity nor impair endothelial properties. CONCLUSION: We show that hiPSC-ECs labelling with low concentration of uSPIONs is feasible and does not show any toxic effects in vitro, which is an important step towards animal studies.

Mutated Huntingtin Causes Testicular Pathology in Transgenic Minipig Boars.

BACKGROUND: Huntington's disease is induced by CAG expansion in a single gene coding the huntingtin protein. The mutated huntingtin (mtHtt) primarily causes degeneration of neurons in the brain, but it also affects peripheral tissues, including testes. OBJECTIVE: We studied sperm and testes of transgenic boars expressing the N-terminal region of human mtHtt. METHODS: In this study, measures of reproductive parameters and electron microscopy (EM) images of spermatozoa and testes of transgenic (TgHD) and wild-type (WT) boars of F1 (24-48 months old) and F2 (12-36 months old) generations were compared. In addition, immunofluorescence, immunohistochemistry, Western blot, hormonal analysis and whole-genome sequencing were done in order to elucidate the effects of mtHtt. RESULTS: Evidence for fertility failure of both TgHD generations was observed at the age of 13 months. Reproductive parameters declined and progressively worsened with age. EM revealed numerous pathological features in sperm tails and in testicular epithelium from 24- and 36-month-old TgHD boars. Moreover, immunohistochemistry confirmed significantly lower proliferation activity of spermatogonia in transgenic testes. mtHtt was highly expressed in spermatozoa and testes of TgHD boars and localized in all cells of seminiferous tubules. Levels of fertility-related hormones did not differ in TgHD and WT siblings. Genome analysis confirmed that insertion of the lentiviral construct did not interrupt any coding sequence in the pig genome. CONCLUSIONS: The sperm and testicular degeneration of TgHD boars is caused by gain-of-function of the highly expressed mtHtt.

Exacerbation of substrate toxicity by IPTG in Escherichia coli BL21(DE3) carrying a synthetic metabolic pathway.

BACKGROUND: Heterologous expression systems based on promoters inducible with isopropyl-ß-D-1-thiogalactopyranoside (IPTG), e.g., Escherichia coli BL21(DE3) and cognate LacI(Q)/P(lacUV5)-T7 vectors, are commonly used for production of recombinant proteins and metabolic pathways. The applicability of such cell factories is limited by the complex physiological burden imposed by overexpression of the exogenous genes during a bioprocess. This burden originates from a combination of stresses that may include competition for the expression machinery, side-reactions due to the activity of the recombinant proteins, or the toxicity of their substrates, products and intermediates. However, the physiological impact of IPTG-induced conditional expression on the recombinant host under such harsh conditions is often overlooked. RESULTS: The physiological responses to IPTG of the E. coli BL21(DE3) strain and three different recombinants carrying a synthetic metabolic pathway for biodegradation of the toxic anthropogenic pollutant 1,2,3-trichloropropane (TCP) were investigated using plating, flow cytometry, and electron microscopy. Collected data revealed unexpected negative synergistic effect of inducer of the expression system and toxic substrate resulting in pronounced physiological stress. Replacing IPTG with the natural sugar effector lactose greatly reduced such stress, demonstrating that the effect was due to the original inducer's chemical properties. CONCLUSIONS: IPTG is not an innocuous inducer; instead, it exacerbates the toxicity of haloalkane substrate and causes appreciable damage to the E. coli BL21(DE3) host, which is already bearing a metabolic burden due to its content of plasmids carrying the genes of the synthetic metabolic pathway. The concentration of IPTG can be effectively tuned to mitigate this negative effect. Importantly, we show that induction with lactose, the natural inducer of P lac , dramatically lightens the burden without reducing the efficiency of the synthetic TCP degradation pathway. This suggests that lactose may be a better inducer than IPTG for the expression of heterologous pathways in E. coli BL21(DE3).

Novosphingobium barchaimii sp. nov., isolated from hexachlorocyclohexane-contaminated soil.

A yellow-pigmented bacterial strain, designated LL02(T), was isolated from hexachlorocyclohexane-contaminated soil from Spolana Neratovice, a former Czech producer of lindane. A neighbour-joining tree based on 16S rRNA gene sequences showed that strain LL02(T) occupied a distinct phylogenetic position in the genus Novosphingobium and showed the highest sequence similarity with Novosphingobium resinovorum NCIMB 8767(T) (98.59 %). DNA-DNA relatedness between strain LL02(T) and its closest phylogenetic neighbours was <70 %, which indicated that strain LL02(T) represented a novel species of the genus Novosphingobium. The DNA G+C content of strain LL02(T) was 67.72±0 mol%. The major respiratory quinone was ubiquinone Q-10. The polar lipid profile of the isolate corresponded to those reported for other members of the genus Novosphingobium (phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylmonomethylethanolamine and sphingoglycolipids), thus supporting its classification in the genus. Spermidine was the major polyamine. The major fatty acids were summed feature 3 (consisting of C(16 : 1)ω7c and/or C(16 : 1)ω6c; 40.13 %), summed feature 8 (consisting of C(18 : 1)ω7c and/or C(18 : 1)ω6c; 31.09 %) and C(14 : 0) 2-OH (23.16 %). The results obtained from DNA-DNA hybridization and biochemical and physiological tests clearly distinguished the isolate from its closest phylogenetic neighbours. Thus, strain LL02(T) represents a novel species of the genus Novosphingobium, for which the name Novosphingobium barchaimii sp. nov. is proposed. The type strain is LL02(T) ( = CCM 7980(T)  = DSM 25411(T)).

Sphingobium baderi sp. nov., isolated from a hexachlorocyclohexane dump site.

A Gram-stain-negative, rod-shaped and white-coloured bacterial strain, designated LL03(T), was isolated from hexachlorocyclohexane-contaminated soil at Spolana Neratovice, Czech Republic, where lindane was formerly produced. Strain LL03(T) was found to be a degrader of α-, γ- and δ-isomers of hexachlorocyclohexane, although no significant degradation activity was observed for the ß-isomer. A neighbour-joining tree based on 16S rRNA gene sequences showed that strain LL03(T) occupied a distinct phylogenetic position in the Sphingobium cluster, showing the highest similarity with Sphingobium wenxiniae JZ-1(T) (99.2 %). The DNA G+C content of strain LL03(T) was 67.0 mol%. DNA-DNA relatedness values of strain LL03(T) with its close phylogenetic neighbours were below the threshold level of 70 %, supporting its identification as a representative of a novel species of the genus Sphingobium. The predominant respiratory quinone was ubiquinone Q-10. The polar lipid profile of strain LL03(T) also corresponded to those reported for other Sphingobium species (phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylmonomethylethanolamine and sphingoglycolipid), supporting its identification as a member of the genus Sphingobium. Spermidine was identified as the major polyamine. The predominant fatty acids were 16 : 0, summed feature 3 (16 : 1ω7c and/or 16 : 1ω6c), summed feature 8 (18 : 1ω7c and/or 18 : 1ω6c) and 14 : 0 2-OH. The polar lipid pattern, the presence of spermidine and ubiquinone Q-10, the predominance of the cellular fatty acids C(18 : 1)ω7c, C(16 : 0) and C(14 : 0) 2-OH and the G+C content of the genomic DNA supported the affiliation of the strain to the genus Sphingobium. The results obtained after DNA-DNA hybridization, biochemical and physiological tests clearly distinguished it from closely related species of the genus Sphingobium. Therefore, strain LL03(T) represents a novel species of the genus Sphingobium for which the name Sphingobium baderi LL03(T) sp. nov. is proposed; the type strain is LL03(T) ( = CCM 7981(T) = DSM 25433(T)).

Sphingobium czechense sp. nov., isolated from a hexachlorocyclohexane dump site.

A yellow-pigmented bacterial strain, designated LL01(T), was isolated from hexachlorocyclohexane (HCH)-contaminated soil at Spolana Neratovice, a former Czech producer of lindane. A neighbour-joining tree based on 16S rRNA gene sequences showed that strain LL01(T) occupied a distinct phylogenetic position in the Sphingobium cluster, showing highest similarity to Sphingobium rhizovicinum CC-FH12-1(T) (98.5 %). The DNA G+C content of strain LL01(T) was 66.1 mol%. The predominant respiratory pigment was ubiquinone Q-10. The polar lipid profile of strain LL01(T) also corresponded to those reported for other Sphingobium species (phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine, sphingoglycolipids), supporting its identification as a member of the genus Sphingobium. Spermidine was the major polyamine observed. The results obtained from DNA-DNA hybridization and biochemical and physiological tests clearly distinguished strain LL01(T) from closely related species of the genus Sphingobium. Therefore, strain LL01(T) represents a novel species of the genus Sphingobium, for which the name Sphingobium czechense sp. nov. is proposed (type strain LL01(T) = CCM 7979(T) = DSM 25410(T)).

Human embryonic stem cells suffer from centrosomal amplification.

Propagation of human embryonic stem cells (hESCs) in culture tends to alter karyotype, potentially limiting the prospective use of these cells in patients. The chromosomal instability of some malignancies is considered to be driven, at least in part, by centrosomal overamplification, perturbing balanced chromosome segregation. Here, we report, for the first time, that very high percentage of cultured hESCs has supernumerary centrosomes during mitosis. Supernumerary centrosomes were strictly associated with an undifferentiated hESC state and progressively disappeared on prolonged propagation in culture. Improved attachment to culture substratum and inhibition of CDK2 and Aurora A (key regulators of centrosomal metabolism) diminished the frequency of multicentrosomal mitoses. Thus, both attenuated cell attachment and deregulation of machinery controlling centrosome number contribute to centrosomal overamplification in hESCs. Linking the excessive number of centrosomes in mitoses to the ploidy indicated that both overduplication within a single cell cycle and mitotic failure contributed to generation of numerical centrosomal abnormalities in hESCs. Collectively, our data indicate that supernumerary centrosomes are a significant risk factor for chromosome instability in cultured hESCs and should be evaluated when new culture conditions are being implemented.

Fatty Acid Supplementation Affects Skin Wound Healing in a Rat Model.

Polyunsaturated fatty acids (PUFA) play an important role in reparative processes. The ratio of PUFAs n-3 to n-6 may affect wound healing. The study aimed to evaluate the effect of dietary supplementation with n-3 and n-6 PUFA in two proportions on skin wounds in laboratory rats. Adult male Wistar rats received 20% fat emulsion with a ratio of 1.4:1 (group A) or 4.3:1 (group B) for n-3:n-6 PUFAs at a daily dose of 1 mL/kg. The control group received water under the same conditions. The animals were supplemented a week before and a week after the skin excision performed on the back. The level of wound closure, various parameters of oxidative stress, and plasma fatty acids composition were evaluated. Wound tissue samples were examined by electron microscopy. The administration of fat emulsions led to significant changes in plasma polyunsaturated fatty acid composition. The increased production of reactive nitrogen species, as well as more numerous newly formed blood vessels and a greater amount of highly organized collagen fibrils in both groups A and B may indicate more intensive healing of the skin wound in rats supplemented with polyunsaturated fatty acids in high n-3:n-6 ratio.

Advantages of nanofibrous membranes for culturing of primary RPE cells compared to commercial scaffolds.

PURPOSE: Dysfunction of the retinal pigment epithelium (RPE) causes numerous forms of retinal degeneration. RPE replacement is a modern option to save vision. We aimed to test the results of transplanting cultured RPEs on biocompatible membranes. METHODS: We cultivated porcine primary RPE cells isolated from cadaver eyes from the slaughterhouse on two types of membranes: commercial polyester scaffolds Transwell (Corning Inc., Kenneburg, ME, USA) with 0.4 µm pore size and prepared Poly (L-lactide-co-DL-lactide) (PDLLA) nanofibrous membranes with an average pore size of 0.4 µm. RESULTS: Five types of assays were used for the analysis: immunocytochemistry (ICC), phagocytosis assay, Western blotting, real-time qPCR (RT-qPCR) and electron microscopy. RT-qPCR demonstrated that RPEs cultured on nanofibrous membranes have higher expressions of BEST1 (bestrophin 1), RLBP1 (retinaldehyde-binding protein 1), RPE65 (retinal pigment epithelium-specific 65 kDa protein), PAX6 (transcription factor PAX6), SOX9 (transcription factor SOX9), DCT (dopachrome tautomerase) and MITF (microphthalmia-associated transcription factor). ICC of the RPEs cultured on nanofibrous membranes showed more intensive staining of markers such as BEST1, MCT1 (monocarboxylate transporter 1), Na+ /K+ ATPase, RPE65 and acetylated tubulin in comparison with commercial ones. Additionally, the absence of α-SMA proved the stability of the RPE polarization state and the absence of epithelial-to-mesenchymal transition. RPE possessed high phagocytic activity. Electron microscopy of both membranes confirmed a confluent layer of RPE cells and their genuine morphological structure, which was comparable to native RPEs. CONCLUSIONS: Retinal pigment epitheliums cultured on polylactide nanofibrous membranes improved the final quality of the cell product by having better maturation and long-term survival of the RPE monolayer compared to those cultured on commercial polyester scaffolds. PDLLA-cultured RPEs are a plausible source for the replacement of non-functioning RPEs during cell therapy.

Dual Targeting of BRAF and mTOR Signaling in Melanoma Cells with Pyridinyl Imidazole Compounds.

BRAF inhibitors can delay the progression of metastatic melanoma, but resistance usually emerges, leading to relapse. Drugs simultaneously targeting two or more pathways essential for cancer growth could slow or prevent the development of resistant clones. Here, we identified pyridinyl imidazole compounds SB202190, SB203580, and SB590885 as dual inhibitors of critical proliferative pathways in human melanoma cells bearing the V600E activating mutation of BRAF kinase. We found that the drugs simultaneously disrupt the BRAF V600E-driven extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activity and the mechanistic target of rapamycin complex 1 (mTORC1) signaling in melanoma cells. Pyridinyl imidazole compounds directly inhibit BRAF V600E kinase. Moreover, they interfere with the endolysosomal compartment, promoting the accumulation of large acidic vacuole-like vesicles and dynamic changes in mTOR signaling. A transient increase in mTORC1 activity is followed by the enrichment of the Ragulator complex protein p18/LAMTOR1 at contact sites of large vesicles and delocalization of mTOR from the lysosomes. The induced disruption of the endolysosomal pathway not only disrupts mTORC1 signaling, but also renders melanoma cells sensitive to endoplasmic reticulum (ER) stress. Our findings identify new activities of pharmacologically relevant small molecule compounds and provide a biological rationale for the development of anti-melanoma therapeutics based on the pyridinyl imidazole core.

Transgenic minipig model of Huntington's disease exhibiting gradually progressing neurodegeneration.

Recently developed therapeutic approaches for the treatment of Huntington's disease (HD) require preclinical testing in large animal models. The minipig is a suitable experimental animal because of its large gyrencephalic brain, body weight of 70-100 kg, long lifespan, and anatomical, physiological and metabolic resemblance to humans. The Libechov transgenic minipig model for HD (TgHD) has proven useful for proof of concept of developing new therapies. However, to evaluate the efficacy of different therapies on disease progression, a broader phenotypic characterization of the TgHD minipig is needed. In this study, we analyzed the brain tissues of TgHD minipigs at the age of 48 and 60-70 months, and compared them to wild-type animals. We were able to demonstrate not only an accumulation of different forms of mutant huntingtin (mHTT) in TgHD brain, but also pathological changes associated with cellular damage caused by mHTT. At 48 months, we detected pathological changes that included the demyelination of brain white matter, loss of function of striatal neurons in the putamen and activation of microglia. At 60-70 months, we found a clear marker of neurodegeneration: significant cell loss detected in the caudate nucleus, putamen and cortex. This was accompanied by clusters of structures accumulating in the neurites of some neurons, a sign of their degeneration that is also seen in Alzheimer's disease, and a significant activation of astrocytes. In summary, our data demonstrate age-dependent neuropathology with later onset of neurodegeneration in TgHD minipigs.

A Comprehensive In Vitro Comparison of Preparation Techniques for Fat Grafting.

BACKGROUND: Lipomodeling is a technique that uses the patient's own fat for tissue regeneration and augmentation. The extent of regenerative effect is reported to be determined by the numbers of adipose-derived stem cells and the viability of cells in processed adipose tissue which, together with other factors, influence the degree of graft retention. This study addresses whether differences exist in properties of fat graft obtained by three commonly used techniques. METHODS: Adipose tissue harvested from the hypogastric regions of 14 patients was processed by decantation, centrifugation, and membrane-based tissue filtration. The morphology of each preparation was assessed by electron microscopy and overall cell viability was assessed by live/dead assay. The number of adipose-derived stem cells was determined and their stem cell character was assessed by the presence of cell surface molecules (i.e., CD105, CD90, CD31, and CD45) and by their capacity to differentiate into adipogenic and osteogenic lineages. RESULTS: First, morphologies of processed fat samples obtained by individual procedures differed, but no preparation caused obvious damage to cellular or acellular components. Second, although the highest numbers of adipose-derived stem cells were contained in the upper fraction of centrifuged lipoaspirates, the difference between preparations was marginal. Third, the maximal concentration of adipose fraction (removal of watery component) of lipoaspirate was achieved by membrane-based tissue filtration. Finally, no significant differences in overall viability were detected. CONCLUSIONS: Properties of processed lipoaspirate were influenced by the preparation procedure. However, the differences were not dramatic; both centrifugation and membrane-based filtration are methods of choice whose selection depends on other criteria (e.g., practicality) for individual surgical settings.

HIF-1alpha Deficiency Attenuates the Cardiomyogenesis of Mouse Embryonic Stem Cells.

Cardiac cell formation, cardiomyogenesis, is critically dependent on oxygen availability. It is known that hypoxia, a reduced oxygen level, modulates the in vitro differentiation of pluripotent cells into cardiomyocytes via hypoxia inducible factor-1alpha (HIF-1α)-dependent mechanisms. However, the direct impact of HIF-1α deficiency on the formation and maturation of cardiac-like cells derived from mouse embryonic stem cells (mESC) in vitro remains to be elucidated. In the present study, we demonstrated that HIF-1α deficiency significantly altered the quality and quantity of mESC-derived cardiomyocytes. It was accompanied with lower mRNA and protein levels of cardiac cell specific markers (myosin heavy chains 6 and 7) and with a decreasing percentage of myosin heavy chain α and ß, and cardiac troponin T-positive cells. As to structural aspects of the differentiated cardiomyocytes, the localization of contractile proteins (cardiac troponin T, myosin heavy chain α and ß) and the organization of myofibrils were also different. Simultaneously, HIF-1α deficiency was associated with a lower percentage of beating embryoid bodies. Interestingly, an observed alteration in the in vitro differentiation scheme of HIF-1α deficient cells was accompanied with significantly lower expression of the endodermal marker (hepatic nuclear factor 4 alpha). These findings thus suggest that HIF-1α deficiency attenuates spontaneous cardiomyogenesis through the negative regulation of endoderm development in mESC differentiating in vitro.

Multimodal holographic microscopy: distinction between apoptosis and oncosis.

Identification of specific cell death is of a great value for many scientists. Predominant types of cell death can be detected by flow-cytometry (FCM). Nevertheless, the absence of cellular morphology analysis leads to the misclassification of cell death type due to underestimated oncosis. However, the definition of the oncosis is important because of its potential reversibility. Therefore, FCM analysis of cell death using annexin V/propidium iodide assay was compared with holographic microscopy coupled with fluorescence detection - "Multimodal holographic microscopy (MHM)". The aim was to highlight FCM limitations and to point out MHM advantages. It was shown that the annexin V+/PI- phenotype is not specific of early apoptotic cells, as previously believed, and that morphological criteria have to be necessarily combined with annexin V/PI for the cell death type to be ascertained precisely. MHM makes it possible to distinguish oncosis clearly from apoptosis and to stratify the progression of oncosis.

Morphology of erythrocytes of patients with ovarian cancer.

AIM OF THE STUDY: To investigate the morphology of erythrocytes in the peripheral blood of patients with ovarian cancer stage II and III. MATERIALS AND METHODS: The patient group consisted of 17 women (age 53 +/- 16 years) diagnosed with stage II or II ovarian cancer. The control group comprised 20 healthy women (age 20 +/- 2 years). Seventeen samples of peripheral venous blood were collected from the cancer patients and 20 from the healthy women. These were then prepared for observation by scanning electron microscopy. RESULTS: The percentages of knizocytes and echinocytes were higher in the blood of patients than in that of the healthy controls. For knizocytes, the values for the mean +/- SD and the range were 2.45 +/- 3.72 and 0-15.7 vs. 0.66 +/- 0.57, 0.1-2.5, with p < 0.01. For echinocytes, these values were 1.94 +/- 1.04 and 0.5-3.6 vs. 1.03 +/- 0.71, 0.1-2.6 and p < 0.01. Acanthocytes were present only in small numbers and were not evaluated. CONCLUSION: The proportion of erythrocytes with abnormal morphology in the blood of cancer patients before cancer therapy was significantly elevated as compared to that healthy controls.

Production of H2O2 in human embryos after long-lasting cryopreservation and subsequent culture.

The production of hydrogen peroxide (H(2)O(2)) was investigated by means of cytochemical reaction with cerium chloride in human embryos cryostoraged for a long time period. The sites of H(2)O(2) generation were demonstrated at submicroscopic level in both freshly thawed embryos and in embryos and blastocysts formed after subsequent culture. The intact blastomeres as well as cells of well developed blastocysts did not produce any H(2)O(2). Two main intracellular sites of H(2)O(2) production were identified: mitochondria and plasma membranes. Some alterations and often destruction of plasma membrane integrity accompanied by massive H(2)O(2) generation are believed to be caused by the freezing and thawing.