The Odad3 Gene Is Necessary for Spermatozoa Development and Male Fertility in Mice.

Odad3 gene loss-of-function mutation leads to Primary Ciliary Dyskinesia (PCD), a disease caused by motile cilia dysfunction. Previously, we demonstrated that knockout of the Odad3 gene in mice replicates several features of PCD, such as hydrocephalus, defects in left-right body symmetry, and male infertility, with a complete absence of sperm in the reproductive tract. The majority of Odad3 knockout animals die before sexual maturation due to severe hydrocephalus and failure to thrive, which precludes fertility studies. Here, we performed the expression analysis of the Odad3 gene during gonad development and in adult testes. We showed that Odad3 starts its expression during the first wave of spermatogenesis, specifically at the meiotic stage, and that its expression is restricted to the germ cells in the adult testes, suggesting that Odad3 plays a role in spermatozoa formation. Subsequently, we conditionally deleted the Odad3 gene in adult males and demonstrated that even partial ablation of the Odad3 gene leads to asthenoteratozoospermia with multiple morphological abnormalities of sperm flagella (MMAF) in mice. The analysis of the seminiferous tubules in Odad3-deficient mice revealed defects in spermatogenesis with accumulation of seminiferous tubules at the spermiogenesis and spermiation phases. Furthermore, analysis of fertility in heterozygous Odad3+/- knockout mice revealed a reduction in sperm count and motility as well as abnormal sperm morphology. Additionally, Odad3+/- males exhibited a shorter fertile lifespan. Overall, these results suggest the important role of Odad3 and Odad3 gene dosage in male fertility. These findings may have an impact on the genetic and fertility counseling practice of PCD patients carrying Odad3 loss-of-function mutations.

Synergistic Antimicrobial Effect of Cold Atmospheric Plasma and Redox-Active Nanoparticles.

Cold argon plasma (CAP) and metal oxide nanoparticles are well known antimicrobial agents. In the current study, on an example of Escherichia coli, a series of analyses was performed to assess the antibacterial action of the combination of these agents and to evaluate the possibility of using cerium oxide and cerium fluoride nanoparticles for a combined treatment of bacterial diseases. The joint effect of the combination of cold argon plasma and several metal oxide and fluoride nanoparticles (CeO2, CeF3, WO3) was investigated on a model of E. coli colony growth on agar plates. The mutagenic effect of different CAP and nanoparticle combinations on bacterial DNA was investigated, by means of a blue-white colony assay and RAPD-PCR. The effect on cell wall damage, using atomic force microscopy, was also studied. The results obtained demonstrate that the combination of CAP and redox-active metal oxide nanoparticles (RAMON) effectively inhibits bacterial growth, providing a synergistic antimicrobial effect exceeding that of any of the agents alone. The combination of CAP and CeF3 was shown to be the most effective mutagen against plasmid DNA, and the combination of CAP and WO3 was the most effective against bacterial genomic DNA. The analysis of direct cell wall damage by atomic force microscopy showed the combination of CAP and CeF3 to be the most effective antimicrobial agent. The combination of CAP and redox-active metal oxide or metal fluoride nanoparticles has a strong synergistic antimicrobial effect on bacterial growth, resulting in plasmid and genomic DNA damage and cell wall damage. For the first time, a strong antimicrobial and DNA-damaging effect of CeF3 nanoparticles has been demonstrated.

A Study on the Planarian Model Confirms the Antioxidant Properties of Tameron against X-ray- and Menadione-Induced Oxidative Stress.

Ionizing radiation and radiation-related oxidative stress are two important factors responsible for the death of actively proliferating cells, thus drastically reducing the regeneration capacity of living organisms. Planarian flatworms are freshwater invertebrates that are rich in stem cells called neoblasts and, therefore, present a well-established model for studies on regeneration and the testing of novel antioxidant and radioprotective substances. In this work, we tested an antiviral and antioxidant drug Tameron (Monosodium α-Luminol or 5-amino-2,3-dihydro-1,4-phthalazinedione sodium salt) for its ability to reduce the harm of X-ray- and chemically induced oxidative stress on a planarian model. Our study has revealed the ability of Tameron to effectively protect planarians from oxidative stress while enhancing their regenerative capacity by modulating the expression of neoblast marker genes and NRF-2-controlled oxidative stress response genes.

Mitogen-like Cerium-Based Nanoparticles Protect Schmidtea mediterranea against Severe Doses of X-rays.

Novel radioprotectors are strongly demanded due to their numerous applications in radiobiology and biomedicine, e.g., for facilitating the remedy after cancer radiotherapy. Currently, cerium-containing nanomaterials are regarded as promising inorganic radioprotectors due to their unrivaled antioxidant activity based on their ability to mimic the action of natural redox enzymes like catalase and superoxide dismutase and to neutralize reactive oxygen species (ROS), which are by far the main damaging factors of ionizing radiation. The freshwater planarian flatworms are considered a promising system for testing new radioprotectors, due to the high regenerative potential of these species and an excessive amount of proliferating stem cells (neoblasts) in their bodies. Using planarian Schmidtea mediterranea, we tested CeO2 nanoparticles, well known for their antioxidant activity, along with much less studied CeF3 nanoparticles, for their radioprotective potential. In addition, both CeO2 and CeF3 nanoparticles improve planarian head blastema regeneration after ionizing irradiation by enhancing blastema growth, increasing the number of mitoses and neoblasts' survival, and modulating the expression of genes responsible for the proliferation and differentiation of neoblasts. The CeO2 nanoparticles' action stems directly from their redox activity as ROS scavengers, while the CeF3 nanoparticles' action is mediated by overexpression of "wound-induced genes" and neoblast- and stem cell-regulating genes.

Effect of weak alternating magnetic fields on planarian regeneration.

We investigated the effect of weak combined magnetic field (CMF) on stem cell proliferation and regeneration of the planarian Schmidtea mediterranea. CMF parameters were set in accordance with Valery Lednev's theory of magnetic parametric resonance. It was shown that CMF with an amplitude of 74 µT and a frequency of 30 Hz accelerated the growth of the planarian head blastema by 25%. Alterations of the frequency in range from 27 to 33 Hz led to a complete disappearance of the effect. A further decrease in the CMF frequency inhibited regeneration. The maximum inhibition (24%) was observed at a frequency of 16 Hz. A further decrease in the CMF frequency (down to 13 Hz) led to disappearance of the described effect. Regeneration rate changes under the CMF are influenced by alterations in stem cell mitotic activity, which in turn depends on the wound-induced gene expression level. Thus, the CMF, preset in accordance to the Lednev's theory, can specifically influence the expression of regeneration-related genes and regeneration itself, what can find biomedical applications.

Planarians as an In Vivo Experimental Model for the Study of New Radioprotective Substances.

Ionising radiation causes the death of the most actively dividing cells, thus leading to depletion of the stem cell pool. Planarians are invertebrate flatworms that are unique in that their stem cells, called neoblasts, constantly replace old, damaged, or dying cells. Amenability to efficient RNAi treatments, the rapid development of clear phenotypes, and sensitivity to ionising radiation, combined with new genomic technologies, make planarians an outstanding tool for the discovery of potential radioprotective agents. In this work, using the well-known antioxidant N-acetylcysteine, planarians are, for the first time, shown to be an excellent model system for the fast and effective screening of novel radioprotective and radio-sensitising substances. In addition, a panel of measurable parameters that can be used for the study of radioprotective effects on this model is suggested.

Distinct and Overlapping Functions of Miscanthus sinensis MYB Transcription Factors SCM1 and MYB103 in Lignin Biosynthesis.

Cell wall recalcitrance is a major constraint for the exploitation of lignocellulosic biomass as a renewable resource for energy and bio-based products. Transcriptional regulators of the lignin biosynthetic pathway represent promising targets for tailoring lignin content and composition in plant secondary cell walls. However, knowledge about the transcriptional regulation of lignin biosynthesis in lignocellulosic feedstocks, such as Miscanthus, is limited. In Miscanthus leaves, MsSCM1 and MsMYB103 are expressed at growth stages associated with lignification. The ectopic expression of MsSCM1 and MsMYB103 in N. benthamiana leaves was sufficient to trigger secondary cell wall deposition with distinct sugar and lignin compositions. Moreover, RNA-seq analysis revealed that the transcriptional responses to MsSCM1 and MsMYB103 overexpression showed an extensive overlap with the response to the NAC master transcription factor MsSND1, but were distinct from each other, underscoring the inherent complexity of secondary cell wall formation. Furthermore, conserved and previously described promoter elements as well as novel and specific motifs could be identified from the target genes of the three transcription factors. Together, MsSCM1 and MsMYB103 represent interesting targets for manipulations of lignin content and composition in Miscanthus towards a tailored biomass.

Dose-Dependent Effects of Cold Atmospheric Argon Plasma on the Mesenchymal Stem and Osteosarcoma Cells In Vitro.

The antimicrobial, anti-inflammatory and tissue-stimulating effects of cold argon atmospheric plasma (CAAP) accelerate its use in various fields of medicine. Here, we investigated the effects of CAAP at different radiation doses on mesenchymal stem cells (MSCs) and human osteosarcoma (MNNG/HOS) cells. We observed an increase in the growth rate of MSCs at sufficiently low irradiation doses (10-15 min) of CAAP, while the growth of MNNG/HOS cells was slowed down to 41% at the same irradiation doses. Using flow cytometry, we found that these effects are associated with cell cycle arrest and extended death of cancer cells by necrosis. Reactive oxygen species (ROS) formation was detected in both types of cells after 15 min of CAAP treatment. Evaluation of the genes' transcriptional activity showed that exposure to low doses of CAAP activates the expression of genes responsible for proliferation, DNA replication, and transition between phases of the cell cycle in MSCs. There was a decrease in the transcriptional activity of most of the studied genes in MNNG/HOS osteosarcoma cancer cells. However, increased transcription of osteogenic differentiation genes was observed in normal and cancer cells. The selective effects of low and high doses of CAAP treatment on cancer and normal cells that we found can be considered in terms of hormesis. The low dose of cold argon plasma irradiation stimulated the vital processes in stem cells due to the slight generation of reactive oxygen species. In cancer cells, the same doses evidently lead to the formation of oxidative stress, which was accompanied by a proliferation inhibition and cell death. The differences in the cancer and normal cells' responses are probably due to different sensitivity to exogenous oxidative stress. Such a selective effect of CAAP action can be used in the combined therapy of oncological diseases such as skin neoplasms, or for the removal of remaining cancer cells after surgical removal of a tumor.

Three-Dimensional X-ray Imaging of ß-Galactosidase Reporter Activity by Micro-CT: Implication for Quantitative Analysis of Gene Expression.

Acquisition of detailed anatomical and molecular knowledge from intact biological samples while preserving their native three-dimensional structure is still a challenging issue for imaging studies aiming to unravel a system's functions. Three-dimensional micro-CT X-ray imaging with a high spatial resolution in minimally perturbed naive non-transparent samples has recently gained increased popularity and broad application in biomedical research. Here, we describe a novel X-ray-based methodology for analysis of ß-galactosidase (lacZ) reporter-driven gene expression in an intact murine brain ex vivo by micro-CT. The method relies on detection of bromine molecules in the product of the enzymatic ß-galactosidase reaction. Enhancement of the X-ray signal is observed specifically in the regions of the murine brain where expression of the lacZ reporter gene is also detected histologically. We performed quantitative analysis of the expression levels of lacZ reporter activity by relative radiodensity estimation of the ß-galactosidase/X-gal precipitate in situ. To demonstrate the feasibility of the method, we performed expression analysis of the Tsen54-lacZ reporter gene in the murine brain in a semi-quantitative manner. Human mutations in the Tsen54 gene cause pontocerebellar hypoplasia (PCH), a group of severe neurodegenerative disorders with both mental and motor deficits. Comparing relative levels of Tsen54 gene expression, we demonstrate that the highest Tsen54 expression is observed in anatomical brain substructures important for the normal motor and memory functions in mice.

YAP Orchestrates Heterotypic Endothelial Cell Communication via HGF/c-MET Signaling in Liver Tumorigenesis.

The oncogene yes-associated protein (YAP) controls liver tumor initiation and progression via cell extrinsic functions by creating a tumor-supporting environment in conjunction with cell autonomous mechanisms. However, how YAP controls organization of the microenvironment and in particular the vascular niche, which contributes to liver disease and hepatocarcinogenesis, is poorly understood. To investigate heterotypic cell communication, we dissected murine and human liver endothelial cell (EC) populations into liver sinusoidal endothelial cells (LSEC) and continuous endothelial cells (CEC) through histomorphological and molecular characterization. In YAPS127A-induced tumorigenesis, a gradual replacement of LSECs by CECs was associated with dynamic changes in the expression of genes involved in paracrine communication. The formation of new communication hubs connecting CECs and LSECs included the hepatocyte growth factor (Hgf)/c-Met signaling pathway. In hepatocytes and tumor cells, YAP/TEA domain transcription factor 4 (TEAD4)-dependent transcriptional induction of osteopontin (Opn) stimulated c-Met expression in EC with CEC phenotype, which sensitized these cells to the promigratory effects of LSEC-derived Hgf. In human hepatocellular carcinoma, the presence of a migration-associated tip-cell signature correlated with poor clinical outcome and the loss of LSEC marker gene expression. The occurrence of c-MET-expressing CECs in human liver cancer samples was confirmed at the single-cell level. In summary, YAP-dependent changes of the liver vascular niche comprise the formation of heterologous communication hubs in which tumor cell-derived factors modify the cross-talk between LSECs and CECs via the HGF/c-MET axis. SIGNIFICANCE: YAP-dependent changes of the liver vascular niche comprise the formation of heterologous communication hubs in which tumor cell-derived factors modify the cross-talk between EC subpopulations. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/24/5502/F1.large.jpg.

Structural basis for the complex DNA binding behavior of the plant stem cell regulator WUSCHEL.

Stem cells are one of the foundational evolutionary novelties that allowed the independent emergence of multicellularity in the plant and animal lineages. In plants, the homeodomain (HD) transcription factor WUSCHEL (WUS) is essential for the maintenance of stem cells in the shoot apical meristem. WUS has been reported to bind to diverse DNA motifs and to act as transcriptional activator and repressor. However, the mechanisms underlying this remarkable behavior have remained unclear. Here, we quantitatively delineate WUS binding to three divergent DNA motifs and resolve the relevant structural underpinnings. We show that WUS exhibits a strong binding preference for TGAA repeat sequences, while retaining the ability to weakly bind to TAAT elements. This behavior is attributable to the formation of dimers through interactions of specific residues in the HD that stabilize WUS DNA interaction. Our results provide a mechanistic basis for dissecting WUS dependent regulatory networks in plant stem cell control.

PVP-stabilized tungsten oxide nanoparticles: pH sensitive anti-cancer platform with high cytotoxicity.

Photochromic tungsten oxide (WO3) nanoparticles stabilized by polyvinylpyrrolidone (PVP) were synthesized to evaluate their potential for biomedical applications. PVP-stabilized tungsten oxide nanoparticles demonstrated a highly selective cytotoxic effect on normal and cancer cells in vitro. WO3 nanoparticles were found to induce substantial cell death in osteosarcoma cells (MNNG/HOS cell line) with a half-maximal inhibitory concentration (IC50) of 5 mg/mL, while producing no, or only minor, toxicity in healthy human mesenchymal stem cells (hMSc). WO3 nanoparticles induced intracellular oxidative stress, which led to apoptosis type cell death. The selective anti-cancer effects of WO3 nanoparticles are due to the pH sensitivity of tungsten oxide and its capability of reactive oxygen species (ROS) generation, which is expressed in the modulation of genes involved in reactive oxygen species metabolism, mitochondrial dysfunction, and apoptosis.

WUSCHEL acts as an auxin response rheostat to maintain apical stem cells in Arabidopsis.

To maintain the balance between long-term stem cell self-renewal and differentiation, dynamic signals need to be translated into spatially precise and temporally stable gene expression states. In the apical plant stem cell system, local accumulation of the small, highly mobile phytohormone auxin triggers differentiation while at the same time, pluripotent stem cells are maintained throughout the entire life-cycle. We find that stem cells are resistant to auxin mediated differentiation, but require low levels of signaling for their maintenance. We demonstrate that the WUSCHEL transcription factor confers this behavior by rheostatically controlling the auxin signaling and response pathway. Finally, we show that WUSCHEL acts via regulation of histone acetylation at target loci, including those with functions in the auxin pathway. Our results reveal an important mechanism that allows cells to differentially translate a potent and highly dynamic developmental signal into stable cell behavior with high spatial precision and temporal robustness.

The first inorganic mitogens: Cerium oxide and cerium fluoride nanoparticles stimulate planarian regeneration via neoblastic activation.

We report the first experimental evidence for the mitogenic action of cerium(IV) oxide and cerium(III) fluoride nanoparticles (CONs and CFNs) on the regeneration of a whole organism - freshwater flatworms Schmidtea mediterranea (planarian). Both types of cerium-containing nanoparticles are shown to be a highly potent mitogen for planaria. Both CONs and CFNs, in micro- and nanomolar concentrations, markedly accelerate planarian blastema growth, due to the enhancement of cellular proliferation, causing an increase in the mitotic index and in the quantity of blastema cells in regenerating planaria. CONs provided maximum activity at concentrations which were two orders of magnitude lower than those for CeF3. The valence state of cerium in cerium-containing nanoparticles plays a significant role in the planarian regeneration mechanism: CeO2 nanoparticles containing predominantly Ce4+ species presumably scavenge wound induced reactive oxygen species and moderately activate gene expression processes, while the regenerative action of CeF3 nanoparticles containing only Ce3+ species is manifested in the pronounced expression of the genes involved in cell division, differentiation and migration. This is the first report on the effect of cerium-containing nanoparticles on tissue regeneration in vivo, further revealing the mechanisms of their biological action, which enhances the possibility of their use in cellular technologies.

Functional loss of Ccdc151 leads to hydrocephalus in a mouse model of primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder affecting normal structure and function of motile cilia, phenotypically manifested as chronic respiratory infections, laterality defects and infertility. Autosomal recessive mutations in genes encoding for different components of the ciliary axoneme have been associated with PCD in humans and in model organisms. The CCDC151 gene encodes for a coiled-coil axonemal protein that ensures correct attachment of outer dynein arm (ODA) complexes to microtubules. A correct arrangement of dynein arm complexes is required to provide the proper mechanical force necessary for cilia beat. Loss-of-function mutations in CCDC151 in humans leads to PCD disease with respiratory distress and defective left-right body asymmetry. In mice with the Ccdc151Snbl loss-of-function mutation (Snowball mutant), left-right body asymmetry with heart defects have been observed. Here, we demonstrate that loss of Ccdc151 gene function via targeted gene deletion in mice leads to perinatal lethality and congenital hydrocephalus. Microcomputed tomography (microCT) X-ray imaging of Ccdc151-ß-galactosidase reporter expression in whole-mount brain and histological analysis show that Ccdc151 is expressed in ependymal cells lining the ventricular brain system, further confirming the role of Ccdc151 dysfunction in hydrocephalus development. Analyzing the features of hydrocephalus in the Ccdc151-knockout animals by microCT volumetric imaging, we observe continuity of the aqueduct of Sylvius, indicating the communicating nature of hydrocephalus in the Ccdc151-knockout animals. Congenital defects in left-right asymmetry and male infertility have been also observed in Ccdc151-null animals. Ccdc151 gene deletion in adult animals results in abnormal sperm counts and defective sperm motility.This article has an associated First Person interview with the joint first authors of the paper.

The Hox transcription factor Ubx stabilizes lineage commitment by suppressing cellular plasticity in Drosophila.

During development cells become restricted in their differentiation potential by repressing alternative cell fates, and the Polycomb complex plays a crucial role in this process. However, how alternative fate genes are lineage-specifically silenced is unclear. We studied Ultrabithorax (Ubx), a multi-lineage transcription factor of the Hox class, in two tissue lineages using sorted nuclei and interfered with Ubx in mesodermal cells. We find that depletion of Ubx leads to the de-repression of genes normally expressed in other lineages. Ubx silences expression of alternative fate genes by retaining the Polycomb Group protein Pleiohomeotic at Ubx targeted genomic regions, thereby stabilizing repressive chromatin marks in a lineage-dependent manner. Our study demonstrates that Ubx stabilizes lineage choice by suppressing the multipotency encoded in the genome via its interaction with Pho. This mechanism may explain why the Hox code is maintained throughout the lifecycle, since it could set a block to transdifferentiation in adult cells.

Equation of State and Amorphization of Ca9R(VO4)7 (R = La, Nd, Gd): A Combined High-Pressure X-ray Diffraction and Raman Spectroscopy Study.

Ca9R(VO4)7 (R = rare earth) multicomponent oxides of a whitlockite-related structure are under consideration for applications in optoelectronics. In this work, the Czochralski-grown Ca9R(VO4)7 crystals were investigated as a function of pressure by powder X-ray diffraction and single-crystal Raman spectroscopy. The diffraction experiments were performed at the ALBA synchrotron under pressures ranging up to 9.22(5), 10.7(1), and 8.55(5) GPa for R = La, Nd, and Gd, respectively, to determine the third order equation of state (EOS) parameters. Fitting of the Birch-Murnaghan EOS provided the isothermal bulk moduli K0 = 63(4), 63(2), and 61(5) GPa for these three orthovanadates. These values are apparently lower than that reported for structurally related tricalcium vanadate Ca3(VO4)2. The compressibility anisotropy was observed; the lattice is markedly stiffer in [001] than in [100] direction. For Ca9Nd(VO4)7, the variation of the diffractograms just above 10 GPa provides an indication on the beginning of amorphization process; during pressure release the whitlockite-like structure is recovered. Raman spectroscopy measurements for single crystals of the above-mentioned rare-earth vanadates and Ca9Y(VO4)7 were performed (the maximum pressures achieved were 16.3(1), 21.2(1), 15.3(1), and 18.6(1) GPa for R = Y, La, Nd, and Gd, respectively). These measurements reveal a partially reversible phase transition interpreted as amorphization, with an onset at the pressure of ∼9-10 GPa, characterized by broadening of the peaks and their shift to lower energies.

Decoding the Regulatory Logic of the Drosophila Male Stem Cell System.

The niche critically controls stem cell behavior, but its regulatory input at the whole-genome level is poorly understood. We elucidated transcriptional programs of the somatic and germline lineages in the Drosophila testis and genome-wide binding profiles of Zfh-1 and Abd-A expressed in somatic support cells and crucial for fate acquisition of both cell lineages. We identified key roles of nucleoporins and V-ATPase proton pumps and demonstrate their importance in controlling germline development from the support side. To make our dataset publicly available, we generated an interactive analysis tool, which uncovered conserved core genes of adult stem cells across species boundaries. We tested the functional relevance of these genes in the Drosophila testis and intestine and found a high frequency of stem cell defects. In summary, our dataset and interactive platform represent versatile tools for identifying gene networks active in diverse stem cell types.

WEADE: A workflow for enrichment analysis and data exploration.

Data analysis based on enrichment of Gene Ontology terms has become an important step in exploring large gene or protein expression datasets and several stand-alone or web tools exist for that purpose. However, a comprehensive and consistent analysis downstream of the enrichment calculation is missing so far. With WEADE we present a free web application that offers an integrated workflow for the exploration of genomic data combining enrichment analysis with a versatile set of tools to directly compare and intersect experiments or candidate gene lists of any size or origin including cross-species data. Lastly, WEADE supports the graphical representation of output data in the form of functional interaction networks based on prior knowledge, allowing users to go from plain expression data to functionally relevant candidate sub-lists in an interactive and consistent manner.

The molecular signature of AML with increased ALDH activity suggests a stem cell origin.

Enrichment of leukemic blasts with a stem cell phenotype correlates with poor survival in acute myeloid leukemia (AML). In this context, measurement of the stem cell marker aldehyde-dehydrogenase (ALDH) activity can distinguish poor prognosis cases with increased fractions of ALDH-positive cells (ALDH-numerous AML) and favorable outcome cases with low percentages (ALDH-rare AML). It has been shown that ALDH-numerous AML favor leukemic engraftment in xenotransplantation assays which suggests increased leukemic stem cell (LSC) potential. To test if this reflects an immature cell of origin, comparative gene-expression studies of CD34+ leukemic blasts were performed. This analysis revealed increased expression of LSC and HSC signatures in ALDH-numerous AML, whereas ALDH-rare AML were enriched for a progenitor signature. The enrichment of stemness-associated transcriptional programs suggests that ALDH-numerous AML derive from immature hematopoietic progenitors and offers an explanation for the poor prognosis and therapy resistance of this subgroup which is likely caused by inherited stem cell properties.