Defect of Mitotic Vimentin Phosphorylation Causes Microophthalmia and Cataract via Aneuploidy and Senescence in Lens Epithelial Cells

Vimentin, a type III intermediate filament (IF) protein, is phosphorylated predominantly in mitosis. The expression of a phosphorylation-compromised vimentin mutant in T24 cultured cells leads to cytokinetic failure, resulting in binucleation (multinucleation). The physiological significance of intermediate filament phosphorylation during mitosis for organogenesis and tissue homeostasis was uncertain. Here, we generated knock-in mice expressing vimentin that have had the serine sites phosphorylated during mitosis substituted by alanine residues. Homozygotic mice (VIM^SA/SA) presented with microophthalmia and cataracts in the lens, whereas heterozygotic mice (VIM^WT/SA) were indistinguishable from WT (VIM^WT/WT) mice. In VIM^SA/SA mice, lens epithelial cell number was not only reduced but the cells also exhibited chromosomal instability, including binucleation and aneuploidy. Electron microscopy revealed fiber membranes that were disorganized in the lenses of VIM^SA/SA, reminiscent of similar characteristic changes seen in age-related cataracts. Because the mRNA level of the senescence (aging)-related gene was significantly elevated in samples from VIM^SA/SA, the lens phenotype suggests a possible causal relationship between chromosomal instability and premature aging.     

Occurrence and characterisation of <Emphasis Type="Italic">Salmonella enterica</Emphasis> subspecies <Emphasis Type="Italic">diarizonae</Emphasis> serovar 61: k: 1, 5, (7) in sheep in the federal state of Thuringia,Germany

Background

The occurrence of Salmonella enterica subspecies diarizonae serovar 61: k: 1, 5, (7) (SASd) and other Salmonella organisms in sheep in the German federal state of Thuringia was examined for the first time. Pooled faecal samples from 90 flocks located in this state were monitored.

Results

Only SASd was detected in 74 (82.2%) out of the 90 sheep herds, other Salmonella serovars were not identified. A positive correlation was found between the flock size and the detection probability of SASd. Despite the agent’s high prevalence, clinical symptoms of a disease exclusively due to SASd have not been observed. The SASd strains were characterised by macrorestriction analysis, antimicrobial testing and the biochemical profile. All strains were sensitive to 13 out of 14 antimicrobial substances and resistant to only sulfamethoxazole. The high number of macrorestriction groups of SASd strains indicated a low clonality of the serovar.

Conclusions

Data from sheep derived foods and public health data in Germany strongly suggest that the significance of SASd for public health is considerably lower than that of serovars belonging to Salmonella enterica subspecies enterica. For this reason and because of the low disease-causing potential of SASd in sheep, it is worthwile to consider a reduction in ongoing activities from combating to monitoring serovar 61: k: 1, 5, (7) in the sheep population.

     

Cytokinetic Failure-induced Tetraploidy Develops into Aneuploidy,Triggering Skin Aging in Phosphovimentin-deficient Mice

Tetraploidy, a state in which cells have doubled chromosomal sets, is observed in ∼20% of solid tumors and is considered to frequently precede aneuploidy in carcinogenesis. Tetraploidy is also detected during terminal differentiation and represents a hallmark of aging. Most tetraploid cultured cells are arrested by p53 stabilization. However, the fate of tetraploid cells in vivo remains largely unknown. Here, we analyze the ability to repair wounds in the skin of phosphovimentin-deficient (VIM^SA/SA) mice. Early into wound healing, subcutaneous fibroblasts failed to undergo cytokinesis, resulting in binucleate tetraploidy. Accordingly, the mRNA level of p21 (a p53-responsive gene) was elevated in a VIM^SA/SA-specific manner. Disappearance of tetraploidy coincided with an increase in aneuploidy. Thereafter, senescence-related markers were significantly elevated in VIM^SA/SA mice. Because our tetraploidy-prone mouse model also exhibited subcutaneous fat loss at the age of 14 months, another premature aging phenotype, our data suggest that following cytokinetic failure, a subset of tetraploid cells enters a new cell cycle and develops into aneuploid cells in vivo, which promote premature aging.     

Endogenous expression of FAD-linked PS1 impairs proliferation,neuronal differentiation and survival of adult hippocampal progenitors

Background

Alzheimer’s disease (AD) is characterized by progressive memory loss and impaired cognitive function. Early-onset familial forms of the disease (FAD) are caused by inheritance of mutant genes encoding presenilin 1 (PS1) variants. We have demonstrated that prion promoter (PrP)-driven expression of human FAD-linked PS1 variants in mice leads to impairments in environmental enrichment (EE)-induced adult hippocampal neural progenitor cell (AHNPC) proliferation and neuronal differentiation, and have provided evidence that accessory cells in the hippocampal niche expressing PS1 variants may modulate AHNPC phenotypes, in vivo. While of significant interest, these latter studies relied on transgenic mice that express human PS1 variant transgenes ubiquitously and at high levels, and the consequences of wild type or mutant PS1 expressed under physiologically relevant levels on EE-mediated AHNPC phenotypes has not yet been tested.

Results

To assess the impact of mutant PS1 on EE-induced AHNPC phenotypes when expressed under physiological levels, we exposed adult mice that constitutively express the PSEN1 M146V mutation driven by the endogenous PSEN1 promoter (PS1 M146V “knock-in” (KI) mice) to standard or EE-housed conditions. We show that in comparison to wild type PS1 mice, AHNPCs in mice carrying homozygous (PS1 ^M146V/M146V ) or heterozygous (PS1 ^M146V/+ ) M146V mutant alleles fail to exhibit EE-induced proliferation and commitment towards neurogenic lineages. More importantly, we report that the survival of newborn progenitors are diminished in PS1 M146V KI mice exposed to EE-conditions compared to respective EE wild type controls.

Conclusions

Our findings reveal that expression at physiological levels achieved by a single PS1 M146V allele is sufficient to impair EE-induced AHNPC proliferation, survival and neuronal differentiation, in vivo. These results and our finding that microglia expressing a single PS1 M146V allele impairs the proliferation of wild type AHNPCs in vitro argue that expression of mutant PS1 in the AHNPC niche impairs AHNPCs phenotypes in a dominant, non-cell autonomous manner.

     

Endogenous Salicylic Acid Levels and Signaling Positively Regulate <Emphasis Type="Italic">Arabidopsis</Emphasis> Response to Polyethylene Glycol-Simulated Drought Stress

Salicylic acid (SA) functions in the plant response to drought stress were assessed using SA-altering Arabidopsis mutants, including snc1 (with constitutively high levels of SA) and its nahG-transformed plants (named as snc1/nahG, with a comparable SA level to the wild type), sid2 and transgenic line nahG (both with SA deficiency), and npr1-1 (with SA signaling blockage). The drought stress was simulated by polyethylene glycol (PEG)-6000 treatment. Compared with wild-type (wt) plants, the snc1 plants displayed obvious easing of PEG-induced growth inhibition, leaf water loss, and photosynthesis-related impairment, whereas in nahG, sid2, and npr1-1 mutants the effect was more severe. PEG stress reduced stomatal conductance, to a higher extent in the snc1 line, whereas it was lower in nahG, sid2, and npr1-1 lines as compared with the wt. The snc1 plants accumulated higher levels of H₂O₂ than the other genotypes tested. PEG stress increased activities of superoxide dismutase and peroxidase, but decreased activities of catalase in all lines tested, to a greater extent in snc1 and less in sid2, nahG, and npr1-1 relative to wt. Proline was significantly increased, especially in snc1 line at 6 % and higher PEG stress. Noticeably, the performance of snc1 under PEG stress was dependent on SA levels, as the expression of nahG in snc1 plants did not only significantly reduce SA levels, but largely reversed the above-mentioned parameters, as well as eliminated the drought tolerance. Based on these data, it was concluded that endogenous SA levels and signaling provided a protective role in the Arabidopsis response to PEG-simulated drought.     

New <Emphasis Type="Italic">slbo</Emphasis>-Gal4 driver lines for the analysis of border cell migration during <Emphasis Type="Italic">Drosophila</Emphasis> oogenesis

Border cell (BC) migration during Drosophila oogenesis is an excellent model for the analysis of the migratory and invasive cell behavior. Most studies on BC migration have exploited a slbo-Gal4 driver to regulate gene expression in these cells or to mark them. Here, we report that the slbo-Gal4 transgene present in the line #6458 from the Bloomington Stock Center is inserted within chickadee (chic), a gene encoding the actin-binding protein Profilin, which promotes actin polymerization and is known to be involved in cell migration. The chic⁶⁴⁵⁸ mutation caused by the transgene insertion behaves as a null chic allele and is homozygous lethal. To evaluate possible effects of chic⁶⁴⁵⁸ on the assessment of BC behavior, we generated new lines bearing the slbo-Gal4 transgene inserted into different second chromosome loci that do not appear to be involved in cell migration. Using these new lines and the slbo-Gal4-chic⁶⁴⁵⁸ line, we defined the functional relationships between the twinfilin (twf) and chic in BC migration. Migration of BCs is substantially reduced by mutations in twf, which encodes an actin-binding protein that inhibits actin filament assembly. The defects caused by twf mutations are significantly suppressed when the slbo-Gal4-chic⁶⁴⁵⁸, but not the new slbo-Gal4 drivers were used. These findings indicate twf and chic interact and function antagonistically during BC migration in Drosophila oogenesis.     

Identification and comparative analysis of <Emphasis Type="Italic">Brassica juncea</Emphasis> pathogenesis-related genes in response to hormonal,biotic and abiotic stresses

Pathogenesis-related proteins (PRs) are the antimicrobial proteins which are commonly used as signatures of defense signaling pathways and systemic acquired resistance. However, in Brassica juncea most of the PR proteins have not been fully characterized and remains largely enigmatic. In this study, full-length cDNA sequences of SA (PR1, PR2, PR5) and JA (PR3, PR12 and PR13) marker genes were isolated from B. juncea and were named as BjPR proteins. BjPR proteins showed maximum identity with known PR proteins of Brassica species. Further, expression profiling of BjPR genes were investigated after hormonal, biotic and abiotic stresses. Pre-treatment with SA and JA stimulators downregulates each other signature genes suggesting an antagonistic relationship between SA and JA in B. juncea. After abscisic acid (ABA) treatment, SA signatures were downregulated while as JA signature genes were upregulated. During Erysiphe cruciferarum infection, SA- and JA-dependent BjPR genes showed distinct expression pattern both locally and systemically, thus suggesting the activation of SA- and JA-dependent signaling pathways. Further, expression of SA marker genes decreases while as JA-responsive genes increases during drought stress. Interestingly, both SA and JA signature genes were induced after salt stress. We also found that BjPR genes displayed ABA-independent gene expression pattern during abiotic stresses thus providing the evidence of SA/JA cross talk. Further, in silico analysis of the upstream regions (1.5 kb) of both SA and JA marker genes showed important cis-regulatory elements related to biotic, abiotic and hormonal stresses.     

Formation of a Z-pinch during electromagnetic compression of a nonquasineutral current filament

A study is made of the structure of a relativistic current filament with the azimuthal magnetic field B_θ in the range 4πn _e m _e c²?B _θ ² 4πn _i m _i c², when the plasma quasineutrality near the filament axis is violated and a narrow peak in electron density is formed there. The ion dynamics in a strong radial electric field of the filament on time scales of about several inverse ion plasma frequencies ω _pi ^?1 is investigated. The initial ion pressure prevents the ion plasma component from compression to infinitely high densities under the action of the electric field and leads to the formation of a dense hot plasma core near the axis of the Z-pinch on time scales of about a dozen ω _pi ^?1 . The compression of the ion component in the axial region gives rise to a collisionless “unloading” shock wave that propagates away from the axis and is accompanied by the vanishing of the radial ion velocity behind the shock front, the accumulation of positive charge near the axis, and the formation of a steady-state ion density profile. It is shown theoretically that ion-ion dissipation manifests itself as the destruction of the hot core of the formed Z-pinch on picosecond time scales. This may serve to explain the explosions of “hot points” in a current-carrying plasma.     

Involvement of Nuclear Receptor REV-ERBβ in Formation of Neurites and Proliferation of Cultured Adult Neural Stem Cells

Neural stem cells (NSCs) serve as the source of both neurons and support cells, and neurogenesis is reportedly linked to the circadian clock. This study aimed to clarify the functional role of the circadian rhythm-related nuclear receptor, REV-ERBβ, in neurogenesis of NSCs from adult brain. Accordingly, Rev-erbβ expression and the effect of Rev-erbβ gene-specific knockdown on neurogenesis in vitro was examined in adult rodent NSCs. Initial experiments confirmed REV-ERBβ expression in cultured adult NSCs, while subsequent gene expression and gene ontogeny analyses identified functional genes upregulated or downregulated by REV-ERBβ. In particular, expression levels of factors associated with proliferation, stemness, and neural differentiation were affected. Knockdown of Rev-erbβ showed involvement of REV-ERBβ in regulation of cellular proliferation and self-renewal of cultured adult NSCs. Moreover, Rev-erbβ-knockdown cells formed neurons with a slightly shrunken morphology, fewer new primary neurites, and reduced length and branch formation of neurites. Altogether, this suggests that REV-ERBβ is involved in neurite formation during neuronal differentiation of cultured adult NSCs. In summary, REV-ERBβ is a known circadian regulatory protein that appears to be involved in neurogenesis via regulation of networks for cell proliferation and neural differentiation/maturation in adult NSCs.     

Adult plant stripe rust resistance gene <Emphasis Type="Italic">Yr71</Emphasis> maps close to <Emphasis Type="Italic">Lr24</Emphasis> in chromosome 3D of common wheat

Australian cultivar Sunco carries three adult plant stripe rust resistance genes. One of these genes corresponded to Yr18 in chromosome 7DS; the second, YrCK, was mapped on chromosome 2D. Here, we describe the characterization of the third adult plant resistance (APR) gene from Sunco. Sunco/2*Avocet S-derived lines SA65 (resistant) and SA67 (susceptible) were crossed and a recombinant inbred line F₆ population was generated. Monogenic segregation among SA65/SA67-derived RIL population was demonstrated and the resistance locus was designated YrSA3. Selective genotyping using an iSelect 90 K Infinium SNP array and SSR markers located YrSA3 on chromosome 3D. Development of KASP markers for SNP loci showing association with YrSA3 allowed construction of a genetic map harboring the resistance gene. Ten KASP markers (KASP_8306, KASP_9142, KASP_10438, KASP_16434, KASP_17207, KASP_20836, KASP_23518, KASP_23615, KASP_57983 and KASP_63653), one SSR marker (gwm114b) and Lr24/Sr24 were mapped 1.8 cM distal to YrSA3. Comparison of marker data indicated that the previously named seedling stripe rust resistance gene Yr45 was located proximal to YrSA3, and therefore the latter was formally designated Yr71. Two recombinants carrying Lr24/Sr24 and Yr71 in combination were identified for use as donor sources in wheat breeding programs. The robustness of gwm114b, KASP_16434, KASP_17207 and KASP_20836 for marker-assisted selection of these genes was demonstrated through tests on 74 Australian wheat cultivars.     

Reduced Proliferation of Oligodendrocyte Progenitor Cells in the Postnatal Brain of <Emphasis Type="Italic">Dystonia Musculorum</Emphasis> Mice

Dystonia musculorum (dt) mice show sensory neurodegeneration and movement disorder, such as dystonia and cerebellar ataxia. The causative gene Dystonin (Dst) encodes a cytoskeleton linker protein. Although sensory neurodegeneration has been well studied, glial cell responses in the central nervous system (CNS) are poorly understood. Here, we investigated cell proliferation in the CNS of Dst ^Gt homozygous mice using newly generated in situ hybridization (ISH) probes—Ki-67 and proliferating cell nuclear antigen (PCNA) probes—both of which effectively detect proliferating cells. We found that Ki-67-positive cells were significantly decreased in the corpus callosum and thalamus of dt brain at postnatal day 21 (P21). There is a similar but not significant tendency at postnatal day 14 (P14) in the dt brain. We also confirmed the reduced proliferation by PCNA ISH and Ki-67 immunohistochemistry. Double staining with cell-type-specific markers revealed that proliferating cells are oligodendrocyte progenitor cells (OPCs) in both wild-type and dt brain. We also observed a reduced number of Olig2-positive cells in the corpus callosum of Dst ^Gt homozygous mice at P21, indicating that reduced proliferation resulted in a reduced number of OPCs. Our data indicate that OPCs proliferation is reduced in the dt mouse brain at the postnatal stage and that it subsequently results in the reduced number of OPCs.     

Characterization of the new human pleomorphic undifferentiated sarcoma <Emphasis Type="Italic">TP53</Emphasis>-<Emphasis Type="Italic">null</Emphasis> cell line <Emphasis Type="Italic">mfh</Emphasis>-<Emphasis Type="Italic">val2</Emphasis>

Pleomorphic undifferentiated sarcoma (PUS), also called malignant fibrous histiocytoma, is a soft tissue sarcoma which occurs predominantly in the extremities. Its origin is a poorly defined mesenchymal cell, which derives to histiocytic and fibroblastic cells. The patient, a 58 year-old man, presented a lesion located in the forearm composed by spindle cells and multinucleated giant cells, which expressed vimentin and adopted a histological pattern formed by irregular-swirling fascicles. Cells were cultured in vitro and a new cell line was established. We characterized this new cell line by histological analyses, cytogenetics (using G-bands and spectral karyotype technique) and cytometric analyses. Cells were grown in culture for more than 100 passages. They had elongated or polygonal morphology. The cells presented a saturation rate of 70,980 cells/cm², a plating efficiency of 21.5% and a mitotic index of 21 mitoses per field. The cell line was tumorigenic in nude mice. The ploidy study using flow cytometry revealed an aneuploid peak with a DNA index of 1.43. A side population was detected, demonstrating the presence of stem and progenitor cells. Cytogenetics showed a hypotriploid range with many clonal unbalanced rearrangements. Loss of p53 gene was evidenced by MLPA. We describe, for the first time, the characterization of a new human PUS TP53-null cell line called mfh-val2. Mfh-val2 presents a wide number of applications as a TP53-null cell line and a great interest in order to characterize genetic alterations influencing the oncogenesis or progression of PUS and to advance in the biological investigation of this tumor.     

Nonquasineutral relativistic current filaments and their X-ray emission

Nonquasineutral electron current filaments with the azimuthal magnetic field are considered that arise due to the generation of electron vorticity in the initial (dissipative) stage of evolution of a current-carrying plasma, when the Hall number is small (σB/en _e c ? 1) because of the low values of the plasma conductivity and magnetic field strength. Equilibrium filamentary structures with both zero and nonzero net currents are considered. Structures with a zero net current type form on time scales of t < t _sk = (r ₀ω _pe /c)² t _st, where t _sk is the skin time, t _st is the typical time of electron-ion collisions, and r ₀ is the radius of the filament. It is shown that, in nonquasineutral filaments in which the current is carried by electrons drifting in the crossed electric (E _r ) and magnetic (B _θ) fields, ultrarelativistic electron beams on the typical charge-separation scale r _B = B/(4πen _e ) (the so-called magnetic Debye radius) can be generated. It is found that, for comparable electron currents, the characteristic electron energy in filaments with a nonzero net current is significantly lower than that in zero-net-current filaments that form on typical time scales of t < t _sk. This is because, in the latter type of filaments, the oppositely directed electron currents repel one another; as a result, both the density and velocity of electrons increase near the filament axis, where the velocities of relativistic electrons are maximum. Filaments with a zero net current can emit X rays with photon energies ? ω up to 10 MeV. The electron velocity distributions in filaments, the X-ray emission spectra, and the total X-ray yield per unit filament length are calculated as functions of the current and the electron number density in the filament. Analytical estimates of the characteristic lifetime of a radiating filament and the typical size of the radiating region as functions of the plasma density are obtained. The results of calculations are compared with the available experimental data.     

Ascorbic acid alters cell fate commitment of human neural progenitors in a WNT/β-catenin/ROS signaling dependent manner

Background

Improving the neuronal yield from in vitro cultivated neural progenitor cells (NPCs) is an essential challenge in transplantation therapy in neurological disorders. In this regard, Ascorbic acid (AA) is widely used to expand neurogenesis from NPCs in cultures although the mechanisms of its action remain unclear. Neurogenesis from NPCs is regulated by the redox-sensitive WNT/β-catenin signaling pathway. We therefore aimed to investigate how AA interacts with this pathway and potentiates neurogenesis.

Methods

Effects of 200 μM AA were compared with the pro-neurogenic reagent and WNT/β-catenin signaling agonist lithium chloride (LiCl), and molecules with antioxidant activities i.e. N-acetyl-L-cysteine (NAC) and ruthenium red (RuR), in differentiating neural progenitor ReNcell VM cells. Cells were supplemented with reagents for two periods of treatment: a full period encompassing the whole differentiation process versus an early short period that is restricted to the cell fate commitment stage. Intracellular redox balance and reactive oxygen species (ROS) metabolism were examined by flow cytometry using redox and ROS sensors. Confocal microscopy was performed to assess cell viability, neuronal yield, and levels of two proteins: Nucleoredoxin (NXN) and the WNT/β-catenin signaling component Dishevelled 2 (DVL2). TUBB3 and MYC gene responses were evaluated by quantitative real-time PCR. DVL2-NXN complex dissociation was measured by fluorescence resonance energy transfer (FRET).

Results

In contrast to NAC which predictably exhibited an antioxidant effect, AA treatment enhanced ROS metabolism with no cytotoxic induction. Both drugs altered ROS levels only at the early stage of the differentiation as no changes were held beyond the neuronal fate commitment stage. FRET studies showed that AA treatment accelerated the redox-dependent release of the initial pool of DVL2 from its sequestration by NXN, while RuR treatment hampered the dissociation of the two proteins. Accordingly, AA increased WNT/β-catenin signaling output i.e. MYC mRNA level, whereas RuR attenuated it. Moreover, AA improved neurogenesis as much as LiCl as both TUBB3-positive cell yield and TUBB3 mRNA level increased, while NAC or RuR attenuated neurogenesis. Markedly, the neurogenesis outputs between the short and the full treatment with either NAC or AA were found unchanged, supporting our model that neuronal yield is altered by events taking place at the early phase of differentiation.

Conclusions

Our findings demonstrate that AA treatment elevates ROS metabolism in a non-lethal manner prior to the NPCs commitment to their neuronal fate. Such effect stimulates the redox-sensitive DVL2 activation and WNT/β-catenin signaling response that would enhance the ensuing neuronal cell differentiation.