Dual plastid targeting of protoporphyrinogen oxidase 2 in Amaranthaceae promotes herbicide tolerance.

Plant tetrapyrrole biosynthesis (TPB) takes place in plastids and provides the chlorophyll and heme required for photosynthesis and many redox processes throughout plant development. TPB is strictly regulated, since accumulation of several intermediates causes photodynamic damage and cell death. Protoporphyrinogen oxidase (PPO) catalyzes the last common step before TPB diverges into chlorophyll and heme branches. Land plants possess two PPO isoforms. PPO1 is encoded as a precursor protein with a transit peptide, but in most dicotyledonous plants PPO2 does not possess a cleavable N-terminal extension. Arabidopsis (Arabidopsis thaliana) PPO1 and PPO2 localize in chloroplast thylakoids and envelope membranes, respectively. Interestingly, PPO2 proteins in Amaranthaceae contain an N-terminal extension that mediates their import into chloroplasts. Here, we present multiple lines of evidence for dual targeting of PPO2 to thylakoid and envelope membranes in this clade and demonstrate that PPO2 is not found in mitochondria. Transcript analyses revealed that dual targeting in chloroplasts involves the use of two transcription start sites and initiation of translation at different AUG codons. Among eudicots, the parallel accumulation of PPO1 and PPO2 in thylakoid membranes is specific for the Amaranthaceae and underlies PPO2-based herbicide resistance in Amaranthus species.

Wind turbines in managed forests partially displace common birds.

Wind turbines are increasingly being installed in forests, which can lead to land use disputes between climate mitigation efforts and nature conservation. Environmental impact assessments precede the construction of wind turbines to ensure that wind turbines are installed only in managed or degraded forests that are of potentially low value for conservation. It is unknown, nevertheless, if animals deemed of minor relevance in environmental impact assessments are affected by wind turbines in managed forests. We investigated the impact of wind turbines on common forest birds, by counting birds along an impact-gradient of wind turbines in 24 temperate forests in Hesse, Germany. During 860 point counts, we counted 2231 birds from 45 species. Bird communities were strongly related to forest structure, season and the rotor diameter of wind turbines, but were not related to wind turbine distance. For instance, bird abundance decreased in structure-poor (-38%) and monocultural (-41%) forests with wind turbines, and in young (-36%) deciduous forests with larger and more wind turbines (-24%). Overall, our findings suggest that wind turbines in managed forests partially displace common forest birds. If these birds are displaced to harsh environments, wind turbines might indirectly contribute to a decline of their populations. Yet, forest bird communities are locally more sensitive to forest quality than to wind turbine presence. To prevent further displacement of forest animals, forests of lowest quality for wildlife should be preferred in spatial planning for wind turbines, for instance small and structure-poor monocultures along highways.

Pathophysiological In Vitro Profile of Neuronal Differentiated Cells Derived from Niemann-Pick Disease Type C2 Patient-Specific iPSCs Carrying the NPC2 Mutations c.58G>T/c.140G>T.

Niemann-Pick type C2 (NP-C2) disease is a rare hereditary disease caused by mutations in the NPC2 gene. NPC2 is a small, soluble protein consisting of 151 amino acids, primarily expressed in late endosomes and lysosomes (LE/LY). Together with NPC1, a transmembrane protein found in these organelles, NPC2 accomplishes the exclusion of cholesterol; thus, both proteins are essential to maintain cellular cholesterol homeostasis. Consequently, mutations in the NPC2 or NPC1 gene result in pathophysiological accumulation of cholesterol and sphingolipids in LE/LY. The vast majority of Niemann-Pick type C disease patients, 95%, suffer from a mutation of NPC1, and only 5% display a mutation of NPC2. The biochemical phenotype of NP-C1 and NP-C2 appears to be indistinguishable, and both diseases share several commonalities in the clinical manifestation. Studies of the pathological mechanisms underlying NP-C2 are mostly based on NP-C2 animal models and NP-C2 patient-derived fibroblasts. Recently, we established induced pluripotent stem cells (iPSCs), derived from a donor carrying the NPC2 mutations c.58G>T/c.140G>T. Here, we present a profile of pathophysiological in vitro features, shared by NP-C1 and NP-C2, of neural differentiated cells obtained from the patient specific iPSCs. Profiling comprised a determination of the NPC2 protein level, detection of cholesterol accumulation by filipin staining, analysis of oxidative stress, and determination of autophagy. As expected, the NPC2-deficient cells displayed a significantly reduced amount of NPC2 protein, and, accordingly, we observed a significantly increased amount of cholesterol. Most notably, NPC2-deficient cells displayed only a slight increase of reactive oxygen species (ROS), suggesting that they do not suffer from oxidative stress and express catalase at a high level. As a site note, comparable NPC1-deficient cells suffer from a lack of catalase and display an increased level of ROS. In summary, this cell line provides a valuable tool to gain deeper understanding, not only of the pathogenic mechanism of NP-C2, but also of NP-C1.

Decreased calcium flux in Niemann-Pick type C1 patient-specific iPSC-derived neurons due to higher amount of calcium-impermeable AMPA receptors.

Niemann-Pick disease type C1 (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene, resulting mainly in the accumulation of cholesterol and the ganglioside GM2. Recently, we described accumulations of these lipids in neuronal differentiated cells derived from NPC1 patient-specific induced pluripotent stem cells (iPSCs). As these lipids are essential for proper cell membrane composition, we were interested in the expression and function of voltage-gated ion channels and excitatory AMPA receptors (AMPARs) in neurons derived from three patient-specific iPSC lines. By means of patch clamp recordings and microfluorimetric measurements of calcium (Ca2+), we examined the expression of voltage-gated ion channels and AMPARs. Cells of the three used cell lines carrying the c.1836A>C/c.1628delC, the c.1180T>C or the c.3182T>C mutation demonstrated a significantly reduced AMPA-induced Ca2+-influx, suggesting an altered expression profile of these receptors. RT-qPCR revealed a significant upregulation of mRNA for the AMPA receptor subunits GluA1 and GluA2 and western blot analysis showed increased protein level of GluA2. Thus, we conclude that the observed reduced Ca2+-influx is based on an increase of GluA2 containing Ca2+-impermeable AMPARs. An attenuated function of GluRs in neurons potentially contributes to the progressive neurodegeneration observed in NPC1 and might represent an objective in regard of the development of new therapeutic approaches in NPC1.

Additive effects of exotic plant abundance and land-use intensity on plant-pollinator interactions.

The continuing spread of exotic plants and increasing human land-use are two major drivers of global change threatening ecosystems, species and their interactions. Separate effects of these two drivers on plant-pollinator interactions have been thoroughly studied, but we still lack an understanding of combined and potential interactive effects. In a subtropical South African landscape, we studied 17 plant-pollinator networks along two gradients of relative abundance of exotics and land-use intensity. In general, pollinator visitation rates were lower on exotic plants than on native ones. Surprisingly, while visitation rates on native plants increased with relative abundance of exotics and land-use intensity, pollinator visitation on exotic plants decreased along the same gradients. There was a decrease in the specialization of plants on pollinators and vice versa with both drivers, regardless of plant origin. Decreases in pollinator specialization thereby seemed to be mediated by a species turnover towards habitat generalists. However, contrary to expectations, we detected no interactive effects between the two drivers. Our results suggest that exotic plants and land-use promote generalist plants and pollinators, while negatively affecting specialized plant-pollinator interactions. Weak integration and high specialization of exotic plants may have prevented interactive effects between exotic plants and land-use. Still, the additive effects of exotic plants and land-use on specialized plant-pollinator interactions would have been overlooked in a single-factor study. We therefore highlight the need to consider multiple drivers of global change in ecological research and conservation management.

Microscopic correlations in the finite-size Kuramoto model of coupled oscillators.

Supercritical Kuramoto oscillators with distributed frequencies can be separated into two disjoint groups: an ordered one locked to the mean field, and a disordered one consisting of effectively decoupled oscillators-at least so in the thermodynamic limit. In finite ensembles, in contrast, such clear separation fails: The mean field fluctuates due to finite-size effects and thereby induces order in the disordered group. This publication demonstrates this effect, similar to noise-induced synchronization, in a purely deterministic system. We start by modeling the situation as a stationary mean field with additional white noise acting on a pair of unlocked Kuramoto oscillators. An analytical expression shows that the cross-correlation between the two increases with decreasing ratio of natural frequency difference and noise intensity. In a deterministic finite Kuramoto model, the strength of the mean-field fluctuations is inextricably linked to the typical natural frequency difference. Therefore, we let a fluctuating mean field, generated by a finite ensemble of active oscillators, act on pairs of passive oscillators with a microscopic natural frequency difference between which we then measure the cross-correlation, at both super- and subcritical coupling.

Generation of the Niemann-Pick type C2 patient-derived iPSC line AKOSi001-A.

Niemann-Pick disease Type C (NPC) is a rare progressive neurodegenerative disorder with an incidence of 1:120,000 caused by mutations in the NPC1 or NPC2 gene. Only 5% of NPC patients suffer from mutations of the NPC2 gene. Here we demonstrate the generation of a Niemann-Pick disease Type C2 (NPC2) patient-derived induced pluripotent stem cell line. This cell line is capable to differentiate into derivatives of the neuronal lineage, providing a valuable tool to study pathogenic mechanisms of NPC2.

Transition to collective oscillations in finite Kuramoto ensembles.

We present an alternative approach to finite-size effects around the synchronization transition in the standard Kuramoto model. Our main focus lies on the conditions under which a collective oscillatory mode is well defined. For this purpose, the minimal value of the amplitude of the complex Kuramoto order parameter appears as a proper indicator. The dependence of this minimum on coupling strength varies due to sampling variations and correlates with the sample kurtosis of the natural frequency distribution. The skewness of the frequency sample determines the frequency of the resulting collective mode. The effects of kurtosis and skewness hold in the thermodynamic limit of infinite ensembles. We prove this by integrating a self-consistency equation for the complex Kuramoto order parameter for two families of distributions with controlled kurtosis and skewness, respectively.

Impact of Reduced Cerebellar EAAT Expression on Purkinje Cell Firing Pattern of NPC1-deficient Mice.

Niemann-Pick disease Type C1 (NPC1) is a rare hereditary neurodegenerative disease. NPC1-patients suffer, amongst others, from ataxia, based on a loss of cerebellar Purkinje cells (PCs). Impaired expression/function of excitatory amino acid transporters (EAATs) are suspected of contributing to PC-degeneration in hereditary spinocerebellar ataxias (SCAs). Thus, we studied EAAT-expression and its impact to PC-activity in NPC1-/-mice. Western blot revealed reduced EAAT1, EAAT2, EAAT4, and ßIII-spectrin levels in NPC1-/-mice. EAATs play a crucial role in synaptic transmission, thus we were interested in the impact of the reduced EAAT-expression on the function of PCs. Patch-clamp recordings of PCs showed no differences in the firing patterns of NPC1+/+and NPC1-/-mice using a low internal chloride concentration. Because EAAT4 also comprises a chloride permeable ion pore, we perturbed the chloride homeostasis using a high internal chloride concentration. We observed differences in the firing patterns of NPC1+/+and NPC1-/-mice, suggesting an impact of the altered EAAT4-expression. Additionally, the EAAT-antagonist DL-TBOA acts differently in NPC1+/+and NPC1-/-mice. Our data support the line of evidence that an altered EAAT-expression/function is involved in neurodegeneration of PCs observed in SCAs. Thus, we suggest that similar pathogenic mechanisms contribute the loss of PCs in NPC1.

Activation of PKC triggers rescue of NPC1 patient specific iPSC derived glial cells from gliosis.

BACKGROUND: Niemann-Pick disease Type C1 (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. The pathological mechanisms, underlying NPC1 are not yet completely understood. Especially the contribution of glial cells and gliosis to the progression of NPC1, are controversially discussed. As an analysis of affected cells is unfeasible in NPC1-patients, we recently developed an in vitro model system, based on cells derived from NPC1-patient specific iPSCs. Here, we asked if this model system recapitulates gliosis, observed in non-human model systems and NPC1 patient post mortem biopsies. We determined the amount of reactive astrocytes and the regulation of the intermediate filaments GFAP and vimentin, all indicating gliosis. Furthermore, we were interested in the assembly and phosphorylation of these intermediate filaments and finally the impact of the activation of protein kinase C (PKC), which is described to ameliorate the pathogenic phenotype of NPC1-deficient fibroblasts, including hypo-phosphorylation of vimentin and cholesterol accumulation. METHODS: We analysed glial cells derived from NPC1 patient specific induced pluripotent stem cells, carrying different NPC1 mutations. The amount of reactive astrocytes was determined by means of immuncytochemical stainings and FACS-analysis. Semi-quantitative western blot was used to determine the amount of phosphorylated GFAP and vimentin. Cholesterol accumulation was analysed by Filipin staining and quantified by Amplex Red Assay. U18666A was used to induce NPC1 phenotype in unaffected cells of the control cell line. Phorbol 12-myristate 13-acetate (PMA) was used to activate PKC. RESULTS: Immunocytochemical detection of GFAP, vimentin and Ki67 revealed that NPC1 mutant glial cells undergo gliosis. We found hypo-phosphorylation of the intermediate filaments GFAP and vimentin and alterations in the assembly of these intermediate filaments in NPC1 mutant cells. The application of U18666A induced not only NPC1 phenotypical accumulation of cholesterol, but characteristics of gliosis in glial cells derived from unaffected control cells. The application of phorbol 12-myristate 13-acetate, an activator of protein kinase C resulted in a significantly reduced number of reactive astrocytes and further characteristics of gliosis in NPC1-deficient cells. Furthermore, it triggered a restoration of cholesterol amounts to level of control cells. CONCLUSION: Our data demonstrate that glial cells derived from NPC1-patient specific iPSCs undergo gliosis. The application of U18666A induced comparable characteristics in un-affected control cells, suggesting that gliosis is triggered by hampered function of NPC1 protein. The activation of protein kinase C induced an amelioration of gliosis, as well as a reduction of cholesterol amount. These results provide further support for the line of evidence that gliosis might not be only a secondary reaction to the loss of neurons, but might be a direct consequence of a reduced PKC activity due to the phenotypical cholesterol accumulation observed in NPC1. In addition, our data support the involvement of PKCs in NPC1 disease pathogenesis and suggest that PKCs may be targeted in future efforts to develop therapeutics for NPC1 disease.

Dataset in support of the generation of Niemann-Pick disease Type C1 patient-specific iPS cell lines carrying the novel NPC1 mutation c.1180T>C or the prevalent c.3182T>C mutation - Analysis of pluripotency and neuronal differentiation.

Data presented in this article demonstrate the generation and characterization of two novel Niemann-Pick disease Type C1 (NPC1) patient-specific induced pluripotent stem cell (iPSC) lines, related to the research article Trilck et al. (Diversity of Glycosphingolipid GM2 and Cholesterol Accumulation in NPC1 Patient-Specific iPSC-Derived Neurons; Brain Res.; 2017; 1657:52-61. doi: 10.1016/j.brainres.2016.11.031). For reprogramming fibroblasts, carrying the novel homozygous mutation c.1180T>C and the prevalent homozygous mutation c.3182T>C, were used. Reprogramming into patient-specific iPSCs was induced by retroviral transduction of the transcription factors Sox2, Klf4, Oct4 and c-Myc, and confirmed according to their pluripotency. The iPSCs were subsequently differentiated into neural progenitor cells, which were terminally differentiated into functional neurons and glial cells. The generation of these cell lines provides further valuable tools to investigate pathogenic mechanism of NPC1 in human neuronal cells carrying different NPC1 mutations.

Diversity of glycosphingolipid GM2 and cholesterol accumulation in NPC1 patient-specific iPSC-derived neurons.

Niemann-Pick disease Type C1 (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. On the cellular level NPC1 mutations lead to an accumulation of cholesterol and gangliosides. As a thorough analysis of the severely affected neuronal cells is unfeasible in NPC1 patients, we recently described the cellular phenotype of neuronal cells derived from NPC1 patient iPSCs carrying the compound heterozygous mutation c.1836A>C/c.1628delC. Here we expanded the analysis to cell lines carrying the prevalent mutation c.3182T>C and the novel mutation c.1180T>C, as well as to the determination of GM2 and GM3 gangliosides in NPC1 patient-specific iPSC-derived neurons and glia cells. Immunocytochemical detection of GM2 revealed punctated staining pattern predominantly localized in neurons. Detection of cholesterol by filipin staining showed a comparable staining pattern, colocalized with GM2, indicating a deposit of GM2 and cholesterol in the same cellular compartments. Accumulations were not only restricted to cell bodies, but were also found in the neuronal extensions. A quantification of the GM2 amount by HPLC-MS/MS confirmed significantly higher amounts in neurons carrying a mutation. Additionally, these cells displayed a lowered activity of the catabolic enzyme Hex A, but not B4GALNT1. Molecular docking simulations indicated binding of cholesterol to Hex A, suggesting cholesterol influences the GM2 degradation pathway and, subsequently, leading to the accumulation of GM2. Taken together, this is the first study showing an accumulation of GM2 in neuronal derivatives of patient-specific iPSCs and thus proving further disease-specific hallmarks in this human in vitro model of NPC1.

Effects of local tree diversity on herbivore communities diminish with increasing forest fragmentation on the landscape scale.

Forest fragmentation and plant diversity have been shown to play a crucial role for herbivorous insects (herbivores, hereafter). In turn, herbivory-induced leaf area loss is known to have direct implications for plant growth and reproduction as well as long-term consequences for ecosystem functioning and forest regeneration. So far, previous studies determined diverging responses of herbivores to forest fragmentation and plant diversity. Those inconsistent results may be owed to complex interactive effects of both co-occurring environmental factors albeit they act on different spatial scales. In this study, we investigated whether forest fragmentation on the landscape scale and tree diversity on the local habitat scale show interactive effects on the herbivore community and leaf area loss in subtropical forests in South Africa. We applied standardized beating samples and a community-based approach to estimate changes in herbivore community composition, herbivore abundance, and the effective number of herbivore species on the tree species-level. We further monitored leaf area loss to link changes in the herbivore community to the associated process of herbivory. Forest fragmentation and tree diversity interactively affected the herbivore community composition, mainly by a species turnover within the family of Curculionidae. Furthermore, herbivore abundance increased and the number of herbivore species decreased with increasing tree diversity in slightly fragmented forests whereas the effects diminished with increasing forest fragmentation. Surprisingly, leaf area loss was neither affected by forest fragmentation or tree diversity, nor by changes in the herbivore community. Our study highlights the need to consider interactive effects of environmental changes across spatial scales in order to draw reliable conclusions for community and interaction patterns. Moreover, forest fragmentation seems to alter the effect of tree diversity on the herbivore community, and thus, has the potential to jeopardize ecosystem functioning and forest regeneration.

Prognostic impact of pretherapeutic laboratory values in head and neck cancer patients.

PURPOSE: The high comorbidity in patients with head and neck cancer (HNC) is mainly caused by the high incidence of tobacco and alcohol abuse and has direct impact on overall survival. We investigated whether HNC and its comorbidity also influence routine laboratory values and whether these values have influence on overall survival. METHODS: A retrospective cohort of 261 patients with primary squamous cell carcinoma of the oral cavity, pharynx, or larynx diagnosed between 2001 and 2006 with a complete set of pretherapeutic laboratory values was identified. The influence of standard oncological parameters, comorbidity, and each laboratory value on overall survival (OS) was investigated in univariate and multivariate analyses. RESULTS: Two-thirds of patients were active smokers and about one half reported high alcohol consumption. 40 % of patients had severe comorbidity according to Charlson comorbidity index. The most frequent laboratory pathologies were elevated C-reactive protein (CRP) values (66 %), impaired liver enzymes (30-50 %), decreased urea levels (33 %), leukocytosis (20 %), and anemia (10 %). In multivariate analysis for OS, a comorbidity index ≥5 (Hazard ratio [HR], 2.008; 95 % confidence interval [CI], 1.117-3.610; p = 0.020), high CRP level (HR, 2.469; CI, 1.414-4.310; p = 0.001), and abnormal low red-cell count (HR, 2.525; CI, 1.250-5.102; p = 0.010) were independent prognostic variables. CONCLUSIONS: Comorbidity reflected by pathologic laboratory values is a major issue in HNC patients. Several pretherapeutic laboratory values have prognostic relevance for overall survival in HNC patients.

Probing the SELEX process with next-generation sequencing.

BACKGROUND: SELEX is an iterative process in which highly diverse synthetic nucleic acid libraries are selected over many rounds to finally identify aptamers with desired properties. However, little is understood as how binders are enriched during the selection course. Next-generation sequencing offers the opportunity to open the black box and observe a large part of the population dynamics during the selection process. METHODOLOGY: We have performed a semi-automated SELEX procedure on the model target streptavidin starting with a synthetic DNA oligonucleotide library and compared results obtained by the conventional analysis via cloning and Sanger sequencing with next-generation sequencing. In order to follow the population dynamics during the selection, pools from all selection rounds were barcoded and sequenced in parallel. CONCLUSIONS: High affinity aptamers can be readily identified simply by copy number enrichment in the first selection rounds. Based on our results, we suggest a new selection scheme that avoids a high number of iterative selection rounds while reducing time, PCR bias, and artifacts.

Increased DHEA and DHEA-S plasma levels in patients with post-traumatic stress disorder and a history of childhood abuse.

Current findings about dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEA-S) in patients with post-traumatic stress disorder (PTSD) have been inconsistent. We investigated whether a history of severe childhood traumatisation affects these steroids in PTSD patients. Patients of 33 with chronic PTSD (15 with and 18 without sexual and/or severe physical abuse before age 12) were studied in a combined low dose dexamethasone/corticotropin-releasing hormone (CRH) test. Mean pre-CRH levels of both plasma DHEA and DHEA-S were significantly increased in the subgroup with childhood abuse, the respective ratios with plasma cortisol were significantly lower. In the entire population of PTSD patients significant amounts of the variation of these parameters could be explained by childhood trauma history. Further studies are needed to clarify the potential role of DHEA and DHEA-S as biomarkers for severe early adverse events in patients suffering from PTSD and in other stress-related disorders.