Owing to the hierarchical organization of biology, from genomes over transcriptomes and proteomes down to metabolomes, there is continuous debate about the extent to which data and interpretations ...derived from one level, e.g. the transcriptome, are in agreement with other levels, e.g. the metabolome. Here, we tested the effect of ocean acidification (OA; 400 vs. 1000 μatm CO2) and its modulation by light intensity (50 vs. 300 μmol photons m-2 s-1) on the biomass composition (represented by 75 key metabolites) of diploid and haploid life-cycle stages of the coccolithophore Emiliania huxleyi (RCC1216 and RCC1217) and compared these data with interpretations from previous physiological and gene expression screenings. The metabolite patterns showed minor responses to OA in both life-cycle stages. Whereas previous gene expression analyses suggested that the observed increased biomass buildup derived from lipid and carbohydrate storage, this dataset suggests that OA slightly increases overall biomass of cells, but does not significantly alter their metabolite composition. Generally, light was shown to be a more dominant driver of metabolite composition than OA, increasing the relative abundances of amino acids, mannitol and storage lipids, and shifting pigment contents to accommodate increased irradiance levels. The diploid stage was shown to contain vastly more osmolytes and mannitol than the haploid stage, which in turn had a higher relative content of amino acids, especially aromatic ones. Besides the differences between the investigated cell types and the general effects on biomass buildup, our analyses indicate that OA imposes only negligible effects on E. huxleyi´s biomass composition.
The control and coordination of a network of geographically and culturally dispersed subsidiaries is one of the most prominent challenges in international management. However, many empirical findings ...on the effectiveness of various control mechanisms and combinations thereof are still counterintuitive. This study uses longitudinal case studies and cross-sectional interview data to extend control theory by examining why, how, and in what sequence large multinational firms (MNCs) implement controls in their networks of foreign subsidiaries. Our analysis draws from literature on institutional theory, embeddedness, and organizational power to demonstrate that MNC headquarters need to overcome institutional duality when implementing their controls abroad. We find that headquarters do so by using social controls, primarily as a way of legitimizing and institutionalizing their process and output controls that are implemented subsequently.
The coccolithophore Emiliania huxleyi is a microalga with biogeochemical and biotechnological relevance, due to its high abundance in the ocean and its ability to form intricate calcium carbonate ...structures. Depletion of macronutrients in oceanic waters is very common and will likely enhance with advancing climate change. We present the first comprehensive metabolome study analyzing the effect of phosphorus (P) and nitrogen (N) starvation on the diploid and haploid life-cycle stage, applying various metabolome analysis methods to gain new insights in intracellular mechanisms to cope with nutrient starvation. P-starvation led to an accumulation of many generic and especially N-rich metabolites, including lipids, osmolytes, and pigments. This suggests that P-starvation primarily arrests cell-cycling due to lacking P for nucleic acid synthesis, but that enzymatic functionality is widely preserved. Also, the de-epoxidation ratio of the xanthophyll cycle was upregulated in the diploid stage under P-starvation, indicating increased nonphotochemical quenching, a response typically observed under high light stress. In contrast, N-starvation resulted in a decrease of most central metabolites, also P-containing ones, especially in the diploid stage, indicating that most enzymatic functionality ceased. The two investigated nutrient starvation conditions caused significantly different responses, contrary to previous assumptions derived from transcriptomic studies. Data highlight that instantaneous biochemical flux is a more dominant driver of the metabolome than the transcriptomically rearranged pathway patterns. Due to the fundamental nature of the observed responses it may be speculated that microalgae with similar nutrient requirements can cope better with P-starvation than with N-starvation.
Developmental programming in the kidney has been recognised for more than two decades, but its contribution to the global burden of kidney diseases remains underappreciated by policy makers.3 In view ...of the many factors known to affect fetal kidney development, including maternal health and nutrition, exposure to stress, poverty, pollutants, drugs, and infections during gestation,3 a holistic strategy to prevent such programming effects is consistent with the life-course approach and aligns with the United Nations (UN) Sustainable Development Goals to foster health.2 Chronic kidney disease is an important contributor to the NCD burden that has been relatively neglected in WHO's Global Action Plan for the Prevention and Control of NCDs, despite chronic kidney disease being a major cause of hypertension and a major risk multiplier of cardiovascular disease.1,4 Although the prevalence of chronic kidney disease in many low-income countries remains unknown, the disease is most prevalent among disadvantaged populations within industrialised nations-eg, African-Americans and Aboriginal Australians.5 The number of people receiving dialysis or transplantation is projected to double, from 2·6 million in 2010 to 5·4 million in 2030.6 In 2010, 2·3-7·1 million adults died from lack of access to dialysis and transplantation in low-income countries.6 In view of the clinical outcomes and often prohibitively high costs of treatment, prevention and early detection are the only sustainable solutions to address this growing global burden. 16
African catfish (Clarias gariepinus) were investigated for physiological stress parameters to assess the influence of different cultivation techniques on fish welfare. The fish were reared in two ...conceptually identical recirculating aquaculture systems (RAS) constructed in a greenhouse. One system was combined with a floating raft hydroponic unit to culture cucumbers (Cucumis sativus) only using the process water. The second system remained without the plant cultivation unit and functioned as the control. Water quality was monitored regularly by measuring dissolved nutrients and physical water parameters. During 87 days, a total of 71 fish from each system (n = 142) were studied, with a final sampling of all fish (n = 107) at the end of the experiment. Blood from the caudal vein was analyzed for the stress hormone cortisol and the glucose concentration. In addition, fish were investigated for external injuries on their skin, fins and barbels. The results demonstrate that the system design had no influence on fish mortalities and growth rates. Furthermore, cortisol and blood glucose levels did not differ significantly between the two systems. However, the number of external injuries was significantly lower in the aquaponics, providing evidence that co-cultivation of fish and plants might offer benefits to the welfare of the fish.
IL-17-producing CD8+ T (Tc17) cells are detectible in multiple sclerosis (MS) lesions; however, their contribution to the disease is unknown. To identify functions of Tc17 cells, we induced EAE, a ...murine model of MS, in mice lacking IFN regulatory factor 4 (IRF4). IRF4-deficient mice failed to generate Tc17 and Th17 cells and were resistant to EAE. After adoptive transfer of WT CD8+ T cells and subsequent immunization for EAE induction in these mice, the CD8+ T cells developed a Tc17 phenotype in the periphery but could not infiltrate the CNS. Similarly, transfer of small numbers of WT CD4+ T cells alone did not evoke EAE, but when transferred together with CD8+ T cells, IL-17-producing CD4+ (Th17) T cells accumulated in the CNS and mice developed severe disease. Th17 accumulation and development of EAE required IL-17A production by CD8+ T cells, suggesting that Tc17 cells are required to promote CD4+ T cell-mediated induction of EAE. Accordingly, patients with early-stage MS harbored a greater number of Tc17 cells in the cerebrospinal fluid than in peripheral blood. Our results reveal that Tc17 cells contribute to the initiation of CNS autoimmunity in mice and humans by supporting Th17 cell pathogenicity.
During CRISPR/Cas9 mediated genome editing, site-specific double strand breaks are introduced and repaired either unspecific by non-homologous end joining (NHEJ) or sequence dependent by homology ...directed repair (HDR). Whereas NHEJ-based generation of gene knock-out is widely performed, the HDR-based knock-in of specific mutations remains a bottleneck. Especially in primary cell lines that are essential for the generation of cell culture and animal models of inherited human diseases, knock-in efficacy is insufficient and needs significant improvement. Here, we tested two different approaches to increase the knock-in frequency of a specific point mutation into the
-gene in porcine fetal fibroblasts. We added a small molecule inhibitor of NHEJ, SCR7 (5,6-bis((E)-benzylideneamino)-2-mercaptopyrimidin-4-ol), during genome editing and screened cell cultures for the point mutation. However, this approach did not yield increased knock-in rates. In an alternative approach, we fused humanized Cas9 (hCas9) to the N-terminal peptide of the Geminin gene (
). The fusion protein is degraded in NHEJ-dominated cell cycle phases, which should increase HDR-rates. Using hCas9-
and point mutation-specific real time PCR screening, we found a two-fold increase in genome edited cell cultures. This increase of HDR by hCas9-
provides a promising way to enrich specific knock-in in porcine fibroblast cultures for somatic cloning approaches.
Leukodystrophies are rare genetic white matter disorders that have been regarded as mainly occurring in childhood. Recent years altered this perception, as a growing number of leukodystrophies was ...described to have an onset at adult ages. Still, many adult patients presenting with white matter changes remain without a specific molecular diagnosis. We describe a novel adult onset leukodystrophy in 16 patients from eight families carrying one of four different stop-gain or frameshift dominant variants in the CST3 gene. Clinical and radiological features differ markedly from the previously described Icelandic Cerebral Amyloid Angiopathy that was found in patients carrying p.Leu68Asn substitution in CST3. The clinical phenotype consists of recurrent episodes of hemiplegic migraine associated with transient unilateral focal deficits and slowly progressing motor symptoms and cognitive decline in mid-old adult ages. In addition, in some cases acute onset clinical deterioration led to a prolonged episode with reduced consciousness and even early death. Radiologically, pathognomonic changes are found at typical predilection sites involving the deep cerebral white matter sparing a periventricular and directly subcortical rim, the middle blade of corpus callosum, posterior limb of the internal capsule, middle cerebellar peduncles, cerebral peduncles, and specifically the globus pallidus. Histopathologic characterization in two autopsy cases did not reveal angiopathy, but instead micro- to macrocystic degeneration of the white matter. Astrocytes were activated at early stages and later on displayed severe degeneration and loss. In addition, despite loss of myelin, elevated numbers of partly apoptotic oligodendrocytes were observed. A structural comparison of the variants in CST3 suggests that specific truncations of Cystatin C result in an abnormal function, possibly by rendering the protein more prone to aggregation. Future studies are required to confirm the assumed effect on the protein and to determine pathophysiologic downstream events at the cellular level.
To evaluate the refractive and biometry results of presbyopic refractive lens exchange (RLE) with trifocal intraocular lens (IOL) implantation in eyes with previous myopic or hyperopic corneal laser ...vision correction (LVC).
Memira AS, Norway, Sweden, and Denmark.
Retrospective case series.
The refractive results included the manifest refraction spherical equivalent, uncorrected near (UNVA) and distance (UDVA) visual acuities, corrected distance visual acuity, safety, efficacy, and precision. The biometry analysis included the refractive prediction error (RPE), median absolute error (MedAE), and percentage of eyes within a certain RPE range for the formulas from the American Society of Cataract and Refractive Surgery (ASCRS) online calculator.
The study comprised 241 eyes. Six months postoperatively, 60.0% of eyes were within ±0.25 diopter (D), 80.9% within ±0.50 D, and 97.9% within ±1.00 D of emmetropia. There were no statistical differences in the mean monocular UDVA (0.87 ± 0.20 SD), safety index (0.98 ± 0.09), or efficacy index (0.81 ± 0.18) between the myopic ablation group and hyperopic ablation group. Binocularly, 85% of patients had simultaneous UDVA and UNVA of 0.9 or better and Jaeger 3, respectively. The ASCRS online calculator formulas gave different performances for previous myopic and hyperopic ablation profiles. Using optimized constants and nomogram for correcting the mean RPE improved the MedAE.
Presbyopic RLE was safe and effective in selected cases with a history of LVC. The use of optimized IOL constants and nomograms can improve the refractive precision of lens-based refractive surgery.
Clinical success of Toll-Like receptor-4 (TLR-4) antagonists in sepsis therapy has thus far been lacking. As inhibition of a receptor can only be useful if the receptor is active, stratification of ...patients with active TLR-4 would be desirable. Our aim was to establish an assay to quantify phosphorylated TLR-4 using the proximity ligation assay (PLA). HEK293 TLR4/MD2/CD14 as well as THP-1 cells were stimulated with LPS and the activation of TLR-4 was measured using the PLA. Furthermore, peripheral blood mononuclear cells (PBMCs) from 25 sepsis patients were used to show the feasibility of this assay in clinical material. Activation of TLR-4 in these samples was compared to the PBMCs of 11 healthy individuals. We could show a transient activation of TLR-4 in both cell lines. Five min after the LPS stimulation, the signal increased 6.7-fold in the HEK293 cells and 4.3-fold in the THP-1 cells. The assay also worked well in the PBMCs of septic patients. Phosphorylation of TLR-4 at study inclusion was 2.9 times higher in septic patients compared to healthy volunteers. To conclude, we established a diagnostic assay that is able to quantify the phosphorylation of TLR-4 in cell culture and in clinical samples of sepsis patients. This makes large-scale stratification of sepsis patients for their TLR-4 activation status possible.
PDF
