Distribution of micro- (Fe,Zn, Cu,and Mn) and risk (Al,As, Cr,Ni, Pb,and Cd) elements in the organs of Rumex alpinus L. in the Alps and Krkonoše Mountains

Plant and Soil - Rumex alpinus is a native plant in the mountains of Europe whose distribution has partly been affected by its utilization as a vegetable and medicinal herb. The distribution of...     

A Critical Period for Postnatal Adaptive Plasticity in a Model of Motor Axon Miswiring

The correct wiring of neuronal circuits is of crucial importance for precise neuromuscular functionality. Therefore, guidance cues provide tight spatiotemporal control of axon growth and guidance. Mice lacking the guidance cue Semaphorin 3F (Sema3F) display very specific axon wiring deficits of motor neurons in the medial aspect of the lateral motor column (LMCm). While these deficits have been investigated extensively during embryonic development, it remained unclear how Sema3F mutant mice cope with these errors postnatally. We therefore investigated whether these animals provide a suitable model for the exploration of adaptive plasticity in a system of miswired neuronal circuitry. We show that the embryonically developed wiring deficits in Sema3F mutants persist until adulthood. As a consequence, these mutants display impairments in motor coordination that improve during normal postnatal development, but never reach wildtype levels. These improvements in motor coordination were boosted to wildtype levels by housing the animals in an enriched environment starting at birth. In contrast, a delayed start of enriched environment housing, at 4 weeks after birth, did not similarly affect motor performance of Sema3F mutants. These results, which are corroborated by neuroanatomical analyses, suggest a critical period for adaptive plasticity in neuromuscular circuitry. Interestingly, the formation of perineuronal nets, which are known to close the critical period for plastic changes in other systems, was not altered between the different housing groups. However, we found significant changes in the number of excitatory synapses on limb innervating motor neurons. Thus, we propose that during the early postnatal phase, when perineuronal nets have not yet been formed around spinal motor neurons, housing in enriched environment conditions induces adaptive plasticity in the motor system by the formation of additional synaptic contacts, in order to compensate for coordination deficits.     

Conventional protein kinase C-α (PKC-α) and PKC-β negatively regulate RIG-I antiviral signal transduction

Retinoic acid-inducible gene I (RIG-I) is a key sensor for viral RNA in the cytosol, and it initiates a signaling cascade that leads to the establishment of an interferon (IFN)-mediated antiviral state. Because of its integral role in immune signaling, RIG-I activity must be precisely controlled. Recent studies have shown that RIG-I CARD-dependent signaling function is regulated by the dynamic balance between phosphorylation and TRIM25-induced K₆₃-linked ubiquitination. While ubiquitination of RIG-I is critical for RIG-I''s ability to induce an antiviral IFN response, phosphorylation of RIG-I at S₈ or T₁₇₀ suppresses RIG-I signal-transducing activity under normal conditions. Here, we not only further define the roles of S₈ and T₁₇₀ phosphorylation for controlling RIG-I activity but also identify conventional protein kinase C-α (PKC-α) and PKC-β as important negative regulators of the RIG-I signaling pathway. Mutational analysis indicated that while the phosphorylation of S₈ or T₁₇₀ potently inhibits RIG-I downstream signaling, the dephosphorylation of RIG-I at both residues is necessary for optimal TRIM25 binding and ubiquitination-mediated RIG-I activation. Furthermore, exogenous expression, gene silencing, and specific inhibitor treatment demonstrated that PKC-α/β are the primary kinases responsible for RIG-I S₈ and T₁₇₀ phosphorylation. Coimmunoprecipitation showed that PKC-α/β interact with RIG-I under normal conditions, leading to its phosphorylation, which suppresses TRIM25 binding, RIG-I CARD ubiquitination, and thereby RIG-I-mediated IFN induction. PKC-α/β double-knockdown cells exhibited markedly decreased S₈/T₁₇₀ phosphorylation levels of RIG-I and resistance to infection by vesicular stomatitis virus. Thus, these findings demonstrate that PKC-α/β-induced RIG-I phosphorylation is a critical regulatory mechanism for controlling RIG-I antiviral signal transduction under normal conditions.     

Reassessing the out-of-phase phenomenon in shake flasks by evaluating the angle-dependent liquid distribution relative to the direction of the centrifugal acceleration

Shake flasks are still the most relevant experimental tool in the development of viscous fermentation processes. The phase number, which defines the onset of the unfavorable out-of-phase (OP) phenomenon in shake flasks, was previously defined via specific power input measurements. In the OP state, the bulk liquid no longer follows the orbital movement of the imposed centrifugal force, which is for example, detrimental to oxygen transfer. In this study, an optical fluorescence technique was used to measure the three-dimensional liquid distribution in shake flasks. Four new optically derived evaluation criteria for the phase transition between the in-phase and OP condition were established: (a) thickness of the liquid film left on the glass wall by the rotating bulk liquid, (b) relative slope of the leading edge of bulk liquid (LB) lines, (c) trend of the angular position of LB, and (d) very high angular position of the leading edge. In contrast to the previously applied power input measurements, the new optical evaluation criteria describe the phase transition in greater detailed. Instead of Ph = 1.26, a less conservative value of Ph = 0.91 is now suggested for the phase transfer, which implies a broader operating window for shake flask cultivations with higher viscosities.     

Social instability in adolescence differentially alters dendritic morphology in the medial prefrontal cortex and its response to stress in adult male and female rats

Adolescence is an important period for HPA axis development and synapse maturation and reorganization in the prefrontal cortex (PFC). Thus, stress during adolescence could alter stress‐sensitive brain regions such as the PFC and may alter the impact of future stressors on these brain regions. Given that women are more susceptible to many stress‐linked psychological disorders in which dysfunction of PFC is implicated, and that this increased vulnerability emerges in adolescence, stress during this time could have sex‐dependent effects. Therefore, we investigated the effects of adolescent social instability stress (SIS) on dendritic morphology of Golgi‐stained pyramidal cells in the medial PFC of adult male and female rats. We then examined dendritic reorganization following chronic restraint stress (CRS) with and without a rest period in adult rats that had been stressed in adolescence. Adolescent SIS conferred long‐term alterations in prelimbic of males and females, whereby females show reduced apical length and basilar thin spine density and males show reduced basilar length. CRS in adulthood failed to produce immediate dendritic remodeling in SIS rats. However, CRS followed by a rest period reduced apical dendritic length and increases mushroom spine density in adolescently stressed adult males. Conversely, CRS followed by rest produced apical outgrowth and decreased mushroom spine density in adolescently stressed adult females. These results suggest that stress during adolescence alters development of the PFC and modulates stress‐induced dendritic changes in adulthood.     

Allogeneic tumor lysate can serve as both antigen source and protein supplementation for dendritic cell culture

BACKGROUND: Recent preclinical and clinical evidence suggests the use of allogeneic tumor as a source of antigen for DC-based immunotherapy against cancer. We hypothesized that addition of allogeneic tumor lysate to monocyte-derived DC culture could serve a dual purpose: (1) antigen source and (2) protein supplementation of DC culture media. Protein supplementation whether of known origin (human serum/plasma, fetal bovine serum, human serum albumin) or undeclared origin ("serum-free" media) is a source of variability and bias. We addressed the question whether protein supplementation can be omitted in the presence of allogeneic tumor lysate. MATERIALS AND METHODS: Human DC cultured in the presence of lysate from medullary thyroid carcinoma (MTC) cell line SHER-I (TuLy-DC) and DC pulsed with the same lysate but cultured in the presence of FBS (FBS-DC) were assessed for morphology, phenotype, maturation and functional properties. RESULTS: In comparison of FBS-DC/TuLy-DC no significant differences in morphology, phenotype and maturation could be detected. Both culture conditions produced CD1a(high), CD14(low) DC with high expression of costimulatory molecules and CD83 upon stimulation. TuLy-DC gave significantly better yields and produced more IL12p70. DC showed high (allo)stimulatory capacity toward T-cells. TuLy-DC induced more intracellular IFNgamma in CD8+T-cells of vaccinated MTC patients. Both types of DC induced killing of SHER-I after short in vitro restimulation. Tumor lysate from SHER-I can substitute for further protein supplementation in DC culture. Allogeneic tumor lysates should be taken into consideration as both source of antigen and protein supplementation in monocyte-derived DC culture.     

Use of space and behavior of weaned piglets kept in enriched two-level housing system

In this study, the possibility of introducing an elevated platform to a piglet pen was explored as a way of increasing available space and creating functional areas. On the platform, nine different manipulable materials were offered. In four batches, 40 weaned piglets were kept for five weeks in the two-level pen. Video recordings were taken two days per week. In the afternoon, more piglets were on the platform than in the morning or at night (7.2 ± 0.1 vs. 4.9 ± 0.1 vs. 0.6 ± 0.1 piglets/5 minutes; p < .05). The area under the platform was preferred more in the morning and at night than in the afternoon (18.5 ± 0.1 vs. 21.6 ± 0.2 vs. 12.5 ± 0.1 piglets/5 minutes; p < .05). Up to 36 piglets were counted there simultaneously, mainly in the recumbent position. On and under the platform, air velocity and ammonia concentration were within the recommended ranges. The study concluded that a two-level pen is a feasible option to increase space allowance and to create functional areas in a piglet pen.     

Global Epigenetic Regulation of MicroRNAs in Multiple Myeloma

Epigenetic changes frequently occur during tumorigenesis and DNA hypermethylation may account for the inactivation of tumor suppressor genes in cancer cells. Studies in Multiple Myeloma (MM) have shown variable DNA methylation patterns with focal hypermethylation changes in clinically aggressive subtypes. We studied global methylation patterns in patients with relapsed/refractory MM and found that the majority of methylation peaks were located in the intronic and intragenic regions in MM samples. Therefore, we investigated the effect of methylation on miRNA regulation in MM. To date, the mechanism by which global miRNA suppression occurs in MM has not been fully described. In this study, we report hypermethylation of miRNAs in MM and perform confirmation in MM cell lines using bisulfite sequencing and methylation-specific PCR (MSP) in the presence or absence of the DNA demethylating agent 5-aza-2′-deoxycytidine. We further characterized the hypermethylation-dependent inhibition of miR-152, -10b-5p and -34c-3p which was shown to exert a putative tumor suppressive role in MM. These findings were corroborated by the demonstration that the same miRNAs were down-regulated in MM patients compared to healthy individuals, alongside enrichment of miR-152-, -10b-5p, and miR-34c-3p-predicted targets, as shown at the mRNA level in primary MM cells. Demethylation or gain of function studies of these specific miRNAs led to induction of apoptosis and inhibition of proliferation as well as down-regulation of putative oncogene targets of these miRNAs such as DNMT1, E2F3, BTRC and MYCBP. These findings provide the rationale for epigenetic therapeutic approaches in subgroups of MM.     

Expression of a Catalytically Inactive Mutant Form of Glutathione Peroxidase 4 (Gpx4) Confers a Dominant-negative Effect in Male Fertility

The selenoenzyme Gpx4 is essential for early embryogenesis and cell viability for its unique function to prevent phospholipid oxidation. Recently, the cytosolic form of Gpx4 was identified as an upstream regulator of a novel form of non-apoptotic cell death, called ferroptosis, whereas the mitochondrial isoform of Gpx4 was previously shown to be crucial for male fertility. Here, we generated and analyzed mice with a targeted mutation of the active site selenocysteine of Gpx4 (Gpx4_U46S). Mice homozygous for Gpx4_U46S died at the same embryonic stage (E7.5) as Gpx4^−/− embryos as expected. Surprisingly, male mice heterozygous for Gpx4_U46S presented subfertility. Subfertility was manifested in a reduced number of litters from heterozygous breeding and an impairment of spermatozoa to fertilize oocytes in vitro. Morphologically, sperm isolated from heterozygous Gpx4_U46S mice revealed many structural abnormalities particularly in the spermatozoa midpiece due to improper oxidation and polymerization of sperm capsular proteins and malformation of the mitochondrial capsule surrounding and stabilizing sperm mitochondria. These findings are reminiscent of sperm isolated from selenium-deprived rodents or from mice specifically lacking mitochondrial Gpx4. Due to a strongly facilitated incorporation of Ser in the polypeptide chain as compared with selenocysteine at the UGA codon, expression of the catalytically inactive Gpx4_U46S was found to be strongly increased. Because the stability of the mitochondrial capsule of mature spermatozoa depends on the moonlighting function of Gpx4 both as an enzyme oxidizing capsular protein thiols and as a structural protein, tightly controlled expression of functional Gpx4 emerges as a key for full male fertility.     

High CO2 concentration and iron availability determine the metabolic inventory in an Emiliania huxleyi-dominated phytoplankton community

Ocean acidification (OA), a consequence of anthropogenic carbon dioxide (CO₂) emissions, strongly impacts marine ecosystems. OA also influences iron (Fe) solubility, affecting biogeochemical and ecological processes. We investigated the interactive effects of CO₂ and Fe availability on the metabolome response of a natural phytoplankton community. Using mesocosms we exposed phytoplankton to ambient (390 μatm) or future CO₂ levels predicted for the year 2100 (900 μatm), combined with ambient (4.5 nM) or high (12 nM) dissolved iron (dFe). By integrating over the whole phytoplankton community, we assigned functional changes based on altered metabolite concentrations. Our study revealed the complexity of phytoplankton metabolism. Metabolic profiles showed three stages in response to treatments and phytoplankton dynamics. Metabolome changes were related to the plankton group contributing respective metabolites, explaining bloom decline and community succession. CO₂ and Fe affected metabolic profiles. Most saccharides, fatty acids, amino acids and many sterols significantly correlated with the high dFe treatment at ambient pCO₂. High CO₂ lowered the abundance of many metabolites irrespective of Fe. However, sugar alcohols accumulated, indicating potential stress. We demonstrate that not only altered species composition but also changes in the metabolic landscape affecting the plankton community may change as a consequence of future high-CO₂ oceans.