The inner membrane protein Mdm33 controls mitochondrial morphology in yeast

Mitochondrial distribution and morphology depend on MDM33, a Saccharomyces cerevisiae gene encoding a novel protein of the mitochondrial inner membrane. Cells lacking Mdm33 contain ring-shaped, mostly interconnected mitochondria, which are able to form large hollow spheres. On the ultrastructural level, these aberrant organelles display extremely elongated stretches of outer and inner membranes enclosing a very narrow matrix space. Dilated parts of Delta mdm33 mitochondria contain well-developed cristae. Overexpression of Mdm33 leads to growth arrest, aggregation of mitochondria, and generation of aberrant inner membrane structures, including septa, inner membrane fragments, and loss of inner membrane cristae. The MDM33 gene is required for the formation of net-like mitochondria in mutants lacking components of the outer membrane fission machinery, and mitochondrial fusion is required for the formation of extended ring-like mitochondria in cells lacking the MDM33 gene. The Mdm33 protein assembles into an oligomeric complex in the inner membrane where it performs homotypic protein-protein interactions. Our results indicate that Mdm33 plays a distinct role in the mitochondrial inner membrane to control mitochondrial morphology. We propose that Mdm33 is involved in fission of the mitochondrial inner membrane.     

Importance of detecting numerical versus structural chromosome aberrations

The aim is to review briefly the key questions related to aneuploidy/polyploidy and to compare the advantages and disadvantages of the in vitro micronucleus test to assess aneuploidy/polyploidy in vitro. The key questions that will be addressed, concern the importance of polyploidy for health, and cancer in particular, the mechanisms leading to aneuploidy and polyploidy, and the survival of aneuploid/polyploid cells.The recently recognised contribution of numerical chromosome changes to carcinogenesis triggered the development and the implementation of tests specifically aiming at the detection of aneugens in the test battery for mutagenicity and carcinogenicity. The validation of the in vitro micronucleus test in combination with the identification of in vitro divided cells with the cytokinesis-block methodology and of centromeres with pancentromeric or chromosome specific centromeric probes fluorescence in situ hybridisation (FISH) provides a sensitive, easy to score and powerful test which allows assessment of cell proliferation, the discrimination between chromosome breaks, chromosome loss and chromosome non-disjunction and polyploidy. Moreover, classic histology permits the estimation of necrosis and apoptosis on the same slide. The cytokinesis-blocked micronucleus assay could be considered as a multi-endpoint test for genotoxic responses to clastogens/aneugens. This methodology has also shown to be capable of identifying threshold values for the induction of chromosome loss and/or non-disjunction by microtubule inhibitors, data which are particularly important for risk calculations. Similar approaches were conducted in vivo on bone marrow in mice and rats (except for identification of chromosome non-disjunction), and are in development for gut in mice.     

Lack of Bdnf and TrkB signalling in the postnatal cochlea leads to a spatial reshaping of innervation along the tonotopic axis and hearing loss

Members of the neurotrophin gene family and their high-affinity Trk receptors control innervation of the cochlea during embryonic development. Lack of neurotrophin signalling in the cochlea has been well documented for early postnatal animals, resulting in a loss of cochlear sensory neurones and a region-specific reduction of target innervation along the tonotopic axis. However, how reduced neurotrophin signalling affects the innervation of the mature cochlea is currently unknown. Here, we have analysed the consequences of a lack of the TrkB receptor and its ligand, the neurotrophin brain-derived neurotrophic factor (Bdnf), in the late postnatal or adult cochlea using mouse mutants. During early postnatal development, mutant animals show a lack of afferent innervation of outer hair cells in the apical part of the cochlea, whereas nerve fibres in the basal part are maintained. Strikingly, this phenotype is reversed during subsequent maturation of the cochlea, which results in a normal pattern of outer hair cell innervation in the apex and loss of nerve fibres at the base in adult mutants. Measurements of auditory brain stem responses of these mice revealed a significant hearing loss. The observed innervation patterns correlate with opposing gradients of Bdnf and Nt3 expression in cochlear neurones along the tonotopic axis. Thus, the reshaping of innervation may be controlled by autocrine signalling between neurotrophins and their receptors in cochlear neurones. Our results indicate a substantial potential for re-innervation processes in the mature cochlea, which may also be of relevance for treatment of hearing loss in humans.     

Regression to the Mean and Predictors of MRI Disease Activity in RRMS Placebo Cohorts - Is There a Place for Baseline-to-Treatment Studies in MS?

BackgroundGadolinium-enhancing (GD+) lesions and T2 lesions are MRI outcomes for phase-2 treatment trials in relapsing-remitting Multiple Sclerosis (RRMS). Little is known about predictors of lesion development and regression-to-the-mean, which is an important aspect in early baseline-to-treatment trials.ObjectivesTo quantify regression-to-the-mean and identify predictors of MRI lesion development in placebo cohorts.Methods21 Phase-2 and Phase-3 trials were identified by a systematic literature research. Random-effects meta-analyses were performed to estimate development of T2 and GD+ after 6 months (phase-2) or 2 years (phase-3). Predictors of lesion development were evaluated with mixed-effect meta-regression.ResultsThe mean number of GD+-lesions per scan was similar after 6 months (1.19, 95%CI: 0.87-1.51) and 2 years (1.19, 95%CI: 1.00-1.39). 39% of the patients were without new T2-lesion after 6 month and 19% after 2 years (95%CI: 12-25%). Mean number of baseline GD+-lesions was the best predictor for new lesions after 6 months.ConclusionBaseline GD-enhancing lesions predict evolution of Gd- and T2 lesions after 6 months and might be used to control for regression to the mean effects. Overall, proof-of-concept studies with a baseline to treatment design have to face a regression to 1.2 GD+lesions per scan within 6 months.     

Ecological Validity of Walking Capacity Tests in Multiple Sclerosis

Background

Ecological validity implicates in how far clinical assessments refer to real life. Short clinical gait tests up to ten meters and 2- or 6-Minutes Walking Tests (2MWT/6MWT) are used as performance-based outcomes in Multiple Sclerosis (MS) studies and considered as moderately associated with real life mobility.

Objective

To investigate the ecological validity of 10 Meter Walking Test (10mWT), 2MWT and 6MWT.

Methods

Persons with MS performed 10mWT, 6MWT including 2MWT and 7 recorded days by accelerometry. Ecological validity was assumed if walking tests represented a typical walking sequence in real-life and correlations with accelerometry parameters were strong.

Results

In this cohort (n=28, medians: age=45, EDSS=3.2, disease duration=9 years), uninterrupted walking of 2 or 6 minutes occurred not frequent in real life (2.61 and 0.35 sequences/day). 10mWT correlated only with slow walking speed quantiles in real life. 2MWT and 6MWT correlated moderately with most real life walking parameters.

Conclusion

Clinical gait tests over a few meters have a poor ecological validity while validity is moderate for 2MWT and 6MWT. Mobile accelerometry offers the opportunity to control and improve the ecological validity of MS mobility outcomes.     

T1- Thresholds in Black Holes Increase Clinical-Radiological Correlation in Multiple Sclerosis Patients

Background

Magnetic Resonance Imaging (MRI) is an established tool in diagnosing and evaluating disease activity in Multiple Sclerosis (MS). While clinical-radiological correlations are limited in general, hypointense T1 lesions (also known as Black Holes (BH)) have shown some promising results. The definition of BHs is very heterogeneous and depends on subjective visual evaluation.

Objective

We aimed to improve clinical-radiological correlations by defining BHs using T1 relaxation time (T1-RT) thresholds to achieve best possible correlation between BH lesion volume and clinical disability.

Method

40 patients with mainly relapsing-remitting MS underwent MRI including 3-dimensional fluid attenuated inversion recovery (FLAIR), magnetization-prepared rapid gradient echo (MPRAGE) before and after Gadolinium (GD) injection and double inversion-contrast magnetization-prepared rapid gradient echo (MP2RAGE) sequences. BHs (BH_vis) were marked by two raters on native T1-weighted (T1w)-MPRAGE, contrast-enhancing lesions (CE lesions) on T1w-MPRAGE after GD and FLAIR lesions (total-FLAIR lesions) were detected separately. BH_vis and total-FLAIR lesion maps were registered to MP2RAGE images, and the mean T1-RT were calculated for all lesion ROIs. Mean T1 values of the cortex (CTX) were calculated for each patient. Subsequently, Spearman rank correlations between clinical scores (Expanded Disability Status Scale and Multiple Sclerosis Functional Composite) and lesion volume were determined for different T1-RT thresholds.

Results

Significant differences in T1-RT were obtained between all different lesion types with highest T1 values in visually marked BHs (BH_vis: 1453.3±213.4 ms, total-FLAIR lesions: 1394.33±187.38 ms, CTX: 1305.6±35.8 ms; p<0.05). Significant correlations between BH_vis/total-FLAIR lesion volume and clinical disability were obtained for a wide range of T1-RT thresholds. The highest correlation for BH_vis and total-FLAIR lesion masks were found at T1-RT>1500 ms (Expanded Disability Status Scale vs. lesion volume: r_BHvis = 0.442 and r_total-FLAIR = 0.497, p<0.05; Multiple Sclerosis Functional Composite vs. lesion volume: r_BHvis = -0.53 and r_total-FLAIR = -0.627, p<0.05).

Conclusion

Clinical-radiological correlations in MS patients are increased by application of T1-RT thresholds. With the short acquisition time of the MP2RAGE sequences, quantitative T1 maps could be easily established in clinical studies.     

Comparing mortality in renal patients on hemodialysis versus peritoneal dialysis using a marginal structural model

When comparing the causal effect of peritoneal dialysis (PD) and hemodialysis (HD) treatment on lowering mortality in renal patients, using observational data, it is necessary to adjust for different forms of confounding and informative censoring. Both the type of dialysis treatment that is started with and mortality are affected by baseline covariates. Longitudinal and baseline variables can affect both the probability of switching from one type of dialysis to the other, and mortality. Longitudinal and baseline variables can also affect the probability of receiving a kidney transplant, possibly causing informative censoring. Adjusting for longitudinal variables by including them as covariates in a regression model potentially causes bias, for instance by losing a possible indirect effect of dialysis on mortality via these longitudinal variables. Instead, we fitted a marginal structural model (MSM) to estimate the causal effect of dialysis type, adjusted for confounding and informative censoring. We used the MSM to compare the hazard of death as well as cumulative survival between the potential treatment trajectories "always PD" and "always HD" over time, conditional on age and diabetes mellitus status. We used inverse probability weighting (IPW) to fit the MSM.     

Effect of Nitric Oxide on Anammox Bacteria

The effects of nitrogen oxides on anammox bacteria are not well known. Therefore, anammox bacteria were exposed to 3,500 ppm nitric oxide (NO) in the gas phase. The anammox bacteria were not inhibited by the high NO concentration but rather used it to oxidize additional ammonium to dinitrogen gas under conditions relevant to wastewater treatment.Nitric oxide (NO) has several different roles in bacteria, fungi, and mammals (24). In nitrogen cycle bacteria, it acts as an intermediate and cell communication/signal transduction molecule. On the other hand, NO is a highly reactive and toxic compound that contributes to ozone depletion and air pollution (5). Due to its reactive nature, many bacteria employ an arsenal of proteins (those encoded by norVW, as well as bacterial globins, heme proteins, etc.) that are used to detoxify NO to the less-reactive and more-stable nitrous oxide (N₂O) (24). Still, N₂O is a very effective greenhouse gas and an unfavorable constituent in the off-gases from nitrification/denitrification nitrogen removal systems (4). The presence of gene(s) encoding cytochrome cd₁ nitrite reductase (EMBL accession no. {"type":"entrez-protein","attrs":{"text":"CAJ74898","term_id":"91201838","term_text":"CAJ74898"}}CAJ74898), flavorubredoxin NorVW (accession no. {"type":"entrez-protein","attrs":{"text":"CAJ73918","term_id":"91200863","term_text":"CAJ73918"}}CAJ73918 and {"type":"entrez-protein","attrs":{"text":"CAJ73688","term_id":"91200638","term_text":"CAJ73688"}}CAJ73688), and bacterial hemoglobin (accession no. {"type":"entrez-protein","attrs":{"text":"CAJ72702","term_id":"91203063","term_text":"CAJ72702"}}CAJ72702) in the genome of Kuenenia stuttgartiensis led to the proposal that NO also plays this dual role (metabolic versus toxic) in anammox bacteria (Fig. ​(Fig.1)1) (10, 20). This has ramifications for both application and metabolism of anammox bacteria. The source of NO in an anammox reactor could be the activity of other community members (ammonium-oxidizing or denitrifying bacteria) or high concentrations of nitrite in the influent wastewater stream. Full-scale anammox reactors typically contain a significant population of ammonium-oxidizing bacteria (AOB). In the single nitritation-anammox reactors, these carry out the conversion of 50% of the ammonium in the wastewater to nitrite (6). It has been shown that AOB may produce significant amounts of NO (2, 7), and recently it was reported that NO and N₂O could be emitted from these reactors up to 0.005 and 1.2% of the total nitrogen load to the reactor, respectively (6, 23). NO may inhibit the anammox bacteria and could also be further reduced to N₂O in these reactors (6, 23). It is presently unknown whether anammox bacteria contribute to the NO or N₂O emissions, although it has been suggested previously that anammox bacteria do not produce N₂O under physiologically relevant conditions (10). Nevertheless, if conversion of NO could be coupled to anaerobic ammonium oxidation, the toxic air pollutant NO would facilitate further removal of ammonium in full-scale anammox bioreactors. In the present study, we investigated the effect of very high NO fluxes on anammox bacteria.Open in a separate windowFIG. 1.The hypothetical anammox pathway with possible routes of NO removal. Solid black arrows: anammox pathway, including nitrite oxidation to nitrate; gray arrow, possible detoxification pathway to N₂O (not observed in the bioreactor); dashed gray arrow, NO oxidation to nitrite/nitrate (not possible under anoxic conditions).NO has been described many times as a potent inhibitor of nitrogen cycle bacteria; aerobic ammonium oxidizers, nitrite oxidizers, and denitrifiers were all inhibited by concentrations as low as a few micromolar units (1, 18, 24). In a previous study, it was suggested that “Candidatus Brocadia anammoxidans” could tolerate up to 600 ppm NO (approximately 1 mg NO·day⁻¹ NO load) (16). In the reported experiments, without direct measurement of nitrous oxide (N₂O) in the effluent gas stream, it was postulated that NO was reduced to N₂O (16). In the present study, we used a carefully monitored sequencing batch reactor (SBR) to further our understanding of the effect and fate of NO in a laboratory-scale anammox reactor under conditions which are relevant in wastewater treatment plants.An SBR (working volume, 3.5 liters) consisting of approximately 80% of the anammox bacterium “Candidatus Brocadia fulgida” and no detectable aerobic ammonium oxidizers (determined by fluorescence in situ hybridization (FISH) as described previously [15]) was used in the present study. Before the first introduction of NO into the reactor, the influent (synthetic wastewater) (21) was supplied to the reactor at a flow rate of 1.4 ml·min⁻¹ with nitrite and ammonium concentrations (assayed as previously described [9]) at 45 and 39 mM, respectively (corresponding to a total of 2,370 mg N·day⁻¹). All nitrite was consumed in the reactor, while 2 mM ammonium was still present in the effluent. For every 1 mol of ammonium, 1.22 mol of nitrite was consumed, similar to the previously determined anammox stoichiometry (19). NO was first introduced at a concentration of 400 to 600 ppm in the gas phase at a flow rate of 10 ml/min (CLD 700EL chemiluminescence NOx analyzer, detection limit of 0.1 ppm NO, with 15 ml/min Ar/CO₂ as the dilution gas [a load of 25 to 28 mg NO·day⁻¹]; EcoPhysics, Michigan). During this period, 45% (±6%) of the supplied NO was removed from the system. Initially, there was no detectable change in the ammonium and nitrite removal efficiencies and no detectable nitrous oxide (N₂O) in the flue gas (analyzed with an Agilent 6890 gas chromatograph). It is most likely that NO was converted to N₂, but the increase in the N₂ concentrations in the off-gas was below the detection limit (1,000 ppm).At day 49, the influent NO concentration was increased to 3,500 ppm (640 mg NO·day⁻¹ load). Simultaneously, the stirring speed of the reactor was increased from 200 to 600 rpm to enable better mass transfer to the flocculent anammox biomass. The increase in the stirring speed did not result in any disturbance in the floc size and settling ability of the biomass but did lead to a much higher level of NO removal (128 mg NO·day⁻¹) by the anammox bacteria. The converted NO could theoretically be converted to N₂O via detoxification enzymes or coupled to ammonium oxidation (Fig. ​(Fig.1).1). Surprisingly, there was no change in the nitrite removal capacity of the bioreactor, suggesting that NO was not a substrate preferred over nitrite. Nitrate concentrations (assayed according to the method in reference 9) were stable around 7.2 mM (±0.7 mM). Theoretically, as anammox bacteria reduce NO, they could oxidize a larger proportion of nitrite to nitrate (Fig. ​(Fig.1)1) to increase their capacity for CO₂ fixation; however, such an increase in nitrate production was not observed (or could not be discriminated by the method used [sensitivity, 100 μM]). During this phase of the experiment, the effluent ammonium concentration gradually decreased to below the detection limit (Fig. ​(Fig.2).2). There was only a minimal N₂O (0.6 ppm) emission from the system, and the total N₂ production increased from 3,060 to 3,680 mg N₂·day⁻¹. This indicated that NO reduction was coupled to the catabolism of the anammox bacteria rather than being detoxified by anammox or other community members. To the best of our knowledge, this was the first time that such a high load of NO was not found to be toxic to the nitrogen cycle bacteria. In a previous study, an NO load of 1 mg NO·day⁻¹ was reported to be toxic to anammox bacteria, most probably due to the fact that the experiments were conducted with biomass that had a 100-fold lower cell density and 10-fold lower activity compared to the current enrichment cultures. Furthermore, the NO conversion in the current experiments was stoichiometrically coupled to ammonium oxidation and not converted to N₂O, indicating that the previously reported N₂O emissions from full-scale anammox bioreactors originated not with the anammox bacteria but rather with other community members as hypothesized previously (8).Open in a separate windowFIG. 2.Ammonium concentration in the effluent of the anammox bioreactor. Dashed lines indicate the trend of effluent ammonium concentration during different phases of the reactor operation. Black arrows indicate the manipulations to influent NO stream, and the gray arrow points to an increase in the influent ammonium concentration. d, day.To determine if there could be more NO-dependent ammonium removal, the influent ammonium concentration was first increased to 41 mM (day 80) and then to 43 mM (day 81). This resulted in a slow but gradual increase in the effluent ammonium concentration, and additional ammonium did not appear to be completely converted, most probably due to NO mass transfer limitations. As a result of the higher level of ammonium removal, the observed anammox stoichiometry in the reactor decreased from 1.22 to 0.91 (nitrite/ammonium). Between days 95 and 131, the NO supply to the reactor was turned off, which resulted in an average ammonium concentration of 3.3 mM (±0.9 mM) in the effluent. Following this period, on day 132, the NO load on the reactor was increased back to 640 mg NO·day⁻¹ (Fig. ​(Fig.2).2). As a result, the effluent ammonium concentration gradually decreased again to an average of 1.5 mM (±0.36 mM). The highest level of NO removal achieved in this period was 371 mg NO·day⁻¹. When the NO supply was turned off on day 165, ammonium concentrations increased back to 3.5 mM (±0.71 mM).During the course of the experiment, the biodiversity of the reactor was monitored using FISH and 16S rRNA gene sequence analysis as described previously (15) with probes specific to eubacteria (3), Planctomycetes (13), anammox bacteria (15), “Ca. Brocadia fulgida” (11), and a variety of aerobic ammonium-oxidizing bacteria (12, 22). Before the experiments started and throughout the cultivation of the anammox bacteria with NO, the only detectable anammox species (with FISH and 16S rRNA gene sequence analysis) was “Candidatus Brocadia fulgida.”In the present study, we showed that 2 mM ammonium (4.5% of the influent concentration) could be removed by anammox bacteria via direct coupling to NO reduction. These observations support the proposal of NO as an intermediate of the anammox reaction and have two consequences for application of the anammox process for nitrogen removal. First, we obtained strong indications that previously reported N₂O emissions (6, 8) from full-scale anammox reactors were not generated by anammox bacteria. In our experiments, even under a very high load of NO, there was hardly any detectable N₂O in the effluent gas stream. The competition for nitrogen oxides by denitrifying and anammox bacteria needs further study but may ultimately be used to design operational conditions that would reduce or even prevent NO and N₂O emissions from full-scale nitritation-anammox reactors. Second, by implementing the results of this study, in the future the anammox process could be designed to remove NO from flue gases. Since NO is mostly emitted together with O₂, this could be achieved by the combination of anammox and aerobic ammonium-oxidizing bacteria, for example, with CANON (completely autotrophic nitrogen removal over nitrite)- or OLAND (oxygen-limited autotrophic nitrification-denitrification)-type reactor systems (14, 17).     

Citrullinated vimentin as an important antigen in immune complexes from synovial fluid of rheumatoid arthritis patients with antibodies against citrullinated proteins

Introduction  

Rheumatoid arthritis (RA) is an inflammatory disease, which results in destruction of the joint. The presence of immune complexes (IC) in serum and synovial fluid of RA patients might contribute to this articular damage through different mechanisms, such as complement activation. Therefore, identification of the antigens from these IC is important to gain more insight into the pathogenesis of RA. Since RA patients have antibodies against citrullinated proteins (ACPA) in their serum and synovial fluid (SF) and since elevated levels of citrullinated proteins are detected in the joints of RA patients, citrullinated antigens are possibly present in IC from RA patients.