The Sec63p J-Domain Is Required for ERAD of Soluble Proteins in Yeast

How misfolded proteins are exported from the ER to the cytosol for degradation (ER-associated Degradation, ERAD) and which proteins are participating in this process is not understood. Several studies using a single, leaky mutant indicated that Sec63p might be involved in ERAD. More recently, Sec63p was also found strongly associated with proteasomes attached to the protein-conducting channel in the ER membrane which presumably form part of the export machinery. These observations prompted us to reinvestigate the role of Sec63p in ERAD by generating new mutants which were selected in a screen monitoring the intracellular accumulation of the ERAD substrate CPY*. We show that a mutation in the DnaJ-domain of Sec63p causes a defect in ERAD, whereas mutations in the Brl, acidic, and transmembrane domains only affect protein import into the ER. Unexpectedly, mutations in the acidic domain which mediates interaction of Sec63p with Sec62p also caused defects in cotranslational import. In contrast to mammalian cells where SEC63 expression levels affect steady-state levels of multi-spanning transmembrane proteins, the sec63 J-domain mutant was only defective in ERAD of soluble substrates.     

Canadian polar bear population structure using genome‐wide markers

Predicting the consequences of environmental changes, including human‐mediated climate change on species, requires that we quantify range‐wide patterns of genetic diversity and identify the ecological, environmental, and historical factors that have contributed to it. Here, we generate baseline data on polar bear population structure across most Canadian subpopulations (n = 358) using 13,488 genome‐wide single nucleotide polymorphisms (SNPs) identified with double‐digest restriction site‐associated DNA sequencing (ddRAD). Our ddRAD dataset showed three genetic clusters in the sampled Canadian range, congruent with previous studies based on microsatellites across the same regions; however, due to a lack of sampling in Norwegian Bay, we were unable to confirm the existence of a unique cluster in that subpopulation. These data on the genetic structure of polar bears using SNPs provide a detailed baseline against which future shifts in population structure can be assessed, and opportunities to develop new noninvasive tools for monitoring polar bears across their range.     

Adapting test timing to the sleep-wake schedule: effects on diurnal neurobehavioral performance changes in young evening and older morning chronotypes

The synchrony effect refers to the beneficial impact of temporal matching between the timing of cognitive task administration and preferred time-of-day for diurnal activity. Aging is often associated with an advance in sleep-wake timing and concomitant optimal performance levels in the morning. In contrast, young adults often perform better in the evening hours. So far, the synchrony effect has been tested at fixed clock times, neglecting the individual's sleep-wake schedule and thus introducing confounds, such as differences in accumulated sleep pressure or circadian phase, which may exacerbate synchrony effects. To probe this hypothesis, the authors tested older morning and young evening chronotypes with a psychomotor vigilance and a Stroop paradigm once at fixed morning and evening hours and once adapting testing time to their preferred sleep-wake schedule in a within-subject design. The authors observe a persistence of synchrony effects for overall median reaction times during a psychomotor vigilance task, even when testing time is adapted to the specific individual's sleep-wake schedule. However, data analysis also indicates that time-of-day modulations are weakened under those conditions for incongruent trials on Stroop performance and the slowest reaction times on the psychomotor vigilance task. The latter result suggests that the classically observed synchrony effect may be partially mediated by a series of parameters, such as differences in socio-professional timing constraints, the amount of accumulated sleep need, or circadian phase, all leading to differential arousal levels at testing.     

Achromobacter xylosoxidans: An Emerging Pathogen Carrying Different Elements Involved in Horizontal Genetic Transfer

In the last few years, numerous cases of multidrug-resistant Achromobacter xylosoxidans infections have been documented in immunocompromised and cystic fibrosis patients. To gain insights into the molecular mechanisms and mobile elements related to multidrug resistance in this bacterium, we studied 24 non-epidemiological A. xylosoxidans clinical isolates from Argentina. Specific primers for plasmids, transposons, insertion sequences, bla _ampC, intI1, and intI2 genes were used in PCR reactions. The obtained results showed the presence of wide host range IncP plasmids in ten isolates and a high dispersion of class 1 integrons (n?=?10) and class 2 integrons (n?=?3). Four arrays in the variable region (vr) of class 1 integrons were identified carrying different gene cassettes as the aminoglycoside resistance aac(6′)-Ib and aadA1, the trimethoprim resistance dfrA1 and dfrA16, and the β-lactamase bla _OXA-2. In only one of the class 2 integrons, a vr was amplified that includes sat2-aadA1. The bla _ampC gene was found in all isolates, confirming its ubiquitous nature. Our results show that A. xylosoxidans clinical isolates contain a rich variety of genetic elements commonly associated with resistance genes and their dissemination. This supports the hypothesis that A. xylosoxidans is becoming a reservoir of horizontal genetic transfer elements commonly involved in spreading antibiotic resistance.     

Decreased oxidative stress during glycolytic inhibition enables maintenance of ATP production and astrocytic survival

Depressed energy metabolism and oxidative stress are common features in many pathological situations in the brain, including stroke. In order to investigate astrocytic responses to such stress, we induced metabolic depression in cultured rat astrocytes. Iodoacetate (IA), an inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used and resulted in a rapid inhibition of GAPDH activity. After 1h of GAPDH inhibition the ATP levels started to decrease and were completely abolished at 4h. In parallel, the activity of reactive oxygen species (ROS) was significantly increased, followed by extensive cell death involving flipping of phosphatidylserine and translocation of apoptosis-inducing factor, but not caspase-3 activation. When IA was combined with azide, a respiratory chain complex IV inhibitor, the ATP levels decreased immediately. Interestingly, with azide present, the ROS activity remained low and the astrocytes remained viable even at very low ATP levels. Addition of exogenous ROS-scavengers prevented the IA-induced ROS activity, the ATP levels were maintained and cell death was prevented. Similar protection could be obtained when astrocytes, prior to addition of IA, were incubated with substances known to activate the nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated endogenous antioxidant system. When IA was washed out, after a relatively moderate ATP depression, massive cell death occurred. This was efficiently prevented by addition of azide or ROS scavengers during the IA treatment or by pre-activation of the Nrf2 system. Our results demonstrate that astrocytes in culture can endure and recover from glycolytic inhibition if the ROS activity remained at a low level and suggest that oxidative stress can be an important component for astrocytic cell death following metabolic stress.     

Hearing dysfunction in heterozygous MitfMi‐wh/+ mice,a model for Waardenburg syndrome type 2 and Tietz syndrome

The human deafness‐pigmentation syndromes, Waardenburg syndrome (WS) type 2a, and Tietz syndrome are characterized by profound deafness but only partial cutaneous pigmentary abnormalities. Both syndromes are caused by mutations in MITF. To illuminate differences between cutaneous and otic melanocytes in these syndromes, their development and survival in heterozygous Microphthalmia‐White (Mitf^Mi‐wh/+) mice were studied and hearing function of these mice characterized. Mitf^Mi‐wh/+ mice have a profound hearing deficit, characterized by elevated auditory brainstem response thresholds, reduced distortion product otoacoustic emissions, absent endocochlear potential, loss of outer hair cells, and stria vascularis abnormalities. Mitf^Mi‐wh/+ embryos have fewer melanoblasts during embryonic development than their wild‐type littermates. Although cochlear melanocytes are present at birth, they disappear from the Mitf^Mi‐wh/+ cochlea between P1 and P7. These findings may provide insight into the mechanism of melanocyte and hearing loss in human deafness‐pigmentation syndromes such as WS and Tietz syndrome and illustrate differences between otic and follicular melanocytes.     

Science in Risk Assessment and Policy (SciRAP): An Online Resource for Evaluating and Reporting In Vivo (Eco)Toxicity Studies

(Eco)toxicity studies conducted according to internationally standardized test guidelines are often considered reliable by default and preferred as key evidence in regulatory risk assessment. At the same time regulatory agencies emphasize the use of all relevant (eco)toxicity data in the risk assessment process, including non-standard studies. However, there is a need to facilitate the use of such studies in regulatory risk assessment. Therefore, we propose a framework that facilitates a systematic and transparent evaluation of the reliability and relevance of (eco)toxicity in vivo studies for health and environmental risk assessment. The framework includes specific criteria to guide study evaluation, as well as a color-coding tool developed to aid the application of these criteria. In addition we provide guidance intended for researchers on how to report non-standard studies to ensure that they meet regulatory requirements. The intention of the evaluating and reporting criteria is to increase the usability of all relevant data that may fill information gaps in chemical risk assessments. The framework is publically available online, free of charge, at the Science in Risk Assessment and Policy (SciRAP) website: www.scirap.org. The aim of this article is to present the framework and resources available at the SciRAP website.