Cytosolic thioredoxin reductase 1 is required for correct disulfide formation in the ER

Folding of proteins entering the secretory pathway in mammalian cells frequently requires the insertion of disulfide bonds. Disulfide insertion can result in covalent linkages found in the native structure as well as those that are not, so‐called non‐native disulfides. The pathways for disulfide formation are well characterized, but our understanding of how non‐native disulfides are reduced so that the correct or native disulfides can form is poor. Here, we use a novel assay to demonstrate that the reduction in non‐native disulfides requires NADPH as the ultimate electron donor, and a robust cytosolic thioredoxin system, driven by thioredoxin reductase 1 (TrxR1 or TXNRD1). Inhibition of this reductive pathway prevents the correct folding and secretion of proteins that are known to form non‐native disulfides during their folding. Hence, we have shown for the first time that mammalian cells have a pathway for transferring reducing equivalents from the cytosol to the ER, which is required to ensure correct disulfide formation in proteins entering the secretory pathway

Exercise Training to Target Gait Unsteadiness in People with Diabetes

Balance impairment and an associated high fall rate in people with diabetes is common, and a huge burden to quality of life and healthcare systems. Causes of impaired balance are commonly attributed to both sensory and motor deficits, which includes impaired muscle strength and function. This study investigated the effects of resistance exercise training on balance control during walking over level ground and on stairs. Ten DM people (age: 62 years, BMI: 29kg/m2, VPT: 9V) and 6 DM people with DPN (age: 59 years, BMI: 27kg/m2, VPT: 31V) performed a 16-week intervention of weekly resistance exercise training to increase ankle and knee extensor muscle strength. Six DM controls did not take part in the intervention (age: 50 years, BMI: 26kg/m2, VPT: 12V). Balance during gait was quantified before and after the intervention, by separation between the body centre-of-mass and centre-of-pressure under the feet during both level and stair walking. Knee and ankle extensor muscle strength was assessed using a dynamometer. The exercise intervention increased strength of ankle plantar flexors (22%) and knee extensors (30%). Despite the increases in lower limb muscle strength produced by the intervention, no improvements in balance were seen post training. However, gait speed did increase by 8%, which previous research has shown to be associated with quality of life. Controls showed no training effects in any variables. Although this exercise intervention had a positive effect on gait by increasing walking speed, there was no effect on the control of balance. Previous research has identified that medio-lateral (side-to-side) balance is impaired in people with DPN. The muscles exercised in the present study mainly control the major sagittal plane (forwards-backwards) movements that occur during gait. Interventions targeting the lateral stabilising muscles of the hip and trunk, may show greater potential efficacy in redressing the balance impairment of this population

The RING-CH ligase K5 antagonizes restriction of KSHV and HIV-1 particle release by mediating ubiquitin-dependent endosomal degradation of tetherin

Tetherin (CD317/BST2) is an interferon-induced membrane protein that inhibits the release of diverse enveloped viral particles. Several mammalian viruses have evolved countermeasures that inactivate tetherin, with the prototype being the HIV-1 Vpu protein. Here we show that the human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) is sensitive to tetherin restriction and its activity is counteracted by the KSHV encoded RING-CH E3 ubiquitin ligase K5. Tetherin expression in KSHV-infected cells inhibits viral particle release, as does depletion of K5 protein using RNA interference. K5 induces a species-specific downregulation of human tetherin from the cell surface followed by its endosomal degradation. We show that K5 targets a single lysine (K18) in the cytoplasmic tail of tetherin for ubiquitination, leading to relocalization of tetherin to CD63-positive endosomal compartments. Tetherin degradation is dependent on ESCRT-mediated endosomal sorting, but does not require a tyrosine-based sorting signal in the tetherin cytoplasmic tail. Importantly, we also show that the ability of K5 to substitute for Vpu in HIV-1 release is entirely dependent on K18 and the RING-CH domain of K5. By contrast, while Vpu induces ubiquitination of tetherin cytoplasmic tail lysine residues, mutation of these positions has no effect on its antagonism of tetherin function, and residual tetherin is associated with the trans-Golgi network (TGN) in Vpu-expressing cells. Taken together our results demonstrate that K5 is a mechanistically distinct viral countermeasure to tetherin-mediated restriction, and that herpesvirus particle release is sensitive to this mode of antiviral inhibition

Species-Specific Activity of HIV-1 Vpu and Positive Selection of Tetherin Transmembrane Domain Variants

Tetherin/BST-2/CD317 is a recently identified antiviral protein that blocks the release of nascent retrovirus, and other virus, particles from infected cells. An HIV-1 accessory protein, Vpu, acts as an antagonist of tetherin. Here, we show that positive selection is evident in primate tetherin sequences and that HIV-1 Vpu appears to have specifically adapted to antagonize variants of tetherin found in humans and chimpanzees. Tetherin variants found in rhesus macaques (rh), African green monkeys (agm) and mice were able to inhibit HIV-1 particle release, but were resistant to antagonism by HIV-1 Vpu. Notably, reciprocal exchange of transmembrane domains between human and monkey tetherins conferred sensitivity and resistance to Vpu, identifying this protein domain as a critical determinant of Vpu function. Indeed, differences between hu-tetherin and rh-tetherin at several positions in the transmembrane domain affected sensitivity to antagonism by Vpu. Two alterations in the hu-tetherin transmembrane domain, that correspond to differences found in rh- and agm-tetherin proteins, were sufficient to render hu-tetherin completely resistant to HIV-1 Vpu. Interestingly, transmembrane and cytoplasmic domain sequences in primate tetherins exhibit variation at numerous codons that is likely the result of positive selection, and some of these changes coincide with determinants of HIV-1 Vpu sensitivity. Overall, these data indicate that tetherin could impose a barrier to viral zoonosis as a consequence of positive selection that has been driven by ancient viral antagonists, and that the HIV-1 Vpu protein has specialized to target the transmembrane domains found in human/chimpanzee tetherin proteins

Proximal femoral fracture in a man resulting from modern clipless pedals: a case report

<p>Abstract</p> <p>Introduction</p> <p>The use of clipless pedals amongst recreational cyclists has become increasingly popular in recent years. We describe a hip fracture, that was sustained due to inadequate set up of such pedals. To the best of our knowledge, this has only been described once before, and this was in the non-English language medical literature.</p> <p>Case Report</p> <p>A 38-year-old Caucasian man who was a club cyclist sustained a displaced intracapsular fracture of the hip whilst cycling. As a direct result of the incorrect set-up of his clipless pedals he was unable to release his feet whilst slowing to a halt. This resulted in a loss of balance and subsequent fall with a direct impact onto his left hip. The resulting fracture was managed successfully with early closed reduction and fixation. At six month review he was walking unaided without pain but, as yet, has been unable to return to cycling.</p> <p>Conclusion</p> <p>This case highlights the dangers of clipless pedals even in experienced cyclists, and underlines the importance of proper information for their correct setup to minimise the risk of potentially serious injuries, especially in the region of the hip.</p

Neuropathy-Related Unsteadiness and Psychosocial Outcomes in Diabetes—Preliminary Findings

Diabetes is a well-established risk factor for psychological distress and reduced quality of life (QoL). This may be part due to biomechanical challenges posed by diabetic peripheral neuropathy (DPN)-related unsteadiness leading to increased risk of falling and reduced physical activity (PA). This cross-sectional study explores relationships between physical (DPN-unsteadiness and PA) and psychosocial outcomes (depression, fear of falling [FoF], and QoL). The preliminary results of 15 type 2 DM people with DPN (age: 67 years; 13M; VPT 24V) indicate that quality of life (NeuroQoL) and depression (Hospital Anxiety and Depression Scale) are strongly associated with objective DPN-unsteadiness (Berg Balance test: r=-.64, p=.01 and r=.63, p=.01; respectively) and FoF (Falls Self-Efficacy Scale: r=.61, p=.02 and r=-.55 p=.03; respectively). Moreover, DPN-unsteadiness (Berg balance score: 47 ±6) and FoF are associated with reduced vigorous exercise PA levels (r=.53, p=.04 and r=-.51, p=.05; respectively), as well as total moderate PA levels (r=-.45, p=.09 and r=.45, p=.09; respectively); measured by General Practice Assessment Questionnaire. Finally, FoF correlates strongly with DPN-unsteadiness (r=-0.79, p<0.001), demonstrating a potential reason why balance impairment may have the negative impact upon PA and QoL. Whilst prospective data are needed to solidify these findings, the preliminary results are robust and support the strong links between the biomechanical impact of DPN and psychosocial outcomes, including depression and fear of falling, and reduced QoL. These data indicate that there is an unmet need for the development of multifaceted interventions that address both psychological distress and biomechanical challenges experienced by patients with this debilitating complication of diabetes

Assisted evolution enables HIV-1 to overcome a high trim5α-imposed genetic barrier to rhesus macaque tropism

Diversification of antiretroviral factors during host evolution has erected formidable barriers to cross-species retrovirus transmission. This phenomenon likely protects humans from infection by many modern retroviruses, but it has also impaired the development of primate models of HIV-1 infection. Indeed, rhesus macaques are resistant to HIV-1, in part due to restriction imposed by the TRIM5α protein (rhTRIM5α). Initially, we attempted to derive rhTRIM5α-resistant HIV-1 strains using two strategies. First, HIV-1 was passaged in engineered human cells expressing rhTRIM5α. Second, a library of randomly mutagenized capsid protein (CA) sequences was screened for mutations that reduced rhTRIM5α sensitivity. Both approaches identified several individual mutations in CA that reduced rhTRIM5α sensitivity. However, neither approach yielded mutants that were fully resistant, perhaps because the locations of the mutations suggested that TRIM5α recognizes multiple determinants on the capsid surface. Moreover, even though additive effects of various CA mutations on HIV-1 resistance to rhTRIM5α were observed, combinations that gave full resistance were highly detrimental to fitness. Therefore, we employed an 'assisted evolution' approach in which individual CA mutations that reduced rhTRIM5α sensitivity without fitness penalties were randomly assorted in a library of viral clones containing synthetic CA sequences. Subsequent passage of the viral library in rhTRIM5α-expressing cells resulted in the selection of individual viral species that were fully fit and resistant to rhTRIM5α. These viruses encoded combinations of five mutations in CA that conferred complete or near complete resistance to the disruptive effects of rhTRIM5α on incoming viral cores, by abolishing recognition of the viral capsid. Importantly, HIV-1 variants encoding these CA substitutions and SIVmac239 Vif replicated efficiently in primary rhesus macaque lymphocytes. These findings demonstrate that rhTRIM5α is difficult to but not impossible to evade, and doing so should facilitate the development of primate models of HIV-1 infection

PCB-Related Alteration of Thyroid Hormones and Thyroid Hormone Receptor Gene Expression in Free-Ranging Harbor Seals (Phoca vitulina)

Persistent organic pollutants are environmental contaminants that, because of their lipophilic properties and long half-lives, bioaccumulate within aquatic food webs and often reach high concentrations in marine mammals, such as harbor seals (Phoca vitulina). Exposure to these contaminants has been associated with developmental abnormalities, immunotoxicity, and reproductive impairment in marine mammals and other high-trophic-level wildlife, mediated via a disruption of endocrine processes. The highly conserved thyroid hormones (THs) represent one vulnerable endocrine end point that is critical for metabolism, growth, and development in vertebrates. We characterized the relationship between contaminants and specific TH receptor (TR ) gene expression in skin/blubber biopsy samples, as well as serum THs, from free-ranging harbor seal pups (n = 39) in British Columbia, Canada, and Washington State, USA. We observed a contaminant-related increase in blubber TR-α gene expression [total polychlorinated biphenyls (∑PCBs); r = 0.679; p < 0.001] and a concomitant decrease in circulating total thyroxine concentrations (∑PCBs; r = −0.711; p < 0.001). Consistent with results observed in carefully controlled laboratory and captive feeding studies, our findings suggest that the TH system in harbor seals is highly sensitive to disruption by environmental contaminants. Such a disruption not only may lead to adverse effects on growth and development but also could have important ramifications for lipid metabolism and energetics in marine mammals

You turn me cold: evidence for temperature contagion

Introduction During social interactions, our own physiological responses influence those of others. Synchronization of physiological (and behavioural) responses can facilitate emotional understanding and group coherence through inter-subjectivity. Here we investigate if observing cues indicating a change in another's body temperature results in a corresponding temperature change in the observer. Methods Thirty-six healthy participants (age; 22.9±3.1 yrs) each observed, then rated, eight purpose-made videos (3 min duration) that depicted actors with either their right or left hand in visibly warm (warm videos) or cold water (cold videos). Four control videos with the actors' hand in front of the water were also shown. Temperature of participant observers' right and left hands was concurrently measured using a thermistor within a Wheatstone bridge with a theoretical temperature sensitivity of <0.0001°C. Temperature data were analysed in a repeated measures ANOVA (temperature × actor's hand × observer's hand). Results Participants rated the videos showing hands immersed in cold water as being significantly cooler than hands immersed in warm water, F(1,34) = 256.67, p0.1). There was however no evidence of left-right mirroring of these temperature effects p>0.1). Sensitivity to temperature contagion was also predicted by inter-individual differences in self-report empathy. Conclusions We illustrate physiological contagion of temperature in healthy individuals, suggesting that empathetic understanding for primary low-level physiological challenges (as well as more complex emotions) are grounded in somatic simulation

Robust automated detection of microstructural white matter degeneration in Alzheimer’s disease using machine learning classification of multicenter DTI data

Diffusion tensor imaging (DTI) based assessment of white matter fiber tract integrity can support the diagnosis of Alzheimer’s disease (AD). The use of DTI as a biomarker, however, depends on its applicability in a multicenter setting accounting for effects of different MRI scanners. We applied multivariate machine learning (ML) to a large multicenter sample from the recently created framework of the European DTI study on Dementia (EDSD). We hypothesized that ML approaches may amend effects of multicenter acquisition. We included a sample of 137 patients with clinically probable AD (MMSE 20.6±5.3) and 143 healthy elderly controls, scanned in nine different scanners. For diagnostic classification we used the DTI indices fractional anisotropy (FA) and mean diffusivity (MD) and, for comparison, gray matter and white matter density maps from anatomical MRI. Data were classified using a Support Vector Machine (SVM) and a Naïve Bayes (NB) classifier. We used two cross-validation approaches, (i) test and training samples randomly drawn from the entire data set (pooled cross-validation) and (ii) data from each scanner as test set, and the data from the remaining scanners as training set (scanner-specific cross-validation). In the pooled cross-validation, SVM achieved an accuracy of 80% for FA and 83% for MD. Accuracies for NB were significantly lower, ranging between 68% and 75%. Removing variance components arising from scanners using principal component analysis did not significantly change the classification results for both classifiers. For the scanner-specific cross-validation, the classification accuracy was reduced for both SVM and NB. After mean correction, classification accuracy reached a level comparable to the results obtained from the pooled cross-validation. Our findings support the notion that machine learning classification allows robust classification of DTI data sets arising from multiple scanners, even if a new data set comes from a scanner that was not part of the training sample