Uncoupling protein-1 (UCP1) contributes to the basal proton conductance of brown adipose tissue mitochondria

Proton leak pathways uncouple substrate oxidation from ATP synthesis in mitochondria. These pathways are classified as basal (not regulated) or inducible (activated and inhibited). Previously it was found that over half of the basal proton conductance of muscle mitochondria was catalyzed by the adenine nucleotide translocase (ANT), an abundant mitochondrial anion carrier protein. To determine whether ANT is the unique protein catalyst, or one of many proteins that catalyze basal proton conductance, we measured proton leak kinetics in mitochondria isolated from brown adipose tissue (BAT). BAT can express another mitochondrial anion carrier, UCP1, at concentrations similar to ANT. Basal proton conductance was measured under conditions where UCP1 and ANT were catalytically inactive and was found to be lower in mitochondria from UCP1 knockout mice compared to wild-type. Ablation of another abundant inner membrane protein, nicotinamide nucleotide transhydrogenase, had no effect on proton leak kinetics in mitochondria from liver, kidney or muscle, showing that basal proton conductance is not catalyzed by all membrane proteins. We identify UCP1 as a second protein propagating basal proton leak, lending support to the hypothesis that basal leak pathways are perpetrated by members of the mitochondrial anion carrier family but not by other mitochondrial inner membrane proteins

Genome-Wide Association Studies in Dogs and Humans Identify ADAMTS20 as a Risk Variant for Cleft Lip and Palate

Cleft lip with or without cleft palate (CL/P) is the most commonly occurring craniofacial birth defect. We provide insight into the genetic etiology of this birth defect by performing genome-wide association studies in two species: dogs and humans. In the dog, a genome-wide association study of 7 CL/P cases and 112 controls from the Nova Scotia Duck Tolling Retriever (NSDTR) breed identified a significantly associated region on canine chromosome 27 (unadjusted p=1.1 x 10-13; adjusted p= 2.2 x 10-3). Further analysis in NSDTR families and additional full sibling cases identified a 1.44 Mb homozygous haplotype (chromosome 27: 9.29 – 10.73 Mb) segregating with a more complex phenotype of cleft lip, cleft palate, and syndactyly (CLPS) in 13 cases. Whole-genome sequencing of 3 CLPS cases and 4 controls at 15X coverage led to the discovery of a frameshift mutation within ADAMTS20 (c.1360_1361delAA (p.Lys453Ilefs*3)), which segregated concordant with the phenotype. In a parallel study in humans, a family-based association analysis (DFAM) of 125 CL/P cases, 420 unaffected relatives, and 392 controls from a Guatemalan cohort, identified a suggestive association (rs10785430; p =2.67 x 10-6) with the same gene, ADAMTS20. Sequencing of cases from the Guatemalan cohort was unable to identify a causative mutation within the coding region of ADAMTS20, but four coding variants were found in additional cases of CL/P. In summary, this study provides genetic evidence for a role of ADAMTS20 in CL/P development in dogs and as a candidate gene for CL/P development in humans

Non-classical ProIL-1beta activation during mammary gland infection is pathogen-dependent but caspase-1 independent

Infection of the mammary gland with live bacteria elicits a pathogen-specific host inflammatory response. To study these host-pathogen interactions wild type mice, NF-kappaB reporter mice as well as caspase-1 and IL-1beta knockout mice were intramammarily challenged with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The murine mastitis model allowed to compare the kinetics of the induced cytokine protein profiles and their underlying pathways. In vivo and ex vivo imaging showed that E. coli rapidly induced NF-kappaB inflammatory signaling concomitant with high mammary levels of TNF-alpha, IL-1 alpha and MCP-1 as determined by multiplex analysis. In contrast, an equal number of S. aureus bacteria induced a low NF-kappaB activity concomitant with high mammary levels of the classical IL-1beta fragment. These quantitative and qualitative differences in local inflammatory mediators resulted in an earlier neutrophil influx and in a more extensive alveolar damage post-infection with E. coli compared to S. aureus. Western blot analysis revealed that the inactive proIL-1beta precursor was processed into pathogen-specific IL-1beta fragmentation patterns as confirmed with IL-1beta knockout animals. Additionally, caspase-1 knockout animals allowed to investigate whether IL-1beta maturation depended on the conventional inflammasome pathway. The lack of caspase-1 did not prevent extensive proIL-1beta fragmentation by either of S. aureus or E. coli. These non-classical IL-1beta patterns were likely caused by different proteases and suggest a sentinel function of IL-1beta during mammary gland infection. Thus, a key signaling nodule can be defined in the differential host innate immune defense upon E. coli versus S. aureus mammary gland infection, which is independent of caspase-1

The Penny’s Dropped: Renegotiating the contemporary coin deposit

This is the Accepted Manuscript of the following article: Ceri Houlbrook, “The penny’s dropped: Renegotiating the contemporary coin deposit”, Journal of Material Culture, Vol. 20(2): 173-189, March 2015. The final published version is available at: http://journals.sagepub.com/doi/pdf/10.1177/1359183515577120#articleCitationDownloadContainer © 2015, © SAGE Publications.This article examines the status of coins as contemporary deposits in the British Isles. With a focus on both historical and contemporary sites, from the Neolithic long barrow of Wayland’s Smithy, Oxfordshire, to the plethora of wishing-wells and coin-trees distributed across the British Isles, it demonstrates the popularity of coins as ritual deposits. The author considers how they are perceived and treated by site custodians, and concludes with a case study of an archaeological excavation, the 2013 Ardmaddy Wishing-Tree Project, which recovered a large amount of contemporary coin deposits. This article does not aim to locate itself within the debates of site custodianship and accessibility, nor does it propose to address the broader dilemmas of a site’s ritual continuity or resurgence. Instead, its aim is to encourage archaeologists to consider the contemporary deposit as an integral part of the ritual narrative of a site, rather than as disposable ‘ritual litter’.Peer reviewedFinal Accepted Versio

Uncoupling Protein-4 (UCP4) Increases ATP Supply by Interacting with Mitochondrial Complex II in Neuroblastoma Cells

Mitochondrial uncoupling protein-4 (UCP4) protects against Complex I deficiency as induced by 1-methyl-4-phenylpyridinium (MPP+), but how UCP4 affects mitochondrial function is unclear. Here we investigated how UCP4 affects mitochondrial bioenergetics in SH-SY5Y cells. Cells stably overexpressing UCP4 exhibited higher oxygen consumption (10.1%, p<0.01), with 20% greater proton leak than vector controls (p<0.01). Increased ATP supply was observed in UCP4-overexpressing cells compared to controls (p<0.05). Although state 4 and state 3 respiration rates of UCP4-overexpressing and control cells were similar, Complex II activity in UCP4-overexpressing cells was 30% higher (p<0.05), associated with protein binding between UCP4 and Complex II, but not that of either Complex I or IV. Mitochondrial ADP consumption by succinate-induced respiration was 26% higher in UCP4-overexpressing cells, with 20% higher ADP:O ratio (p<0.05). ADP/ATP exchange rate was not altered by UCP4 overexpression, as shown by unchanged mitochondrial ADP uptake activity. UCP4 overexpression retained normal mitochondrial morphology in situ, with similar mitochondrial membrane potential compared to controls. Our findings elucidate how UCP4 overexpression increases ATP synthesis by specifically interacting with Complex II. This highlights a unique role of UCP4 as a potential regulatory target to modulate mitochondrial Complex II and ATP output in preserving existing neurons against energy crisis

Frequency-Invariant Representation of Interaural Time Differences in Mammals

Interaural time differences (ITDs) are the major cue for localizing low-frequency sounds. The activity of neuronal populations in the brainstem encodes ITDs with an exquisite temporal acuity of about . The response of single neurons, however, also changes with other stimulus properties like the spectral composition of sound. The influence of stimulus frequency is very different across neurons and thus it is unclear how ITDs are encoded independently of stimulus frequency by populations of neurons. Here we fitted a statistical model to single-cell rate responses of the dorsal nucleus of the lateral lemniscus. The model was used to evaluate the impact of single-cell response characteristics on the frequency-invariant mutual information between rate response and ITD. We found a rough correspondence between the measured cell characteristics and those predicted by computing mutual information. Furthermore, we studied two readout mechanisms, a linear classifier and a two-channel rate difference decoder. The latter turned out to be better suited to decode the population patterns obtained from the fitted model

Toll-like receptor 9 polymorphisms are associated with severity variables in a cohort of meningococcal meningitis survivors

BACKGROUND: Genetic variation in immune response genes is associated with susceptibility and severity of infectious diseases. Toll-like receptor (TLR) 9 polymorphisms are associated with susceptibility to develop meningococcal meningitis (MM). The aim of this study is to compare genotype distributions of two TLR9 polymorphisms between clinical severity variables in MM survivors. METHODS: We used DNA samples of a cohort of 390 children who survived MM. Next, we determined the genotype frequencies of TLR9 -1237 and TLR9 +2848 polymorphisms and compared these between thirteen clinical variables associated with prognostic factors predicting adverse outcome of bacterial meningitis in children. RESULTS: The TLR9 -1237 TC and CC genotypes were associated with a decreased incidence of a positive blood culture for Neisseria (N.) meningitidis (p = 0.014, odds ratio (OR) 0.5. 95% confidence interval (CI) 0.3 – 0.9). The TLR9 +2848 AA mutant was associated with a decreased incidence of a positive blood culture for N. meningitidis (p = 0.017, OR 0.6, 95% CI 0.3 – 0.9). Cerebrospinal fluid (CSF) leukocytes per μL were higher in patients carrying the TLR9 -1237 TC or CC genotypes compared to carriers of the TT wild type (WT) (p = 0.024, medians: 2117, interquartile range (IQR) 4987 versus 955, IQR 3938). CSF blood/glucose ratios were lower in TLR9 -1237 TC or CC carriers than in carriers of the TT WT (p = 0.017, medians: 0.20, IQR 0.4 versus 0.35, IQR 0.5). CSF leukocytes/μL were higher in patients carrying the TLR9 +2848 AA mutant compared to carriers of GG or GA (p = 0.0067, medians: 1907, IQR 5221 versus 891, IQR 3952). CONCLUSIONS: We identified TLR9 genotypes associated with protection against meningococcemia and enhanced local inflammatory responses inside the central nervous system, important steps in MM pathogenesis and defense

Sirtuin 3, a New Target of PGC-1α, Plays an Important Role in the Suppression of ROS and Mitochondrial Biogenesis

Sirtuin 3 (SIRT3) is one of the seven mammalian sirtuins, which are homologs of the yeast Sir2 gene. SIRT3 is the only sirtuin with a reported association with the human life span. Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) plays important roles in adaptive thermogenesis, gluconeogenesis, mitochondrial biogenesis and respiration. PGC-1alpha induces several key reactive oxygen species (ROS)-detoxifying enzymes, but the molecular mechanism underlying this is not well understood.Here we show that PGC-1alpha strongly stimulated mouse Sirt3 gene expression in muscle cells and hepatocytes. Knockdown of PGC-1alpha led to decreased Sirt3 gene expression. PGC-1alpha activated the mouse SIRT3 promoter, which was mediated by an estrogen-related receptor (ERR) binding element (ERRE) (-407/-399) mapped to the promoter region. Chromatin immunoprecipitation and electrophoretic mobility shift assays confirmed that ERRalpha bound to the identified ERRE and PGC-1alpha co-localized with ERRalpha in the mSirt3 promoter. Knockdown of ERRalpha reduced the induction of Sirt3 by PGC-1alpha in C(2)C(12) myotubes. Furthermore, Sirt3 was essential for PGC-1alpha-dependent induction of ROS-detoxifying enzymes and several components of the respiratory chain, including glutathione peroxidase-1, superoxide dismutase 2, ATP synthase 5c, and cytochrome c. Overexpression of SIRT3 or PGC-1alpha in C(2)C(12) myotubes decreased basal ROS level. In contrast, knockdown of mSIRT3 increased basal ROS level and blocked the inhibitory effect of PGC-1alpha on cellular ROS production. Finally, SIRT3 stimulated mitochondrial biogenesis, and SIRT3 knockdown decreased the stimulatory effect of PGC-1alpha on mitochondrial biogenesis in C(2)C(12) myotubes.Our results indicate that Sirt3 functions as a downstream target gene of PGC-1alpha and mediates the PGC-1alpha effects on cellular ROS production and mitochondrial biogenesis. Thus, SIRT3 integrates cellular energy metabolism and ROS generation. The elucidation of the molecular mechanisms of SIRT3 regulation and its physiological functions may provide a novel target for treating ROS-related disease