A Non-Coding Genomic Duplication at the HMX1 Locus Is Associated with Crop Ears in Highland Cattle

Highland cattle with congenital crop ears have notches of variable size on the tips of both ears. In some cases, cartilage deformation can be seen and occasionally the external ears are shortened. We collected 40 cases and 80 controls across Switzerland. Pedigree data analysis confirmed a monogenic autosomal dominant mode of inheritance with variable expressivity. All affected animals could be traced back to a single common ancestor. A genome-wide association study was performed and the causative mutation was mapped to a 4 Mb interval on bovine chromosome 6. The H6 family homeobox 1 (HMX1) gene was selected as a positional and functional candidate gene. By whole genome re-sequencing of an affected Highland cattle, we detected 6 non-synonymous coding sequence variants and two variants in an ultra-conserved element at the HMX1 locus with respect to the reference genome. Of these 8 variants, only a non-coding 76 bp genomic duplication (g.106720058_106720133dup) located in the conserved region was perfectly associated with crop ears. The identified copy number variation probably results in HMX1 misregulation and possible gain-of-function. Our findings confirm the role of HMX1 during the development of the external ear. As it is sometimes difficult to phenotypically diagnose Highland cattle with slight ear notches, genetic testing can now be used to improve selection against this undesired trait.     

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca²⁺ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca²⁺ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca²⁺ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca²⁺ indicator and a hydrophilic fluorescent dye/Ca²⁺ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F₀.     

Crystal Structure of the Human Short Coiled Coil Protein and Insights into SCOC-FEZ1 Complex Formation

The short coiled coil protein (SCOC) forms a complex with fasciculation and elongation protein zeta 1 (FEZ1). This complex is involved in autophagy regulation. We determined the crystal structure of the coiled coil domain of human SCOC at 2.7 Å resolution. SCOC forms a parallel left handed coiled coil dimer. We observed two distinct dimers in the crystal structure, which shows that SCOC is conformationally flexible. This plasticity is due to the high incidence of polar and charged residues at the core a/d-heptad positions. We prepared two double mutants, where these core residues were mutated to either leucines or valines (E93V/K97L and N125L/N132V). These mutations led to a dramatic increase in stability and change of oligomerisation state. The oligomerisation state of the mutants was characterized by multi-angle laser light scattering and native mass spectrometry measurements. The E93V/K97 mutant forms a trimer and the N125L/N132V mutant is a tetramer. We further demonstrate that SCOC forms a stable homogeneous complex with the coiled coil domain of FEZ1. SCOC dimerization and the SCOC surface residue R117 are important for this interaction.     

NADPH Oxidase Deficient Mice Develop Colitis and Bacteremia upon Infection with Normally Avirulent,TTSS-1- and TTSS-2-Deficient Salmonella Typhimurium

Infections, microbe sampling and occasional leakage of commensal microbiota and their products across the intestinal epithelial cell layer represent a permanent challenge to the intestinal immune system. The production of reactive oxygen species by NADPH oxidase is thought to be a key element of defense. Patients suffering from chronic granulomatous disease are deficient in one of the subunits of NADPH oxidase. They display a high incidence of Crohn’s disease-like intestinal inflammation and are hyper-susceptible to infection with fungi and bacteria, including a 10-fold increased risk of Salmonellosis. It is not completely understood which steps of the infection process are affected by the NADPH oxidase deficiency. We employed a mouse model for Salmonella diarrhea to study how NADPH oxidase deficiency (Cybb ^−/−) affects microbe handling by the large intestinal mucosa. In this animal model, wild type S. Typhimurium causes pronounced enteropathy in wild type mice. In contrast, an avirulent S. Typhimurium mutant (S.Tm^avir; invGsseD), which lacks virulence factors boosting trans-epithelial penetration and growth in the lamina propria, cannot cause enteropathy in wild type mice. We found that Cybb ^−/− mice are efficiently infected by S.Tm^avir and develop enteropathy by day 4 post infection. Cell depletion experiments and infections in Cybb ^−/− Myd88 ^−/− mice indicated that the S.Tm^avir-inflicted disease in Cybb ^−/− mice hinges on CD11c⁺CX₃CR1⁺ monocytic phagocytes mediating colonization of the cecal lamina propria and on Myd88-dependent proinflammatory immune responses. Interestingly, in mixed bone marrow chimeras a partial reconstitution of Cybb-proficiency in the bone marrow derived compartment was sufficient to ameliorate disease severity. Our data indicate that NADPH oxidase expression is of key importance for restricting the growth of S.Tm^avir in the mucosal lamina propria. This provides important insights into microbe handling by the large intestinal mucosa and the role of NADPH oxidase in maintaining microbe-host mutualism at this exposed body surface.     

Effects of Sustained Sleep Restriction on Mitogen-Stimulated Cytokines,Chemokines and T Helper 1/ T Helper 2 Balance in Humans

Background

Recent studies suggest that acute sleep deprivation disrupts cellular immune responses by shifting T helper (Th) cell activity towards a Th2 cytokine profile. Since little is known about more long-term effects, we investigated how five days of sleep restriction would affect pro-inflammatory, chemotactic, Th1- and Th2 cytokine secretion.

Methods

Nine healthy males participated in an experimental sleep protocol with two baseline sleep-wake cycles (sleep 23.00 – 07.00 h) followed by 5 days with restricted sleep (03.00 – 07.00 h). On the second baseline day and on the fifth day with restricted sleep, samples were drawn every third hour for determination of cytokines/chemokines (tumor necrosis factor alpha (TNF-α), interleukin (IL) -1β, IL-2, IL-4 and monocyte chemoattractant protein-1 (MCP-1)) after in vitro stimulation of whole blood samples with the mitogen phytohemagglutinin (PHA). Also leukocyte numbers, mononuclear cells and cortisol were analysed.

Results

5-days of sleep restriction affected PHA-induced immune responses in several ways. There was a general decrease of IL-2 production (p<.05). A shift in Th1/Th2 cytokine balance was also evident, as determined by a decrease in IL2/IL4 ratio. No other main effects of restricted sleep were shown. Two significant interactions showed that restricted sleep resulted in increased TNF-α and MCP-1 in the late evening and early night hours (p’s<.05). In addition, all variables varied across the 24 h day.

Conclusions

5-days of sleep restriction is characterized by a shift towards Th2 activity (i.e. lower 1L-2/IL-4 ratio) which is similar to the effects of acute sleep deprivation and psychological stress. This may have implications for people suffering from conditions characterized by excessive Th2 activity like in allergic disease, such as asthma, for whom restricted sleep could have negative consequences.     

The Incubation Period of Buruli Ulcer (Mycobacterium ulcerans Infection)

Introduction

Buruli Ulcer (BU) is caused by the environmental microbe Mycobacterium ulcerans. Despite unclear transmission, contact with a BU endemic region is the key known risk factor. In Victoria, Australia, where endemic areas have been carefully mapped, we aimed to estimate the Incubation Period (IP) of BU by interviewing patients who reported defined periods of contact with an endemic area prior to BU diagnosis.

Method

A retrospective review was undertaken of 408 notifications of BU in Victoria from 2002 to 2012. Telephone interviews using a structured questionnaire and review of notification records were performed. Patients with a single visit exposure to a defined endemic area were included and the period from exposure to disease onset determined (IP).

Results

We identified 111 of 408 notified patients (27%) who had a residential address outside a known endemic area, of whom 23 (6%) reported a single visit exposure within the previous 24 months. The median age of included patients was 30 years (range: 6 to 73) and 65% were male. 61% had visited the Bellarine Peninsula, currently the most active endemic area. The median time from symptom onset to diagnosis was 71 days (range: 34–204 days). The midpoint of the reported IP range was utilized to calculate a point estimate of the IP for each case. Subsequently, the mean IP for the cohort was calculated at 135 days (IQR: 109–160; CI 95%: 113.9–156), corresponding to 4.5 months or 19.2 weeks. The shortest IP recorded was 32 days and longest 264 days (Figure 1 & 2). IP did not vary for variables investigated.Open in a separate windowFigure 1Geographic representation of Bellarine Peninsula, considered endemic for BU as of 2012.Bellarine Peninsula – east of line from Geelong to Torquay. Mornington and Westernport – southwest of line from Hampton to Tooradin (including Phillip Island).Open in a separate windowFigure 2Geographic representation of East Gippsland, considered endemic for BU as of 2012.East Gippsland: East of Sale and south of the great divide.

Conclusions

The estimated mean IP of BU in Victoria is 135 days (IQR: 109–160 days), 4.5 months. The shortest recorded was IP 34 days and longest 264 days. A greater understanding of BU IP will aid clinical risk assessment and future research.     

Calcium ions and osteoclastogenesis initiate the induction of bone formation by coral‐derived macroporous constructs

Coral-derived calcium carbonate/hydroxyapatite macroporous constructs of the genus Goniopora with limited hydrothermal conversion to hydroxyapatite (7% HA/CC) initiate the induction of bone formation. Which are the molecular signals that initiate pattern formation and the induction of bone formation? To evaluate the role of released calcium ions and osteoclastogenesis, 7% HA/CC was pre-loaded with either 500 μg of the calcium channel blocker, verapamil hydrochloride, or 240 μg of the osteoclast inhibitor, biphosphonate zoledronate, and implanted in the rectus abdominis muscle of six adult Chacma baboons Papio ursinus. Generated tissues on days 15, 60 and 90 were analysed by histomorphometry and qRT-PCR. On day 15, up-regulation of type IV collagen characterized all the implanted constructs correlating with vascular invasion. Zoledronate-treated specimens showed an important delay in tissue patterning and morphogenesis with limited bone formation. Osteoclastic inhibition yielded minimal, if any, bone formation by induction. 7% HA/CC pre-loaded with the Ca⁺⁺ channel blocker verapamil hydrochloride strongly inhibited the induction of bone formation. Down-regulation of bone morphogenetic protein-2 (BMP-2) together with up-regulation of Noggin genes correlated with limited bone formation in 7% HA/CC pre-loaded with either verapamil or zoledronate, indicating that the induction of bone formation by coral-derived macroporous constructs is via the BMPs pathway. The spontaneous induction of bone formation is initiated by a local peak of Ca⁺⁺ activating stem cell differentiation and the induction of bone formation.     

Relative Contribution of Th1 and Th17 Cells in Adaptive Immunity to Bordetella pertussis: Towards the Rational Design of an Improved Acellular Pertussis Vaccine

Whooping cough caused by Bordetella pertussis is a re-emerging infectious disease despite the introduction of safer acellular pertussis vaccines (Pa). One explanation for this is that Pa are less protective than the more reactogenic whole cell pertussis vaccines (Pw) that they replaced. Although Pa induce potent antibody responses, and protection has been found to be associated with high concentrations of circulating IgG against vaccine antigens, it has not been firmly established that host protection induced with this vaccine is mediated solely by humoral immunity. The aim of this study was to examine the relative contribution of Th1 and Th17 cells in host immunity to infection with B. pertussis and in immunity induced by immunization with Pw and Pa and to use this information to help rationally design a more effective Pa. Our findings demonstrate that Th1 and Th17 both function in protective immunity induced by infection with B. pertussis or immunization with Pw. In contrast, a current licensed Pa, administered with alum as the adjuvant, induced Th2 and Th17 cells, but weak Th1 responses. We found that IL-1 signalling played a central role in protective immunity induced with alum-adsorbed Pa and this was associated with the induction of Th17 cells. Pa generated strong antibody and Th2 responses, but was fully protective in IL-4-defective mice, suggesting that Th2 cells were dispensable. In contrast, Pa failed to confer protective immunity in IL-17A-defective mice. Bacterial clearance mediated by Pa-induced Th17 cells was associated with cell recruitment to the lungs after challenge. Finally, protective immunity induced by an experimental Pa could be enhanced by substituting alum with a TLR agonist that induces Th1 cells. Our findings demonstrate that alum promotes protective immunity through IL-1β-induced IL-17A production, but also reveal that optimum protection against B. pertussis requires induction of Th1, but not Th2 cells.