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Mutations of SLC26A4 cause an enlarged vestibular aqueduct, nonsyndromic deafness, and deafness as part of Pendred syndrome. SLC26A4 encodes pendrin, an anion exchanger located in the cochlea, thyroid, and kidney. The goal of the present study was to determine whether developmental delays, possibly mediated by systemic or local hypothyroidism, contribute to the failure to develop hearing in mice lacking Slc26a4 (Slc26a4−/−). We evaluated thyroid function by voltage and pH measurements, by array-assisted gene expression analysis, and by determination of plasma thyroxine levels. Cochlear development was evaluated for signs of hypothyroidism by microscopy, in situ hybridization, and quantitative RT-PCR. No differences in plasma thyroxine levels were found in Slc26a4−/− and sex-matched Slc26a4+/− littermates between postnatal day 5 (P5) and P90. In adult Slc26a4−/− mice, the transepithelial potential and the pH of thyroid follicles were reduced. No differences in the expression of genes that participate in thyroid hormone synthesis or ion transport were observed at P15, when plasma thyroxine levels peaked. Scala media of the cochlea was 10-fold enlarged, bulging into and thereby displacing fibrocytes, which express Dio2 to generate a cochlear thyroid hormone peak at P7. Cochlear development, including tunnel opening, arrival of efferent innervation at outer hair cells, endochondral and intramembraneous ossification, and developmental changes in the expression of Dio2, Dio3, and Tectb were delayed by 1–4 days. These data suggest that pendrin functions as a HCO3− transporter in the thyroid, that Slc26a4−/− mice are systemically euthyroid, and that delays in cochlear development, possibly due to local hypothyroidism, lead to the failure to develop hearing.
- Pendred syndrome
- thyroid
- Tectb
- bicarbonate
mutation of the gene SLC26A4, which leads to a loss of function of the protein pendrin, causes an enlargement of the vestibular aqueduct as well as nonsyndromic or syndromic hearing loss including Pendred syndrome (22, 58). Hearing loss can be congenital or progressive during childhood (14, 16). This implies that a reduction or lack of pendrin function is compatible with hearing, but that this deficiency facilitates hearing loss in conjunction with other genetic and/or epigenetic factors.
Hypothyroidism and goiter are two variable manifestations of Pendred syndrome that may depend on nutritional iodine intake (33). Interestingly, the coincidence of goiter and deafness led to the first description of the syndrome (43). Hypothyroidism has variably been observed in patients with Pendred syndrome, which raises the question whether hypothyroidism is an epigenetic factor that contributes to the development of hearing loss in carriers of SLC26A4 mutations (8, 55, 57).
Studies of the consequences of a congenital loss of pendrin have recently been facilitated by the establishment of a mouse model (Slc26a4−/− mice) (21). Slc26a4−/− mice develop prenatally an enlargement of the endolymphatic compartment in the cochlea and the vestibular labyrinth that is consistent with the enlargement of the vestibular aqueduct and cochlear malformations that are seen in human patients (21). Compared with humans, the life span of mice is much compressed and mice are born developmentally immature. Wild-type and Slc26a4+/− mice are born deaf and begin to hear at postnatal day 12 (P12). Slc26a4−/− mice, however, fail to develop hearing (63). The present study was designed to determine whether developmental delays, possibly mediated by systemic or local cochlear hypothyroidism, may be an epigenetic factor that contributes to the failure to develop hearing in Slc26a4−/− mice and possibly in human carriers of mutations that lead to a loss of pendrin function.
Thyroid epithelial cells synthesize and secrete thyroglobulin (Tg) into the lumen of the follicle and use this protein as a matrix for the synthesis of the prohormone thyroxine (T4). Specific tyrosine residues are iodinated and coupled together by thyroperoxidase (Tpo) (5, 17). T4 synthesis requires normal ion transport including the transport of I− across the follicular epithelial cells into the lumen (42). I− is taken up across the basolateral membrane via the Na+/I− cotransporter (NIS), the product of the Slc5a5 gene. The mechanism of I− transport across the apical membrane is not yet fully understood (7, 33). Pendrin, which is an exchanger that transports anions such as Cl−, I−, and HCO3−, is involved in the secretion of I− across the apical membrane of thyroid follicular epithelial cells (24, 50, 51, 53, 56, 66). In addition to pendrin, several other I−-permeable transporters may contribute to I− transport including the anion/proton exchangers, ClC-3 and ClC-5, and the anion channels Cftr and Slc26a7 (6, 19, 40, 61, 65). Expression levels of pendrin in the murine thyroid have been found to be ∼50-fold less than in the kidney (61). In the kidney, cochlea, and in the vestibular labyrinth of the inner ear, pendrin has been found to secrete HCO3− and a lack of pendrin has been shown to reduce the luminal pH, consistent with a reduced rate of HCO3− secretion (37, 48, 63). It is therefore conceivable that pendrin also secretes HCO3− in the thyroid. These findings led to the first goal of the present study, which is to determine whether pendrin has a functional role in the murine thyroid, by measuring pH in thyroid follicles of Slc26a4+/− and Slc26a4−/− mice. In addition, we analyzed expression levels of genes involved in ion transport by gene array.
The thyroid gland releases mainly T4 and, to a much lesser extent, the active hormone tri-iodo-thyronine (T3). Plasma levels of T4 in mice surge and reach a peak at P15 (2, 11) to support the many processes that depend on thyroid hormone during postnatal development. Plasma levels of T4 have been found to be normal in adult Slc26a4−/− mice (21, 30). Adult mice, however, generate much smaller amounts of T4 compared with developing animals. It is conceivable that the importance of pendrin for T4 synthesis has been underestimated. Therefore, the second goal of the present study was to determine whether lack of pendrin compromises the surge in plasma T4 during early postnatal development. This goal was addressed by measurement of total T4 levels in plasma of Slc26a4+/− and Slc26a4−/− mice during early postnatal development.
Thyroid hormone is essential for normal growth and development of the cochlea, brain, and bone, among others. Consequently, hypothyroidism during early development leads to deafness, retarded neuronal development, and reduced bone mineralization and growth. Data from models of severe and prolonged hypothyroidism have established a catalog of events in the developing cochlea, brain, and bone that depend on thyroid hormone. This catalog includes opening of the tunnel in the organ of Corti of the cochlea at P5-P8 (18, 49), shaping of the tectorial membrane in the cochlea through the expression of the tectorial membrane-specific proteins α-tectorin (coded by the gene Tecta) and β-tectorin (coded by the gene Tectb) (31, 45), arrival of efferent innervation at cochlear outer hair cells (12, 60), expression of the neuron-specific gene synaptotagmin Srg1 (20, 44), and the maturation and mineralization of bone-forming chondrocytes and mesenchymal cells (1, 52). The cochlea controls T3 levels locally through the expression of two deiodinases, Dio2 and Dio3, and is therefore somewhat independent of circulating levels of T3 if sufficient amounts of T4 are provided (11, 39). Dio2 forms the active hormone T3 from the prohormone T4. Dio3 inactivates and reduces T3 levels. Dio3 forms the inactive T2 from T3 and furthermore depletes T4 by forming the inactive reverse tri-iodo-thyronine (rT3). In the inner ear, Dio3 gene expression is high during prenatal development and expression is largely lost postnatally. In contrast, Dio2 is not expressed during prenatal development but highly expressed in fibrocytes of the modiolus and the spiral ligament during early postnatal development, with a peak of Dio2 activity being reached at P7 (11, 39). Changes in the local expression of the two deiodinases can be expected to produce a locally confined hypo- or hyperthyroidism. Fibrocyte-containing tissues in the spiral ligament and spiral limbus of Slc26a4−/− mice have been reported to be reduced in thickness, which raises the question whether local T3 concentrations are reduced, thus leading to a locally confined hypothyroidism (62). The third goal of the present study was to determine whether the cochlea of Slc26a4−/− mice bears signs of hypothyroidism. This goal was addressed by localizing Dio2 expression with in situ hybridization, by monitoring the opening of the tunnel in the organ of Corti and the arrival of efferent innervation at outer hair cells by confocal microscopy, and by monitoring tissue mineralization in alizarin-stained tissue sections with laser-scanning microscopy. In addition, the expression of Dio2 and Dio3, which are potential effectors of hypothyroidism, and the expression of the Tectb and Srg1, which are potential reporters of hypothyroidism, were monitored by quantitative RT-PCR in Slc26a4+/− and Slc26a4−/− mice during early postnatal development.
A colony of Slc26a4−/− and Slc26a4+/− mice was established at Kansas State University from breeders kindly provided by Dr. Susan Wall (Emory University). Genetic drift of the colony was limited by back-crossing to the original strain (129SvEvTac obtained from Taconic, Germantown, NY). The colony was freed from helicobacter rodentium through a fostering approach and is maintained free of known and suspected murine pathogens. Serologic tests (Radil, Columbia, MO) that were consistently and repeatedly negative included the following antibodies: EDIM (epizootic diarrhea of infant mice virus–a mouse rotavirus), TMEV (Theiler's murine encephalomyelitis virus–mouse poliovirus, strain GDVII), MHV (mouse hepatitis virus–a mouse coronavirus), MVM (minute virus of mice–a mouse parvovirus), MNV (murine norovirus–a mouse calicivirus), M. pulmonis (mycoplasma pulmonis–the agent of murine mycoplasmosis), MPV (a mouse parvovirus), Parvo NS-1 (a conserved recombinant parvoviral protein, rNS1), and Sendai (Sendai virus–a type 1 paramyxovirus).
Mice used in this study were deeply anesthetized with 4% tri-bromo-ethanol and killed by decapitation or by transcardial perfusion. Preweaning mice received 0.013 ml/g body wt ip and adult mice 0.014 ml/g body wt ip of 4% tri-bromo-ethanol. Anesthesia of neonatal mice (P2) was supported by rapid cooling on an ice slush. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Kansas State University.
Thyroxine measurements in plasma.
Plasma was collected from sex-matched Slc26a4−/− and Slc26a4+/− littermates. Thyroxine levels were measured at the Diagnostic Laboratory in the College of Veterinary Medicine using the Immulite/Immulite 1000 Total T4 kit (Siemens Healthcare Diagnostics, Deerfield, IL).
Electrophysiological pH measurements.
The interstitial and follicular pH and the transepithelial potential were measured with double-barreled microelectrodes in situ. Thyroid glands were surgically exposed via a ventral approach. The slope of the pH-sensitive electrodes was 56 ± 3 mV/pH unit (n = 12). Procedures for in situ measurements of transepithelial potentials and pH were reported earlier (37, 63). Electrodes were calibrated in situ using three different pH values. Calibration solutions contained (in mM) 130 NaCl, 20 MES for pH 6, 130 NaCl, 20 HEPES for pH 7; and 130 NaCl, 20 tricine for pH 8. Data were recorded analog (Flat Bed Chart recorder, Kipp & Zonen, The Netherlands) for annotation and digital for presentation and data analysis (DIGIDATA 1322A and AxoScope 9, Axon Instruments). Data were analyzed using custom software written by P. W. in LabTalk (Origin 6.0, The Origin, Northhampton, MA).
Double-barreled glass microelectrodes were manufactured from filament-containing glass tubing (World Precision Instruments 1B100F-4, Sarasota, FL) using a micropipette puller (Narishige PD-5, Tokyo, Japan). Before silanization, microelectrodes were baked at 180°C for 2 h to ensure dryness. The longer ion-selective barrel was mounted in the lid of a beaker. The beaker was heated to 210°C and the electrodes were silanized by a 90-s exposure to 0.08 ml di-methyl-di-chloro-silane (Fluka 40136) at room temperature. The shorter reference barrel was protected from silanization by sealing the open end with Parafilm (Alcan Packaging, Chicago, IL). After silanization, microelectrodes were baked at 180°C for 3 h and tips were broken to a final outer diameter of ∼3 μm. The reference barrel was filled with 1 M KCl and the ion-selective barrel was filled at the tip with liquid ion exchanger (Hydrogen ionophore II–Cocktail A, Fluka 95297) and back-filled with buffer solution (500 mM KCl, 20 mM HEPES, pH 7.34). Each barrel was connected to an input of a grounded dual electrometer (World Precision Instruments FD223) via Ag-AgCl wire electrodes. The animal was grounded via a Ag-AgCl wire electrode inserted into the neck musculature. Current pulses (1 nA) were injected via the reference barrel to monitor electrode resistance.
Confocal microscopy.
Cochleae were fixed by transcardial and cochlear perfusion and postfixation. The transcardial perfusate was first NaCl solution (6 ml, 1 min) followed by NaCl solution containing 4% paraformaldehyde (24 ml, 4 min). Cochleae were removed and scala tympani and scala vestibuli were individually perfused with NaCl solution containing 4% paraformaldehyde (50 μl, 5 min). For experiments that only required phalloidin and DAPI staining, 0.5% glutaraldehyde was added to the paraformaldehyde solution. NaCl solution contained (in mM) 150 NaCl, 1.6 K2HPO4, 0.4 KH2PO4, 0.7 CaCl2, 1 MgCl2, and 5 glucose, pH 7.4. Postfixation consisted of 1- to 2-h incubation in paraformaldehyde solution at 4°C with rotation.
Temporal bones were decalcified in 10% EDTA, processed through a sucrose gradient, and infiltrated with polyethylene glycol. Midmodiolar cryosections (12 μm, CM3050S, Leica, Nussloch, Germany) were cut such that four cross-sections of the organ of Corti became visible. Sections from Slc26a4+/− and Slc26a4−/− littermates were put on the same slide to aid side-by-side evaluation. Images were taken from equivalent cross-sections. Sections on slides were blocked in 137 mM NaCl, 10.1 mM Na2HPO4, 1.8 mM KH2PO4, 2.7 mM KCl, pH 7.4 with 0.2% Triton X-100 (PBS-TX) and 5% BSA. Slides were incubated overnight at 4°C with primary antibody, rabbit anti-synaptophysin (1:100, Dako, Carpinteria, CA) in PBS-TX with 1–3% BSA. Slides were washed in PBS-TX and incubated for 1 h at room temperature with secondary antibodies, donkey anti-rabbit Alexa 488 (1:1,000, Invitrogen-Molecular Probes, Eugene, OR) in PBS-TX with 1–3% BSA. After incubation, slides were washed with PBS-TX and coverslipped with FluorSave (Calbiochem, La Jolla, CA). Cryosections were viewed by confocal laser-scanning microscopy (LSM 510 Meta, Carl Zeiss, Göttingen, Germany).
Laser-scanning microscopy.
Temporal bones were fixed by immersion into Cl−-free solution containing 4% paraformaldehyde (1 h), processed through a sucrose gradient, and infiltrated with polyethylene glycol. Cl−-free solution contained (in mM) 150 Na-gluconate, 1.6 K2HPO4, 0.4 KH2PO4, 4 Ca-(gluconate)2, 1 MgSO4, and 5 glucose, pH 7.4. Midmodiolar cryosections (10 μm, CM3050S, Leica) were cut and stained with alizarin-red (2% in water, 5 s) and washed under running water (20 s). Cryosections were viewed by laser-scanning microscopy using similar intensities of red (633 nm), green (543 nm), and blue (488 nm) laser lights (LSM 510 Meta, Carl Zeiss). Transmitted light intensities were recorded and color images were generated by addition of red, green, and blue images.
Array-assisted gene expression analysis.
Total RNA was isolated from the thyroid of P15 sex-matched Slc26a4−/− and Slc26a4+/− littermates (RNeasy micro kit, Qiagen, Valencia, CA). Total RNA was amplified and converted to cDNA (Ovation RNA Amplification System V2, NuGen Technology, San Carlos, CA). This amplification system has earlier been evaluated in our laboratory (54). The cDNA was fragmented and biotinylated (FL-Ovation cDNA Biotin Module V2, NuGen Technologies) and hybridized to high-density oligonucleotide gene chips (mouse 430 2.0 gene chip, Affymetrix, Santa Clara, CA). RNA was isolated in our lab and processed for gene chip analysis at the Gene Expression Facility at Kansas State University. A total of six chips were run. Three chips each were used to analyze expression in the thyroid of Slc26a4+/− and Slc26a4−/− mice. Raw data were analyzed using commercial (GCOS, Affymetrix) and custom-written software (Excel, Microsoft, Redmond, WA). Quality metrics conformed with MIAME standards (Table 1). Data were submitted to the Geo database (accession number: GSE10589). Average intensities were calculated and the ratio (Slc26a4+/−/Slc26a4−/−) was computed. Ratios >0.7 and <1.3 were considered insignificant.
Quantitative RT-PCR.
Quantitative RT-PCR was performed on total RNA isolated from the whole cochlea. The whole cochlea, rather than microdissected fractions, was chosen because it represents a well-defined preparation throughout development from P2 to P15, which includes the transition from a cartilaginous to a bony structure. The associated changes in the mechanical properties would present challenges to the preparation of microdissected fractions that would introduce unnecessary variations in the data. Thus, total RNA was isolated from the whole cochlea (RNeasy micro kit, Qiagen). Quantity and quality of total RNA were evaluated by microfluidic electrophoresis (BioAnalyzer, Agilent, Santa Clara, CA) and by microliter absorption photometry (Nanodrop, Wilmington, DE). RNA samples were accepted for quantitative RT-PCR only when they were free of contamination and had matching electrophoretic and photometric quantifications and excellent RNA quality. Concentrations of RNA samples were adjusted to 10 ng/μl using RNA storage solution (Applied Biosystems/Ambion, Austin, TX) and stored at −80°C before quantitative RT-PCR. Quantitative RT-PCR was carried out in 96-well plates (QuantiTect SYBR Green RT-PCR Kit, Qiagen; iCycler, Bio-Rad, Hercules, CA) using 10 ng of total RNA per well and gene-specific primers (Table 2). RNA samples from sex-matched Slc26a4+/− and Slc26a4−/− littermates were analyzed and referenced to 18S rRNA. Each reaction was carried out in duplicate. The average difference in Ct values between duplicates was 0.36 ± 0.1 SD (average of 21 plates, 96-well that examined high, intermediate, and low expressed genes). Reactions were assembled with the assistance of an automatic pipetting station (Biomek NXp, Beckman Coulter, Fullerton, CA) with hardware modifications and software programming by P. W. RT was performed for 30 min at 50°C and terminated by heating to 95°C for 15 min. PCR consisted of 40 cycles of 30-s annealing at 60°C, 30-s elongation at 72°C, 15-s hot measurement at 78°C, and 15-s denaturation at 94°C. Amplification of a single product of the appropriate size was verified by microfluidic electrophoresis (BioAnalyzer, Agilent). The number of template molecules (T) was estimated according to T = 10 ^log number of molecules at Ct/Ct * log (PCR efficiency). Ct was defined as the cycle at which the fluorescence of the product molecules reached a common threshold chosen in the middle of the log-linear part of the growth curve. The number of molecules at Ct was calibrated by amplifying 18S rRNA present in the samples. PCR efficiency was obtained from the slope of the log-linear phase of the growth curve (46).
In situ hybridization.
Temporal bones were fixed in PBS containing 4% paraformaldehyde (overnight), washed (PBS, 3 × 3 min), cryoprotected (30% sucrose, 2 h, 4°C), and embedded in polyethylene glycol (TissueTek, Torrance, CA). Cryosections (12 μm) were air-dried, stored at −80°C, brought to room temperature, washed (PBS, 3 × 5 min), treated with proteinase K (1 g/ml in PBS, 2 min), postfixed in PBS containing 4% paraformaldehyde (5 min), washed (PBS, 3 × 5 min), acetylated (0.1 M tri-ethanol-amine containing 0.25% acetic anhydride, 10 min), permeabilized (0.1% Triton X-100 in PBS, 10 min), and again washed (PBS, 3 × 5 min). Slides were then overlaid with 200 μl of hybridization buffer, coverslipped, and incubated (20 h, 68°C). Hybridization buffer contained 500 ng/ml digoxygenin-labeled Dio2 antisense or sense probe (39) in 4× Na-citrate buffer (600 mM NaCl, 60 mM Na-citrate, pH 7.0), 50% formamide, 10% dextran sulfate, 1× Denhardt's reagent, and 50 g/ml herring fish sperm DNA. After hybridization, slides were washed at 70°C first with 1× Na-citrate buffer (1×, 15 min) and then with 0.2× Na-citrate buffer (4×, 15 min). Further washes were performed at room temperature. Slides were washed first with 0.2× Na-citrate buffer and then with MABT buffer (2×, 30 min). MABT buffer contained 150 mM NaCl, 100 mM maleic acid, and 0.1% Tween 20, pH 7.5. Slides were blocked (MABT buffer with 2% blocking reagent; Roche, Mannheim, Germany; 2 h, room temperature), incubated with alkaline-phosphatase-conjugated sheep anti-digoxygenin FAB fragments [1:2,000 in MABT buffer with 2% blocking reagent (Roche), overnight at room temperature], and washed with MABT buffer (2×, 30 min). For color development, slides were washed (2×) and incubated (16 h, room temperature) with AP-staining buffer. AP-staining buffer contained 0.38 mg/ml 5-bromo-4-chloro-3-indolyl phosphate, 0.19 mg/ml p-nitroblue tetrazolium chloride, 100 mM NaCl, 5 mM MgCl2, 100 mM Tris·HCl, 0.1% Tween 20, pH 9.5. The color reaction was stopped by washing slides with PBS (2×, 5 min). Slides were then mounted in an aqueous mounting media (Aqua-Poly/Mount, Polysciences, Warrington, PA) and imaged (Nikon 80i microscope, NIS Elements imaging software, Nikon, Melville, NY).
Data are generally presented as average ± SE or, when indicated, as average ± SD with n being the number of animals. Data acquired in paired experiments using sex-matched littermates were evaluated by paired t-test; other data were compared by unpaired t-test. Significance was assumed when P < 0.05.
Lack of pendrin causes acidification of thyroid follicles and a reduced transepithelial potential.
Lack of pendrin reduces the luminal pH in the cochlea and the vestibular labyrinth, consistent with a reduced rate of HCO3− secretion (37, 63). The presence of a similar acidification of the lumen in the thyroid would indicate that pendrin has a physiological role in the murine thyroid. Thus, we measured the follicular pH and the interstitial pH as well as the transepithelial potential in the thyroid of adult Slc26a4+/− (P51 ± 9 SD, n = 8) and Slc26a4−/− (P53 ± 11 SD, n = 6) mice (Fig. 1). Lack of pendrin caused a reduction in the transepithelial potential and an acidification of the follicular pH. No significant difference was found in the pH of the interstitial fluid surrounding the follicles. These observations suggest that pendrin in the murine thyroid is functional and that pendrin in the thyroid, as previously shown for the kidney and the inner ear, is involved in the secretion of HCO3−.
Mice lacking pendrin generate a normal T4 surge during postnatal development.
T4 was measured in plasma collected from Slc26a4−/− and Slc26a4+/− mice at age P5, P10, P15, P30, and P80-P100. A pronounced surge of plasma T4 was observed during early postnatal development (Fig. 2). No significant differences in T4 levels between Slc26a4−/− and Slc26a4+/− mice were found. These observations underscore that pendrin is not essential for T4 production and not necessary for the generation of a normal T4 surge.
Lack of pendrin does not change expression levels of genes involved in T4 synthesis and epithelial ion transport in the thyroid.
Gene expression was evaluated by gene array analysis at P15, which is at the peak of the T4 surge. Total RNA was collected from thyroids of sex-matched P15 Slc26a4−/− and Slc26a4+/− littermates. A selected dataset focused on genes involved in T4 synthesis, ion transport, and epithelial barrier function is given in Fig. 3. Neither the expression of genes involved in T4 synthesis nor the expression of genes involved in epithelial ion transport was changed in the thyroid of Slc26a4−/− mice. No information on the expression levels of pendrin (Slc26a4), Cftr, and Clcn5 was provided since expression of these genes was below detection level in the array. Interestingly, however, the expression of the deiodinase Dio2 was increased. This finding was supported by three probes. It is tempting to speculate whether maintenance of thyroid function in the absence of pendrin requires some level of autocrine compensation. A similar increase in Dio2 expression has recently been observed in a Pendred syndrome patient (41).
Lack of pendrin leads to an enlargement of epithelia-lined scala media and results in a displacement of adjacent fibrocytes and mesenchymal cells.
Endolymphatic spaces in the cochlea and the vestibular labyrinth of Slc26a4−/− mice are known to be significantly enlarged from embryonic day 15.5 onward and this enlargement persists postnatally (21). We quantified the enlargement in the postnatal cochlea by measuring cross-sectional areas of scala media, scala vestibuli, and scala tympani in the lower three cross-sections of the cochlear duct. Data from equivalent cross-sections of Slc26a4+/− and Slc26a4−/− littermates (n = 5) aged P3-P7 were pooled. The cross-sectional area of scala media was 10.4-fold enlarged in Slc26a4−/− mice. In contrast, the cross-sectional area of scala vestibuli was 4.8-fold reduced and of scala tympani was 2.7-fold reduced (Fig. 4). The enlargement of scala media was mainly mediated by a distention of Reissner's membrane that included an increase in the number of cells. In contrast, the organ of Corti and stria vascularis appeared to contain the same number of cells. No enlargements of apical surface areas of cells in the organ of Corti had been noticed (21). Apical surface areas of marginal cells, however, are enlarged by a factor of 2 (27), which is consistent with a distention of stria vascularis roughly by a factor of 2 (Fig. 4).
The enlarged scala media not only bulged in apical direction into scala vestibuli and in basal direction into scala tympani, but also in centripetal direction into the modiolus and in centrifugal direction into the spiral ligament (Fig. 4). Bulging led to a displacement or a distortion of fibrocytes and mesenchymal cells in the spiral ligament and the modiolus, since the otic capsule roughly maintained normal dimensions. The space occupied by the spiral ligament, which is located in the lateral wall behind stria vascularis and reaches from scala vestibuli to scala tympani, was distended by roughly a factor of 2 (Fig. 4). Fibrocytes remained concentrated at the lower portion of the spiral ligament; however, the areas behind stria vascularis and the upper portion of the spiral ligament were depleted of fibrocytes. Displacement and local depletion of mesenchymal cells or fibrocytes may compromise cell signaling mediated by secreted molecules or requiring cell-to-cell contact. It is conceivable that regions of local hypothyroidism occurred due to increased diffusional distances between cells bearing thyroid hormone (TH) receptors and fibrocytes that transiently express Dio2 to generate T3 during early postnatal development. Hypothyroidism has well-known consequences on cochlear development. These consequences, in turn, can be taken as evidence for hypothyroidism.
Lack of pendrin leads to a local depletion of Dio2 expression.
Fibrocytes in the spiral ligament, underneath the spiral limbus, and surrounding the spiral ganglion in the modiolus are known to express Dio2, which encodes Dio2, the enzyme that generates T3 during early postnatal development of the cochlea (39). The observed displacement of fibrocytes implies that regions of local hypothyroidism may arise in the cochlea of Slc26a4−/− mice. To verify this concept, we localized Dio2 expression by in situ hybridization in P7 sex-matched Slc26a4+/− and Slc26a4−/− littermates. Expression of Dio2 was found in fibrocytes of Slc26a4+/− and Slc26a4−/− mice but was notably depleted in Slc26a4−/− mice in the space between stria vascularis and bone (Fig. 5). The observed depletion of Dio2 expression supports the concept of a local cochlear hypothyroidism.
Lack of pendrin delays opening of the tunnel during cochlear development and causes hypertrophy of the tectorial membrane.
Among the most easily observable effects of cochlear hypothyroidism is a delay of the opening of the tunnel in the organ of Corti (18, 49). Opening of the tunnel in the organ of Corti was evaluated in cryosections prepared from Slc26a4+/− and Slc26a4−/− littermates (Fig. 6). Sections were treated with phalloidin to stain actin filaments and with DAPI to stain nuclei. Transmitted light (bright field) images were recorded to visualize unstained structures such as the tectorial membrane. A delay in tunnel opening by ∼1 day and a persistent hypertrophy of the tectorial membrane were observed. Both observations are consistent with hypothyroidism.
Lack of pendrin delays arrival of efferent innervation in the cochlea.
Another easily observable consequence of hypothyroidism is a delay in the arrival of efferent nerve terminals at outer hair cells (12, 60). Arrival of efferents was evaluated in cryosections prepared from Slc26a4+/− and Slc26a4−/− littermates. Efferent nerve terminals were labeled using anti-synaptophysin antibodies (Fig. 7). A delay in the arrival of efferent innervation was observed. This observation provides further evidence for the hypothesis that the cochlea of Slc26a4−/− mice is hypothyroid.
Lack of pendrin alters Dio2 and Dio3 expression in the cochlea.
The cochlea controls T3 levels locally through the expression of two deiodinases, Dio2 and Dio3 (11, 39). Expression levels of Dio2 and Dio3 were monitored by quantitative RT-PCR in total RNA isolated from whole cochleae of sex-matched Slc26a4−/− and Slc26a4+/− littermates (Fig. 8A). Expression of Dio2 was found to peak between P6 and P8, which is consistent with the reported peak of cochlear Dio2 activity at P7 (11). Significant differences between Slc26a4+/− and Slc26a4−/− mice were observed at P6 and P8. Dio2 expression in Slc26a4−/− mice was at P6 lower and at P8 higher than in Slc26a4+/− mice. These differences are consistent with the peak of Dio2 expression in Slc26a4−/− mice being delayed by ∼1 day. Subsequently, Dio2 expression declined and this decline was slower in Slc26a4−/− mice compared with Slc26a4+/− littermates. Expression of Dio3 was found to drop low at P6 and to then to rise again till P10 (Fig. 8B). The low level expression found at P6 coincides with the peak in Dio2 expression and is consistent with peak levels of T3 at this time. Between P6 and P10, Dio3 expression rose again and this rise was delayed in Slc26a4−/− mice by ∼1 day. Taken together, these data suggest that the peak in T3 levels in the cochlea of Slc26a4−/− mice is delayed by ∼1 day.
Lack of pendrin alters Tectb but not Srg1 expression in the cochlea.
A small number of T3-sensitive genes is solely expressed in the organ of Corti or the modiolus of the cochlea and thereby can be used as a marker for thyroid hormone status in these regions. We selected two genes, Tectb and Srg1, to serve as reporters for T3 levels. Tectb (tectorin b) is exclusively expressed in a structure, the greater epithelial ridge, that develops into the spiral limbus and inner sulcus of the organ of Corti (31). Tectb was chosen as a reporter of T3 levels in the organ of Corti. Srg1 (synaptotagmin XII) is a neuron-specific gene (44). Although expression of Srg1 has not yet been localized in the cochlea, it is reasonable to expect that expression is limited to the neurons of the spiral ganglion cells in the modiolus. Thus, Srg1 was chosen as a reporter of T3 levels in the modiolus of the cochlea.
Expression of Tectb has been shown to strongly depend on thyroid hormone (31). In euthyroid rats, expression of Tectb steeply rises from P2 to a peak at P6 and rapidly declines between P8 and P12. In contrast, Tectb expression in severely hypothyroid rats was found to rise slower, reaching a lower level peak in expression at P8 and decline much more slowly thereafter (31). In sex-matched Slc26a4−/− and Slc26a4+/− littermates, we found high expression levels at P2, P6, and P8 (Fig. 8C). The following decline in Tectb expression was significantly slower in Slc26a4−/− mice compared with Slc26a4+/− littermates. These data suggest that the organ of Corti of the cochlea of Slc26a4−/− mice is hypothyroid.
Expression of Srg1 in the cerebellum has been shown to be reduced in hypothyroidism (20, 44). Thus, we used the expression Srg1 as a reporter of hypothyroidism in the spiral ganglion, where Srg1 is suspected to be expressed. No difference in expression was found between Slc26a4+/− mice and sex-matched littermates (Fig. 8D). This observation suggests that spiral ganglion cells in the modiolus are euthyroid.
Lack of pendrin delays tissue mineralization in the cochlea.
Bone formation in the cochlea consists of endochondral and intramembraneous ossification. Both ossification mechanisms, based on data from other bones, are known to depend on thyroid hormone (4, 13). A delayed or reduced tissue mineralization may therefore provide additional evidence for local cochlear hypothyroidism. Mineralization of the otic capsule occurs by endochondral bone formation and mineralization of the modiolus by intramembraneous bone formation. Tissue mineralization was visualized between P1 and P10 in alizarin-stained cochlear cryosections. Zones of resting, proliferative, hypertrophic chondrocytes, and chondrocytes that mineralized the extracellular matrix to form a primary spongiosum, were observed at P1 in Slc26a4+/− and Slc26a4−/− mice at the base of otic capule (Fig. 9A). Ensuing chondrocyte apoptosis compacts the primary spongiosum that is then remodeled into bone. At P1, the otic capsule of Slc26a4+/− and Slc26a4−/− mice consisted of a thick layer of chondrocytes (Fig. 10). At P4, a primary spongiosum was found throughout the otic capsule of Slc26a4+/− and Slc26a4−/− mice, although it appeared thinner in Slc26a4−/− mice compared with Slc26a4+/− mice. Interestingly, between P4 and P8, the primary spongiosum in parts of the otic capsule condensed in Slc26a4+/− mice into two layers of bone that provide in their middle a space for bone marrow (Fig. 10). Only one layer of bone was formed in Slc26a4−/− mice (Fig. 11A). At P6, the inner layer in Slc26a4+/− mice retained the structure of a primary spongiosum, whereas the outer layer appeared to be already more compacted (Fig. 11B). These data demonstrate a compromise in endochondral bone formation that is consistent with hypothyroidism.
Intramembraneous bone formation was observed to begin at P4 in the modiolus of the cochlea of Slc26a4+/− mice where mesenchymal cells surrounding the spiral ganglia of the cochlear nerve began to mineralize the extracellular matrix (Fig. 9B). Intramembraneous bone formation in the modiolus of Slc26a4−/− mice appeared to be delayed by ∼4 days (Fig. 10). The observed delay in intramembraneous bone formation is consistent with hypothyroidism.
Lack of pendrin does not alter tissue mineralization in phalangeal and metatarsal bones.
Bone formation of long bones such as phalangeal and metatarsal bones occurs by endochondral bone formation. A compromise of endochondral bone formation would be expected to lead to a reduced length of the ossified zone between the two growth plates located at opposite ends of the bone. Lengths of the mineralized bones were measured in alizarin-stained phalangeal and metatarsal bones from P8 Slc26a4+/− and Slc26a4−/− littermates (n = 4–5). No differences were found in the lengths of intermediate phalanges 2–5, proximal phalanges 1–5, and metatarsals 1–5 (data not shown). These findings demonstrate that the compromised endochondral bone formation that was observed in the cochlea is not a manifestation of a systemic defect.
The most salient findings of the present study on mice lacking pendrin are 1) that the thyroid has a more acidic follicular pH, generates a lower transepithelial potential but produces a normal plasma T4 surge during early postnatal development, and expresses normal levels of genes involved in T4 synthesis, epithelial ion transport, and barrier function, 2) that the cochlea develops a 10-fold enlargement of scala media which displaces fibrocytes and thereby may distort reciprocal epithelial-mesenchymal interactions that are necessary for normal cochlear development, and 3) that the cochlea shows signs of local hypothyroidism including delayed opening of the tunnel, delayed arrival of efferent innervation, and delayed changes in the expression levels of Dio2, Dio3, and Tectb, as well as reduced endochondral and delayed intramembraneous bone formation.
Pendrin mediates HCO3− secretion into the follicular lumen of the thyroid but is not essential for I− secretion or T4 generation.
The observations that the follicular pH in the thyroid of Slc26a4+/− mice is more alkaline than the surrounding interstitial fluid (Fig. 1) and that loss of pendrin leads to an acidification of the follicular pH suggest that alkaline equivalents such as HCO3− are among the anions transported by pendrin in the thyroid. HCO3− may be the most readily available anion since it is generated from metabolically produced CO2 through carbonic anhydrase-mediated conversion. HCO3− secretion as the significant function of pendrin in the thyroid is consistent with similar functions in the cochlea, the vestibular labyrinth, and the kidney (37, 48, 63). The physiologic relevance of HCO3− secretion in the thyroid may lie in an alkalinization of the apical membrane, which has been shown to facilitate binding of thyroglobulin before endocytosis (25). The observations, however, that Slc26a4−/− mice generated a normal surge in T4 (Fig. 2) and maintained normal expression levels of genes involved in T4 synthesis (Fig. 3) underscore that pendrin is not essential for I− transport or T4 synthesis.
The observations that the thyroid epithelium of mice lacking pendrin generated a smaller lumen-negative transepithelial potential (Fig. 1) but maintained normal expression levels of genes involved in ion transport and barrier function (Fig. 3) suggest that the ion transporters are controlled by translational or posttranslational mechanisms. Ion transport has been extensively studied in porcine primary cultures of the thyroid follicular epithelium, which secrete Cl− and absorb Na+ (3, 9). Primary cultures generate a lumen-negative transepithelial potential that is similar in magnitude to the transepithelial potential that we observe in situ in the murine thyroid. The lumen-negative transepithelial potential is most likely generated by a combination of K+ channels expressed in the basolateral membrane and Na+ and Cl− channels expressed in the apical membrane (9, 10, 36). A possible cause for the reduced transepithelial potential in mice lacking pendrin is an inhibition of the apical Na+ channels or the basolateral K+ channels by an acidic cytosolic pH (47). Whether the more acidic pH in the follicular lumen is associated with a more acidic cytosolic pH, however, is currently unknown.
Cochlear development is compromised by the enlargement of scala media.
Scala media of Slc26a4−/− mice develops an enlargement at embryonic day 15.5, which is shortly after the onset of pendrin expression (21). During early postnatal development, cross-sectional areas of scala media were found to be 10.4-fold enlarged and this enlargement persists into adulthood. It is currently unknown whether this enlargement is due to hypersecretion or hypoabsorption of osmolytes followed by water flux. The finding that the otic capsule roughly maintained normal dimensions is interesting given its formation is induced at embryonic day 12–13 of development, which is immediately before the onset of pendrin expression and also before the onset of the enlargement (35). It appears that the size of the otic capsule is defined at the time of induction of the otic capsule.
The enlargement of scala media within the confines of the otic capsule leads to a displacement and local depletion of mesenchymal cells or fibrocytes that may compromise cell signaling mediated by secreted molecules or requiring cell-to-cell contact. This possible compromise of reciprocal cell-to-cell interactions may be responsible for the delayed development of the cochlear epithelium and the delayed and incomplete ossification of the otic capsule. Although we focused our study on the hypothesis that a local hypothyroidism may contribute to the delayed development, it is conceivable that other cell signaling mechanisms are compromised by the distortion and that these mechanisms contribute to the observed developmental delays that result in the failure to develop hearing.
Cochlear development is possibly compromised by local hypothyroidism.
Development of the cochlea depends on a well-orchestrated sequence of events that include a narrow window during early postnatal development during which T3-mediated cell signaling must take place (32). Invariably, lack of T3 signaling at this critical period leads to a delay in the opening of the tunnel, a thickening of the tectorial membrane, a delay in the arrival of efferent innervation, together resulting in deafness. These hallmarks of hypothyroidism are observed regardless of whether hypothyroidism is due to reduced levels of the prohormone T4, as in Pax8−/− mice (15), Tshrhyt/hyt mice (34), Duox2−/− mice (28), and rats treated with propylthiouracil (59) or methimazole (32), or due to an inability to convert T4 to T3, as in Dio2−/− mice (38), or due to a lack of the T3 receptor, as in Thrb−/− mice (23).
Cochlear development depends on the local production of T3 by Dio2 that is temporarily expressed by fibrocytes located in the spiral ligament and the modiolus (11, 39). The observations that Dio2-expressing fibrocytes are displaced due to the bulging of the epithelial-lined scala media (Fig. 5) suggest that local cochlear hypothyroidism may occur as a consequence of a dilution in fibrocyte density and increased diffusional distances between fibrocytes and cells that bear TH receptors. Evidence for cochlear hypothyroidism in Slc26a4−/− mice comes from the observations of a delayed opening of the tunnel in the organ of Corti (Fig. 6), a hypertrophy of the tectorial membrane (Fig. 6), and a delayed arrival of efferent innervation at outer hair cells (Fig. 7). Delayed changes in the developmentally controlled expression levels of Tectb are also consistent with cochlear hypothyroidism (Fig. 8). A similar delay in the decline of Tectb expression has been observed in methimazole-treated rats (31). In rats, however, the primary finding was a delayed onset of Tectb expression, which occurs in rats at P5-7 and thereby can easily be monitored postnatally. The onset of Tectb expression in mice is apparently earlier such that only the delay in the decline of expression could be observed in the present study. A similar difference between rats and mice in the onset of cochlear genes has been reported for Kcnq4 (64).
The finding of no differences in expression levels of Srg1 appears on first sight to argue against the presence of cochlear hypothyroidism. Srg1 expression has been shown in the cerebellum to be sensitive to hypothyroidism (20, 44). It is conceivable, however, that cerebellar genes such as Srg1 are less sensitive indicators of hypothyroidism, which would be consistent with the general view that cochlear function is more sensitive to hypothyroidism than cerebellar function.
A caveat in the argument for the presence of cochlear hypothyroidism is that the delayed tunnel opening, the hypertrophy of the tectorial membrane, the delayed downregulation of Tectb expression, and the delayed arrival of efferent innervation could all be consequences of alterations in the epithelial cells that, in Slc26a4−/− mice, face a more acidic pH at their apical membrane (63). We therefore looked for markers of hypothyroidism in tissues that are not in contact with scala media and decided to investigate differences in cochlear bone formation. Precursor cells of bone are in contact with perilymph but not with endolymph. Unlike endolymph, no differences in the ionic composition or the pH of perilymph, or blood for this matter, have so far been found (62, 63). Both endochondral and intramembraneous bone formation are dependent on thyroid hormone (4, 13) and both forms of bone formation contribute to the ossification of the cochlea. Cochlear ossification may thus serve as an indicator of hypothyroidism. Endochondral bone formation is a two-step mineralization process that begins with chondrocytes proliferating, differentiating, mineralizing their extracellular matrix, and undergoing apoptosis. Osteoclasts and osteoblasts consequently remodel the mineralized extracellular matrix into bone. In contrast, intramembraneous ossification does not involve chondrocytes but depends on mesenchymal cells congregating and differentiating into osteoblasts that mineralize the extracellular matrix. Endochondral bone formation is the process leading to the ossification of the otic capsule and intramembraneous ossification is the process leading to the ossification of the modiolus. Consistent with cochlear hypothyroidism, endochondral bone formation of the otic capsule was reduced and intramembraneous ossification of the modiolus was delayed (Figs. 10 and 11). The use of these findings as arguments for hypothyroidism rests on the assumption that cochlear bone development resembles the formation of parietal and long bones, where the consequences of hypothyroidism have mainly been studied (1, 4, 52). It is conceivable that factors other than thyroid hormone are secreted by the fibrocytes of the spiral ligament and that the depletion of the fibrocytes leads to the depletion of these unknown factors and thereby to the reduced endochondral bone formation of the otic capsule.
Another argument for the presence of cochlear hypothyroidism comes from the finding that cochlear expression of the deiodinases Dio2 and Dio3 was delayed (Fig. 8). The observed delays in expression are most likely underestimations since expression was measured in the whole cochlea and cochlea development progresses from base to apex. The cause for the delays in Dio2 and Dio3 expression in Slc26a4−/− mice remains undetermined. The observed delays in expression changes of Dio2 and Dio3 can be expected to lead to a delay in T3 generation, which in conjunction with a dilution of Dio2 expressing fibrocytes and increased diffusion distances between these fibrocytes and T3 target cells would contribute to the manifestation of hypothyroidism is specific parts of the cochlea. The observed delay in downregulation of Dio2 expression is conceivably due to the delayed arrival of a negative feedback signal that is returned upon T3 stimulation from TH receptor-bearing target cells.
The pathway for T3 between fibrocytes that transiently express Dio2 and the cells in the organ of Corti that express TH receptors involves most likely mechanisms of T3 transport and two networks of gap junctions. The first network of gap junctions connects fibrocytes among each other. The second network connects epithelial cells of the outer sulcus with epithelial cells in the organ of Corti (29). Epithelial cells of the outer sulcus are also called root cells since they project long basolateral "roots" into the spiral ligament. It is conceivable that the function of these roots is to collect T3 from fibrocytes. Interestingly, prenatal disruption of these gap junction networks leads to a delayed opening of the tunnel, consistent with local hypothyroidism in the organ of Corti, and results in a failure to develop hearing (26). Postnatal disruption of these gap junction networks after P6, however, permits the development of hearing, which is consistent with the essential but transient requirement of T3 for cochlear development (X. Lin, Emory University, Abstract #686, "Mechanisms of deafness caused by connexin 26 and connexin 30 mutations studied in mouse models." 32nd Midwinter Research meeting of the Association for Research in Otolaryngology, February 2009, http://www.aro.org/abstracts/abstracts.html).
In conclusion, hypothyroidism has repeatedly been observed in patients with Pendred syndrome, which raised the question whether hypothyroidism contributes to the development of hearing loss in people bearing mutations of SLC26A4. Our data suggest that pendrin functions as a HCO3− transporter in the murine thyroid and that Slc26a4−/− mice are systemically euthyroid but develop an enlargement of scala media, which leads to a displacement of fibrocytes and conceivably distorts reciprocal epithelial-mesenchymal interactions. This is possibly leading to a local cochlear hypothyroidism independent of the systemic thyroid status. Systemic hypothyroidism, which can be expected to aggravate local hypothyroidism, is likely a factor that promotes congenital deafness in people bearing mutation of SLC26A4.
We gratefully acknowledge the support by grants from the National Institute on Deafness and Other Communication Disorders National Institutes of Health (NIH)-R01-DC01098 to P. Wangemann and NIH-R01-DC00212 to D. C. Marcus, grants from the National Institute for Research Resources NIH-P20-RR017686-Project 2 to P. Fong, NIH-P20-RR017686-Core B to D. C. Marcus, and NIH-P20-RR017686-Core C to D. C. Marcus, and grants from College of Veterinary Medicine at Kansas State University SFYI to P. Fong and SMILE to P. Wangemann. Furthermore, the support of the intermural program at National Institute of Diabetes and Digestive and Kidney Diseases at the NIH to D. Forrest is gratefully acknowledged.
This work would not have been possible without the dedication of Dr. T. Miesner, S. Rose, J. Dille, and Dr. B. Carter from the Animal Resource Facility at Kansas State University, College of Veterinary Medicine. The authors thank Dr. J. Bai (Gene Expression Facility at Kansas State University), K. Smith (Diagnostic Laboratory in the College of Veterinary Medicine), and C. Linsenmayer (Laboratory Technician) for excellent services.
- Copyright © 2009 American Physiological Society


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In 1919, eleven states, the Territory of Hawaii, the Canadian Province of British Columbia and the Mexico District of Lower California met and organized the Western Plant Board for the purpose of discussing and developing uniformity in conducting plant protection work. The Western Plant Board later grew to its current size. The remaining states and the Commonwealth of Puerto Rico were organized into three separate regional plant boards in 1925. The National Plant Board was created the same year.
Central Plant Board
- President: Collin Wamsley, MO
- Vice-President: Brian Kuhn, WI
- Secretary/Treasurer: Dan Kenny, OH
Eastern Plant Board
- President: Dana Rhodes, PA
- Vice-President: Christopher Logue, NY
- Secretary/Treasurer: Piera Siegert, NH
Southern Plant Board
- President:Larry Nichols, VA
- Vice-President: Kenneth Calcote, MS
- Secretary/Treasurer: Phil Wilson, NC
Western Plant Board
- President: Brad White, WA
- Vice-President: G. John Caravetta, AZ
- Secretary: Brad Lewis, NM
- Treasurer:
Last Modified: August 24, 2016

subset: commoncrawl


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Golf with a mountain view!
Tee off in a beautiful landscape with views of the majestic Allgäu Alps - a truly holistic experience for your body and senses.
The Golf Resort Sonnenalp - Oberallgäu is a golf centre at the heart of the Allgäu region that is virtually unmatched in Germany.
At the Golf Resort Sonnenalp - Oberallgäu you will enjoy golf at its best: Attractive levels of difficulty, perfectly maintained, sweeping grounds framed by the majestic Alpine panorama make for an unforgettable experience at every hole.
Situated on a mountain plateau at the heart of the Upper Allgäu region between Fischen, Bolsterlang and Ofterschwang, the resort combines sportive challenges with romantic rural charm. Enjoy the peace and quiet, and play in one of the most beautiful regions of Germany.
Two challenging 18-hole championship courses, and one 9-hole short-game course await, which blend perfectly into the idyllic surroundings. Natural obstacles like ponds, cultivated areas and flower-dotted meadows offer ample opportunity for competitive gameplay situations. Each course is full of surprises - the signature of renowned golf course architect Kurt Rossknecht, who created the layout concept for the resort.
When it comes to deciding where you would like to stay, you can choose an accommodation directly adjacent to the new golf course, or in one of the neighbouring Hörnerdörfer villages. Wellness hotels, cosy bed & breakfasts, and 3, 4, and 5 star holiday apartments cater to every taste and budget.
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@alastor To my knowledge the only ones who will have "early" access to the cloaked demo will be those who have the SCC disk. At some time after (rumors of 1 day to 1 week only rumors so don't be spreading them), all those who have a key or code from Gamestop or other retail shops will have access. I do not know how long after nor do I know if you got a key directly from Ubi. I am sure we will get more instructions as we closer to the date.
I got my beta key from gamestop for pre ordering the game. It said go to www.gamestop.com/code to see how to redeem the code. I went through the process and from the Uplay website, it said that my beta key would be emailed to me. Still have not recieved it.


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AlienVault® Unified Security Management™ (USM™) is an innovative approach to security monitoring, delivered in a unified platform. The USM platform includes five essential security capabilities that provide resource-constrained organizations with everything they need for effective threat detection, incident response, and compliance management, in a single pane of glass.Advertisement
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Boards Client/Server
Boards is a commonly referred to as an Digital Asset Management Server and is intended to acquire, process, manage and distribute multimedia content.
JPEG Image repair tool
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Java Cave Maps
This project will be a project that will be used by Speleologist (cavers) to help aid in the mapping of the underground world. It will be simple at first.. only allowing the entry of survey (dist/azmith/V-angle) and give back a line plot..
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QTed (Questions and Tests editor) is a web-based editor that allows to create items and tests in agreement with standard IMS - QTI v2.1. and to maintain a personal repository (of items and tests) for each user.
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Java library of chart. It supports 2d and 3d visualization of line charts,pie charts,bar charts etc.
Global survey system


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break out in a sweat
Definition of break out in a sweat
: to begin to sweat I broke out in a sweat as I struggled up the hill.
Word by Word Definitions
: to separate into parts with suddenness or violence
: fracture
: to fracture a bone of (a bodily part)
: in a direction away from the inside or center
: outside
: from among others
: eject, oust
: to identify publicly as being such secretly
: to identify as being a closet homosexual
: used as a function word to indicate an outward movement
: situated outside : external
: out-of-bounds
: situated at a distance : outlying
: outside
: one who is out of office or power or on the outside
: an act or instance of putting a player out or of being put out in baseball
: to excrete moisture in visible quantities through the openings of the sweat glands : perspire
: to labor or exert oneself so as to cause perspiration
: to emit or exude moisture
: hard work : drudgery
: the fluid excreted from the sweat glands of the skin : perspiration
: moisture issuing from or gathering in drops on a surface
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NOAH Summer Festival 2015 was a Professional wrestling event produced by Pro Wrestling NOAH, which took place on August 16, 2015 at the Differ Ariake in Tokyo.
- Numbers in parentheses indicate the length of the match.
- (c) refers to the champion(s) heading into the match.
- Super Crazy vs. Taiji Ishimori - Time Limit Draw (10:00)
- Quiet Storm defeated Genba Hirayanagi (6:41)
- Cho Kibou-Gun (Hajime Ohara & Kenou) defeated Atsushi Kotoge & Hitoshi Kumano (11:10)
- No Mercy (Daisuke Harada & Takashi Sugiura) defeated Cho Kibou-Gun (Maybach Taniguchi & Mitsuhiro Kitamiya) (9:53)


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One would think that Barack Obama would have at least some chance at a decent birthday, which is this upcoming Sunday. The type where he would relax with his family, probably out in Hawaii, enjoy the surf and random press photos. It would make sense to believe this anyway: August is usually a quiet month in the West, given its late summer and a general lull has settled in. Some countries, like France, use this period to take the entire month off. Most people go on vacation around this time. It is not unusual to suggest that President Obama take it easy on this particular birthday weekend.
Unfortunately, that will not be the case for President Obama this birthday. The State Department today announced a travel alert worldwide on news of increased militant activity. The activity and communications increased followed Eid al-Fitr, the end of the Muslim fasting month of Ramadan, which occured Wednesday. Consequently, the State Department will be closing the American embassies in Libya, Iraq, Israel, and other Middle Eastern countries on Sunday. The travel alert also indicates unsafe travel for Americans in key areas of the Middle East, northern Africa, and central Asia over the weekend, based on what the State Department deems a terrorist threat likely to be enacted Sunday. While the State Department declined to indicate the source of the threat, House Foreign Affairs Committee Chairman Ed Royce (R-CA) told CNN the source was al-Qaeda based from groups in the Middle East and central Asia.
While the situation may turn out to be nothing, the State Department still issued the alert out of concern for fellow travelers. It also did so in the aftermath of an attack on a diplomatic mission in Benghazi, Libya last year that killed the American ambassador there and three others. Still, this has got to be an annoying birthday for President Obama, especially given what he has had to contend with in recent days, especially with leaks from the NSA regarding terrorism-related spying practices and a tepid jobs report. In any event, expect a quiet, if slightly tedious, weekend if you're traveling to those places, especially if you're going on a vacation yourself.


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Where to Eat in Los Angeles Right Now
Josh Ozersky claimed the only person he trusted for L.A. restaurant recommendations was Veronica Rogov, who was then a server at Chi Spacca. Now she's our authority on the Los Angeles dining scene.
Broken Spanish
Ray Garcia's food is unique but comfortable, and the service is soulful but efficient. Michael Lay, one of the best beverage directors in the city, designed the cocktail program. 1050 South Flower Street; 213-749-1460
Nicole Rucker's Doughnuts at Cofax Coffee
Rucker just became a partner at Cofax Coffee, where she collaborates with local tastemakers, like Compartés chocolate, to capture L.A. 440 North Fairfax Avenue; 323-424-7485
At Travis Lett's new spot, an army of cooks makes whipped burrata and rabbit-pheasant terrine and oil-cured anchovies and just incredible porchetta—it's an orchestra of delectables. 320 Sunset Avenue; 310-314-0320


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Cuyahoga County Looks At Renewing Sin Tax For Cleveland Stadiums
Ohio's most populous county is considering whether to put a sin tax renewal before voters to benefit three Cleveland stadiums.
The Cuyahoga County Council has until Feb. 5 to put a 20-year renewal of the tax on alcohol and cigarettes on the May 6 ballot. Otherwise, the tax will expire in 2015.
The council began hearing arguments on both sides at packed meetings this week and could come to a vote as soon as Tuesday.
The News-Herald of Willoughby reports that renewing the tax could produce a projected $260 million over 20 years.
The county first approved of the tax in 1990, drawing just more than $350 million for construction and maintenance of the stadiums for the Cleveland Indians, Cavaliers and Browns.
©2017 by The Associated Press. All rights reserved. This material may not be published, broadcast, rewritten or redistributed.


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Definition of unpleased
: not happy or satisfied : not pleased unpleased with the children's behavior … was not unpleased to see how well his charms worked. — Herman Melville
First Known Use of unpleased
15th century
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LONDON ISSUE 295 Sep 2011
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Chevelle + All That Remains Lead New Additions for 2015 Loudwire Music Festival
Chevelle and All That Remains lead the latest wave of acts joining the 2015 Loudwire Music Festival.
Chevelle and All That Remains lead the latest wave of acts joining the 2015 Loudwire Music Festival.
If you missed it earlier this week, here are some pictures of All That Remains in concert at Club Patron in Odessa.
Phil Labonte never shies away from expressing his political and social views, so we gave him yet another forum to speak on a number of topics.
As the Feb. 24 release of All That Remains' new album 'The Order of Things' draws closer, the band has unveiled the track 'Tru-Kvlt-Metal.'
All That Remains rock amongst the ruins in the 'This Probably Won't End Well' video.
Robb Flynn certainly does not agree with the All That Remains singer's viewpoint, calling Labonte "ignorant."
All That Remains frontman Phil Labonte is no stranger to ruffling feathers, but the vocalist is still dealing with the backlash of his use of homophobic slurs.
All That Remains are back with their brand new single 'This Probably Won't End Well.'
All That Remains frontman Philip Labonte explains his harsh critique of the band's 'A War You Cannot Win' album and shares his hopes for their upcoming disc.
In Flames will return stateside in 2015 with a North American trek that also includes All That Remains and Wovenwar.


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Child Malnutrition
Malnutrition is central to many of the health issues in Bangladesh, as well as in many developing countries, for instance low birth weight, poor growth, slow cognitive development and susceptibility to infections.
But what is malnutrition?
"Malnutrition" is a condition that results from an inadequate or excessive intake of nutrients. Obesity caused by excessive intake of refined carbohydrates and fat, is also a form of malnutrition which is more common in industrialised countries, but recent studies have shown that this 'western' problem is now seeping into the developing nations leading to the situation of a "double burden" of under- and over-nutrition. [1]
There are 2 types of malnutrition:
- Primary malnutrition - occurs in parts of the world wherein most of its population is undernourished because of famine, poverty and limited crop production. This affects mostly children in underdeveloped countries.
- Secondary malnutrition is the result of an underlying disease which includes: eating disorder like anorexia or bulimia, cancer etc which can eventually cause malnutrition because of the inability to digest food or absorb the nutrients derived from food. People who have an alcohol or drug addiction may lose interest in food, which can lead to severe malnutrition.
From 2003 data the World undernourished situation is alarming, as evident from the map below [2]:
General Statistics [3]:
In March 2009, Unicef made their year long observation and stats public and stated that 10million more children around the globe face starvation because of the global financial meltdown, with 400,000 expected to die this year (2009). Unicef goes on to say that up to 2.7 million youngsters are acutely malnourished and nine times more likely to die in Africa. Whilst in South Asia, up to 4.7million are suffering.
- According to the Global Hunger Index, South Asia has the highest child malnutrition rate.
- The 2006 report mentioned that "the low status of women in South Asian countries and their lack of nutritional knowledge are important determinants of high prevalence of underweight children in the region" and was concerned that South Asia has "inadequate feeding and caring practices for young children".
- Half of children in India are underweight, one of the highest rates in the world and nearly double the rate of Sub-Saharan Africa.
- Bangladesh ranks 3rd in the countries with the highest number of undernourished people.
- One child dies every 5 seconds as a result of hunger - 700 every hour - 16 000 each day - 6 million each year - 60% of all child deaths. [4]
This problem is especially prevalent amongst children who are most vulnerable to diseases and adverse environmental conditions, with no assistance or guidance on how to maintain a well-balanced diet or even the guarantee of seeing their next source of sustenance: the street children.
- Street dwellers are often exposed to, and have easy access to non-prescriptive drugs, such as alcohol, tobacco, sniffing glue/petrol. Although alcohol has calories and provides energy it can also prevent the absorption of necessary vitamins and other nutrients. In general, a person taking substances can easily overlook the importance of good nutrition. Street children use substances to relieve hunger and this can lead to malnutrition. [5]
- And though some street children may get enough to eat, they do not have nutritious diets. Which inevitably leads to malnutrition, anaemia and vitamin deficiencies.
- Street children experience many common diseases such as tuberculosis, chest and respiratory infections, typhoid/cholera and dental problems largely due to the lack of personal hygiene and access to clean food and water. [6]
Below is a snapshot on the issue of malnourishment around the world, something that is still clearly the most important Millennium Development Goal that needs to be met.
Calcutta City, India: Results revealed that 69.4% of pavement-dwelling children are undernourished and that 16% of them suffer from severe malnutrition. Prevalence of severe grade malnutrition was noted to be 3 times higher in females. Moreover, children of illiterate parents and non-working mothers had a higher incidence of severe Protein Energy Malnutrition (PEM). [7]
Manila, Philippines: About 30% of Manila's 50,000-75,000 street children are estimated to be moderately or severely malnourished. According to international standards, both male and female respondents are seriously underweight and under-height. About half the street children reported regular drug use, primarily glue sniffing. [8]
Jakarta, Indonesia: Children scavenge food, i.e. at train stations, where some of them stroll through train cars looking for unfinished rice packs from travellers, and from little restaurants around the market, where the children watched for customers who had not finished their meals. Some owners of these food stalls call the children. However, most owners chased the children away. When the children purchased food, they usually buy their meals from the numerous food stalls along the roads or from food pedlars. The staple food of the children is, as everywhere in Indonesia, rice. This is usually consumed with different kinds of oily sauces and local vegetables. The consumption of fish and meat is normally rare, as it is expensive. "...more children were too small for their age ... 52% of the children suffered from stunting" caused by malnutrition at birth. [9]
Eldoret, Kenya: The malnutrition rates are high with 31.1% and 41.9% of the children being stunted and underweight, respectively. Abandoned children staying in shelters have the highest rate of malnutrition with 51.8% and 64.3% being stunted and underweight. Street children have a higher incidence of childhood diseases than their counterparts. [10]
Guatemala: As one might expects, street children suffer profoundly and face enormous economic, political and social challenges. In addition to economic poverty, which often leads to malnutrition and even starvation, these children are exploited and victimised by their own governments, usually by a police force. [11]
Dhaka, Bangladesh: About 73% of the street children in the Dhaka city suffer from chronic malnutrition while mortality and morbidity status among the street dwellers has reached an alarming level due to lack of basic healthcare services. This was revealed at a seminar organised in Dhaka by ICDDR,B [13] to release the findings of a study on 'Health Needs and Health Seeking Behaviours of Street Dwellers in Dhaka City'. [12] In Bangladesh 45% of newborn infants weigh less than 2500 grams; this proportion is the highest in the world. The high incidence of low birth weight is viewed as a major obstacle to improving child health and development. Because so many children are small, PEM is not recognised as abnormal. Similarly, healthcare providers do not recognise children as malnourished when they come for treatment of common illnesses. [14] In Dhaka, Bangladesh only few of the street dwellers are aware of the existence of government health facilities or Extended Programme on Immunization (EPI). The ICDDR,B study showed an alarmingly 69% of men taking drugs daily in addition to sharing syringes and needles among injecting drug users. [13]
Actions to be taken by governments and humanitarian organisations:
- Free and accessible health services should be made available for street children to help curb the incidence of common diseases.
- Introduction of 'static clinics' at identified locations reinforced by mobile outreach sites in evening hours in order to make the available health services accessible.
- Street children should be given free access to cleaning facilities (bathrooms and toilets) to reduce incidence of preventable diseases.
- Introducing nutrition programmes in existing health care services for street children. To inform the street dwellers about the existing health services, drug vendors can be explored as potential advocates to increase awareness among street dwellers.
- Improving child feeding including increasing rates of exclusive breastfeeding and appropriate and adequate complementary feeding.
- Provision of vocational training and micro-loans for street children to break the cycle of poverty.
- Provision of free psycho-social counselling and drug-rehabilitation services for street children.
- Rigorous lobbying of government to implement street children friendly health policies and to commit to allocating budgetary resources.
- Food-based approaches to combat the double burden among the poor: challenges in the Asian context. Asia Pac J Clin Nutr. 2008;17 Suppl 1:111-5.
- Percentage population undernourished world map
- 2008 Global Hunger Index Key Findings & Facts
- Human Rights Council
- WHO. Working With Street Children
- WHO. Working With Street Children
- Nutritional status of pavement dweller children of Calcutta City. Indian J Public Health. 1999 Jan-Mar;43(1):49-54
- Social correlates of malnutrition among Filipino street children. Conn Med. 1993 Jun;57(6):373-6
- Height and weight as a reflection of the nutritional situation of school-aged children working and living in the streets of Jakarta. Gross et al. 1996. Soc. Sci. Med. Vol. 43. No. 4, pp. 453-458
- Health problems of street children in Eldoret, Kenya. East Afr Med J. 2001 Dec;78(12):624-9
- Morales Case Focused International Attention on Plight, Rights of Street Children
- 73pc street children face chronic malnutrition: study
- ICDDR, B: Dissemination Seminar on: Health Needs and Health Seeking Behaviours of Street Dwellers in Dhaka City
- ICDDR,B. Nutrition Research


text:
Discover How 4,781
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World of Warcraft
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Bedford's Republican Party headquarters is open for business on North Bridge Street in Bedford. The site they chose is in the store front most recently occupied by Ranch Road. According to Nate Boyer, chairman of the Bedford Republican Committee, the site was chosen because its Centertown location gives them better exposure to foot traffic. This was especially important as they opened in time for Centerfest and this location placed them in the middle of the festival area. Boyer said that it also gives them space for larger events.
The turnout was less than what local officials had hoped for, but those attending last Thursday's meeting on reversion in Bedford City Council Chambers had ample opportunity to get their questions answered.
"The city is not in economic distress," Bedford City Manager Charles Kolakowski said about Bedford's decision to revert from a city to a town. "We've looked at this as a positive step for both parties."
At its last meeting in August, the Bedford County School Board voted unanimously to amend the school division's staffing standards to add an additional teacher to each school with less than 250 students. This will result in hiring seven additional teachers.
In order to pay for that, the school board had to ask the board of supervisors to move $300,000 from the school maintenance category to the operations category to cover the cost. The supervisors unanimously approved that transfer at their regular meeting Monday night.
Sometimes you just have to play the hand you're dealt.
That was certainly the case in Bedford County Circuit Court last week for Commonwealth's Attorney Randy Krantz who had two different defendants—claiming two different accounts about the disposal of the body of Sean Placko—sentenced for the same crime.
While some county agricultural operations got hammered by dry weather this summer, orchards have been an exception.
By Congressman Robert Hurt
Just days ago President Obama addressed a joint session of Congress to lay out his plan to get Americans back to work and following that speech he sent his legislative proposal to Congress. I appreciate the President's willingness to address this critical situation and like the President I am committed to finding meaningful ways to deal with our nation's unemployment crisis. However, we cannot tax and spend our way out of this crisis.
By District 2 Supervisor Chuck Neudorfer
I will be holding my quarterly Town Meeting at the Moneta Library on Saturday morning, Sept. 24, from 10 to 11:30. There are a lot of topics we can discuss.
Campaign signs have popped up everywhere in the Bedford, Roanoke and Lynchburg areas as state legislative races begin to come into focus. Or at least we hope so.
It's a shame that the public waits until the last minute to pay attention to Virginia General Assembly campaigns. Then when it finally does, not enough people actually vote.
Tensions in the relations between Turkey and Israel, which have been increasingly sour over the past six years, took a turn for the worse last month after the United Nations affirmed Israel's right to defend itself by maintaining a blockade on Hamas-controlled Gaza.
Copyright © 2017
The Bedford Bulletin is your source for local news, sports, events, and information in Bedford County and Bedford, VA and the surrounding area.


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can I use amazing mld putty to make beer bottles (mold)?
Okay so for the past year there have been posts about Amazing Mold Putty in this forum and I haven't responded because I have thought that for some people this is a readily available product in their area and if they like it great. But now it seems that this forum is becoming a place where the virtues of Amazing Mold Putty are repeatedly discussed. Though I may be making a mistake responding, I am going to give it a try.
I would like to create an analogy that may be useful in communicating my thoughts. Okay so lets imagine that you have your own cake business and down the road there is a high volume retail baking operation that competes with you for the wedding cake business in your area. You are known for quality cakes and they are known for cheap cakes. You explain to your customers that you use high quality ingredients and that your cake tastes better, looks better and impresses your friends and family in a way that cakes from the other operation cannot. The high volume retail bakery on the other hand can bang out a cake very quickly, it will be iced with buttercream from a bucket and the decorations are mass produced and so hard they will break your teeth. And it is CHEAP. Now we all know that no matter how much you promote your quality some customers will go for the cheap cakes and some customers will choose yours. The similarities and differences between Amazing Mold Putty and Silicone Plastique are as obvious as the differences described above between the cakes made by two different operations.
Let me identify the differences between Silicone Plastique and Amazing Mold Putty. Silicone Plastique is a stronger silicone that will make a mold that will last longer and be more resistant to tearing. Silicone Plastique has less oil in its formulation which makes it better for chocolate and sugar casting. Silicone Plastique gives you more time to make your mold so that you can take your time and work the material into every detail. Silicone Plastique is also firmer which makes it impress details into gumpaste and fondant that are sharper and more defined.
Amazing Mold Putty on the other hand is cheap and it will work for small projects that are not too big.
Okay so lets go back to the analogy I was describing before between your high quality cake business and the cheap high volume retail bakery down the street. How would you feel if during a consultation with one of your customers, someone barged into your shop and tried to convince your future customer to save money and get a cheap cake down the street. I am sure this is a situation you would like to avoid, yet some of the posts in this forum recommending Amazing Mold Putty have the same effect as the scenario I just described. You might feel that this forum is about mold making in general and since Amazing Mold Putty can be used to make molds it is fair game. In actuality this is a vendor forum where questions can be answered about MYOM products as they pertain to mold making. Here is how Jackie (Founder and President of CC introduced the MYOM forum back in November 2009.
Jackie Wrote, "Cake Central is excited to announce our partnership with Chef Dominic Palazzolo and his company www.MakeYourOwnMolds.com
MakeYourOwnMolds.com is your source for food grade silicone mold making materials that are safe and particularly well suited for creating food molds. These unique silicone mold rubber compounds have been engineered to comply with FDA regulations and designed to be easy to use. Because of the unique nature of the product and its endless possibilities, Chef Palazzolo will be making his team available on the Cake Central forums to help our members learn the process of mold making and answer any questions they may have about their products and their applications.
Additionally Chef Palazzolo will be writing a blog on Cake Central where he will share tutorial videos and various projects that can be accomplished with the MakeYourOwnMolds products.
MakeYourOwnMolds.com will also be contributing a percentage of all sales generated via Cake Central to help keep CakeCentral.com freely available."
This last sentence is particularly important because it describes a relationship that exists between MYOM and CC where purchases generated from this forum directly benefit Cake Central with financial contributions.
In closing, I hope you understand when I ask that recommendations for Amazing Mold Putty not be made in this forum and instead those recommendations can be taken and posted to the Non-Vendor forums that cover all things cake.
Thank You for Your Consideration,


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Funk Keyboards - The Complete Method
By Johnson Gail
A Contemporary Guide to Chords, Rhythms, and Licks. Musicians Institute Press. Funk and Instructional. Instructional book (softcover) and accompaniment CD. With chord names, introductory text, instructional text and illustrations. 56 pages. Published by Musicians Institute Press (HL.695336).
Item Number: HL.695336
ISBN 0793598702. 9x12 inches.
Explore the chords, rhythms, and techniques used by the greatest funk keyboardists! Subjects covered include: common chords and progressions; classic funk rhythms, licks and patterns; synth bass & multiple keyboard playing; and pitch wheel and modulation. The accompanying CD includes 81 full-band tracks.


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Here's the group form: Blue Care Network Primary Care Physician Selection form - Group (PDF)
What you'll need:
- A printer to print the form
- An envelope and postage to mail the form, or a fax machine. Each form includes instructions, a mailing address and a fax number.
Step by step instructions:
- Select your primary care physician. If you need help finding one, use bcbsm.com to find a list of doctors that participate in Blue Care Network.
- Fill out the form completely. Check with your employer to find out if you should return the form to them first before mailing it to us.
- Complete the form, sign and return it to us along with your Subscriber New Enrollment form (PDF). Return this form as soon as you enroll so that we can notify your doctor of your membership.
Fax or mail the form to:
|Membership and Billing – M.C. C411
Blue Care Network
P.O. Box 5043
Southfield, MI 48086
|Fax: 1-877-218-1466
If you have any questions, please contact your employer.


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Posts tagged "bethesda"
New Wolfenstein II trailer is all about trusting "Big Brother"
"Trust In Big Brother"Read More
Wolfenstein 2: The New Colossus gets season pass with three expansions
The Season Pass includes three expansions, each based on a different character.Read More
Doom getting major patch alongside free weekend
Bethesda is overhauling the game's multiplayer, and you can try it all out for free.Read More
God of War to Resident Evil 7: All the big games revealed at E3 2016
With all the major press conferences completed, we now have a complete list of all the new games that were announced at E3 2016, and here they are with trailers/gameplay.Read More
Review: Doom
Doom is a return to form for id software after the lackluster Doom 3 and one that brings to the table an amazing campaign that is worthy of the Doom name.Read More
Dishonored 2 set for Novermber 11 release, gameplay debut at E3
A full gameplay reveal is planned for Bethesda's E3 2016 showcase on June 13, which will be streamed live via Twitch and YouTube.Read More
Dishonored: Definitive Edition up for pre-order
The Definitive Edition will bundle all DLC that's been released for the game so far along with updated visuals for the new consoles.Read More
Battlecry: Bethesda's online action combat title enters global beta
The free-to-play game has been in beta for Australia and New Zealand for a while and is now entering global beta, with a full release later this year.Read More
E3 2015: Bethesda shows the rest how it's done
Bethesda's press conference was a success largely because it had some great games to show, but also because it was an example of great time management.Read More
Dishonored 2 releases spring 2016, Dishonored Definitive Edition coming to PS4, Xbox One
Dishonored 2 will be set 15 years after the first game in the city of Karnaca, and will feature two playable characters – Corvo Attano and Emily Kaldwin.Read More
Doom single, multiplayer revealed at E3, user creation tools detailed
The reboot is staying true to the franchise, with fast movement, lots of big powerful weapons, and plenty of demons to shoot in the face.Read More
Fallout 4 releases 10th November, Fallout Shelter for iOS out now
Fallout 4 has been in development since the release of Fallout 3 in 2008, and features several firsts for the storied franchise.Read More
Video: Bethesda confirms Fallout 4, and this one will make it to India
Fallout 4 is expected to release in India, after the release of Fallout 3 was bizarrely cancelled by Microsoft, citing the "cultural sensitivities in India".Read More
Bethesda releases Doom teaser ahead of E3 reveal
The teaser does not reveal a lot aside from the fact that the game will get a gameplay reveal at Bethesda's first ever E3 press conference next month.Read More
Valve axes paid mods from Steam Workshop, offers full refunds
Valve admitted to having made a mistake and not fully understanding the requirements of the community, and is offering refunds to anyone who paid for mods.Read More
Review: Wolfenstein: The New Order
The New Order is a fun ride if you can stomach the cost of admission and quirks that go along with it. It's refreshingly old school, much like the choice of vinyl over MP3.Read More
Bethesda announces Battlecry, a free-to-play, third-person multiplayer combat game
Battlecry is all about gory, third-person combat, except that the game has almost no guns, instead focussing on melee combat and 'transformative' long-ranged weapons.Read More
The Evil Within gets August release date
The new survival-horror game from Resident Evil creator Shinji Mikami.Read More
The Elder Scrolls Online priced for India
The PC version releases on 4th April at Rs 3,499, with the PS4 version to follow in June at Rs 4,799.Read More
The Elder Scrolls V: Skyrim gets Legendary Edition
Bethesda announces the definitive version of Skyrim for the Xbox 360, PS3 and PC.Read More
Rage 2 cancelled, Doom 4 being reworked for next-gen consoles
id Software to rebuild Doom 4 from scratch for next gen consoles. Read More
Bethesda announces Hearthfire add-on for Skyrim
Ever wanted to build your own house in Skyrim? If so, Hearthfire could be the add-on for you. Read More
Dawnguard out now for PC, delayed for PS3
Bethesda not satisfied with Dawnguard's performance on PS3.Read More
Review: The Elder Scolls V Skyrim: Dawnguard
At twice the price of Bethesda's other DLC releases, Dawnguard does give you a lot to see and do, but it ends up being redundant and unnecessary.Read More


text: I'm 23. When I was 18 and first started doing my own grocery shopping, it was easy to find ground beef for sale for around $2.50 a pound, or sometimes as low as $1.99. Now the prices are between $3.50-$4.00 most weeks. I have always been a fan of chicken and my local grocery stores have boneless skinless chicken breasts for $1.99 a pound on a regular basis. I didn't have many meal ideas to use with chicken so I … Read the rest


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DENNIS TOWNSHIP – A new café has opened in the township for fourth-eighth grade students serving up hot chocolate and career advice in the STEM fields, school officials said.
The Dennis Township Career Café got its start in November with Monica DiVito, guidance counselor, working alongside Julie Factor, media specialist, to help students take an online career assessment to help them find potential career fields to explore.
According to a statement, a "virtual job shadow" computer program also allows students to explore and understand different careers or career paths by following a day in the life of a professional.
DiVito says she and Factor are planning follow-up activities in the Career Cafe that will focus on specific "career clusters," as well as preparing for job interviews and writing resumes for seventh and eighth grade students.
According to the federal Department of Education, there are 16 career clusters that link to more than 70 specific career pathways. Within those pathways, the DOE defines another 1,800 career specialties.
Career clusters include: agriculture, food and natural resources; architecture and construction; arts, audio visual technology and communications; business management and administration; education and training; finance; government and public administration; health science; hospitality and tourism; human services; information technology; law, public safety, corrections and security; manufacturing; marketing; science, technology, engineering and mathematics; and transportation, distribution and logistics.
School officials said that the Career Cafe is a prelude to the planned Feb. 24 Dennis Township School Job Fair. They said that the next cafe is set Dec 6, and will focus on careers in the medical field.


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Words starting with roam, the most common words first
6 words found.
The most frequently occuring words starting with roam
The number indicates the approximate number of times the word occurs per million wordsroam (7)
roaming (2)
roamed (1)
There are 3 less common words:roamer
List all words starting with roam: sorted by length, sorted alphabetically.
Some random words: twa hwan ceviche ka eosin shy soever
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