text:
Whole-genome analysis for early infant epilepsy
September 4, 2018 by dkoboldt Leave a Comment
Early infantile epileptic encephalopathy (EIEE) is a devastating syndrome of intractable seizures that strike in the first months of life. According to Orphanet, it affects 1 in 50,000-100,000 births. Infants with EIEE may suffer hundreds of tonic spasms per day, both during sleep and wakefulness. The prognosis is not good. Most patients die within two years; those who survive are severely impaired.
Most cases of EIEE arise sporadically (i.e. without a family history), though autosomal recessive inheritance has also been reported. Clinical diagnosis can be challenging, since infantile epilepsy is also associated with certain brain malformations and metabolic syndromes. Genetic testing for EIEE is made challenging by the fact that upwards of 50 genes have been associated with the disorder (current gene panels test ~50-130 genes). Even so, 40% of patients tested by gene panel or exome sequencing fail to achieve a diagnosis.
Earlier this month, a group at the University of Utah published a study demonstrating the power of whole-genome sequencing as a diagnostic tool for EIEE. Their cohort comprised 14 EIEE patients (recruited in 2015-2016) who had undergone extensive prior testing without receiving a diagnosis. The probands were tested, along with their parents, by deep whole genome sequencing (~65x coverage) and comprehensive variant analysis.
Comprehensive Whole Genome Analysis
One showpiece of this study is the bioinformatics methodology the authors employed to maximize their chances of detecting causal variants. They applied well-established tools to call SNVs/indels detector (GATK) and SVs/CNVs (LUMPY/SVtyper). Because they were particularly interested in de novo mutations — present in the child but not either parent — they also utilized a reference-free k-mer analysis algorithm called RUFUS to reveal sequences present in the proband that were not found in either parent, suggestive of a de novo event.
This is important because most of the apparent de novo mutations identified by routine variant analysis tools (e.g. GATK) are not real: they're either alignment artifacts in the child, or variants that were missed in one of the parents. By performing a k-mer analysis, you avoid the former. It's a cool idea, really. Wish I had thought of it myself.
The authors prioritized variants using what one might call the "lowest hanging fruit" strategy. They only looked at de novo events, and their search tiers were as follows:
Nonsynonymous coding mutations in known or candidate EIEE-associated genes
SVs predicted to disrupt EIEE-associated genes
Nonsynonymous coding mutations in genes not associated with EIEE
SVs affecting genes not associated with EIEE
De Novo Mutations in Infant Epilepsy Genes
Notably, the first tier search returned a diagnosis — a pathogenic/likely pathogenic mutation in an EIEE-associated gene — for 10/14 subjects (71%). That's close to the expected diagnostic rate, and thus not terribly surprising. What is a bit surprising is that three of these subjects reportedly had negative epilepsy gene panels prior to enrollment, which raises the question of how such obvious pathogenic mutations were missed.
One case was solved in search tier 2, when the proband was found to harbor a de novo 63-kb duplication within CDKL5. The duplication was predicted to cause a frameshift and early stop, and it was on the X-chromosome (so hemizygous) in a male patient.
Mutations in Strong Candidate Genes
Two cases were solved in search tier 3. One harbored a mutation in DEAF1 (associated with dominant intellectual disability and recessive epilepsy). The variant (p.G212S) lies in the SAND domain, in which virtually all pathogenic / likely pathogenic missense variants in ClinVar reside:
ClinVar P/LP missense (blue), nonsense (red), and inframe (light blue) variants in DEAF1.
As you may have noticed from the above, the p.G212S variant is already in ClinVar, as it was reported in a different patient with dominant intellectual disability and seizures.
Another harbored a missense mutation in CAMK2G, which the authors propose as a novel EIEE gene. CAMK2G encodes the gamma subunit of the calcium/calmodulin-dependent protein kinase II complex, which plays an essential role in synaptic function. The missense variant is predicted to be damaging by about half of in silico algorithms (our lab would not apply PP3 here, but this doesn't change the interpretation). There is one reported heterozygote for this variant in gnomAD, but read data are not available and in my opinion that's within the margin of error. Missense variants in CAMK2G appear to be under constraint. Overall, a pretty good candidate.
A de novo balanced translocation
So now there's only one case to solve, and for that one the authors plunge into search tier 4 (SVs not affecting EIEE genes), also called the bucket of crap. I have spent countless hours running down candidates in tier 4 for our rare disease cases with little to show for it. Unlike me, however, the authors of this study actually found something that may be relevant: a de novo balanced translocation between chromosome 2 (2p16.1) and the X-chromosome (Xq28). A 92-gene segment from chromosome X thus ends up on chromosome 2, where the authors speculate that X-inactivation may be disrupted, altering their transcription.
Translocation in patient 2 (Ostrander et al, NJP 2018)
One of the translocated genes is MECP2, which also harbored a de novo noncoding mutation in this patient. Mutations in MECP2 can cause Rett syndrome, an X-linked dominant disorder that manifests with a rather specific phenotype. From OMIM:
Rett syndrome is a neurodevelopmental disorder that occurs almost exclusively in females. It is characterized by arrested development between 6 and 18 months of age, regression of acquired skills, loss of speech, stereotypic movements (classically of the hands), microcephaly, seizures, and mental retardation.
Rett, a Viennese pediatrician, first described Rett syndrome after observing 2 girls who exhibited the same unusual behavior who happened to be seated next to each other in the waiting room.
The patient in this study had microcephaly, seizures, and global delay, which are all prominent features of the Rett phenotype. I'd be very curious to learn if she also develops the characteristic hand movements. While not definitive, the fact that these alterations are de novo, and affect multiple highly relevant genes, suggests that these are somehow responsible for the EIEE phenotype.
Molecular Diagnosis versus Candidate Gene
In the abstract, Ostrander et al write that "the detection of a pathogenic or likely pathogenic mutation in all 14 subjects demonstrates the utility of WGA." If I wanted to nitpick, I'd argue that they have P/LP variants in 12 patients, since CAMK2G is not yet established as an EIEE gene and the causal nature of the chr2-chrX translocation in patient 2 is not established. More accurately, the authors have uncovered a P/LP variant or strong candidate variant in all 14 patients.
The cohort in this study does not entirely represent clinical reality, particularly because only subjects for which both parents were willing and available to participate were included. That's an ideal setup if one wants to pursue only de novo mutations. A patient with only one available parent (or no available parents) is a common situation in genetics clinic and would present considerable challenges to this type of analysis.
Whole Genome Sequencing Cost and Speed
Most geneticists recognize the power of WGS to comprehensively detect genomic variants, and to return those results relatively quickly. The cost of WGS, however, remains a significant barrier. Clinical WGS currently costs around $15,000 per trio, roughly twice the cost of clinical exome sequencing. Yet, as Ostrander et al point out, the patients in this cohort underwent a minimum of 24 diagnostic tests, costing (on average) $30,866 per patient.
Perhaps just as importantly, at least three of the patients in this study had undergone gene panel and/or exome sequencing which failed to detect a rather obvious pathogenic mutation. The authors did not explore this further, at least in the paper. Yet it emphasizes that WGS may provide faster and more comprehensive results as a diagnostic tool.
Filed Under: Clinical Sequencing, Rare Diseases
Sequencing or Array Testing for Genetic Diseases?
July 19, 2018 by dkoboldt Leave a Comment
When a child is born with a suspected genetic condition, an increasing number of tools are available to the clinician: newborn screening panels, metabolic testing, cytogenetic testing, gene panels for certain conditions… and more recently, comprehensive sequencing of the exome (i.e. all protein-coding genes) or the genome. Yet chromosomal microarray (CMA) is often the frontline diagnostic tool.
In fact, many clinical laboratories offering clinical exome sequencing require or strongly recommend that an array be performed. There are good reasons for this. CMA testing:
Has a faster turnaround time than WES
Costs less than WES/WGS
Diagnoses (or excludes) many prevalent genetic conditions such as Down syndrome, 22q (deletion) syndrome, Smith-Magenis syndrome, etc.
Many clinical testing labs actually require that microarray testing to have been performed (when appropriate) prior to exome sequencing. There's sound reasoning behind this: large-scale chromosomal aberrations should generally be ruled out before one goes looking for small variants that might cause syndromic changes.
Will Sequencing Supplant Arrays for Testing?
As the cost and turnaround time of sequencing-based tests improve, there's a growing interest in ordering these tests either in parallel or instead of chromosomal microarray. Some of that interest comes from Illumina, whose representatives have been touting whole-genome sequencing as the "one test to rule them all" for the diagnosis of rare disorders.
Of course, Illumina has a monopoly on the next-gen sequencing market, so they have financial incentives for encouraging the switch to sequencing-based tests.
One requirement, if we move in that direction, is a solid understanding of the diagnostic and clinical utility of each test. In other words, it's useful to know how often array, exome, or genome sequencing tests provide a molecular diagnosis (diagnostic utility) and whether or not that changes patient care (clinical utility).
Diagnostic Success of Array, Exome, and WGS
A study published this month in NPJ Genomic Medicine examines the diagnostic success and clinical utility of whole genome sequencing (WGS), whole exome sequencing (WES), and chromosomal microarray (CMA) over the past five years (see the nice summary on GenomeWeb). Michelle Clark et al looked at 37 studies (published from 2013 to 2017) describing a total of 20,068 patients who underwent genetic testing by WGS, WES, and/or CMA.
Clark et al, NPJ 2018
The studies ranged in size from 22 to 1,745 probands, and the number was modestly correlated with diagnostic success: on average, an increase of 1,000 probands decreased diagnostic utility by 28% (see right). So large studies tend to have a lower diagnostic rate, but I should point out that there are a number of factors at play here. One obvious consideration is that it's easier to perform deeper analysis/interpretation on a small cohort than a large one. Another is that only a subset of laboratories have the capacity to test thousands of samples. More on that in a minute.
The Setting Effect: Hospitals versus Reference Labs
The studies in this meta-analysis took place in one of three settings:
Research studies to identify new disease genes or test novel methodology (8 studies totaling 1,885 probands; the majority coming from the DDD study)
Hospital-based sequencing and interpretation (19 studies totaling 1,597 probands)
Clinical testing by reference laboratories (8 studies totaling 7,065 probands)
The authors found that the diagnostic utility of hospital-based testing (0.42) was significantly higher than that of reference laboratories (0.29). They speculate that this, in view of the number of probands reported, may influence the inverse correlation between study size and diagnostic success. Consider some of the well-known studies in the "Reference Laboratory" category:
Baylor (2014): 1,745 probands, 26% diagnostic success
Ambry (2015): 417 probands, 31% diagnostic success
GeneDx (2016): 3,040 probands, 29% diagnostic success
Despite the lower-than-hospital diagnostic utility, reference laboratory publications are important for a number of reasons. First, they were large studies that helped set expectations in the medical and research communities about the expected "yield" of clinical WES. In essence, they're the basis of the widely-accepted figure that exome sequencing provides an answer in about 1/3 of cases.
Second, their underlying metrics provided guidance for groups who carried out their own sequencing and interpretation. For example, they highlighted the major contribution of de novo mutations, which account for ~45-50% of successful diagnoses. Third, these studies served as a form of accreditation for major commercial providers of clinical WES.
The Value of Trios
It's well known among sequencing laboratories that there's a higher chance of success when testing a trio (proband and both parents) than a proband alone (singleton). Trios provide valuable information, such as the origin of a variant, that factor into interpretation. The meta-analysis by Clark et al has put some numbers behind it: Across five studies totaling 3,613 children, the diagnostic rate for trios was double the diagnostic rate of singletons.
This supports the notion that, when possible, a full trio should be used for genetic testing. Some laboratories accomplish this by sequencing the proband and then testing parents for a limited number of promising variants. Other labs, like ours, prefer to sequence all three samples right off the bat.
It's good that we can quantify the benefit of testing a full family trio. That being said, we have to recognize that this will not always be possible in our modern world. Some children won't have both biological parents around, available, and willing to participate in genetic testing. That's just a fact, and we have to do our best to serve them even in non-ideal testing conditions.
Sequencing Versus Array
Now we come to the meat of it: comparing diagnostic and clinical utility between WES, WGS, and microarray. Only two studies compared the diagnostic rate between WES and WGS, and it was not significantly different. Thus, WES and WGS together were compared to microarray.
On average across 28 studies totaling 10,392 probands, the diagnostic rate by WES/WGS was 31%.
On average across 13 studies totaling 11,429 probands, the diagnostic rate by array was 11%.
That's the main answer, but we can dig deeper using studies of children that received both WES/WGS and array testing. There were four such studies that provided sufficient information to calculate the odds ratios, which revealed that the odds of a diagnosis by WGS/WES was 8.3 times greater than array.
Array Utility (Clark et al, NPJ 2018)
The authors also noticed a interesting trend over the five years of their meta-analysis: the odds of diagnosis increased 16% each year for WES/WGS but decreased 14% each year for array. They point out that the former is probably due to the maturation of sequencing-based tests over time. The latter, however, is initially surprising since array testing was already fairly mature in 2013.
It turns out that the decrease in array diagnostic utility is probably due to reimbursement. Once it started being routinely reimbursed by insurance, array testing got ordered much more often and the diagnostic utility decreased as a result. I expect we might see a similar effect as WES becomes the frequent go-to for clinicians.
It isn't necessarily a bad thing to have WES accepted and reimbursed as a frontline diagnostic tool, but we should be prepared to explain such possible outcomes.
Clinical Utility of WES, WGS, and Array
Last we come to the vital question of clinical utility, i.e. whether a patient's management is changed based on the results of molecular testing. Here there were differences between sequencing strategies: clinical utility was significantly higher for WGS (0.27) than for WES (0.17). Yet both of them had substantially better clinical utility than array (0.06).
Recommendations for Genetic Testing Practice
Under current guidelines, chromosomal microarray testing is the first-line genomic test for children with intellectual disability, developmental delay, autism spectrum disorder, and congenital anomalies. There are no guidelines yet established for WES/WGS testing.
It may be time for that to change.
Rare Disease Research Collaboration: RLIM in X-linked Intellectual Disability
May 7, 2018 by dkoboldt Leave a Comment
Access to samples is one of the main challenges of studying rare inherited conditions, particularly those whose molecular basis is unknown. Every individual harbors 3-5 million genetic variants compared to the human reference sequence. Even if we winnow these down to only rare variants likely to disrupt a protein-coding gene, hundreds still remain.
The Power of Multiple Patients
One of the most efficient ways to isolate a disease-causing variant is to search for genes that are disrupted in multiple patients. Better yet, genes disrupted in multiple unrelated patients. This is nicely illustrated by Miller syndrome, one of the first disorders whose gene was identified by exome sequencing in 2010. Here's a table I've adapted from that paper showing the number of candidate genes at each stage of the analysis:
Adapted from Ng et al, Nature 2010 (doi:10.1048/ng499)
The first family kindred had two affected siblings, each harboring several thousand nonsynonymous (NS), splice site (SS), or indel (I) variants in coding regions. Filtering against population allele frequency databases and looking at both siblings simultaneously winnowed this down to 237 candidate genes, which is a lot. However, incorporating data from unrelated kindreds 2 and 3 rapidly narrowed it down to less than ten genes, of which one was DHODH, the ultimate cause of disease.
Clearly, searching for recurrently altered genes in multiple patients/kindreds with a rare disorder provides enormous discriminatory power. The challenge is finding two or more willing participants with a rare condition.
The Challenge of Rare Disorders
A rare disorder that occurs in 1 out of every 100,000 individuals will affect about 40 patients per year in the United States. Most of those will go through a battery of diagnostic testing once they come to medical attention. Only a fraction will be referred to a research study, and an even smaller fraction will be enrolled. That's effectively a handful of new cases per year. These will likely be spread out across dozens of research institutions.
Given the wide spectrum of rare conditions, it's extremely unlikely that two patients with the same disorder would be identified (and recognized as such) at a single institution. Programs like the NIH Undiagnosed Disease Network aim to centralize the recruitment, sequencing, and analysis of rare disease cases. However, the vast majority of undiagnosed disorders are being studies by research institutions outside of such programs.
Efforts such as MyGene2 and the MatchMaker Exchange aim to facilitate data sharing among families with rare disorders and the investigators who study them. However, a number of rare disorders are still solved by good old-fashioned academic collaboration.
The RLIM/RNF12 Gene in X-linked Intellectual Disability
Case in point: in late 2016, our rare disease research program enrolled a patient with small size, hypotonia, and developmental delay — three of the most common phenotypes of patients referred to genetics. This patient, however, also had hypospadias and ambiguous genitalia. There was also a family history of congenital diaphragmatic hernia (a birth defect involving the diaphragm), which initially did not seem very important.
We performed whole-genome sequencing on the patient and his affected parents, which yielded about 6.5 million genetic variants. After running these through our comprehensive annotation/analysis pipeline, I narrowed the list to 12 recessive genes and three X-linked genes. The most interesting candidate was a hemizygous missense variant in X-linked gene RLIM, also known as RNF12. In a recent paper in the journal Molecular Psychiatry, a European-led team had reported a similar variant (p.P587R) in the same domain in two families with non-specific mild to profound ID, behavior problems, ID, microcephaly, micrognathia and cryptorchidism.
Our clinicians thought it was an interesting fit, but noted that our patient's phenotype seemed more severe than the cases described in the paper. We discussed testing additional family members and possibly doing X-chromosome inactivation studies (XCI) on the patient's mother. At the same time, one of our genetics clinicians reached out to the group in Germany who'd published the RLIM paper. We were hoping, at the very least, to get their impression of whether our patient resembled the RLIM cases in their cohort.
The response was unexpected but exciting: the German team was working on a follow-up paper with clinical details and functional studies on 3 large families with affected males in several generations.
They'd already performed functional studies in zebrafish and examined XCI in heterozygous females. The phenotypes were quite similar to those of our family, including (in some cases) congenital diaphragmatic hernia. The authors graciously invited us to be part of the manuscript.
The resulting paper, Pathogenic variants in E3 ubiquitin ligase RLIM/RNF12 lead to a syndromic X-linked intellectual disability and behavior disorder, is now online at Molecular Psychiatry. Its publication not only provides definitive diagnosis for our patient, but expands the phenotypic spectrum of RLIM mutation carriers and establishes the gene's role in normal development, cognition, and behavior.
On a broader scale, this demonstrates the power of open communication and data sharing in the rare disease research community.
Filed Under: Rare Diseases
The Power of Sequencing Data Re-analysis
April 6, 2018 by dkoboldt Leave a Comment
The clinical genetics group at our hospital holds a weekly conference to discuss patients recently seen by the clinicians and/or genetic counselors. I began attending this "post-clinic conference" when I moved here a year and a half ago. It's been very educational for me, both in learning about some of the rare conditions affecting our pediatric patients and in how clinicians diagnose them.
In one of the first post-clinic conferences I attended (early September 2016), one of the clinicians presented a case of two sister with a severe disorder of unknown cause. They've apparently been followed by our team for a number of years. Both have severe intellectual disability, hypotonia, seizures, and a distinctive craniofacial appearance. Various clinical tests had been ordered, but none had provided a diagnosis.
It came up in the course of discussion that research exome sequencing had been performed on both girls and their (healthy) parents some time ago. There had been no finding at the time, but it was a few years back. Since I'd been recruited to work on rare disease genomics, I offered to revisit the analysis. We ran the case through our current pipeline, and found an interesting result:
Exome data for the ASXL3 mutation
Both sisters appeared to share a de novo (i.e. newly arisen) nonsense mutation in a gene called Additional Sex Combs Like 3 (ASXL3).
ASXL Mutations and Disease
A few years ago, Matt Bainbridge and his colleagues at Baylor had sequenced four unrelated patients with a disorder resembling Bohring-Opitz syndrome. Bohring-Opitz is a severe malformation disorder characterized by intrauterine growth retardation, severe intellectual disability, flexion deformities of the upper limbs, forehead hirsutism, and some craniofacial features. In 2011, a group in Europe had found that Bohring-Opitz was caused by de novo nonsense mutations in Additional Sex Combs Like 1 (ASXL1), which is required for maintenance of both activation and silencing of Hox genes.
The four unrelated patients in the Bainbridge study had de novo nonsense and frameshift mutations in ASXL3 (not ASXL1). Moreover, they were clinically distinct from Bohring-Opitz syndrome, with features including severe feeding difficulties, failure to thrive, and neurologic abnormalities with significant developmental delay. OMIM entered it as a new syndrome in their database, and named it Bainbridge-Ropers syndrome (BRPS).
The two sisters from our case also had a de novo ASXL3 mutation. I was excited, and presented the finding at post-clinic in mid-September 2016. However, it got a lukewarm reception. Our mutation was in the middle of the ASXL3 gene (residue 1036), and predicted to truncate it to 46% of its wild-type length. The Bainbridge et al mutations were closer to the front of the gene (residues 404-466), which would remove a lot more. The phenotype was not a perfect match, either. Bottom line, they were unconvinced.
New Reports of Bainbridge-Ropers Syndrome
Over the next year, however, other groups identified de novo truncating ASXL3 mutations in other individuals with severe intellectual disability. A 2017 study by Balasubramanian et al described 12 previously unreported patients who were part of the DDD Consortium study (a large-scale intellectual disability cohort based in the UK). They delineated the six emerging common features of BRPS:
Severe intellectual disability (11/12 patients)
Poor or absent speech (12/12)
Autistic traits (9/12)
Distinct face, including arched eyebrows, prominent forehead, high-arched palate, hypertelorism and downslanting palpebral fissures (9/12)
Hypotonia (11/12)
Significant feeding difficulties when young (9/12)
Our patients, you might recall, had all of these features. Also, the new studies reported de novo truncating mutations in the 1,000-1,100 region of the ASXL3 protein, similar to ours. With this new information, everyone was convinced and we started writing the paper. In the interim, one of our clinicians saw another (unrelated) patient who got a clinical diagnosis of BRPS.
A Chat with Bainbridge
In October 2017, I went to the ASHG annual meeting, where I bumped into Matt Bainbridge. We've known each other for years (since the 1,000 Genomes Project). Naturally, I gave him some trouble about having a syndrome named after him. Then I asked him about our patients' mutation in ASXL3. He basically said, that's right in the other cluster of disease-causing mutations.
I asked Matt if he thought it strange that our institution had three patients with a super-rare syndrome (<30 cases in the literature). He said that it's probably more common than people realized. In fact, there might be as many as 200 cases diagnosed in the US.
"How do you know?" I asked.
"Because there's a Facebook support group for the families," he said. "In fact, they'd probably like to talk to your family, if they were interested, because most of their kids are younger."
A Shared de novo Mutation
We reported the ASXL3 mutation to ClinVar in October 2017. By then, we'd performed independent Sanger sequencing of all four family members to confirm that it was de novo. The paper was accepted in December, and is available as an early access publication at Molecular Case Studies.
One obvious question is how two siblings born over a year apart could share the same de novo mutation if it arose after conception. Although this could theoretically happen by chance, the odds are extremely low. More likely, one of the parents is a mosaic for the mutation. The family declined further testing to determine the origin. They were not planning to have more children, anyway.
Unfortunately, there's not much else we can do to help the family. However, we did refer them to the Facebook support group, and that may offer some support.
The genomic landscape of pediatric cancers
March 9, 2018 by dkoboldt Leave a Comment
Although pediatric cancer is rare, it's the leading cause of disease-related death among children who survive past infancy (in the Western world). Around 1 in 500 children will be diagnosed with cancer by age 15. Like most rare diseases, childhood cancer is difficult to study because so few patients are available. Even for the most common types — leukemia, brain/CNS tumors, and lymphomas — only a few hundred cases have been sequenced over the past decade. But we're making progress.
Two consortium-led studies published last month in the journal Nature offer the most comprehensive surveys to-date of pediatric cancer genomes.
Landscape of Genomic Alterations
Study 1, led by researchers in Germany, systematically analyzed 961 tumors representing 24 molecular subtypes of pediatric cancer, including 547 whole genome sequences (~37x coverage genome-wide) and 414 whole-exome sequences (~121x coverage of coding regions only). Their extensive analysis is presented in a full-length article that includes six figures and TEN extended data figures. Here are some highlights
Mutation rates vary across cancers and are lower than adult tumors
The mutation rates in pediatric tumors ranged from 0.02 mutations per megabase in hepatoblastoma to 0.49 in Burkitt's lymphoma.
Mutation rates of pediatric cancer (Gröbner et al, Nature 2018)
Yet overall the mutation rate in pediatric tumors was 14 times lower than that of adult tumors profiled by TCGA. Mutation rate was also correlated with the patient's age, which is a trend we've seen before.
8% of pediatric cancer patients had an inherited predisposition
Consistent with previous studies, the authors found pathogenic germline variants in 7.6% of their pediatric cases. Most of these were in DNA repair genes, i.e. mismatch repair (MSH2, MSH6, PMS2) or double-stranded break repair (TP53, BRCA2, CHEK2). Predisposition was most common in adrenocortical carcinomas (50%) and hypodiploid B-ALL (28%), followed by high-grade gliomas, medulloblastomas, and retinoblastomas (15-25%).
Significantly mutated genes point to regulation
Using the Mutation Significance In Cancer package, the authors identified 77% significantly mutated genes (SMGs). About half of all cases harbored at least one mutation in an SMG, and the frequencies of SMG mutations suggested that they were early, driver events in tumorigenesis. Most SMGs also showed mutual exclusivity with one another across cancer types — meaning that a tumor with a mutation in gene A almost never had a mutation in gene B — suggesting that a single mutation event is often enough to drive childhood cancer.
TP53 was the most commonly altered gene, which is unsurprising. Interestingly, the most commonly mutated set of genes were those involved in epigenetic modification (25% of tumors) followed by transcriptional regulators and MAP kinase-associated genes (12-15%).
Druggable mutated genes
The authors identified 453 potentially druggable events (PDEs) in 59 genes, 3% of which were germline variants. Most commonly, PDEs were detected in Burkitt's lymphomas (the highest mutated) and pilocytic astrocytomas. After adjusting for relative incidence, 52% of pediatric tumors had a potential druggable event. This supports the idea that genomic profiling should be a routine part of pediatric cancer care.
Pan-cancer Genomes and Transcriptomes
Study 2, led by researchers in the U.S., analyzed the genomes and transcriptomes of 1,699 pediatric leukemias and solid tumors representing six histotypes. All tumor samples were obtained at initial diagnosis via the Children's Oncology Group (COG) trials. This is a landmark study not only due to the size, but the systematic characterization of each case using whole-genome sequencing (WGS), exome sequencing (WES), and transcriptome sequencing (RNA-seq).
11 distinct mutational signatures
A key advantage of whole-genome sequencing in cancer is that it captures the full scope of somatic alterations (from SNVs to large structural variants) throughout the genome. The authors assigned somatic mutations to 96 different bins (based on the substitution and sequence context) to assess the "mutational signature" of each tumor. There were 11 distinct mutational signatures, henceforth referred to as T-1 through T-11.
Endogenous mutational process signatures (T1 and T4) were present in all tumors and accounted for a large proportion of the burden in T-ALL (97%), AML (63%), B-ALL (36%), and Wilms tumor (28%).
APOBEC family mRNA editing signatures (T2 and T7) were highly enriched in B-ALLs that had ETV6-RUNX1 fusions.
Homologous recombination deficiency (T-3) was evident in many tumors, especially osteosarcomas and NBLs.
Oxidating DNA damage (T-8) was present in 36% of neuroblastoma signatures, and associated with age at diagnosis.
The UV-light exposure signature (T-5) was present in all eight B-ALL samples.
This last one was surprising, since we think of ultraviolet exposure as contributing to melanoma idults, but not B-ALL (a leukemia of B-cells) in children. But the signature was 110-fold enriched in B-ALL tumors compared to other types in this study, and other observations, such as transcriptional strand bias, suggest it's not an artifact.
Unique cancer driver genes
The authors identified 142 "driver genes" that were significantly mutated within a histotype or across the cohort. More than half of these were specific to a single histotype, and 83% of driver genes were not shared between liquid and solid tumors. In other words, different pediatric tumor types seem to arise from different mutated genes and pathways.
Childhood cancer genes also differed from adult cancer genes: of 142 significant genes in this study, 78 (55%) were not found in adult pan-cancer studies and 43 (30%) were not in the Cancer Gene Consensus.
Structural variants and clonality
Some of the earliest studies of cancer genomes in AML noted that, within a single patient, there were subsets of mutations present at lower allele frequencies, suggesting that they're not present in all cells. These "tumor subclones" are often very relevant as the tumor evolves (especially during treatment). In this study, the authors noted that 40-50% of somatic point mutations in leukemias showed lower allele frequencies, suggesting that subclonality is also an important feature of childhood leukemia.
Another key finding of this study is the substantial contribution of large variants (copy number alterations and structural variants), which are often missed by exome sequencing. NBL offers a good example. Exome sequencing alone detected driver gene alterations in 26% of tumors, compared to 72% by genome sequencing. This trend of superior sensitivity for SMG alterations holds across all tumor types:
Tumors with 1+ driver alterations, by seq type (Ma et al, Nature 2018)
I've always been a proponent of WGS but it seems especially warranted in genomic studies of childhood cancers.
Expression of Mutant Alleles
Transcriptome sequencing is incredibly valuable for cancer genomics, because it offers insights into the immediate molecular effects of somatic mutations. In this study the authors analyzed 6,959 coding mutations that had genome and transcriptome sequencing coverage. The mutant allele was expressed (detected in the RNA) in 34%. Detection correlated with both the relative expression level of the gene and the allele frequency of the mutation in the genome.
Of the 2,477 somatic coding mutations with good coverage by both WGS and RNA-seq, 486 (20%) showed showed allele-specific expression. For 279 of them, the mutant allele does not appear to be expressed. For the remaining 207 candidate ASE mutations, some interesting patterns emerged:
76% of truncating (loss-of-function) mutations showed suppression of the mutated allele, likely via nonsense-mediated decay.
87% of hotspot (gain-of-function) mutations showed elevation of the mutated allele, likely because they conferred an advantage to the tumor.
In other words, the expression of a somatic mutation generally reflects its purpose for the tumor: loss-of-function mutations are suppressed, but activating hotspot mutations are over-expressed. What a great story.
The Need for Pediatric Cancer Genomics
These two studies represent landmark achievements for the field of childhood cancer genomics. They also demonstrate that pediatric cancers are unique from one another and from adult cancers. In short, genomic analysis (and especially whole-genome sequencing) is deeply informative for childhood cancers, and we should do a lot more of it.
Filed Under: Childhood Cancer


Home » CSI book "Social progress in Britain" now published » Do British Muslims face employment penalties?
Ethnic minorities face well-established penalties in the labour market. When attempting to understand the employment experiences of different religious groups, it is important to disentangle the separate roles of religion and ethnicity. Doing so helps us identify whether differences in unemployment and access to the top progressions between groups are due to ethnic or religious differences, which can help shed light on the best ways of reducing the disadvantages faced by certain groups.


Order Hay & Herbs for your Degoe easily and quickly at DRD Rodent Shop!
For hay, herbs and seeds for your Degu you are in the right place in our rodent shop. Would you like to spoil your Degu with healthy herbs and seeds? You will find various types of hay and herbs here at an attractive price. Ordering is easy and fast in our rodent webshop.


text:
Who is Andrew Charalambous
By admin September 12, 2016 July 18, 2018
Mr Andrew Charalambous is a private landlord and entrepreneur from London in the United Kingdom.
In 2014, he won the award of Outstanding Entrepreneur in 2014 at the Global Business Excellence Awards and was described in the Times Newspaper as different kind of social entrepreneur.
Andrew feels that the UK Government is not doing enough to help the near two Million people on the waiting list for housing and has his company has made it a little easier for people to rent a property.
There are a number of obstacles when trying to rent a property and if you claim housing benefit it is possible that you will not be in favour as much as a person that does claim it. As a result, you could possibly lose out.
Andrew thinks that you should not be treated unfairly or discriminated against if you need to claim housing benefit and wants a level playing field.
Andrew and his company has helped by making the application process so much easier, meaning that if you are in need of a property you will not have the obstacles that you will come across using traditional methods by using a small letting firm or landlord not attached to a business.
The process does not ask you for financial records or a reference from one or more people, most often a past landlord.
Also no deposit is needed as security od accidental damage or non-payment of rent and bills. Mr Charalambous thinks that this can actually improve the relationship of the tenant and landlord. As a result, he claims that people stay in his accommodation twice as much the duration of the national average due to the ease of the whole process. He feels that this also reduces the chance of late or non-payment of rent.
Andrew is currently the spokesperson for the United Kingdom Independence Party on the Environment and housing. You can find out what he is up to currently doing and follow him on his personal blog at www.andrewcharalambous.com or the official Andrew Charalambous Pinterest page
A Guide To Digital Marketing
Why You Should Have EHIC Card When Traveling In Europe For Studies/Business?

redpajama_set_name: RedPajamaCommonCrawl


25 Best Ideas About Sofas For Small Spaces On Pinterest Couches Amazing Comfy Armchairs Pertaining To 17 is one of pictures thet are related with the picture before in the collection gallery. If you would like to see the 25 Best Ideas About Sofas For Small Spaces On Pinterest Couches Amazing Comfy Armchairs Pertaining To 17 in High Resolution [HD Resolution] version, please press the right click on picures/image then choose "Save as Image" option, and done. You will get 25 Best Ideas About Sofas For Small Spaces On Pinterest Couches Amazing Comfy Armchairs Pertaining To 17 pictures that you want. The exactly dimension of 25 Best Ideas About Sofas For Small Spaces On Pinterest Couches Amazing Comfy Armchairs Pertaining To 17 was 540x752 pixels. You can also look for some pictures by collection on below this picture. Find the other picture or article about Comfy Armchairs For Small Spaces here. We hope it can help you to get information of the picture.


Big Tobacco is increasingly using social media to find new ways to hook young people on smoking, circumventing decades of laws restricting the marketing of traditional cigarettes to minors.
In major cities around the world such as Rio de Janeiro, Cairo, Jakarta and Milan, tobacco companies have been holding extravagant events with names like "K_Player" and "RedMoveNow" that were designed to connect with young people. Often featuring alcohol, live music and attractive hosts, these lavish events spare no expense as they seek find new buyers for their tobacco products.
The problem? Those party-goers are carefully targeted young influencers, who are encouraged to share photos of their glamorous tobacco-sponsored adventures with friends and followers on social media using appealing hashtags like #iamonthemove, #decideyourflow and #mydaynow. And although the influencers are over 18, their social media followers can be much younger.
This exploitation of social media's organic reach is one of the findings from a global research project I've been working on since 2016 with more than a dozen different scholars. The anti-smoking advocacy group Tobacco-Free Kids noticed a lot of photos of young people with cigarettes turning up in their online scans of global social media and asked me to look into it.
My own research focuses on how to rigorously research online culture using natural observational techniques, something that this study definitely required.
My team's task was to monitor, report upon and analyze the programs behind the hashtagged social media posts of young people smoking. What we learned about tobacco company's current advertising surprised us.
Tobacco ads were much bigger in 1996 – literally.
Tobacco companies have always had a knack for finding creative ways to skirt regulations intended to curb marketing to young people.
In 1971, the U.S. Congress banned tobacco ads from television and radio. In response, companies invested heavily in outdoor advertising and magazines. In 1997, the Tobacco Master Settlement Agreement banned tobacco on outdoor and billboard ads. In response, tobacco money flowed into sponsorships of sports, music and other events. These type of event sponsorships were banned, with some exceptions, in 2010, at the same time wider restrictions on youth marketing were also introduced.
No matter the medium, the messaging was often the same: find ways to reach new and young potential smokers. As documents from the Legacy Tobacco Documents Library reveal, tobacco executives have long believed that the continued survival and success of their companies depends on one thing: convincing young people to buy their products.
In 2005, the World Health Organization banned tobacco advertising in 168 signatory countries. By 2010, the U.S. had closed a lot of Big Tobacco's favorite advertising and tobacco loopholes.
With conventional media mostly off-limits, what was Big Tobacco to do? Like the Marlboro Man, the unregulated Wild West of social media rode to the rescue.
Social media fits Big Tobacco's advertising needs to a tee.
At least 88 percent of American youth say they use social media apps like Facebook and Instagram regularly, and the technologies are notoriously difficult to regulate.
With Tobacco-Free Kids' financial support, I assembled a growing team of researchers to investigate. Our work is ongoing.
My team collected a plethora of social media data and also conducted interviews with a range of tobacco brand ambassadors, party attendees, influencers and industry insiders from around the world. What we found was an astoundingly effective use of social media by a range of different tobacco companies to connect with the next generation of potential cigarette smokers.
While tobacco companies were careful to abide by the letter of the law – the influencers involved in these posts were all of legal smoking age in their countries – social media has a public setting that makes it an effective and largely unregulated form of broadcast.
Legally, anyone age 13 or over can have an Instagram or Facebook account. Our "netnography" – a type of qualitative social media inquiry that focuses on cultural contexts, social structures and deeper meanings – only looked at public posts, images that any 13-year-old with an account could see.
Our investigation uncovered a range of promotional activities and a web of public relations and advertising agencies that cleverly leveraged the strengths of social media to keep tobacco advertising under the radar of existing regulation.
We found tobacco companies in countries such as Indonesia and the Philippines recruiting "nano-influencers" of just 2,000-3,000 followers on Facebook and Instagram and encouraging them to post about their tobacco-sponsored adventures.
In Indonesia, we found brand ambassador training camps that lasted two full weeks and were run by the domestic tobacco company Gudang Garam. At these camps, young nano-influencers were paid generous fees, taught about cigarette brands images and then provided lessons about how to better maintain their social media feeds.
Public relations agencies in Uruguay taught their influencers how to take pictures of cigarette packages in ways that best accentuated their brands, offering tips on lighting, hashtags and the best time to post them for maximum impact.
At those parties, young people were greeted by attractive attendants who offered them cigarettes and encouraged them to pose with floor designs modeled after cigarette brand logos. After snapping pictures, they were encouraged to post them on their social media feed using the party's decisiveness and action-oriented hashtags. The result was unquestionably a new form of cigarette promotion.
These activities clearly violate the spirit of the existing agreements not to indirectly advertise to young people. You can call it stealth, undercover or guerrilla marketing if you wish. Whatever its name, this is 21st-century cigarette advertising that reaches millions of young people around the world.
Our research has not only helped shine a light on Big Tobacco's unchecked use of social media, it has also informed a recent petition to the U.S. Federal Trade Commission requesting it to investigate and enforce these novel forms of cigarette advertising.
Although it might be difficult for governments to keep on top of media in these rapidly changing times, they must do so if they hope to prevent global smoking rates and their consequent health problems from rising once again. Indeed, with leadership change in the Food and Drug Administration, new and tighter regulations on tobacco and vaping in the United States are already being cast into doubt.
Social media provide an incredible advance in communications that democratize communications in unprecedented ways.
However, that openness is easy to exploit by marketers with dubious motives.


text:
35 The Ideal Gas Law and Some Applications
Learn the ideal gas law.
Apply the ideal gas law to any set of conditions of a gas.
Apply the ideal gas law to molar volumes, density, and stoichiometry problems.
So far, the gas laws we have considered have all required that the gas change its conditions; then we predict a resulting change in one of its properties. Are there any gas laws that relate the physical properties of a gas at any given time?
Consider a further extension of the combined gas law to include n. By analogy to Avogadro's law, n is positioned in the denominator of the fraction, opposite the volume. So
Because pressure, volume, temperature, and amount are the only four independent physical properties of a gas, the constant in the above equation is truly a constant; indeed, because we do not need to specify the identity of a gas to apply the gas laws, this constant is the same for all gases. We define this constant with the symbol R, so the previous equation is written as
which is usually rearranged as
PV = nRT
This equation is called the ideal gas law. It relates the four independent properties of a gas at any time. The constant R is called the ideal gas law constant. Its value depends on the units used to express pressure and volume. Table 6.1 "Values of the Ideal Gas Law Constant R" lists the numerical values of R.
Table 6.1 Values of the Ideal Gas Law Constant R
Numerical Value
0.08205 L·atm/mol·K
62.36 L·torr/mol·K = L·mmHg/mol·K
8.314 J/mol·K
The ideal gas law is used like any other gas law, with attention paid to the units and making sure that temperature is expressed in kelvins. However, the ideal gas law does not require a change in the conditions of a gas sample. The ideal gas law implies that if you know any three of the physical properties of a gas, you can calculate the fourth property.
A 4.22 mol sample of Ar has a pressure of 1.21 atm and a temperature of 34°C. What is its volume?
The first step is to convert temperature to kelvins:
34 + 273 = 307 K
Now we can substitute the conditions into the ideal gas law:
The atm unit is in the numerator of both sides, so it cancels. On the right side of the equation, the mol and K units appear in the numerator and the denominator, so they cancel as well. The only unit remaining is L, which is the unit of volume we are looking for. We isolate the volume variable by dividing both sides of the equation by 1.21:
Then solving for volume, we get
V = 87.9 L
A 0.0997 mol sample of O2 has a pressure of 0.692 atm and a temperature of 333 K. What is its volume?
At a given temperature, 0.00332 g of Hg in the gas phase has a pressure of 0.00120 mmHg and a volume of 435 L. What is its temperature?
We are not given the number of moles of Hg directly, but we are given a mass. We can use the molar mass of Hg to convert to the number of moles.
Pressure is given in units of millimeters of mercury. We can either convert this to atmospheres or use the value of the ideal gas constant that includes the mmHg unit. We will take the second option. Substituting into the ideal gas law,
The mmHg, L, and mol units cancel, leaving the K unit, the unit of temperature. Isolating T all by itself on one side, we get
Then solving for K, we get
T = 507 K
For a 0.00554 mol sample of H2, P = 23.44 torr and T = 557 K. What is its volume?
The ideal gas law can also be used in stoichiometry problems.
What volume of H2 is produced at 299 K and 1.07 atm when 55.8 g of Zn metal react with excess HCl?
Zn(s) + 2 HCl(aq) → ZnCl2(aq) + H2(g)
Here we have a stoichiometry problem where we need to find the number of moles of H2 produced. Then we can use the ideal gas law, with the given temperature and pressure, to determine the volume of gas produced. First, the number of moles of H2 is calculated:
Now that we know the number of moles of gas, we can use the ideal gas law to determine the volume, given the other conditions:
All the units cancel except for L, for volume, which means
What pressure of HCl is generated if 3.44 g of Cl2 are reacted in 4.55 L at 455 K?
H2(g) + Cl2(g) → 2HCl(g)
0.796 atm
It should be obvious by now that some physical properties of gases depend strongly on the conditions. What we need is a set of standard conditions so that properties of gases can be properly compared to each other. Standard temperature and pressure (STP) is defined as exactly 100 kPa of pressure (0.986 atm) and 273 K (0°C). For simplicity, we will use 1 atm as standard pressure. Defining STP allows us to compare more directly the properties of gases that differ from each other.
One property shared among gases is a molar volume. The molar volume is the volume of 1 mol of a gas. At STP, the molar volume of a gas can be easily determined by using the ideal gas law:
All the units cancel except for L, the unit of volume. So
Note that we have not specified the identity of the gas; we have specified only that the pressure is 1 atm and the temperature is 273 K. This makes for a very useful approximation: any gas at STP has a volume of 22.4 L per mole of gas; that is, the molar volume at STP is 22.4 L/mol (Figure 6.3 "Molar Volume"). This molar volume makes a useful conversion factor in stoichiometry problems if the conditions are at STP. If the conditions are not at STP, a molar volume of 22.4 L/mol is not applicable. However, if the conditions are not at STP, the combined gas law can be used to calculate the volume of the gas at STP; then the 22.4 L/mol molar volume can be used.
Figure 6.3 Molar Volume
A mole of gas at STP occupies 22.4 L, the volume of a cube that is 28.2 cm on a side.
How many moles of Ar are present in 38.7 L at STP?
We can use the molar volume, 22.4 L/mol, as a conversion factor, but we need to reverse the fraction so that the L units cancel and mol units are introduced. It is a one-step conversion:
What volume does 4.87 mol of Kr have at STP?
What volume of H2 is produced at STP when 55.8 g of Zn metal react with excess HCl?
This is a stoichiometry problem with a twist: we need to use the molar volume of a gas at STP to determine the final answer. The first part of the calculation is the same as in a previous example:
Now we can use the molar volume, 22.4 L/mol, because the gas is at STP:
Alternatively, we could have applied the molar volume as a third conversion factor in the original stoichiometry calculation.
What volume of HCl is generated if 3.44 g of Cl2 are reacted at STP?
H2(g) + Cl2(g) → 2 HCl(g)
The ideal gas law can also be used to determine the densities of gases. Recall that density is defined as the mass of a substance divided by its volume:
Assume that you have exactly 1 mol of a gas. If you know the identity of the gas, you can determine the molar mass of the substance. Using the ideal gas law, you can also determine the volume of that mole of gas, using whatever the temperature and pressure conditions are. Then you can calculate the density of the gas by using
What is the density of N2 at 25°C and 0.955 atm?
First, we must convert the temperature into kelvins:
If we assume exactly 1 mol of N2, then we know its mass: 28.0 g. Using the ideal gas law, we can calculate the volume:
Knowing the molar mass and the molar volume, we can determine the density of N2 under these conditions:
What is the density of CO2 at a pressure of 0.0079 atm and 227 K? (These are the approximate atmospheric conditions on Mars.)
0.019 g/L
Chemistry Is Everywhere: Breathing
Breathing (more properly called respiration) is the process by which we draw air into our lungs so that our bodies can take up oxygen from the air. Let us apply the gas laws to breathing.
Start by considering pressure. We draw air into our lungs because the diaphragm, a muscle underneath the lungs, moves down to reduce pressure in the lungs, causing external air to rush in to fill the lower-pressure volume. We expel air by the diaphragm pushing against the lungs, increasing pressure inside the lungs and forcing the high-pressure air out. What are the pressure changes involved? A quarter of an atmosphere? A tenth of an atmosphere? Actually, under normal conditions, a pressure difference of only 1 or 2 torr makes us breathe in and out.
Figure 6.4 Breathing Mechanics
Breathing involves pressure differences between the inside of the lungs and the air outside. The pressure differences are only a few torr.
A normal breath is about 0.50 L. If room temperature is about 22°C, then the air has a temperature of about 295 K. With normal pressure being 1.0 atm, how many moles of air do we take in for every breath? The ideal gas law gives us an answer:
Solving for the number of moles, we get
n = 0.021 mol air
This ends up being about 0.6 g of air per breath—not much but enough to keep us alive.
The ideal gas law relates the four independent physical properties of a gas at any time.
The ideal gas law can be used in stoichiometry problems in which chemical reactions involve gases.
Standard temperature and pressure (STP) are a useful set of benchmark conditions to compare other properties of gases.
At STP, gases have a volume of 22.4 L per mole.
The ideal gas law can be used to determine densities of gases.
What is the ideal gas law? What is the significance of R?
Why does R have different numerical values (see Table 6.1 "Values of the Ideal Gas Law Constant R")?
A sample of gas has a volume of 3.91 L, a temperature of 305 K, and a pressure of 2.09 atm. How many moles of gas are present?
A 3.88 mol sample of gas has a temperature of 28°C and a pressure of 885 torr. What is its volume?
A 0.0555 mol sample of Kr has a temperature of 188°C and a volume of 0.577 L. What pressure does it have?
If 1.000 mol of gas has a volume of 5.00 L and a pressure of 5.00 atm, what is its temperature?
A sample of 7.55 g of He has a volume of 5,520 mL and a temperature of 123°C. What is its pressure in torr?
A sample of 87.4 g of Cl2 has a temperature of −22°C and a pressure of 993 torr. What is its volume in milliliters?
A sample of Ne has a pressure of 0.772 atm and a volume of 18.95 L. If its temperature is 295 K, what mass is present in the sample?
A mercury lamp contains 0.0055 g of Hg vapor in a volume of 15.0 mL. If the operating temperature is 2,800 K, what is the pressure of the mercury vapor?
Oxygen is a product of the decomposition of mercury(II) oxide:
2 HgO(s) → 2 Hg(ℓ) + O2(g)
What volume of O2 is formed from the decomposition of 3.009 g of HgO if the gas has a pressure of 744 torr and a temperature of 122°C?
Lithium oxide is used to absorb carbon dioxide:
Li2O(s) + CO2(g) → Li2CO3(s)
What volume of CO2 can 6.77 g of Li2O absorb if the CO2 pressure is 3.5 × 10−4 atm and the temperature is 295 K?
What is the volume of 17.88 mol of Ar at STP?
How many moles are present in 334 L of H2 at STP?
How many liters of CO2 at STP are produced from 100.0 g of C8H18, the approximate formula of gasoline?
2 C8H18(ℓ) + 25 O2(g) → 16 CO2(g) + 18 H2O(ℓ)
How many liters of O2 at STP are required to burn 3.77 g of butane from a disposable lighter?
2 C4H10(g) + 13 O2(g) → 8 CO2(g) + 10 H2O(ℓ)
What is the density of each gas at STP?
a) He
b) Ne
c) Ar
d) Kr
18. What is the density of each gas at STP?
a) H2
b) O2
c) N2
19. What is the density of SF6 at 335 K and 788 torr?
20. What is the density of He at −200°C and 33.9 torr?
1. The ideal gas law is PV = nRT. R is the ideal gas law constant, which relates the other four variables.
3. 0.327 mol
5. 3.64 atm
7. 8,440 torr
9. 12.2 g
11. 0.230 L
13. 401 L
a) 0.179 g/L
b) 0.901 g/L
c) 1.78 g/L
d) 3.74 g/L
19. 5.51 g/L
Previous: Introduction to Gases
Next: End-of-Chapter Material


WatchWear's goal is to provide 100% CUSTOMER SATISFACTION.
Your purchase should make you happy. If for some reason you need to return an item, please follow the procedures below.
1. If you are not 100% satisfied with your purchase, you MUST contact us within 60 days from the time you receive your purchase to obtain a RETURN MERCHANDISE AUTHORIZATION NUMBER (RMA). This must be clearly marked on the outside of the package. All returns must be accompanied with a Return Authorization number. To provide the customer with proper and quick service, the Return Authorization number will allow our staff to follow your instructions for exchange or credit. Without the Return Authorization we are unable to service the buyer with the correct actions. Please contact our customer service department via email or toll free telephone number for a Return Authorization number. If the return has no number it could be subject to a surcharge. We understand the need of returns and are happy to provide 100% CUSTOMER SATISFACTION however, please help us to serve you better. We want you to be a happy returning customer.
3. THE WATCH CAN BE RETURNED ONLY IF IT HAS NEVER BEEN WORN. We only sell new watches. It is impossible to wear a watch, even for a short time and not get it scratched or blemished. PLEASE understand that we can not accept a return if it is not in the EXACT same condition as we shipped to you. RESIZED or MODIFIED watches with links removed will not be eligible for returns.
4. If you have any questions regarding your return, please feel free to contact us at anytime, we are always happy to help. CUSTOMER SATISFACTION IS IMPORTANT TO WatchWear.


text:
Q: How to maximize application in over the Taskbar at Windows Forms? I need to maximize my application in C# that it will be over the taskbar. I have change the property WindowState = Maximized. But the lower part of the application appears under the taskbar. I want the status bar to appear above with the taskbar also shown below it.

It's maximized but where is my statusbar :)

This is Normal State of Window; you can see status bar in this pic.

A: As I indicated in my comment, you've done something odd to your window, which is why you're getting this behaviour. I can reproduce this. I created a new WinForms project, put a button on the form that has this code:
private void button1_Click(object sender, EventArgs e)
{
    this.WindowState = FormWindowState.Maximized;
}

Added a status bar, and set the MaximizeBox property of the form to false. Running the code, pressing my custom button, the form maximizes, but the status bar disappears behind the task bar.
If I then close the form, re-enable the MaximizeBox property, and re-run the project, then maximizing the form gets the behaviour you want (the status bar's bottom is aligned with the top of the task bar). So re-enable your MaximizeBox and you should be done.
(Once the Maximize box is available, maximizing works whether using that or the custom button)

A: First, make sure that you have no ControlBox, but text in the Form Text - this adds the title bar. Then, set your form WindowState to Maximized.
The last thing you do in the Form_Load event is to set the form Text property to an empty string. The Title bar is removed, the form is maximized, and the Task Bar is visible.

A: I am guessing that you have TopMost property set to 'True'.  If you need this setting then you can get taskbar information from a Win32 API call.  There is some information about it here:
Taskbar Height
Otherwise you could just disable the TopMost setting.  You may want to check that your program runs in 64bit os (if you are not already using this).  I use VirtualBox with a 64 bit guest o/s for testing 64bit on my 32 bit host. (Requires a 64 bit processor though.)
I hope that this helps.

redpajama_set_name: RedPajamaStackExchange


It's Monday, and Monday means music fun! And hello?! It's Halloween! So naturally our theme this week is all Halloween all the time. Find your spookiest tune, and let's dance, shall we?
Let's start with a song that was this artist's breakout single with a circus theme where you won't be able to shake off the feeling that an evil clown is about to sneak up behind you while watching.
This next song opens with the artist eerily humming, and the sound becomes the centerpiece to the Hunger Games soundtrack song. The dissonant harmonies on the chorus add to its Halloween-ness, but the song's beat can still get party-goers groovin'.
No Halloween themed music post would be complete without this next tune. Just ask the kids, they'll tell you.
Last, but not least, is one of mine and Princess Nagger's favorite Halloween tunes. It's simply not Halloween without it.
This entry was posted in Monday's Music Moves Me, Music Monday and tagged Halloween, halloween theme, Monday Music Moves Me, Music, Music Monday, tunes by StacyUncorked. Bookmark the permalink.
Stacy, well here we are at the end of another month. This year is zipping by like a bat out of …! I managed to align with the 4M crew's mewsic theme today without eve a peek at Marie's line up. Your selection of Halloween tunes are all new to me, except for I Want Candy. I appreciated the exposure to these new songs. One of the Hungar Games movies is on Amazon Prime. It might be the second one in the series. I started watching it but decided to start it again with DH but just haven't gotten around to it. The only thing is we like watching a series in the right order, so I'm waiting to see if the first one shows up. Oh well, y'all be careful if you go out trick or treating. PN is all grown up now so handing out candy is a good way for her to spend the evening. Keep that candy from Little Dude or you'll never get him to bed. 😀 Thanks for hosting the best Halloween dance party in Blogosphere, my friend!
What a great bunch of Halloween tunes. Some are very new to me as I have never seen the Hunger Games.
Love "I want candy" and "Nightmare Before Christmas" those are classics. Thanks for sharing.
Wow! That Bow wow song is definitely candy! Long lived!
Love the new ones you've share here Stacy!
I wonder how many people were walking around singing "I Want Candy" last night! Really enjoyed that Lorde song as well.


Reading matters. Somebody should tell Queensland Premier Campbell Newman.
WHY does Australia need literary prizes? I never thought this would be an argument anyone would have to make, but I was too complacent. One of the first things Campbell Newman did when he came to power was to axe the Queensland Premier's Literary Awards.
This is a small saving (just under $250,000) for Queensland taxpayers, but a huge blow to our culture. True, some writers and publishers have valiantly pledged to keep the 2012 awards going - but without government support or prizemoney.
More worrying than the axing itself is what it might mean for books. It legitimises the idea that not just literary prize funding but arts funding in general is a waste of government money. Which leads in turn to the idea that nobody should bother supporting art: let the market decide.
But we need our literary prizes. For a start, writers need money. While a few who work in such popular genres as romance fiction make a good living, most are paid a pittance. According to the Australian Society of Authors, the average income of a writer in Australia is $11,000 a year. And it doesn't follow that the better writer you are, the more you earn. Some of our most admired authors have shockingly modest incomes.
You don't hear them complain much. In other ways, they are lucky people, and the economics of publishing mean they won't get a pay rise. So most do other work to earn a living, fit their writing around it, and hope for whatever fillip they can get in competition with other writers. A $20,000 prize can buy a huge amount of writing time.
Even more importantly, writers need recognition. Not so much the Careys and Wintons and Garners, perhaps, but it was prizes that helped them become known in the first place. With Victoria, Queensland was one of two states that offered a prize for an unpublished manuscript. It also offered the only prize for unpublished indigenous writers, the David Unaipon award. These awards kick-start careers in a way almost impossible for an unknown writer to achieve otherwise.
It's not just writers who need awards: readers need them too. At a time when the simple act of sitting down to read a book (whether an e-book or the old-fashioned paper kind) is becoming ever more squeezed by the pressure of work and the lure of the internet and social media, government-sponsored award ceremonies help to reassure us that reading still matters. And reading our own writers is one way of asserting that our own country matters.
When you go into a bookshop, you will find plenty of Australian books. But you will have to look for them. They will be competing with piles of bestsellers by huge international names, masses of hype trying to persuade you that these are the best books in the shop. A little sticker to say that this book has won or has been shortlisted for an Australian prize might just tip the balance, and give you a wonderful read you might never have discovered otherwise.
Last week, Melbourne writer Jon Bauer knocked Jonathan Franzen's Freedom and Emma Donoghue's Room out of contention to win a place on the shortlist for one of the richest international book awards, the Dublin IMPAC prize. I bet many keen Australian readers who know all about Franzen and Donoghue have never heard of Bauer and his first novel, Rocks in the Belly. Now it's on the IMPAC list, I hope they will read it.
It takes a long time to set up a literary prize. The committee seeking to establish a new Australian prize for women writers, the Stella Prize, is going through months of negotiation, raising awareness, replying to vigorous criticism of the concept, and above all, trying to get enough sponsors on board. Once you have a prize, you need money to sustain it; it may take decades to build up prestige and reputation, which can be shaken at any time by controversy or scandal - and there's always plenty of that.
But to destroy support for a whole raft of awards, you don't even need a specific mandate from voters, just the stroke of a pen. It doesn't bode well for books.
Jane Sullivan writes the Age Saturday books column Turning Pages.


text:
Heather Lyke is the athletic director at the University of Pittsburgh. She was previously the athletic director and vice president at Eastern Michigan University, and has also served in administrative roles at Ohio State University and the University of Cincinnati.

Career
During her tenure at Eastern Michigan University, Lyke oversaw significant growth in the school's athletic programs thanks to a 51% increase in athletic donations. This increased funding was used for capital upgrades to athletic facilities. The school won seventeen MAC titles during her tenure. The football team made its first bowl appearance since 1987, with a 20–24 loss to Old Dominion in the 2019 Bahamas Bowl under head coach Chris Creighton.

She was announced as the athletic director for the University of Pittsburgh in March 2017 following the departure of Scott Barnes.

References

External links
Pittsburgh Panthers bio

Living people
Eastern Michigan Eagles athletic directors
Michigan Wolverines softball players
Pittsburgh Panthers athletic directors
Sportspeople from Canton, Ohio
Women college athletic directors in the United States
University of Akron alumni
1970 births

redpajama_set_name: RedPajamaWikipedia


Meyer Distributing has announced a new partnership with performance drivetrain parts supplier Nitro Gear & Axle.
Meyer Distributing has announced a new partnership with performance parts supplier Nitro Gear & Axle.
A well-known name in the diesel, off-road and overlanding communities, Meyer will add Nitro's differential and axle parts to its ever-growing lineup of drivetrain, axle and gear products.


text:
Laughanstown Cross
N 53° 14' 50.8" W 006° 09' 22.4"
Grid Ref.
O 23332 23464
Map No.
Elevation a.s.l. (m)
GPS Accuracy (m)
Show Google Map Show Monuments in the area
The cross seen from the field where the other Laughanstown cross is.
On the northeast side of a narrow lane in the townland of Laughanstown there's a stone cross set onto a stone and concrete plinth. It is said that it was placed in this position by James F. Grehan in 1897 when the road was lowered. Grehan had this plinth built in order to keep the cross at the original position and height.
The plinth can be climbed via a set of narrow and steep steps on the southeast side of it.
The cross is 2.24 metres tall, the arms are 94 centimetres wide, the shaft measures 36 centimetres in width and 20 centimetres in depth. The plinth is about 4.40 metres in height. It faces the road to the west (275°).
About 100 metres down the same lane is the Tully church, and in the field across the lane to the south is the other Laughanstown cross.
Browse by Monument Type
Browse by Date of Visit
Browse by Map Number
Clickable Counties
Clickable OS Maps Grid
Find a Map
Multimap
The days before GPS
The Stones in the Movies
Site view counter: 12573505
Copyright © 2003-2021 Antonio D'Imperio
All the photos, the graphics and the texts on this website are automatically copyrighted to me under the Berne Convention for the Protection of Literary and Artistic Works 1886. Any violation of the copyright will be pursued according to the applicable laws.
info@irishstones.org
Powered by AxeCMS/CustomEngine(V0.25.00 build 999) by Sergio "Axeman" Lorenzetti. (C) 2009-2015


Our A4 Portrait Multi Pocket Universal Freestanding Literature Holder comes with three tiers, each with a 32mm capacity. Injection moulded in clear styrene for additional style and design, these literature holders are ideal for any commercial environment.
Personalised branding is also available. If you require specific branding please call our acrylic display experts for details.