SUMMARY The development of the nervous system involves a coordinated succession of events including the migration of GABAergic neurons from ventral to dorsal forebrain and their integration into cortical circuits. However, these interregional interactions have not yet been modelled with human cells. Here, we generate from human pluripotent cells three-dimensional spheroids resembling either the dorsal or ventral forebrain and containing cortical glutamatergic or GABAergic neurons. These subdomain-specific forebrain spheroids can be assembled to recapitulate the saltatory migration of interneurons similar to migration in fetal forebrain. Using this system, we find that in Timothy syndrome– a neurodevelopmental disorder that is caused by mutations in the CaV1.2 calcium channel, interneurons display abnormal migratory saltations. We also show that after migration, interneurons functionally integrate with glutamatergic neurons to form a microphysiological system. We anticipate that this approach will be useful for studying development and disease, and for deriving spheroids that resemble other brain regions to assemble circuits in vitro.

These parameters were developed by the Joint Task Force on Practice Parameters, representing the American Academy of Allergy, Asthma & Immunology (AAAAI); the American College of Allergy, Asthma & Immunology (ACAAI); and the Joint Council of Allergy, Asthma and Immunology. The AAAAI and the ACAAI have jointly accepted responsibility for establishing "The Diagnosis and Management of Anaphylaxis Practice Parameter: 2010 Update." This is a complete and comprehensive document at the current time. The medical environment is a changing environment, and not all recommendations will be appropriate for all patients. Because this document incorporated the efforts of many participants, no single individual, including those who served on the Joint Task Force, is authorized to provide an official AAAAI or ACAAI interpretation of these practice parameters. Any request for information about or an interpretation of these practice parameters by the AAAAI or ACAAI should be directed to the Executive Offices of the AAAAI, the ACAAI, or the Joint Council of Allergy, Asthma and Immunology. These parameters are not designed for use by pharmaceutical companies in drug promotion.

Much of the information available about factors that affect mRNA decay in Escherichia coli, and by inference in other bacteria, has been gleaned from study of less than 25 of the Ϸ4,300 predicted E. coli messages. To investigate these factors more broadly, we examined the half-lives and steady-state abundance of known and predicted E. coli mRNAs at single-gene resolution by using two-color fluorescent DNA microarrays. An rRNA-based strategy for normalization of microarray data was developed to permit quantitation of mRNA decay after transcriptional arrest by rifampicin. We found that globally, mRNA half-lives were similar in nutrient-rich media and defined media in which the generation time was approximately tripled. A wide range of stabilities was observed for individual mRNAs of E. coli, although Ϸ80% of all mRNAs had half-lives between 3 and 8 min. Genes having biologically related metabolic functions were commonly observed to have similar stabilities. Whereas the half-lives of a limited number of mRNAs correlated positively with their abundance, we found that overall, increased mRNA stability is not predictive of increased abundance. Neither the density of putative sites of cleavage by RNase E, which is believed to initiate mRNA decay in E. coli, nor the free energy of folding of 5 or 3 untranslated region sequences was predictive of mRNA half-life. Our results identify previously unsuspected features of mRNA decay at a global level and also indicate that generalizations about decay derived from the study of individual gene transcripts may have limited applicability.RNA degradation ͉ RNA half-life ͉ transcript abundance T he rate of mRNA degradation plays a central role in the metabolism of nucleic acids in both prokaryotic and eukaryotic cells (for reviews see refs. 1-5). mRNA decay has been studied in a range of organisms, and much has been learned about the substrate features and ribonucleolytic enzymes that influence mRNA stability. In the case of Escherichia coli, experimenters have demonstrated that extensive variation exists in the rate of decay of individual mRNAs and that specific features of the mRNA sequence as well as transcript secondary structure can be important determinants of such variation (1, 3). Additionally, although the half-lives of some RNAs depend on the physiological state of the cell, as influenced by genetic and environmental factors, the half-lives of other RNAs seem to be independent of cell physiology (6, 7).Much of the information available about RNA decay has been derived from study of RNAs encoded by a very limited number of genes. In fact, we can find published reports of RNA half-life for less than 25 of the 4,288 predicted ORFs in the E. coli genome. Current models of mRNA decay are based on this relatively limited experimental sampling of message turnover. To gain a broader understanding of the fate of mRNAs in E. coli we adapted DNA microarray methodology to measure the decay of each chromosomally encoded mRNA simultaneously.DNA microarrays containing sequences derived ...

BackgroundWe published the Canadian 2003 International Consensus Algorithm for the Diagnosis, Therapy, and Management of Hereditary Angioedema (HAE; C1 inhibitor [C1-INH] deficiency) and updated this as Hereditary angioedema: a current state-of-the-art review: Canadian Hungarian 2007 International Consensus Algorithm for the Diagnosis, Therapy, and Management of Hereditary Angioedema.ObjectiveTo update the International Consensus Algorithm for the Diagnosis, Therapy and Management of Hereditary Angioedema (circa 2010).MethodsThe Canadian Hereditary Angioedema Network (CHAEN)/Réseau Canadien d'angioédème héréditaire (RCAH) http://www.haecanada.com and cosponsors University of Calgary and the Canadian Society of Allergy and Clinical Immunology (with an unrestricted educational grant from CSL Behring) held our third Conference May 15th to 16th, 2010 in Toronto Canada to update our consensus approach. The Consensus document was reviewed at the meeting and then circulated for review.ResultsThis manuscript is the 2010 International Consensus Algorithm for the Diagnosis, Therapy and Management of Hereditary Angioedema that resulted from that conference.ConclusionsConsensus approach is only an interim guide to a complex disorder such as HAE and should be replaced as soon as possible with large phase III and IV clinical trials, meta analyses, and using data base registry validation of approaches including quality of life and cost benefit analyses, followed by large head-to-head clinical trials and then evidence-based guidelines and standards for HAE disease management.

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Variant pathogenicity classifiers such as SIFT, PolyPhen-2, CADD, and MetaLR assist in interpretation of the hundreds of rare, missense variants in the typical patient genome by deprioritizing some variants as likely benign. These widely used methods misclassify 26 to 38% of known pathogenic mutations, which could lead to missed diagnoses if the classifiers are trusted as definitive in a clinical setting. We developed M-CAP, a clinical pathogenicity classifier that outperforms existing methods at all thresholds and correctly dismisses 60% of rare, missense variants of uncertain significance in a typical genome at 95% sensitivity.

Individuals with congenital or acquired prolongation of the QT interval, or long QT syndrome (LQTS), are at risk of life threatening ventricular arrhythmia 1, 2. LQTS is commonly genetic in origin but can also be caused or exacerbated by environmental factors1, 3. A missense mutation in the L-type calcium channel CaV1.2 leads to LQTS in patients with Timothy syndrome (TS)4, 5. To explore the effect of the TS mutation on the electrical activity and contraction of human cardiomyocytes (CMs), we reprogrammed human skin cells from TS patients to generate induced pluripotent stem cells (iPSCs), and differentiated these cells into CMs. Electrophysiological recording and calcium (Ca2+) imaging studies of these cells revealed irregular contraction, excess Ca2+ influx, prolonged action potentials, irregular electrical activity and abnormal calcium transients in ventricular-like cells. We found that roscovitine (Ros), a compound that increases the voltage-dependent inactivation (VDI) of CaV1.26–8, restored the electrical and Ca2+ signaling properties of CMs from TS patients. This study opens new avenues for studying the molecular and cellular mechanisms of cardiac arrhythmias in humans, and provides a robust assay for developing new drugs to treat these diseases.

Monogenic neurodevelopmental disorders provide key insights into the pathogenesis of disease and help us understand how specific genes control the development of the human brain. Timothy syndrome is caused by a missense mutation in the L-type calcium channel Cav1.2 that is associated with developmental delay and autism 1. We generated cortical neuronal precursor cells and neurons from induced pluripotent stem cells derived from individuals with Timothy syndrome. Cells from these individuals have defects in calcium (Ca2+) signaling and activity-dependent gene expression. They also show abnormalities in differentiation, including decreased expression of genes that are expressed in lower cortical layers and in callosal projection neurons. In addition, neurons derived from individuals with Timothy syndrome show abnormal expression of tyrosine hydroxylase and increased production of norepinephrine and dopamine. This phenotype can be reversed by treatment with roscovitine, a cyclin-dependent kinase inhibitor and atypical L-type–channel blocker 2, 3, 4. These findings provide strong evidence that Cav1.2 regulates the differentiation of cortical neurons in humans and offer new insights into the causes of autism in individuals with Timothy syndrome.