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Nonsense and missense mutations in the transcription factor PAX6 cause a wide range of eye development defects, including aniridia, microphthalmia and coloboma. To understand how changes of PAX6:DNA binding cause these phenotypes, we combined saturation mutagenesis of the paired domain of PAX6 with a yeast one-hybrid (Y1H) assay in which expression of a PAX6-GAL4 fusion gene drives antibiotic resistance. We quantified binding of more than 2700 single amino-acid variants to two DNA sequence elements. Mutations in DNA-facing residues of the N-terminal subdomain and linker region were most detrimental, as were mutations to prolines and to negatively charged residues. Many variants caused sequence-specific molecular gain-of-function effects, including variants in position 71 that increased binding to the LE9 enhancer but decreased binding to a SELEX-derived binding site. In the absence of antibiotic selection, variants that retained DNA binding slowed yeast growth, likely because such variants perturbed the yeast transcriptome. Benchmarking against known patient variants and applying ACMG/AMP guidelines to variant classification, we obtained supporting-to-moderate evidence that 977 variants are likely pathogenic and 1306 are likely benign. Our analysis shows that most pathogenic mutations in the paired domain of PAX6 can be explained simply by the effects of these mutations on PAX6:DNA association, and establishes Y1H as a generalisable assay for the interpretation of variant effects in transcription factors. Synopsis: A deep mutational scanning (DMS) assay using the yeast-one-hybrid system as the DNA binding readout reveals the effects of more than 2700 single amino-acid mutations in the human transcription factor PAX6. The assay shows: PAX6 variants with sequence-specific effects on DNA binding. Gain-of-function variants in PAX6. Highly accurate prediction of benign and pathogenic variants. Discrimination between patient phenotypes. A deep mutational scanning (DMS) assay using the yeast-one-hybrid system as the DNA binding readout reveals the effects of more than 2700 single amino-acid mutations in the human transcription factor PAX6. [ABSTRACT FROM AUTHOR]

Background and aims: On 1 May 2018, Scotland introduced a minimum unit price (MUP) of £0.50 for alcohol, with one UK unit of alcohol being 10 ml of pure ethanol. This study measured the association between MUP and changes in the volume of alcohol‐related ambulance call‐outs in the overall population and in call‐outs subsets (night‐time call‐outs and subpopulations with higher incidence of alcohol‐related harm). Design: An interrupted time−series (ITS) was used to measure variations in the daily volume of alcohol‐related call‐outs. We performed uncontrolled ITS on both the intervention and control group and a controlled ITS built on the difference between the two series. Data were from electronic patient clinical records from the Scottish Ambulance Service. Setting and cases: Alcohol‐related ambulance call‐outs (intervention group) and total ambulance call‐outs for people aged under 13 years (control group) in Scotland, from December 2017 to March 2020. Measurements Call‐outs were deemed alcohol‐related if ambulance clinicians indicated that alcohol was a 'contributing factor' in the call‐out and/or a validated Scottish Ambulance Service algorithm determined that the call‐out was alcohol‐related. Findings No statistically significant association in the volume of call‐outs was found in both the uncontrolled series [step change = 0.062, 95% confidence interval (CI) = −0.012, 0.0135 P = 0.091; slope change = −0.001, 95% CI = −0.001, 0.1 × 10−3P = 0.139] and controlled series (step change = −0.01, 95% CI = −0.317, 0.298 P = 0.951; slope change = −0.003, 95% CI = −0.008, 0.002 P = 0.257). Similarly, no significant changes were found for the night‐time series or for any population subgroups. Conclusions: There appears to be no statistically significant association between the introduction of minimum unit pricing for alcohol in Scotland and the volume of alcohol‐related ambulance call‐outs. This was observed overall, across subpopulations and at night‐time. [ABSTRACT FROM AUTHOR]

Decomposition of lignin‐rich wood by fungi drives nutrient recycling in woodland ecosystems. Fluctuating abiotic conditions are known to promote the functioning of ecological communities and ecosystems. In the context of wood decay, fluctuating temperature increases decomposition rates. Metabolomics, in tandem with other 'omics tools, can highlight the metabolic processes affected by experimental treatments, even in the absence of genome sequences and annotations. Globally, natural wood decay communities are dominated by the phylum Basidiomycota. We examined the metabolic responses of Mucidula mucida, a dominant constituent of pioneer communities in beech branches in British woodlands, and Exidia glandulosa, a stress‐selected constituent of the same communities, in response to constant and diurnally cycling temperature.We applied untargeted metabolomics and proteomics to beech wood blocks, colonised by M. mucida or E. glandulosa and exposed to either diurnally cycling (mean 15 ± 10°C) or constant (15°C) temperature, in a fully factorial design.Metabolites and proteins linked to lignin breakdown, the citric acid cycle, pentose phosphate pathway, carbohydrate metabolism, fatty acid metabolism and protein biosynthesis and turnover were under‐enriched in fluctuating, compared to stable temperatures, in the generalist M. mucida. Conversely, E. glandulosa showed little differential response to the experimental treatments.Synthesis. By demonstrating temperature‐dependant metabolic signatures related to nutrient acquisition in a generalist wood decay fungus, we provide new insights into how abiotic conditions can affect community‐mediated decomposition and carbon turnover in forests. We show that mechanisms underpinning important biogeochemical processes can be highlighted using untargeted metabolomics and proteomics in the absence of well‐annotated genomes. [ABSTRACT FROM AUTHOR]

Background: The incidence of Glioblastoma Multiforme (GBM) is increasing among the older population and is associated with poor prognosis. Management guidelines are lacking in this group. The purpose of this study was to analyze survival data and determine predictors of survival in patients aged ≥70 years treated with radiotherapy (RT) and/or Temozolomide. Materials and Methods: A retrospective analysis of all GBM patients treated at our institution between January 2011 and January 2017 was carried out. Results: One‐hundred and four patients were eligible. Median age was 73.8 years (70‐87). Thirty‐three patients received radical RT and 71 palliative RT. Overall median survival (MS) was 6 months. The MS was 10.6 months for radical patients and 4.9 months for palliative patients (P < 0.0005). The MS was 6.9 months in patients aged 70‐75 years and 5.2 months in those aged 76‐80 years (P = 0.004). The debulked group had a statistically significantly longer survival (8.0 months) than the biopsy only group (4.9 months). Biopsy only (hazard ratio [HR] 2.4), ECOG performance status 3 vs 0 (HR 6.4), and increasing age (HR 1.06) were associated with statistically significant shorter survival after adjustment for the effects of concurrent chemo, delay in starting RT, and RT dose. Conclusion: The MS for radical patients was favorable and approaching current literature for the under 70 age group. Radical treatment should be considered for good performance patients aged 70‐75 years. Increasing age was associated with shorter MS in patients aged ≥76 years. Debulking and good performance status were associated with improved survival. [ABSTRACT FROM AUTHOR]

Intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate the pupillary light reflex, circadian entrainment, and may contribute to luminance and color perception. The diversity of ipRGCs varies from rodents to primates, suggesting differences in their contributions to retinal output. To further understand the variability in their organization and diversity across species, we used immunohistochemical methods to examine ipRGCs in tree shrew (Tupaia belangeri). Tree shrews share membership in the same clade, or evolutionary branch, as rodents and primates. They are highly visual, diurnal animals with a cone‐dominated retina and a geniculo‐cortical organization resembling that of primates. We identified cells with morphological similarities to M1 and M2 cells described previously in rodents and primates. M1‐like cells typically had somas in the ganglion cell layer, with 23% displaced to the inner nuclear layer (INL). However, unlike M1 cells, they had bistratified dendritic fields ramifying in S1 and S5 that collectively tiled space. M2‐like cells had dendritic fields restricted to S5 that were smaller and more densely branching. A novel third type of melanopsin immunopositive cell was identified. These cells had somata exclusively in the INL and monostratified dendritic fields restricted to S1 that tiled space. Surprisingly, these cells immunolabeled for tyrosine hydroxylase, a key component in dopamine synthesis. These cells immunolabeled for an RGC marker, not amacrine cell markers, suggesting that they are dopaminergic ipRGCs. We found no evidence for M4 or M5 ipRGCs, described previously in rodents. These results identify some organizational features of the ipRGC system that are canonical versus species‐specific. We identify and distinguish multiple types of intrinsically photosensitive retinal ganglion cells in tree shrew. The tree shrew is a highly visual diurnal mammal with a cone‐dominated retina, and is an evolutionary intermediate between rodents and primates. Thus, these animals provide insight in the evolution of mammalian and primate vision. One population of melanopsin expressing cells (cyan in accompanied image) also expresses tyrosine hydroxylase (yellow). This expression pattern suggests an intrinsically photosensitive ganglion cell that is dopaminergic. [ABSTRACT FROM AUTHOR]

Introduction: We reviewed local control (LC) and overall survival (OS) post intracranial SRS to cavity post resection of brain metastases at one institution, and factors affecting LC.Methods: A retrospective review was conducted of adjuvant SRS at one institution from 2013 to 2016. Patient records, treatment plans and diagnostic images were reviewed. Local failure was MRI defined. Categorical variables were analysed using chi-square and Fisher's exact tests. Continuous variables were analysed using Mann-Whitney tests. The Kaplan-Meier method was used to estimate survival times and the log-rank test was used to compare differences in survival.Results: Forty-seven patients with 48 cavities were treated with SRS post operatively. LC rate was 69%, and the distant intracranial failure rate was 47% for entirety of the follow-up period. The 12-month freedom from local recurrence (FFLR) was 77% (63-91%). Median OS (95% CI) was 22.7 (14.6-30.8) months. Patients with a single metastasis had longer FFLR (30.1 vs 14.4 months; P = 0.014). Median interval from surgery to SRS was 6.3 weeks. Patients with interval >7 weeks had increased local recurrence (LR) (62%) than <7 weeks (37%), P = 0.025. Patients with a margin < 2 mm were more likely to experience LR (48%) than those with margin equal to 2 mm (20%); this approached statistical significance (P = 0.063). The median follow-up for all patients was 15.4 months (2-41).Conclusions: We determined LC and OS post adjuvant SRS at our institution. Based on the findings of this retrospective review SRS should be given promptly post operatively with a 2 mm PTV margin. [ABSTRACT FROM AUTHOR]

Horstick et al. (2013) previously reported a homozygous p.Trp284Ser variant in STAC3 as the cause of Native American myopathy (NAM) in 5 Lumbee Native American families with congenital hypotonia and weakness, cleft palate, short stature, ptosis, kyphoscoliosis, talipes deformities, and susceptibility to malignant hyperthermia (MH). Here we present two non-Native American families, who were found to have STAC3 pathogenic variants. The first proband and her affected older sister are from a consanguineous Qatari family with a suspected clinical diagnosis of Carey-Fineman-Ziter syndrome (CFZS) based on features of hypotonia, myopathic facies with generalized weakness, ptosis, normal extraocular movements, cleft palate, growth delay, and kyphoscoliosis. We identified the homozygous c.851G>C;p.Trp284Ser variant in STAC3 in both sisters. The second proband and his affected sister are from a non-consanguineous, Puerto Rican family who was evaluated for a possible diagnosis of Moebius syndrome (MBS). His features included facial and generalized weakness, minimal limitation of horizontal gaze, cleft palate, and hypotonia, and he has a history of MH. The siblings were identified to be compound heterozygous for STAC3 variants c.851G>C;p.Trp284Ser and c.763_766delCTCT;p.Leu255IlefsX58. Given the phenotypic overlap of individuals with CFZS, MBS, and NAM, we screened STAC3 in 12 individuals diagnosed with CFZS and in 50 individuals diagnosed with MBS or a congenital facial weakness disorder. We did not identify any rare coding variants in STAC3. NAM should be considered in patients presenting with facial and generalized weakness, normal or mildly abnormal extraocular movement, hypotonia, cleft palate, and scoliosis, particularly if there is a history of MH. [ABSTRACT FROM AUTHOR]

SMC1A encodes one of the proteins of the cohesin complex. SMC1A variants are known to cause a phenotype resembling Cornelia de Lange syndrome (CdLS). Exome sequencing has allowed recognizing SMC1A variants in individuals with encephalopathy with epilepsy who do not resemble CdLS. We performed an international, interdisciplinary study on 51 individuals with SMC1A variants for physical and behavioral characteristics, and compare results to those in 67 individuals with NIPBL variants. For the Netherlands all known individuals with SMC1A variants were studied, both with and without CdLS phenotype. Individuals with SMC1A variants can resemble CdLS, but manifestations are less marked compared to individuals with NIPBL variants: growth is less disturbed, facial signs are less marked (except for periocular signs and thin upper vermillion), there are no major limb anomalies, and they have a higher level of cognitive and adaptive functioning. Self-injurious behavior is more frequent and more severe in the NIPBL group. In the Dutch group 5 of 13 individuals (all females) had a phenotype that shows a remarkable resemblance to Rett syndrome: epileptic encephalopathy, severe or profound intellectual disability, stereotypic movements, and (in some) regression. Their missense, nonsense, and frameshift mutations are evenly spread over the gene. We conclude that SMC1A variants can result in a phenotype resembling CdLS and a phenotype resembling Rett syndrome. Resemblances between the SMC1A group and the NIPBL group suggest that a disturbed cohesin function contributes to the phenotype, but differences between these groups may also be explained by other underlying mechanisms such as moonlighting of the cohesin genes. [ABSTRACT FROM AUTHOR]

Cornelia de Lange syndrome (CdLS) is a multisystem genetic disorder associated with unusual facial features, limb abnormalities, a wide range of health conditions, and intellectual disability. Mutations in five genes that encode ( SMC1A, SMC3, RAD21) or regulate ( NIPBL, HDAC8) the cohesin complex have been identified in up to 70% of individuals. Genetic cause remains unknown for a proportion of individuals. There is substantial heterogeneity in all aspects of CdLS but very little is known about what predicts phenotypic heterogeneity. In this study, we evaluated genotype-phenotype associations in 34 individuals with CdLS. Participants with NIPBL mutations had significantly lower self help skills and were less likely to have verbal skills relative to those who were negative for the NIPBL mutation. No significant differences were identified between the groups in relation to repetitive behavior, mood, interest and pleasure, challenging behavior, activity, impulsivity, and characteristics of autism spectrum disorder whilst controlling differences in self help skills. Significant correlations indicating lower mood, interest and pleasure, and increased insistence on sameness with older age were identified for those who were NIPBL mutation positive. The findings suggest similarities in the behavioral phenotype between those with and without the NIPBL mutation once differences in self help skills are controlled for. However, there may be subtle differences in the developmental trajectory of these behaviors according to genetic mutation status in CdLS. [ABSTRACT FROM AUTHOR]

Objective The phenotype of seizure clustering with febrile illnesses in infancy/early childhood is well recognized. To date the only genetic epilepsy consistently associated with this phenotype is PCDH19, an X-linked disorder restricted to females, and males with mosaicism. The SMC1A gene, which encodes a structural component of the cohesin complex is also located on the X chromosome. Missense variants and small in-frame deletions of SMC1A cause approximately 5% of Cornelia de Lange Syndrome (Cd LS). Recently, protein truncating mutations in SMC1A have been reported in five females, all of whom have been affected by a drug-resistant epilepsy, and severe developmental impairment. Our objective was to further delineate the phenotype of SMC1A truncation. Method Female cases with de novo truncation mutations in SMC1A were identified from the Deciphering Developmental Disorders ( DDD) study (n = 8), from postmortem testing of an affected twin (n = 1), and from clinical testing with an epilepsy gene panel (n = 1). Detailed information on the phenotype in each case was obtained. Results Ten cases with heterozygous de novo mutations in the SMC1A gene are presented. All 10 mutations identified are predicted to result in premature truncation of the SMC1A protein. All cases are female, and none had a clinical diagnosis of Cd LS. They presented with onset of epileptic seizures between <4 weeks and 28 months of age. In the majority of cases, a marked preponderance for seizures to occur in clusters was noted. Seizure clusters were associated with developmental regression. Moderate or severe developmental impairment was apparent in all cases. Significance Truncation mutations in SMC1A cause a severe epilepsy phenotype with cluster seizures in females. These mutations are likely to be nonviable in males. [ABSTRACT FROM AUTHOR]

Background Exome sequencing has led to the discovery of mutations in novel causative genes for epilepsy. One such gene is EEF1A2, encoding a neuromuscular specific translation elongation factor, which has been found to be mutated de novo in five cases of severe epilepsy. We now report on a further seven cases, each with a different mutation, of which five are newly described. Methods New cases were identified and sequenced through the Deciphering Developmental Disabilities project, via direct contact with neurologists or geneticists, or recruited via our website. Results All the mutations cause epilepsy and intellectual disability, but with a much wider range of severity than previously identified. All new cases share specific subtle facial dysmorphic features. Each mutation occurs at an evolutionarily highly conserved amino acid position indicating strong structural or functional selective pressure. Conclusions EEF1A2 should be considered as a causative gene not only in cases of epileptic encephalopathy but also in children with less severe epilepsy and intellectual disability. The emergence of a possible discernible phenotype, a broad nasal bridge, tented upper lip, everted lower lip and downturned corners of the mouth may help in identifying patients with mutations in EEF1A2. [ABSTRACT FROM AUTHOR]

De novo mutations (DNM) in SYNGAP1, encoding Ras/Rap GTPase-activating protein SynGAP, have been reported in individuals with nonsyndromic intellectual disability (ID). We identified 10 previously unreported individuals with SYNGAP1 DNM; seven via the Deciphering Developmental Disorders (DDD) Study, one through clinical analysis for copy number variation and the remaining two (monozygotic twins) via a research multi-gene panel analysis. Seven of the nine heterozygous mutations are likely to result in loss-of-function (3 nonsense; 3 frameshift; 1 whole gene deletion). The remaining two mutations, one of which affected the monozygotic twins, were missense variants. Each individual carrying a DNM in SYNGAP1 had moderate-to-severe ID and 7/10 had epilepsy; typically myoclonic seizures, absences or drop attacks. 8/10 had hypotonia, 5/10 had significant constipation, 7/10 had wide-based/unsteady gait, 3/10 had strabismus, and 2/10 had significant hip dysplasia. A proportion of the affected individuals had a similar, myopathic facial appearance, with broad nasal bridge, relatively long nose and full lower lip vermilion. A distinctive behavioral phenotype was also observed with aggressive/challenging behavior and significant sleep problems being common. 7/10 individuals had MR imaging of the brain each of which was reported as normal. The clinical features of the individuals reported here show significant overlap with those associated with 6p21.3 microdeletions, confirming that haploinsufficiency for SYNGAP1 is responsible for both disorders. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Microdeletions of the 5q11.2 region are rare; in literature only two patients with a deletion in this region have been reported so far. In this study, we describe four additional patients and further define this new 5q11.2 microdeletion syndrome. A comparison of the features observed in all six patients with overlapping 5q11.2 deletions showed a phenotypic spectrum that overlaps with CHARGE syndrome and 22q11.2 deletion syndrome including choanal atresia, developmental delay, heart defects, external ear abnormalities, and short stature. No colobomas or abnormalities of semicircular canals and olfactory nerves were reported. Two male patients had genital abnormalities. We estimated a 2.0 Mb (53.0-55.0 Mb) Shortest Region of Overlap (SRO) for the main clinical characteristics of the syndrome. This region contains nine genes and two non-coding microRNAs. In this region DHX29 serves as the candidate gene as it encodes an ATP-dependent RNA-helicase that is involved in the initiation of RNA translation. Screening a small cohort of 14 patients who presented the main features, however, did not reveal any pathogenic abnormalities of DHX29. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The gene network that specifies flower shape in Antirrhinum majus (bilateral floral symmetry or zygomorphy) includes two MYB-class genes - RADIALIS ( RAD) and DIVARICATA ( DIV). RAD is involved in establishing the dorsal identity program and its role is to regulate the domain of activity of DIV (the ventral identity program) by restricting it to ventral regions of the flower., Plantago is in the same family as Antirrhinum but has small, radially symmetrical (actinomorphic) flowers derived from a zygomorphic ancestral state. Here we investigate the MYB-class floral symmetry genes and the role they have played in the evolution of derived actinomorphy in Plantago lanceolata., A DIV ortholog ( Pl DIV) but no RAD ortholog was identified in P. lanceolata. Pl DIV is expressed across all petals and stamens later in flower development, which is consistent with the loss of RAD gene function. Pl DIV expression in anther sporogenous tissue also suggests that Pl DIV was co-opted to regulate cell proliferation during the early stages of pollen development., These results indicate that evolution of derived actinomorphy in Plantago involved complete loss of dorsal gene function, resulting in expansion of the domain of expression of the ventral class of floral symmetry genes. [ABSTRACT FROM AUTHOR]

Purpose To develop and validate short axial and sagittal MRI scans (<1min) to assess in vivo patellofemoral contact areas in loaded knee flexion. Materials and Methods Contact area was assessed in four cadaver knee specimens from axial and sagittal scans using two contact area extraction techniques (delineation and intersection) and three calculation techniques (slice thickness multiplication, linear interpolation, and spline interpolation). Error was expressed as the mean absolute and percentage difference from a dye staining-based reference standard. Intrareader and intrasubject repeatability, expressed as the mean standard deviation, was determined. Results Contact area assessments from the sagittal MRI scans using the delineation and slice thickness multiplication technique had the smallest error (47.7 ± 38.1 mm2 or 10.7%). The intrareader repeatability from assessments using the sagittal scans was smaller than those using the axial scans when the delineation method was used (<9.4 ± 4.3 mm2 and <15.4 ± 14.1 mm2, respectively). The intrasubject repeatability of the assessment from the sagittal scan was less than 39.9 ± 23.0 mm2. Conclusion This protocol yields assessments of contact area in less than 1 minute that have errors similar to those made using scans many times longer and can be used in series with kinematic scans to carry out simultaneous assessments in vivo to study patellofemoral joint disease. J. Magn. Reson. Imaging 2014;39:978-987. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background Outbreaks of equine influenza ( EI) in endemic populations cause disruption and economic loss. Objectives To identify (i) factors involved in the spread of EI (ii) virus strains responsible for outbreaks (iii) single radial haemolysis ( SRH) antibody levels correlating with protection against current virus strains (iv) evidence of vaccination breakdown. Methods RT- PCR, virus isolation and SRH were carried out on nasopharyngeal swabs and blood samples collected from horses, ponies and donkeys on affected premises. Data relating to 629 samples from 135 equidae were analysed. Results and conclusions Outbreaks were sporadic, self limiting and associated with the movement of horses. Vaccination status and age influenced clinical signs of disease while housing and fomites contributed to virus spread. Subclinical infection as defined as a horse which tested positive by one or more of the following; RT- PCR, virus isolation and seroconversion in the absence of clinical signs, was identified in 9% of animals. Of the horses with up to date vaccination records 32% developed clinical signs. Vaccine breakdown occurred among horses vaccinated with all four commercially available vaccines. Analysis of HA1 sequence data generated for 26 viruses indicated that they all belonged to clade 2 of the Florida sublineage. Higher SRH antibody levels were required for both clinical and virological protection than reported in studies where vaccine strains were antigenically and genetically similar to those circulating in the field. The results of this study therefore support the OIE recommendations that vaccines be updated to include representatives of both clades of the Florida sublineage. [ABSTRACT FROM AUTHOR]

Do most academic historians of twentieth-century Ireland conform to a 'constitutional narrative', driven by a moral imperative to subvert republican interpretations? Has their shared political agenda led to widespread distortion, suppression, 'elision' and falsification of the evidential record? Is it high time to root out 'the insidious influence of Irish public histories presented as objective historical evidence'? In the view of John M. Regan, the answer in all cases is an emphatic 'yes'. This riposte to his recent article in History examines the credibility of Regan's critique, with particular attention to his claim that the late Peter Hart was guilty of 'academic fraud' in his controversial analysis of republican sectarianism and 'ethnic cleansing' in the Irish revolution. When closely examined, Regan's analysis seems perverse and untenable, implicitly relying on a reductionist conspiracy theory which ignores the rich variety and sound scholarship displayed by several generations of historians of modern Ireland. [ABSTRACT FROM AUTHOR]

Nablus mask-like facial syndrome (NMLFS) has many distinctive phenotypic features, particularly tight glistening skin with reduced facial expression, blepharophimosis, telecanthus, bulky nasal tip, abnormal external ear architecture, upswept frontal hairline, and sparse eyebrows. Over the last few years, several individuals with NMLFS have been reported to have a microdeletion of 8q21.3q22.1, demonstrated by microarray analysis. The minimal overlapping region is 93.98-96.22 Mb (hg19). Here we present clinical and microarray data from five singletons and two mother-child pairs who have heterozygous deletions significantly overlapping the region associated with NMLFS. Notably, while one mother and child were said to have mild tightening of facial skin, none of these individuals exhibited reduced facial expression or the classical facial phenotype of NMLFS. These findings indicate that deletion of the 8q21.3q22.1 region is necessary but not sufficient for development of the NMLFS. We discuss possible genetic mechanisms underlying the complex pattern of inheritance for this condition. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background: DNA variation in Interferon Regulatory Factor 6 ( IRF6) contributes risk for orofacial clefting, including a common DNA variant rs642961. This DNA variant is located in a multi-species conserved sequence that is 9.7 kb upstream from the IRF6 transcriptional start site ( MCS9.7). The MCS9.7 element was shown to possess enhancer activity that mimicked the expression of endogenous Irf6 at embryonic day 11.5 in transient transgenic embryos, and also contains a p63 binding site that transactivates IRF6 expression. To analyze whether the MCS9.7 enhancer is sufficient to drive IRF6 expression, we generated stable transgenic murine lines that carry a MCS9.7-lacZ transgene. We hypothesized that MCS9.7 was sufficient to recapitulate the endogenous expression of Irf6 at other time-points during embryonic development. Results: We observed that MCS9.7 activity recapitulated endogenous Irf6 expression in most tissues, but not in the medial edge epithelium (MEE) at E14.5, when Irf6 expression was high during secondary palatal fusion. Also, while MCS9.7 activity and Irf6 expression were associated with p63 expression, we observed MCS9.7 activity and Irf6 expression in periderm, although p63 was absent. Conclusion: These data suggest that MCS9.7 enhancer activity is not sufficient to recapitulate IRF6 expression, and that p63 expression is not always necessary nor sufficient for transactivation of IRF6. Developmental Dynamics 241:340-349, 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Summary Cryptococcus neoformans is a human fungal pathogen that is the causative agent of cryptococcosis and fatal meningitis in immuno-compromised hosts. Recent studies suggest that copper (Cu) acquisition plays an important role in C. neoformans virulence, as mutants that lack Cuf1, which activates the Ctr4 high affinity Cu importer, are hypo-virulent in mouse models. To understand the constellation of Cu-responsive genes in C. neoformans and how their expression might contribute to virulence, we determined the transcript profile of C. neoformans in response to elevated Cu or Cu deficiency. We identified two metallothionein genes ( CMT1 and CMT2), encoding cysteine-rich Cu binding and detoxifying proteins, whose expression is dramatically elevated in response to excess Cu. We identified a new C. neoformans Cu transporter, CnCtr1, that is induced by Cu deficiency and is distinct from CnCtr4 and which shows significant phylogenetic relationship to Ctr1 from other fungi. Surprisingly, in contrast to other fungi, we found that induction of both Cn CTR1 and Cn CTR4 expression under Cu limitation, and CMT1 and CMT2 in response to Cu excess, are dependent on the CnCuf1 Cu metalloregulatory transcription factor. These studies set the stage for the evaluation of the specific Cuf1 target genes required for virulence in C. neoformans. [ABSTRACT FROM AUTHOR]

A treatment process which integrates simulation, planning, and delivery in one single session of <=30 min on a treatment unit capable of cone-beam CT imaging (CBCT) is under development in our institution for palliation of spinal metastases. The objective of this work is to develop and validate a semiautomatic vertebra detection and identification algorithm to streamline the target definition process and improve the consistency of online planning on cone-beam CT data sets while the patient is on the treatment couch. Key issues pertaining to this work are the limited field of view and image quality of CBCT, the inter- and intrapatient variation of vertebra morphology, and the spine curvature. An initial library of ten patient CBCT data sets was used to derive the vertebra detection and identification method and set the parameters used by the algorithm. In this method, sagittal and coronal “curved” digitally reconstructed radiographs (cDRRs) are first created by projecting a subvolume of the CBCT data orthogonally to the centerline of a cylinder model positioned manually. The detection of the vertebra centers is then performed on the cDRRs based on an edge detection algorithm. The identification of the vertebrae by name is based on the detection of one or more of four different reference anatomical landmarks on cDRRs. The validation of the vertebra detection and identification algorithm was performed on a library of 27 patient CBCT data sets with an average detection success rate of 92.8% and 89.9% for sagittal and coronal cDRRs, respectively, for three different users. The entire process including manual steps and user approval was performed on average in 3.23–3.45 min (n=37, three users), with only 0.14 min for the automatic detection and identification of the vertebrae. The semiautomatic identification and segmentation of vertebrae on CBCT images was shown to be robust and effective. The next step will be the clinical implementation of the algorithm within the online planning and delivery treatment technique for patients with spinal bone metastases. [ABSTRACT FROM AUTHOR]

Purpose: Seizures are noted in a significant proportion of cases of de novo, heterozygous, loss-of-function mutations in SOX2, ascertained because of severe bilateral eye malformations. We wished to determine the underlying cerebral phenotype in SOX2 mutation and to test the candidacy of SOX2 as a gene contributing to human epilepsies. Methods: We examined high-resolution MRI scans in four patients with SOX2 mutations, two of whom had seizures. We determined the Sox2 expression pattern in developing murine brain. We searched for SOX2 mutation in 24 patients with typical hippocampal sclerosis and for common variations in SOX2 in 655 patients without eye disease but with epilepsy, including 91 patients with febrile seizures, 93 with hippocampal sclerosis, and 258 with temporal lobe epilepsy. Results: Striking hippocampal and parahippocampal malformations were seen in all cases, with a history of febrile seizures or epilepsy in two of four cases. The Sox2 expression pattern in developing mouse brain supports the pattern of malformations observed. Mutation screening in patients with epilepsy did not reveal any abnormalities in SOX2. No associations were found between any clinical epilepsy phenotype and common variation in SOX2. Conclusions: SOX2 haploinsufficiency causes mesial temporal malformation in humans, making SOX2 dysfunction a candidate mechanism for mesial temporal abnormalities associated with chronic epilepsy. However, although mutation of SOX2 in humans causes hippocampal malformation, SOX2 mutation or variation is unlikely to contribute commonly to mesial temporal lobe epilepsy or its structural (hippocampal sclerosis) or historic (febrile seizures) associations in humans. [ABSTRACT FROM AUTHOR]

Programmed death-1 ligand 2 (PD-L2) is a ligand for programmed death-1 (PD-1), a receptor that plays an inhibitory role in T cell activation. Since previous studies have shown up-regulation of PD-L2 expression by Th2 cytokines, and asthma is driven by a Th2 response, we hypothesized that PD-L2 might be involved in regulation of the immune response in this disease. We have found that lungs from asthmatic mice had sustained up-regulation of PD-1 and PD-L2, with PD-L2 primarily on dendritic cells. Although addition of PD-L2-Fc in vitro led to decreased T cell proliferation and cytokine production, administration of PD-L2-Fc in vivo in a mouse asthma model resulted in elevated serum IgE levels, increased eosinophilic and lymphocytic infiltration into bronchoalveolar lavage fluid, higher number of cells in the draining lymph nodes, and production of IL-5 and IL-13 from these cells. Although PD-1 was expressed on regulatory T cells, PD-L2-Fc did not affect regulatory T cell activity in vitro. This study provides in vivo evidence of an exacerbated inflammatory response following PD-L2-Fc administration and indicates a potential role for this molecule in Th2-mediated diseases such as asthma. [ABSTRACT FROM AUTHOR]

Illustrates the compounds present in grapes and wines responsible for endothelium-dependent relaxation (EDR) activity of blood vessels. Several cardiovascular protective effects of nitric oxide (NO); Some EDR impairments; How the compounds were separated by high-performance liquid chromatography (HPLC); Discussion of the EDR activity in wine.

Ca-dependent degradation of triphosphoinositide has been postulated to regulate levels of membrane-bound Ca and to generate a 1,2-diacylglycerol fusogen in cell fusion. Triphosphoinositide metabolism was therefore studied during Ca-induced fusion of cultured chick embryo myoblasts. Using a frequently cited extraction procedure, it was found that apparent Ca-dependent triphosphoinositide degradation was actually due to inhibition of extraction. A new procedure using the ion-pairing reagent tetrabutylammonium sulfate was developed which was unaffected by Ca and gave 2- to 20-fold greater extraction of triphosphoinositide than existing procedures. With this procedure, no changes in triphosphoinositide metabolism were found during myoblast fusion. [ABSTRACT FROM AUTHOR]