Diagnostic Alleles From Electrophoresis Distinguish Two Noctuid Pest Species, \u3ci\u3eHydraecia Immanis\u3c/i\u3e and \u3ci\u3eH. Micacea\u3c/i\u3e (Lepidoptera: Noctuidae).

Native hop vine borer (Hydraecia immanis) and introduced potato stem borer (H. micacea) populations in Midwest corn have reached noticeable levels near the Great Lakes plant community ecotone between boreal forests and temperate deciduous forests. The hop vine borer is more specialized in its diet and occurs in corn generally south of the plant community ecotone, whereas the potato stem borer is polyphagous and occurs in corn mostly north of the Great Lakes plant transition zone. We analyzed the genetic composition of each species using cellulose acetate electrophoresis and resolved 19 loci of which 6 exhibited fixed or nearly fixed allelic differences. We expect that this will be useful in determining the degree of hybridization where the two species become sympatric due to expected continued range expansions in Michigan, Wisconsin, and New York State

Radiometric observations of atmospheric attenuation at 20.6 and 31.65 GHz: The Wave Propagation Laboratory data base

The National Oceanic and Atmospheric Administration (NOAA) Wave Propagation Laboratory (WPL) presently operates five dual-channel microwave radiometers, one triple-channel microwave radiometer, and one six-channel microwave radiometer. The dual-channel radiometers operate at frequencies of 20.6 or 23.87 GHz and 31.4 or 31.65 GHz. The triple-channel radiometer operates at 20.6, 31.65, and 90.0 GHz. The six-channel radiometer operates at frequencies of 20.6, 31.65, 52.85, 53.85, 55.45, and 58.8 GHz. Recent brightness temperature measurements and attenuation values from some of the above radiometers are presented. These radiometric measurements, taken in different locations throughout the world, have given WPL a diverse set of measurements under a variety of atmospheric conditions. We propose to do a more complete attenuation analysis on these measurements in the future. In addition, a new spinning reflector was installed recently for the dual-channel radiometer at the Platteville, Colorado site. This reflector will extend our measurement capabilities during precipating conditions. Locating the three-channel and portable dual-channel radiometers at or near Greeley, Colorado to support the Advanced Communications Technology Satellite (ACTS) program is discussed

Dissecting a Charged Network at the Active Site of Orotidine-5′-phosphate Decarboxylase

The crystal structure of yeast orotidine-5'-phosphate decarboxylase in complex with the postulated transition state analog, 6-hydroxyuridine-5'-phosphate, reveals contacts between this inhibitor and a novel quartet of charged residues (Lys-59, Asp-91, Lys-93, and Asp-96) within the active site. The structure also suggests a possible interaction between O2 of the 6-hydroxyuridine-5'-phosphate pyrimidine ring and Gln-215. Here we report the results of mutagenesis of each of the charged active site residues and Gln-215. The activities of the Q215A and wild-type enzymes were equal indicating that any interactions between this residue and the pyrimidine ring are dispensable for efficient decarboxylation. For the D91A and K93A mutant enzymes, activity was reduced by more than 5 orders of magnitude and substrate binding could not be detected by isothermal calorimetry. For the D96A mutant enzyme, k(cat) was reduced by more than 5 orders of magnitude, and isothermal calorimetry indicated an 11-fold decrease in the affinity of this enzyme for the substrate in the ground state. For the K59A enzyme, k(cat) was reduced by a factor of 130, and K(m) had increased by a factor of 900. These results indicate that the integrity of the network of charged residues is essential for transition state stabilization

Biological constraints limit the use of rapamycin-inducible FKBP12-Inp54p for depleting PIP2 in dorsal root ganglia neurons

Background: Rapamycin-induced translocation systems can be used to manipulate biological processes with precise temporal control. These systems are based on rapamycin-induced dimerization of FK506 Binding Protein 12 (FKBP12) with the FKBP Rapamycin Binding (FRB) domain of mammalian target of rapamycin (mTOR). Here, we sought to adapt a rapamycin-inducible phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phosphatase (Inp54p) system to deplete PIP2 in nociceptive dorsal root ganglia (DRG) neurons. Results: We genetically targeted membrane-tethered CFP-FRBPLF (a destabilized FRB mutant) to the ubiquitously expressed Rosa26 locus, generating a Rosa26-FRBPLF knockin mouse. In a second knockin mouse line, we targeted Venus-FKBP12-Inp54p to the Calcitonin gene-related peptide-alpha (CGRPα) locus. We hypothesized that after intercrossing these mice, rapamycin treatment would induce translocation of Venus-FKBP12-Inp54p to the plasma membrane in CGRP+ DRG neurons. In control experiments with cell lines, rapamycin induced translocation of Venus-FKBP12-Inp54p to the plasma membrane, and subsequent depletion of PIP2, as measured with a PIP2 biosensor. However, rapamycin did not induce translocation of Venus-FKBP12-Inp54p to the plasma membrane in FRBPLF-expressing DRG neurons (in vitro or in vivo). Moreover, rapamycin treatment did not alter PIP2-dependent thermosensation in vivo. Instead, rapamycin treatment stabilized FRBPLF in cultured DRG neurons, suggesting that rapamycin promoted dimerization of FRBPLF with endogenous FKBP12. Conclusions: Taken together, our data indicate that these knockin mice cannot be used to inducibly deplete PIP2 in DRG neurons. Moreover, our data suggest that high levels of endogenous FKBP12 could compete for binding to FRBPLF, hence limiting the use of rapamycin-inducible systems to cells with low levels of endogenous FKBP12

An Autism-Linked Mutation Disables Phosphorylation Control of UBE3A

Deletion of UBE3A causes the neurodevelopmental disorder Angelman syndrome (AS) while duplication or triplication of UBE3A is linked to autism. These genetic findings suggest that the ubiquitin ligase activity of UBE3A must be tightly maintained to promote normal brain development. Here, we found that protein kinase A (PKA) phosphorylates UBE3A in a region outside the catalytic domain, at residue T485, and inhibits UBE3A activity towards itself and other substrates. A de novo autism-linked missense mutation disrupts this phosphorylation site, causing enhanced UBE3A activity in vitro, enhanced substrate turnover in patient-derived cells, and excessive dendritic spine development in the brain. Our study identifies PKA as an upstream regulator of UBE3A activity, and shows that an autism-linked mutation disrupts this phosphorylation control. Moreover, our findings implicate excessive UBE3A activity and the resulting synaptic dysfunction to autism pathogenesis

An Autism-Linked Mutation Disables Phosphorylation Control of UBE3A

Deletion of UBE3A causes the neurodevelopmental disorder Angelman syndrome (AS) while duplication or triplication of UBE3A is linked to autism. These genetic findings suggest that the ubiquitin ligase activity of UBE3A must be tightly maintained to promote normal brain development. Here, we found that protein kinase A (PKA) phosphorylates UBE3A in a region outside the catalytic domain, at residue T485, and inhibits UBE3A activity towards itself and other substrates. A de novo autism-linked missense mutation disrupts this phosphorylation site, causing enhanced UBE3A activity in vitro, enhanced substrate turnover in patient-derived cells, and excessive dendritic spine development in the brain. Our study identifies PKA as an upstream regulator of UBE3A activity, and shows that an autism-linked mutation disrupts this phosphorylation control. Moreover, our findings implicate excessive UBE3A activity and the resulting synaptic dysfunction to autism pathogenesis

Aromatase inhibition remodels the clonal architecture of estrogen-receptor-positive breast cancers

Resistance to oestrogen-deprivation therapy is common in oestrogen-receptor-positive (ER+) breast cancer. To better understand the contributions of tumour heterogeneity and evolution to resistance, here we perform comprehensive genomic characterization of 22 primary tumours sampled before and after 4 months of neoadjuvant aromatase inhibitor (NAI) treatment. Comparing whole-genome sequencing of tumour/normal pairs from the two time points, with coincident tumour RNA sequencing, reveals widespread spatial and temporal heterogeneity, with marked remodelling of the clonal landscape in response to NAI. Two cases have genomic evidence of two independent tumours, most obviously an ER− ‘collision tumour', which was only detected after NAI treatment of baseline ER+ disease. Many mutations are newly detected or enriched post treatment, including two ligand-binding domain mutations in ESR1. The observed clonal complexity of the ER+ breast cancer genome suggests that precision medicine approaches based on genomic analysis of a single specimen are likely insufficient to capture all clinically significant information

A comparison of PAM50 intrinsic subtyping with immunohistochemistry and clinical prognostic factors in tamoxifen-treated estrogen receptor-positive breast cancer

Purpose: To compare clinical, immunohistochemical (IHC), and gene expression models of prognosis applicable to formalin-fixed, paraffin-embedded blocks in a large series of estrogen receptor (ER)–positive breast cancers from patients uniformly treated with adjuvant tamoxifen. Experimental Design: Quantitative real-time reverse transcription-PCR (qRT-PCR) assays for 50 genes identifying intrinsic breast cancer subtypes were completed on 786 specimens linked to clinical (median follow-up, 11.7 years) and IHC [ER, progesterone receptor (PR), HER2, and Ki67] data. Performance of predefined intrinsic subtype and risk-of-relapse scores was assessed using multivariable Cox models and Kaplan-Meier analysis. Harrell's C-index was used to compare fixed models trained in independent data sets, including proliferation signatures. Results: Despite clinical ER positivity, 10% of cases were assigned to nonluminal subtypes. qRT-PCR signatures for proliferation genes gave more prognostic information than clinical assays for hormone receptors or Ki67. In Cox models incorporating standard prognostic variables, hazard ratios for breast cancer disease-specific survival over the first 5 years of follow-up, relative to the most common luminal A subtype, are 1.99 [95% confidence interval (CI), 1.09-3.64] for luminal B, 3.65 (95% CI, 1.64-8.16) for HER2-enriched subtype, and 17.71 (95% CI, 1.71-183.33) for the basal-like subtype. For node-negative disease, PAM50 qRT-PCR–based risk assignment weighted for tumor size and proliferation identifies a group with >95% 10-year survival without chemotherapy. In node-positive disease, PAM50-based prognostic models were also superior. Conclusion: The PAM50 gene expression test for intrinsic biological subtype can be applied to large series of formalin-fixed, paraffin-embedded breast cancers, and gives more prognostic information than clinical factors and IHC using standard cut points