GABI-Kat SimpleSearch: new features of the Arabidopsis thaliana T-DNA mutant database

T-DNA insertion mutants are very valuable for reverse genetics in Arabidopsis thaliana. Several projects have generated large sequence-indexed collections of T-DNA insertion lines, of which GABI-Kat is the second largest resource worldwide. User access to the collection and its Flanking Sequence Tags (FSTs) is provided by the front end SimpleSearch (http://www.GABI-Kat.de). Several significant improvements have been implemented recently. The database now relies on the TAIRv10 genome sequence and annotation dataset. All FSTs have been newly mapped using an optimized procedure that leads to improved accuracy of insertion site predictions. A fraction of the collection with weak FST yield was re-analysed by generating new FSTs. Along with newly found predictions for older sequences about 20 000 new FSTs were included in the database. Information about groups of FSTs pointing to the same insertion site that is found in several lines but is real only in a single line are included, and many problematic FST-to-line links have been corrected using new wet-lab data. SimpleSearch currently contains data from ∼71 000 lines with predicted insertions covering 62.5% of the 27 206 nuclear protein coding genes, and offers insertion allele-specific data from 9545 confirmed lines that are available from the Nottingham Arabidopsis Stock Centre

Protein Kinase A Governs Oxidative Phosphorylation Kinetics and Oxidant Emitting Potential at Complex I

he mitochondrial electron transport system (ETS) is responsible for setting and maintaining both the energy and redox charges throughout the cell. Reversible phosphorylation of mitochondrial proteins, particularly via the soluble adenylyl cyclase (sAC)/cyclic AMP (cAMP)/Protein kinase A (PKA) axis, has recently been revealed as a potential mechanism regulating the ETS. However, the governance of cAMP/PKA signaling and its implications on ETS function are incompletely understood. In contrast to prior reports using exogenous bicarbonate, we provide evidence that endogenous CO2 produced by increased tricarboxylic acid (TCA) cycle flux is insufficient to increase mitochondrial cAMP levels, and that exogenous addition of membrane permeant 8Br-cAMP does not enhance mitochondrial respiratory capacity. We also report important non-specific effects of commonly used inhibitors of sAC which preclude their use in studies of mitochondrial function. In isolated liver mitochondria, inhibition of PKA reduced complex I-, but not complex II-supported respiratory capacity. In permeabilized myofibers, inhibition of PKA lowered both the Km and Vmax for complex I-supported respiration as well as succinate-supported H2O2 emitting potential. In summary, the data provided here improve our understanding of how mitochondrial cAMP production is regulated, illustrate a need for better tools to examine the impact of sAC activity on mitochondrial biology, and suggest that cAMP/PKA signaling contributes to the governance of electron flow through complex I of the ETS

DETERMINATION OF MISCONCEPTIONS REGARDING "COMPOUNDS'' CHAPTER IN SECONDARY EDUCATION 9th GRADE BY TWO-TIER CONCEPTUAL UNDERSTANDING TEST

Bu çalışmada ortaöğretim 9. sınıf öğrencilerinin kimya dersi "Bileşikler" ünitesi ile ilgili kavram yanılgılarına sahip olup olmadıklarını ve sahip oldukları kavram yanılgıları varsa bu yanılgıların derecesini tespit edebilmek için iki aşamalı kavramsal anlama testini geliştirmek amaçlanmıştır. Çalışmada ayrıca geliştirilen bu test yoluyla öğrencilerin kavram yanılgılarının belirlenmesi hedeflenmiştir. Toplam 34 maddeden oluşan test İzmir ilinde bulunan ortaöğretim kurumlarında öğrenim görmekte olan ve konuyu bilen toplam 175 öğrenciye uygulanmıştır. Yapılan madde analizleri sonucunda testin güvenirliği, maddelerin güçlük ve ayırt edicilik indeksleri ve çeldirici fonksiyonları elde edilmiştir. Madde analizlerinin sonucunda iki aşamalı kavramsal anlama testinden 10 madde çıkarılmıştır. Testin güvenirlik katsayısı (Cronbach Alfa) 0.80 olarak bulunmuştur. Testin madde güçlük indeksleri 0.24-0.90 aralığında, ayırt edicilik indeksleri ise 0.22-0.50 aralığında bulunmuştur. Testte her bir maddeye verilen yanıtların incelenmesi sonucunda, ünite ile ilgili öğrencilerin bazı kavram yanılgılarına sahip oldukları belirlenmiştir. The aim of this study is to determine whether 9th grade students in secondary schools have misconceptions regarding ‘'compounds'' chapter and to develope a two-tier conceptual understanding test to be able to determine the degree of the misconceptions if there are any. Furthermore; this study aims determining the the misconceptions of the students by means of this test. The test, consisting of a total of 34 items, was carried out with 175 students familiar with the subject and receiving education in secondary schools in Izmir. As a result of item analyses made, the reliability of the test, the difficulty and distinctiveness indices and the distractor functions of the items were acquired. As a result of the analysis item two-tier conceptual understanding test 10 items were removed.The relaibility coefficient of the test (Cronbach Alfa) was found as 0.796. As a result of item analyses, the item difficulty indices were found to be between 0.24-0.90 and the item discrimination index were between 0.20-0.50. Some misconceptions about compounds chapter were determined after the analysis of the items in the tes

PCP-B class pollen coat proteins are key regulators of the hydration checkpoint in Arabidopsis thaliana pollen-stigma interactions

The establishment of pollen–pistil compatibility is strictly regulated by factors derived from both male and female reproductive structures. Highly diverse small cysteine-rich proteins (CRPs) have been found to play multiple roles in plant reproduction, including the earliest stages of the pollen–stigma interaction. Secreted CRPs found in the pollen coat of members of the Brassicaceae, the pollen coat proteins (PCPs), are emerging as important signalling molecules that regulate the pollen–stigma interaction. Using a combination of protein characterization, expression and phylogenetic analyses we identified a novel class of Arabidopsis thaliana pollen-borne CRPs, the PCP-Bs (for pollen coat protein B-class) that are related to embryo surrounding factor (ESF1) developmental regulators. Single and multiple PCP-B mutant lines were utilized in bioassays to assess effects on pollen hydration, adhesion and pollen tube growth. Our results revealed that pollen hydration is severely impaired when multiple PCP-Bs are lost from the pollen coat. The hydration defect also resulted in reduced pollen adhesion and delayed pollen tube growth in all mutants studied. These results demonstrate that AtPCP-Bs are key regulators of the hydration ‘checkpoint’ in establishment of pollen–stigma compatibility. In addition, we propose that interspecies diversity of PCP-Bs may contribute to reproductive barriers in the Brassicaceae

Directed evolution of and structural insights into antibody-mediated disruption of a stable receptor-ligand complex.

Antibody drugs exert therapeutic effects via a range of mechanisms, including competitive inhibition, allosteric modulation, and immune effector mechanisms. Facilitated dissociation is an additional mechanism where antibody-mediated "disruption" of stable high-affinity macromolecular complexes can potentially enhance therapeutic efficacy. However, this mechanism is not well understood or utilized therapeutically. Here, we investigate and engineer the weak disruptive activity of an existing therapeutic antibody, omalizumab, which targets IgE antibodies to block the allergic response. We develop a yeast display approach to select for and engineer antibody disruptive efficiency and generate potent omalizumab variants that dissociate receptor-bound IgE. We determine a low resolution cryo-EM structure of a transient disruption intermediate containing the IgE-Fc, its partially dissociated receptor and an antibody inhibitor. Our results provide a conceptual framework for engineering disruptive inhibitors for other targets, insights into the failure in clinical trials of the previous high affinity omalizumab HAE variant and anti-IgE antibodies that safely and rapidly disarm allergic effector cells