Characterization of Cre recombinase mouse lines enabling cell type-specific targeting of postnatal intervertebral discs

Cre/loxP technology is an important tool for studying cell type-specific gene functions. Cre recombinase mouse lines, including Agc1-CreER^T2, Col2a1-Cre; Col2a1-CreER^T2, Shh-Cre, Shh-CreER^T2, and Osx-Cre, have been proven to be valuable tools to elucidate the biology of long bones, yet the information for their activity in postnatal intervertebral disc (IVD) tissues was very limited. In this study, we used R26-mTmG fluorescent reporter to systematically analyze cell specificity and targeting efficiency of these six mouse lines in IVD tissues at postnatal growing and adult stages. We found that Agc1-CreER^T2 is effective to direct recombination in all components of IVDs, including annulus fibrosus (AF), nucleus pulposus (NP), and cartilaginous endplate (CEP), upon tamoxifen induction at either 2 weeks or 2 months of ages. Moreover, Col2a1-Cre targets most of the cells in IVDs, except for some cells in the outer AF (OAF) and NP. In contrast, the activity of Col2a1-CreER^T2 is mainly limited to the IAF of IVD tissues at either stage of tamoxifen injection. Similarly, Shh-Cre directs recombination specifically in all NP cells, whereas Shh-CreER^T2 is active only in a few NP cells when tamoxifen is administered at either stage. Finally, Osx-Cre targets cells in the CEP, but not in the NP or AF of IVDs tissues at these two stages. Thus, our data demonstrated that all these Cre lines can direct recombination in IVD tissues at postnatal stages with different cell type specificity and/or targeting efficiency, and can, therefore, serve as valuable tools to dissect cell type-specific gene functions in IVD development and homeostasis.     

Exploring the Factor Structure of Neurocognitive Measures in Older Individuals

Here we focus on factor analysis from a best practices point of view, by investigating the factor structure of neuropsychological tests and using the results obtained to illustrate on choosing a reasonable solution. The sample (n=1051 individuals) was randomly divided into two groups: one for exploratory factor analysis (EFA) and principal component analysis (PCA), to investigate the number of factors underlying the neurocognitive variables; the second to test the “best fit” model via confirmatory factor analysis (CFA). For the exploratory step, three extraction (maximum likelihood, principal axis factoring and principal components) and two rotation (orthogonal and oblique) methods were used. The analysis methodology allowed exploring how different cognitive/psychological tests correlated/discriminated between dimensions, indicating that to capture latent structures in similar sample sizes and measures, with approximately normal data distribution, reflective models with oblimin rotation might prove the most adequate.     

Connecting Proline and γ-Aminobutyric Acid in Stressed Plants through Non-Enzymatic Reactions

The accumulation of proline (Pro) in plants exposed to biotic/abiotic stress is a well-documented and conserved response in most vegetal species. Stress conditions induce the overproduction of reactive oxygen species which can lead to cellular damage. In vitro assays have shown that enzyme inactivation by hydroxyl radicals (^·OH) can be avoided in presence of Pro, suggesting that this amino acid could act as an ^·OH scavenger. We applied Density Functional Theory coupled with a polarizable continuum model to elucidate how Pro reacts with ^·OH. In this work we suggest that Pro reacts favourably with ^·OH by H–abstraction on the amine group. This reaction produces the spontaneous decarboxylation of Pro leading to the formation of pyrrolidin-1-yl. In turn, pyrrolidin-1-yl can easily be converted to Δ¹-pyrroline, the substrate of the enzyme Δ¹-pyrroline dehydrogenase, which produces γ-aminobutyric acid (GABA). GABA and Pro are frequently accumulated in stressed plants and several protective roles have been assigned to these molecules. Thereby we present an alternative non-enzymatic way to synthetize GABA under oxidative stress. Finally this work sheds light on a new beneficial role of Pro accumulation in the maintenance of photosynthetic activity.     

Rare species habitat suitability assessment and reliability evaluation of an expert-based model: A case study of the insectivorous plant Pinguicula crystallina Sibth. et Smith subsp. hirtiflora (Ten.) Strid (Lentibulariaceae)

The development of habitat suitability models requires a large amount of data which are rarely available. In this case, researchers need to get information on the ecological features of the studied species, based on the opinion of experts or on the literature, to construct a qualitative model. However, such models cannot be rigorously evaluated, as in most cases absence points are not available. In this paper, we assess the habitat suitability for a vulnerable insectivorous plant, Pinguicula crystallina Sibth. et Smith subsp. hirtiflora (Ten.) Strid (Lentibulariaceae) in the Campania region. Our aim was to develop an expert-based, presence-only model in support of possible conservation actions. Topographic and geological features of this species suggested by the literature were used in our model. Both the Boyce index and field surveys were chosen to evaluate the model's reliability. During field surveys, 31 absence sites and 1 new presence site were identified, and differences between sites with regard to water chemistry and quality were investigated, water being an element in the species habitat. Factors that affect reliability of the model, such as the lack of a large amount of information on the species and the limited spatial resolution of geographical information system data, are discussed.     

In vitro induction of haploid,diploid and triploid plantlets by anther culture of Iochroma warscewiczii Regel

Pollen of Iochroma warscewiczii Regel (Solanaceae) produced embryogenic calli or embryos inside anthers cultured on Nitsch & Nitsch medium. Two distinct pathways could be recognized in this process, one involving mainly the vegetative cell, and the second starting with two equal cells in the pollen grains.In all media tested, androgenesis initiation was highest when anthers contained pollen at the first mitosis, or close to it, at inoculation. High sucrose (7%) and calcium (11.3 mM) concentrations were found to be highly desirable for the induction of androgenesis in this species. Addition of benzylaminopurine (0.5 mg l^–1) to the culture medium seems to slightly improve callus or embryo production. When all three factors were present at optimal concentrations as much as 13.9% of inoculated anthers were found to be embryogenic.Plantlet development from pollen embryos required lower sucrose (3%) and a combination of 0.1 mg l^–1 benzylaminopurine and 0.5 mg l^–1 gibberellic acid in the culture medium. Cytological analysis of 55 regenerated plantlets showed that about 49% were haploids, but diploid (ca. 49%) and triploid (ca. 2%) plants were also obtained.     

Data quality aware analysis of differential expression in RNA-seq with NOISeq R/Bioc package

As the use of RNA-seq has popularized, there is an increasing consciousness of the importance of experimental design, bias removal, accurate quantification and control of false positives for proper data analysis. We introduce the NOISeq R-package for quality control and analysis of count data. We show how the available diagnostic tools can be used to monitor quality issues, make pre-processing decisions and improve analysis. We demonstrate that the non-parametric NOISeqBIO efficiently controls false discoveries in experiments with biological replication and outperforms state-of-the-art methods. NOISeq is a comprehensive resource that meets current needs for robust data-aware analysis of RNA-seq differential expression.     

Anatomy of the antennal dorsal organ in female of Neodryinus typhlocybae (Hymenoptera: Dryinidae): A peculiar sensory structure possibly involved in perception of host vibration

Neodryinus typhlocybae (Hymenoptera: Dryinidae) is a natural enemy of the planthopper Metcalfa pruinosa, which was introduced from North America into Europe and has become established in various regions as a pest species. Vibrational signals play a crucial role in the communication of M. pruinosa, which appears to be exploited by N. typhlocybae. Scanning and transmission electron microscopy have shown that the antennae of N. typhlocybae females have peculiar and complex sensory structures: deep longitudinal grooves that house long sensilla trichodea, termed here “Antennal Dorsal Organs.” Such structures were not present on male antennae. These sensilla extend for the length of the grooves, without contact with the groove cuticle. Their hair shaft is empty and aporous, and inserted into a specialized socket, underneath which there is a cuticular ampulla‐like chamber. Each sensillum is associated with two sensory neurons: one terminates at the proximal end of the dendritic sheath; the other continues into the sensillum sinus and is enclosed in the dendritic sheath. This second sensory neuron then enters the ampulla‐like chamber through the circular opening, and then terminates with a conspicuous tubular body at the shaft base. The possible involvement of this peculiar structure in the context of host recognition mechanism is discussed. J. Morphol. 277:128–137, 2016. © 2015 Wiley Periodicals, Inc.     

Theory of allosteric effects in serine proteases.

The classical Botts-Morales theory for the action of a modifier on the catalytic properties of an enzyme has been extended to deal with allosteric effects in serine proteases. The exact analytical solution derived for the linkage scheme at steady state provides a rigorous framework for the study of many biologically relevant systems, including enzymes activated by monovalent cations and cofactor-controlled protease-zymogen interactions in blood coagulation. When the enzyme obeys Michaelis-Menten kinetics, the exact solution of the kinetic linkage scheme simplifies considerably. Of particular importance for practical applications is a simple equation expressing the dependence of the specificity constant of the enzyme, kcat/Km, on the concentration of the modifier, from which the equilibrium binding constant for the formation of the enzyme-modifier complex can be estimated. Analysis of the allosteric changes in thrombin activity induced by thrombomodulin and Na+ in terms of this equation yields accurate determinations of the equilibrium binding constants for both effectors.     

Deposition of BaSO4 in the tight junctions of amphibian epithelia causes their opening; apical Ca2+ reverses this effect

Selective deposition of BaSO₄ in the tight junctions (TJs) of frog skins led to profound and reversible functional alterations of these structures, as revealed by changes of tissue conductance (G), clamping current (I), and fluxes of extracellular markers (sulfate (J^SO ₄) and sucrose (J^SUC)). Experiments were performed with nominally Ca²⁺ -free simple salt solutions on the apical side (usually KCl) and Na₂SO₄-Ringer on the inner side of skins. The deposition of BaSO₄ in the TJs was obtained by diffusion and/or migration through the paracellular path of Ba²⁺ from the apical solution and SO ₄ ^2– from the inner solution. A brief presence (2 to 6 min) of apical Ba²⁺ (Ba²⁺ pulse) is followed (i.e., when Ba²⁺ is removed from the apical fluid) by a large increase of G, I, J^SO ₄ and J^SUC, above pre-Ba²⁺ levels. These attain a steady state within 15 to 30 min (overshoot phase), characterizing a conspicuous increase of the paracellular permeability. During the overshoot phase, a second Ba²⁺ pulse blocks the paracellular route while apical Ba²⁺ is present, leading to a new and larger overshoot when the Ba²⁺ pulse is terminated. Addition of apical Ca²⁺ triggers the resealing of the TJs, resulting in a full recovery of G, I, J^SO ₄ and J^SUC. This Ca²⁺ -induced recovery persists when apical Ca²⁺ is removed. The presence of a normal Ca²⁺ concentration in the inner bathing Ringer does not induce the recovery process. Tissues remain viable after being submitted to the Ba²⁺ treatment and the subsequent overshoot. Experiments performed in the urinary bladder of Rana catesbeiana and skins and urinary bladders of Bufo marinus indicate that Ba²⁺ effect can also be elicited in these tissues. The above results seem to report general properties of the TJs. Incidentally, they warn about the use of Ba²⁺ as an ion channel blocker in epithelial membranes in association with SO ₄ ^2– -containing solutions on the contralateral side.This project was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (91/0293-7 to F.L.V., and 90/1788-1 to A.S.), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (410068/90-0 and 303633-85/BF to F.L.V.). J.A.C. received a doctoral fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/Fundação Universidade do Rio Grande. We thank Dr. Alice T. Ferreira for help in the measurements of free Ca²⁺ concentration.