Custom Distinctions in the Interaction of G-protein β Subunits with N-type (CaV2.2) Versus P/Q-type (CaV2.1) Calcium Channels

Inhibition of N- (Cav2.2) and P/Q-type (Cav2.1) calcium channels by G-proteins contribute importantly to presynaptic inhibition as well as to the effects of opiates and cannabinoids. Accordingly, elucidating the molecular mechanisms underlying G-protein inhibition of voltage-gated calcium channels has been a major research focus. So far, inhibition is thought to result from the interaction of multiple proposed sites with the Gβγ complex (Gβγ). Far less is known about the important interaction sites on Gβγ itself. Here, we developed a novel electrophysiological paradigm, “compound-state willing-reluctant analysis,” to describe Gβγ interaction with N- and P/Q-type channels, and to provide a sensitive and efficient screen for changes in modulatory behavior over a broad range of potentials. The analysis confirmed that the apparent (un)binding kinetics of Gβγ with N-type are twofold slower than with P/Q-type at the voltage extremes, and emphasized that the kinetic discrepancy increases up to ten-fold in the mid-voltage range. To further investigate apparent differences in modulatory behavior, we screened both channels for the effects of single point alanine mutations within four regions of Gβ1, at residues known to interact with Gα. These residues might thereby be expected to interact with channel effectors. Of eight mutations studied, six affected G-protein modulation of both N- and P/Q-type channels to varying degrees, and one had no appreciable effect on either channel. The remaining mutation was remarkable for selective attenuation of effects on P/Q-, but not N-type channels. Surprisingly, this mutation decreased the (un)binding rates without affecting its overall affinity. The latter mutation suggests that the binding surface on Gβγ for N- and P/Q-type channels are different. Also, the manner in which this last mutation affected P/Q-type channels suggests that some residues may be important for “steering” or guiding the protein into the binding pocket, whereas others are important for simply binding to the channel

Obtaining deeper insights into microbiome diversity using a simple method to block host and nontargets in amplicon sequencing

Abstract Profiling diverse microbiomes is revolutionizing our understanding of biological mechanisms and ecologically relevant problems, including metaorganism (host + microbiome) assembly, functions and adaptation. Amplicon sequencing of multiple conserved, phylogenetically informative loci has therefore become an instrumental tool for many researchers. Investigations in many systems are hindered, however, since essential sequencing depth can be lost by amplification of nontarget DNA from hosts or overabundant microorganisms. Here, we introduce “blocking oligos”, a low‐cost and flexible method using standard oligonucleotides to block amplification of diverse nontargets and software to aid their design. We apply them primarily in leaves, where exceptional challenges with host amplification prevail. A . thaliana ‐specific blocking oligos applied in eight different target loci reduce undesirable host amplification by up to 90%. To expand applicability, we designed universal 16S and 18S rRNA gene plant blocking oligos for targets that are conserved in diverse plant species and demonstrate that they efficiently block five plant species from five orders spanning monocots and dicots ( Bromus erectus , Plantago lanceolata , Lotus corniculatus , Amaranth sp., Arabidopsis thaliana ). These can increase alpha diversity discovery without biasing beta diversity patterns and do not compromise microbial load information inherent to plant‐derived 16S rRNA gene amplicon sequencing data. Finally, we designed and tested blocking oligos to avoid amplification of 18S rRNA genes of a sporulating oomycete pathogen, demonstrating their effectiveness in applications well beyond plants. Using these tools, we generated a survey of the A . thaliana leaf microbiome based on eight loci targeting bacterial, fungal, oomycete and other eukaryotic microorganisms and discuss complementarity of commonly used amplicon sequencing regions for describing leaf microbiota. This approach has potential to make questions in a variety of study systems more tractable by making amplicon sequencing more targeted, leading to deeper, systems‐based insights into microbial discovery. For fast and easy design for blocking oligos for any nontarget DNA in other study systems, we developed a publicly available R package

Applications of Hilbert Module Approach to Multivariable Operator Theory

A commuting $n$-tuple $(T_1, \ldots, T_n)$ of bounded linear operators on a Hilbert space \clh associate a Hilbert module $\mathcal{H}$ over $\mathbb{C}[z_1, \ldots, z_n]$ in the following sense: $$\mathbb{C}[z_1, \ldots, z_n] \times \mathcal{H} \rightarrow \mathcal{H}, \quad \quad (p, h) \mapsto p(T_1, \ldots, T_n)h,$$where $p \in \mathbb{C}[z_1, \ldots, z_n]$ and $h \in \mathcal{H}$. A companion survey provides an introduction to the theory of Hilbert modules and some (Hilbert) module point of view to multivariable operator theory. The purpose of this survey is to emphasize algebraic and geometric aspects of Hilbert module approach to operator theory and to survey several applications of the theory of Hilbert modules in multivariable operator theory. The topics which are studied include: generalized canonical models and Cowen-Douglas class, dilations and factorization of reproducing kernel Hilbert spaces, a class of simple submodules and quotient modules of the Hardy modules over polydisc, commutant lifting theorem, similarity and free Hilbert modules, left invertible multipliers, inner resolutions, essentially normal Hilbert modules, localizations of free resolutions and rigidity phenomenon. This article is a companion paper to "An Introduction to Hilbert Module Approach to Multivariable Operator Theory".Comment: 46 pages. This is a companion paper to arXiv:1308.6103. To appear in Handbook of Operator Theory, Springe

Site-specific cleavage of bacterial MucD by secreted proteases mediates antibacterial resistance in Arabidopsis

Plant innate immunity restricts growth of bacterial pathogens that threaten global food security. However, the mechanisms by which plant immunity suppresses bacterial growth remain enigmatic. Here we show that Arabidopsis thaliana secreted aspartic protease 1 and 2 (SAP1 and SAP2) cleave the evolutionarily conserved bacterial protein MucD to redundantly inhibit the growth of the bacterial pathogen Pseudomonas syringae. Antibacterial activity of SAP1 requires its protease activity in planta and in vitro. Plants overexpressing SAP1 exhibit enhanced MucD cleavage and resistance but incur no penalties in growth and reproduction, while sap1 sap2 double mutant plants exhibit compromised MucD cleavage and resistance against P. syringae. P. syringae lacking mucD shows compromised growth in planta and in vitro. Notably, growth of ΔmucD complemented with the non-cleavable MucD(F106Y) is not affected by SAP activity in planta and in vitro. Our findings identify the genetic factors and biochemical process underlying an antibacterial mechanism in plants

Some comments on developments in exact solutions in statistical mechanics since 1944

Lars Onsager and Bruria Kaufman calculated the partition function of the Ising model exactly in 1944 and 1949. Since then there have been many developments in the exact solution of similar, but usually more complicated, models. Here I shall mention a few, and show how some of the latest work seems to be returning once again to the properties observed by Onsager and Kaufman.Comment: 28 pages, 5 figures, section on six-vertex model revise

Operator theory and function theory in Drury-Arveson space and its quotients

The Drury-Arveson space $H^2_d$, also known as symmetric Fock space or the $d$-shift space, is a Hilbert function space that has a natural $d$-tuple of operators acting on it, which gives it the structure of a Hilbert module. This survey aims to introduce the Drury-Arveson space, to give a panoramic view of the main operator theoretic and function theoretic aspects of this space, and to describe the universal role that it plays in multivariable operator theory and in Pick interpolation theory.Comment: Final version (to appear in Handbook of Operator Theory); 42 page

Invariant Distances

In this chapter we shall define the (invariant) distance we are going to use, and collect some of its main properties we shall need later on