Allosteric activation of the 5-HT_3AB receptor by mCPBG

The 5-HT_3AB receptor contains three A and two B subunits in an A-A-B-A-B order. However, serotonin function at the 5-HT_3AB receptor has been shown to depend solely on the A-A interface present in the homomeric receptor. Using mutations at sites on both the primary (E122) and complementary (Y146) faces of the B subunit, we demonstrate that meta-chlorophenyl biguanide (mCPBG), a 5-HT_3 selective agonist, is capable of binding to and activating the 5-HT_3AB receptor at all five subunit interfaces of the heteromer. Further, mCPBG is capable of allosterically modulating the activity of serotonin from these sites. While these five binding sites are similar enough that they form to a monophasic dose – response relationship, we uncover subtle differences in the heteromeric binding sites. We also find that the A-A interface appears to contribute disproportionately to the efficacy of 5-HT_3AB receptor activation

The 5-HT3AB Receptor Shows an A3B2 Stoichiometry at the Plasma Membrane

AbstractThe 5-HT3AB receptor is the best-characterized heteropentameric 5-HT3 receptor. Under conditions of heterologous expression, the 5-HT3AB receptor shows a single functionally resolvable population, suggesting the presence of a unique subunit stoichiometry; however, conflicting previous reports have suggested two different possible stoichiometries. Here we isolate plasma membrane sheets containing assembled receptors from individual HEK293T cells. We then determine the stoichiometry of 5-HT3AB receptors on the plasma membrane by fluorescence methods, employing meCFP- and meYFP-labeled A and B subunits. Over a wide range of cDNA transfection ratios, fluorescence intensity ratios are closest to values that correspond to a subunit ratio of A3B2. Förster resonance energy transfer (family FRET) efficiencies provide minor corrections (3–6%) to the subunit ratios and provide independent support for a predominantly A3B2 stoichiometry on the plasma membrane sheets. Twin FRET efficiencies support these data, also suggesting that the two B subunits are nonadjacent in most of the heteropentamers. The high-frequency variant HTR3B p.Y129S (c.386A>C, rs11767445), linked to psychiatric disease, also forms A3B2 receptors on the plasma membrane. The 5-HT3B Y129S, subunit incorporates in a slightly (11–14%) more efficient manner than the common variant. In general, most of the subunits reside within the cell. In contrast to the findings for the plasma membrane, the relative abundances and FRET characteristics of intracellular subunits depend strongly on the transfection ratio. The straightforward and unambiguous combination of plasma membrane-sheet isolation, fluorescence intensity ratios, and FRET is a generally promising procedure for determining stoichiometry of proteins on the plasma membrane

The 5-HT_3AB Receptor Shows an A_3B_2 Stoichiometry at the Plasma Membrane

The 5-HT_3AB receptor is the best-characterized heteropentameric 5-HT_3 receptor. Under conditions of heterologous expression, the 5-HT_3AB receptor shows a single functionally resolvable population, suggesting the presence of a unique subunit stoichiometry; however, conflicting previous reports have suggested two different possible stoichiometries. Here we isolate plasma membrane sheets containing assembled receptors from individual HEK293T cells. We then determine the stoichiometry of 5-HT_3AB receptors on the plasma membrane by fluorescence methods, employing meCFP- and meYFP-labeled A and B subunits. Over a wide range of cDNA transfection ratios, fluorescence intensity ratios are closest to values that correspond to a subunit ratio of A_3B_2. Förster resonance energy transfer (family FRET) efficiencies provide minor corrections (3–6%) to the subunit ratios and provide independent support for a predominantly A_3B_2 stoichiometry on the plasma membrane sheets. Twin FRET efficiencies support these data, also suggesting that the two B subunits are nonadjacent in most of the heteropentamers. The high-frequency variant HTR3B p.Y129S (c.386A>C, rs11767445), linked to psychiatric disease, also forms A_3B_2 receptors on the plasma membrane. The 5-HT_3B Y129S, subunit incorporates in a slightly (11–14%) more efficient manner than the common variant. In general, most of the subunits reside within the cell. In contrast to the findings for the plasma membrane, the relative abundances and FRET characteristics of intracellular subunits depend strongly on the transfection ratio. The straightforward and unambiguous combination of plasma membrane-sheet isolation, fluorescence intensity ratios, and FRET is a generally promising procedure for determining stoichiometry of proteins on the plasma membrane

Cement brand and preparation effects cement-in-cement mantle shear strength

Creating bi-laminar cement mantles as part of revision hip arthroplasty is well-documented but there is a lack of data concerning the effect of cement brand on the procedure. The aim of this study was to compare the shear strength of bi-laminar cement mantles using various combinations of two leading bone cement brands. Bi-laminar cement mantles were created using Simplex P with Tobramycin, and Palacos R+G: Simplex-Simplex (SS); Simplex-Palacos (SP); Palacos-Simplex (PS); and Palacos-Palacos (PP). Additionally, specimens were produced by rasping (R) the surface of the original mantle, or leaving it unrasped (U), leading to a total of eight groups (n = 10). Specimens were loaded in shear, at 0.1 mm/min, until failure, and the maximum shear strength calculated. The highest mean shear strength was found in the PSU and PSR groups (23.69 and 23.89 MPa respectively), and the lowest in the PPU group (14.70 MPa), which was significantly lower than all but two groups. Unrasped groups generally demonstrated greater standard error than rasped groups. In a further comparison to assess the effect of the new cement mantle brand, irrespective of the brand of the original mantle, Simplex significantly increased the shear strength compared to Palacos with equivalent preparation. It is recommended that the original mantle is rasped prior to injection of new cement, and that Simplex P with Tobramycin be used in preference to Palacos R+G irrespective of the existing cement type. Further research is needed to investigate more cement brands, and understand the underlying mechanisms relating to cement-in-cement procedures. </jats:p

Adeno-Associated Virus Toolkit to Target Diverse Brain Cells

Recombinant adeno-associated viruses (AAVs) are commonly used gene delivery vehicles for neuroscience research. They have two engineerable features: the capsid (outer protein shell) and cargo (encapsulated genome). These features can be modified to enhance cell type or tissue tropism and control transgene expression, respectively. Several engineered AAV capsids with unique tropisms have been identified, including variants with enhanced central nervous system transduction, cell type specificity, and retrograde transport in neurons. Pairing these AAVs with modern gene regulatory elements and state-of-the-art reporter, sensor, and effector cargo enables highly specific transgene expression for anatomical and functional analyses of brain cells and circuits. Here, we discuss recent advances that provide a comprehensive (capsid and cargo) AAV toolkit for genetic access to molecularly defined brain cell types

Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types

Recombinant adeno-associated viruses (rAAVs) are efficient gene delivery vectors via intravenous delivery; however, natural serotypes display a finite set of tropisms. To expand their utility, we evolved AAV capsids to efficiently transduce specific cell types in adult mouse brains. Building upon our Cre-recombination-based AAV targeted evolution (CREATE) platform, we developed Multiplexed-CREATE (M-CREATE) to identify variants of interest in a given selection landscape through multiple positive and negative selection criteria. M-CREATE incorporates next-generation sequencing, synthetic library generation and a dedicated analysis pipeline. We have identified capsid variants that can transduce the central nervous system broadly, exhibit bias toward vascular cells and astrocytes, target neurons with greater specificity or cross the blood–brain barrier across diverse murine strains. Collectively, the M-CREATE methodology accelerates the discovery of capsids for use in neuroscience and gene-therapy applications

TESS asteroseismology of the known red-giant host stars HD 212771 and HD 203949

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