Here, we present the draft genome of the endosymbiont "Candidatus Ruthia magnifica" UCD-CM, a member of the phylum Proteobacteria, found from the gills of a deep-sea giant clam, Calyptogena magnifica. The assembly consists of 1,160,249 bp contained in 18 contigs.

The POU genes represent a diverse class of animal-specific transcription factors that play important roles in neurogenesis, pluripotency, and cell-type specification. Although previous attempts have been made to reconstruct the evolution of the POU class, these studies have been limited by a small number of representative taxa, and a lack of sequences from basally branching organisms. In this study, we performed comparative analyses on available genomes and sequences recovered through "gene fishing" to better resolve the topology of the POU gene tree. We then used ancestral state reconstruction to map the most likely changes in amino acid evolution for the conserved domains. Our work suggests that four of the six POU families evolved before the last common ancestor of living animals-doubling previous estimates-and were followed by extensive clade-specific gene loss. Amino acid changes are distributed unequally across the gene tree, consistent with a neofunctionalization model of protein evolution. We consider our results in the context of early animal evolution, and the role of POU5 genes in maintaining stem cell pluripotency.

Here, we present the draft genome sequences of Vibrio splendidus UCD-SED7 and UCD-SED10 (phylum Proteobacteria). These strains were isolated from sediment surrounding Zostera marina roots near the UC Davis Bodega Marine Laboratory (Bodega, Bay, California). These assemblies contain 5,334,236 bp and 5,904,824 bp, respectively.

Here, we present the draft genome sequence of Pseudoalteromonas tetraodonis UCD-SED8, a marine bacterium normally associated with the production of tetrodotoxin in pufferfish. This strain was isolated from sediment samples surrounding Zostera marina roots collected from Bodega Marine, California. The assembly consists of 4,017,727 bp contained in 35 contigs.

Here, we present the draft genome sequence of Bacillus vietnamensis UCD-SED5 (phylum Firmicutes). This strain was isolated from sediment surrounding Zostera marina roots near the UC Davis Bodega Marine Laboratory (Bodega Bay, California) and represents the second genome of this species. The assembly consists of 4,325,707 bp, in 108 contigs.

Tetramethylenedisulfotetramine (TETS) is a so-called "caged" convulsant that is responsible for thousands of accidental and malicious poisonings. Similar to the widely used GABA receptor type A (GABA_A) antagonist picrotoxinin, TETS has been proposed to bind to the noncompetitive antagonist (NCA) site in the pore of the receptor channel. However, the TETS binding site has never been experimentally mapped, and we here set out to gain atomistic level insights into how TETS inhibits the human α ₂ β ₃ γ ₂ GABA_A receptor. Using the Rosetta molecular modeling suite, we generated three homology models of the α ₂ β ₃ γ ₂ receptor in the open, desensitized, and closed/resting state. Three different ligand-docking algorithms (RosettaLigand, Glide, and Swissdock) identified two possible TETS binding sites in the channel pore. Using a combination of site-directed mutagenesis, electrophysiology, and modeling to probe both sites, we demonstrate that TETS binds at the T6' ring in the closed/resting-state model, in which it shows perfect space complementarity and forms hydrogen bonds or makes hydrophobic interactions with all five pore-lining threonine residues of the pentameric receptor. Mutating T6' in either the α ₂ or β ₃ subunit reduces the IC₅₀ of TETS by ∼700-fold in whole-cell patch-clamp experiments. TETS is thus interacting at the NCA site in the pore of the GABA_A receptor at a location that is overlapping but not identical to the picrotoxinin binding site. SIGNIFICANCE STATEMENT: Our study identifies the binding site of the highly toxic convulsant tetramethylenedisulfotetramine (TETS), which is classified as a threat agent by the World Health Organization. Using a combination of homology protein modeling, ligand docking, site-directed mutagenesis, and electrophysiology, we show that TETS is binding in the pore of the α₂β₃γ₂ GABA receptor type A receptor at the so-called T6' ring, wherein five threonine residues line the permeation pathway of the pentameric receptor channel.

Here, we present the draft genome sequences of Pseudoalteromonas porphyrae UCD-SED9 and UCD-SED14 (phylum Proteobacteria). These strains were isolated from sediment surrounding the roots of the seagrass, Zostera marina, collected near the UC, Davis Bodega Marine Laboratory (Bodega Bay, California). The assemblies contain 4,847,456 bp and 4,817,752 bp, respectively.

Introduction: Mechanical thrombectomy has improved treatment options and outcomes for acute ischemic stroke with large artery occlusion. However, as the time window of endovascular thrombectomy is extended there is an increasing need to develop immunocytoprotective therapies that can reduce inflammation in the penumbra and prevent reperfusion injury. We previously demonstrated, that by reducing neuroinflammation, K_V1.3 inhibitors can improve outcomes not only in young male rodents but also in female and aged animals. To further explore the therapeutic potential of K_V1.3 inhibitors for stroke therapy, we here directly compared a peptidic and a small molecule K_V1.3 blocker and asked whether K_V1.3 inhibition would still be beneficial when started at 72 hours after reperfusion. Methods: Transient middle cerebral artery occlusion (tMCAO, 90-min) was induced in male Wistar rats and neurological deficit assessed daily. On day-8 infarction was determined by T2-weighted MRI and inflammatory marker expression in the brain by quantitative PCR. Potential interactions with tissue plasminogen activator (tPA) were evaluated in-vitro with a chromogenic assay. Results: In a direct comparison with administration started at 2 hours after reperfusion, the small molecule PAP-1 significantly improved outcomes on day-8, while the peptide ShK-223 failed to reduce infarction and neurological deficits despite reducing inflammatory marker expression. PAP-1 still provided benefits when started 72 hours after reperfusion. PAP-1 does not reduce the proteolytic activity of tPA. Discussion: Our studies suggest that K_V1.3 inhibition for immunocytoprotection after ischemic stroke has a wide therapeutic window for salvaging the inflammatory penumbra and requires brain-penetrant small molecules.

Objective

Royal demolition explosive (RDX) can induce seizures in wildlife and humans following release into the environment or after voluntary consumption. During the Vietnam War, RDX intoxication was the most common cause of generalized seizures in US service personnel, and in some sections of the armed forces, eating of RDX has continued as "a dare" to this day. After its mechanism of action was long unknown, RDX was recently shown to be a GABA_A receptor antagonist. We here determined the GABA_A receptor subtype-selectivity of RDX and mapped its functional binding site.

Methods

We used whole-cell patch-clamp to determine the potency of RDX on 10 recombinantly expressed GABA_A receptors and mapped the RDX binding site using a combination of Rosetta molecular modeling and site-directed mutagenesis.

Results

RDX was found to reversibly inhibit the α1β2γ2 GABA_A receptor with an IC₅₀ of 23 μmol/L (95% CI 15.1-33.3 μmol/L), whereas α4 and α6 containing GABA_A receptor combinations were 4-10-fold less sensitive. RDX is binding to the noncompetitive antagonist (NCA) site in the pore. In a molecular model based on the cryo-EM structure of the resting state of the α1β2γ2 receptor, RDX forms two hydrogen bonds with the threonines at the T6' ring and makes hydrophobic interactions with the valine and alanine in 2' position of the α1 or β2 subunits.

Interpretation

Our findings characterize the mechanism of action of RDX at the atomistic level and suggest that RDX-induced seizures should be susceptible to treatment with GABA_A modulating drugs such as benzodiazepines, barbiturates, propofol, or neurosteroids.