Acinetobacter baumannii Catabolizes Ethanolamine in the Absence of a Metabolosome and Converts Cobinamide into Adenosylated Cobamides.

Acinetobacter baumannii is an opportunistic pathogen typically associated with hospital-acquired infections. Our understanding of the metabolism and physiology of A. baumannii is limited. Here, we report that A. baumannii uses ethanolamine (EA) as the sole source of nitrogen and can use this aminoalcohol as a source of carbon and energy if the expression of the eutBC genes encoding ethanolamine ammonia-lyase (EAL) is increased. A strain with an ISAba1 element upstream of the eutBC genes efficiently used EA as a carbon and energy source. The A. baumannii EAL (AbEAL) enzyme supported the growth of a strain of Salmonella lacking the entire eut operon. Remarkably, the growth of the above-mentioned Salmonella strain did not require the metabolosome, the reactivase EutA enzyme, the EutE acetaldehyde dehydrogenase, or the addition of glutathione to the medium. Transmission electron micrographs showed that when Acinetobacter baumannii or Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 synthesized AbEAL, the protein localized to the cell membrane. We also report that the A. baumannii genome encodes all of the enzymes needed for the assembly of the nucleotide loop of cobamides and that it uses these enzymes to synthesize different cobamides from the precursor cobinamide and several nucleobases. In the absence of exogenous nucleobases, the most abundant cobamide produced by A. baumannii was cobalamin. IMPORTANCE Acinetobacter baumannii is a Gram-negative bacterium commonly found in soil and water. A. baumannii is an opportunistic human pathogen, considered by the CDC to be a serious threat to human health due to the multidrug resistance commonly associated with this bacterium. Knowledge of the metabolic capabilities of A. baumannii is limited. The importance of the work reported here lies in the identification of ethanolamine catabolism occurring in the absence of a metabolosome structure. In other bacteria, this structure protects the cell against damage by acetaldehyde generated by the deamination of ethanolamine. In addition, the ethanolamine ammonia-lyase (EAL) enzyme of this bacterium is unique in that it does not require a reactivase enzyme to remain active. Importantly, we also demonstrate that the A. baumannii genome encodes the functions needed to assemble adenosylcobamide, the coenzyme of EAL, from the precursor cobinamide.

A method for the efficient adenosylation of corrinoids.

Adenosylcobamides (AdoCbas) are coenzymes required by organisms from all domains of life to perform challenging chemical reactions. AdoCbas are characterized by a cobalt-containing tetrapyrrole ring, where an adenosyl group is covalently attached to the cobalt ion via a unique Co-C organometallic bond. During catalysis, this bond is homolytically cleaved by AdoCba-dependent enzymes to form an adenosyl radical that is critical for intra-molecular rearrangements. The formation of the Co-C bond is catalyzed by a family of enzymes known as ATP:Co(I)rrinoid adenosyltransferases (ACATs). ACATs adenosylate Cbas in two steps: (I) they generate a planar, Co(II) four-coordinate Cba to facilitate the reduction of Co(II) to Co(I), and (II) they transfer the adenosyl group from ATP to the Co(I) ion. To synthesize adenosylated corrinoids in vitro, it is imperative that anoxic conditions are maintained to avoid oxidation of Co(II) or Co(I) ions. Here we describe a method for the enzymatic synthesis and quantification of specific AdoCbas.

HSP70 chaperones RNA-free TDP-43 into anisotropic intranuclear liquid spherical shells.

The RNA binding protein TDP-43 forms intranuclear or cytoplasmic aggregates in age-related neurodegenerative diseases. In this study, we found that RNA binding-deficient TDP-43 (produced by neurodegeneration-causing mutations or posttranslational acetylation in its RNA recognition motifs) drove TDP-43 demixing into intranuclear liquid spherical shells with liquid cores. These droplets, which we named "anisosomes", have shells that exhibit birefringence, thus indicating liquid crystal formation. Guided by mathematical modeling, we identified the primary components of the liquid core to be HSP70 family chaperones, whose adenosine triphosphate (ATP)-dependent activity maintained the liquidity of shells and cores. In vivo proteasome inhibition within neurons, to mimic aging-related reduction of proteasome activity, induced TDP-43-containing anisosomes. These structures converted to aggregates when ATP levels were reduced. Thus, acetylation, HSP70, and proteasome activities regulate TDP-43 phase separation and conversion into a gel or solid phase.

A photoactivable multi-inhibitor nanoliposome for tumour control and simultaneous inhibition of treatment escape pathways.

Nanoscale drug delivery vehicles can facilitate multimodal therapies of cancer by promoting tumour-selective drug release. However, few are effective because cancer cells develop ways to resist and evade treatment. Here, we introduce a photoactivable multi-inhibitor nanoliposome (PMIL) that imparts light-induced cytotoxicity in synchrony with a photoinitiated and sustained release of inhibitors that suppress tumour regrowth and treatment escape signalling pathways. The PMIL consists of a nanoliposome doped with a photoactivable chromophore (benzoporphyrin derivative, BPD) in the lipid bilayer, and a nanoparticle containing cabozantinib (XL184)--a multikinase inhibitor--encapsulated inside. Near-infrared tumour irradiation, following intravenous PMIL administration, triggers photodynamic damage of tumour cells and microvessels, and simultaneously initiates release of XL184 inside the tumour. A single PMIL treatment achieves prolonged tumour reduction in two mouse models and suppresses metastatic escape in an orthotopic pancreatic tumour model. The PMIL offers new prospects for cancer therapy by enabling spatiotemporal control of drug release while reducing systemic drug exposure and associated toxicities.

Dynein achieves processive motion using both stochastic and coordinated stepping.

Processivity, the ability of single molecules to move continuously along a track, is a fundamental requirement of cargo-transporting molecular motors. Here, we investigate how cytoplasmic dynein, a homodimeric, microtubule-based motor, achieves processive motion. To do this, we developed a versatile method for assembling Saccharomyces cerevisiae dynein heterodimers, using complementary DNA oligonucleotides covalently linked to dynein monomers labeled with different organic fluorophores. Using two-color, single-molecule microscopy and high-precision, two-dimensional tracking, we find that dynein has a highly variable stepping pattern that is distinct from all other processive cytoskeletal motors, which use 'hand-over-hand' mechanisms. Uniquely, dynein stepping is stochastic when its two motor domains are close together. However, coordination emerges as the distance between motor domains increases, implying that a tension-based mechanism governs these steps. This plasticity may allow tuning of dynein for its diverse cellular functions.

Cryo-EM structure and rRNA model of a translating eukaryotic 80S ribosome at 5.5-A resolution.

Protein biosynthesis, the translation of the genetic code into polypeptides, occurs on ribonucleoprotein particles called ribosomes. Although X-ray structures of bacterial ribosomes are available, high-resolution structures of eukaryotic 80S ribosomes are lacking. Using cryoelectron microscopy and single-particle reconstruction, we have determined the structure of a translating plant (Triticum aestivum) 80S ribosome at 5.5-Šresolution. This map, together with a 6.1-Šmap of a Saccharomyces cerevisiae 80S ribosome, has enabled us to model ∼98% of the rRNA. Accurate assignment of the rRNA expansion segments (ES) and variable regions has revealed unique ES-ES and r-protein-ES interactions, providing insight into the structure and evolution of the eukaryotic ribosome.

Structural insight into nascent polypeptide chain-mediated translational stalling.

Expression of the Escherichia coli tryptophanase operon depends on ribosome stalling during translation of the upstream TnaC leader peptide, a process for which interactions between the TnaC nascent chain and the ribosomal exit tunnel are critical. We determined a 5.8 angstrom-resolution cryo-electron microscopy and single-particle reconstruction of a ribosome stalled during translation of the tnaC leader gene. The nascent chain was extended within the exit tunnel, making contacts with ribosomal components at distinct sites. Upon stalling, two conserved residues within the peptidyltransferase center adopted conformations that preclude binding of release factors. We propose a model whereby interactions within the tunnel are relayed to the peptidyltransferase center to inhibit translation. Moreover, we show that nascent chains adopt distinct conformations within the ribosomal exit tunnel.

Structural model and excitonic properties of the dimeric RC-LH1-PufX complex from Rhodobacter sphaeroides.

The light-harvesting apparatus of the purple bacterial photosynthetic unit consists of a pool of peripheral light-harvesting complexes that transfer excitation energy to a reaction center (RC) via the surrounding pigment-protein complex LH1. Recent electron microscopy and atomic force microscopy studies have revealed that RC-LH1 units of Rhodobacter sphaeroides form membrane-bending dimeric complexes together with the polypeptide PufX. We present a structural model for these RC-LH1-PufX dimeric complexes constructed using the molecular dynamics flexible fitting method based on an EM density map. The arrangement of the LH1 BChls displays a distortion near the proposed location of the PufX polypeptide. The resulting atomic model for BChl arrays is used to compute the excitonic properties of the dimeric RC-LH1 complex. A comparison is presented between the structural and excitonic features of the S-shaped dimeric BChl array of Rhodobacter sphaeroides and the circular BChl arrangement found in other purple bacteria.

Changes in compulsion and anxiety symptoms with nicotine transdermal patches in non-smoking obsessive-compulsive disorder patients.

OBJECTIVE: Some of the obsessive-compulsive disorder (OCD) symptoms can be elicited in rats by the administration of quinpirole (D2/D3 dopaminergic agonist). Nicotine administration blocked some aspect of checking behavior in that model. The main goal of this study was to determine if the clinical manifestations of OCD non smoking patients change with the administration of transdermal nicotine patches. MATERIAL AND METHODS: Eleven patients were studied (6 female and 5 males), average age 29.7 +/- 5.5 years. All of them were OCD according to DSM-IV criteria. Clinical scorings were done with Yale-Brown Obsessive Compulsive Scale (Y-BOCS), Beck Depression Inventory (BDI) and Beck Anxiety Inventory (BAI). Nicotine (17.5 mg/day) or placebo transdermal patches were randomly administered for five continuous days. RESULTS: Nicotine administration reduced the total Y-BOCS and the compulsive score of that scale, but did not reduce obsessions. Also anxiety was reduced as was shown by the BAI scores, when patients were on nicotine patches, no changes were observed in BDI. CONCLUSIONS: The present results replicated the animal findings about reduction in compulsive behavior after nicotine administration. Also suggest that nicotine depending on the dysfunction of the different neurotransmitter systems may produce different behavior or cognitive effects, also that even nicotine showed some beneficial effect in OCD patients, the low rate of nicotine smoking in this type of patients, may show other mechanisms than may protect to smoke in some psychiatric patients.