Structure of the Pseudomonas aeruginosa transamidosome reveals unique aspects of bacterial tRNA-dependent asparagine biosynthesis

Many prokaryotes lack a tRNA synthetase to attach asparagine to its cognate tRNA^Asn, and instead synthesize asparagine from tRNA^Asn-bound aspartate. This conversion involves two enzymes: a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) that forms Asp-tRNA^Asn, and a heterotrimeric amidotransferase GatCAB that amidates Asp-tRNA^Asn to form Asn-tRNA^Asn for use in protein synthesis. ND-AspRS, GatCAB, and tRNA^Asn may assemble in an ∼400-kDa complex, known as the Asn-transamidosome, which couples the two steps of asparagine biosynthesis in space and time to yield Asn-tRNA^Asn. We report the 3.7-Å resolution crystal structure of the Pseudomonas aeruginosa Asn-transamidosome, which represents the most common machinery for asparagine biosynthesis in bacteria. We show that, in contrast to a previously described archaeal-type transamidosome, a bacteria-specific GAD domain of ND-AspRS provokes a principally new architecture of the complex. Both tRNA^Asn molecules in the transamidosome simultaneously serve as substrates and scaffolds for the complex assembly. This architecture rationalizes an elevated dynamic and a greater turnover of ND-AspRS within bacterial-type transamidosomes, and possibly may explain a different evolutionary pathway of GatCAB in organisms with bacterial-type vs. archaeal-type Asn-transamidosomes. Importantly, because the two-step pathway for Asn-tRNA^Asn formation evolutionarily preceded the direct attachment of Asn to tRNA^Asn, our structure also may reflect the mechanism by which asparagine was initially added to the genetic code.Accurate translation of the genetic code into a protein sequence relies on a covalent attachment of amino acids to cognate tRNAs that are later used in protein synthesis (1). This attachment is catalyzed by aminoacyl-tRNA synthetases (aaRSs), each specific to one amino acid and a set of tRNA isoacceptors (2). However, the majority of prokaryotes lack several tRNA synthetases, particularly asparaginyl-tRNA synthetase (AsnRS), which ligates asparagine to tRNA^Asn (3, 4). In these organisms, asparagine is synthesized in a two-step, tRNA-dependent pathway (5). First, a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) attaches aspartate to tRNA^Asn to form Asp-tRNA^Asn (6, 7). Then the tRNA-bound aspartate is converted to asparagine by the amidotransferase (AdT) GatCAB to yield the final product, Asn-tRNA^Asn (6, 8–13). Likewise, in prokaryotes lacking glutaminyl-tRNA synthetase (GlnRS), Gln-tRNA^Gln is formed by the sequential actions of a nondiscriminating glutamyl-tRNA synthetase (ND-GluRS) (14) and an AdT (5). In bacteria, the role of AdT is played by GatCAB (15), whereas in archaea, it is played by GatDE (16, 17).More than 25 years ago (18), it was proposed that ND-aaRSs and AdTs may form a complex—now called a transamidosome—to couple the two steps of Asn-tRNA^Asn formation in space and time and allow efficient transfer of Asp-tRNA^Asn from the aaRS to the AdT. The first characterized transamidosome was the Asn-transamidosome from Thermus thermophilus (TtAsn-transamidosome) (19, 20). This complex was identified as a tRNA-dependent association of AspRS2 (TtAspRS2) and GatCAB in a 2:2:2 ratio. It was shown that transamidosome formation stabilizes interactions between subunits of GatCAB (21) and protects Asn-tRNA^Asn from hydrolysis, with product release being rate-limiting (19). In the complex, the AspRS forms a dimer with only one catalytic site active at a time (21). It was suggested that the key advantages of asparagine formation by the transamidosome compared with separate enzymes are enhanced aspartylation of tRNA^Asn and better prevention of the misacylated Asp-tRNA^Asn from use in translation, because this would compromise the fidelity of protein synthesis (19–21).Importantly, TtAspRS2 was acquired through horizontal gene transfer from archaea (10), and lacks the GAD domain typical of bacterial AspRSs (22). The TtAsn-transamidosome crystal structure suggests that a complex between bacterial ND-AspRS and GatCAB should be less stable and more structurally distinct than the T. thermophilus complex, owing to the presence of the GAD domain in bacterial ND-AspRS (21, 23). Consistent with this notion, the stable association of the Helicobacter pylori ND-AspRS (HpND-AspRS) with GatCAB requires the presence of an auxiliary factor, Hp0100 (23, 24). In the complex, the activity of the HpND-AspRS is unchanged, although the activity of GatCAB increases (23); however, Hp0100 is phylogenetically limited to ε-proteobacteria (23). Therefore, most bacteria have a structurally and, possibly, functionally distinct class of transamidosomes than those described by the T. thermophilus and H. pylori complexes.In the bacterium Pseudomonas aeruginosa, Asn-tRNA^Asn formation is catalyzed by GatCAB and bacterial ND-AspRS, and thus represents the most common type of bacterial Asn-transamidosome (25). Here we report the crystal structure of the P. aeruginosa Asn-transamidosome (PaAsn-transamidosome), which represents the transamidation state of the Asn-tRNA^Asn formation. The structure suggests that the additional GAD domain within the ND-AspRS changes the overall architecture of the complex relative to the previously described TtAsn-transamidosome. Consistent with the structure, our in vitro measurements show that PaAsn-transamidosome has unique kinetic properties and functions primarily to enhance tRNA^Asn turnover and facilitate Asp-tRNA^Asn handoff from AspRS to GatCAB.     

Iodine-125 seed brachytherapy for early stage prostate cancer: a single-institution review

Purpose

We are reporting the five-year biochemical control, toxicity profile and dosimetric parameters using iodine-125 low dose rate brachytherapy (BT) as monotherapy for early stage prostate cancer at a single institution.

Material and methods

Between April 2006 and December 2010, 169 men with early stage prostate cancer were treated with BT. Biochemical failure was defined using the Phoenix definition (nadir?+?2 ng/mL). Treatment-related morbidities, including urinary, rectal and sexual function, were measured, applying the International Prostate Symptom Score (IPSS), the 7-grade Quality of Life Scale (QoL) and medical status, the International Consultation on Incontinence Modular Questionnaire (ICIQ), the International Index of Erectile Function (IIEF-5) and the Common Terminology Criteria for Adverse Events (CTCAE v4.03). Seed migration and loss, dosimetric parameters and learning effects were also analyzed.

Results

Medium follow-up time was 50 months (range, 1–85 months). The five-year biochemical failure rate was 7%. Acute proctitis rates were 19% (grade 1) and 1% (grade 2), respectively. The overall incidence of incontinence was 19% (mild), 16% (moderate) and?<?1% (severe). An increase in IPSS?≥?5 points was detected in 59% of patients, with 38% regaining their baseline. Seed dislocation was found in 24% of patients and correlated with D90 and V100. A learning curve was found for seed migration, D90 and V100. QoL correlated with the general health condition of patient, incontinence symptoms and IPSS.

Conclusions

BT for early stage prostate cancer offers excellent five-year biochemical control with low toxicities. QoL aspects are favorable. A learning curve was detected for procedural aspects but its impact on patient relevant endpoints remains inconclusive.

     

Impact of manual control point selection accuracy on automated surface matching of digital dental models

Clinical Oral Investigations - Treatment outcomes are frequently evaluated based on the superimposition of digital dental models. However, errors from surface matching may distort these findings....     

Recapitulating bone development events in a customised bioreactor through interplay of oxygen tension,medium pH,and systematic differentiation approaches

Bone development and homeostasis are intricate processes that require co‐existence and dynamic interactions among multiple cell types. However, controlled dynamic niches that derive and support stable propagation of these cells from single stem cell source is not sustainable in conventional culturing vessels. In bioreactor cultures that support dynamic niches, the limited source and stability of growth factors are often a major limiting factor for long‐term in vitro cultures. Hence, alternative growth factor‐free differentiation approaches are designed and their efficacy to achieve different osteochondral cell types is investigated. Briefly, a dynamic niche is achieved by varying medium pH, oxygen tension (pO₂) distribution in bioreactor, initiating chondrogenic differentiation with chondroitin sulphate A (CSA), and implementing systematic differentiation regimes. In this study, we demonstrated that CSA is a potent chondrogenic inducer, specifically in combination with acidic medium and low pO₂. Further, endochondral ossification is recapitulated through a systematic chondrogenic–osteogenic (ch‐os) differentiation regime, and multiple osteochondral cell types are derived. Chondrogenic hypertrophy was also enhanced specifically in high pO₂ regions. Consequently, mineralised constructs with higher structural integrity, volume, and tailored dimensions are achieved. In contrast, a continuous osteogenic differentiation regime (os‐os) has derived compact and dense constructs, whereas a continuous chondrogenic differentiation regime (ch‐ch) has attenuated construct mineralisation and impaired development. In conclusion, a growth factor‐free differentiation approach is achieved through interplay of pO₂, medium pH, and systematic differentiation regimes. The controlled dynamic niches have recapitulated endochondral ossification and can potentially be exploited to derive larger bone constructs with near physiological properties.     

Photocatalytic coatings based on a zinc(ii) phthalocyanine derivative immobilized on nanoporous gold leafs with various pore sizes

A series of singlet oxygen sensitizing hybrid materials is reported consisting of a zinc(ii) phthalocyanine (ZnPc) derivative immobilized on nanoporous gold leafs (npAu) with various pore sizes. The resulting photocatalytic coatings exhibit a thickness of around 100 nm and pore sizes between 9–50 nm. Herein, we report the synthesis and characterization of those hybrid materials which were synthesized by functionalization of npAu leafs by an azide terminated alkanethiol self-assembled monolayer (SAM) and subsequent copper catalyzed azide–alkyne cycloaddition (CuAAC). The characterization of the samples morphology included scanning electron microscopy (SEM), UV-Vis spectroscopy as well as energy dispersive X-ray spectroscopy (EDX). The morphology–reactivity relationship was investigated employing the hybrid photocatalysts in the photooxidation of diphenylisobenzofuran (DPBF) as selective singlet oxygen quencher. An increasing photocatalytic activity was found for smaller pore sizes up to 15 nm, due to the gain in specific surface area concomitant with an increasing amount of immobilized photosensitizer, completely dominating the effect of the higher spectral overlap caused by the shift of the plasmon resonance of npAu, until mass transport and diffusion limitation gets predominant for pore sizes below 15 nm.

A series of hybrid materials consisting of a zinc(ii) phthalocyanine derivative immobilized on nanoporous gold leafs with various pore sizes was prepared and investigated regarding its singlet oxygen sensitization activity.     

Which concentration to choose in dual flow cardiac CT?: Dual flow cardiac CT

Purpose

An extensive number of protocols have been suggested to allow for functional diagnostics; however, no data is available about the minimal amount of contrast medium to achieve reliable imaging properties. None of the plethora of existing studies report a rational why the specific concentration was chosen.

Materials and methods

A total of 40 patients were included in this prospective, controlled study. They were divided up into four equal groups getting a different concentration (10%, 20%, 30% or 40%) of a second contrast medium bolus. Corresponding septal and right ventricular ROIs were compared. A visual score was established. Coronary attenuation was measured in the right and left coronary artery. Streak artifacts in the right atrium/ventricle were assessed.

Results

In the 10% contrast medium (CM) group only in 5/10 (50%) patients full septal delineation was reached. In all other groups full septal visualization was obtained.No group showed a relevant difference of mean density measured in HU units of the left ventricle or the coronary arteries. All study groups except of group 1 (10% CM) showed streak artifacts in the right atrium.

Conclusion

The dual flow protocol with a minimum concentration of 20% improves septal visualization as basis for left ventricular functional assessment, however, does not allow for reliable right ventricular or atrial visualization.There is no significant difference between the different concentration protocols in terms of coronary attenuation.     

Fluorine-18 labeling of three novel D-peptides by conjugation with N-succinimidyl-4-[18F]fluorobenzoate and preliminary examination by postmortem whole-hemisphere human brain autoradiography

Introductionβ-Amyloid (Aβ) plaques and neurofibrillary tangles are the main characteristics of Alzheimer's disease (AD). Positron emission tomography (PET), a high-resolution, sensitive, and noninvasive imaging technique, has been widely utilized in visualizing the localization of plaques and tangles and thereby distinguishing between AD and healthy controls. A small 12-mer d-enantiomeric peptide (amino acid sequence=QSHYRHISPAQV), denoted as D1, has high binding affinity to Aβ in vitro in the sub-micromolar range, and consequently, its radiolabeled analogues have a potential as radioligands for visualizing amyloid plaques in vivo by PET.AimThe aims of the present work were to develop three different potent D1 derivative peptides labeled with fluorine-18 and to examine them in the AD and control postmortem human brain by autoradiography (ARG).MethodsThree different D1 derivative peptides were radiolabeled with fluorine-18 ([¹⁸F]ACI-87, [¹⁸F]ACI-88, [¹⁸F]ACI-89) using the prosthetic group N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB) and purified by high performance liquid chromatography (HPLC). Preliminary ARG measurements were performed in AD and control brains.ResultsThe three fluorine-18-labeled d-peptides were obtained in a total synthesis time of 140 min with radiochemical purity higher than 98%. The specific radioactivities of the three different D1 derivative peptides were between 9 and 113 GBq/μmol. ARG demonstrated a higher radioligand uptake in the cortical gray matter and the hippocampus in the AD brain as compared to age-matched control brain.ConclusionsFluorine-18 labeling of the three novel D1 derivative peptides using [¹⁸F]SFB was successfully accomplished. Higher contrast between AD and control brain slices demonstrates their potential applicability for further use in vivo by PET.     

Comparison of Two Objective Monitors for Assessing Physical Activity and Sedentary Behaviors in Bariatric Surgery Patients

Background  

Objective quantification of physical activity (PA) is needed to understand PA and sedentary behaviors in bariatric surgery patients, yet it is unclear whether PA estimates produced by different monitors are comparable and can be interpreted similarly across studies.