A redox regulatory system critical for mycobacterial survival in macrophages and biofilm development.

Survival of M. tuberculosis in host macrophages requires the eukaryotic-type protein kinase G, PknG, but the underlying mechanism has remained unknown. Here, we show that PknG is an integral component of a novel redox homeostatic system, RHOCS, which includes the ribosomal protein L13 and RenU, a Nudix hydrolase encoded by a gene adjacent to pknG. Studies in M. smegmatis showed that PknG expression is uniquely induced by NADH, which plays a key role in metabolism and redox homeostasis. In vitro, RenU hydrolyses FAD, ADP-ribose and NADH, but not NAD+. Absence of RHOCS activities in vivo causes NADH and FAD accumulation, and increased susceptibility to oxidative stress. We show that PknG phosphorylates L13 and promotes its cytoplasmic association with RenU, and the phosphorylated L13 accelerates the RenU-catalyzed NADH hydrolysis. Importantly, interruption of RHOCS leads to impaired mycobacterial biofilms and reduced survival of M. tuberculosis in macrophages. Thus, RHOCS represents a checkpoint in the developmental program required for mycobacterial growth in these environments.

Quantification of Bone Marrow Involvement in Treated Gaucher Disease With Proton MR Spectroscopy: Correlation With Bone Marrow MRI Scores and Clinical Status.

OBJECTIVE: The objective of our study was to use proton MR spectroscopy (MRS) to quantitatively evaluate bone marrow infiltration by measuring the fat fraction (FF) and to compare the FF with semiquantitative bone marrow MRI scores and clinical status in children treated for type 1 Gaucher disease (GD). SUBJECTS AND METHODS: Over a 2-year period, we prospectively evaluated 10 treated GD patients (six males, four females; median age, 15.1 years) and 10 healthy age-matched control subjects (five males, five females; median age, 15.3 years) using 3-T proton MRS of L5 and the femoral neck. Water and lipid AUCs were measured to calculate the FF. Two blinded pediatric musculoskeletal radiologists performed a semiquantitative analysis of the conventional MR images using the bone marrow burden score and modified Spanish MRI score. We evaluated symptoms, spleen and liver volumes, platelet levels, hemoglobin levels, and bone complications. RESULTS: In the femur, the FF was higher in the control subjects (median, 0.71) than the GD patients (0.54) (p = 0.02). In L5, the difference in FF--higher FF in control subjects (0.37) than in GD patients (0.26)--was not significant (p = 0.16). In both groups and both regions, the FF increased with patient age (p < 0.02). Semiquantitative scores showed no differences between control subjects and treated GD patients (p > 0.11). Eight of 10 GD patients were asymptomatic and two had chronic bone pain. The median age of patients at symptom onset was 4.0 years, the median age of patients at the initiation of enzyme replacement therapy was 4.3 years, and the median treatment duration was 10.2 years. Hemoglobin level, platelet count, and liver volume at MRI were normal. Mean pretreatment spleen volume (15.4-fold above normal) decreased to 2.8-fold above normal at the time of MRI (p = 0.01). CONCLUSION: Proton MRS detected FF differences that were undetectable using conventional MRI; for that reason, proton MRS can be used to optimize treatment of GD patients.

Multimodality imaging manifestations of the Meckel diverticulum in children.

Meckel diverticulum is the most common congenital abnormality of the gastrointestinal (GI) tract, occurring in approximately 2% of the general population. The lifetime complication rate from a Meckel diverticulum is 0.5%-2%. The most common complications include bleeding, obstruction, inflammation and perforation. However, the clinical manifestations of a Meckel diverticulum are frequently nonspecific. As a result, complications secondary to Meckel diverticulitis can mimic a variety of more common intra-abdominal processes, such as appendicitis, inflammatory bowel disease and any other cause of small bowel inflammation or obstruction. The radiologist should be aware of potential manifestations of the disease on different imaging modalities. In this pictorial essay, we illustrate several complications related to the Meckel diverticulum, in multiple modalities.

The YΦ motif defines the structure-activity relationships of human 20S proteasome activators.

The 20S proteasome (20S) facilitates turnover of most eukaryotic proteins. Substrate entry into the 20S first requires opening of gating loops through binding of HbYX motifs that are present at the C-termini of certain proteasome activators (PAs). The HbYX motif has been predominantly characterized in the archaeal 20S, whereas little is known about the sequence preferences of the human 20S (h20S). Here, we synthesize and screen ~120 HbYX-like peptides, revealing unexpected differences from the archaeal system and defining the h20S recognition sequence as the Y-F/Y (YФ) motif. To gain further insight, we create a functional chimera of the optimized sequence, NLSYYT, fused to the model activator, PA26E102A. A cryo-EM structure of PA26E102A-h20S is used to identify key interactions, including non-canonical contacts and gate-opening mechanisms. Finally, we demonstrate that the YФ sequence preferences are tuned by valency, allowing multivalent PAs to sample greater sequence space. These results expand the model for termini-mediated gating and provide a template for the design of h20S activators.

Botulinum toxin A injection of salivary glands in children with drooling and chronic aspiration.

PURPOSE: To review outcomes of ultrasound (US)-guided percutaneous submandibular gland injection of botulinum toxin A (BTX-A) in the treatment of drooling and chronic aspiration. MATERIALS AND METHODS: A 3-year retrospective review was performed of 220 US-guided salivary gland injections in 36 patients. There were 21 male patients and 15 female patients with an age range of 1.4 to 19.8 years (mean, 8.6 y) and a weight range of 7.8 to 73 kg (mean, 24.4 kg). The mean pretreatment analysis period was 48 months and the mean follow-up period was 21 months. The study group was divided into groups with anterior (n = 9) and posterior (n = 27) drooling, with those with both (n = 10) included in the posterior group. RESULTS: All procedures were technically successful. Bilateral submandibular injections were performed in 34 procedures and bilateral submandibular and parotid injections were performed in 38 procedures. Of the 27 patients with posterior drooling, improvement occurred in 24 patients (88%), no improvement was seen in two (8%), and one (4%) was lost to follow-up. Of the nine patients with anterior drooling, six (66%) showed improvement, there was no response in two (22%), and one (12%) was lost to follow-up. The total number of hospitalizations for respiratory issues and presumed aspiration pneumonia decreased by 56.4% per year in the patients with posterior drooling. There was one procedure-related complication: an episode of self-limited oral bleeding. CONCLUSION: Salivary gland BTX-A injection for salivary control shows promising results in decreasing saliva production and frequency of respiratory symptoms in children with drooling and chronic aspiration.

Diagnosis of osteomyelitis in children: utility of fat-suppressed contrast-enhanced MRI.

OBJECTIVE: The purpose of this study was to determine whether the use of fat-suppressed contrast-enhanced MRI, compared with unenhanced MRI alone, increases reader confidence in the diagnosis of osteomyelitis and its complications in children. MATERIALS AND METHODS: MRI studies of 78 skeletally immature children and adolescents (median age, 3.6 years) with suspected nonspinal osteomyelitis were reviewed in consensus by two readers. Unenhanced images were evaluated first and then contrast-enhanced MR images. Images were scored for the presence or absence of osteomyelitis, abscess, septic arthritis, and physeal involvement on a 5-point scale ranging from definitely absent to definitely present. Forty-two additional studies were evaluated to test interobserver agreement. RESULTS: Osteomyelitis was clinically diagnosed in 40 cases (51%). There was no significant difference between the sensitivity and specificity of unenhanced MRI (p = 1.0) and those of contrast-enhanced MRI (p = 0.77) for the diagnosis of osteomyelitis. Nonetheless, there was a significant (p < 0.001) increase in confidence in the diagnosis of osteomyelitis and its complications. This increase in confidence was most pronounced for the diagnosis of abscess (46%). The addition of contrast enhancement was least useful in findings deemed definitely absent on unenhanced MR images. CONCLUSION: Although it does not increase the sensitivity or specificity of the diagnosis, use of contrast-enhanced MRI does increase reader confidence in the diagnosis of osteomyelitis and its complications in cases in which bone or soft-tissue edema is found on unenhanced images. In the clear absence of edema on unenhanced images, however, contrast enhancement is not needed.

Partial splenic embolization in a child with Gaucher disease, massive splenomegaly and severe thrombocytopenia.

A 13-month-old boy with Gaucher disease presented with severe thrombocytopenia, anemia and massive splenomegaly. In addition he had significant respiratory compromise caused by abdominal compartment syndrome, requiring mechanical ventilation. Because of the degree of respiratory compromise and his existing bone marrow suppression, splenic artery embolization was chosen as an alternative to splenectomy. Splenic artery embolization was performed using 355-500-microm polyvinyl alcohol particles, with 70% ablation achieved. Within 24 h of the procedure the platelet count had risen to greater than 70,000/mm(3) and to more than 170,000/mm(3) on postoperative day 4. At the 8-month follow-up the splenic size had decreased from 18 cm to 8 cm, with a platelet count of 578,000/mm(3). Partial splenic embolization provides a minimally invasive alternative to splenectomy in patients with Gaucher disease with massive splenomegaly and bone marrow suppression.

Specific lid-base contacts in the 26s proteasome control the conformational switching required for substrate degradation.

The 26S proteasome is essential for proteostasis and the regulation of vital processes through ATP-dependent degradation of ubiquitinated substrates. To accomplish the multi-step degradation process, the proteasome's regulatory particle, consisting of lid and base subcomplexes, undergoes major conformational changes whose origin is unknown. Investigating the Saccharomyces cerevisiae proteasome, we found that peripheral interactions between the lid subunit Rpn5 and the base AAA+ ATPase ring are important for stabilizing the substrate-engagement-competent state and coordinating the conformational switch to processing states upon substrate engagement. Disrupting these interactions perturbs the conformational equilibrium and interferes with degradation initiation, while later processing steps remain unaffected. Similar defects in early degradation steps are observed when eliminating hydrolysis in the ATPase subunit Rpt6, whose nucleotide state seems to control proteasome conformational transitions. These results provide important insight into interaction networks that coordinate conformational changes with various stages of degradation, and how modulators of conformational equilibria may influence substrate turnover.

Substrate-engaged 26S proteasome structures reveal mechanisms for ATP-hydrolysis-driven translocation.

The 26S proteasome is the primary eukaryotic degradation machine and thus is critically involved in numerous cellular processes. The heterohexameric adenosine triphosphatase (ATPase) motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo-electron microscopy structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26S proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination and how ATP-binding, -hydrolysis, and phosphate-release events are coordinated within the AAA+ (ATPases associated with diverse cellular activities) motor to induce conformational changes and propel the substrate through the central pore.

What's the Key to Unlocking the Proteasome's Gate?

In this issue of Structure, Bolten et al. (2016) describe the organization of the mycobacterial proteasome in complex with the ATP-independent bacterial proteasome activator (Bpa, PafE). They confirm several activation motifs employed by archaea and eukaryotes and highlight differences that pose Bpa as a novel architectural class of proteasome activators.

Ectopic craniopharyngioma of the fourth ventricle in a patient with Gardner syndrome.

Ectopic craniopharyngioma is uncommon and a craniopharyngioma confined purely within the fourth ventricle is extremely rare. We report a craniopharyngioma of the fourth ventricle in a 20-year-old man with Gardner syndrome. Imaging characteristics of craniopharyngiomas and fourth ventricle lesions are discussed with a review of the literature regarding the pathogenesis of craniopharyngiomas and the possible association with Gardner syndrome.

DSF Guided Refolding As A Novel Method Of Protein Production.

The Anfinsen hypothesis, the demonstration of which led to the Nobel prize in Chemistry, posits that all information required to determine a proteins' three dimensional structure is contained within its amino acid sequence. This suggests that it should be possible, in theory, to fold any protein in vitro. In practice, however, protein production by refolding is challenging because suitable refolding conditions must be empirically determined for each protein and can be painstaking. Here we demonstrate, using a variety of proteins, that differential scanning fluorimetry (DSF) can be used to determine and optimize conditions that favor proper protein folding in a rapid and high-throughput fashion. The resulting method, which we deem DSF guided refolding (DGR), thus enables the production of aggregation-prone and disulfide-containing proteins by refolding from E. coli inclusion bodies, which would not normally be amenable to production in bacteria.

Structural and Enzymatic Characterization of a Nucleoside Diphosphate Sugar Hydrolase from Bdellovibrio bacteriovorus.

Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 -- a Nudix hydrolase from Bdellovibrio bacteriovorus-that we show localized in the periplasmic space of this obligate Gram-negative predator. We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase) and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Å Cα RMSD respectively). Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the α-ß-α NDPSase fold differentiates NDPSases from ADPRases.