Mining Disaggregase Sequence Space to Safely Counter TDP-43, FUS, and α-Synuclein Proteotoxicity.

Hsp104 is an AAA+ protein disaggregase, which can be potentiated via diverse mutations in its autoregulatory middle domain (MD) to mitigate toxic misfolding of TDP-43, FUS, and α-synuclein implicated in fatal neurodegenerative disorders. Problematically, potentiated MD variants can exhibit off-target toxicity. Here, we mine disaggregase sequence space to safely enhance Hsp104 activity via single mutations in nucleotide-binding domain 1 (NBD1) or NBD2. Like MD variants, NBD variants counter TDP-43, FUS, and α-synuclein toxicity and exhibit elevated ATPase and disaggregase activity. Unlike MD variants, non-toxic NBD1 and NBD2 variants emerge that rescue TDP-43, FUS, and α-synuclein toxicity. Potentiating substitutions alter NBD1 residues that contact ATP, ATP-binding residues, or the MD. Mutating the NBD2 protomer interface can also safely ameliorate Hsp104. Thus, we disambiguate allosteric regulation of Hsp104 by several tunable structural contacts, which can be engineered to spawn enhanced therapeutic disaggregases with minimal off-target toxicity.

Determinants of Severe Metabolic Bone Disease in Very Low-Birth-Weight Infants with Severe Bronchopulmonary Dysplasia Admitted to a Tertiary Referral Center.

OBJECTIVE: Nonrespiratory comorbidities are common among preterm infants with severe bronchopulmonary dysplasia (BPD) referred to tertiary perinatal centers. We evaluated the incidence, severity, and risk factors for metabolic bone disease (MBD) in this population. STUDY DESIGN: We conducted a retrospective cohort study of all infants born ≤ 1,500 g who were diagnosed with severe BPD in our single, tertiary referral center between September 2010 and October 2012. MBD severity was classified by serial radiography. RESULTS: Among the 83 infants diagnosed with severe BPD, 26 (31%) developed severe MBD (rickets). Male gender and lower gestational age and birth weight were associated with increased odds of severe MBD. After adjustment for these potential confounders, cytomegalovirus infection, postnatal growth restriction, surgical necrotizing enterocolitis, and blood culture confirmed sepsis were associated with increased odds of severe MBD. The cumulative duration of therapy with furosemide, hydrocortisone, and prednisolone each correlated with significantly greater probability of severe MBD. CONCLUSIONS: Severe MBD was common in this referral-based cohort with severe BPD. The high incidence in this population is likely explained by the coexistence of multiple exposures and comorbidities associated with bone demineralization.

Disparate Mutations Confer Therapeutic Gain of Hsp104 Function.

Hsp104, a protein disaggregase from yeast, can be engineered and potentiated to counter TDP-43, FUS, or α-synuclein misfolding and toxicity implicated in neurodegenerative disease. Here, we reveal that extraordinarily disparate mutations potentiate Hsp104. Remarkably, diverse single missense mutations at 20 different positions interspersed throughout the middle domain (MD) and small domain of nucleotide-binding domain 1 (NBD1) confer a therapeutic gain of Hsp104 function. Moreover, potentiation emerges from deletion of MD helix 3 or 4 or via synergistic missense mutations in the MD distal loop and helix 4. We define the most critical aspect of Hsp104 potentiation as enhanced disaggregase activity in the absence of Hsp70 and Hsp40. We suggest that potentiation likely stems from a loss of a fragilely constrained autoinhibited state that enables precise spatiotemporal regulation of disaggregase activity.

The evolution of the Japanese medical education system: a historical perspective.

The Japanese Medical Education system has been influenced by political events throughout the country's history. From long periods of isolation from the western world to the effect of world wars, Japan's training system for physicians has had to adapt in many ways and will continue to change. The Japanese medical education system was recently compared to the "Galapagos Islands" for its unusual and singular evolution, in a speech by visiting professor Dr. Gordon L. Noel at the University of Tokyo International Research center.1 Japanese medical schools are currently working to increase their students' clinical hours or else these students may not be able to train in the United States for residencies. Knowing the history of the Japanese Medical education system is paramount to understanding the current system in place today. Studying the historical foundation of this system will also provide insight on how the system must change in order to produce better clinicians. This article provides a glimpse into the medical system of another nation that may encourage needed reflection on the state of current healthcare training in the United States.

Isolating potentiated Hsp104 variants using yeast proteinopathy models.

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and α-synuclein, implicated in Parkinson's disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and α-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.

Pulmonary hemosiderosis in children with bronchopulmonary dysplasia.

We describe a possible association between pulmonary hemosiderosis (PH) and a history of bronchopulmonary dysplasia (BPD). Both patients were born at 28-week gestation and presented with PH at ages 22 months and 6 years, respectively. Both initially presented with cough and tachypnea, and bronchoalveolar lavage showed evidence of hemosiderin-laden macrophages. Initial hemoglobin levels were < 4 g/dL and chest radiographs showed diffuse infiltrates that cleared dramatically within days after initiation of intravenous corticosteroids. In the first case, frank pulmonary blood was observed upon initial intubation, prompting the need for high frequency ventilation, immediate corticosteroids, and antibiotics. The mechanical ventilation wean was made possible by the addition of mycophenolate mofetil (MMF) and hydroxychloroquine. Slow tapering off of medications was accomplished over 6 years. These cases represent a possible correlation between prematurity-associated BPD and PH. We present a review of the literature regarding this possible association. In addition, MMF proved to be life-saving in one of the PH cases, as it has been in pulmonary hemorrhage related to systemic lupus erythematosus. Further studies are warranted to investigate the possible association between PH and prematurity-related BPD, as well as the use of MMF in the treatment of PH.

An intra-articular ganglion cyst in a patient with juvenile idiopathic arthritis.

We report an intra-articular ganglion cyst (IAGC) presenting as knee pain and a mass in a patient with longstanding Juvenile Idiopathic Arthritis (JIA). We could not find a similar case of an IAGC occurring in the knee of JIA patients in the literature. IAGC may need to be included as a possibility in patients with inflammatory arthritis with new-onset knee pain, especially in those with a palpable mass. MRI was useful in distinguishing IAGC from more worrisome causes of a knee mass. Orthopedic input was helpful in diagnosis and treatment. In addition, methotrexate therapy was effective in bringing about a long-lasting remission.