DELE1 oligomerization promotes integrated stress response activation.

Mitochondria are dynamic organelles that continually respond to cellular stress. Recent studies have demonstrated that mitochondrial stress is relayed from mitochondria to the cytosol by the release of a proteolytic fragment of DELE1 that binds to the eIF2α kinase HRI to initiate integrated stress response (ISR) signaling. We report the cryo-electron microscopy structure of the C-terminal cleavage product of human DELE1, which assembles into a high-order oligomer. The oligomer consists of eight DELE1 monomers that assemble with D4 symmetry via two sets of hydrophobic inter-subunit interactions. We identified the key residues involved in DELE1 oligomerization, and confirmed their role in stabilizing the octamer in vitro and in cells using mutagenesis. We further show that assembly-impaired DELE1 mutants are compromised in their ability to induce HRI-dependent ISR activation in cell culture models. Together, our findings provide molecular insights into the activity of DELE1 and how it signals to promote ISR activity following mitochondrial insult.

Cryo-EM structure of hexameric yeast Lon protease (PIM1) highlights the importance of conserved structural elements.

Lon protease is a conserved ATP-dependent serine protease composed of an AAA+ domain that mechanically unfolds substrates and a serine protease domain that degrades these unfolded substrates. In yeast, dysregulation of Lon protease (PIM1) attenuates lifespan and leads to gross mitochondrial morphological perturbations. Although structures of the bacterial and human Lon protease reveal a hexameric assembly, yeast PIM1 was speculated to form a heptameric assembly and is uniquely characterized by a ∼50-residue insertion between the ATPase and protease domains. To further understand the yeast-specific properties of PIM1, we determined a high-resolution cryo-electron microscopy structure of PIM1 in a substrate-translocating state. Here, we reveal that PIM1 forms a hexamer, conserved with that of bacterial and human Lon proteases, wherein the ATPase domains form a canonical closed spiral that enables pore loop residues to translocate substrates to the protease chamber. In the substrate-translocating state, PIM1 protease domains form a planar protease chamber in an active conformation and are uniquely characterized by a ∼15-residue C-terminal extension. These additional C-terminal residues form an α-helix located along the base of the protease domain. Finally, we did not observe density for the yeast-specific insertion between the ATPase and protease domains, likely due to high conformational flexibility. Biochemical studies to investigate the insertion using constructs that truncated or replaced the insertion with a glycine-serine linker suggest that the yeast-specific insertion is dispensable for PIM1's enzymatic function. Altogether, our structural and biochemical studies highlight unique components of PIM1 machinery and demonstrate evolutionary conservation of Lon protease function.

Structures of the human LONP1 protease reveal regulatory steps involved in protease activation.

The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the underlying molecular mechanisms governing substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. Unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate as it is translocated toward the proteolytic chamber. Intriguingly, and in contrast to its bacterial ortholog, substrate binding within the central ATPase channel of LONP1 alone is insufficient to induce the activated conformation of the protease domains. To successfully induce the active protease conformation in substrate-bound LONP1, substrate binding within the protease active site is necessary, which we demonstrate by adding bortezomib, a peptidomimetic active site inhibitor of LONP1. These results suggest LONP1 can decouple ATPase and protease activities depending on whether AAA+ or both AAA+ and protease domains bind substrate. Importantly, our structures provide a molecular framework to define the critical importance of LONP1 in regulating mitochondrial proteostasis in health and disease.

The area method for measuring acetabular cup anteversion: An accurate and autonomous solution.

Several radiological methods of measuring anteversion of the acetabular component after total hip arthroplasty have been described, all time-consuming and with varying reproducibility. This study aimed to compare the recently proposed Area method to true cup anteversion as determined by an accelerometer. This study further applied this method programmatically to autonomously determine radiographic cup orientation using two computer programs, then compared these results to hand and accelerometer measurements. 160 anteroposterior pelvis radiographs were taken of a standard Sawbones® pelvis fitted with a total hip arthroplasty system. The acetabular cup was re-oriented between each radiograph, with anteversion ranging from 0° to 90°. An accelerometer was mounted to the cup to measure true cup anteversion. Radiographic anteversion was independently measured via three methods: by hand, linear image processing, and machine learning. Measurements were compared to triaxial accelerometer recordings. Coefficient of determination (R2) was found to be 0.997, 0.991, and 0.989 for hand measurements, the machine learning, and linear image processing, respectively. The machine learning program and hand measurements overestimated anteversion by 0.70° and 0.02° respectively. The program using linear techniques underestimated anteversion by 5.02°. Average runtime was 0.03 and 0.59 s for the machine learning and linear image processing program, respectively. The machine learning program averaged within 1° of cup orientation given a true cup anteversion less than 51°, and within 2° given an anteversion less than 85°. The Area method showed great accuracy and reliability with hand measurements compared to true anteversion. The results of this study support the use of machine learning for accurate, timely, autonomous assessment of cup orientation.

Artificial intelligence and machine learning in orthopedic surgery: a systematic review protocol.

BACKGROUND: Artificial intelligence (AI) and machine learning (ML) are interwoven into our everyday lives and have grown enormously in some major fields in medicine including cardiology and radiology. While these specialties have quickly embraced AI and ML, orthopedic surgery has been slower to do so. Fortunately, there has been a recent surge in new research emphasizing the need for a systematic review. The primary objective of this systematic review will be to provide an update on the advances of AI and ML in the field of orthopedic surgery. The secondary objectives will be to evaluate the applications of AI and ML in providing a clinical diagnosis and predicting post-operative outcomes and complications in orthopedic surgery. METHODS: A systematic search will be conducted in PubMed, ScienceDirect, and Google Scholar databases for articles written in English, Italian, French, Spanish, and Portuguese language articles published up to September 2020. References will be screened and assessed for eligibility by at least two independent reviewers as per PRISMA guidelines. Studies must apply to orthopedic interventions and acute and chronic orthopedic musculoskeletal injuries to be considered eligible. Studies will be excluded if they are animal studies and do not relate to orthopedic interventions or if no clinical data were produced. Gold standard processes and practices to obtain a clinical diagnosis and predict post-operative outcomes shall be compared with and without the use of ML algorithms. Any case reports and other primary studies assessing the prediction rate of post-operative outcomes or the ability to identify a diagnosis in orthopedic surgery will be included. Systematic reviews or literature reviews will be examined to identify further studies for inclusion, and the results of meta-analyses will not be included in the analysis. DISCUSSION: Our findings will evaluate the advances of AI and ML in the field of orthopedic surgery. We expect to find a large quantity of uncontrolled studies and a smaller subset of articles describing actual applications and outcomes for clinical care. Cohort studies and large randomized control trial will likely be needed. TRIAL REGISTRATION: The protocol will be registered on PROSPERO international prospective register of systematic reviews prior to commencement.

Unique Structural Features of the Mitochondrial AAA+ Protease AFG3L2 Reveal the Molecular Basis for Activity in Health and Disease.

Mitochondrial AAA+ quality-control proteases regulate diverse aspects of mitochondrial biology through specialized protein degradation, but the underlying mechanisms of these enzymes remain poorly defined. The mitochondrial AAA+ protease AFG3L2 is of particular interest, as genetic mutations localized throughout AFG3L2 are linked to diverse neurodegenerative disorders. However, a lack of structural data has limited our understanding of how mutations impact enzymatic function. Here, we used cryoelectron microscopy (cryo-EM) to determine a substrate-bound structure of the catalytic core of human AFG3L2. This structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins. Many disease-relevant mutations localize to these unique structural features of AFG3L2 and distinctly influence its activity and stability. Our results provide a molecular basis for neurological phenotypes associated with different AFG3L2 mutations and establish a structural framework to understand how different members of the AAA+ superfamily achieve specialized biological functions.

Update on Imaging of Knee Arthroplasties: Normal Findings and Hardware Complications.

Total knee arthroplasty (TKA) is the most common joint replacement performed. This article reviews the normal appearance of TKA including the most common types of arthroplasties as well as complications. Common complications at the present time are infection, aseptic loosening, and instability. Rarer complications such as polyethylene wear, periprosthetic fracture, and soft tissue pathology are also discussed. Although the mainstay of imaging is radiographs, newer techniques in TKA imaging such as computed tomography and magnetic resonance imaging are also reviewed.

Conformational ensemble of the human TRPV3 ion channel.

Transient receptor potential vanilloid channel 3 (TRPV3), a member of the thermosensitive TRP (thermoTRPV) channels, is activated by warm temperatures and serves as a key regulator of normal skin physiology through the release of pro-inflammatory messengers. Mutations in trpv3 have been identified as the cause of the congenital skin disorder, Olmsted syndrome. Unlike other members of the thermoTRPV channel family, TRPV3 sensitizes upon repeated stimulation, yet a lack of structural information about the channel precludes a molecular-level understanding of TRPV3 sensitization and gating. Here, we present the cryo-electron microscopy structures of apo and sensitized human TRPV3, as well as several structures of TRPV3 in the presence of the common thermoTRPV agonist 2-aminoethoxydiphenyl borate (2-APB). Our results show α-to-π-helix transitions in the S6 during sensitization, and suggest a critical role for the S4-S5 linker π-helix during ligand-dependent gating.

Practicality of exchanging transparent 3D CT for radiography for pelvic fractures.

OBJECTIVE: We assess the utility of transparent 3D reconstructed CT images for evaluation of traumatic pelvic bony injuries compared to traditional radiographs. METHODS: Radiographs and 3D reconstructed CT were anonymized and randomized before review by 4 board certified physicians using a standardized questionnaire and compared to a gold-standard axial CT by a fifth board certified physician. RESULTS: 49 patients were included. We found significant agreement (K=[0.5-0.92], p<0.001) and comparable accuracy (K=[0.36-0.38], p<0.02) and ghost images of radiographs and transparent 3D reconstructed CT without a difference in confidence (p=0.38). CONCLUSION: Transparent 3D reconstructed CT images may be sufficient for pelvic trauma injury without the use of radiographs.

Metabolic relationship between diabetes and Alzheimer's Disease affected by Cyclo(His-Pro) plus zinc treatment.

BACKGROUND: Association of Alzheimer's Disease (AD) with Type 2 Diabetes (T2D) has been well established. Cyclo(His-Pro) plus zinc (Cyclo-Z) treatment ameliorated diabetes in rats and similar improvements have been seen in human patients. Treatment of amyloid precursor protein (APP) transgenic mice with Cyclo-Z exhibited memory improvements and significantly reduced Aß-40 and Aß-42 protein levels in the brain tissues of the mice. SCOPE OF REVIEW: Metabolic relationship between AD and T2D will be described with particular attention to insulin sensitivity and Aß degradation in brain and plasma tissues. Mechanistic effect of insulin degrading enzyme (IDE) in decreasing blood glucose and brain Aß levels will be elucidated. Cyclo-Z effects on these biochemical parameters will be discussed. MAJOR CONCLUSION: Stimulation of IDE synthesis is effective for the clinical treatment of metabolic diseases including AD and T2D. GENERAL SIGNIFICANCE: Cyclo-Z might be the effective treatment of AD and T2D by stimulating IDE synthesis.

Erdheim Chester disease with appendicular skeletal, renal and pleural involvement responding to Zelboraf (BRAF inhibitor) treatment: case report.

Erdheim Chester disease is a rare non-Langerhans cell histiocytosis which may involve multiple organs including bone, soft tissue, lungs, cardiovascular system, kidneys (retroperitoneum), skin, and central nervous system. Bone involvement is most common followed by other organs. This case report describes a 58-year-old man who presented with progressive renal dysfunction presumed due to obstruction. The patient failed multiple urinary tract interventions, and clinical course was complicated by recurrent low-grade fevers, and bilateral knee pain. Advanced imaging and histopathological features on bone biopsy were consistent with Erdheim Chester disease. Molecular studies of tissue showed BRAF V600 mutation. This patient was treated with Zelboraf (vemurafenib) BRAF inhibitor with subsequent improvement in renal and pleural dysfunction as well as decreased histiocytic soft tissue masses on CT.

Immobilization of the N-terminal helix stabilizes prefusion paramyxovirus fusion proteins.

Parainfluenza virus 5 (PIV5) is an enveloped, single-stranded, negative-sense RNA virus of the Paramyxoviridae family. PIV5 fusion and entry are mediated by the coordinated action of the receptor-binding protein, hemagglutinin-neuraminidase (HN), and the fusion protein (F). Upon triggering by HN, F undergoes an irreversible ATP- and pH-independent conformational change, going down an energy gradient from a metastable prefusion state to a highly stable postfusion state. Previous studies have highlighted key conformational changes in the F-protein refolding pathway, but a detailed understanding of prefusion F-protein metastability remains elusive. Here, using two previously described F-protein mutations (S443D or P22L), we examine the capacity to modulate PIV5 F stability and the mechanisms by which these point mutants act. The S443D mutation destabilizes prefusion F proteins by disrupting a hydrogen bond network at the base of the F-protein globular head. The introduction of a P22L mutation robustly rescues destabilized F proteins through a local hydrophobic interaction between the N-terminal helix and a hydrophobic pocket. Prefusion stabilization conferred by a P22L-homologous mutation is demonstrated in the F protein of Newcastle disease virus, a paramyxovirus of a different genus, suggesting a conserved stabilizing structural element within the paramyxovirus family. Taken together, the available data suggest that movement of the N-terminal helix is a necessary early step for paramyxovirus F-protein refolding and presents a novel target for structure-based drug design.

Rapid expansion of the protein disulfide isomerase gene family facilitates the folding of venom peptides.

Formation of correct disulfide bonds in the endoplasmic reticulum is a crucial step for folding proteins destined for secretion. Protein disulfide isomerases (PDIs) play a central role in this process. We report a previously unidentified, hypervariable family of PDIs that represents the most diverse gene family of oxidoreductases described in a single genus to date. These enzymes are highly expressed specifically in the venom glands of predatory cone snails, animals that synthesize a remarkably diverse set of cysteine-rich peptide toxins (conotoxins). Enzymes in this PDI family, termed conotoxin-specific PDIs, significantly and differentially accelerate the kinetics of disulfide-bond formation of several conotoxins. Our results are consistent with a unique biological scenario associated with protein folding: The diversification of a family of foldases can be correlated with the rapid evolution of an unprecedented diversity of disulfide-rich structural domains expressed by venomous marine snails in the superfamily Conoidea.

On the stability of parainfluenza virus 5 F proteins.

The crystal structure of the F protein (prefusion form) of the paramyxovirus parainfluenza virus 5 (PIV5) WR isolate was determined. We investigated the basis by which point mutations affect fusion in PIV5 isolates W3A and WR, which differ by two residues in the F ectodomain. The P22 stabilizing site acts through a local conformational change and a hydrophobic pocket interaction, whereas the S443 destabilizing site appears sensitive to both conformational effects and amino acid charge/polarity changes.

Acute fibular sesamoid fracture: one part of the spectrum of sesamoid pathologies.

A 35-year-old male athlete presented with acute pain plantar to the hallux metatarsophalangeal joint following jumping to catch a basketball.

Fusion activation through attachment protein stalk domains indicates a conserved core mechanism of paramyxovirus entry into cells.

UNLABELLED: Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [HN, H, or G]) mediate a concerted process of host receptor recognition, followed by the fusion of viral and cellular membranes, resulting in viral nucleocapsid entry into the cytoplasm. However, the sequence of events that closely links the timing of receptor recognition by HN, H, or G and the "triggering" interaction of the attachment protein with F is unclear. F activation results in F undergoing a series of irreversible conformational rearrangements to bring about membrane merger and virus entry. By extensive study of properties of multiple paramyxovirus HN proteins, we show that key features of F activation, including the F-activating regions of HN proteins, flexibility within this F-activating region, and changes in globular head-stalk interactions are highly conserved. These results, together with functionally active "headless" mumps and Newcastle disease virus HN proteins, provide insights into the F-triggering process. Based on these data and very recently published data for morbillivirus H and henipavirus G proteins, we extend our recently proposed "stalk exposure model" to other paramyxoviruses and propose an "induced fit" hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirus-mediated membrane fusion. IMPORTANCE: Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [HN, H, or G]) mediate a concerted process of host receptor recognition, followed by the fusion of viral and cellular membranes. We describe here the molecular mechanism by which HN activates the F protein such that virus-cell fusion is controlled and occurs at the right time and the right place. We extend our recently proposed "stalk exposure model" first proposed for parainfluenza virus 5 to other paramyxoviruses and propose an "induced fit" hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirus-mediated membrane fusion.

Metaphyseal and diaphyseal chondroblastomas.

OBJECTIVE: Epiphyseal/apophyseal locations are important diagnostic radiological features of chondroblastomas (CB). Although the tumor may secondarily involve the metaphysis, reports of primary metaphyseal or diaphyseal CB without any epiphyseal or apophyseal involvement are exceptionally rare and frequently present as a diagnostic dilemma. The purpose of this study was to present seven cases of pure metaphyseal and/or diaphyseal CB along with a review of pertinent literature. METHODS: A retrospective review of databases at two major referral centers revealed 390 cases of CB between 1960 and 2009. Out of these, seven histologically proven CB cases (1.8%) were found to be radiologically located in metaphysis and/or diaphysis, without involving the epiphysis and/or apophysis, and formed the study cohort. RESULTS: There were four males and three females (age range 2-25 years). Locations included proximal femur (n = 1), distal femur (2), proximal humerus (2), clavicle (1), and proximal radius (1). All lesions showed marginal sclerosis. A periosteal reaction was seen in five cases (71%), cortical expansion in four cases (57%), and chondroid matrix in four cases (57%). A CT (two cases) demonstrated a matrix in both cases. An MR (one case) showed extensive perilesional edema. Bone scan (one case) showed intense uptake. CONCLUSION: Pure metaphyseal and/or diaphyseal CB are exceedingly rare. A presumptive diagnosis may be considered in the appropriate age group in the presence of chondroid matrix, perilesional edema, periosteal reaction, and marginal sclerosis. Regardless of all the diagnostic possibilities, biopsy may still be required. However, knowledge of this entity will help make the final diagnosis and guide the correct treatment.