A new paradigm for enzymatic control of α-cleavage and ß-cleavage of the prion protein.

The cellular form of the prion protein (PrP(C)) is found in both full-length and several different cleaved forms in vivo. Although the precise functions of the PrP(C) proteolytic products are not known, cleavage between the unstructured N-terminal domain and the structured C-terminal domain at Lys-109↓His-110 (mouse sequence), termed α-cleavage, has been shown to produce the anti-apoptotic N1 and the scrapie-resistant C1 peptide fragments. ß-Cleavage, residing adjacent to the octarepeat domain and N-terminal to the α-cleavage site, is thought to arise from the action of reactive oxygen species produced from redox cycling of coordinated copper. We sought to elucidate the role of key members of the ADAM (a disintegrin and metalloproteinase) enzyme family, as well as Cu(2+) redox cycling, in recombinant mouse PrP (MoPrP) cleavage through LC/MS analysis. Our findings show that although Cu(2+) redox-generated reactive oxygen species do produce fragmentation corresponding to ß-cleavage, ADAM8 also cleaves MoPrP in the octarepeat domain in a Cu(2+)- and Zn(2+)-dependent manner. Additional cleavage by ADAM8 was observed at the previously proposed location of α-cleavage, Lys-109↓His-110 (MoPrP sequencing); however, upon addition of Cu(2+), the location of α-cleavage shifted by several amino acids toward the C terminus. ADAM10 and ADAM17 have also been implicated in α-cleavage at Lys-109↓His-110; however, we observed that they instead cleaved MoPrP at a novel location, Ala-119↓Val-120, with additional cleavage by ADAM10 at Gly-227↓Arg-228 near the C terminus. Together, our results show that MoPrP cleavage is far more complex than previously thought and suggest a mechanism by which PrP(C) fragmentation responds to Cu(2+) and Zn(2+).

Combined EXAFS and DFT structure calculations provide structural insights into the 1:1 multi-histidine complexes of Cu(II) , Cu(I) , and Zn(II) with the tandem octarepeats of the mammalian prion protein.

The metal-coordinating properties of the prion protein (PrP) have been the subject of intense focus and debate since the first reports of its interaction with copper just before the turn of the century. The picture of metal coordination to PrP has been improved and refined over the past decade, but structural details of the various metal coordination modes have not been fully elucidated in some cases. In the present study, we have employed X-ray absorption near-edge spectroscopy as well as extended X-ray absorption fine structure (EXAFS) spectroscopy to structurally characterize the dominant 1:1 coordination modes for Cu(II) , Cu(I) , and Zn(II) with an N-terminal fragment of PrP. The PrP fragment corresponds to four tandem repeats representative of the mammalian octarepeat domain, designated as OR4 , which is also the most studied PrP fragment for metal interactions, making our findings applicable to a large body of previous work. Density functional theory (DFT) calculations have provided additional structural and thermodynamic data, and candidate structures have been used to inform EXAFS data analysis. The optimized geometries from DFT calculations have been used to identify potential coordination complexes for multi-histidine coordination of Cu(II) , Cu(I) , and Zn(II) in an aqueous medium, modelled using 4-methylimidazole to represent the histidine side chain. Through a combination of in silico coordination chemistry as well as rigorous EXAFS curve-fitting, using full multiple scattering on candidate structures derived from DFT calculations, we have characterized the predominant coordination modes for the 1:1 complexes of Cu(II) , Cu(I) , and Zn(II) with the OR4 peptide at pH 7.4 at atomic resolution, which are best represented as square-planar [Cu(II) (His)4 ](2+) , digonal [Cu(I) (His)2 ](+) , and tetrahedral [Zn(II) (His)3 (OH2 )](2+) , respectively.

The Perfect Step is the first one: Improving the quality of life for patients with spinal cord injuries through structured exercise.

CONTEXT: More than 1 million people live with spinal cord injuries (SCI) in the United States alone. Despite research suggesting improvement in functional activities in patients who participate in regular physical activity, there is limited data on the specific impact of exercise as it pertains to patient-reported quality-of-life (QoL) measures of mood, pain, independence, or medical complications. Patients with SCI participating in a longitudinal exercise program at a community-based fitness facility (The Perfect Step) in Pomona, California were invited to participate in this study. This program consisted of at least two hours a week of exercise-based therapy, loadbearing, and neuroplasticity retraining for two years. Participants were asked to retrospectively rate patient-reported quality-of-life outcomes at the start of participation in the program, and at 6, 12, and 24 months after the program start as measured by an 89-item SCI QoL questionnaire covering domains including mood (39 items), pain (16 items), independence (7 items), bladder function (14 items), and pressure ulcers (13 items). 24 patients with SCI (16 cervical, 4 thoracic, 3 lumbar, 1 unspecified) were included in the final analysis. FINDINGS: Wilcoxon rank sum tests were used to compare patient-reported outcome measures over time. Patients reported statistically significantly improved outcomes across all domains at all timepoints to varying degrees, with improvement in 73/89 items at 6 months, 57/89 at 12 months, and 65/89 at 24 months after participation start (P < 0.05, all). CONCLUSION/CLINICAL RELEVANCE: Patients with SCI engaging in a structured exercise program demonstrated sustainable, longitudinal improvements in mood, pain control, independence, and fewer impairments related to bladder function and pressure ulcers. Physicians should consider the integration of structured exercise into the rehabilitation program alongside traditional medical care to optimize the quality of life for those with SCI. Future research should involve a direct comparison of QoL in patients with SCI participating in structured exercise versus those who are not.

Experimental results of a 330 GW impedance-matched Marx generator.

Impedance-matched Marx generators (IMGs) are considered next generation pulsed-power drivers because of their long lifetime (> 10,000 shots), repetition rate (> 0.1-Hz), fast rise time (~ 100-ns), and high-energy-delivery efficiency (~ 90%). "TITAN" is a 14-stage IMG designed to deliver 1-TW to a 2-Ω matched load. In this paper, design, simulation, and experimental results for six stages of TITAN including its triggering system, air delivery system, and pulse shaping are presented. To achieve efficiency over 85% and maximize the capability of an IMG, synchronized triggering, reduced pre-fire rate, and pulse shaping ability are crucial. In this paper, novel engineering solutions are introduced, tested, and proven to overcome those challenges. 6-stage TITAN, powered by 102 identical bricks and 102 field-distortion-triggered gas switches, could generate ~ 600-kA and ~ 700-kV across a ~ 0.9-Ω matched load when fully charged to ± 100-kV. In these experiments, 6-stage TITAN is tested up to ± 70-kV charge voltage which delivers a peak power of 330-GW to a 1.2-Ω resistive load.

Primary Care-Based Interventional Procedures for Chronic Pain.

Chronic pain interventions in the primary care setting can provide temporal relief and are best used as part of a comprehensive treatment plan. Interventional therapies may use steroids, local anesthetic, saline, prolotherapy, no medication at all (dry needling), acupuncture, or transcutaneous electrical nerve stimulation. These interventions may include adjuvant modalities, such as ultrasound, to improve precision and accuracy of injection. Choice of interventional therapy for chronic pain in the primary care setting is highly dependent on the clinician, location, and cause of the pain as well as a multitude of patient factors, which are discussed in this article.

Copper redox cycling in the prion protein depends critically on binding mode.

The prion protein (PrP) takes up 4-6 equiv of copper in its extended N-terminal domain, composed of the octarepeat (OR) segment (human sequence residues 60-91) and two mononuclear binding sites (at His96 and His111; also referred to as the non-OR region). The OR segment responds to specific copper concentrations by transitioning from a multi-His mode at low copper levels to a single-His, amide nitrogen mode at high levels (Chattopadhyay et al. J. Am. Chem. Soc. 2005, 127, 12647-12656). The specific function of PrP in healthy tissue is unclear, but numerous reports link copper uptake to a neuroprotective role that regulates cellular stress (Stevens, et al. PLoS Pathog.2009, 5 (4), e1000390). A current working hypothesis is that the high occupancy binding mode quenches copper's inherent redox cycling, thus, protecting against the production of reactive oxygen species from unregulated Fenton type reactions. Here, we directly test this hypothesis by performing detailed pH-dependent electrochemical measurements on both low and high occupancy copper binding modes. In contrast to the current belief, we find that the low occupancy mode completely quenches redox cycling, but high occupancy leads to the gentle production of hydrogen peroxide through a catalytic reduction of oxygen facilitated by the complex. These electrochemical findings are supported by independent kinetic measurements that probe for ascorbate usage and also peroxide production. Hydrogen peroxide production is also observed from a segment corresponding to the non-OR region. Collectively, these results overturn the current working hypothesis and suggest, instead, that the redox cycling of copper bound to PrP in the high occupancy mode is not quenched, but is regulated. The observed production of hydrogen peroxide suggests a mechanism that could explain PrP's putative role in cellular signaling.

What States Can Do to Address Out-of-Network Air Ambulance Bills.

Out-of-network air ambulance bills are a pernicious and financially devastating type of surprise medical bill. Courts have broadly interpreted the Airline Deregulation Act to preempt most state attempts to regulate air ambulance billing abuses, so a federal solution is ultimately needed. However, in the absence of a federal fix, states have experimented with a variety of approaches that may survive preemption and provide some protections for their citizens.

Selected Musculoskeletal Issues in Adolescents.

Musculoskeletal care of the adolescent patient involves unique knowledge of their rapidly changing physical and psychological health. In this article, the importance of preventing early sports specialization is elucidated, and an encouragement of the safety and necessity of resistance training in adolescents is undertaken. It also explores two common conditions, one affecting the immature skeleton (apophysitis), and one affecting the improperly developed muscular system (patellofemoral syndrome), both of which are diagnosed clinically, and require little advanced imaging. Finally, a brief overview of relative energy deficiency in sport is given.

Anti-NMDA receptor encephalitis: a case study and illness overview.

Anti-N-methyl D-aspartate (NMDA) receptor (anti-NMDAR) encephalitis is among one of the most common autoimmune encephalitides. However, variations in clinical presentation and nonsequential multiphasic course often lead to delays in diagnosis. The mild encephalitis (ME) hypothesis suggests a pathogenetic mechanism of low-level neuroinflammation sharing symptom overlap between anti-NMDAR encephalitis and other psychiatric disorders including schizophrenia. Clinical symptoms of anti-NMDAR encephalitis may mimic schizophrenia and psychotic spectrum disorders or substance-induced psychosis. Although initially described in association with ovarian teratomas in women, anti-NMDAR encephalitis has been reported in individuals without paraneoplastic association, as well as in males. It can affect all age groups but is usually lower in prevalence in individuals greater than 50 years old, and it affects females more than males. Clinical evaluation is supported by laboratory workup, which includes cerebrospinal fluid (CSF) assays. The latter often reveals lymphocytic pleocytosis or oligoclonal bands with normal to elevated CSF protein. CSF testing for anti-NMDAR antibodies facilitates diagnostic confirmation. Serum anti-NMDAR antibody assays are not as sensitive as CSF assays. Management includes symptomatic treatment and immunotherapy.

Altered Domain Structure of the Prion Protein Caused by Cu2+ Binding and Functionally Relevant Mutations: Analysis by Cross-Linking, MS/MS, and NMR.

The cellular isoform of the prion protein (PrPC) serves as precursor to the infectious isoform (PrPSc), and as a cell-surface receptor, which binds misfolded protein oligomers as well as physiological ligands such as Cu2+ ions. PrPC consists of two domains: a flexible N-terminal domain and a structured C-terminal domain. Both the physiological and pathological functions of PrP depend on intramolecular interactions between these two domains, but the specific amino acid residues involved have proven challenging to define. Here, we employ a combination of chemical cross-linking, mass spectrometry, NMR, molecular dynamics simulations, and functional assays to identify residue-level contacts between the N- and C-terminal domains of PrPC. We also determine how these interdomain contacts are altered by binding of Cu2+ ions and by functionally relevant mutations. Our results provide a structural basis for interpreting both the normal and toxic activities of PrP.

An inter-domain regulatory mechanism controls toxic activities of PrPC.

The normal function of PrPC, the cellular prion protein, has remained mysterious since its first description over 30 years ago. Amazingly, although complete deletion of the gene encoding PrPC has little phenotypic consequence, expression in transgenic mice of PrP molecules carrying certain internal deletions produces dramatic neurodegenerative phenotypes. In our recent paper, 1 we have demonstrated that the flexible, N-terminal domain of PrPC possesses toxic effector functions, which are regulated by a docking interaction with the structured, C-terminal domain. Disruption of this inter-domain interaction, for example by deletions of the hinge region or by binding of antibodies to the C-terminal domain, results in abnormal ionic currents and degeneration of dendritic spines in cultured neuronal cells. This mechanism may contribute to the neurotoxicity of PrPSc and possibly other protein aggregates, and could play a role in the physiological activity of PrPC. These results also provide a warning about the potential toxic side effects of PrP-directed antibody therapies for prion and Alzheimer's diseases.

Prevalence of Injuries during Brazilian Jiu-Jitsu Training.

Brazilian jiu-jitsu (BJJ) is a martial art that focuses on groundwork, joint locks, and chokeholds. The purpose of this study is to determine the prevalence of injuries sustained during BJJ training. A 27-question research survey was e-mailed to 166 BJJ gyms in the United States. Demographic information, belt level, weight class, training hours, competition experience, and injury prevalence data were collected. The majority of respondents were Caucasian (n = 96) males (n = 121) with an average age of 30.3 years. Overall, the most common injury locations were to the hand and fingers (n = 70), foot and toes (n = 52), and arm and elbow (n = 51). The most common medically diagnosed conditions were skin infections (n = 38), injuries to the knee (n =26), and foot and toes (n = 19). The most common non-medically diagnosed injuries occurred to the hand and fingers (n = 56), arm and elbow (n = 40), and foot and toes (n = 33). In general, athletes were more likely to sustain distal rather than proximal injuries. Athletes reported more frequent medically diagnosed injuries to the lower extremity and more frequent self-diagnosed injuries to the upper extremity. Upper extremity injuries appear to be more frequent but less severe than lower extremity injuries with the opposite being true for lower extremity injuries.

The N-terminus of the prion protein is a toxic effector regulated by the C-terminus.

PrPC, the cellular isoform of the prion protein, serves to transduce the neurotoxic effects of PrPSc, the infectious isoform, but how this occurs is mysterious. Here, using a combination of electrophysiological, cellular, and biophysical techniques, we show that the flexible, N-terminal domain of PrPC functions as a powerful toxicity-transducing effector whose activity is tightly regulated in cis by the globular C-terminal domain. Ligands binding to the N-terminal domain abolish the spontaneous ionic currents associated with neurotoxic mutants of PrP, and the isolated N-terminal domain induces currents when expressed in the absence of the C-terminal domain. Anti-PrP antibodies targeting epitopes in the C-terminal domain induce currents, and cause degeneration of dendrites on murine hippocampal neurons, effects that entirely dependent on the effector function of the N-terminus. NMR experiments demonstrate intramolecular docking between N- and C-terminal domains of PrPC, revealing a novel auto-inhibitory mechanism that regulates the functional activity of PrPC.

Octarepeat region flexibility impacts prion function, endoproteolysis and disease manifestation.

The cellular prion protein (PrP(C)) comprises a natively unstructured N-terminal domain, including a metal-binding octarepeat region (OR) and a linker, followed by a C-terminal domain that misfolds to form PrP(S) (c) in Creutzfeldt-Jakob disease. PrP(C) ß-endoproteolysis to the C2 fragment allows PrP(S) (c) formation, while α-endoproteolysis blocks production. To examine the OR, we used structure-directed design to make novel alleles, 'S1' and 'S3', locking this region in extended or compact conformations, respectively. S1 and S3 PrP resembled WT PrP in supporting peripheral nerve myelination. Prion-infected S1 and S3 transgenic mice both accumulated similar low levels of PrP(S) (c) and infectious prion particles, but differed in their clinical presentation. Unexpectedly, S3 PrP overproduced C2 fragment in the brain by a mechanism distinct from metal-catalysed hydrolysis reported previously. OR flexibility is concluded to impact diverse biological endpoints; it is a salient variable in infectious disease paradigms and modulates how the levels of PrP(S) (c) and infectivity can either uncouple or engage to drive the onset of clinical disease.

Neurosyphilis in newly admitted psychiatric patients: three case reports.

BACKGROUND: Neurosyphilis, also known as "general paresis of the insane," at one time accounted for a large portion of admissions to state psychiatric facilities. With the introduction of antibiotics, neurosyphilis is now considered very rare. METHOD: Chart review was performed on patients diagnosed with neurosyphilis who were admitted to a state psychiatric hospital in Raleigh, N.C., during 2002. RESULTS: We identified 3 cases of confirmed neurosyphilis, representing 0.1% of adult admissions, diagnosed in newly admitted psychiatric patients. None of the patients were immunocompromised. Response to antibiotic treatment was poor. CONCLUSIONS: Given the increase in primary and secondary syphilis reported in the 1980s and early 1990s, routine screening of psychiatric patients for the presence of syphilis should be considered.

Unerupted mandibular second primary molar with an unusual histopathological finding: a case report.

This is the case of a healthy 6-year old female with a clinically absent right mandibular second primary molar with no history of that tooth ever being present. Radiographic examination revealed a well-circumscribed pericoronal radiolucency surrounding the mandibular right primary second molar. The mandibular right second premolar was displaced mesially. Treatment consisted of enucleation of the lesion with removal of both the unerupted primary second molar and second premolar. The histopathology of the excised lesion revealed a hyperplastic dental follicle with a focal proliferation of odontogenic epithelium and duct-like structures, probably representing an incipient adenomatoid odontogenic tumor.

PrP overdrive: does inhibition of α-cleavage contribute to PrP(C) toxicity and prion disease?

Knockout of the cellular prion protein (PrP(C)) in mice is tolerated, as is complete elimination of the protein's N-terminal domain. However, deletion of select short segments between the N- and C-terminal domains is lethal. How can one reconcile this apparent paradox? Research over the last few years demonstrates that PrP(C) undergoes α-cleavage in the vicinity of residue 109 (mouse sequence) to release the bioactive N1 and C1 fragments. In biophysical studies, we recently characterized the action of relevant members of the ADAM (A Disintegrin And Metalloproteinase) enzyme family (ADAM8, 10, and 17) and found that they all produce α-cleavage, but at 3 distinct cleavage sites, with proteolytic efficiency modulated by the physiologic metals copper and zinc. Remarkably, the shortest lethal deletion segment in PrP(C) fully encompasses the 3 α-cleavage sites. Analysis of all reported PrP(C) deletion mutants suggests that elimination of α-cleavage, coupled with retention of the protein's N-terminal residues, segments 23-31 and longer, confers the lethal phenotype. Interestingly, these N-terminal residues are implicated in the activation of several membrane proteins, including synaptic glutamate receptors. We propose that α-cleavage is a general mechanism essential for downregulating PrP(C)'s intrinsic activity, and that blockage of proteolysis leads to constitutively active PrP(C) and consequent dyshomeostasis.

New insights into metal interactions with the prion protein: EXAFS analysis and structure calculations of copper binding to a single octarepeat from the prion protein.

Copper coordination to the prion protein (PrP) has garnered considerable interest for almost 20 years, due in part to the possibility that this interaction may be part of the normal function of PrP. The most characterized form of copper binding to PrP has been Cu(2+) interaction with the conserved tandem repeats in the N-terminal domain of PrP, termed the octarepeats, with many studies focusing on single and multiple repeats of PHGGGWGQ. Extended X-ray absorption fine structure (EXAFS) spectroscopy has been used in several previous instances to characterize the solution structure of Cu(2+) binding into the peptide backbone in the HGGG portion of the octarepeats. All previous EXAFS studies, however, have benefitted from crystallographic structure information for [Cu(II) (Ac-HGGGW-NH2)(-2H)] but have not conclusively demonstrated that the complex EXAFS spectrum represents the same coordination environment for Cu(2+) bound to the peptide backbone. Density functional structure calculations as well as full multiple scattering EXAFS curve fitting analysis are brought to bear on the predominant coordination mode for Cu(2+) with the Ac-PHGGGWGQ-NH2 peptide at physiological pH, under high Cu(2+) occupancy conditions. In addition to the structure calculations, which provide a thermodynamic link to structural information, methods are also presented for extensive deconvolution of the EXAFS spectrum. We demonstrate how the EXAFS data can be analyzed to extract the maximum structural information and arrive at a structural model that is significantly improved over previous EXAFS characterizations. The EXAFS spectrum for the chemically reduced form of copper binding to the Ac-PHGGGWGQ-NH2 peptide is presented, which is best modeled as a linear two-coordinate species with a single His imidazole ligand and a water molecule. The extent of in situ photoreduction of the copper center during standard data collection is also presented, and EXAFS curve fitting of the photoreduced species reveals an intermediate structure that is similar to the Cu(2+) form with reduced coordination number.

Copper Sensing with a Prion Protein Modified Nanopipette.

Protein-metal interactions determine and regulate many biological functions. Nanopipettes functionalized with peptide moieties can be used as sensors for metal ions in solution.