The E46K mutation modulates α-synuclein prion replication in transgenic mice.

In multiple system atrophy (MSA), the α-synuclein protein misfolds into a self-templating prion conformation that spreads throughout the brain, leading to progressive neurodegeneration. While the E46K mutation in α-synuclein causes familial Parkinson's disease (PD), we previously discovered that this mutation blocks in vitro propagation of MSA prions. Recent studies by others indicate that α-synuclein adopts a misfolded conformation in MSA in which a Greek key motif is stabilized by an intramolecular salt bridge between residues E46 and K80. Hypothesizing that the E46K mutation impedes salt bridge formation and, therefore, exerts a selective pressure that can modulate α-synuclein strain propagation, we asked whether three distinct α-synuclein prion strains could propagate in TgM47+/- mice, which express human α-synuclein with the E46K mutation. Following intracranial injection of these strains, TgM47+/- mice were resistant to MSA prion transmission, whereas recombinant E46K preformed fibrils (PFFs) transmitted neurological disease to mice and induced the formation of phosphorylated α-synuclein neuropathology. In contrast, heterotypic seeding following wild-type (WT) PFF-inoculation resulted in preclinical α-synuclein prion propagation. Moreover, when we inoculated TgM20+/- mice, which express WT human α-synuclein, with E46K PFFs, we observed delayed transmission kinetics with an incomplete attack rate. These findings suggest that the E46K mutation constrains the number of α-synuclein prion conformations that can propagate in TgM47+/- mice, expanding our understanding of the selective pressures that impact α-synuclein prion replication.

Does psychological distress predict risk of orthopaedic surgery and postoperative opioid prescribing in patients with hip pain? A retrospective study.

BACKGROUND: Clinicians and public health professionals have allocated resources to curb opioid over-prescription and address psychological needs among patients with musculoskeletal pain. However, associations between psychological distress, risk of surgery, and opioid prescribing among those with hip pathologies remain unclear. METHODS: Using a retrospective cohort study design, we identified patients that were evaluated for hip pain from January 13, 2020 to October 27, 2021. Patients' surgical histories and postoperative opioid prescriptions were extracted via chart review. Risk of hip surgery within one year of evaluation was analyzed using multivariable logistic regression. Multivariable linear regression was employed to predict average morphine milligram equivalents (MME) per day of opioid prescriptions within the first 30 days after surgery. Candidate predictors included age, gender, race, ethnicity, employment, insurance type, hip function and quality of life on the International Hip Outcome Tool (iHOT-12), and psychological distress phenotype using the OSPRO Yellow Flag (OSPRO-YF) Assessment Tool. RESULTS: Of the 672 patients, n = 350 (52.1%) underwent orthopaedic surgery for hip pain. In multivariable analysis, younger patients, those with TRICARE/other government insurance, and those with a high psychological distress phenotype had higher odds of surgery. After adding iHOT-12 scores, younger patients and lower iHOT-12 scores were associated with higher odds of surgery, while Black/African American patients had lower odds of surgery. In multivariable analysis of average MME, patients with periacetabular osteotomy (PAO) received opioid prescriptions with significantly higher average MME than those with other procedures, and surgery type was the only significant predictor. Post-hoc analysis excluding PAO found higher average MME for patients undergoing hip arthroscopy (compared to arthroplasty or other non-PAO procedures) and significantly lower average MME for patients with public insurance (Medicare/Medicaid) compared to those with private insurance. Among those only undergoing arthroscopy, older age and having public insurance were associated with opioid prescriptions with lower average MME. Neither iHOT-12 scores nor OSPRO-YF phenotype assignment were significant predictors of postoperative mean MME. CONCLUSIONS: Psychological distress characteristics are modifiable targets for rehabilitation programs, but their use as prognostic factors for risk of orthopaedic surgery and opioid prescribing in patients with hip pain appears limited when considered alongside other commonly collected clinical information such as age, insurance, type of surgery pursued, and iHOT-12 scores.

Heparin activation of protein Z-dependent protease inhibitor (ZPI) allosterically blocks protein Z activation through an extended heparin-binding site.

Protein Z (PZ)-dependent protease inhibitor (ZPI) is a plasma anticoagulant protein of the serpin superfamily, which is activated by its cofactor, PZ, to rapidly inhibit activated factor X (FXa) on a procoagulant membrane surface. ZPI is also activated by heparin to inhibit free FXa at a physiologically significant rate. Here, we show that heparin binding to ZPI antagonizes PZ binding to and activation of ZPI. Virtual docking of heparin to ZPI showed that a heparin-binding site near helix H close to the PZ-binding site as well as a previously mapped site in helix C was both favored. Alanine scanning mutagenesis of the helix H and helix C sites demonstrated that both sites were critical for heparin activation. The binding of heparin chains 72 to 5-saccharides in length to ZPI was similarly effective in antagonizing PZ binding and in inducing tryptophan fluorescence changes in ZPI. Heparin binding to variant ZPIs with either the helix C sites or the helix H sites mutated showed that heparin interaction with the higher affinity helix C site most distant from the PZ-binding site was sufficient to induce these tryptophan fluorescence changes. Together, these findings suggest that heparin binding to a site on ZPI extending from helix C to helix H promotes ZPI inhibition of FXa and allosterically antagonizes PZ binding to ZPI through long-range conformational changes. Heparin antagonism of PZ binding to ZPI may serve to spare limiting PZ and allow PZ and heparin cofactors to target FXa at different sites of action.

Discovery of novel furanylbenzamide inhibitors that target oncogenic tyrosine phosphatase SHP2 in leukemia cells.

Disturbance of the dynamic balance between tyrosine phosphorylation and dephosphorylation of signaling molecules, controlled by protein tyrosine kinases and protein tyrosine phosphatases (PTPs), is known to lead to the development of cancer. While most approved targeted cancer therapies are tyrosine kinase inhibitors, PTPs have long been stigmatized as undruggable and have only recently gained renewed attention in drug discovery. One PTP target is the Src-homology 2 domain-containing phosphatase 2 (SHP2). SHP2 is implicated in tumor initiation, progression, metastasis, and treatment resistance, primarily because of its role as a signaling nexus of the extracellular signal-regulated kinase pathway, acting upstream of the small GTPase Ras. Efforts to develop small molecules that target SHP2 are ongoing, and several SHP2 allosteric inhibitors are currently in clinical trials for the treatment of solid tumors. However, while the reported allosteric inhibitors are highly effective against cells expressing WT SHP2, none have significant activity against the most frequent oncogenic SHP2 variants that drive leukemogenesis in several juvenile and acute leukemias. Here, we report the discovery of novel furanylbenzamide molecules as inhibitors of both WT and oncogenic SHP2. Importantly, these inhibitors readily cross cell membranes, bind and inhibit SHP2 under physiological conditions, and effectively decrease the growth of cancer cells, including triple-negative breast cancer cells, acute myeloid leukemia cells expressing either WT or oncogenic SHP2, and patient-derived acute myeloid leukemia cells. These novel compounds are effective chemical probes of active SHP2 and may serve as starting points for therapeutics targeting WT or mutant SHP2 in cancer.

Critical Risk Factors for Opioid Demand after Pelvic and Acetabular Fracture Surgery.

The characteristics that contribute to opioid demand in pelvic and acetabular fracture surgery are not well understood. We hypothesize that fracture pattern and psychiatric comorbidities will be associated with increased opioid demand. This study evaluated perioperative opioid prescription filling in 743 patients undergoing operative fixation of pelvic and acetabular injuries. Multivariable linear and logistic regression models were used to evaluate associations between baseline factors and opioid outcomes. Patients filled prescriptions for 111.2, 89.3, and 200.3 oxycodone 5-mg pills at the 1-month preop to 90-days postop, 3-months postop to 1-year postop, and 1-month preop to 1-year postop timeframes. Operatively treated wall, transverse and two-column acetabular fractures were associated with the highest opioid demand. Drug abuse and pre-injury opioid use were the primary non-surgical drivers of opioid demand. Acetabular fractures, pre-injury opioid filling, and drug abuse were the main risk factors for increased perioperative opioid prescription filling. Level of Evidence: Level III, retrospective, prognostic cohort study. (Journal of Surgical Orthopaedic Advances 32(1):041-046, 2023).

Kinetics of α-synuclein prions preceding neuropathological inclusions in multiple system atrophy.

Multiple system atrophy (MSA), a progressive neurodegenerative disease characterized by autonomic dysfunction and motor impairment, is caused by the self-templated misfolding of the protein α-synuclein. With no treatment currently available, we sought to characterize the spread of α-synuclein in a transgenic mouse model of MSA prion propagation to support drug discovery programs for synucleinopathies. Brain homogenates from MSA patient samples or mouse-passaged MSA were inoculated either by standard freehand injection or stereotactically into TgM83+/- mice, which express human α-synuclein with the A53T mutation. Following disease onset, brains from the mice were tested for biologically active α-synuclein prions using a cell-based assay and examined for α-synuclein neuropathology. Inoculation studies using homogenates prepared from brain regions lacking detectable α-synuclein neuropathology transmitted neurological disease to mice. Terminal animals contained similar concentrations of α-synuclein prions; however, a time-course study where mice were terminated every five days through disease progression revealed that the kinetics of α-synuclein prion replication in the mice were variable. Stereotactic inoculation into the thalamus reduced variability in disease onset in the mice, although incubation times were consistent with standard inoculations. Using human samples with and without neuropathological lesions, we observed that α-synuclein prion formation precedes neuropathology in the brain, suggesting that disease in patients is not limited to brain regions containing neuropathological lesions.

Multiple system atrophy prions transmit neurological disease to mice expressing wild-type human α-synuclein.

In multiple system atrophy (MSA), the protein α-synuclein misfolds into a prion conformation that self-templates and causes progressive neurodegeneration. While many point mutations in the α-synuclein gene, SNCA, have been identified as the cause of heritable Parkinson's disease (PD), none have been identified as causing MSA. To examine whether MSA prions can transmit disease to mice expressing wild-type (WT) human α-synuclein, we inoculated transgenic (Tg) mice denoted TgM20+/- with brain homogenates prepared from six different deceased MSA patients. All six samples transmitted CNS disease to the mice, with an average incubation period of ~ 280 days. Interestingly, TgM20+/- female mice developed disease > 60 days earlier than their male counterparts. Brains from terminal mice contained phosphorylated α-synuclein throughout the hindbrain, consistent with the distribution of α-synuclein inclusions in MSA patients. In addition, using our α-syn-YFP cell lines, we detected α-synuclein prions in brain homogenates prepared from terminal mice that retained MSA strain properties. To our knowledge, the studies described here are the first to show that MSA prions transmit neurological disease to mice expressing WT SNCA and that the rate of transmission is sex dependent. By comparison, TgM20+/- mice inoculated with WT preformed fibrils (PFFs) developed severe neurological disease in ~ 210 days and exhibited robust α-synuclein neuropathology in both limbic regions and the hindbrain. Brain homogenates from these animals exhibited biological activities that are distinct from those found in MSA-inoculated mice when tested in the α-syn-YFP cell lines. Differences between brains from MSA-inoculated and WT PFF-inoculated mice potentially argue that α-synuclein prions from MSA patients are distinct from the PFF inocula and that PFFs do not replicate MSA strain biology.

Paramount Importance of Core Conformational Changes for Heparin Allosteric Activation of Antithrombin.

Antithrombin is unique among serpin family protein protease inhibitors with respect to the major reactive center loop (RCL) and core conformational changes that mediate allosteric activation of its anticoagulant function by heparin. A critical role for expulsion of the RCL hinge from a native stabilizing interaction with the hydrophobic core in the activation mechanism has been proposed from reports that antithrombin variants that block this change through engineered disulfide bonds block activation. However, the sufficiency of core conformational changes for activation without expulsion of the RCL from the core is suggested by variants that are activated without the need for heparin and retain the native RCL-core interaction. To resolve these apparently conflicting findings, we engineered variants in which disulfides designed to block the RCL conformational change were combined with constitutively activating mutations. Our findings demonstrate that while a reversible constitutive activation can be engineered in variants that retain the native RCL-core interaction, engineered disulfides that lock the RCL native conformation can also block heparin allosteric activation. Such findings support a three-state allosteric activation model in which constitutive activating mutations stabilize an intermediate-activated state wherein core conformational changes and a major activation have occurred without the release of the RCL from the core but with a necessary repositioning of the RCL to allow productive engagement with an exosite. Rigid disulfide bonds that lock the RCL native conformation block heparin activation by preventing both RCL repositioning in the intermediate-activated state and the release of the RCL from the core in the fully activated state.

Familial Parkinson's point mutation abolishes multiple system atrophy prion replication.

In the neurodegenerative disease multiple system atrophy (MSA), α-synuclein misfolds into a self-templating conformation to become a prion. To compare the biological activity of α-synuclein prions in MSA and Parkinson's disease (PD), we developed nine α-synuclein-YFP cell lines expressing point mutations responsible for inherited PD. MSA prions robustly infected wild-type, A30P, and A53T α-synuclein-YFP cells, but they were unable to replicate in cells expressing the E46K mutation. Coexpression of the A53T and E46K mutations was unable to rescue MSA prion infection in vitro, establishing that MSA α-synuclein prions are conformationally distinct from the misfolded α-synuclein in PD patients. This observation may have profound implications for developing treatments for neurodegenerative diseases.

New Developments in Hip Fracture Treatment to Reduce Morbidity and Mortality.

There have been major changes in the treatment of various hip fracture patterns in the proximal femur. The orthopaedic surgeon should be up to date on device management, current guidelines, and techniques in the care of hip fracture patterns.

Selectivity in the Addition of Electron Deficient Radicals to the C2 Position of Indoles.

The addition of electron deficient radicals to the C2 position of indoles has been described in the literature as opposed to electrophilic addition at the C3 position. Density functional theory calculations were used to understand the switch in regioselectivity from C3 to C2 for indole to undergo radical additions. Electron deficient radicals have a lower barrier for reaction at C2 and a lower energy radical intermediate that benefits from benzylic radical stabilization. Trifluoromethyl radical addition has a lower energy barrier than acetonitrile radical, and the C3 addition transition state is just 0.8 kcal/mol higher than C2. This is supported by experimental observations.

Multiple system atrophy prions retain strain specificity after serial propagation in two different Tg(SNCA*A53T) mouse lines.

Previously, we reported that intracranial inoculation of brain homogenate from multiple system atrophy (MSA) patient samples produces neurological disease in the transgenic (Tg) mouse model TgM83+/-, which uses the prion protein promoter to express human α-synuclein harboring the A53T mutation found in familial Parkinson's disease (PD). In our studies, we inoculated MSA and control patient samples into Tg mice constructed using a P1 artificial chromosome to express wild-type (WT), A30P, and A53T human α-synuclein on a mouse α-synuclein knockout background [Tg(SNCA+/+)Nbm, Tg(SNCA*A30P+/+)Nbm, and Tg(SNCA*A53T+/+)Nbm]. In contrast to studies using TgM83+/- mice, motor deficits were not observed by 330-400 days in any of the Tg(SNCA)Nbm mice after inoculation with MSA brain homogenates. However, using a cell-based bioassay to measure α-synuclein prions, we found brain homogenates from Tg(SNCA*A53T+/+)Nbm mice inoculated with MSA patient samples contained α-synuclein prions, whereas control mice did not. Moreover, these α-synuclein aggregates retained the biological and biochemical characteristics of the α-synuclein prions in MSA patient samples. Intriguingly, Tg(SNCA*A53T+/+)Nbm mice developed α-synuclein pathology in neurons and astrocytes throughout the limbic system. This finding is in contrast to MSA-inoculated TgM83+/- mice, which develop exclusively neuronal α-synuclein aggregates in the hindbrain that cause motor deficits with advanced disease. In a crossover experiment, we inoculated TgM83+/- mice with brain homogenate from two MSA patient samples or one control sample first inoculated, or passaged, in Tg(SNCA*A53T+/+)Nbm animals. Additionally, we performed the reverse experiment by inoculating Tg(SNCA*A53T+/+)Nbm mice with brain homogenate from the same two MSA samples and one control sample first passaged in TgM83+/- animals. The TgM83+/- mice inoculated with mouse-passaged MSA developed motor dysfunction and α-synuclein prions, whereas the mouse-passaged control sample had no effect. Similarly, the mouse-passaged MSA samples induced α-synuclein prion formation in Tg(SNCA*A53T+/+)Nbm mice, but the mouse-passaged control sample did not. The confirmed transmission of α-synuclein prions to a second synucleinopathy model and the ability to propagate prions between two distinct mouse lines while retaining strain-specific properties provides compelling evidence that MSA is a prion disease.

Tau prions from Alzheimer's disease and chronic traumatic encephalopathy patients propagate in cultured cells.

Tau prions are thought to aggregate in the central nervous system, resulting in neurodegeneration. Among the tauopathies, Alzheimer's disease (AD) is the most common, whereas argyrophilic grain disease (AGD), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), Pick's disease (PiD), and progressive supranuclear palsy (PSP) are less prevalent. Brain extracts from deceased individuals with PiD, a neurodegenerative disorder characterized by three-repeat (3R) tau prions, were used to infect HEK293T cells expressing 3R tau fused to yellow fluorescent protein (YFP). Extracts from AGD, CBD, and PSP patient samples, which contain four-repeat (4R) tau prions, were transmitted to HEK293 cells expressing 4R tau fused to YFP. These studies demonstrated that prion propagation in HEK cells requires isoform pairing between the infecting prion and the recipient substrate. Interestingly, tau aggregates in AD and CTE, containing both 3R and 4R isoforms, were unable to robustly infect either 3R- or 4R-expressing cells. However, AD and CTE prions were able to replicate in HEK293T cells expressing both 3R and 4R tau. Unexpectedly, increasing the level of 4R isoform expression alone supported the propagation of both AD and CTE prions. These results allowed us to determine the levels of tau prions in AD and CTE brain extracts.

Evaluation of the safety and efficacy of a new hemostatic powder using a quantitative surface bleeding severity scale.

AIMS OF THE STUDY: The safety and efficacy of a hemostatic powder (HP) versus a control agent, absorbable gelatin sponge and thrombin (G + T), were assessed, using a validated, quantitative bleeding severity scale. METHODS: Subjects were randomized to receive HP (256 subjects) or G + T (132 subjects) for treatment of minimal, mild, or moderate bleeding at 20 investigational sites. The primary efficacy endpoint was non-inferiority of HP relative to G + T for success at achieving hemostasis within 6 minutes. Secondary endpoints in rank order included: superiority of HP relative to G + T in mean preparation time; non-inferiority of HP relative to G + T for achieving hemostasis within 3 min; superiority of HP relative to G + T for achieving hemostasis within 6 min; and superiority of HP relative to G + T for success for achieving hemostasis within 3 min. RESULTS: A total of 388 subjects were included in the primary efficacy analysis. At 6 min, hemostasis was achieved in 93.0% (238/256) of the HP group compared to 77.3% (102/132) of the G + T group (non-inferiority P < 0.0001, superiority P < 0.0001). All secondary endpoints were met. Complications were comparable between treatment groups. CONCLUSIONS: HP had superior rates of hemostasis, shorter preparation time, and a similar safety profile compared to G + T in this prospective, randomized trial using quantitative bleeding severity criteria.

Avoiding and Managing Complications After Hip Fracture Fixation.

There is an enormous burden of disease associated with the management of a failed hip fracture fixation. The goal of surgical management is to facilitate an early return to mobilization with the retention of as much independence as possible. Despite numerous studies that are focused on the care of patients with proximal femur fractures, complication rates remain high. Surgeons should review current strategies to avoid and manage complications after hip fracture fixation. This will have important implications given the detrimental consequences of failed management of hip fractures, including permanent disability, life-threatening medical complications, and an increased risk of death.

Cooperative Interactions of Three Hotspot Heparin Binding Residues Are Critical for Allosteric Activation of Antithrombin by Heparin.

Heparin allosterically activates the anticoagulant serpin, antithrombin, by binding through a sequence-specific pentasaccharide and inducing activating conformational changes in the protein. Three basic residues of antithrombin, Lys114, Lys125, and Arg129, have been shown to be hotspots for binding the pentasaccharide, but the molecular basis for such hotspot binding has been unclear. To determine whether this results from cooperative interactions, we analyzed the effects of single, double, and triple mutations of the hotspot residues on pentasaccharide binding and activation of antithrombin. Double-mutant cycles revealed that the contribution of each residue to pentasaccharide binding energy was progressively reduced when one or both of the other residues were mutated, indicating strong coupling between each pair of residues that was dependent on the third residue and reflective of the three residues acting as a cooperative unit. Rapid kinetic studies showed that the hotspot residue mutations progressively abrogated the ability of the pentasaccharide to bind productively to native antithrombin and to conformationally activate the serpin by engaging the hotspot residues in an induced-fit interaction. Examination of the antithrombin-pentasaccharide complex structure revealed that the hotspot residues form two adjoining binding pockets for critical sulfates of the pentasaccharide that structurally link these residues. Together, these findings demonstrate that cooperative interactions of Lys114, Lys125, and Arg129 are critical for the productive induced-fit binding of the heparin pentasaccharide to antithrombin that allosterically activates the anticoagulant function of the serpin.

Disease-causing mutations in the serpin antithrombin reveal a key domain critical for inhibiting protease activities.

Antithrombin mainly inhibits factor Xa and thrombin. The reactive center loop (RCL) is crucial for its interactions with its protease targets and is fully inserted into the A-sheet after its cleavage, causing translocation of the covalently linked protease to the opposite end of the A-sheet. Antithrombin variants with altered RCL hinge residues behave as substrates rather than inhibitors, resulting in stoichiometries of inhibition greater than one. Other antithrombin residues have been suggested to interfere with RCL insertion or the stability of the antithrombin-protease complex, but available crystal structures or mutagenesis studies have failed to identify such residues. Here, we characterized two mutations, S365L and I207T, present in individuals with type II antithrombin deficiency and identified a new antithrombin functional domain. S365L did not form stable complexes with thrombin or factor Xa, and the I207T/I207A variants inhibited both proteases with elevated stoichiometries of inhibition. Close proximity of Ile-207 and Ser-365 to the inserted RCL suggested that the preferred reaction of these mutants as protease substrates reflects an effect on the rate of the RCL insertion and protease translocation. However, both residues lie within the final docking site for the protease in the antithrombin-protease complex, supporting the idea that the enhanced substrate reactions may result from an increased dissociation of the final complexes. Our findings demonstrate that the distal end of the antithrombin A-sheet is crucial for the last steps of protease inhibition either by affecting the rate of RCL insertion or through critical interactions with proteases at the end of the A-sheet.

Lipid oxidation inactivates the anticoagulant function of protein Z-dependent protease inhibitor (ZPI).

Lipid oxidation due to oxidative stress plays an important role in the pathogenesis of inflammatory and thrombotic cardiovascular diseases. Several findings suggest that lipid peroxidation can alter the function of coagulation proteins and contribute to a hypercoagulable state, but the molecular mechanisms are unclear. Here, we report that oxidized phospholipids suppress the anticoagulant function of the serpin, protein Z-dependent protease inhibitor (ZPI), a specific inhibitor of membrane-associated factor Xa (FXa) that requires protein Z (PZ), phospholipid, and calcium as cofactors. We found that this suppression arises from a diminished ability of the oxidized membrane to function as a cofactor to promote ZPI inhibition of membrane-bound FXa, due fully or in part to the susceptibility of the bound ZPI-PZ complex to oxidative inactivation. Surprisingly, free ZPI was also susceptible to inactivation by oxidized membrane vesicles in the absence of calcium. Oxidized vesicles containing both phosphatidylserine and polyunsaturated fatty acids were required to promote inactivation of the ZPI-PZ complex or free ZPI, indicating that binding of the PZ-complexed or free ZPI to peroxide-modified phospholipid vesicles mediates the inactivation. Heparin protected the ZPI-PZ complex and free ZPI from inactivation, suggesting that blocking the heparin-binding site on ZPI interferes with ZPI binding to lipid or to PZ. This was confirmed by direct lipid-binding experiments. Native PAGE indicated that oxidization induced dissociation of the ZPI-PZ complex and increased the negative charge of ZPI. We conclude that compromised ZPI anticoagulant function could contribute to thrombus initiation and growth in oxidative stress-induced cardiovascular diseases.

MSA prions exhibit remarkable stability and resistance to inactivation.

In multiple system atrophy (MSA), progressive neurodegeneration results from the protein α-synuclein misfolding into a self-templating prion conformation that spreads throughout the brain. MSA prions are transmissible to transgenic (Tg) mice expressing mutated human α-synuclein (TgM83+/-), inducing neurological disease following intracranial inoculation with brain homogenate from deceased patient samples. Noting the similarities between α-synuclein prions and PrP scrapie (PrPSc) prions responsible for Creutzfeldt-Jakob disease (CJD), we investigated MSA transmission under conditions known to result in PrPSc transmission. When peripherally exposed to MSA via the peritoneal cavity, hind leg muscle, and tongue, TgM83+/- mice developed neurological signs accompanied by α-synuclein prions in the brain. Iatrogenic CJD, resulting from PrPSc prion adherence to surgical steel instruments, has been investigated by incubating steel sutures in contaminated brain homogenate before implantation into mouse brain. Mice studied using this model for MSA developed disease, whereas wire incubated in control homogenate had no effect on the animals. Notably, formalin fixation did not inactivate α-synuclein prions. Formalin-fixed MSA patient samples also transmitted disease to TgM83+/- mice, even after incubating in fixative for 244 months. Finally, at least 10% sarkosyl was found to be the concentration necessary to partially inactivate MSA prions. These results demonstrate the robustness of α-synuclein prions to denaturation. Moreover, they establish the parallel characteristics between PrPSc and α-synuclein prions, arguing that clinicians should exercise caution when working with materials that might contain α-synuclein prions to prevent disease.

Photoredox Cyanomethylation of Indoles: Catalyst Modification and Mechanism.

The direct cyanomethylation of indoles at the 2- or 3-position was achieved via photoredox catalysis. The versatile nitrile synthon is introduced as a radical generated from bromoacetonitrile, a photocatalyst, and blue LED as a light source. The mechanism of the reaction is explored by determination of the Stern-Volmer quenching constants. By combining photophysical data and mass spectrometry to follow the catalyst decomposition, the catalyst ligands were tuned to enable synthetically useful yields of radical coupling products. A range of indole substrates with alkyl, aryl, halogen, ester, and ether functional groups participate in the reaction, affording products in 16-90% yields. The reaction allows the rapid construction of synthetically useful cyanomethylindoles, products that otherwise require several synthetic steps.