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Parkinson’s disease (PD) and Multiple System Atrophy (MSA) are clinically distinctive diseases that feature a common neuropathological hallmark of aggregated α-synuclein. Little is known about how differences in α-synuclein aggregate structure affect disease phenotype. Here, we amplified α-synuclein aggregates from PD and MSA brain extracts and analyzed the conformational properties using fluorescent probes, NMR spectroscopy and electron paramagnetic resonance. We also generated and analyzed several in vitro α-synuclein polymorphs. We found that brain-derived α-synuclein fibrils were structurally different to all of the in vitro polymorphs analyzed. Importantly, there was a greater structural heterogeneity among α-synuclein fibrils from the PD brain compared to those from the MSA brain, possibly reflecting on the greater variability of disease phenotypes evident in PD. Our findings have significant ramifications for the use of non-brain-derived α-synuclein fibrils in PD and MSA studies, and raise important questions regarding the one disease-one strain hypothesis in the study of α-synucleinopathies. Parkinson’s disease (PD) and Multiple System Atrophy (MSA) are characterized by the pathological accumulation of α-synuclein. Here the authors employ fluorescent probes, electron microscopy and NMR spectroscopy to study the properties of α-synuclein aggregates that were amplified from patient brain extracts and observe a greater structural diversity among PD patients compared to MSA patients.

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways. Protein complexes at single-molecule resolution Analysis of protein interactions is crucial for understanding cellular function and regulation. Here, Taekjip Ha and colleagues develop a novel method for elucidating the identity and stoichiometry of protein complexes from cells and tissues at single-molecule resolution. The method, called single molecule pull-down or SiMPull, can discriminate between multiple association states of a protein, and simultaneously allows determination of complex stoichiometry through photobleaching step analysis. The potential of the assay is demonstrated in a variety of contexts, including endogenous proteins from tissue extracts, organelles and membrane proteins.

The stereotypical distribution of TAR DNA-binding 43 protein (TDP-43) aggregates in frontotemporal lobar degeneration (FTLD-TDP) suggests that pathological TDP-43 spreads throughout the brain via cell-to-cell transmission and correlates with disease progression, but no in vivo experimental data support this hypothesis. We first develop a doxycycline-inducible cell line expressing GFP-tagged cytoplasmic TDP-43 protein (iGFP-NLSm) as a cell-based system to screen and identify seeding activity of human brain-derived pathological TDP-43 isolated from sporadic FTLD-TDP and familial cases with Granulin (FTLD-TDP-GRN) or C9orf72 repeat expansion mutations (FTLD-TDP-C9+). We demonstrate that intracerebral injections of biologically active pathogenic FTLD-TDP seeds into transgenic mice expressing cytoplasmic human TDP-43 (lines CamKIIa-hTDP-43 NLSm , rNLS8, and CamKIIa-208) and non-transgenic mice led to the induction of de-novo TDP-43 pathology. Moreover, TDP-43 pathology progressively spreads throughout the brain in a time-dependent manner via the neuroanatomic connectome. Our study suggests that the progression of FTLD-TDP reflects the templated cell-to-cell transneuronal spread of pathological TDP-43. Cell-to-cell transmission of TDP43 occurs in cell cultures and may contribute to pathological TDP43 propagation in FTLD-TDP. In this study, the authors demonstrate using mouse models that a single intracerebral injection of human brain-derived pathological TDP43 from FTLD-TDP cases initiates the process of seeding and spreading of TDP43 pathology in a spatio-temporal dependent manner in the brain.

Background: Maintenance of the ratio of glutathione in the reduced (GSH) and oxidised (GSSG) state in cells is important in redox control, signal transduction and gene regulation, factors that are altered in many diseases. The accurate and reliable determination of GSH and GSSG simultaneously is a useful tool for oxidative stress determination. Measurement is limited primarily to the underestimation of GSH and overestimation GSSG as a result of auto-oxidation of GSH. The aim of this study was to overcome this limitation and develop, optimise and validate a reverse-phase high performance liquid chromatographic (HPLC) assay of GSH and GSSG for the determination of oxidant status in cardiac and chronic kidney diseases. Methods: Fluorescence detection of the derivative, glutathione-O-pthaldialdehyde (OPA) adduct was used. The assay was validated by measuring the stability of glutathione and glutathione-OPA adduct under conditions that could affect the reproducibility including reaction time and temperature. Linearity, concentration range, limit of detection (LOD), limit of quantification (LOQ), recovery and extraction efficiency and selectivity of the method were assessed. Results: There was excellent linearity for GSH (r2 = 0.998) and GSSG (r2 = 0.996) over concentration ranges of 0.1 µM–4 mM and 0.2 µM–0.4 mM respectively. The extraction of GSH from tissues was consistent and precise. The limit of detection for GSH and GSSG were 0.34 µM and 0.26 µM respectively whilst their limits of quantification were 1.14 µM and 0.88 µM respectively. Conclusion: These data validate a method for the simultaneous measurement of GSH and GSSG in samples extracted from biological tissues and offer a simple determination of redox status in clinical samples.

In the cartilage matrix, complex interactions occur between angiogenic and anti-angiogenic components, growth factors, and environmental stressors to maintain a proper cartilage phenotype that allows for effective load bearing and force distribution. However, as seen in both degenerative disease and tissue engineering, cartilage can lose its vascular resistance. This vascularization then leads to matrix breakdown, chondrocyte apoptosis, and ossification. Research has shown that articular cartilage inflammation leads to compromised joint function and decreased clinical potential for regeneration. Unfortunately, few articles comprehensively summarize what we have learned from previous investigations. In this review, we summarize our current understanding of the factors that stabilize chondrocytes to prevent terminal differentiation and applications of these factors to rescue the cartilage phenotype during cartilage engineering and osteoarthritis treatment. Inhibiting vascularization will allow for enhanced phenotypic stability so that we are able to develop more stable implants for cartilage repair and regeneration.

Dynamic nuclear polarization of ¹³C-labeled cell substrates has been shown to massively increase their sensitivity to detection in NMR experiments. The sensitivity gain is sufficiently large that if these polarized molecules are injected intravenously, their spatial distribution and subsequent conversion into other cell metabolites can be imaged. We have used this method to image the conversion of fumarate to malate in a murine lymphoma tumor in vivo after i.v. injection of hyperpolarized [1,4-¹³C₂]fumarate. In isolated lymphoma cells, the rate of labeled malate production was unaffected by coadministration of succinate, which competes with fumarate for transport into the cell. There was, however, a correlation with the percentage of cells that had lost plasma membrane integrity, suggesting that the production of labeled malate from fumarate is a sensitive marker of cellular necrosis. Twenty-four hours after treating implanted lymphoma tumors with etoposide, at which point there were significant levels of tumor cell necrosis, there was a 2.4-fold increase in hyperpolarized [1,4-¹³C₂]malate production compared with the untreated tumors. Therefore, the formation of hyperpolarized ¹³C-labeled malate from [1,4-¹³C₂]fumarate appears to be a sensitive marker of tumor cell death in vivo and could be used to detect the early response of tumors to treatment. Given that fumarate is an endogenous molecule, this technique has the potential to be used clinically.

Background: Kallikreins (KLKs) are a group of 15 secreted serine proteases. Some KLKs are established or candidate cancer biomarkers, but for most the physiological function is unknown. We characterized the protein and mRNA abundance patterns of all 15 KLKs in multiple panels of human tissues and biological fluids. Methods: We used sensitive and specific sandwich-type ELISAs for each KLK. Reverse transcription PCR was used for transcript amplification. Multiple panels of human tissue extracts (adult and fetal) were tested, along with various biological fluids. Results: Quantitative protein expression data on 7 sets of adult and 3 sets of fetal tissues were collected for all 15 KLKs. KLKs were also quantified in the following biological fluids: seminal plasma, breast milk, follicular fluid, breast cyst fluid, breast cancer cytosol, amniotic fluid, ovarian cancer ascites, cerebrospinal fluid, cervicovaginal fluid, and urine. The data were used to generate heat maps of KLK concentrations in tissues and fluids and categorize KLK abundance as highly restricted (KLK2 and KLK3 in prostate), restricted (KLK5 in skin, salivary gland, breast, and esophagus; KLK6 in brain and central nervous system; KLK7 in esophagus, heart, liver, and skin; KLK8 in breast, esophagus, skin, and tonsil; KLK13 in esophagus and tonsil), or wide (KLKs 1, 4, 9, 10, 11, 12, 14, and 15). Conclusions: Quantitative KLK concentrations in tissues and fluids aid in the elucidation of KLK function, and coexpression patterns provide clues for KLK participation in proteolytic cascades.

The induction of adequate vascularization, a major challenge in tissue engineering, has been tried with numerous methods but with unsatisfactory results. Adipose tissue, an active endocrine organ with dense vasculature, secretes a wide number of angiogenic and adipogenic factors and seems an attractive source for these bioactive factors. We produced a novel cell-free extract from mature human adipose tissue (adipose tissue extract [ATE]) and analyzed the ability of this extract to induce angiogenesis and adipogenesis in vitro and studied the cytokine and growth factor composition of ATE with ELISA and cytokine array. We demonstrate that ATE, when added as cell culture supplement, effectively induced triglyceride accumulation in human adipose stem cells at concentrations from 200 μg/mL upward in less than a week and caused elevated levels of adipocyte differentiation markers (proliferator-activated receptor gamma and acyl-CoA-binding protein) when treated with at least 350 μg/mL of ATE. ATE induced angiogenesis from 450 μg/mL upward after a week in vitro . ATE contained numerous angiogenic and adipogenic factors, for example, vascular endothelial growth factor, basic fibroblast growth factor, interleukin-6, adiponectin, angiogenin, leptin, and insulin-like growth factor-I, as well as lower levels of a wide variety of other cytokines. We here present a novel cell-free angiogenesis- and adipogenesis-inducing agent that is cell-free and easy to produce, and its effect is dose dependent and its composition can be easily modified. Therefore, ATE is a promising novel agent to be used for angiogenesis induction to overcome the challenge of vascularization and for adipogenesis induction in a wide variety of tissue engineering applications in vitro and in vivo . ATE is also efficient for reproduction and modeling of natural adipogenesis in vitro for, for example, obesity and diabetes studies.

Radix Inulae is endemic to China and has been used in traditional medicine to treat upper body pain, emesis and diarrhoea, and to eliminate parasites. Here, an UPLC-MS/MS method was developed and applied to study the pharmacokinetics, distribution and excretion of isoalantolactone and alantolactone, which are two main active sesquiterpene lactones in Radix Inulae, in Sprague-Dawley rats following oral administration of total Radix Inulae extract. Isoalantolactone, alantolactone and osthole (internal standard) were prepared using acetonitrile precipitation, and the separation of isoalantolactone and alantolactone was achieved by isocratic elution using water (containing 0.1% formic acid) and acetonitrile as the mobile phase using a ZORBAX Eclipse Plus C18 column. The total run time was 6.4 min. The present study showed poor absorption of isoalantolactone and alantolactone in vivo. The apparent Cmax, Tmax, T1/2 and total exposure (AUC0–12h) in rat plasma were 37.8 ng/mL, 120 min, 351.7 min and 6112.3 ng-min/mL for isoalantolactone and 25.9 ng/mL, 90 min, 321.0 min and 4918.9 ng-min/mL for alantolactone, respectively. It was shown that the highest concentration was achieved in the small intestine and feces clearance was shown to be the dominant elimination pathway of the lactones.

Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively by the misfolded prion protein (PrP Sc ). Disease is transmitted by the autocatalytic propagation of PrP Sc misfolding at the expense of the normal prion protein. The biggest challenge of the prion hypothesis has been to explain the molecular mechanism by which prions can exist as different strains, producing diseases with distinguishable characteristics. Here, we show that PrP Sc generated in vitro by protein misfolding cyclic amplification from five different mouse prion strains maintains the strain‐specific properties. Inoculation of wild‐type mice with in vitro ‐generated PrP Sc caused a disease with indistinguishable incubation times as well as neuropathological and biochemical characteristics as the parental strains. Biochemical features were also maintained upon replication of four human prion strains. These results provide additional support for the prion hypothesis and indicate that strain characteristics can be faithfully propagated in the absence of living cells, suggesting that strain variation is dependent on PrP Sc properties.