A Kinetic Map of the Influence of Biomimetic Lipid Model Membranes on Aß42 Aggregation.

The aggregation of the amyloid ß (Aß) peptide is one of the molecular hallmarks of Alzheimer's disease (AD). Although Aß deposits have mostly been observed extracellularly, various studies have also reported the presence of intracellular Aß assemblies. Because these intracellular Aß aggregates might play a role in the onset and progression of AD, it is important to investigate their possible origins at different locations of the cell along the secretory pathway of the amyloid precursor protein, from which Aß is derived by proteolytic cleavage. Senile plaques found in AD are largely composed of the 42-residue form of Aß (Aß42). Intracellularly, Aß42 is produced in the endoplasmatic reticulum (ER) and Golgi apparatus. Since lipid bilayers have been shown to promote the aggregation of Aß, in this study, we measure the effects of the lipid membrane composition on the in vitro aggregation kinetics of Aß42. By using large unilamellar vesicles to model cellular membranes at different locations, including the inner and outer leaflets of the plasma membrane, late endosomes, the ER, and the Golgi apparatus, we show that Aß42 aggregation is inhibited by the ER and Golgi model membranes. These results provide a preliminary map of the possible effects of the membrane composition in different cellular locations on Aß aggregation and suggest the presence of an evolutionary optimization of the lipid composition to prevent the intracellular aggregation of Aß.

An antibody scanning method for the detection of α-synuclein oligomers in the serum of Parkinson's disease patients.

Misfolded α-synuclein oligomers are closely implicated in the pathology of Parkinson's disease and related synucleinopathies. The elusive nature of these aberrant assemblies makes it challenging to develop quantitative methods to detect them and modify their behavior. Existing detection methods use antibodies to bind α-synuclein aggregates in biofluids, although it remains challenging to raise antibodies against α-synuclein oligomers. To address this problem, we used an antibody scanning approach in which we designed a panel of 9 single-domain epitope-specific antibodies against α-synuclein. We screened these antibodies for their ability to inhibit the aggregation process of α-synuclein, finding that they affected the generation of α-synuclein oligomers to different extents. We then used these antibodies to investigate the size distribution and morphology of soluble α-synuclein aggregates in serum and cerebrospinal fluid samples from Parkinson's disease patients. Our results indicate that the approach that we present offers a promising route for the development of antibodies to characterize soluble α-synuclein aggregates in biofluids.

Dry needling for the treatment of acute myofascial pain syndrome in general practitioners' clinics: a cohort study.

BACKGROUND: Musculoskeletal pain is one of the leading complaints in the ambulatory setting. There are many ways to treat it, including pharmacologic and non-pharmacologic approaches. Dry needling (DN) is an option that is easy to learn, cheap and has a good safety profile. The aim of this study was to assess the association between DN performed by GPs for acute myofascial pain syndrome (MPS) and pain relief and to evaluate factors associated with treatment success. METHODS: In this prospective cohort study, two GPs performed DN in their clinics. Patients were asked to rank their pain using the Short-Form McGill Pain Questionnaire (SF-MPQ) before, 10-min and 1-week after the procedure. The SF-MPQ index consists of 3 parts; visual analog scale (VAS), pain rating index (PRI) and present pain intensity (PPI). Logistic regressions were performed to assess the variables associated with short- and medium- term success. RESULTS: Fifty two patients were recruited from September 2019 until August 2020. VAS was 6.0 ± 2.3 (before), 4.1 ± 2.5 (10-min after) and 2.6 ± 2.71 (1-week after), P < 0.05. PRI was 17 ± 9.1 (before), 10.8 ± 8.5 (10-min after) and 5.1 ± 6.5 (1-week after), P < 0.05. PPI was 2.6 ± 1.0 (before), 1.7 ± 1.0 (10-min after) and 1.1 ± 1.2 (1-week after), P < 0.05. Short-term success was associated with the physician who performed the procedure (OR 10.08, 95% CI 1.15,88.4) and with the use of a single needle (vs. multiple needles inserted) (OR 4.55, 95% CI 1.03,20.11). Medium-term success was associated with being a native born (non-immigrant), OR 8.59, 95% CI 1.11,66.28 and with high level of initial pain, OR 11.22, 95% CI 1.82,69.27. CONCLUSION: Our study demonstrated improvement in acute pain 10-min and 1-week after DN performed by a GP, in all parts of the SF-MPQ. Therefore, we believe DN is a good therapeutic option for GPs to aid patients suffering from MPS.

TrypOx, a Novel Eukaryotic Homolog of the Redox-Regulated Chaperone Hsp33 in Trypanosoma brucei.

ATP-independent chaperones are widespread across all domains of life and serve as the first line of defense during protein unfolding stresses. One of the known crucial chaperones for bacterial survival in a hostile environment (e.g., heat and oxidative stress) is the highly conserved, redox-regulated ATP-independent bacterial chaperone Hsp33. Using a bioinformatic analysis, we describe novel eukaryotic homologs of Hsp33 identified in eukaryotic pathogens belonging to the kinetoplastids, a family responsible for lethal human diseases such as Chagas disease as caused by Trypanosoma cruzi, African sleeping sickness caused by Trypanosoma brucei spp., and leishmaniasis pathologies delivered by various Leishmania species. During their pathogenic life cycle, kinetoplastids need to cope with elevated temperatures and oxidative stress, the same conditions which convert Hsp33 into a powerful chaperone in bacteria, thus preventing aggregation of a wide range of misfolded proteins. Here, we focused on a functional characterization of the Hsp33 homolog in one of the members of the kinetoplastid family, T. brucei, (Tb927.6.2630), which we have named TrypOx. RNAi silencing of TrypOx led to a significant decrease in the survival of T. brucei under mild oxidative stress conditions, implying a protective role of TrypOx during the Trypanosomes growth. We then adopted a proteomics-driven approach to investigate the role of TrypOx in defining the oxidative stress response. Depletion of TrypOx significantly altered the abundance of proteins mediating redox homeostasis, linking TrypOx with the antioxidant system. Using biochemical approaches, we identified the redox-switch domain of TrypOx, showing its modularity and oxidation-dependent structural plasticity. Kinetoplastid parasites such as T. brucei need to cope with high levels of oxidants produced by the innate immune system, such that parasite-specific antioxidant proteins like TrypOx - which are depleted in mammals - are highly promising candidates for drug targeting.

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry.

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of reactive oxygen species, and more. These fluctuations can lead to a widespread protein unfolding, aggregation, and cell death. Therefore, cells have evolved a dynamic and stress-specific network of molecular chaperones, which maintain a "healthy" proteome during stress conditions. ATP-independent chaperones constitute one major class of molecular chaperones, which serve as first-line defense molecules, protecting against protein aggregation in a stress-dependent manner. One feature these chaperones have in common is their ability to utilize structural plasticity for their stress-specific activation, recognition, and release of the misfolded client. In this paper, we focus on the functional and structural analysis of one such intrinsically disordered chaperone, the bacterial redox-regulated Hsp33, which protects proteins against aggregation during oxidative stress. Here, we present a toolbox of diverse techniques for studying redox-regulated chaperone activity, as well as for mapping conformational changes of the chaperone, underlying its activity. Specifically, we describe a workflow which includes the preparation of fully reduced and fully oxidized proteins, followed by an analysis of the chaperone anti-aggregation activity in vitro using light-scattering, focusing on the degree of the anti-aggregation activity and its kinetics. To overcome frequent outliers accumulated during aggregation assays, we describe the usage of Kfits, a novel graphical tool which allows easy processing of kinetic measurements. This tool can be easily applied to other types of kinetic measurements for removing outliers and fitting kinetic parameters. To correlate the function with the protein structure, we describe the setup and workflow of a structural mass spectrometry technique, hydrogen-deuterium exchange mass spectrometry, that allows the mapping of conformational changes on the chaperone and substrate during different stages of Hsp33 activity. The same methodology can be applied to other protein-protein and protein-ligand interactions.

Kfits: a software framework for fitting and cleaning outliers in kinetic measurements.

Motivation: Kinetic measurements have played an important role in elucidating biochemical and biophysical phenomena for over a century. While many tools for analysing kinetic measurements exist, most require low noise levels in the data, leaving outlier measurements to be cleaned manually. This is particularly true for protein misfolding and aggregation processes, which are extremely noisy and hence difficult to model. Understanding these processes is paramount, as they are associated with diverse physiological processes and disorders, most notably neurodegenerative diseases. Therefore, a better tool for analysing and cleaning protein aggregation traces is required. Results: Here we introduce Kfits, an intuitive graphical tool for detecting and removing noise caused by outliers in protein aggregation kinetics data. Following its workflow allows the user to quickly and easily clean large quantities of data and receive kinetic parameters for assessment of the results. With minor adjustments, the software can be applied to any type of kinetic measurements, not restricted to protein aggregation. Availability and implementation: Kfits is implemented in Python and available online at http://kfits.reichmannlab.com, in source at https://github.com/odedrim/kfits/, or by direct installation from PyPI (`pip install kfits`). Contact: danare@mail.huji.ac.il. Supplementary information: Supplementary data are available at Bioinformatics online.

A Role of Metastable Regions and Their Connectivity in the Inactivation of a Redox-Regulated Chaperone and Its Inter-Chaperone Crosstalk.

AIMS: A recently discovered group of conditionally disordered chaperones share a very unique feature; they need to lose structure to become active as chaperones. This activation mechanism makes these chaperones particularly suited to respond to protein-unfolding stress conditions, such as oxidative unfolding. However, the role of this disorder in stress-related activation, chaperone function, and the crosstalk with other chaperone systems is not yet clear. Here, we focus on one of the members of the conditionally disordered chaperones, a thiol-redox switch of the bacterial proteostasis system, Hsp33. RESULTS: By modifying the Hsp33's sequence, we reveal that the metastable region has evolved to abolish redox-dependent chaperone activity, rather than enhance binding affinity for client proteins. The intrinsically disordered region of Hsp33 serves as an anchor for the reduced, inactive state of Hsp33, and it dramatically affects the crosstalk with the synergetic chaperone system, DnaK/J. Using mass spectrometry, we describe the role that the metastable region plays in determining client specificity during normal and oxidative stress conditions in the cell. Innovation and Conclusion: We uncover a new role of protein plasticity in Hsp33's inactivation, client specificity, crosstalk with the synergistic chaperone system DnaK/J, and oxidative stress-specific interactions in bacteria. Our results also suggest that Hsp33 might serve as a member of the house-keeping proteostasis machinery, tasked with maintaining a "healthy" proteome during normal conditions, and that this function does not depend on the metastable linker region. Antioxid. Redox Signal. 27, 1252-1267.

Video-otoscopy in children and patient-centered care: A randomized, controlled study.

OBJECTIVES: Medicine today is moving towards patient centered care and patient empowerment. This enhances patient autonomy, allows shared decision making and increases satisfaction. Current technology enables the caregiver to share visual data with the patients, making them more active participants in a medical encounter. It has been shown that higher patient satisfaction rate has a positive effect on outcome and patient compliance. Otoscopic examination is one of the most common procedures performed in the pediatric population. Video-otoscopy uses endoscopic technology to project the image of the tympanic membrane onto a monitor visible to both the physician and the patient. The objective of this study is to assess whether video-otoscopy used in a pediatric emergency department can improve patient-centeredness and parental satisfaction with treatment. METHODS: A randomized, controlled study comparing video-otoscopy with conventional otoscopy was performed. Patients were randomized into 2 groups. In one, ear examination was performed by video-otoscopy and in the other by conventional otoscopy. Following examination, parents in both groups were asked to fill a questionnaire regarding their satisfaction with the otoscopic examination and the patient-centeredness of the encounter. RESULTS: 60 children were recruited and were randomized into two groups: 30 in the video-otoscopy group and 30 in the conventional otoscopy group. Parental satisfaction was significantly higher in the video-otoscopy group. The level of patient-centeredness was significantly higher in the video-otoscopy group. There was a positive correlation between patient-centeredness and parental satisfaction in both groups, with a significantly higher correlation in the video-otoscopy group. CONCLUSIONS: Video-otoscopy was found to encourage patient-centered care and increase parental satisfaction with otoscopy. There is a significantly higher positive correlation between patient-centeredness and parental satisfaction when video-otoscopy is used.