The effect of oxidative stress induced by neurotoxic metal ions on the properties of the brain 20S proteasome or multicatalytic proteinase complex (MPC) has been studied. Exposure of the 20S ...proteasome to increasing amounts of Fe(III), Fe(II), Cu(II) or Zn(II) affects its main hydrolytic activities: trypsin-like (T-L), chymotrypsin-like (ChT-L), peptidylglutamyl-peptide hydrolase (PGPH), branched-chain amino acid preferring (BrAAP) and caseinolytic activities, although in different ways. T-L activity showed gradual activation by both iron ions but inhibition by Cu(II) and Zn(II). ChT-L and PGPH activities were inhibited whereas BrAAP activity was widely activated by all the tested metal salts except for zinc ions. Moreover, the exposure to ferrous salt increased the degradation rate of casein. The functional effects appear to be linked to oxidation-induced modifications, as demonstrated by an increase of carbonyl groups following the exposure to metal ions. In addition, modifications induced by ferrous salt on the catalytic subunits were also supported by western blot analyses performed using anti-X, anti-Y and anti-Z antibodies. The results obtained clearly indicate that metal-catalyzed oxidation strongly affects the functions of the brain 20S proteasome, even though the catalytic subunits seem to be differently influenced by oxidative phenomena.
Abstract The role of amyloid-β protein (Aβ) in the pathogenesis of Alzheimer's disease (AD) has been widely investigated and amyloid aggregates are considered a major cause of neuronal dysfunction. ...Increasing evidence has identified a correlation between this protein and the proteasome, the cellular proteolytic machinery, in particular the ubiquitin–proteasome system. The 20S proteasome is the catalytic core of a complex, known as 26S proteasome, and is the main responsible for the clearance of misfolded and oxidized proteins. In this work we have investigated the effects of different assembly states of two major amyloid peptides, Aβ (1–40) and Aβ (1–42) on the 20S proteasome functionality and on the ubiquitin-dependent pathway of protein degradation. In particular, we have tested proteasome activities after Aβ treatment on purified 20S complexes and on lysates of a human neuroblastoma cell line. Our findings show a significant decrease in proteasome activity, more evident in cell lysates than in isolated complexes, and an increased amount of ubiquitin–protein conjugates and of a known proteasome substrate (p27). Furthermore, the altered proteasome functionality is not associated with a decrease in cell viability, but is linked with increased levels of protein oxidation.
Abstract
Background
Physical activity (PA) is a core component of cardiac rehabilitation (CR) and is crucial for enhancing cardiovascular (CV) fitness1. In the UK, CR programmes involve 1-2 hours of ...face-to-face contact per week over 6-8 weeks, implying that 98-99% of the intervention period is currently unmonitored and not optimised for potential positive lifestyle interventions. Consequently, CR staff are blind to PA that occurs outside of the clinical setting, which could be of too low or high intensity and/or duration, according to the Association of Certified Physiotherapists in Cardiac Rehabilitation (ACPICR) guidelines2. A previous pilot study showed a novel digital system to be feasible, acceptable and effective for CR patients3, making it a promising intervention to assess in a randomised control trial (RCT).
Purpose
To conduct a RCT to examine whether contextualised data feedback delivered using a novel digital service, in conjunction with usual care (UC) promotes positive PA behavioural change, with direct comparison to UC alone.
Methods
This prospective, parallel group, open-label, RCT enrolled 130 patients eligible for CR and consisted of an 8-week intervention period with follow-up at 8-weeks. The intervention group received the novel digital CR service plus UC, with the control group receiving UC alone. The primary outcome was changes in PA to achieve the ACPICR guidelines. Both groups wore a PA monitor to collect minute-level energy expenditure data that underwent processing using a patented method to extract the specific PA metrics required for analysis. The secondary outcome was changes in CV fitness between pre and post assessments, measured using Incremental Shuttle Walk Test (ISWT).
Results
Patient characteristics are shown in Table 1. Adherence to data collection was good, with an average of 37 complete days (≥768 minutes per day) of PA data per person. Fifty-six intervention group patients and 49 control group patients were analysed for the primary outcome. A significant difference (p<0.025) in the probability of achieving the ACPICR ‘Daily Activity’ guideline was found between groups, with the intervention group more likely to achieve the guideline at week-8 versus control. Despite Covid-19 challenges, pre and post ISWT measurements were obtained for 33 intervention group patients and 37 control group patients. No significant differences between groups were found. However, the average improvement seen in the intervention group, +89 metres (m), exceeded the minimum clinically important difference for the ISWT (+70 m), and was more than double that seen in the control group, +44 m.
Conclusion
This RCT builds on previous evidence supporting the use of the novel digital service in CR3. Engagement with, and completion of, the digital CR service was excellent. Participation was associated with statistically significant improved PA in relation to ACPICR guidelines and clinically important increases in CV fitness versus UC alone.Table 1
Mutations in SNCA, the gene encoding α-synuclein (αSyn), cause familial Parkinson's disease (PD) and aberrant αSyn is a key pathological hallmark of idiopathic PD. This α-synucleinopathy leads to ...mitochondrial dysfunction, which may drive dopaminergic neurodegeneration. PARKIN and PINK1, mutated in autosomal recessive PD, regulate the preferential autophagic clearance of dysfunctional mitochondria ("mitophagy") by inducing ubiquitylation of mitochondrial proteins, a process counteracted by deubiquitylation via USP30. Here we show that loss of USP30 in Usp30 knockout mice protects against behavioral deficits and leads to increased mitophagy, decreased phospho-S129 αSyn, and attenuation of SN dopaminergic neuronal loss induced by αSyn. These observations were recapitulated with a potent, selective, brain-penetrant USP30 inhibitor, MTX115325, with good drug-like properties. These data strongly support further study of USP30 inhibition as a potential disease-modifying therapy for PD.
Purpose
Evaluate whether parents have ever discussed sexuality with their children with anorectal malformations (ARM), which sexuality issues they think should be addressed and who, in their opinion, ...should address these issues.
Methods
Parents from the Italian Parents’ and Patients’ Association for Anorectal Malformation participated in meetings organized by the Association together with local Pediatric Surgical Units and were asked to fill in a questionnaire.
Results
103 parents participated. Overall, 66% of parents had never talked about sex with their children. Children’s age was marginally correlated with occasions to talk about sexuality (
r
= .202,
p
= 0.53) indicating that the older were the children, the more the parents talked about sexuality. The majority of parents reported that their children should have the possibility to talk about sex with them (72%), psychologists (57%), gynecologists/andrologists (47%), pediatric surgeons (33.5%), surgeons specialized in ARM (39.8%), friends (28%), nurses (24.7%) and teachers (20.4%), respectively. The most important topic they thought their children should address was handling serene sexuality, although the most common topic effectively discussed with them was the conception.
Conclusion
Psychologists, gynecologists/andrologists, and pediatric surgeons are seen as key resources for talking about sexuality. A great number of parents express the wish that their children had more opportunities to discuss sexual topics with pediatric surgeons.
Transcriptional changes happen within minutes; however, RNAi or genetic deletion requires days to weeks before transcription networks can be analyzed. This limitation has made it challenging to ...distinguish direct from indirect targets of sequence-specific transcription factors. This inability to define direct transcriptional targets hinders detailed studies of transcriptional mechanisms. This protocol combines rapid degradation of endogenous transcription factors with nascent transcript analysis to define the earliest, and likely direct, regulatory targets of transcription factors.
For complete details on the use and execution of this protocol, please refer to Stengel et al., 2021).
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•A chemical genetic approach for small-molecule-induced degradation of endogenous proteins•Genomic analysis identifies direct targets of transcriptional regulators•Powerful system for interrogating mechanisms of transcriptional control•Step-by-step protocol from sgRNA design to integration of genomic data sets
Transcriptional changes happen within minutes; however, RNAi or genetic deletion requires days to weeks before transcription networks can be analyzed. This limitation has made it challenging to distinguish direct from indirect targets of sequence-specific transcription factors. This inability to define direct transcriptional targets hinders detailed studies of transcriptional mechanisms. This protocol combines rapid degradation of endogenous transcription factors with nascent transcript analysis to define the earliest, and likely direct, regulatory targets of transcription factors.
•Male infertility and adverse pregnancy outcomes are relevant human health problems.•Radiofrequency electromagnetic fields are widespread in the human environment.•A link between radiofrequency and ...adverse reproductive outcomes is controversial.•This is the protocol of WHO-funded systematic review and meta-analysis on this issue.
Radiofrequency Electromagnetic Fields (RF-EMF) at environmental level have been reported to induce adverse effects on the male reproductive system and developing embryos. However, despite the number of experiments conducted since the 1970s, the diversity of testing approaches and exposure conditions, inconsistencies among results, and dosimetric flaws have not yet permitted a solid assessment of the relationship between RF-EMF exposure and such effects, warranting a more systematic and methodologically rigorous approach to the evaluation of available data.
This study aims at evaluating the effects of RF-EMF exposure on male fertility and pregnancy outcomes by a systematic review (SR) of experimental studies, conducted in compliance with international guidelines. The evidence will be organized into three streams: 1) Studies evaluating the impact of RF-EMF on the male reproductive system of experimental mammals; 2) studies evaluating the impact of RF-EMF on human sperm exposed in vitro; 3) studies evaluating the impact of RF-EMF on adverse pregnancy, birth outcomes and delayed effects in experimental mammals exposed in utero.
Eligible studies will include peer-reviewed articles reporting of original results about effects of controlled exposures to RF-EMF in the frequency range 100 kHz–300 GHz on the selected outcomes without any language or year-of-publication restrictions. Eligible studies will be retrieved by calibrated search strings applied to three electronic databases, PubMed, Scopus and EMF Portal and by manual search of the list of references of included papers and published reviews.
The internal validity of the studies will be evaluated using the Risk of Bias (RoB) Rating Tool developed by National Toxicology Program/Office of Health Assessment and Translation (NTP/OHAT) integrated with input from the SYRCLE RoB tool. Given sufficient commensurate data, meta-analyses will be performed, otherwise narrative syntheses will be produced. Finally, the certainty of the effects of RF-EMF exposure on male fertility and pregnancy and birth outcomes will be established following GRADE.
The study is financially supported by the World Health Organization.
OSF Registration DOI https://doi.org/10.17605/OSF.IO/7MUS3; PROSPERO CRD42021227729, CRD42021227746.
The effect of several polyphenols on the 20S proteasomes, both the constitutive and the LMP proteasomes, isolated from bovine tissues, has been investigated. Polyphenolic compounds show many ...biological activities such as antiviral, antibacterial, antifungal, anti-inflammatory, antimutagenic, and antiallergic activities. However, the molecular mechanism underlying these effects has not been identified. It is well established that polyphenols possess inhibitory activities on several enzymes and among them the 20S proteasome. In the present work, the ChT-L, BrAAP, PGPH, and T-L activities of the isolated constitutive and immuno-proteasomes were assayed in order to get an overall information on the polyphenols binding to the complexes. The effects of the polyphenols on the proteasomal activities were analyzed, taking into account the different subunits composition of the two complexes. Furthermore the same activities were measured on whole extracts from cancer cells exposed to EGCG and gallic acid, evaluating, also, their antioxidant action under oxidative stress. EGCG and gallic acid are able to affect the 20S proteasomes functionality, depending on the complex subunit composition and, in cell extracts, they behave both as antioxidants and proteasome effectors.
There is no treatment for the myelin loss in multiple sclerosis, ultimately resulting in the axonal degeneration that leads to the progressive phase of the disease. We established a multi-tiered ...platform for the sequential screening of drugs that could be repurposed as remyelinating agents. We screened a library of 2,000 compounds (mainly Food and Drug Administration (FDA)-approved compounds and natural products) for cellular metabolic activity on mouse oligodendrocyte precursors (OPC), identifying 42 molecules with significant stimulating effects. We then characterized the effects of these compounds on OPC proliferation and differentiation in mouse glial cultures, and on myelination and remyelination in organotypic cultures. Three molecules, edaravone, 5-methyl-7-methoxyisoflavone and lovastatin, gave positive results in all screening tiers. We validated the results by retesting independent stocks of the compounds, analyzing their purity, and performing dose-response curves. To identify the chemical features that may be modified to enhance the compounds' activity, we tested chemical analogs and identified, for edaravone, the functional groups that may be essential for its activity. Among the selected remyelinating candidates, edaravone appears to be of strong interest, also considering that this drug has been approved as a neuroprotective agent for acute ischemic stroke and amyotrophic lateral sclerosis in Japan.
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