NRDE2 negatively regulates exosome functions by inhibiting MTR4 recruitment and exosome interaction.

The exosome functions in the degradation of diverse RNA species, yet how it is negatively regulated remains largely unknown. Here, we show that NRDE2 forms a 1:1 complex with MTR4, a nuclear exosome cofactor critical for exosome recruitment, via a conserved MTR4-interacting domain (MID). Unexpectedly, NRDE2 mainly localizes in nuclear speckles, where it inhibits MTR4 recruitment and RNA degradation, and thereby ensures efficient mRNA nuclear export. Structural and biochemical data revealed that NRDE2 interacts with MTR4's key residues, locks MTR4 in a closed conformation, and inhibits MTR4 interaction with the exosome as well as proteins important for MTR4 recruitment, such as the cap-binding complex (CBC) and ZFC3H1. Functionally, MID deletion results in the loss of self-renewal of mouse embryonic stem cells. Together, our data pinpoint NRDE2 as a nuclear exosome negative regulator that ensures mRNA stability and nuclear export.

Recent advances in bioanalytical methods to measure proteome stability in cells.

Proteome stability constitutes an essential aspect of protein homeostasis (proteostasis). Proteostasis networks maintain proteins and their interactors in a defined conformation for their activity, localisation, and function. However, endogenous or exogenous stressors can perturb proteostasis integrity and deplete folding capacity, generating destabilized folding intermediates and deleterious aggregated species. Over the years, protein unfolding, misfolding and aggregation have been reported to be associated with aging and many diseases such as neurodegenerative diseases, diabetes, cardiac disease and toxicity, and cancers. Therefore, monitoring proteome stability is central to understanding underlying biological processes and mechanisms of disease progression. Herein, we review the recent bioanalytical methods to measure protein stability in cells on a proteome-wide scale.

Correction: Recent advances in bioanalytical methods to measure proteome stability in cells.

Correction for 'Recent advances in bioanalytical methods to measure proteome stability in cells' by Shouxiang Zhang et al., Analyst, 2021, DOI: .

Measuring Cysteine Exposure in Unfolded Proteins with Tetraphenylethene Maleimide and its Analogs.

When proteostasis is challenged and becomes unbalanced, unfolded proteins can accumulate in the cells. Protein unfolding causes conformational changes and subsequent differentials in side-chain solvent accessibility and reactivity. In particular, when protein unfolds, non-disulfide-bonded cysteines that are usually buried in the native state can become surface exposed and thus accessible. A series of fluorogenic dyes including tetraphenylethene maleimide (TPE-MI) and its analogs were developed to capture cysteine exposure in unfolded proteins as a measure of unfolded protein load and proteostasis capacity in cells. These dyes are inherently non-fluorescent but show fluorescence turn-on effect when conjugated to unfolded proteins via reacting with exposed cysteines on the protein. Reacting with small biothiols such as glutathione does not induce fluorescence of these dyes. Here we describe the routine workflow to characterize unfolded proteins in vitro or unfolded proteomes in cells by TPE-MIs.

Synthesis of a ß-Arylethenesulfonyl Fluoride-Functionalized AIEgen for Activity-Based Urinary Trypsin Detection.

Trypsin is one of the most important enzymes of the digestive system produced by the pancreatic acinar cells. Abnormal trypsin activity will affect pancreatic function, resulting in the corresponding pathological changes in the human body. Herein, we present a strategy based on the ensemble of a novel dual warhead probe HPC-ESF and the natural trypsin substrate bovine serum albumin (BSA) for the detection of trypsin activity including in real urine samples. The ß-arylethenesulfonyl bearing HPC-ESF is nonemissive when dissolved in aqueous solution but becomes highly fluorescent upon conjugation to BSA through covalent bond formation with nucleophilic amino acids to create the HPC-ESF:BSA sensing system. The HPC-ESF:BSA complex can be hydrolyzed in the presence of trypsin, which results in a distinct fluorescence decrease in correlation with trypsin concentration and thus allows the detection of trypsin. Compared to previous methods, our covalent approach is simple to prepare and highly reliable. Our work will provide a different avenue for researchers to design fluorescent sensors based on a covalent labeling strategy, enriching the small library of functional groups available for such applications.

Extracorporeal shock wave therapy for bone marrow edema syndrome in patients with osteonecrosis of the femoral head: a retrospective cohort study.

BACKGROUND: There is now ample evidence suggesting that extracorporeal shock wave therapy (ESWT) can improve hip mobility and reduce pain in patients with osteonecrosis of the femoral head (ONFH). The ability of ESWT to cure bone marrow edema syndrome (BMES) in patients with ONFH, 12 weeks after the initial course of ESWT, needs to be verified further and more relevant clinical research-based evidence should be consolidated. This study aimed to evaluate the efficacy of ESWT for BMES caused by ONFH. METHODS: This retrospective cohort study included 67 patients with BMES caused by ONFH who were participating in a rehabilitation program as outpatients. Before and after ESWT, the area of femoral bone marrow edema was evaluated by magnetic resonance imaging (MRI), and the Harris score and Charnley score were evaluated as hip pain and function indicators. RESULTS: After ESWT, MRI revealed that the area of bone marrow edema decreased from 984.6 ± 433.2 mm2 to 189.7 ± 214.4 mm2 (P < 0.0001). The Harris score increased from 42.2 ± 9.1 to 77.7 ± 10.8 points (P < 0.0001). The Charnley score increased from 7.3 ± 1.4 to 12.0 ± 1.7 (P < 0.0001). ESWT was effective in treating BMES in 98.5% of the cases. CONCLUSIONS: This study demonstrated that ESWT can effectively treat BMES caused by ONFH and can aid in pain relief and functional recovery in patients with ONFH. Thus, ESWT should be included in the classic physical therapy regimen for patients with ONFH and BMES.

Construction of a Highly Sensitive Thiol-Reactive AIEgen-Peptide Conjugate for Monitoring Protein Unfolding and Aggregation in Cells.

Impairment of the protein quality control network leads to the accumulation of unfolded and aggregated proteins. Direct detection of unfolded protein accumulation in the cells may provide the possibility for early diagnosis of neurodegenerative diseases. Here a new platform based on a peptide-conjugated thiol-reactive aggregation-induced emission fluorogen (AIEgen), named MI-BTD-P (or D1), for labeling and tracking unfolded proteins in cells is reported. In vitro experiments with model proteins show that the non-fluorescent D1 only becomes highly fluorescent when reacted with the thiol group of free cysteine (Cys) residues on unfolded proteins but not glutathione or folded proteins with buried or surface exposed Cys. When the labeled unfolded proteins form aggregates, D1 fluorescence intensity is further increased, and fluorescence lifetime is prolonged. D1 is then used to measure unfolded protein loads in cells by flow cytometry and track the aggregate formation of the D1 labeled unfolded proteins using confocal microscopy. In combination with fluorescence lifetime imaging technique, the proteome at different folding statuses can be better differentiated, demonstrating the versatility of this new platform. The rational design of D1 demonstrates the outlook of incorporation of diverse functional groups to achieve maximal sensitivity and selectivity in biological samples.

A Maleimide-functionalized Tetraphenylethene for Measuring and Imaging Unfolded Proteins in Cells.

Collapse of the protein homeostasis (proteostasis) can lead to accumulation and aggregation of unfolded proteins, which has been found to associate with a number of disease conditions including neurodegenerative diseases, diabetes and inflammation. Here we report a maleimide-functionalized tetraphenylethene (TPE)-derivatized fluorescent dye, TPE-NMI, which shows fluorescence turn-on property upon reacting with unfolded proteins in vitro and in live cells under proteostatic stress conditions. The level of unfolded proteins can be measured by flow cytometry and visualized with confocal microscopy.

Effect of extracorporeal shock-wave therapy for treating patients with chronic rotator cuff tendonitis.

BACKGROUND: This study aimed to determine the efficacy and safety of extracorporeal shock-wave therapy (ESWT) for treating patients with chronic rotator cuff tendonitis (CRCT). METHODS: In this study, 84 patients with CRCT were randomly divided into intervention and control groups in a ratio of 1:1. Patients in the intervention group received ESWT, whereas those in the control group received placebo. The primary outcome was measured by Numeric Rating Scale (NRS). The secondary outcomes were measured by the Constant-Murley score (CMS), simple shoulder test (SST) score, and adverse events. RESULTS: Compared with placebo, ESWT showed greater efficacy in shoulder pain relief with regard to NRS score and shoulder function as measured by using CMS and SST score at 4 weeks (P < .05) and 8 weeks (P < .01) after treatment. However, no adverse events occurred in both groups. CONCLUSION: ESWT was efficacious and safe for treating patients with CRCT.

Discovery of N-(3-(5-((3-acrylamido-4-(morpholine-4-carbonyl)phenyl)amino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-methylphenyl)-4-(tert-butyl)benzamide (CHMFL-BTK-01) as a highly selective irreversible Bruton's tyrosine kinase (BTK) inhibitor.

Currently there are several irreversible BTK inhibitors targeting Cys481 residue under preclinical or clinical development. However, most of these inhibitors also targeted other kinases such as BMX, JAK3, and EGFR that bear the highly similar active cysteine residues. Through a structure-based drug design approach, we discovered a highly potent (IC50: 7 nM) irreversible BTK inhibitor compound 9 (CHMFL-BTK-01), which displayed a high selectivity profile in KINOMEscan (S score (35) = 0.00) among 468 kinases/mutants at the concentration of 1 µM. Compound 9 completely abolished BMX, JAK3 and EGFR's activity. Both X-ray crystal structure and cysteine-serine mutation mediated rescue experiment confirmed 9's irreversible binding mode. 9 also potently inhibited BTK Y223 auto-phosphorylation (EC50: <30 nM), arrested cell cycle in G0/G1 phase and induced apoptosis in U2932 and Pfeiffer cells. We believe these features would make 9 a good pharmacological tool to study the BTK related pathology.