Hsp90α forms condensate engaging client proteins with RG motif repeats.

Hsp90α, a pivotal canonical chaperone, is renowned for its broad interaction with numerous protein clients to maintain protein homeostasis, chromatin remodeling, and cell growth. Recent studies indicate its role in modifying various components of membraneless organelles (MLOs) such as stress granules and processing bodies, suggesting its participation in the regulation of protein condensates. In this study, we found that Hsp90α possesses an inherent ability to form dynamic condensates in vitro. Utilizing LC-MS/MS, we further pinpointed proteins in cell lysates that preferentially integrate into Hsp90α condensates. Significantly, we observed a prevalence of RG motif repeats in client proteins of Hsp90α condensates, many of which are linked to various MLOs. Moreover, each of the three domains of Hsp90α was found to undergo phase separation, with numerous solvent-exposed negatively charged residues on these domains being crucial for driving Hsp90α condensation through multivalent weak electrostatic interactions. Additionally, various clients like TDP-43 and hnRNPA1, along with poly-GR and PR dipeptide repeats, exhibit varied impacts on the dynamic behavior of Hsp90α condensates. Our study spotlights various client proteins associated with Hsp90α condensates, illustrating its intricate adaptive nature in interacting with diverse clients and its functional adaptability across multiple MLOs.

Diagnostic value of contrast-enhanced ultrasound and shear-wave elastography for small breast nodules.

Background: The study aims to evaluate the diagnostic efficacy of contrast-enhanced ultrasound (CEUS) and shear-wave elastography (SWE) in detecting small malignant breast nodules in an effort to inform further refinements of the Breast Imaging Reporting and Data System (BI-RADS) classification system. Methods: This study retrospectively analyzed patients with breast nodules who underwent conventional ultrasound, CEUS, and SWE at Gongli Hospital from November 2015 to December 2019. The inclusion criteria were nodules ≤ 2 cm in diameter with pathological outcomes determined by biopsy, no prior treatments, and solid or predominantly solid nodules. The exclusion criteria included pregnancy or lactation and low-quality images. Imaging features were detailed and classified per BI-RADS. Diagnostic accuracy was assessed using receiver operating characteristic curves. Results: The study included 302 patients with 305 breast nodules, 113 of which were malignant. The diagnostic accuracy was significantly improved by combining the BI-RADS classification with CEUS and SWE. The combined approach yielded a sensitivity of 88.5%, specificity of 87.0%, positive predictive value of 80.0%, negative predictive value of 92.8%, and accuracy of 87.5% with an area under the curve of 0.877. Notably, 55.8% of BI-RADS 4A nodules were downgraded to BI-RADS 3 and confirmed as benign after pathological examination, suggesting the potential to avoid unnecessary biopsies. Conclusion: The integrated use of the BI-RADS classification, CEUS, and SWE enhances the accuracy of differentiating benign and malignant small breast nodule, potentially reducing the need for unnecessary biopsies.

VAMP2 chaperones α-synuclein in synaptic vesicle co-condensates.

α-Synuclein (α-Syn) aggregation is closely associated with Parkinson's disease neuropathology. Physiologically, α-Syn promotes synaptic vesicle (SV) clustering and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly. However, the underlying structural and molecular mechanisms are uncertain and it is not known whether this function affects the pathological aggregation of α-Syn. Here we show that the juxtamembrane region of vesicle-associated membrane protein 2 (VAMP2)-a component of the SNARE complex that resides on SVs-directly interacts with the carboxy-terminal region of α-Syn through charged residues to regulate α-Syn's function in clustering SVs and promoting SNARE complex assembly by inducing a multi-component condensed phase of SVs, α-Syn and other components. Moreover, VAMP2 binding protects α-Syn against forming aggregation-prone oligomers and fibrils in these condensates. Our results suggest a molecular mechanism that maintains α-Syn's function and prevents its pathological amyloid aggregation, the failure of which may lead to Parkinson's disease.

Phase-separated ParB enforces diverse DNA compaction modes and stabilizes the parS-centered partition complex.

The tripartite ParABS system mediates chromosome segregation in the majority of bacterial species. Typically, DNA-bound ParB proteins around the parS sites condense the chromosomal DNA into a higher-order multimeric nucleoprotein complex for the ParA-driven partition. Despite extensive studies, the molecular mechanism underlying the dynamic assembly of the partition complex remains unclear. Herein, we demonstrate that Bacillus subtilis ParB (Spo0J), through the multimerization of its N-terminal domain, forms phase-separated condensates along a single DNA molecule, leading to the concurrent organization of DNA into a compact structure. Specifically, in addition to the co-condensation of ParB dimers with DNA, the engagement of well-established ParB condensates with DNA allows for the compression of adjacent DNA and the looping of distant DNA. Notably, the presence of CTP promotes the formation of condensates by a low amount of ParB at parS sites, triggering two-step DNA condensation. Remarkably, parS-centered ParB-DNA co-condensate constitutes a robust nucleoprotein architecture capable of withstanding disruptive forces of tens of piconewton. Overall, our findings unveil diverse modes of DNA compaction enabled by phase-separated ParB and offer new insights into the dynamic assembly and maintenance of the bacterial partition complex.

Machine learning predictor PSPire screens for phase-separating proteins lacking intrinsically disordered regions.

The burgeoning comprehension of protein phase separation (PS) has ushered in a wealth of bioinformatics tools for the prediction of phase-separating proteins (PSPs). These tools often skew towards PSPs with a high content of intrinsically disordered regions (IDRs), thus frequently undervaluing potential PSPs without IDRs. Nonetheless, PS is not only steered by IDRs but also by the structured modular domains and interactions that aren't necessarily reflected in amino acid sequences. In this work, we introduce PSPire, a machine learning predictor that incorporates both residue-level and structure-level features for the precise prediction of PSPs. Compared to current PSP predictors, PSPire shows a notable improvement in identifying PSPs without IDRs, which underscores the crucial role of non-IDR, structure-based characteristics in multivalent interactions throughout the PS process. Additionally, our biological validation experiments substantiate the predictive capacity of PSPire, with 9 out of 11 chosen candidate PSPs confirmed to form condensates within cells.

Phosphorylation-Regulated Dynamic Phase Separation of HIP-55 Protects Against Heart Failure.

BACKGROUND: Heart failure (HF), which is the terminal stage of many cardiovascular diseases, is associated with low survival rates and a severe financial burden. The mechanisms, especially the molecular mechanism combined with new theories, underlying the pathogenesis of HF remain elusive. We demonstrate that phosphorylation-regulated dynamic liquid-liquid phase separation of HIP-55 (hematopoietic progenitor kinase 1-interacting protein of 55 kDa) protects against HF. METHODS: Fluorescence recovery after photobleaching assay, differential interference contrast analysis, pull-down assay, immunofluorescence, and immunohistochemical analysis were used to investigate the liquid-liquid phase separation capacity of HIP-55 and its dynamic regulation in vivo and in vitro. Mice with genetic deletion of HIP-55 and mice with cardiac-specific overexpression of HIP-55 were used to examine the role of HIP-55 on ß-adrenergic receptor hyperactivation-induced HF. Mutation analysis and mice with specific phospho-resistant site mutagenesis were used to identify the role of phosphorylation-regulated dynamic liquid-liquid phase separation of HIP-55 in HF. RESULTS: Genetic deletion of HIP-55 aggravated HF, whereas cardiac-specific overexpression of HIP-55 significantly alleviated HF in vivo. HIP-55 possesses a strong capacity for phase separation. Phase separation of HIP-55 is dynamically regulated by AKT-mediated phosphorylation at S269 and T291 sites, failure of which leads to impairment of HIP-55 dynamic phase separation by formation of abnormal aggregation. Prolonged sympathetic hyperactivation stress induced decreased phosphorylation of HIP-55 S269 and T291, dysregulated phase separation, and subsequent aggregate formation of HIP55. Moreover, we demonstrated the important role of dynamic phase separation of HIP-55 in inhibiting hyperactivation of the ß-adrenergic receptor-mediated P38/MAPK (mitogen-activated protein kinase) signaling pathway. A phosphorylation-deficient HIP-55 mutation, which undergoes massive phase separation and forms insoluble aggregates, loses the protective activity against HF. CONCLUSIONS: Our work reveals that the phosphorylation-regulated dynamic phase separation of HIP-55 protects against sympathetic/adrenergic system-mediated heart failure.

Bioactive compounds and biological functions of medicinal plant-derived extracellular vesicles.

Extracellular vesicles (EVs) are tiny lipid bilayer-enclosed membrane particles released from a variety of cell types into the surrounding environment. These EVs have massive participated in cell-to-cell communication and interspecies communication. In recent years, plant-derived extracellular vesicles (PDEVs) and "exosome-like" EVs populations found in distinct plants have attracted widespread attention. Especially, research on medicinal plant-derived extracellular vesicles (MPDEVs) are increasing, which are considered a kind of promising natural compound. This review summarizes current knowledge on MPDEVs in terms of bioactive compounds, including small RNA, protein, lipid, and metabolite, have been found on the surface and/or in the lumen of MPDEVs. Moreover, both in vitro and in vivo experiments have shown that MPDEVs exert broad biomedical functions, such as anti-inflammatory, anticancer, antioxidant, modulate microbiota, etc. MPDEVs may be a better substitute than animal-derived extracellular vesicles (ADEVs) because of safety and biocompatibility in the future.

Lack of causal association between epilepsy and dementia: A Mendelian randomization analysis.

OBJECTIVE: Epidemiological studies have reported an association between epilepsy and dementia. However, the causal relationship between epilepsy and the risk of dementia is not clear. We aimed to inspect the causal effect of epilepsy on memory loss and dementia. METHODS: We analyzed summary data of epilepsy, memory loss, and dementia from the genome-wide association study (GWAS) using the two-sample Mendelian randomization (MR) method. We used the estimated odds ratio of memory loss and dementia associated with each of the genetically defined traits to infer evidence for a causal relationship with the following exposures: all epilepsy, focal epilepsy (including focal epilepsy with hippocampal sclerosis, lesion-negative focal epilepsy, and focal epilepsy with other lesions), and genetic generalized epilepsy (including childhood absence epilepsy, generalized tonic-clonic seizures alone, Juvenile absence epilepsy, and Juvenile myoclonic epilepsy). RESULTS: According to the result of MR using the inverse variance weighted method (IVW), we found that genetically predicted epilepsy did not causally increase the risk of memory loss and dementia (p > 0.05). Results of the MR-Egger and weighted median method were consistent with the IVW method. CONCLUSIONS: No evidence has been found to support the notion that epilepsy can result in memory loss and dementia. The associations observed in epidemiological studies could be attributed, in part, to confounding or nongenetic determinants.

The validity and IRT psychometric analysis of Chinese version of Difficult Doctor-Patient Relationship Questionnaire (DDPRQ-10).

OBJECTIVE: The doctor-patient relationship (DPR) plays a crucial role in the Chinese healthcare system, functioning to improve medical quality and reduce medical costs. This study examined the psychometric properties of the Chinese version of the Difficult Doctor-Patient Relationship Questionnaire (DDPRQ-10) among general hospital inpatients in China. METHODS: The research recruited 38 resident doctors responsible for 120 participants, and factor analyses were used to assess the construct validity of the scale. Convergent validity was evaluated by examining the correlation between DDPRQ-10 and depressive symptoms, burnout, and self-efficacy, using the Patient Health Questionnaire Depression Scale-9 item (PHQ-9), and the Maslach Burnout Inventory (MBI). Both multidimensional item response theory (MIRT) and unidimensional item response theory (IRT) frameworks were used to estimate the parameters of each item. RESULTS: The Chinese version of DDPRQ-10 showed satisfactory internal consistency (Cronbach's alpha = 0.931), and fitted in a modified two-factor model of positive feelings and negative feelings (χ2/df = 1.494, GFI = 0.925, RMSEA = 0.071, SRMR = 0.008, CFI = 0.985, NFI = 0.958, NNFI = 0.980, TLI = 0.980, IFI = 0.986). Significant correlations with PHQ-9 with DDPRQ-10 and both subscales were revealed (r = 0.293 ~ 0.333, p < .001), while DDPRQ-10 score also significantly correlated with doctors' MBI score (r = -0.467, p < .001). The MIRT model of full scale and IRT models of both subscales showed high discrimination of all items (a = 2.30 ~ 10.18), and the test information within the range of low-quality relationship was relatively high. CONCLUSION: The Chinese version of DDPRQ-10 displayed satisfactory reliability and validity and thus was appropriate for measuring the DPR in Chinese medical settings.

Superhard bulk high-entropy carbides with enhanced toughness via metastable in-situ particles.

Despite the extremely high hardness of recently proposed high-entropy carbides (HECs), the low fracture toughness limits their applications in harsh mechanical environment. Here, we introduce a metastability engineering strategy to achieve superhard HECs with enhanced toughness via in-situ metastable particles. This is realized by developing a (WTaNbZrTi)C HEC showing a solid solution matrix with uniformly dispersed in-situ tetragonal and monoclinic ZrO2 particles. Apart from a high hardness of 21.0 GPa, the HEC can obtain an enhanced fracture toughness of 5.89 MPa·m1/2, significantly exceeding the value predicted by rule of mixture and that of other reported HECs. The toughening effect is primarily attributed to the transformation of the metastable tetragonal ZrO2 particles under mechanical loading, which promotes crack tip shielding mechanisms including crack deflection, crack bridging and crack branching. The work demonstrates the concept of using in-situ metastable particles for toughening bulk high-entropy ceramics by taking advantage of their compositional flexibility.

Development and psychometric validation of the hospitalized patients' expectations for treatment scale-patient version.

Objectives: A general expectation measurement of inpatients across wards is needed in the patient safety management systems of general hospitals. This study developed and psychometrically validated a new scale fulfilling the requirements above: the Hospitalized Patients' Expectations for Treatment Scale-Patient version (HOPE-P). Methods: A total of 35 experts and ten inpatients were interviewed during the formulation of the HOPE-P scale, which was initially designed with three dimensions: doctor-patient communication expectations, treatment outcome expectations, and disease management expectancy. We recruited 210 inpatients from a general hospital in China and explored the reliability, validity, and psychometric characteristics of the questionnaire. Item analysis, construct validity, internal consistency and 7-day test-retest reliability analysis were applied. Results: Exploratory and confirmatory analyses supported a 2-dimension (doctor-patient communication expectation and treatment outcome expectation) structure with satisfactory model fit parameters (root mean square residual (RMR) = 0.035, a root-mean-square-error of approximation (RMSEA) = 0.072, comparative fit index (CFI) = 0.984, Tucker-Lewis index (TLI) = 0.970). Item analysis revealed an appropriate item design (r = 0.573-0.820). The scale exhibited good internal consistency, with Cronbach's α of 0.893, 0.761, and 0.919 for the overall scale, the doctor-patient communication expectation subscale, and the treatment outcome expectation subscale, respectively. The 7-day test-retest reliability was 0.782 (p < .001). Conclusion: Our results indicated that the HOPE-P is a reliable and valid assessment tool to measure the expectations of general hospital inpatients, with a strong capacity to recognize patients' expectations regarding doctor-patient communication and treatment outcomes.

The causal relationship between circulating biomarkersand the risk of bipolar disorder: A two-sample Mendelian randomization study.

OBJECTIVE: To identify susceptible biomarkers for the development of bipolar disorder (BD), we conducted a Mendelian Randomization (MR) design to screen circulating proteins for the potential risk of bipolar disorder systematically. METHODS: We performed a two-sample Mendelian randomization (MR) analysis to estimate the causality of 4782 human circulating proteins on the risk of bipolar disorder. 376 circulating biomarkers were selected in MR estimation (4406 circulating proteins with less than 3 SNPs were excluded) with 5368 European descents. GWAS meta-analysis of the potential role of all-cause bipolar disorder arose from the Psychiatric Genomics Consortium (41,917 cases, 371,549 controls). RESULTS: After IVW and sensitivity analysis, 4 circulating proteins having causal effects on bipolar disorder were identified. ISG15, as a key player in the innate immune response, decreased the risk of bipolar disorder causally (OR = 0.92, 95% CI = 0.89-0.94, P = 1.46e-09). Furthermore, MLN decreased the risk of bipolar disorder causally (OR = 0.94, 95% CI = 0.91-0.97, P = 1.04e-04). In addition, SFTPC (OR = 0.91, 95% CI = 0.86-0.96, P = 4.47e-04) and VCY (OR = 0.86, 95% CI = 0.77-0.96, P = 8.55e-03) presented a suggestive association with bipolar disorder. CONCLUSIONS: Our findings indicated that ISG15 and MLN showed evidence of causality in bipolar disorder and provided a promising target for the diagnosis and treatment of diseases.

ß2-Microglobulin coaggregates with Aß and contributes to amyloid pathology and cognitive deficits in Alzheimer's disease model mice.

Extensive studies indicate that ß-amyloid (Aß) aggregation is pivotal for Alzheimer's disease (AD) progression; however, cumulative evidence suggests that Aß itself is not sufficient to trigger AD-associated degeneration, and whether other additional pathological factors drive AD pathogenesis remains unclear. Here, we characterize pathogenic aggregates composed of ß2-microglobulin (ß2M) and Aß that trigger neurodegeneration in AD. ß2M, a component of major histocompatibility complex class I (MHC class I), is upregulated in the brains of individuals with AD and constitutes the amyloid plaque core. Elevation of ß2M aggravates amyloid pathology independent of MHC class I, and coaggregation with ß2M is essential for Aß neurotoxicity. B2m genetic ablation abrogates amyloid spreading and cognitive deficits in AD mice. Antisense oligonucleotide- or monoclonal antibody-mediated ß2M depletion mitigates AD-associated neuropathology, and inhibition of ß2M-Aß coaggregation with a ß2M-based blocking peptide ameliorates amyloid pathology and cognitive deficits in AD mice. Our findings identify ß2M as an essential factor for Aß neurotoxicity and a potential target for treating AD.

The Chinese version of patient-doctor-relationship questionnaire (PDRQ-9): Factor structure, validation, and IRT psychometric analysis.

Objective: The patient-doctor relationship has been considered as a crucial concept in primary healthcare, while the medical reform launched by the Chinese government in 2009 has brought significant changes to the healthcare system, which made it urgent to introduce reliable measurement instruments for assessing today's doctor-patient relationship in China. This study examined the psychometric properties of the Chinese version of the Patient-Doctor-Relationship Questionnaire-9 item (PDRQ-9) scale among general hospital inpatients in China. Materials and methods: A total of 203 participants responded to the survey, of which 39 completed retest after 7 days. Factor analyses were used to test the construct validity of the scale. Convergent validity was evaluated by the correlation between PDRQ-9 and depressive symptoms measured using PHQ-9 (Patient Health Questionnaire Depression Scale-9 item). Both multidimensional item response theory (MIRT) and unidimensional item response theory (IRT) framework were used to estimate the parameters of each item. Results: The two-factor model of relationship quality and treatment quality was supported (χ2/df = 1.494, GFI = 0.925, RMSEA = 0.071, RMR = 0.008, CFI = 0.985, NFI = 0.958, NNFI = 0.980, TLI = 0.980, IFI = 0.986). The PDRQ-9 and both subscales showed significant correlation with PHQ-9 (r = -0.196∼-0.309) and good internal consistency (Cronbach's alpha = 0.865∼0.933). ANCOVA analysis adjusted with age revealed significant difference in PDRQ-9 ratings between patients with or without significant depressive symptoms (P = 0.019). The 7-day test-retest reliability of the scale was 0.730. The MIRT model of full scale and IRT models of both subscales showed high discrimination of all items (a = 2.46∼38.46), and the test information within the range of low-quality relationship was relatively high. Conclusion: The Chinese version of PDRQ-9 is a valid and reliable rating scale, which can measure the doctor-patient relationship among Chinese patients.

Emotional distress and burnout at a fever clinic in China: Comparison between different periods of COVID-19.

Background: Frontline healthcare workers (FHWs) experienced psychological stress and heavy workload during COVID-19 pandemic. This study examined the psychological symptoms and occupational burnout of FHWs in a fever clinic during different periods of the pandemic. Methods: A cross-sectional survey of FHWs in the fever clinic of a tertiary hospital was carried out during both the outbreak period and regular period of COVID-19. Psychological measurement instruments including Generalized Anxiety Disorder 7-item, the 9-Question Patient Health Questionnaire, the Maslach Burnout Inventory-Human Service Survey, and the General Self-Efficacy Scale were used to evaluate anxiety, depression, burnout, and self-efficacy, respectively. The correlation between clinical variables was explored. Results: A total of 162 participants were involved in this study, including 118 FHWs during the outbreak period (Group 1) and 44 FHWs during the regular period (Group 2). Anxiety symptoms were more prevalent in Group 2 (x 2 = 27.477) while depressive symptoms were significantly more prevalent in Group 1 (x 2 = 69.538). Burnout rate was higher in Group 2 (x 2 = 29.526). Self-efficacy was higher in Group 1 (t = 3.194). Burnout was positively correlated with anxiety symptoms (r 2 = 0.424) and negatively correlated with self-efficacy (r 2 = -0.312). Conclusion: Anxiety, depressive symptoms and burnout were prevalent in FHWs during different periods of the COVID-19 pandemic. There is a tendency to be less depressed, but more anxious and burned out over time, although the severity of the pandemic is decreasing. Self-efficacy may be an important factor in protecting FHWs from occupational burnout. Support and intervention plans for FHWs should be made at the institutional level.

HPV 16 E6 promotes growth and metastasis of esophageal squamous cell carcinoma cells in vitro.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies worldwide. Increasing evidence suggests that human papillomavirus (HPV) infection may be associated with the etiology of ESCC. However, the precise role of HPV in ESCC remains unclear. METHODS AND RESULTS: Proliferation and apoptosis of ESCC cells upon infection with HPV16 E6 were detected using CCK-8 assays and Western blot analyses. The migration rate was measured with a wound healing assay, and a Transwell Matrigel invasion assay was used to detect the invasive ability. RT-qPCR was performed to detect the expression of E6AP, p53, and miR-34a. The proliferation rates were significantly higher in HPV16E6-transfected cell groups compared with the negative control groups. Bax protein expression was downregulated in HPV16E6-treated groups compared to the controls. The wound healing and Transwell Matrigel invasion assays indicated that HPV16 E6 infection could increase ESCC cell migration and invasion. Furthermore, E6AP, p53 and miR-34a expression were decreased in HPV16 E6-transfected cell lines. CONCLUSION: Our results not only provide evidence that HPV16 E6 promotes cell proliferation, migration, and invasion in ESCC, but also suggests a correlation between HPV infection and E6AP, p53 and miR-34a expression. Consequently, HPV16 E6 may play an important role in ESCC development.

Development and psychometric validation of the hospitalized patients' expectations for treatment scale-clinician version.

Objective: Patient safety management systems in general hospitals require a comprehensive tool for assessing the expectations of inpatients across different wards. This study aimed to develop and psychometrically validate a new scale, the hospitalized patients' expectations for treatment scale-clinician version (HOPE-C), to meet this requirement. Methods: We interviewed 35 experts and 10 inpatients while developing the HOPE-C scale. The scale was initially designed with three dimensions: clinicians' expectations regarding doctor-patient communication, clinicians' expectations regarding treatment outcome, and clinicians' expectations regarding disease management. We recruited 200 inpatients from a general hospital in China. At the same time, 51 clinicians were assigned to the enrolled patients who completed the HOPE-C to examine the reliability, validity, and psychometric characteristics of the questionnaire. We applied item analysis, assessed construct validity, evaluated internal consistency, and conducted a test-retest reliability analysis over 7 days. Results: Both exploratory and confirmatory analyses supported a 2-dimensional structure, comprising doctor-patient communication expectations and treatment outcome expectations, with favorable model fit parameters (root mean square residual [RMR] = 0.042, root mean square error of approximation [RMSEA] = 0.049, comparative fit index [CFI] = 0.989, Tucker-Lewis index [TLI] = 0.984). Item analysis demonstrated appropriate item design (r = 0.744-0.961). The scale exhibited strong internal consistency, with Cronbach's α values of 0.884, 0.816, and 0.840 for the overall scale, the doctor-patient communication expectation subscale, and the treatment outcome expectation subscale, respectively. The 7-day test-retest reliability was 0.996 (p < 0.001). Conclusion: Our findings suggest that the HOPE-C is a reliable and valid assessment tool for measuring the expectations of inpatients in general hospitals. It effectively identifies patients' expectations concerning doctor-patient communication and treatment outcomes.

A redox switch regulates the assembly and anti-CRISPR activity of AcrIIC1.

Anti-CRISPRs (Acrs) are natural inhibitors of bacteria's CRISPR-Cas systems, and have been developed as a safeguard to reduce the off-target effects of CRISPR gene-editing technology. Acrs can directly bind to CRISPR-Cas complexes and inhibit their activities. However, whether this process is under regulation in diverse eukaryotic cellular environments is poorly understood. In this work, we report the discovery of a redox switch for NmeAcrIIC1, which regulates NmeAcrIIC1's monomer-dimer interconversion and inhibitory activity on Cas9. Further structural studies reveal that a pair of conserved cysteines mediates the formation of inactive NmeAcrIIC1 dimer and directs the redox cycle. The redox switch also applies to the other two AcrIIC1 orthologs. Moreover, by replacing the redox-sensitive cysteines, we generated a robust AcrIIC1 variant that maintains potent inhibitory activity under various redox conditions. Our results reveal a redox-dependent regulation mechanism of Acr, and shed light on the design of superior Acr for CRISPR-Cas systems.

Heat-shock chaperone HSPB1 regulates cytoplasmic TDP-43 phase separation and liquid-to-gel transition.

While acetylated, RNA-binding-deficient TDP-43 reversibly phase separates within nuclei into complex droplets (anisosomes) comprised of TDP-43-containing liquid outer shells and liquid centres of HSP70-family chaperones, cytoplasmic aggregates of TDP-43 are hallmarks of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Here we show that transient oxidative stress, proteasome inhibition or inhibition of the ATP-dependent chaperone activity of HSP70 provokes reversible cytoplasmic TDP-43 de-mixing and transition from liquid to gel/solid, independently of RNA binding or stress granules. Isotope labelling mass spectrometry was used to identify that phase-separated cytoplasmic TDP-43 is bound by the small heat-shock protein HSPB1. Binding is direct, mediated through TDP-43's RNA binding and low-complexity domains. HSPB1 partitions into TDP-43 droplets, inhibits TDP-43 assembly into fibrils, and is essential for disassembly of stress-induced TDP-43 droplets. A decrease in HSPB1 promotes cytoplasmic TDP-43 de-mixing and mislocalization. HSPB1 depletion was identified in spinal motor neurons of patients with ALS containing aggregated TDP-43. These findings identify HSPB1 to be a regulator of cytoplasmic TDP-43 phase separation and aggregation.

Bloom Syndrome Helicase Compresses Single-Stranded DNA into Phase-Separated Condensates.

Bloom syndrome protein (BLM) is a conserved RecQ family helicase involved in the maintenance of genome stability. BLM has been widely recognized as a genome "caretaker" that processes structured DNA. In contrast, our knowledge of how BLM behaves on single-stranded (ss) DNA is still limited. Here, we demonstrate that BLM possesses the intrinsic ability for phase separation and can co-phase separate with ssDNA to form dynamically arrested protein/ssDNA co-condensates. The introduction of ATP potentiates the capability of BLM to condense on ssDNA, which further promotes the compression of ssDNA against a resistive force of up to 60 piconewtons. Moreover, BLM is also capable of condensing replication protein A (RPA)- or RAD51-coated ssDNA, before which it generates naked ssDNA by dismantling these ssDNA-binding proteins. Overall, our findings identify an unexpected characteristic of a DNA helicase and provide a new angle of protein/ssDNA co-condensation for understanding the genomic instability caused by BLM overexpression under diseased conditions.