Correction: Dynein and Dynactin Leverage Their Bivalent Character to Form a High-Affinity Interaction.

[This corrects the article DOI: 10.1371/journal.pone.0059453.].

Reconstitution of Arp2/3-Nucleated Actin Assembly with CP, V-1 and CARMIL.

Actin polymerization is often associated with membrane proteins containing capping-protein-interacting (CPI) motifs, such as CARMIL, CD2AP, and WASHCAP/Fam21. CPI motifs bind directly to actin capping protein (CP), and this interaction weakens the binding of CP to barbed ends of actin filaments, lessening the ability of CP to functionally cap those ends. The protein V-1 / myotrophin binds to the F-actin binding site on CP and sterically blocks CP from binding barbed ends. CPI-motif proteins also weaken the binding between V-1 and CP, which decreases the inhibitory effects of V-1, thereby freeing CP to cap barbed ends. Here, we address the question of whether CPI-motif proteins on a surface analogous to a membrane lead to net activation or inhibition of actin assembly nucleated by Arp2/3 complex. Using reconstitution with purified components, we discovered that CARMIL at the surface promotes and enhances actin assembly, countering the inhibitory effects of V-1 and thus activating CP. The reconstitution involves the presence of an Arp2/3 activator on the surface, along with Arp2/3 complex, V-1, CP, profilin and actin monomers in solution, recreating key features of cell physiology.

A register-based study describing time trends in risk factor control and serious hypoglycaemia together with the effects of starting continuous glucose monitoring in people with type 1 diabetes in Norway.

AIMS: To describe trends in risk factor control and serious hypoglycaemia in people with type 1 diabetes and to assess the effect of starting continuous glucose monitoring (CGM) in the real-world setting. METHODS: Two cross-sectional surveys including 5746 individuals in 2012 and 18,984 individuals in 2020 based on data recorded in the Norwegian Diabetes Register for Adults (NDR-A) and an analysis of a longitudinal cohort of 2057 individuals where data on CGM and HbA1c were available in the NDR-A in 2012 and 2020. RESULTS: In the cross-sectional surveys mean HbA1c decreased from 66 mmol/mol (99% CI 65, 66) (8.2%) in 2012 to 61 mmol/mol (99% CI 61, 61) (7.7%) in 2020 (p < 0.0001). The proportion reporting serious hypoglycaemia decreased from 16.9 to 6.2% in 2020 (p < 0.0001). Mean LDL-cholesterol decreased from 2.80 (99% CI 2.78, 2.83) to 2.63 (99% CI 2.61, 2.65) mmol/l in 2020 (p < 0.0001). Mean blood pressure increased slightly. In the CGM cohort, we found a 3 mmol/mol (0.3%) greater improvement in mean HbA1c and a greater reduction in serious hypoglycaemia (-12.3% vs. -6.2%) among individuals that had started using CGM between 2013 and 2020 when compared with individuals that had not started using CGM. CONCLUSIONS: Between 2012 and 2020, we found marked improvements in glycaemic control and a considerable decrease in the proportion of individuals reporting serious hypoglycaemia. The proportion of individuals using CGM increased substantially and individuals that had started using CGM by 2020 showed greater improvement in glycaemic control and less serious hypoglycaemia.

Mammalian Antigen Display for Pandemic Countermeasures.

Pandemic countermeasures require the rapid design of antigens for vaccines, profiling patient antibody responses, assessing antigen structure-function landscapes, and the surveillance of emerging viral lineages. Cell surface display of a viral antigen or its subdomains can facilitate these goals by coupling the phenotypes of protein variants to their DNA sequence. Screening surface-displayed proteins via flow cytometry also eliminates time-consuming protein purification steps. Prior approaches have primarily relied on yeast as a display chassis. However, yeast often cannot express large viral glycoproteins, requiring their truncation into subdomains. Here, we describe a method to design and express antigens on the surface of mammalian HEK293T cells. We discuss three use cases, including screening of stabilizing mutations, deep mutational scanning, and epitope mapping. The mammalian antigen display platform described herein will accelerate ongoing and future pandemic countermeasures.

High prevalence of retinopathy in young-onset type 2 diabetes and possible sex differences: insights from Norwegian general practice.

INTRODUCTION: People with young-onset type 2 diabetes (YOD), defined as diabetes diagnosis before age 40, have a high lifetime risk of vascular complications. We aimed to estimate the prevalence of YOD among adults with type 2 diabetes (T2D) in Norwegian general practice and explore associations between age at diabetes diagnosis and retinopathy overall and in men and women. RESEARCH DESIGN AND METHODS: We collected cross-sectional data from general practice electronic medical records of 10 241 adults with T2D in 2014, and repeated measurements of hemoglobin A1c (HbA1c) from 2012 to 2014. Using multivariate logistic regression, we assessed associations between YOD and later-onset T2D, sex and retinopathy. RESULTS: Of all individuals with T2D, 10% were diagnosed before 40 years of age in both sexes. Compared with later-onset T2D, HbA1c increased faster in YOD, and at the time of diagnosis HbA1c was higher in men, particularly in YOD. Retinopathy was found in 25% with YOD, twice as frequently as in later onset. After adjustments for confounders (age, country of origin, education, body mass index), OR of retinopathy was increased in both men with YOD (OR 2.6 (95% CI 2.0 to 3.5)) and women with YOD (OR 2.2 (1.5 to 3.0)). After further adjustments for potential mediators (diabetes duration and HbA1c), the higher OR persisted in men with YOD (OR 1.8 (1.3 to 2.4)) but was attenuated and no longer significant for women with YOD. CONCLUSIONS: Retinopathy prevalence was more than twice as high in YOD as in later-onset T2D. The increased likelihood of retinopathy in YOD was partly mediated by higher HbA1c and longer T2D duration, but after accounting for these factors it remained higher in men with YOD.

Quantification of Antiviral Drug Tenofovir (TFV) by Surface-Enhanced Raman Spectroscopy (SERS) Using Cumulative Distribution Functions (CDFs).

Surface-enhanced Raman spectroscopy (SERS) is an ultrasensitive spectroscopic technique that generates signal-enhanced fingerprint vibrational spectra of small molecules. However, without rigorous control of SERS substrate active sites, geometry, surface area, or surface functionality, SERS is notoriously irreproducible, complicating the consistent quantitative analysis of small molecules. While evaporatively prepared samples yield significant SERS enhancement resulting in lower detection limits, the distribution of these enhancements along the SERS surface is inherently stochastic. Acquiring spatially resolved SERS spectra of these dried surfaces, we have shown that this enhancement is governed by a power law as a function of analyte concentration. Consequently, by definition, there is no true mean of SERS enhancement, requiring an alternative approach to achieve reproducible quantitative results. In this study, we introduce a new method of analysis of SERS data using a cumulative distribution function (CDF). The antiviral drug tenofovir (TFV) in an aqueous matrix was quantified down to a clinically relevant concentration of 25 ng/mL using hydroxylamine-reduced silver colloids evaporated to dryness. The data presented in this study provide a rationale for the benefits of combining a novel statistical approach using CDFs with simple and inexpensive experimental techniques to increase the precision, accuracy, and analytical sensitivity of aqueous TFV quantification by SERS. TFV calibration curves generated using CDF analysis showed higher analytical sensitivity (in the form of a normalized calibration curve average slope increase of 0.25) compared to traditional SERS intensity calculations. A second aliquot of nanoparticles and analyte dried on the SERS surface followed by CDF analysis showed further analytical sensitivity with a normalized calibration curve slope increase of 0.23 and decreased variation among replicates represented by an average standard deviation decrease of 0.02 with a second aliquot. The quantitative analysis of SERS data using CDFs presented here shows promise to be a reproducible method for quantitative analysis of SERS data, a significant step toward implementing SERS as an analytical method in clinical and industrial settings.

Do Phosphodiesterase Type 5 Inhibitors Increase the Risk of Biochemical Recurrence After Radical Prostatectomy?

PURPOSE: There have been conflicting studies on the association between phosphodiesterase type 5 inhibitor (PDE5i) use and biochemical recurrence (BCR) following radical prostatectomy (RP). Our aim was to determine whether PDE5i drug exposure after RP increases the risk of BCR in patients undergoing RP. MATERIALS AND METHODS: An institutional database of prostate cancer patients treated between January 2009 and December 2020 was reviewed. BCR was defined as 2 PSA measurements greater than 0.1 ng/mL. PDE5i exposure was defined using a 0 to 3 scale, with 0 representing never use, 1 sometimes use, 2 regularly use, and 3 routinely use. The risk of BCR with any PDE5i exposure, the quantity of exposure, and the duration of PDE5i exposure were assessed by multivariable Cox proportional hazards models. RESULTS: The sample size included 4630 patients to be analyzed, with 776 patients having BCR. The median follow-up for patients without BCR was 27 (IQR 12, 49) months. Eighty-nine percent reported taking a PDE5i at any time during the first 12 months after RP, and 60% reported doing so for 6 or more months during the year after RP. There was no evidence of an increase in the risk of BCR associated with any PDE5i use (HR 1.05, 95% CI 0.84, 1.31, P = .7) or duration of PDE5i use in the first year (HR 0.98 per 1 month duration, 95% CI 0.96, 1.00, P = .055). Baseline oncologic risk was lower in patients using PDE5i, but differences between groups were small, suggesting that residual confounding is unlikely to obscure any causal association with BCR. CONCLUSIONS: Prescription of PDE5i to men after RP can be based exclusively on quality of life considerations. Patients receiving PDE5is can be reassured that their use does not increase the risk of BCR.

Diabetes Distress and Associations With Demographic and Clinical Variables: A Nationwide Population-Based Registry Study of 10,186 Adults With Type 1 Diabetes in Norway.

OBJECTIVE: To estimate diabetes distress prevalence and associations with demographic and clinical variables among adults with type 1 diabetes in Norway. RESEARCH DESIGN AND METHODS: In this nationwide population-based registry study, the 20-item Problem Areas in Diabetes (PAID-20) questionnaire was sent to 16,255 adults with type 1 diabetes. Linear regression models examined associations of demographic and clinical variables with distress. RESULTS: In total, 10,186 individuals (62.7%) completed the PAID-20, with a mean score of 25.4 (SD 18.4) and 21.7% reporting high distress. Respondents endorsed worrying about the future and complications as the most problematic item (23.0%). Female sex, younger age, non-European origin, primary education only, unemployment, smoking, continuous glucose monitoring use, more symptomatic hypoglycemia, reduced foot sensitivity, treated retinopathy, and higher HbA1c were associated with higher distress. CONCLUSIONS: Diabetes distress is common among adults with type 1 diabetes and associated with clinically relevant factors, underlining that regular care should include efforts to identify and address distress.

Capping protein regulators of actin assembly in budding yeast.

Cellular functions of actin capping protein (CP) regulators are poorly understood. Di Pietro and colleagues (https://doi.org/10.1083/jcb.202306154) shed unprecedented light on this topic using budding yeast. Two proteins with CPI (capping protein interacting) motifs recruit CP to sites of actin assembly, while a third contributes to CP turnover.

Insulin initiation in patients with type 2 diabetes is often delayed, but access to a diabetes nurse may help-insights from Norwegian general practice.

Objective: We opted to study how support staff operational capacity and diabetes competences may impact the timeliness of basal insulin-initiation in general practice patients with type 2 diabetes (T2D).Design/Setting/Outcomes: This was an observational and retrospective study on Norwegian primary care patients with T2D included from the ROSA4-dataset. Exposures were (1) support staff size, (2) staff size relative to number of GPs, (3) clinic access to a diabetes nurse and (4) share of staff with diabetes course (1 and 2 both relate to staff operational capacity, whereas 3 and 4 are both indicatory of staff diabetes competences). Outcomes were 'timely basal insulin-initiation' (primary) and 'attainment of HbA1c<7%' after insulin start-up (secondary). Associations were analyzed using multiple linear regression, and directed acyclic graphs guided statistical adjustments.Subjects: Insulin naïve patients with 'timely' (N = 294), 'postponed' (N = 219) or 'no need of' (N = 3,781) basal insulin-initiation, respectively.Results: HbA1c [median (IQR)] increased to 8.8% (IQR, 8.0, 10.2) prior to basal insulin-initiation, which reduced HbA1c to 7.3 (6.8-8.1) % by which only 35% of the subjects reached HbA1c <7%. Adjusted risk of 'timely basal insulin-initiation' was more than twofold higher if access to a diabetes nurse (OR = 2.40, [95%CI, 1.68, 3.43]), but related only vaguely to staff size (OR = 1.01, [95%CI, 1.00, 1.03]). No other staff factors related significantly to neither the primary nor the secondary outcome.Conclusion: In Norwegian general practice, insulin initiation in people with T2D may be affected by therapeutic inertia but access to a diabetes nurse may help facilitating more timely insulin start-up.

In patients with type 2 diabetes (T2D) cared for by their general practice physician (GP), insulin therapy was susceptible to therapeutic inertia.In Norwegian general practice, chance of timely basal insulin-initiation was found more than two-fold higher if the GP had access to a diabetes nurse.In contrast, the timeliness of basal insulin-initiation in general practice patients with T2D seemed unaffected by share of support staff with diabetes course and by factors indicatory of support staff overall operational capacity.In Norwegian general practice, a diabetes nurse seems to offer unique clinical benefits to the care of insulin treated patients with T2D.

Privacy Concerns Related to Data Sharing for European Diabetes Devices.

BACKGROUND: Individuals with diabetes rely on medical equipment (eg, continuous glucose monitoring (CGM), hybrid closed-loop systems) and mobile applications to manage their condition, providing valuable data to health care providers. Data sharing from this equipment is regulated via Terms of Service (ToS) and Privacy Policy documents. The introduction of the Medical Devices Regulation (MDR) and In Vitro Diagnostic Medical Devices Regulation (IVDR) in the European Union has established updated rules for medical devices, including software. OBJECTIVE: This study examines how data sharing is regulated by the ToS and Privacy Policy documents of approved diabetes medical equipment and associated software. It focuses on the equipment approved by the Norwegian Regional Health Authorities. METHODS: A document analysis was conducted on the ToS and Privacy Policy documents of diabetes medical equipment and software applications approved in Norway. RESULTS: The analysis identified 11 medical equipment and 12 software applications used for diabetes data transfer and analysis in Norway. Only 3 medical equipment (OmniPod Dash, Accu-Chek Insight, and Accu-Chek Solo) were registered in the European Database on Medical Devices (EUDAMED) database, whereas none of their respective software applications were registered. Compliance with General Data Protection Regulation (GDPR) security requirements varied, with some software relying on adequacy decisions (8/12), whereas others did not (4/12). CONCLUSIONS: The study highlights the dominance of non-European Economic Area (EEA) companies in medical device technology development. It also identifies the lack of registration for medical equipment and software in the EUDAMED database, which is currently not mandatory. These findings underscore the need for further attention to ensure regulatory compliance and improve data-sharing practices in the context of diabetes management.

Biophysical Mechanism of Allosteric Regulation of Actin Capping Protein.

Actin capping protein (CP) can be regulated by steric and allosteric mechanisms. The molecular mechanism of the allosteric regulation at a biophysical level includes linkage between the binding sites for three ligands: F-actin, Capping-Protein-Interacting (CPI) motifs, and V-1/myotrophin, based on biochemical functional studies and solvent accessibility experiments. Here, we investigated the mechanism of allosteric regulation at the atomic level using single-molecule Förster resonance energy transfer (FRET) and molecular dynamics (MD) to assess the conformational and structural dynamics of CP in response to linked-binding site ligands. In the absence of ligand, both single-molecule FRET and MD revealed two distinct conformations of CP in solution; previous crystallographic studies revealed only one. Interaction with CPI-motif peptides induced conformations within CP that bring the cap and stalk closer, while interaction with V-1 moves them away from one another. Comparing CPI-motif peptides from different proteins, we identified variations in CP conformations and dynamics that are specific to each CPI motif. MD simulations for CP alone and in complex with a CPI motif and V-1 reveal atomistic details of the conformational changes. Analysis of the interaction of CP with wild-type (wt) and chimeric CPI-motif peptides using single-molecule FRET, isothermal calorimetry (ITC) and MD simulation indicated that conformational and affinity differences are intrinsic to the C-terminal portion of the CPI motif. We conclude that allosteric regulation of CP involves changes in conformation that disseminate across the protein to link distinct binding-site functions. Our results provide novel insights into the biophysical mechanism of the allosteric regulation of CP.

Real-World Pitfalls of Analyzing Real-World Data: A Cautionary Note and Path Forward.

PURPOSE: Real-world data (RWD) are pervasive in oncology research and offer insights into clinical trends and patient outcomes. However, RWD have shortcomings, making them prone to pitfalls during survival analyses. The American Society of Clinical Oncology (ASCO) CancerLinQ Discovery (CLQD) multiple myeloma (MM) data set was used to demonstrate some common pitfalls when analyzing survival from RWD: using incorrect surrogate markers for missing data and/or classification errors, ignoring deaths at time zero, and failing to account for guarantee-time bias. METHODS: The ASCO CLQD MM data set (July 19, 2021, release) was used to compare overall survival (OS) in patients with a known versus presumed date of MM diagnosis, in patients with secondary AML (sAML) with early deaths (ie, 0 months) included versus dropped, and in patients with second primary malignancies (SPMs) matched versus unmatched to control for time-related confounding factors (ie, guarantee-time bias). Analyses were conducted using STATA Version 17.0 (College Station, TX). RESULTS: In the CLQD MM data set, 28% of patients were missing a diagnosis date. Attempts to use the presumed diagnosis date (ie, first bortezomib or lenalidomide administration) as a surrogate marker for missing diagnosis dates were not successful as median OS was significantly different in patients with a recorded versus presumed diagnosis date (107 v 40 months, hazard ratio [HR], 2.5; 95% CI, 2.39 to 2.64; P < .001). Dropping deaths within 1 month of sAML diagnosis resulted in an exaggerated median OS (46 v 39 months). OS in patients with MM with SPMs differed substantially before and after incorporation of matching methods to account for guarantee-time bias (HR, 0.73; 95% CI, 0.67 to 0.78; P < .001 before matching, HR, 1.30; 95% CI, 1.18 to 1.43; P < .001 after matching). CONCLUSION: To fully maximize the benefits of RWD in oncology research, clinicians must be aware of analytic methods that can overcome pitfalls in survival analyses.

Biophysical Mechanism of Allosteric Regulation of Actin Capping Protein.

Actin capping protein (CP) can be regulated by steric and allosteric mechanisms. The molecular mechanism of the allosteric regulation at a biophysical level includes linkage between the binding sites for three ligands: F-actin, Capping-Protein-Interacting (CPI) motifs, and V-1/myotrophin, based on biochemical functional studies and solvent accessibility experiments. Here, we investigated the mechanism of allosteric regulation at the atomic level using single-molecule Förster resonance energy transfer (FRET) and molecular dynamics (MD) to assess the conformational and structural dynamics of CP in response to linked-binding site ligands. In the absence of ligand, both single-molecule FRET and MD revealed two distinct conformations of CP in solution; previous crystallographic studies revealed only one. CPI-motif peptide association induced conformational changes within CP that propagate in one direction, while V-1 association induced conformational changes in the opposite direction. Comparing CPI-motif peptides from different proteins, we identified variations in CP conformations and dynamics that are specific to each CPI motif. MD simulations for CP alone and in complex with a CPI motif and V-1 reveal atomistic details of the conformational changes. Analysis of the interaction of CP with wildtype (wt) and chimeric CPI-motif peptides using single-molecule FRET, isothermal calorimetry (ITC) and MD simulation indicated that conformational and affinity differences are intrinsic to the C-terminal portion of the CPI-motif. We conclude that allosteric regulation of CP involves changes in conformation that disseminate across the protein to link distinct binding-site functions. Our results provide novel insights into the biophysical mechanism of the allosteric regulation of CP.

EBNA1 Inhibitors Block Proliferation of Spontaneous Lymphoblastoid Cell Lines From Patients With Multiple Sclerosis and Healthy Controls.

BACKGROUND AND OBJECTIVES: Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that establishes lifelong latency in memory B cells and has been identified as a major risk factor of multiple sclerosis (MS). B cell depletion therapies have disease-modifying benefit in MS. However, it is unclear whether this benefit is partly attributable to the elimination of EBV+ B cells. Currently, there are no EBV-specific antiviral therapies available for targeting EBV latent infection in MS and limited experimental models to study EBV in MS. METHODS: In this study, we describe the establishment of spontaneous lymphoblastoid cell lines (SLCLs) generated ex vivo with the endogenous EBV of patients with MS and controls and treated with either an Epstein-Barr virus nuclear antigen 1 (EBNA1) inhibitor (VK-1727) or cladribine, a nucleoside analog that eliminates B cells. RESULTS: We showed that a small molecule inhibitor of EBNA1, a critical regulator of the EBV life cycle, blocks the proliferation and metabolic activity of these SLCLs. In contrast to cladribine, a highly cytotoxic B cell depleting therapy currently used in MS, the EBNA1 inhibitor VK-1727 was cytostatic rather than cytotoxic and selective for EBV+ cells, while having no discernible effects on EBV- cells. We validate that VK-1727 reduces EBNA1 DNA binding at known viral and cellular sites by ChIP-qPCR. DISCUSSION: This study shows that patient-derived SLCLs provide a useful tool for interrogating the role of EBV+ B cells in MS and suggests that a clinical trial testing the effect of EBNA1 inhibitors in MS may be warranted.

Intensified follow-up of patients with type 1 diabetes and poor glycaemic control: a multicentre quality improvement collaborative based on data from the Norwegian Diabetes Register for Adults.

BACKGROUND: Patients with type 1 diabetes mellitus (T1DM) and poor glycaemic control are at high risk of developing microvascular and macrovascular complications. The aim of this study was to determine if a quality improvement collaborative (QIC) initiated by the Norwegian Diabetes Register for adults (NDR-A) could reduce the proportion of patients with T1DM with poor glycaemic control (defined as glycated haemoglobin (HbA1c)≥75 mmol/mol) and reduce mean HbA1c at participating clinics compared with 14 control clinics. METHOD: Multicentre study with controlled before and after design. Representatives of 13 diabetes outpatient clinics (n=5145 patients with T1DM) in the intervention group attended four project meetings during an 18-month QIC. They were required to identify areas requiring improvement at their clinic and make action plans. Continuous feedback on HbA1c outcomes was provided by NDR-A during the project. In total 4084 patients with type 1 diabetes attended the control clinics. RESULTS: Between 2016 and 2019, the overall proportion of patients with T1DM and HbA1c≥75 mmol/mol in the intervention group were reduced from 19.3% to 14.1% (p<0.001). Corresponding proportions in the control group were reduced from 17.3% (2016) to 14.4% (2019) (p<0.001). Between 2016 and 2019, overall mean HbA1c decreased by 2.8 mmol/mol (p<0.001) at intervention clinics compared with 2.3 mmol/mol (p<0.001) at control clinics. After adjusting for the baseline differences in glycaemic control, there were no significant differences in the overall improvement in glycaemic control between intervention and control clinics. CONCLUSIONS: The registry linked QIC did not result in a significantly greater improvement in glycaemic control at intervention clinics compared with control clinics. However, there has been a sustained improvement in glycaemic control and importantly a significant reduction in the proportion of patients with poor glycaemic control at both intervention and control clinics during and after the QIC time frame. It is possible that some of this improvement may be due to a spillover effect from the QIC.

Layered metals as polarized transparent conductors.

The quest to improve transparent conductors balances two key goals: increasing electrical conductivity and increasing optical transparency. To improve both simultaneously is hindered by the physical limitation that good metals with high electrical conductivity have large carrier densities that push the plasma edge into the ultra-violet range. Technological solutions reflect this trade-off, achieving the desired transparencies only by reducing the conductor thickness or carrier density at the expense of a lower conductance. Here we demonstrate that highly anisotropic crystalline conductors offer an alternative solution, avoiding this compromise by separating the directions of conduction and transmission. We demonstrate that slabs of the layered oxides Sr2RuO4 and Tl2Ba2CuO6+δ are optically transparent even at macroscopic thicknesses >2 µm for c-axis polarized light. Underlying this observation is the fabrication of out-of-plane slabs by focused ion beam milling. This work provides a glimpse into future technologies, such as highly polarized and addressable optical screens.