Afucosylated IgG responses in humans - structural clues to the regulation of humoral immunity.

Healthy immune responses require efficient protection without excessive inflammation. Recent discoveries on the degree of fucosylation of a human N-linked glycan at a conserved site in the immunoglobulin IgG-Fc domain might add an additional regulatory layer to adaptive humoral immunity. Specifically, afucosylation of IgG-Fc enhances the interaction of IgG with FcγRIII and thereby its activity. Although plasma IgG is generally fucosylated, afucosylated IgG is raised in responses to enveloped viruses and Plasmodium falciparum proteins expressed on infected erythrocytes, as well as during alloimmune responses. Moreover, while afucosylation can exacerbate some infectious diseases (e.g., COVID-19), it also correlates with traits of protective immunity against malaria and HIV-1. Herein we discuss the implications of IgG afucosylation for health and disease, as well as for vaccination.

Assessment of IgG-Fc glycosylation from individual RhD-specific B cell clones reveals regulation at clonal rather than clonotypic level.

The type and strength of effector functions mediated by immunoglobulin G (IgG) antibodies rely on the subclass and the composition of the N297 glycan. Glycosylation analysis of both bulk and antigen-specific human IgG has revealed a marked diversity of the glycosylation signatures, including highly dynamic patterns as well as long-term stability of profiles, yet information on how individual B cell clones would contribute to this diversity has hitherto been lacking. Here, we assessed whether clonally related B cells share N297 glycosylation patterns of their secreted IgG. We differentiated single antigen-specific peripheral IgG+ memory B cells into antibody-secreting cells and analysed Fc glycosylation of secreted IgG. Furthermore, we sequenced the variable region of their heavy chain, which allowed the grouping of the clones into clonotypes. We found highly diverse glycosylation patterns of culture-derived IgG, which, to some degree, mimicked the glycosylation of plasma IgG. Each B cell clone secreted IgG with a mixture of different Fc glycosylation patterns. The majority of clones produced fully fucosylated IgG. B cells producing afucosylated IgG were scattered across different clonotypes. In contrast, the remaining glycosylation traits were, in general, more uniform. These results indicate IgG-Fc fucosylation to be regulated at the single-clone level, whereas the regulation of other glycosylation traits most likely occurs at a clonotypic or systemic level. The discrepancies between plasma IgG and culture-derived IgG, could be caused by the origin of the B cells analysed, clonal dominance or factors from the culture system, which need to be addressed in future studies.

Revaccination of patients with systemic lupus erythematosus or rheumatoid arthritis without an initial COVID-19 vaccine response elicits seroconversion in half of the patients.

OBJECTIVES: To investigate the effect of COVID-19 mRNA revaccination (two doses) on the antibody response in patients with rheumatic diseases (RD) who were initial vaccine non-responders. Further, to examine if B-cell levels or T-cell responses before revaccination predicted seroconversion. METHODS: From a RD cohort vaccinated with the standard two-dose COVID-19 vaccinations, we enrolled cases without detectable antibody responses (n=17) and controls with detectable antibody response (n=29). Blood donors (n=32) were included as additional controls. Samples were collected before and six weeks after completed revaccination. Total antibodies and specific IgG, IgA, and IgM against SARS-CoV-2 spike protein, SARS-CoV-2 neutralising antibodies, and SARS-CoV-2 reacting CD4+ and CD8+ T-cells were measured before and after revaccination. B-cells (CD19+CD45+) were quantified before revaccination. RESULTS: Forty-seven percent of cases had detectable neutralising antibodies after revaccination. However, antibody levels were significantly lower than in controls and blood donors. Revaccination induced an antibody class switch in cases with a decrease in IgM and increase in IgG. No significant difference was observed in T-cell responses before and after revaccination between the three groups. Only 29% of cases had measurable B-cells compared to 100% of controls and blood donors. Fifty percent of revaccinated cases who seroconverted had measurable B-cells before revaccination. CONCLUSIONS: Forty-seven percent of initial non-responders seroconverted after two-dose revaccination but still had lower levels of SARS-CoV-2 antibodies compared with controls and blood donors. RD patients without a detectable serological response after the initial COVID-19 mRNA vaccine had a T-cell response similar to immunocompetent controls and blood donors.

The diagnosis of cerebral palsy in two Danish national registries: a validation study.

AIMS: To determine the quality of prospectively collected data from the highly specialized Danish Cerebral Palsy Follow-up Program (CPOP), and to establish the validity of a reported cerebral palsy (CP) diagnosis in the Danish National Patient Registry (NPR), regularly used as a proxy for neurodevelopmental disorders in epidemiological research. METHODS: We compared data from the two registries on children with registered CP, born in Denmark between 2008 and 2009, with information from medical records verified by two experienced physicians specializing in pediatric neurology. Data accuracy was estimated by completeness, correctness, and reliability. Completeness was calculated as the number of cases with correctly registered CP diagnoses divided by the total number of true CP diagnoses (similar to sensitivity). Correctness was calculated as the number of cases with correct registrations divided by the total number of cases (similar to positive predictive value). Reliability was estimated using kappa statistics. RESULTS: Registered CP diagnoses in the CPOP had high accuracy, with 94% correctness and 91% completeness. Furthermore, most key variables in the CPOP showed excellent reliability, especially variables defining the severity of the condition. In the Danish NPR, only 225 of 348 children with a noted CP diagnosis fulfilled the diagnostic criteria for CP, resulting in 65% correctness. CONCLUSIONS: Danish CPOP data are a valid source for epidemiological research. Conversely, a noted CP diagnosis in the Danish NPR was, at best, correct in only two out of three patients.

Differentiating between activation via the lectin or the classical complement pathway in patients with systemic lupus erythematosus.

Complement activation is a hallmark of systemic lupus erythematosus (SLE) and can proceed through the classical (CP), lectin (LP), or alternative pathway (AP). When managing SLE patients, pathway-specific complement activation is rarely monitored as clinical assays are unavailable. In this study, we aim to differentiate between CP- or LP-mediated complement activation in SLE patients by quantifying pathway-specific protein complexes, namely C1s/C1-inhibitor (C1-INH) (CP-specific activation) and MASP-1/C1-INH (LP-specific activation). Levels for both complexes were assessed in 156 SLE patients and 50 controls using two newly developed ELISAs. We investigated whether pathway-specific complement activation was associated with disease activity and lupus nephritis (LN). Disease activity stratification was performed using SLEDAI scores assessed at inclusion. C1s/C1-INH concentrations were significantly increased in active SLE patients (SLEDAI ≥6) when compared with SLE patients with low disease activity (SLEDAI <6, P < 0.01) and correlated with SLEDAI score (r = .29, P < 0.01). In active LN, MASP-1/C1-INH plasma concentrations were significantly increased compared with nonactive LN (P = 0.02). No differences in MASP-1/C1-INH plasma concentrations were observed between active SLE patients and patients with low disease activity (P = 0.11) nor did we observe a significant correlation with disease activity (r = 0.12, P = 0.15). Our data suggest that the CP and the LP are activated in SLE. The CP is activated in active SLE disease, whereas activation of the LP might be more specific to disease manifestations like LN. Our results warrant further research into specific complement pathway activation in SLE patients to potentially improve specific-targeted and tailored-treatment approaches.

Rituximab-treated rheumatic patients: B cells predict seroconversion after COVID-19 boost or revaccination in initial vaccine non-responders.

OBJECTIVES: To investigate the effect of either a booster vaccine (one dose) or revaccination (two doses 3 weeks apart) on the antibody response to the COVID-19 mRNA vaccines in patients with rheumatic disease (RD) treated with rituximab (RTX) who had not produced vaccine-reactive antibodies after the initial two vaccine doses. Further, to examine if B cell levels in peripheral blood predicted seroconversion. METHODS: We included 91 RTX-treated RD patients previously vaccinated against COVID-19. Patients were offered revaccination or a single booster vaccination with an mRNA vaccine. Serum total antibodies against SARS-CoV-2 spike protein were measured before and 6 weeks after the last vaccine dose. B cells (CD19+CD45+) were measured by flow cytometry at inclusion. RESULTS: Of RD patients with undetectable SARS-CoV-2 antibody levels before inclusion, seroconversion was seen in 38% 6 weeks after the booster dose and 32% after revaccination. Patients receiving revaccination had significantly higher antibody levels than patients receiving a booster dose (P < 0.001). In both univariate and multivariate logistic regression analysis, only B cells higher than 10/µl before boost or revaccination were associated with seroconversion (P = 0.009 and P = 0.01, respectively). Seroconversion was independent of age, gender, diagnosis, cumulative RTX dose, RTX treatment time and time since last RTX treatment. CONCLUSION: Continuously impaired humoral response to mRNA vaccines was found in most RTX-treated patients after a booster dose or revaccination. Seroconversion was observed in approximately one-third of the patients. Measurable B cells before boosting or revaccination was the strongest predictor of antibody response after boost or revaccination.

Dose-Response of Myofibrillar Protein Synthesis To Ingested Whey Protein During Energy Restriction in Overweight Postmenopausal Women: A Randomized, Controlled Trial.

BACKGROUND: Diet-induced weight loss is associated with a decline in lean body mass, as mediated by an impaired response of muscle protein synthesis (MPS). The dose-response of MPS to ingested protein, with or without resistance exercise, is well characterized during energy balance but limited data exist under conditions of energy restriction in clinical populations. OBJECTIVE: To determine the dose-response of MPS to ingested whey protein following short-term diet-induced energy restriction in overweight, postmenopausal, women at rest and postexercise. DESIGN: Forty middle-aged (58.6±0.4 y), overweight (BMI: 28.6±0.4), postmenopausal women were randomly assigned to 1 of 4 groups: Three groups underwent 5 d of energy restriction (∼800 kcal/d). On day 6, participants performed a unilateral leg resistance exercise bout before ingesting either a bolus of 15g (ERW15, n = 10), 35g (ERW35, n = 10) or 60g (ERW60, n = 10) of whey protein. The fourth group (n = 10) ingested a 35g whey protein bolus after 5 d of an energy balanced diet (EBW35, n = 10). Myofibrillar fractional synthetic rate (FSR) was calculated under basal, fed (FED) and postexercise (FED-EX) conditions by combining an L-[ring-13C6] phenylalanine tracer infusion with the collection of bilateral muscle biopsies. RESULTS: Myofibrillar FSR was greater in ERW35 (0.043±0.003%/h, P = 0.013) and ERW60 (0.042±0.003%/h, P = 0.026) than ERW15 (0.032 ± 0.003%/h), with no differences between ERW35 and ERW60 (P = 1.000). Myofibrillar FSR was greater in FED (0.044 ± 0.003%/h, P < 0.001) and FED-EX (0.048 ± 0.003%/h, P < 0.001) than BASAL (0.027 ± 0.003%/h), but no differences were detected between FED and FED-EX (P = 0.732) conditions. No differences in myofibrillar FSR were observed between EBW35 (0.042 ± 0.003%/h) and ERW35 (0.043 ± 0.003%/h, P = 0.744). CONCLUSION: A 35 g dose of whey protein, ingested with or without resistance exercise, is sufficient to stimulate a maximal acute response of MPS following short-term energy restriction in overweight, postmenopausal women, and thus may provide a per serving protein recommendation to mitigate muscle loss during a weight loss program. TRIAL REGISTRY: clinicaltrials.gov (ID: NCT03326284).

A Fbxo48 inhibitor prevents pAMPKα degradation and ameliorates insulin resistance.

The adenosine monophosphate (AMP)-activated protein kinase (Ampk) is a central regulator of metabolic pathways, and increasing Ampk activity has been considered to be an attractive therapeutic target. Here, we have identified an orphan ubiquitin E3 ligase subunit protein, Fbxo48, that targets the active, phosphorylated Ampkα (pAmpkα) for polyubiquitylation and proteasomal degradation. We have generated a novel Fbxo48 inhibitory compound, BC1618, whose potency in stimulating Ampk-dependent signaling greatly exceeds 5-aminoimidazole-4-carboxamide-1-ß-ribofuranoside (AICAR) or metformin. This compound increases the biological activity of Ampk not by stimulating the activation of Ampk, but rather by preventing activated pAmpkα from Fbxo48-mediated degradation. We demonstrate that, consistent with augmenting Ampk activity, BC1618 promotes mitochondrial fission, facilitates autophagy and improves hepatic insulin sensitivity in high-fat-diet-induced obese mice. Hence, we provide a unique bioactive compound that inhibits pAmpkα disposal. Together, these results define a new pathway regulating Ampk biological activity and demonstrate the potential utility of modulating this pathway for therapeutic benefit.

Children in Denmark with cerebral palsy rarely complete elementary school.

AIM: To investigate how children with cerebral palsy (CP) perform in the Danish school system and which factors are associated with school performance. METHOD: This was a population-based cohort study including 463 126 children born from 1997 to 2003. Data were extracted from seven national registries. The study encompassed 818 children with CP (483 [59.0%] males, 335 [41.0%] females) and 417 731 without CP (214 535 [51.4%] males, 203 196 [48.6%] females). We evaluated two primary outcomes: not completing 10 years of elementary school, defined as attending fewer than eight final mandatory exams; and grade point averages (GPAs). Mann-Whitney U tests were used to analyse differences in GPAs and logistic regressions were used to calculate odds ratios (ORs). RESULTS: Among children with and without CP, 62.6% and 12.4% did not complete elementary school respectively (OR = 11.85 [10.28-13.66]). Additionally, children with CP who attended all final exams achieved lower overall GPAs than children without CP (6.6 vs 7.3, p = 0.001). In children with CP, comorbidities, maternal education, severity of motor impairments, and intellectual deficits were associated with increased odds of not completing elementary school. Notably, one-third of children with CP with apparent normal intelligence did not complete school, despite special educational measures. INTERPRETATION: Danish children with CP rarely complete elementary school despite initiatives for a more supportive educational system. The complexity of individual needs in children with CP may be challenging for an inclusive school environment. WHAT THIS PAPER ADDS: Children with cerebral palsy (CP) have a high risk of not completing elementary school. Children with CP achieve lower overall grades than children without CP. Motor impairment, comorbidities, and maternal education are associated with poor school performance. Intellectual impairment is the most important predictor of poor school performance.

Surface temperature determination using long range thermal emission spectroscopy based on a first order scanning Fabry-Pérot interferometer.

Determination of the surface temperature of different materials based on thermographic imaging is a difficult task as the thermal emission spectrum is both temperature and emissivity dependent. Without prior knowledge of the emissivity of the object under investigation, it makes up a temperature-emissivity underdetermined system. This work demonstrates the possibility of recognizing specific materials from hyperspectral thermal images (HSTI) in the wavelength range from 8-14 µm. The hyperspectral images were acquired using a microbolometer sensor array in combination with a scanning 1st order Fabry-Pérot interferometer acting as a bandpass filter. A logistic regression model was used to successfully differentiate between polyimide tape, sapphire, borosilicate glass, fused silica, and alumina ceramic at temperatures as low as 34.0 ± 0.05 °C. Each material was recognized with true positive rates above 94% calculated from individual pixel spectra. The surface temperature of the samples was subsequently predicted using pre-fitted partial least squares (PLS) models, which predicted all surface temperature values with a common root mean square error (RMSE) of 1.10 °C and thereby outperforming conventional thermography. This approach paves the way for a practical solution to the underdetermined temperature-emissivity system.

The effect of gestational age on major neurodevelopmental disorders in preterm infants.

BACKGROUND: Preterm infants have an increased risk of neurodevelopmental disorders. We established a direct quantitative comparison of the association between the degree of prematurity and three different neurodevelopmental disorders. METHODS: In this cohort study, we combined data from 995,498 children in the Danish Medical Birth Register, from birth years 1997-2013, with information on cerebral palsy, epilepsy, and special educational needs. We estimated the gestational week-specific prevalence and risk for each of the disorders. RESULTS: The risk ratio of cerebral palsy at gestational weeks 21-24, compared to term birth, was more than ten times higher than for the two other disorders. The prevalence of epilepsy and special educational needs declined almost parallel, with 9.2% (4.6%-13.5%) and 12.5% (11.2%-13.7%), respectively, per week of gestation toward term birth. Cerebral palsy did not decline similarly: from gestational weeks 21-24 until week 29 the prevalence declined insignificantly by 0.6% (-11.1%-11.0%) per week; whereas from week 29 until term, the prevalence declined markedly by 36.7% (25.9%-45.9%) per week. CONCLUSIONS: The prevalence and risk of cerebral palsy are affected differently by the degree of prematurity compared with epilepsy and special educational needs, possibly reflecting important differences in cerebral pathophysiology. IMPACT: For each week of gestation toward term birth, there was a clear log-linear decline in the prevalence of early childhood epilepsy and special educational needs. In contrast, the risk of cerebral palsy was high at the earliest gestational age, and the prevalence did not decline significantly until gestational week 29, from where it declined notably by nearly 40% for each week of gestation until term birth. Our results indicate important differences in the pathophysiological processes that associate preterm birth with these three neurodevelopmental disorders.

Declining prevalence of cerebral palsy in children born at term in Denmark.

AIM: To investigate reasons for the declining prevalence of cerebral palsy (CP) in children born at term in Denmark by evaluating obstetric and neonatal factors associated with CP, and their changes over time. METHOD: In this cohort study, we included 987 495 children (504 600 [51.1%] males and 482 895 [48.9%] females) born after 37 completed gestational weeks during birth years 1997 to 2013. Risk ratios of CP for each factor were calculated with log-binominal regression analyses. Significant factors were evaluated concerning their development in prevalence over time. RESULTS: In the antenatal period, there were significant associations with an increased risk of CP and high maternal body mass index (BMI), smoking during pregnancy, nulliparity, male sex, gestational age, and low birthweight. In the study period, fewer females smoked during pregnancy and fewer children were born post-term, dropping from 22.6% to 11.4% and 9.4% to 2.5% respectively. Conversely, the proportion of females with high BMI increased. Most significant risk factors were found in the neonatal period, with an increase in children with diagnosed birth defects and children admitted to neonatal care. INTERPRETATION: Reasons for the declining prevalence of CP appear to be multifactorial and likely include the decline in maternal smoking and children born post-term along with centralization and advances in neonatal treatment.

Kelch-like protein 42 is a profibrotic ubiquitin E3 ligase involved in systemic sclerosis.

Systemic scleroderma (SSc) is an autoimmune disease that affects over 2.5 million people globally. SSc results in dysfunctional connective tissues with excessive profibrotic signaling, affecting skin, cardiovascular, and particularly lung tissue. Over three-quarters of individuals with SSc develop pulmonary fibrosis within 5 years, the main cause of SSc mortality. No approved medicines to manage lung SSc currently exist. Recent research suggests that profibrotic signaling by transforming growth factor ß (TGF-ß) is directly tied to SSc. Previous studies have also shown that ubiquitin E3 ligases potently control TGF-ß signaling through targeted degradation of key regulatory proteins; however, the roles of these ligases in SSc-TGF-ß signaling remain unclear. Here we utilized primary SSc patient lung cells for high-throughput screening of TGF-ß signaling via high-content imaging of nuclear translocation of the profibrotic transcription factor SMAD family member 2/3 (SMAD2/3). We screened an RNAi library targeting ubiquitin E3 ligases and observed that knockdown of the E3 ligase Kelch-like protein 42 (KLHL42) impairs TGF-ß-dependent profibrotic signaling. KLHL42 knockdown reduced fibrotic tissue production and decreased TGF-ß-mediated SMAD activation. Using unbiased ubiquitin proteomics, we identified phosphatase 2 regulatory subunit B'ϵ (PPP2R5ϵ) as a KLHL42 substrate. Mechanistic experiments validated ubiquitin-mediated control of PPP2R5ϵ stability through KLHL42. PPP2R5ϵ knockdown exacerbated TGF-ß-mediated profibrotic signaling, indicating a role of PPP2R5ϵ in SSc. Our findings indicate that the KLHL42-PPP2R5ϵ axis controls profibrotic signaling in SSc lung fibroblasts. We propose that future studies could investigate whether chemical inhibition of KLHL42 may ameliorate profibrotic signaling in SSc.

Separating the contributions of SLC26A9 and CFTR to anion secretion in primary human bronchial epithelia.

Aberrant anion secretion across the bronchial epithelium is associated with airway disease, most notably in cystic fibrosis. Although the cystic fibrosis transmembrane conductance regulator (CFTR) is recognized as the primary source of airway anion secretion, alternative anion transport mechanisms play a contributing role. An alternative anion transporter of growing interest is SLC26A9, a constitutively active chloride channel that has been shown to interact with CFTR and may also contribute to bicarbonate secretion. Interest in SLC26A9 has been fueled by genome-wide association studies that suggest it is a significant modifier of CF disease severity. Despite this growing evidence that SLC26A9 plays an important role in the airway, its presence and function in bronchial epithelia remain poorly understood, in part, because its activity is difficult to separate from the activity of CFTR. Here, we present results using primary human bronchial epithelia (HBE) from multiple patient sources to confirm that SLC26A9 mRNA is present in HBE and that its constitutive channel activity is unaffected by knockdown of CFTR. Furthermore, SLC26A9 and CFTR show differential responses to common inhibitors of anion secretion. Finally, we assess the impact of bicarbonate on the activity of SLC26A9 and CFTR. These results confirm that SLC26A9 is the primary source of constitutive anion secretion across HBE, and should inform future studies focused on activation of SLC26A9 as an alternative anion channel in CF. These results should provide a strong foundation to investigate how single-nucleotide polymorphisms in SLC26A9 modulate airway disease.

Improved correction of F508del-CFTR biogenesis with a folding facilitator and an inhibitor of protein ubiquitination.

A growing number of diseases are linked to the misfolding of integral membrane proteins, and many of these proteins are targeted for ubiquitin-proteasome-dependent degradation. One such substrate is a mutant form of the Cystic Fibrosis Transmembrane Conductance Regulator (F508del-CFTR). Protein folding "correctors" that repair the F508del-CFTR folding defect have entered the clinic, but they are unlikely to protect the entire protein from degradation. To increase the pool of F508del-CFTR protein that is available for correction by existing treatments, we determined a structure-activity relationship to improve the efficacy and reduce the toxicity of an inhibitor of the E1 ubiquitin activating enzyme that facilitates F508del-CFTR maturation. A resulting lead compound lacked measurable toxicity and improved the ability of an FDA-approved corrector to augment F508del-CFTR folding, transport the protein to the plasma membrane, and maintain its activity. These data support a proof-of-concept that modest inhibition of substrate ubiquitination improves the activity of small molecule correctors to treat CF and potentially other protein conformational disorders.

Solvent-Controlled Self-Assembled Oligopyrrolic Receptor.

We report a fully organic pyridine-tetrapyrrolic U-shaped acyclic receptor 10, which prefers a supramolecular pseudo-macrocyclic dimeric structure (10)2 in a less polar, non-coordinating solvent (e.g., CHCl3). Conversely, when it is crystalized from a polar, coordinating solvent (e.g., N,N-dimethylformamide, DMF), it exhibited an infinite supramolecular one-dimensional (1D) "zig-zag" polymeric chain, as inferred from the single-crystal X-ray structures. This supramolecular system acts as a potential receptor for strong acids, e.g., p-toluenesulfonic acid (PTSA), methane sulfonic acid (MSA), H2SO4, HNO3, and HCl, with a prominent colorimetric response from pale yellow to deep red. The receptor can easily be recovered from the organic solution of the host-guest complex by simple aqueous washing. It was observed that relatively stronger acids with pKa < -1.92 in water were able to interact with the receptor, as inferred from 1H NMR titration in tetrahydrofuran-d8 (THF-d8) and ultraviolet-visible (UV-vis) spectroscopic titrations in anhydrous THF at 298 K. Therefore, this new dynamic supramolecular receptor system may have potentiality in materials science research.

The RING-type E3 ligase RNF186 ubiquitinates Sestrin-2 and thereby controls nutrient sensing.

Nutrient sensing is a critical cellular process controlling metabolism and signaling. mTOR complex 1 (mTORC1) is the primary signaling hub for nutrient sensing and, when activated, stimulates anabolic processes while decreasing autophagic flux. mTORC1 receives nutrient status signals from intracellular amino acid sensors. One of these sensors, Sestrin-2, functions as an intracellular sensor of cytosolic leucine and inhibitor of mTORC1 activity. Genetic studies of Sestrin-2 have confirmed its critical role in regulating mTORC1 activity, especially in the case of leucine starvation. Sestrin-2 is known to be transcriptionally controlled by several mechanisms; however, the post-translational proteolytic regulation of Sestrin-2 remains unclear. Here, we explored how Sestrin-2 is regulated through the ubiquitin proteasome system. Using an unbiased screening approach of an siRNA library targeting ubiquitin E3 ligases, we identified a RING-type E3 ligase, ring finger protein 186 (RNF186), that critically mediates the Sestrin-2 ubiquitination and degradation. We observed that RNF186 and Sestrin-2 bind each other through distinct C-terminal motifs and that Lys-13 in Sestrin-2 is a putative ubiquitin acceptor site. RNF186 knockdown increased Sestrin-2 protein levels and decreased mTORC1 activation. These results reveal a new mechanism of E3 ligase control of mTORC1 activity through the RNF186-Sestrin-2 axis, suggesting that RNF186 inhibition may be a potential strategy to increase levels of the mTORC1 inhibitor Sestrin-2.

Semiconducting Supramolecular Organic Frameworks Assembled from a Near-Infrared Fluorescent Macrocyclic Probe and Fullerenes.

We report here a new extended tetrathiafulvalene (exTTF)-porphyrin scaffold, 2, that acts as a ball-and-socket receptor for C60 and C70. Supramolecular interactions between 2 and these fullerenes serve to stabilize 3D supramolecular organic frameworks (SOFs) in the solid state formally comprising peapod-like linear assemblies. The SOFs prepared via self-assembly in this way act as "tunable functional materials", wherein the complementary geometry of the components and the choice of fullerene play crucial roles in defining the conductance properties. The highest electrical conductivity (σ = 1.3 × 10-8 S cm-1 at 298 K) was observed in the case of the C70-based SOF. In contrast, low conductivity was seen for the SOF based on pristine 2 (σ = 5.9 × 10-11 S cm-1 at 298 K). The conductivity seen for the C70-based SOF approaches that seen for other TTF- and fullerene-based supramolecular materials despite the fact that the present systems are metal-free and constructed entirely from neutral building blocks. Transient absorption spectroscopic measurements corroborated the formation of charge-transfer states (i.e., 2δ+/C60δ- and 2δ+/C70δ-, respectively) rather than fully charge separated states (i.e., 2•+/C60•- and 2•+/C70•-, respectively) both in solution (toluene and benzonitrile) and in the solid state at 298 K. Such findings are considered consistent with an ability to transfer charges effectively over long distances within the present SOFs, rather than, for example, the formation of energetically trapped ionic species.

Benefits and Harm of Treatment with P2Y12 Inhibitors beyond 12 Months in Patients with Coronary Artery Disease.

The trade-off between the benefits and harm of long-term (> 12 months) treatment with P2Y12 inhibitors in patients with coronary artery disease (CAD) after percutaneous coronary intervention (PCI) remains controversial. This review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. PubMed and Embase were searched without time restrictions to identify randomized controlled trials comparing > 12-month P2Y12 inhibition versus ≤ 12-month treatment in patients with acute coronary syndrome (ACS) or stable CAD undergoing PCI. A qualitative assessment was performed using the assessment tool from the National Heart, Lung, and Blood Institute of the National Institutes of Health. We performed a meta-analysis of the following endpoints: primary outcome (primarily major cardiovascular events), all-cause death, and major bleeding. Eight trials, comprising 40,218 patients, were included. Five studies were rated "good," two studies "fair," and one study "poor." The meta-analysis showed that > 12-month P2Y12 inhibition significantly reduced the primary outcomes compared with ≤ 12-month treatment (hazard ratio [HR]: 0.85; 95% confidence interval (CI): 0.75-0.97; p = 0.01). No significant difference was demonstrated between groups in all-cause death (HR: 1.02; 95% CI: 0.76-1.36; p = 0.91) or major bleedings (HR: 1.26; 95% CI: 0.93-1.70; p = 0.14). I 2 test showed low to moderate heterogeneity among the included studies (21.6-62.3%). This systematic review and meta-analysis therefore demonstrates a reduction in major cardiovascular events during extended P2Y12-inhibitor treatment beyond 12 months compared with ≤ 12 months in patients with ACS or stable CAD undergoing PCI. There was no significant difference in all-cause death or major bleedings.

Platelet microRNA expression and association with platelet maturity and function in patients with essential thrombocythemia.

Essential thrombocythemia (ET) is characterized by persistently elevated platelet counts and an increased risk of thromboembolic events. Dysregulated expression of small noncoding microRNAs (miRNAs) have been shown in ET and may influence platelet maturity and function in ET patients. In this study, we included 22 ET patients and 19 healthy controls to investigate the expression of 12 platelet miRNAs previously reported to be dysregulated in ET. Further, we investigated the correlation between the expression of selected miRNAs and platelet maturity and platelet function. Total RNA was isolated from platelets, and expression analyses were performed using TaqMan quantitative PCR (qPCR). Mean platelet volume (MPV) and immature platelet count and -fraction (IPC and IPF) were measured using the Sysmex XE-5000 automated haematology system. Platelet function was investigated by multiple electrode aggregometry (agonists: arachidonic acid (AA), thrombin-receptor-activating-peptide (TRAP) and adenosine diphosphate (ADP)), while platelet activation was determined by multi-colour flow cytometry (antibodies: bound-fibrinogen, CD63 and P-selectin (CD62p), agonists: AA, TRAP and ADP). We showed that miR-9 and miR-490 were significantly upregulated in ET patients compared with healthy controls (p-values < 0.01), while miR-10a, miR-28, miR-126, miR-155, miR-221, miR-222, miR-223 and miR-431 were significantly downregulated in ET patients (all p-values < 0.001). A significant positive correlation was observed between miR-431 and MPV, IPC and IPF (all p-values < 0.05). The expression of miR-126 was negatively correlated with platelet aggregation induced by AA and TRAP (p < 0.05). In addition, we found the expression of miR-9 and miR-490 to be negatively correlated with the percentage of fibrinogen-, CD63- and P-selectin- positive platelets using TRAP as agonist (p < 0.05). In conclusion, our data indicate that platelet microRNAs may play a role in ET and that specific microRNAs are correlated with platelet maturity and platelet function.