Quantification of Binding of Small Molecules to Native Proteins Overexpressed in Living Cells.

The affinity and selectivity of small molecules for proteins drive drug discovery and development. We report a fluorescent probe cellular binding assay (FPCBA) for determination of these values for native (untagged) proteins overexpressed in living cells. This method uses fluorophores such as Pacific Blue (PB) linked to cell-permeable protein ligands to generate probes that rapidly and reversibly equilibrate with intracellular targets, as established by kinetic assays of cellular uptake and efflux. To analyze binding to untagged proteins, an internal ribosomal entry site (IRES) vector was employed that allows a single mRNA to encode both the protein target and a separate orthogonal fluorescent protein (mVenus). This enabled cellular uptake of the probe to be correlated with protein expression by flow cytometry, allowing measurement of cellular dissociation constants (Kd) of the probe. This approach was validated by studies of the binding of allosteric activators to eight different Protein Kinase C (PKC) isozymes. Full-length PKCs expressed in transiently transfected HEK293T cells were used to measure cellular Kd values of a probe comprising PB linked to the natural product phorbol via a carbamate. These values were further used to determine competitive binding constants (cellular Ki values) of the nonfluorescent phorbol ester PDBu and the anticancer agent bryostatin 1 for each isozyme. For some PKC-small molecule pairs, these cellular Ki values matched known biochemical Ki values, but for others, altered selectivity was observed in cells. This approach can facilitate quantification of interactions of small molecules with physiologically relevant native proteins.

N-substituted-4-(pyridin-4-ylalkyl)piperazine-1-carboxamides and related compounds as Leishmania CYP51 and CYP5122A1 inhibitors.

CYP5122A1, an enzyme involved in sterol biosynthesis in Leishmania, was recently characterized as a sterol C4-methyl oxidase. Screening of a library of compounds against CYP5122A1 and CYP51 from Leishmania resulted in the identification of two structurally related classes of inhibitors of these enzymes. Analogs of screening hit N-(3,5-dimethylphenyl)-4-(pyridin-4-ylmethyl)piperazine-1-carboxamide (4a) were generally strong inhibitors of CYP51 but were less potent against CYP5122A1 and typically displayed weak inhibition of L. donovani promastigote growth. Analogs of screening hit N-(4-(benzyloxy)phenyl)-4-(2-(pyridin-4-yl)ethyl)piperazine-1-carboxamide (18a) were stronger inhibitors of both CYP5122A1 and L. donovani promastigote proliferation but also remained selective for inhibition of CYP51. Two compounds in this series, N-(4-((3,5-bis(trifluoromethyl)benzyl)oxy)phenyl)-4-(2-(pyridin-4-yl)ethyl)piperazine-1-carboxamide (18e) and N-(4-((3,5-di-tert-butylbenzyl)oxy)phenyl)-4-(2-(pyridin-4-yl)ethyl)piperazine-1-carboxamide (18i) showed modest selectivity for inhibiting L. donovani promastigote proliferation compared to J774 macrophages and were effective against intracellular L. donovani with EC50 values in the low micromolar range. Replacement of the 4-pyridyl ring present in 18e with imidazole resulted in a compound (4-(2-(1H-imidazol-1-yl)ethyl)-N-(4-((3,5-bis(trifluoromethyl)benzyl)oxy)phenyl)piperazine-1-carboxamide, 18p) with approximately fourfold selectivity for CYP5122A1 over CYP51 that inhibited both enzymes with IC50 values ≤ 1 µM, although selective potency against L. donovani promastigotes was lost. Compound 18p also inhibited the proliferation of L. major promastigotes and caused the accumulation of 4-methylated sterols in L. major membranes, indicating that this compound blocks sterol demethylation at the 4-position in Leishmania parasites. The molecules described here may therefore be useful for the future identification of dual inhibitors of CYP51 and CYP5122A1 as potential antileishmanial drug candidates and as probes to shed further light on sterol biosynthesis in Leishmania and related parasites.

Development and validation of an early acute kidney injury risk prediction model for patients with sepsis in emergency departments.

In this study, we aimed to develop and validate a nomogram to predicting the risk of sepsis-associated acute kidney injury (SA-AKI) in patients admitted to emergency departments (EDs). We randomly divided a retrospective dataset of 391 patients with sepsis into a 294-person training cohort and a 97-person validation cohort, and developed three predictive models using multivariate logistic regression analysis and clinical insight. No difference was observed between the three models using the DeLong test and Model 3 was selected as the risk prediction model based on the principle of least inclusion indicators. The use of vasopressor drugs, patient age, platelet count, procalcitonin, and D-dimer levels were included. The training and validation cohorts had a consistency index of 0.832 and 0.866, respectively, indicating high accuracy and stability in predicting SA-AKI risk. The area under the receiver operating characteristic curve was 0.832, showing excellent discrimination. The calibration curves for the training and validation cohorts showed excellent calibration. The decision curve and clinical impact curve analyses showed that the net clinical benefit of using the nomogram was greatest over a probability threshold of 0.05-0.90. In addition, the model showed moderate validity in predicting the 30-day survival and the incidence of major adverse renal events within 30 days. The nomogram developed for SA-AKI risk assessment in patients in EDs showed good discriminability and clinical utility. It can provide a theoretical basis for emergency physicians to prevent SA-AKI.

Efficacy of Posterior Scleral Reinforcement in Treating Anisometropic Amblyopia Associated With Unilateral High Myopia in Children.

The aim of this study is to examine the effectiveness and safety of posterior scleral reinforcement (PSR) for treating anisometropic amblyopia and myopia associated with high myopia in children. Children with unilateral high myopia (uHM) and anisometropic amblyopia who underwent PSR in their highly myopic eyes were included in this study. The treated eye was designated as the PSR group, whereas the healthier eye served as the control, and paired comparisons were made. Thirteen patients (13 eyes in each group) were included, with an average age of 5.85±2.08 years. Cross-sectional comparisons between the groups showed that within 2 to 3 years after surgery, the PSR group had a significantly greater increase in best-corrected visual acuity per month compared with the control group (P=0.03), a significantly lower increase in axial length per month (P=0.007), and a significantly smaller absolute value of the increase in SE per month (P=0.002). Temporal longitudinal comparisons within the PSR group indicated that the increase in axial length per month 2 to 3 years after surgery was significantly slower than 1 year before surgery (P=0.045). The increase in best-corrected visual acuity per month was higher than 1 year before surgery, although not significantly (P=0.859). PSR is a safe and effective treatment for refractory anisometropic amblyopia caused by uHM, and for controlling axial length and the degree of myopia.

Effect and safety posterior scleral reinforcement on controlling myopia in children: a meta-analysis.

PURPOSE: The aim of this meta-analysis was to assess the efficacy of posterior scleral reinforcement (PSR) on the control of pediatric myopia. Electronic databases were systematically searched. METHODS: Standardized mean differences (SMDs) of outcomes were calculated. Eight studies with 357 patients with pediatric myopia were included. The SMD for the increase in mean axial length (AL) in the PSR and control group was - 1.19 (95% CI - 1.71, - 0.68). RESULTS: The SMD for decrease of best-corrected visual acuity (BCVA) LogMAR in the PSR and control group was 0.85 (95% CI 0.28, 1.43). The SMD for change in intraocular pressure (IOP) at the time of surgery and at the end of the follow-up period in the PSR group was - 0.01 (95% CI - 0.48, 0.47). CONCLUSION: This meta-analysis indicates that PSR surgery may be an effective therapeutic strategy to control the progression of myopia in childhood with acceptable adverse effects.

Validation of the novel GLAS algorithm as an aid in the detection of liver fibrosis and cirrhosis based on GP73, LG2m, age, and sex.

BACKGROUND: Diagnosis of liver disease at earlier stages can improve outcomes and reduce the risk of progression to malignancy. Liver biopsy is the gold standard for diagnosis of liver disease, but is invasive and sample acquisition errors are common. Serum biomarkers for liver function and fibrosis, combined with patient factors, may allow for noninvasive detection of liver disease. In this pilot study, we tested and validated the performance of an algorithm that combines GP73 and LG2m serum biomarkers with age and sex (GLAS) to differentiate between patients with liver disease and healthy individuals in two independent cohorts. METHODS: To develop the algorithm, prototype immunoassays were used to measure GP73 and LG2m in residual serum samples collected between 2003 and 2016 from patients with staged fibrosis and cirrhosis of viral or non-viral etiology (n = 260) and healthy subjects (n = 133). The performance of five predictive models using combinations of age, sex, GP73, and/or LG2m from the development cohort were tested. Residual samples from a separate cohort with liver disease (fibrosis, cirrhosis, or chronic liver disease; n = 395) and healthy subjects (n = 106) were used to validate the best performing model. RESULTS: GP73 and LG2m concentrations were higher in patients with liver disease than healthy controls and higher in those with cirrhosis than fibrosis in both the development and validation cohorts. The best performing model included both GP73 and LG2m plus age and sex (GLAS algorithm), which had an AUC of 0.92 (95% CI: 0.90-0.95), a sensitivity of 88.8%, and a specificity of 75.9%. In the validation cohort, the GLAS algorithm had an estimated an AUC of 0.93 (95% CI: 0.90-0.95), a sensitivity of 91.1%, and a specificity of 80.2%. In both cohorts, the GLAS algorithm had high predictive probability for distinguishing between patients with liver disease versus healthy controls. CONCLUSIONS: GP73 and LG2m serum biomarkers, when combined with age and sex (GLAS algorithm), showed high sensitivity and specificity for detection of liver disease in two independent cohorts. The GLAS algorithm will need to be validated and refined in larger cohorts and tested in longitudinal studies for differentiating between stable versus advancing liver disease over time.

Pre-analytical considerations in the development of a prototype SARS-CoV-2 antigen ARCHITECT automated immunoassay.

OBJECTIVES: To evaluate pre-analytical challenges related to high-volume central laboratory SARS-CoV-2 antigen testing with a prototype qualitative SARS-CoV-2 antigen immunoassay run on the automated Abbott ARCHITECT instrument. METHODS: Contrived positive and negative specimens and de-identified nasal and nasopharyngeal specimens in transport media were used to evaluate specimen and reagent on-board stability, assay analytical performance and interference, and clinical performance. RESULTS: TCID50/mL values were similar for specimens in various transport media. Inactivated positive clinical specimens and viral lysate (USA-WA1/2020) were positive on the prototype immunoassay. Within-laboratory imprecision was ≤0.10 SD (<1.00 S/C) with a ≤10% CV (≥1.00 S/C). Assay reagents were stable on board the instrument for 14 days. No high-dose hook effect was observed with a SARS-CoV-2 stock of Ct 13.0 (RLU>1.0 × 106). No interference was observed from mucin, whole blood, 12 drugs, and more than 20 cross-reactants. While specimen stability was limited at room temperature for specimens with or without viral inactivation, a single freeze/thaw cycle or long-term storage (>30 days) at -20 °C did not adversely impact specimen stability or assay performance. Specificity of the prototype SARS-CoV-2 antigen immunoassay was ≥98.5% and sensitivity was ≥89.5% across two ARCHITECT instruments. Assay sensitivity was inversely correlated with Ct and was similar to that reported for the Roche Elecsys® SARS-CoV-2 Ag immunoassay. CONCLUSIONS: The prototype SARS-CoV-2 antigen ARCHITECT immunoassay is sensitive and specific for detection of SARS-CoV-2 in nasal and nasopharyngeal specimens. Endogenous proteases in mucus may degrade the target antigen, which limits specimen storage and transport times and complicates assay workflow.

Contrastive pre-training and linear interaction attention-based transformer for universal medical reports generation.

Interpreting medical images such as chest X-ray images and retina images is an essential step for diagnosing and treating relevant diseases. Proposing automatic and reliable medical report generation systems can reduce the time-consuming workload, improve efficiencies of clinical workflows, and decrease practical variations between different clinical professionals. Many recent approaches based on image-encoder and language-decoder structure have been proposed to tackle this task. However, some technical challenges remain to be solved, including the fusion efficacy between the language and visual cues and the difficulty of obtaining an effective pre-trained image feature extractor for medical-specific tasks. In this work, we proposed the weighted query-key interacting attention module, including both the second-order and first-order interactions. Compared with the conventional scaled dot-product attention, this design generates a strong fusion mechanism between language and visual signals. In addition, we also proposed the contrastive pre-training step to reduce the domain gap between the image encoder and the target dataset. To test the generalizability of our learning scheme, we collected and verified our model on the world-first multi-modality retina report generation dataset referred to as Retina ImBank and another large-scale retina Chinese-based report dataset referred to as Retina Chinese. These two datasets will be made publicly available and serve as benchmarks to encourage further research exploration in this field. From our experimental results, we demonstrate that our proposed method has outperformed multiple state-of-the-art image captioning and medical report generation methods on IU X-RAY, MIMIC-CXR, Retina ImBank, and Retina Chinese datasets.

Ultrasound-guided pacemaker implantation at the bedside: A lifesaving technique for cardiac emergencies.

OBJECTIVE: To investigate the safety and effectiveness of implanting temporary pacemakers using ultrasound-guidance at the bedside for rescuing patients in case of cardiac emergencies. METHODS: We enrolled 194 patients with cardiac emergencies requiring temporary pacemakers in this study, and randomly assigned them to either a bedside ultrasound-guided installation group or an electrocardiogram-guided installation group. There were 105 cases in the bedside ultrasound-guided installation group, aged approximately 66.3 ± 10.2 years, and 89 cases in the electrocardiogram-guided installation group, aged approximately 65.8 ± 9.5 years old, and disease composition was similar between the two groups. We then compared the duration of the procedure, success rates, and occurrence of adverse events between the two groups. RESULTS: The two groups showed similar clinical characteristics. The success rates of venipuncture and temporary pacemaker electrode placement were both 100% in the bedside ultrasound-guided installation group, compared to 87.8% and 96.7% respectively, in the electrocardiogram-guided installation group, with a statistically significant difference between the two groups. The duration of puncture was significantly shorter in the bedside ultrasound-guided installation group than in the electrocardiogram-guided installation group, with statistically significant differences. Moreover, no adverse events such as hematoma, pneumothorax and electrode dislodgement occurred in the bedside ultrasound-guided installation group, while 13 cases in the electrocardiogram-guided installation group experienced adverse events, and the difference was statistically significant. CONCLUSIONS: The bedside installation of temporary pacemakers using ultrasound guidance is a simple, safe, effective, and cost-efficient procedure that boasts a high success rate, does not involve radiation, and enables accurate placement of the electrode catheter.

A Study on the Effectiveness of 650-nm Red-Light Feeding Instruments in the Control of Myopia.

INTRODUCTION: This study analyzed the effectiveness of 650-nm red-light feeding instruments in the control of myopia. METHODS: In this study, 164 school-aged participants diagnosed with myopia in the city of Shenzhen were enrolled in a red-light feeding instrument study. Of these, 41 were enrolled in the mild-to-moderate myopia group that received red-light feeding (RLMM group), 65 were enrolled in the mild-to-moderate myopia group that received single-vision spectacle treatment (SVSMM group), and 58 were included in the severe myopia group that received red-light feeding (RLS group). RESULTS: After the baseline values of the three groups were matched, the right eye data were used for statistical analysis. The average return visit time of each group was 60.42 days, and changes in the observation indexes before treatment and after follow-up treatment were compared. As the primary outcome, the axial length changes in the right eye of the SVSMM group (0.08 ± 0.40 mm), the RLMM group (-0.03 ± 0.11 mm), and the RLS group (-0.07 ± 0.11 mm) were compared and showed a statistical result of p < 0.001. CONCLUSION: The study results verified that red light had a noticeable effect on the control of myopia and that low-level red-light therapy played a vital role in the treatment of severe myopia.

The Influence of Parents' Background and Their Perception on the Progression of Myopia in Children.

Purpose: To study the influence of parents' educational backgrounds and understanding on the progress of myopia in their offspring. Methods: Spherical equivalent refraction (SE) of the children (aged 6-14) in China was assessed with cycloplegic autorefraction in a two-year longitudinal study. The parents' background information and myopia-related cognition were collected by questionnaires. Results: The offspring of parents with lower education and more myopic SE had higher myopic progression (mean = -1.42 ± 1.06) than the children of other groups (P < 0.05). The parents' understanding of the proper outdoor activity time, sleep duration, reading distance, and indoor illumination for children was not significantly correlated with the progression of myopia in their offspring. The parent's preference for eye care visit frequency had a significant correlation with the myopia development of their children (r = 0.076, P=0.001∗). The mean SE progression was -0.84 ± 1.37 and -0.58 ± 1.29 in the children whose parents considered that extracurricular classes would negatively affect myopia development progression and the children whose parents believed it would not, respectively (P=0.026∗). Conclusions: Most parents misunderstand the influence of insufficient outdoor sports time and extracurricular classes, which require extra near-vision work. Besides, for parents with low educational background and more myopic SE, their offspring had higher myopia progression and may be the key group for myopia control. Finally, parents may obtain life advice and knowledge related to preventing myopia after their children become myopic. It may be of positive significance if this process could take place before myopia onset.

Survival outcomes and prognostic factors of papillary serous adenocarcinoma and papillary squamous cell carcinoma of the uterine cervix.

The aim of this retrospective population-based study was to investigate the survival outcomes and prognostic factors of patients with the two cervical carcinomas. A cohort of patients diagnosed with papillary serous adenocarcinoma of the uterine cervix (PSAC) and papillary squamous cell carcinoma (PSCC) between 1973 and 2015 were drawn from the National Cancer Institute's Surveillance Epidemiology and End Results (SEER) database. Overall survival (OS) and cancer-specific survival (CSS) were estimated using the Kaplan-Meier method, and prognostic factors were assessed using Cox proportional hazards survival regression analysis. The 5-year and 10-year OS rates were 38.4 and 33.1% for PSAC and 64.6 and 50.8% for PSCC, respectively. The 2-year and 5-year CSS rates were 60.6 and 45.9% for PSAC and 79.6 and 69.0% for PSCC, respectively. Patients with PSCC survive longer than PSAC patients and have other well-described prognostic factors for improved survival rates, including an early cancer stage, a younger patient age and standardised surgery.Impact statementWhat is already known on this subject? Papillary serous adenocarcinoma of the uterine cervix (PSAC) and papillary squamous cell carcinoma (PSCC)are both very rare subtypes of cervical carcinomas.What do the results of this study add? This retrospective population-based analysis has evaluated the survival outcomes and prognostic indicators of patients with PSAC and PSCC.What the implications are of these findings for clinical practice and/or further research? Knowing the survival outcomes and prognostic indicators of PSAC and PSCC patients, we can better follow up patients.

Deletion of a Neuronal Drp1 Activator Protects against Cerebral Ischemia.

Mitochondrial fission catalyzed by dynamin-related protein 1 (Drp1) is necessary for mitochondrial biogenesis and maintenance of healthy mitochondria. However, excessive fission has been associated with multiple neurodegenerative disorders, and we recently reported that mice with smaller mitochondria are sensitized to ischemic stroke injury. Although pharmacological Drp1 inhibition has been put forward as neuroprotective, the specificity and mechanism of the inhibitor used is controversial. Here, we provide genetic evidence that Drp1 inhibition is neuroprotective. Drp1 is activated by dephosphorylation of an inhibitory phosphorylation site, Ser637. We identify Bß2, a mitochondria-localized protein phosphatase 2A (PP2A) regulatory subunit, as a neuron-specific Drp1 activator in vivo Bß2 KO mice of both sexes display elongated mitochondria in neurons and are protected from cerebral ischemic injury. Functionally, deletion of Bß2 and maintained Drp1 Ser637 phosphorylation improved mitochondrial respiratory capacity, Ca2+ homeostasis, and attenuated superoxide production in response to ischemia and excitotoxicity in vitro and ex vivo Last, deletion of Bß2 rescued excessive stroke damage associated with dephosphorylation of Drp1 S637 and mitochondrial fission. These results indicate that the state of mitochondrial connectivity and PP2A/Bß2-mediated dephosphorylation of Drp1 play a critical role in determining the severity of cerebral ischemic injury. Therefore, Bß2 may represent a target for prophylactic neuroprotective therapy in populations at high risk of stroke.SIGNIFICANCE STATEMENT With recent advances in clinical practice including mechanical thrombectomy up to 24 h after the ischemic event, there is resurgent interest in neuroprotective stroke therapies. In this study, we demonstrate reduced stroke damage in the brain of mice lacking the Bß2 regulatory subunit of protein phosphatase 2A, which we have shown previously acts as a positive regulator of the mitochondrial fission enzyme dynamin-related protein 1 (Drp1). Importantly, we provide evidence that deletion of Bß2 can rescue excessive ischemic damage in mice lacking the mitochondrial PKA scaffold AKAP1, apparently via opposing effects on Drp1 S637 phosphorylation. These results highlight reversible phosphorylation in bidirectional regulation of Drp1 activity and identify Bß2 as a potential pharmacological target to protect the brain from stroke injury.

Association between hemoglobin within the normal range and hemoglobin A1c among Chinese non-diabetes adults.

BACKGROUND: Hemoglobin A1c (HbA1c) is the product of a non-enzymatic chemical reaction between hemoglobin (Hb) and glucose. However, the association between Hb and HbA1c remains to be fully elucidated in view of the controversial findings reported to date. Therefore, our aim in this study was to evaluate the association between Hb levels within the normal range and HbA1c levels among Chinese non-diabetes adults using cross-sectional data from the China Health and Nutrition Survey 2009. METHODS: Our analysis was based on the data of 1659 non-diabete adults 20-49 years of age. Multivariable linear models were applied to examine the association between Hb and HbA1c levels. Subgroup analyses stratified by age and sex were also performed. RESULTS: The association between Hb and HbA1c levels was positive in the unadjusted model (ß =0.020, 95% CI: 0.008, 0.032). However, this association did not remain significant when the regression model was minimally adjusted for age and sex (ß =0.006, 95% CI: - 0.014, 0.024); this association became negative when the model was further adjusted for covariates whose effect estimates of HbA1c levels more than 10% (ß = - 0.042, 95% CI: - 0.064, - 0.020). The association remained negative on subgroup analyses stratified by age (20-34 years: ß = - 0.052, 95% CI: - 0.091, - 0.013; 35-49 years: ß = - 0.041, 95% CI: - 0.068, - 0.014) and sex (men: ß = - 0.042, 95% CI: - 0.074, - 0.010; women: ß = - 0.042, 95% CI: - 0.073, - 0.012) when controlling for covariates. CONCLUSIONS: Our findings revealed that Hb levels within the normal range were negatively associated with HbA1c levels among Chinese non-diabetes adults. Confounding factors, such as red blood cell counts can affect the association between Hb and HbA1c levels.

The integrity and barrier function of porcine vocal fold epithelium: its susceptibility to damage by deoxycholic acid compared with pepsin.

PURPOSE: In this study, we aim to systematically evaluate the damaging role of gastric (pepsin and acid) and duodenal ingredients (bile acids) on vocal fold epithelium in excised porcine larynges. METHODS: Fresh ex vivo porcine larynges were exposed to one of five experimental conditions for 1 h. These conditions will be referred to as alkaline deoxycholic acid, acidic pepsin, acid pH3 only, acid pH5 only, and control, respectively. A Franz diffusing cell was used to evaluate the barrier function of vocal fold epithelium by measuring the permeability to fluorescein isothiocyanate dextran of 4 kDa. Histological changes were observed using transmission electron microscopy. RESULTS: After immersing the fresh porcine larynges in the five solution groups, we found that the vocal fold epithelium in the deoxycholic acid group had more permeability to FD4 than the pepsin group (P < 0.001). Fragmentation and desquamation of dead cell layers were observed in both the pepsin and deoxycholic acid groups, but were more severe in the deoxycholic acid group than the pepsin group. The thickness of the dead epithelial cell layer gradually increased with increasing acid concentration (P < 0.05). Additionally, the thickness of the dead epithelial cell layer in the deoxycholic acid group was significantly higher than that in the pepsin group (P < 0.01). CONCLUSION: Deoxycholic acid in a weakly acidic condition is more likely than pepsin to induce apoptosis in ex vivo porcine vocal fold epithelium, destroy the link proteins between epithelial cells, and affect their integrity and barrier function.

ß-Cell-specific ablation of sirtuin 4 does not affect nutrient-stimulated insulin secretion in mice.

Sirtuins are a family of proteins that regulate biological processes such as cellular stress and aging by removing posttranslational modifications (PTMs). We recently identified several novel PTMs that can be removed by sirtuin 4 (SIRT4), which is found in mitochondria. We showed that mice with a global loss of SIRT4 [SIRT4-knockout (KO) mice] developed an increase in glucose- and leucine-stimulated insulin secretion, and this was followed by accelerated age-induced glucose intolerance and insulin resistance. Because whole body SIRT4-KO mice had alterations to nutrient-stimulated insulin secretion, we hypothesized that SIRT4 plays a direct role in regulating pancreatic ß-cell function. Thus, we tested whether ß-cell-specific ablation of SIRT4 would recapitulate the elevated insulin secretion seen in mice with a global loss of SIRT4. Tamoxifen-inducible ß-cell-specific SIRT4-KO mice were generated, and their glucose tolerance and glucose- and leucine-stimulated insulin secretion were measured over time. These mice exhibited normal glucose- and leucine-stimulated insulin secretion and maintained normal glucose tolerance even as they aged. Furthermore, 832/13 ß-cells with a CRISPR/Cas9n-mediated loss of SIRT4 did not show any alterations in nutrient-stimulated insulin secretion. Despite the fact that whole body SIRT4-KO mice demonstrated an age-induced increase in glucose- and leucine-stimulated insulin secretion, our current data indicate that the loss of SIRT4 specifically in pancreatic ß-cells, both in vivo and in vitro, does not have a significant impact on nutrient-stimulated insulin secretion. These data suggest that SIRT4 controls nutrient-stimulated insulin secretion during aging by acting on tissues external to the ß-cell, which warrants further study.

Imidazo[1,2-a]pyridine as an Electron Acceptor to Construct High-Performance Deep-Blue Organic Light-Emitting Diodes with Negligible Efficiency Roll-Off.

Two novel bipolar deep-blue fluorescent emitters, IP-PPI and IP-DPPI, featuring different lengths of the phenyl bridge, were designed and synthesized, in which imidazo[1,2-a]pyridine (IP) and phenanthroimidazole (PI) were proposed as an electron acceptor and an electron donor, respectively. Both of them exhibit outstanding thermal stability and high emission quantum yields. All the devices based on these two materials showed negligible efficiency roll-off with increasing current density. Impressively, non-doped organic light-emitting diodes (OLEDs) based on IP-PPI and IP-DPPI exhibited external quantum efficiencies (EQEs) of 4.85 % and 4.74 % with CIE coordinates of (0.153, 0.097) and (0.154, 0.114) at 10000 cd m-2 , respectively. In addition, the 40 wt % IP-PPI doped device maintained a high EQE of 5.23 % with CIE coordinates of (0.154, 0.077) at 10000 cd m-2 . The doped device based on 20 wt % IP-DPPI exhibited a higher deep-blue electroluminescence (EL) performance with a maximum EQE of up to 6.13 % at CIE of (0.153, 0.078) and maintained an EQE of 5.07 % at 10000 cd m-2 . To the best of our knowledge, these performances are among the state-of-the art devices with CIEy ≤0.08 at a high brightness of 10000 cd m-2 . Furthermore, by doping a red phosphorescent dye Ir(MDQ)2 (MDQ=2-methyldibenzo[f,h]quinoxaline) into the IP-PPI and IP-DPPI hosts, high-performance red phosphorescent OLEDs with EQEs of 20.8 % and 19.1 % were achieved, respectively. This work may provide a new approach for designing highly efficient deep-blue emitters with negligible roll-off for OLED applications.

Deletion of mitochondrial calcium uniporter incompletely inhibits calcium uptake and induction of the permeability transition pore in brain mitochondria.

Ca2+ influx into mitochondria is mediated by the mitochondrial calcium uniporter (MCU), whose identity was recently revealed as a 40-kDa protein that along with other proteins forms the mitochondrial Ca2+ uptake machinery. The MCU is a Ca2+-conducting channel spanning the inner mitochondrial membrane. Here, deletion of the MCU completely inhibited Ca2+ uptake in liver, heart, and skeletal muscle mitochondria. However, in brain nonsynaptic and synaptic mitochondria from neuronal somata/glial cells and nerve terminals, respectively, the MCU deletion slowed, but did not completely block, Ca2+ uptake. Under resting conditions, brain MCU-KO mitochondria remained polarized, and in brain MCU-KO mitochondria, the electrophoretic Ca2+ ionophore ETH129 significantly accelerated Ca2+ uptake. The residual Ca2+ uptake in brain MCU-KO mitochondria was insensitive to inhibitors of mitochondrial Na+/Ca2+ exchanger and ryanodine receptor (CGP37157 and dantrolene, respectively), but was blocked by the MCU inhibitor Ru360. Respiration of WT and MCU-KO brain mitochondria was similar except that for mitochondria that oxidized pyruvate and malate, Ca2+ more strongly inhibited respiration in WT than in MCU-KO mitochondria. Of note, the MCU deletion significantly attenuated but did not completely prevent induction of the permeability transition pore (PTP) in brain mitochondria. Expression level of cyclophilin D and ATP content in mitochondria, two factors that modulate PTP induction, were unaffected by MCU-KO, whereas ADP was lower in MCU-KO than in WT brain mitochondria. Our results suggest the presence of an MCU-independent Ca2+ uptake pathway in brain mitochondria that mediates residual Ca2+ influx and induction of PTP in a fraction of the mitochondrial population.

Reduced Graphene Oxide Nanohybrid-Assembled Microneedles as Mini-Invasive Electrodes for Real-Time Transdermal Biosensing.

A variety of nanomaterial-based biosensors have been developed to sensitively detect biomolecules in vitro, yet limited success has been achieved in real-time sensing in vivo. The application of microneedles (MN) may offer a solution for painless and minimally-invasive transdermal biosensing. However, integration of nanostructural materials on microneedle surface as transdermal electrodes remains challenging in applications. Here, a transdermal H2 O2 electrochemical biosensor based on MNs integrated with nanohybrid consisting of reduced graphene oxide and Pt nanoparticles (Pt/rGO) is developed. The Pt/rGO significantly improves the detection sensitivity of the MN electrode, while the MNs are utilized as a painless transdermal tool to access the in vivo environment. The Pt/rGO nanostructures are protected by a water-soluble polymer layer to avoid mechanical destruction during the MN skin insertion process. The polymer layer can readily be dissolved by the interstitial fluid and exposes the Pt/rGO on MNs for biosensing in vivo. The applications of the Pt/rGO-integrated MNs for in situ and real-time sensing of H2 O2 in vivo are demonstrated both on pigskin and living mice. This work offers a unique real-time transdermal biosensing system, which is a promising tool for sensing in vivo with high sensitivity but in a minimally-invasive manner.

Loss of tau and Fyn reduces compensatory effects of MAP2 for tau and reveals a Fyn-independent effect of tau on calcium.

Microtubule-associated protein tau associates with Src family tyrosine kinase Fyn and is tyrosine phosphorylated by Fyn. The presence of tyrosine phosphorylated tau in AD and the involvement of Fyn in AD has drawn attention to the tau-Fyn complex. In this study, a tau-Fyn double knockout (DKO) mouse was generated to investigate the role of the complex. DKO mice resembled Fyn KO in novel object recognition and contextual fear conditioning tasks and resembled tau KO mice in the pole test and protection from pentylenetetrazole-induced seizures. In glutamate-induced Ca2+ response, Fyn KO was decreased relative to WT and DKO had a greater reduction relative to Fyn KO, suggesting that tau may have a Fyn-independent role. Since tau KO resembled WT in its Ca2+ response, we investigated whether microtubule-associated protein 2 (MAP2) served to compensate for tau, since the MAP2 level was increased in tau KO but decreased in DKO mice. We found that like tau, MAP2 increased Fyn activity. Moreover, tau KO neurons had increased density of dendritic MAP2-Fyn complexes relative to WT neurons. Therefore, we hypothesize that in the tau KO, the absence of tau would be compensated by MAP2, especially in the dendrites, where tau-Fyn complexes are of critical importance. In the DKO, decreased levels of MAP2 made compensation more difficult, thus revealing the effect of tau in the Ca2+ response.