A multi-omics approach to overeating and inactivity-induced muscle atrophy in db/db mice.

BACKGROUND: Overeating and inactivity are associated with type 2 diabetes. This study aimed to investigate its pathological basis using integrated omics and db/db/mice, a model representing this condition. METHODS: The study involved housing 8-week-old db/m and db/db mice for 8 weeks. Various analyses were conducted, including gene expression in skeletal muscle and small intestine using next-generation sequencing; cytokine arrays of serum; assessment of metabolites in skeletal muscle, stool, and serum; and analysis of the gut microbiota. Histone modifications in small intestinal epithelial cells were profiled using CUT&Tag. RESULTS: Compared with db/m mice, db/db mice had 22.4% lower grip strength and approximately five times the visceral fat weight (P < 0.0001). Serum cytokine arrays showed a 2.8-fold relative concentration of VEGF-A in db/db mice (P < 0.0001) and lower concentrations of several other cytokines. mRNA sequencing revealed downregulation of Myh expression in skeletal muscle, upregulation of lipid and glucose transporters, and downregulation of amino acid transporters in the small intestine of db/db/mice. The concentrations of saturated fatty acids in skeletal muscle were significantly higher, and the levels of essential amino acids were lower in db/db mice. Analysis of the gut microbiota, 16S rRNA sequencing, revealed lower levels of the phylum Bacteroidetes (59.7% vs. 44.9%) and higher levels of the phylum Firmicutes (20.9% vs. 31.4%) in db/db mice (P = 0.003). The integrated signal of histone modifications of lipid and glucose transporters was higher, while the integrated signal of histone modifications of amino acid transporters was lower in the db/db mice. CONCLUSIONS: The multi-omics approach provided insights into the epigenomic alterations in the small intestine, suggesting their involvement in the pathogenesis of inactivity-induced muscle atrophy in obese mice.

Optimal Body Weights for Adults with Severe Motor and Intellectual Disabilities.

Determining the optimal body weight for individuals with severe motor and intellectual disabilities (SMID) lacks a standardized approach. In this study, we aimed to develop a formula to estimate the ideal body weight for each SMID patient, considering factors such as reduced muscle and bone mass. We analyzed data from 111 SMID patients (56 male, 55 female; age range 20 to 73 y) who underwent blood tests measuring creatinine (Cr) and cystatin C (cysC) for clinical reasons between Feb. 2018 and Feb. 2023. To create the optimal body weight formula, we utilized three variables: height, estimated glomerular filtration (eGFR)-Cr, and eGFR-cysC. The validity of the formula was assessed by comparing the measured triceps subcutaneous fat thickness (TSF) to the reference TSF (%TSF), evaluating how accurately it reflects the appropriate physique. The derived optimal body weight formula is as follows: Optimal body weight=(height)2×(18.5-25.0)×{1-0.41×(1-eGFR-cysC/eGFR-Cr)}×0.93. Our formula demonstrated validity when using %TSF as an indicator. Establishing a method to determine optimal body weight in SMID patients, considering their low muscle and bone mass, is crucial for accurate nutritional assessment and subsequent nutritional management.

PHF2 regulates sarcomeric gene transcription in myogenesis.

Myogenesis is regulated mainly by transcription factors known as Myogenic Regulatory Factors (MRFs), and the transcription is affected by epigenetic modifications. However, the epigenetic regulation of myogenesis is poorly understood. Here, we focused on the epigenomic modification enzyme, PHF2, which demethylates histone 3 lysine 9 dimethyl (H3K9me2) during myogenesis. Phf2 mRNA was expressed during myogenesis, and PHF2 was localized in the nuclei of myoblasts and myotubes. We generated Phf2 knockout C2C12 myoblasts using the CRISPR/Cas9 system and analyzed global transcriptional changes via RNA-sequencing. Phf2 knockout (KO) cells 2 d post differentiation were subjected to RNA sequencing. Gene ontology (GO) analysis revealed that Phf2 KO impaired the expression of the genes related to skeletal muscle fiber formation and muscle cell development. The expression levels of sarcomeric genes such as Myhs and Mybpc2 were severely reduced in Phf2 KO cells at 7 d post differentiation, and H3K9me2 modification of Mybpc2, Mef2c and Myh7 was increased in Phf2 KO cells at 4 d post differentiation. These findings suggest that PHF2 regulates sarcomeric gene expression via epigenetic modification.

Associations between thyroid hormones and appendicular skeletal muscle index, and hand grip strength in people with diabetes: The KAMOGAWA-A study.

AIM: To assess the effects of thyroid hormones on appendicular skeletal muscle index (SMI) and hand grip strength (HGS) in people with diabetes. METHODS: This cross-sectional cohort included 1,135 participants with diabetes admitted to 3 hospitals in Japan. Multiple regression analysis was performed to determine the associations among thyroid hormone levels, SMI, and HGS. RESULTS: Of the 1,135 participants, 480 were female. Their median (interquartile range) age, body mass index, durations of diabetes, and glycated haemoglobin levels were 68 years, 24.3 kg/m2, 10 years, and 7.6 %, respectively. The median (interquartile range) SMI (kg/m2) and hand grip strength of the cohort were 7.1 kg/m2 and 28.2 kg, respectively. Positive correlations between FT3 and the FT3/FT4 ratio with SMI and HGS was observed after adjusting for covariates in males. A negative correlation was found between the FT3/FT4 ratio and sarcopenia as a result of low SMI and low HGS in the male participants but not in females (p for interaction = 0.02). CONCLUSIONS: FT3/FT4 ratios may impact skeletal muscles in people with diabetes-particularly in males. Assessments of FT3/FT4 ratios may represent key indicators of muscle mass and strength in males.

Association of Elevated Serum Branched-chain Amino Acid Levels With Longitudinal Skeletal Muscle Loss.

Context: Branched-chain amino acids (BCAA) are substrates for protein synthesis. Although their intake may contribute to an increase in skeletal muscle mass, elevated serum BCAA levels have been reported to be associated with insulin resistance, potentially resulting in decreased skeletal muscle mass. Objective: This study aimed to explore the association between elevated serum BCAA levels and longitudinal skeletal muscle loss. Design and Setting: A cohort analysis was conducted, in which serum amino acids were analyzed in healthy individuals who underwent a medical health checkup at Kameoka Municipal Hospital (HOZUGAWA study), Japan. Patients: Seventy-one participants (37 men and 34 women) underwent follow-up checkups after the baseline visit. The follow-up duration was 1.2 ± .4 years. Main Outcome Measures: The relationship between fasting baseline serum BCAA levels and lifestyle factors, body composition, blood test results, dietary history, and changes in skeletal muscle mass was evaluated. Results: In both men and women, serum BCAA levels were positively correlated with body weight, body mass index, skeletal muscle mass index (SMI), and serum triglycerides but inversely correlated with serum high-density lipoprotein cholesterol. In men, fasting serum BCAA levels were inversely associated with the rate of change in SMI (adjusted ß = -.529, P = .006), and elevated BCAA levels were independently associated with a longitudinal decrease in skeletal muscle mass (odds ratio: 1.740; 95% confidence interval: 1.023-2.960 per 50 nmol/mL serum BCAAs increase). Conclusion: Increased circulating BCAAs could be an indicator of skeletal muscle loss in men.

Effects of weight loss on metabolic dysfunction-associated fatty liver disease in Japanese people: Non-alcoholic fatty liver disease in the Gifu area, longitudinal analysis study.

AIM: Metabolic dysfunction-associated fatty liver disease (MAFLD) is a major health concern. This cohort study aimed to evaluate the association between weight loss and remission of MAFLD in the Japanese population to aid the development of efficient treatment strategies. METHODS: This retrospective cohort study was conducted at a Japanese health screening center. Participants included 3309 individuals diagnosed with baseline MAFLD between 2004 and 2016. Logistic regression analysis was used to assess the association between MAFLD remission from baseline to 5 years and weight change. RESULTS: After 5 years, 671 participants achieved MAFLD remission. Weight loss was associated with MAFLD remission for every 1 kg of weight loss over 5 years; the odds ratio for MAFLD remission was 1.24 (95% CI 1.15-1.34) for participants with type 2 diabetes, 1.40 (95% CI 1.35-1.45) for overweight participants, and 1.51 (95% CI 1.33-1.72) for non-overweight participants with metabolic dysfunctions. The cutoff values for weight loss for MAFLD remission were 1.9 kg for all participants, 3.0 kg for participants with type 2 diabetes, 1.9 kg for overweight participants, and 0.8 kg for non-overweight participants with metabolic dysfunctions. CONCLUSIONS: Among participants diagnosed with MAFLD, weight loss was associated with MAFLD remission regardless of the type of metabolic dysfunction in MAFLD. The results of this study may contribute to the development of novel approaches to achieve MAFLD remission.

The role of chicken eggs in modulating sarcopenic obesity and gut microbiota in db/db mice.

Background: Sarcopenia obesity, in which loss of muscle mass and fat accumulation occur simultaneously, is the pathological basis of type 2 diabetes mellitus. The usefulness of chicken eggs in sarcopenia prevention has been reported in several previous studies. The purpose of this study was to determine the beneficial effects of chicken eggs in the prevention of sarcopenic obesity in db/db mice. Methods: We raised 8-week-old db/db male mice, a model of sarcopenia obesity, for 8 weeks and fed them a diet mixed with dried whole eggs. The fecal microbiota transplant (FMT) group was treated with antibiotics for 2 weeks, starting at 6 weeks of age, followed by FMT twice a week until 16 weeks of age. Results: Eggs administered to db/db mice showed increased grip strength (p = 0.022) and muscle mass (p = 0.013), decreased visceral fat mass (p = 0.005), and significantly increased physical activity (p < 0.001). The FMT group of egg-fed mice showed a significant improvement in glucose intolerance and sarcopenic obesity. Sequencing of gene expression in the small intestine showed that the gene expression of amino acid transporters such as Slc6a18, Slc6a19, and Slc38a6 was increased in egg-fed mice. 16S rRNA sequencing of the gut microbiota showed an increase in the genus Vampirovibrio in both the egg-fed and FMT groups compared to that in egg-fed mice. Conclusion: The results of this study indicate that egg consumption not only increases the intake of amino acids and other nutrients but also alters the intestinal microbiota and increases amino acid absorption from the intestinal tract, suggesting that eggs might contribute to the ameliorative mechanism of sarcopenic obesity.

Evaluation of eGFRs to Calculate Kidney Function in Adults with Severe Motor and Intellectual Disabilities.

Introduction: The gold standard for evaluating kidney function is kidney inulin clearance (Cin). However, this procedure is difficult to perform in patients with neuromuscular disabilities and/or bladder dysfunction. We aimed to develop a simpler method for determining the estimated glomerular filtration rate (eGFR) using equations and values for three biomarkers: serum creatinine (sCr), serum cystatin C (cysC), and serum beta-2 microglobulin (ß2MG). This study evaluated three eGFR equations in patients with severe motor and intellectual disabilities (SMID). Methods: We evaluated the equations using data of 18 adult SMID patients with a clinical need for creatinine clearance (Ccr). We compared the results of each equation with Ccr-based eGFR instead of Cin using mean error (ME), root mean square error (RMSE), and P30. Results: Based on eGFR, the ME values of Cr, cysC, ß2MG, and Ccr were 74.5, 2.3, and 6.5 mL/min/1.73 m2, RMSE values, 92.3, 25.7, and 33.4 mL/min/1.73 m2; and P30, 16.7%, 77.8%, and 72.2%, respectively. Conclusions: eGFR-Cr cannot be used to reliably assess kidney function in adult SMID patients. It is better to use eGFR-cysC to evaluate kidney function in this patient population.

Metabolic outcomes and changes in innate immunity induced by diesel exhaust particles airway exposure and high-fat high-sucrose diet.

AIMS: Epidemiological studies have shown that exposure to diesel exhaust particles (DEP) is associated with metabolic diseases. We used mice with nonalcoholic fatty liver disease (NAFLD) caused by a high-fat, high-sucrose diet (HFHSD), which mimics a Western diet, to investigate the mechanism of NAFLD exacerbation via changes in innate immunity in the lungs by airway exposure to DEP. MAIN METHODS: Six-week-old C57BL6/J male mice were fed HFHSD, and DEP was administered endotracheally once a week for eight weeks. The histology, gene expression, innate immunity cells in the lung and liver, and the serum inflammatory cytokine levels, were investigated. KEY FINDINGS: Under the HFHSD, DEP increased blood glucose levels, serum lipid levels, and NAFLD activity scores, and also the expression of genes associated with inflammation in the lungs and liver. DEP caused an increase in ILC1s, ILC2s, ILC3s, and M1 macrophages in the lungs and a marked increase in ILC1s, ILC3s, M1 macrophages, and natural killer cells in the liver, while ILC2 levels were not changed. Furthermore, DEP caused high levels of inflammatory cytokines in the serum. SIGNIFICANCE: Chronic exposure to DEP in HFHSD-fed mice increased inflammatory cells involved in innate immunity in the lungs and raised local inflammatory cytokine levels. This inflammation spread throughout the body, suggesting the association with the progression of NAFLD via increased inflammatory cells involved in innate immunity and inflammatory cytokine levels in the liver. These findings contribute to a better understanding of the role of innate immunity in air pollution-related systemic diseases, especially metabolic diseases.

Oral Exposure to Polystyrene Microplastics of Mice on a Normal or High-Fat Diet and Intestinal and Metabolic Outcomes.

BACKGROUND: Microplastics (MPs) are small particles of plastic (≤5mm in diameter). In recent years, oral exposure to MPs in living organisms has been a cause of concern. Leaky gut syndrome (LGS), associated with a high-fat diet (HFD) in mice, can increase the entry of foreign substances into the body through the intestinal mucosa. OBJECTIVES: We aimed to evaluate the pathophysiology of intestinal outcomes associated with consuming a high-fat diet and simultaneous intake of MPs, focusing on endocrine and metabolic systems. METHODS: C57BL6/J mice were fed a normal diet (ND) or HFD with or without polystyrene MP for 4 wk to investigate differences in glucose tolerance, intestinal permeability, gut microbiota, as well as metabolites in serum, feces, and liver. RESULTS: In comparison with HFD mice, mice fed the HFD with MPs had higher blood glucose, serum lipid concentrations, and nonalcoholic fatty liver disease (NAFLD) activity scores. Permeability and goblet cell count of the small intestine (SI) in HFD-fed mice were higher and lower, respectively, than in ND-fed mice. There was no obvious difference in the number of inflammatory cells in the SI lamina propria between mice fed the ND and mice fed the ND with MP, but there were more inflammatory cells and fewer anti-inflammatory cells in mice fed the HFD with MPs in comparison with mice fed the HFD without MPs. The expression of genes related to inflammation, long-chain fatty acid transporter, and Na+/glucose cotransporter was significantly higher in mice fed the HFD with MPs than in mice fed the HFD without MPs. Furthermore, the genus Desulfovibrio was significantly more abundant in the intestines of mice fed the HFD with MPs in comparison with mice fed the HFD without MPs. Muc2 gene expression was decreased when palmitic acid and microplastics were added to the murine intestinal epithelial cell line MODE-K cells, and Muc2 gene expression was increased when IL-22 was added. DISCUSSION: Our findings suggest that in this study, MP induced metabolic disturbances, such as diabetes and NAFLD, only in mice fed a high-fat diet. These findings suggest that LGS might have been triggered by HFD, causing MPs to be deposited in the intestinal mucosa, resulting in inflammation of the intestinal mucosal intrinsic layer and thereby altering nutrient absorption. These results highlight the need for reducing oral exposure to MPs through remedial environmental measures to improve metabolic disturbance under high-fat diet conditions. https://doi.org/10.1289/EHP11072.

Reliable identification of cardiac conduction abnormalities in drug discovery using automated patch clamp II: Best practices for Nav1.5 peak current in a high throughput screening environment.

INTRODUCTION: For reliable identification of cardiac safety risk, compounds should be screened for activity on cardiac ion channels in addition to hERG, including NaV1.5 and CaV1.2. We identified different parameters that might affect IC50s of compounds on NaV1.5 peak and late currents recorded using automated patch clamp (APC) and suggest outlines for best practices. METHODS: APC instruments SyncroPatch 384 and Patchliner were used to record NaV1.5 peak and late current. Up to 24 CiPA compounds were used to investigate effects of voltage protocol, holding potential (-80 mV or - 95 mV) and temperature (23 ± 1 °C or 36 ± 1 °C) on IC50 values on hNaV1.5 overexpressed in HEK or CHO cells either as frozen cells or running cultures. RESULTS: The IC50s of 18 compounds on the NaV1.5 peak current recorded on the SyncroPatch 384 using the CiPA step-ramp protocol correlated well with the literature. The use of frozen or cultured cells did not affect IC50s but voltage protocol and holding potential did cause differences in IC50 values. Temperature can affect Vhalf of inactivation and also compound potency. A compound incubation time of 5-6 min was sufficient for most compounds, however slow acting compounds such as terfenadine required longer to reach maximum effect. DISCUSSION: We conclude that holding potential, voltage protocol and temperature can affect IC50 values and recommend the use of the CiPA step-ramp protocol at physiological temperature to record NaV1.5 peak and late currents for cardiac safety. Further recommendations include: a minimum compound incubation time of 5 min, a replicate number of 4 and the use of positive and negative controls for reliable IC50s.

Effect of COVID-19 Pandemic on the Change in Skeletal Muscle Mass in Older Patients with Type 2 Diabetes: A Retrospective Cohort Study.

Background: The aim of this study was to investigate the effect of the coronavirus disease (COVID-19) pandemic restrictions on the change in muscle mass in older patients with type 2 diabetes (T2D), who were not infected with COVID-19. Methods: In this retrospective cohort study, data were obtained from outpatients who underwent bioelectrical impedance analysis at least twice before April 2020 and at least once thereafter. Skeletal muscle mass index (SMI, kg/m2) was calculated as appendicular muscle mass (kg) divided by height squared (m2). Change in SMI (kg/m2/year) was calculated as (follow-up SMI-baseline SMI/follow-up period). The differences between the changes in SMI before and after the start of the COVID-19 pandemic were evaluated using paired t test. Results: This study recruited 56 patients, with a mean (SD) age of 75.2 (7.1) years. SMI changed from 6.7 (0.9) to 6.8 (0.9) kg/m2 before the COVID-19 pandemic, whereas SMI changed from 6.8 (0.9) to 6.6 (0.9) kg/m2 after the start of the COVID-19 pandemic. SMI decreased after the start of the COVID-19 pandemic compared with before the pandemic (-0.117 (0.240) vs. 0.005 (0.289) kg/m2/year, p = 0.049). This decrease was observed in men (-0.159 (0.257) vs. 0.031 (0.325) kg/m2/year, p = 0.038), patients with poor glycemic control (-0.170 (0.264) vs. 0.031 (0.285) kg/m2/year, p = 0.042), and those with a long diabetes duration (-0.153 (0.229) vs. 0.082 (0.291) kg/m2, p = 0.049). Conclusions: The COVID-19 pandemic restrictions caused muscle mass loss in older patents with T2D. Actions, including recommendation of exercise and adequate diet intake, are needed to prevent loss of muscle mass.

Reliable identification of cardiac liability in drug discovery using automated patch clamp: Benchmarking best practices and calibration standards for improved proarrhythmic assessment.

INTRODUCTION: Screening compounds for activity on the hERG channel using patch clamp is a crucial part of safety testing. Automated patch clamp (APC) is becoming widely accepted as an alternative to manual patch clamp in order to increase throughput whilst maintaining data quality. In order to standardize APC experiments, we have investigated the effects on IC50 values under different conditions using several devices across multiple sites. METHODS: APC instruments SyncroPatch 384i, SyncroPatch 384PE and Patchliner, were used to record hERG expressed in HEK or CHO cells. Up to 27 CiPA compounds were used to investigate effects of voltage protocol, incubation time, labware and time between compound preparation and experiment on IC50 values. RESULTS: All IC50 values of 21 compounds recorded on the SyncroPatch 384PE correlated well with IC50 values from the literature (Kramer et al., 2013) regardless of voltage protocol or labware, when compounds were used immediately after preparation, but potency of astemizole decreased if prepared in Teflon or polypropylene (PP) compound plates 2-3 h prior to experiments. Slow acting compounds such as dofetilide, astemizole, and terfenadine required extended incubation times of at least 6 min to reach steady state and therefore, stable IC50 values. DISCUSSION: Assessing the influence of different experimental conditions on hERG assay reliability, we conclude that either the step-ramp protocol recommended by CiPA or a standard 2-s step-pulse protocol can be used to record hERG; a minimum incubation time of 5 min should be used and although glass, Teflon, PP or polystyrene (PS) compound plates can be used for experiments, caution should be taken if using Teflon, PS or PP vessels as some adsorption can occur if experiments are not performed immediately after preparation. Our recommendations are not limited to the APC devices described in this report, but could also be extended to other APC devices.

Involvement of Inner Choroidal Layer in Choroidal Thinning during Regression of Multiple Evanescent White Dot Syndrome.

PURPOSE: To investigate relationships between total thickness and the thickness of inner and outer layers in the choroid during regression in patients with multiple evanescent white dot syndrome (MEWDS). METHODS: This retrospective observational case series included 15 unilaterally affected eyes and 13 unaffected fellow eyes from 15 MEWDS patients (4 men and 11 women; mean age, 37.6 ± 17.6 years). Using enhanced depth imaging optical coherence tomography, whole, inner, and outer choroidal layer thicknesses at the fovea and perifovea were manually measured at the initial visit and at 1 and 3 months after the initial visit. The mean thickness values of the layers were compared at each stage. RESULTS: With regression of MEWDS, the mean subfoveal whole and inner choroidal layer thicknesses significantly decreased at 1 and 3 months compared to baseline values in MEWDS eyes (P=0.01 and P < 0.0001, respectively), but not in fellow eyes. The outer layer in MEWDS eyes tended to thin. Changes in the inner and outer layers at the perifovea in MEWDS eyes also showed the same trends. Simple linear regression analysis revealed significant positive correlations in choroidal thickness changes between the whole and inner layers (R = 0.53, P=0.04) and between the whole and outer layers (R = 0.91, P < 0.0001) from baseline to 3 months. Multiple linear regression analysis revealed that choroidal thickness changes in the whole layer were significantly correlated with those in the inner (ß = 0.51, P < 0.0001) and outer (ß = 0.73, P < 0.0001) layers. CONCLUSION: The inner choroidal layer significantly thinned with regression of MEWDS, correlating with the thinning of total choroidal thickness. These results suggest that MEWDS lesions in the choroid are likely to lie mainly in the inner layer.

Increased thickness and decreased blood flow velocity of the choroid in a patient with acute macular neuroretinopathy.

BACKGROUND: The involvement of choroidal lesions in acute macular neuroretinopathy (AMN) is not yet fully understood. We quantitatively examined sequential changes in the morphology and circulation hemodynamics of the choroid using enhanced depth imaging optical coherence tomography (EDI-OCT) and laser speckle flowgraphy (LSFG) in a patient with AMN. CASE PRESENTATION: A 15-year-old boy was referred to our hospital due to AMN in his right eye alone. The next day AMN developed in his left eye. Three months later, AMN lesions in both eyes spontaneously resolved and the morphology of macular photoreceptors improved. Using EDI-OCT, central choroidal thickness (CCT) was examined for a period of three months, starting from the initial visit. Using LSFG, macular mean blur rate (MBR) was examined for three months, starting 1 week after the initial visit. At the first visit, CCT of the right eye with AMN was 82 µm higher than that of the left eye, which had not yet developed AMN, and decreased by 86 µm after three months. In the left eye, similarly, CCT increased by 16 µm after the AMN onset at 1 week compared with a pre-onset value at the first visit and thereafter decreased by 57 µm at 3 months. Macular MBR increased by 20-55% OD and 51-71% OS during the follow-up until 3 months. CONCLUSIONS: We found that the choroid at the macula thickened at the onset of AMN and became thin with the regression of disease. Therefore, in concert with MBR data, these results further strengthened our hypothesis that choroidal circulation impairment plays a role in the pathogenesis of AMN.

Inhibition of GCN2 sensitizes ASNS-low cancer cells to asparaginase by disrupting the amino acid response.

General control nonderepressible 2 (GCN2) plays a major role in the cellular response to amino acid limitation. Although maintenance of amino acid homeostasis is critical for tumor growth, the contribution of GCN2 to cancer cell survival and proliferation is poorly understood. In this study, we generated GCN2 inhibitors and demonstrated that inhibition of GCN2 sensitizes cancer cells with low basal-level expression of asparagine synthetase (ASNS) to the antileukemic agent l-asparaginase (ASNase) in vitro and in vivo. We first tested acute lymphoblastic leukemia (ALL) cells and showed that treatment with GCN2 inhibitors rendered ALL cells sensitive to ASNase by preventing the induction of ASNS, resulting in reduced levels of de novo protein synthesis. Comprehensive gene-expression profiling revealed that combined treatment with ASNase and GCN2 inhibitors induced the stress-activated MAPK pathway, thereby triggering apoptosis. By using cell-panel analyses, we also showed that acute myelogenous leukemia and pancreatic cancer cells were highly sensitive to the combined treatment. Notably, basal ASNS expression at protein levels was significantly correlated with sensitivity to combined treatment. These results provide mechanistic insights into the role of GCN2 in the amino acid response and a rationale for further investigation of GCN2 inhibitors for the treatment of cancer.

Non-invasive detection of language-related prefrontal high gamma band activity with beamforming MEG.

High gamma band (>50 Hz) activity is a key oscillatory phenomenon of brain activation. However, there has not been a non-invasive method established to detect language-related high gamma band activity. We used a 160-channel whole-head magnetoencephalography (MEG) system equipped with superconducting quantum interference device (SQUID) gradiometers to non-invasively investigate neuromagnetic activities during silent reading and verb generation tasks in 15 healthy participants. Individual data were divided into alpha (8-13 Hz), beta (13-25 Hz), low gamma (25-50 Hz), and high gamma (50-100 Hz) bands and analysed with the beamformer method. The time window was consecutively moved. Group analysis was performed to delineate common areas of brain activation. In the verb generation task, transient power increases in the high gamma band appeared in the left middle frontal gyrus (MFG) at the 550-750 ms post-stimulus window. We set a virtual sensor on the left MFG for time-frequency analysis, and high gamma event-related synchronization (ERS) induced by a verb generation task was demonstrated at 650 ms. In contrast, ERS in the high gamma band was not detected in the silent reading task. Thus, our study successfully non-invasively measured language-related prefrontal high gamma band activity.

Relationship between Choroidal Thickness and Visual Field Impairment in Acute Zonal Occult Outer Retinopathy.

PURPOSE: To evaluate sequential changes in choroidal thickness at the affected area in patients with acute zonal occult outer retinopathy (AZOOR). METHODS: This retrospective observational case series included 14 affected eyes and 6 unaffected fellow eyes from 10 AZOOR patients with impaired macular area. Using enhanced depth imaging optical coherence tomography, choroidal thicknesses at the subfovea and at nasal and temporal sites 1000 µm away from the fovea were manually measured at baseline and 3 and 6 months thereafter. Changes in the choroidal thicknesses and the average threshold at the affected area on Humphrey perimetry were compared during the 6-month follow-up. RESULTS: In AZOOR eyes, the average threshold at the affected area significantly increased over time, while outer retinal structure ameliorated. The mean choroidal thicknesses at all the sites measured significantly decreased at 3 and 6 months compared with baseline values in AZOOR eyes, but not in fellow eyes. There was an inverse correlation between the changing rates of the average threshold and the subfoveal choroidal thickness at 6 months from baseline. CONCLUSION: The current data suggest that choroidal thickness at AZOOR-affected area significantly decreased with regression of AZOOR and this anatomical change correlated with the functional recovery.

Studies of CDK 8/19 inhibitors: Discovery of novel and selective CDK8/19 dual inhibitors and elimination of their CYP3A4 time-dependent inhibition potential.

In this article, synthetic studies around a pyridylacrylamide-based hit compound (1), utilizing structure-based drug design guided by CDK8 docking models, is discussed. Modification of the pendant 4-fluorophenyl group to various heteroaromatic rings was conducted aiming an interaction with the proximal amino acids, and then replacement of the morpholine ring was targeted for decreasing potential of time-dependent CYP3A4 inhibition. These efforts led to the compound 4k, with enhanced CDK8 inhibitory activity and no apparent potential for time-dependent CYP3A4 inhibition (CDK8 IC50: 2.5nM; CYP3A4 TDI: 99% compound remaining). Compound 4k was found to possess a highly selective kinase inhibition profile, and also showed favorable pharmacokinetic profile. Oral administration of 4k (15mg/kg, bid. for 2weeks) suppressed tumor growth (T/C 29%) in an RPMI8226 mouse xenograft model.