Inhibition of Mitochondria- and Endoplasmic Reticulum Stress-Mediated Autophagy Augments Temozolomide-Induced Apoptosis in Glioma Cells

Autophagy is a crucial process for cells to maintain homeostasis and survival through degradation of cellular proteins and organelles, including mitochondria and endoplasmic reticula (ER). We previously demonstrated that temozolomide (TMZ), an alkylating agent for brain tumor chemotherapy, induced reactive oxygen species (ROS)/extracellular signal-regulated kinase (ERK)-mediated autophagy to protect glioma cells from apoptosis. In this study, we investigated the role of mitochondrial damage and ER stress in TMZ-induced cytotoxicity. Mitochondrial depolarization and mitochondrial permeability transition pore (MPTP) opening were observed as a prelude to TMZ-induced autophagy, and these were followed by the loss of mitochondrial mass. Electron transport chain (ETC) inhibitors, such as rotenone (a complex I inhibitor), sodium azide (a complex IV inhibitor), and oligomycin (a complex V inhibitor), or the MPTP inhibitor, cyclosporine A, decreased mitochondrial damage-mediated autophagy, and therefore increased TMZ-induced apoptosis. TMZ treatment triggered ER stress with increased expression of GADD153 and GRP78 proteins, and deceased pro-caspase 12 protein. ER stress consequently induced autophagy through c-Jun N-terminal kinases (JNK) and Ca2+ signaling pathways. Combination of TMZ with 4-phenylbutyrate (4-PBA), an ER stress inhibitor, augmented TMZ-induced cytotoxicity by inhibiting autophagy. Taken together, our data indicate that TMZ induced autophagy through mitochondrial damage- and ER stress-dependent mechanisms to protect glioma cells. This study provides evidence that agents targeting mitochondria or ER may be potential anticancer strategies

Evodiamine Induces Transient Receptor Potential Vanilloid-1-Mediated Protective Autophagy in U87-MG Astrocytes

Cerebral ischemia is a leading cause of mortality and morbidity worldwide, which results in cognitive and motor dysfunction, neurodegenerative diseases, and death. Evodiamine (Evo) is extracted from Evodia rutaecarpa Bentham, a plant widely used in Chinese herbal medicine, which possesses variable biological abilities, such as anticancer, anti-inflammation, antiobesity, anti-Alzheimer’s disease, antimetastatic, antianoxic, and antinociceptive functions. But the effect of Evo on ischemic stroke is unclear. Increasing data suggest that activation of autophagy, an adaptive response to environmental stresses, could protect neurons from ischemia-induced cell death. In this study, we found that Evo induced autophagy in U87-MG astrocytes. A scavenger of extracellular calcium and an antagonist of transient receptor potential vanilloid-1 (TRPV-1) decreased the percentage of autophagy accompanied by an increase in apoptosis, suggesting that Evo may induce calcium-mediated protective autophagy resulting from an influx of extracellular calcium. The same phenomena were also confirmed by a small interfering RNA technique to knock down the expression of TRPV1. Finally, Evo-induced c-Jun N-terminal kinases (JNK) activation was reduced by a TRPV1 antagonist, indicating that Evo-induced autophagy may occur through a calcium/c-Jun N-terminal kinase (JNK) pathway. Collectively, Evo induced an influx of extracellular calcium, which led to JNK-mediated protective autophagy, and this provides a new option for ischemic stroke treatment

Development of Risk Prediction Equations for Incident Chronic Kidney Disease

IMPORTANCE ‐ Early identification of individuals at elevated risk of developing chronic kidney disease  could improve clinical care through enhanced surveillance and better management of underlying health  conditions.  OBJECTIVE – To develop assessment tools to identify individuals at increased risk of chronic kidney  disease, defined by reduced estimated glomerular filtration rate (eGFR).  DESIGN, SETTING, AND PARTICIPANTS – Individual level data analysis of 34 multinational cohorts from  the CKD Prognosis Consortium including 5,222,711 individuals from 28 countries. Data were collected  from April, 1970 through January, 2017. A two‐stage analysis was performed, with each study first  analyzed individually and summarized overall using a weighted average. Since clinical variables were  often differentially available by diabetes status, models were developed separately within participants  with diabetes and without diabetes. Discrimination and calibration were also tested in 9 external  cohorts (N=2,253,540). EXPOSURE Demographic and clinical factors.  MAIN OUTCOMES AND MEASURES – Incident eGFR <60 ml/min/1.73 m2.  RESULTS – In 4,441,084 participants without diabetes (mean age, 54 years, 38% female), there were  660,856 incident cases of reduced eGFR during a mean follow‐up of 4.2 years. In 781,627 participants  with diabetes (mean age, 62 years, 13% female), there were 313,646 incident cases during a mean follow‐up of 3.9 years. Equations for the 5‐year risk of reduced eGFR included age, sex, ethnicity, eGFR, history of cardiovascular disease, ever smoker, hypertension, BMI, and albuminuria. For participants  with diabetes, the models also included diabetes medications, hemoglobin A1c, and the interaction  between the two. The risk equations had a median C statistic for the 5‐year predicted probability of  0.845 (25th – 75th percentile, 0.789‐0.890) in the cohorts without diabetes and 0.801 (25th – 75th percentile, 0.750‐0.819) in the cohorts with diabetes. Calibration analysis showed that 9 out of 13 (69%) study populations had a slope of observed to predicted risk between 0.80 and 1.25. Discrimination was  similar in 18 study populations in 9 external validation cohorts; calibration showed that 16 out of 18 (89%) had a slope of observed to predicted risk between 0.80 and 1.25. CONCLUSIONS AND RELEVANCE – Equations for predicting risk of incident chronic kidney disease developed in over 5 million people from 34 multinational cohorts demonstrated high discrimination and  variable calibration in diverse populations

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Supercritical carbon dioxide extraction of omega-3 oil compounds from Ficus awkeotsang Makino achenes

This work investigated the production of linolenic-rich oil from Ficus awkeotsang Makino achenes using supercritical carbon dioxide extraction. The 16 h Soxhlet n-hexane extraction of these achenes produced total yield of 12.91% and concentration of 686.3 mg/g of the extracted oil. Supercritical carbon dioxide extractions were performed with pressures ranged from 325 to 375 bar, temperatures ranged from 323 to 343 K, and solvent to solid ratios ranged from 80 to 160 by using a three-factor experimental designed response surface methodology. Three responses of total yield (TY), concentration of triglycerides (CTG) and recovery of triglycerides (RTG) were measured. Under a suitable condition, the values of TY, CTG, and RTG were 9.24%, 893.5 mg/g and 93.2%, respectively. This study demonstrated that environmental benign supercritical carbon dioxide extraction of linolenic-rich oil from F. awkeotsang M. achenes is a feasible and sustainable green process, alternative to traditional organic solvent extraction

Recovery of vitamins B from supercritical carbon dioxide-defatted rice bran powder using ultrasound water extraction

This study investigates the ultrasonic water extraction of vitamins B from supercritical carbon dioxide-defatted rice bran powder. Experimental data show that when the vitamins B content in the defatted extract reached 0.63% (wt), the extract was healthy. The effects of the solvent to solid ratio and the extraction time on the total yield, extraction efficiency, purification factor and purity of vitamins B in the extract were also determined. A two-factor center composite response surface method was adopted to optimize the condition for ultrasonic water extraction. Both parameters are important. The solvent to solid ratio has a greater influence on total yield than the extraction time. However, the extraction time is the most important in increasing the efficiency of extraction of vitamins B. Given an appropriate solvent to solid ratio, the vitamins B content in the defatted rice bran extract increased substantially with extraction time, exceeding that of the raw rice bran extract. Ultrasonic water extraction at a solvent to solid ratio of 10.0 at 323 K for 1.5 h yields a vitamin B content of 0.63%, a purification factor of 4.55 and a recovery rate of vitamins B of 92.8% in the dried defatted rice bran extract

IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells to temozolomide by targeting the NEDD4L-inhibited Wnt/β-catenin pathway.

Temozolomide (TMZ) is a first-line alkylating agent for glioblastoma multiforme (GBM). Clarifying the mechanisms inducing TMZ insensitivity may be helpful in improving its therapeutic effectiveness against GBM. Insulin-like growth factor (IGF)-1 signaling and micro (mi)RNAs are relevant in mediating GBM progression. However, their roles in desensitizing GBM cells to TMZ are still unclear. We aimed to identify IGF-1-mediated miRNA regulatory networks that elicit TMZ insensitivity for GBM. IGF-1 treatment attenuated TMZ cytotoxicity via WNT/β-catenin signaling, but did not influence glioma cell growth. By miRNA array analyses, 93 upregulated and 148 downregulated miRNAs were identified in IGF-1-treated glioma cells. miR-513a-5p from the miR-513a-2 gene locus was upregulated by IGF-1-mediated phosphoinositide 3-kinase (PI3K) signaling. Its elevated levels were also observed in gliomas versus normal cells, in array data of The Cancer Genome Atlas (TCGA), and the GSE61710, GSE37366, and GSE41032 datasets. In addition, lower levels of neural precursor cell-expressed developmentally downregulated 4-like (NEDD4L), an E3 ubiquitin protein ligase that inhibits WNT signaling, were found in gliomas by analyzing cells, arrays, and RNA sequencing data of TCGA glioma patients. Furthermore, a negative correlation was identified between miR-513a-5p and NEDD4L in glioma. NEDD4L was also validated as a direct target gene of miR-513a-5p, and it was reduced by IGF-1 treatment. Overexpression of NEDD4L inhibited glioma cell viability and reversed IGF-1-repressed TMZ cytotoxicity. In contrast, miR-513a-5p significantly affected NEDD4L-inhibited WNT signaling and reduced TMZ cytotoxicity. These findings demonstrate a distinct role of IGF-1 signaling through miR-513a-5p-inhibited NEDD4L networks in influencing GBM's drug sensitivity to TMZ

The impact of ambient temperature on HbA1c in Taiwanese type 2 diabetic patients: The most vulnerable subgroup

The relationship between temperature variability and HbA1c has been reported in Caucasians, but not for Asians of Taiwanese origin. This study investigated the impact of temperature on HbA1c in various groups of Taiwanese with type 2 diabetes in Taiwan. Methods: For this longitudinal follow-up study which started in 2006, we recruited a total of 4399 patients with type 2 diabetes who had been regularly followed up at Chi Mei Medical Center and obtained local temperature data for 2006 to 2011 from Taiwan's Central Weather Bureau. We used a generalized estimated equation (GEE) to analyze the HbA1c level and its change over time with temperature and temperature changes, respectively. Results: We found a negative correlation between HbA1c and temperature (R = −0.475, p = 0.001). For every 1°C decrement in temperature, there was an increase in the risk of having a HbA1c level >7% [p  7% among those in the lowest quartile of temperatures than the highest quartile (p = 0.0038, adjusted OR: 1.13). Patients with diabetic patients were at higher risk of HbA1C > 7% in the winter and spring than those in the summer (adjusted OR: 1.13, p = 0.0027; adjusted OR: 1.14, p = 0.0022). After adjusting for various confounders, we found people who were younger than 65 years old, people who had diabetes for longer than 6 years, and people who had a body mass index (BMI) < 24 to be more susceptible to temperature changes (p = 0.0022, β: 0.0095; p < 0.0001, β: 0.0125; p < 0.0001, β: 0.016, respectively). Conclusion: Our study suggests cold weather may adversely affect HbA1c levels in Taiwanese people with type 2 diabetes, especially in people under 65 years old, people with diabetes for longer than 6 years, and those with a BMI < 24