Acetylation of yeast AMPK controls intrinsic aging independently of caloric restriction.

Acetylation of histone and nonhistone proteins is an important posttranslational modification affecting many cellular processes. Here, we report that NuA4 acetylation of Sip2, a regulatory ß subunit of the Snf1 complex (yeast AMP-activated protein kinase), decreases as cells age. Sip2 acetylation, controlled by antagonizing NuA4 acetyltransferase and Rpd3 deacetylase, enhances interaction with Snf1, the catalytic subunit of Snf1 complex. Sip2-Snf1 interaction inhibits Snf1 activity, thus decreasing phosphorylation of a downstream target, Sch9 (homolog of Akt/S6K), and ultimately leading to slower growth but extended replicative life span. Sip2 acetylation mimetics are more resistant to oxidative stress. We further demonstrate that the anti-aging effect of Sip2 acetylation is independent of extrinsic nutrient availability and TORC1 activity. We propose a protein acetylation-phosphorylation cascade that regulates Sch9 activity, controls intrinsic aging, and extends replicative life span in yeast.

The deficient CLEC5A ameliorates the behavioral and pathological deficits via the microglial Aß clearance in Alzheimer's disease mouse model.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disease that causes cognitive dysfunction in older adults. One of the AD pathological factors, ß-Amyloid (Aß), triggers inflammatory responses and phagocytosis of microglia. C-type lectin domain family 5 member A (CLEC5A) induces over-reactive inflammatory responses in several virus infections. Yet, the role of CLEC5A in AD progression remains unknown. This study aimed to elucidate the contribution of CLEC5A to Aß-induced microglial activation and behavioral deficits. METHODS: The AD mouse model was crossed with Clec5a knockout mice for subsequent behavioral and pathological tests. The memory deficit was revealed by the Morris water maze, while the nociception abnormalities were examined by the von Frey filament and hotplate test. The Aß deposition and microglia recruitment were identified by ELISA and immunohistochemistry. The inflammatory signals were identified by ELISA and western blotting. In the Clec5a knockdown microglial cell model and Clec5a knockout primary microglia, the microglial phagocytosis was revealed using the fluorescent-labeled Aß. RESULTS: The AD mice with Clec5a knockout improved Aß-induced memory deficit and abnormal nociception. These mice have reduced Aß deposition and increased microglia coverage surrounding the amyloid plaque, suggesting the involvement of CLEC5A in AD progression and Aß clearance. Moreover, the phagocytosis was also increased in the Aß-stressed Clec5a knockdown microglial cell lines and Clec5a knockout primary microglia. CONCLUSION: The Clec5a knockout ameliorates AD-like deficits by modulating microglial Aß clearance. This study implies that targeting microglial Clec5a could offer a promising approach to mitigate AD progression.

Protein acetylation microarray reveals that NuA4 controls key metabolic target regulating gluconeogenesis.

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) conduct many critical functions through nonhistone substrates in metazoans, but only chromatin-associated nonhistone substrates are known in Saccharomyces cerevisiae. Using yeast proteome microarrays, we identified and validated many nonchromatin substrates of the essential nucleosome acetyltransferase of H4 (NuA4) complex. Among these, acetylation sites (Lys19 and 514) of phosphoenolpyruvate carboxykinase (Pck1p) were determined by tandem mass spectrometry. Acetylation at Lys514 was crucial for enzymatic activity and the ability of yeast cells to grow on nonfermentable carbon sources. Furthermore, Sir2p deacetylated Pck1p both in vitro and in vivo. Loss of Pck1p activity blocked the extension of yeast chronological life span caused by water starvation. In human hepatocellular carcinoma (HepG2) cells, human Pck1 acetylation and glucose production were dependent on TIP60, the human homolog of ESA1. Our findings demonstrate a regulatory function for the NuA4 complex in glucose metabolism and life span by acetylating a critical metabolic enzyme.

Functional dissection of lysine deacetylases reveals that HDAC1 and p300 regulate AMPK.

First identified as histone-modifying proteins, lysine acetyltransferases (KATs) and deacetylases (KDACs) antagonize each other through modification of the side chains of lysine residues in histone proteins. Acetylation of many non-histone proteins involved in chromatin, metabolism or cytoskeleton regulation were further identified in eukaryotic organisms, but the corresponding enzymes and substrate-specific functions of the modifications are unclear. Moreover, mechanisms underlying functional specificity of individual KDACs remain enigmatic, and the substrate spectra of each KDAC lack comprehensive definition. Here we dissect the functional specificity of 12 critical human KDACs using a genome-wide synthetic lethality screen in cultured human cells. The genetic interaction profiles revealed enzyme-substrate relationships between individual KDACs and many important substrates governing a wide array of biological processes including metabolism, development and cell cycle progression. We further confirmed that acetylation and deacetylation of the catalytic subunit of the adenosine monophosphate-activated protein kinase (AMPK), a critical cellular energy-sensing protein kinase complex, is controlled by the opposing catalytic activities of HDAC1 and p300. Deacetylation of AMPK enhances physical interaction with the upstream kinase LKB1, leading to AMPK phosphorylation and activation, and resulting in lipid breakdown in human liver cells. These findings provide new insights into previously underappreciated metabolic regulatory roles of HDAC1 in coordinating nutrient availability and cellular responses upstream of AMPK, and demonstrate the importance of high-throughput genetic interaction profiling to elucidate functional specificity and critical substrates of individual human KDACs potentially valuable for therapeutic applications.

Foetal bovine serum can reduce toxicity of indocyanine green, brilliant blue G and trypan blue in ARPE-19 cellular model that suggests new surgical staining protocols for internal limiting membrane peeling procedure.

BACKGROUND: To investigate and compare the cytotoxicity of indocyanine green (ICG), brilliant blue G (BBG) and trypan blue (TB) using ARPE-19 cells that have been pre-treated/post-treated with balanced salt solution (BSS) or foetal bovine serum (FBS). METHODS: The cultured human retina pigment epithelium ARPE-19 cells were pre-treated/post-treated with BSS or FBS (represent the autologous serum in clinic) in parallel with cells being soaked with various concentrations of ICG, BBG and TB. The cells were then assessed for viability, growth rate, reactive oxygen species (ROS) level, mitochondrial membrane potential (Δψ) and mitochondrial mass as cytotoxic indices. For the FBS pre-treated cells, only ROS was examined. RESULTS: Using the MTT assay, cytotoxicity seemed to appear when the dye concentration was above 2.5 mg/mL for ICG but no cytotoxicity for BBG and TB at the concentrations used. Cell growth was arrested at a concentration 1 mg/mL when ICG or BBG were present but no arrest at any of the tested concentrations was found for TB with the cell-growth curve was slowest for ICG. Cellular ROS levels increased at all concentrations of all dyes, but the increasing slopes were decreased after FBS post-treatment washout. CONCLUSIONS: As a rinse buffer FBS performs much better than BSS in terms of cell rescue, which agrees with a clinical report when autologous whole blood was applied to macular hole surgery. However, FBS pre-treatment seems to be much better than FBS use as washout buffer in post-treatment.

Motor skill learning enhances the expression of activity-regulated cytoskeleton-associated protein in the rat cerebellum.

Motor skill learning is essential for environmental adaptations during everyday life. It has been shown that the cerebellum plays an important role in both the adaptation of eye movements and the motor skill learning. However, the neuronal substrates responsible for consolidation of complex motor skills rather than simple reflexes are still uncertain. Because the induction of immediate-early genes activity-regulated cytoskeleton-associated protein (Arc) and zinc finger binding protein clone 268 (Zif268) has been regarded as a marker for recent neuronal activity, therefore, in the present study, a rat paradigm of motor skill learning was used to investigate the protein expression of Arc and zif268 in the cerebellum after motor skill learning. Rats were trained to traverse the runway apparatus for 5 days. Protein samples were collected from the cerebellar cortices 1 hour after the training on days 1, 3, and 5, and analyzed by western blotting. The results showed that the expression of Arc, but not zif268, was significantly increased in the cerebellum following motor skill learning. These findings suggest that motor skill learning induces Arc expression in the cerebellum, which may play a role in acquiring complex motor skills.

Profiling lipid-protein interactions using nonquenched fluorescent liposomal nanovesicles and proteome microarrays.

Fluorescent liposomal nanovesicles (liposomes) are commonly used for lipid research and/or signal enhancement. However, the problem of self-quenching with conventional fluorescent liposomes limits their applications because these liposomes must be lysed to detect the fluorescent signals. Here, we developed a nonquenched fluorescent (NQF)1 liposome by optimizing the proportion of sulforhodamine B (SRB) encapsulant and lissamine rhodamine B-dipalmitoyl phosphatidylethanol (LRB-DPPE) on a liposomal surface for signal amplification. Our study showed that 0.3% of LRB-DPPE with 200 µm of SRB provided the maximal fluorescent signal without the need to lyse the liposomes. We also observed that the NQF liposomes largely eliminated self-quenching effects and produced greatly enhanced signals than SRB-only liposomes by 5.3-fold. To show their application in proteomics research, we constructed NQF liposomes that contained phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and profiled its protein interactome using a yeast proteome microarray. Our profiling led to the identification of 162 PI(3,5)P2-specific binding proteins (PI(3,5)P2-BPs). We not only recovered many proteins that possessed known PI(3,5)P2-binding domains, but we also found two unknown Pfam domains (Pfam-B_8509 and Pfam-B_10446) that were enriched in our dataset. The validation of many newly discovered PI(3,5)P2-BPs was performed using a bead-based affinity assay. Further bioinformatics analyses revealed that the functional roles of 22 PI(3,5)P2-BPs were similar to those associated with PI(3,5)P2, including vesicle-mediated transport, GTPase, cytoskeleton, and kinase. Among the 162 PI(3,5)P2-BPs, we found a novel motif, HRDIKP[ES]NJLL that showed statistical significance. A docking simulation showed that PI(3,5)P2 interacted primarily with lysine or arginine side chains of the newly identified PI(3,5)P2-binding kinases. Our study showed that this new tool would greatly benefit profiling lipid-protein interactions in high-throughput studies.

Disparities in Survival Outcomes Between Locally Advanced Cervical Squamous Cell Carcinoma and Adenocarcinoma Treated with Chemoradiotherapy.

Purpose: To determine the disparities in survival outcomes between stage IIB-IVA cervical squamous cell carcinoma (SCC) and adenocarcinoma (AC) treated with chemoradiotherapy. Methods: Patients diagnosed between 2004 and 2015 were retrospectively included from the Surveillance, Epidemiology, and End Results databases. Propensity score matching (PSM) was used in this study. The primary endpoints were cervical cancer-specific survival (CCSS) and overall survival (OS). Results: A total of 2752 patients were identified, including 87.5% (n=2408) were SCC and 12.5% (n=344) were AC. Patients with AC had inferior 5-year CCSS (67.5% vs 54.8%, P<0.001) and OS (58.4% vs 47.2%, P<0.001) compared to those with the SCC subtype. The hazard curve of cervical cancer-related death in AC peaked at 2 years (19%) and still small peaks in the 7 and 11 years of follow-up. Regarding SCC, cervical cancer-related deaths peaked at 2 years (15%) and the hazard rate was 2.0% during the six years of follow-up. The multivariate Cox regression analyses indicated that histology was an independent prognostic factor associated with survival outcomes. Patients with AC had significantly poor CCSS (P<0.001) and OS (P<0.001). Similar results were found after PSM. Conclusion: Our study demonstrates a significantly better prognosis for cervical SCC patients compared to those with cervical AC undergoing chemoradiotherapy. These results highlight the importance of histological subtyping in predicting treatment outcomes and tailoring therapeutic strategies.

Utilization and outcomes of the 21-gene recurrence score in de novo metastatic breast cancer.

BACKGROUND: Limited data exist regarding the utility and validity of the 21-gene recurrence score (RS) in patients with de novo metastatic breast cancer (dnMBC). This study aimed to investigate the practice patterns as well as associated survival outcomes based on 21-gene RS in dnMBC. RESEARCH DESIGN AND METHODS: The Surveillance, Epidemiology, and End Results Oncotype database was queried for women with hormone receptor-positive and Her2-negative dnMBC. RESULTS: A total of 153 patients were identified, including 62.7% and 37.3% of patients who had RS < 26 and ≥ 26, respectively. Patients with RS ≥ 26 were more likely to receive chemotherapy compared to those with RS < 26 (61.4% vs. 28.1%, p < 0.001). Patients with RS ≥ 26 had an inferior breast cancer-specific survival (BCSS) (2-year BCSS: 84.3% vs. 89.5, p = 0.067) and overall survival (OS) compared to those with RS < 26 (2-year OS: 76.9% vs. 87.4%, p = 0.018). The multivariate Cox proportional hazard models showed that those with RS ≥ 26 had a significantly inferior BCSS (hazard ratio [HR] 2.251, 95% confidence interval [CI] 1.056-4.799, p = 0.036) and OS (HR 2.151, 95%CI 1.123-4.120, p = 0.021) compared to those with RS < 26. CONCLUSIONS: The 21-gene RS assay is an important prognostic factor in patients with dnMBC.

Prediction of Visual Impairment in Epiretinal Membrane and Feature Analysis: A Deep Learning Approach Using Optical Coherence Tomography.

PURPOSE: The aim was to develop a deep learning model for predicting the extent of visual impairment in epiretinal membrane (ERM) using optical coherence tomography (OCT) images, and to analyze the associated features. METHODS: Six hundred macular OCT images from eyes with ERM and no visually significant media opacity or other retinal diseases were obtained. Those with best-corrected visual acuity ≤20/50 were classified as "profound visual impairment," while those with best-corrected visual acuity >20/50 were classified as "less visual impairment." Ninety percent of images were used as the training data set and 10% were used for testing. Two convolutional neural network models (ResNet-50 and ResNet-18) were adopted for training. The t-distributed stochastic neighbor-embedding approach was used to compare their performances. The Grad-CAM technique was used in the heat map generative phase for feature analysis. RESULTS: During the model development, the training accuracy was 100% in both convolutional neural network models, while the testing accuracy was 70% and 80% for ResNet-18 and ResNet-50, respectively. The t-distributed stochastic neighbor-embedding approach found that the deeper structure (ResNet-50) had better discrimination on OCT characteristics for visual impairment than the shallower structure (ResNet-18). The heat maps indicated that the key features for visual impairment were located mostly in the inner retinal layers of the fovea and parafoveal regions. CONCLUSIONS: Deep learning algorithms could assess the extent of visual impairment from OCT images in patients with ERM. Changes in inner retinal layers were found to have a greater impact on visual acuity than the outer retinal changes.

Detection of gastroesophageal reflux esophagitis using 2-fluoro-2-deoxy-d-glucose positron emission tomography.

BACKGROUND: Gastroesophageal reflux disease (GERD) is a common disease and a major upper gastrointestinal problem. The purpose of the present study is to evaluate the use of noninvasive 2-fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET) to detect gastroesophageal reflux esophagitis. MATERIALS AND METHODS: This is a retrospective study reviewing 408 healthy check-up subjects (169 females and 239 men), who underwent both FDG-PET and upper gastrointestinal endoscopy during September 2008 to December 2009. Quantitative analysis of FDG uptake in the distal part of the esophagus was performed by calculating the maximum standard uptake value (SUVmax). This indicated the degree of esophagitis. FDG-PET findings were compared with endoscopic (modified version of the Los Angeles classification) diagnoses as the gold standard. RESULTS: The SUVmax ranged from 1.30 to 3.40 in normal subjects and from 1.30 to 4.00 in subjects with gastroesophageal reflux esophagitis. In the esophagitis group, the SUVmax was 2.13 ± 0.42 in subjects with modified LA grade M, 2.21 ± 0.45 in subjects with LA grade A, and 2.48 ± 0.44 in subjects with LA grade B and C gastroesophageal reflux esophagitis. One-way ANOVA and post-hoc comparison with Bonferroni correction (P value = 0.003) identified statistical differences between the three groups. CONCLUSION: Noninvasive FDG-PET may be useful in the detection and evaluation of various degrees of gastroesophageal reflux esophagitis.

Phonetic posteriorgram-based voice conversion system to improve speech intelligibility of dysarthric patients.

BACKGROUND AND OBJECTIVE: Most dysarthric patients encounter communication problems due to unintelligible speech. Currently, there are many voice-driven systems aimed at improving their speech intelligibility; however, the intelligibility performance of these systems are affected by challenging application conditions (e.g., time variance of patient's speech and background noise). To alleviate these problems, we proposed a dysarthria voice conversion (DVC) system for dysarthric patients and investigated the benefits under challenging application conditions. METHOD: A deep learning-based voice conversion system with phonetic posteriorgram (PPG) features, called the DVC-PPG system, was proposed in this study. An objective-evaluation metric of Google automatic speech recognition (Google ASR) system and a listening test were used to demonstrate the speech intelligibility benefits of DVC-PPG under quiet and noisy test conditions; besides, the well-known voice conversion system using mel-spectrogram, DVC-Mels, was used for comparison to verify the benefits of the proposed DVC-PPG system. RESULTS: The objective-evaluation metric of Google ASR showed the average accuracy of two subjects in the duplicate and outside test conditions while the DVC-PPG system provided higher speech recognitions rate (83.2% and 67.5%) than dysarthric speech (36.5% and 26.9%) and DVC-Mels (52.9% and 33.8%) under quiet conditions. However, the DVC-PPG system provided more stable performance than the DVC-Mels under noisy test conditions. In addition, the results of the listening test showed that the speech-intelligibility performance of DVC-PPG was better than those obtained via the dysarthria speech and DVC-Mels under the duplicate and outside conditions, respectively. CONCLUSIONS: The objective-evaluation metric and listening test results showed that the recognition rate of the proposed DVC-PPG system was significantly higher than those obtained via the original dysarthric speech and DVC-Mels system. Therefore, it can be inferred from our study that the DVC-PPG system can improve the ability of dysarthric patients to communicate with people under challenging application conditions.

Plasmid-chromosome shuffling for non-deletion alleles in yeast.

Here we describe a facile plasmid-chromosome shuffling technique for generating and analyzing non-deletion alleles in the yeast Saccharomyces cerevisiae. This technique takes advantage of an existing set of genome-wide haploid-convertible heterozygous diploid yeast knockout mutants. This simple method will facilitate characterization of essential gene functions and genome-wide investigation of protein structure-function relationships.

[Application of laser speckle contrast imaging technology to researches on acupuncture and microcirculation].

The therapeutic effect of acupuncture is often closely related to the improvement of microcirculation. Acupuncture stimulation can adjust pathophysiological changes of microcirculation in the human body through multiple ways, multiple links and multiple levels, healing disorders at last. Laser speckle contrast imaging (LSCI), an emerging technology, can be used to study the effect of acupuncture on microcirculation. In this paper, retrieving the related literature published in recent years, we found that LSCI technology has already been applied to the following aspects 1) acupoint-viscera and microcirculation, 2) stimulation-induced transmission of sensations along the meridian and microcirculation, 3) non-meridians or non-acupoints and microcirculation, 4) specific acupoints and microcirculation, as well as 5) different acupuncture needle manipulations and microcirculation. However, most of the studies focused on animal experiments and healthy subjects, observing microcirculatory effects under normal physiological conditions, and the sample size of participants was smaller. In the future, we highly recommend that increasing the sample size and further studying the local microcirculation are necessary in the pathological state of diseases.

Deep-learning-based human motion tracking for rehabilitation applications using 3D image features.

Motion rehabilitation is increasingly required owing to an aging population and suffering of stroke, which means human motion analysis must be valued. Based on the concept mentioned above, a deep-learning-based system is proposed to track human motion based on three-dimensional (3D) images in this work; meanwhile, the features of traditional red green blue (RGB) images, known as two-dimensional (2D) images, were used as a comparison. The results indicate that 3D images have an advantage over 2D images due to the information of spatial relationships, which implies that the proposed system can be a potential technology for human motion analysis applications.

In Vivo Real-Time Discrimination Among Glioma, Infiltration Zone, and Normal Brain Tissue via Autofluorescence Technology.

BACKGROUND: Surgery is the first-line therapy for glioblastoma. There is evidence that extent of resection is significantly associated with patient survival. Unfortunately, optimal surgical resection is usually limited because of the difficulty in discriminating tumor-infiltrated region and normal brain tissue. This study aimed to develop a tool to distinguish between infiltration zone and normal tissue in real time during glioma surgery. METHODS: In an in vivo study, C6 glioma cells were implanted into the left cerebral hemispheres of 6 rats to mimic tumorigenesis. A newly designed optical fiber-embedded needle probe was used to measure the autofluorescence of both cerebral hemispheres at various depths 5 days after the implantation. These rats were then sacrificed, and both cerebral hemispheres were removed for histopathologic analysis. RESULTS: Comparative analyses of corresponding areas by histopathology and autofluorescence revealed highly significant (P < 0.001) differences among the normal tissue, infiltration zone, tumors, and the contralateral cerebral hemispheres. The area of the receiver operating characteristic curve was 0.978, and the sensitivity and specificity of tumor delineation were 93.9% and 94.4%, respectively. CONCLUSIONS: The newly designed in vivo fiber-optic probe can distinguish tumor-infiltration zones from normal brain tissue in this in vivo study. Therefore, it may help neurosurgeons to increase extent of resection without damaging normal brain tissue and thus potentially improve the patients' survival and quality of life.

Curcuminoid submicron particle ameliorates cognitive deficits and decreases amyloid pathology in Alzheimer's disease mouse model.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder and is triggered via abnormal accumulation of amyloid-ß peptide (Aß). Aggregated Aß is responsible for disrupting calcium homeostasis, inducing neuroinflammation, and promoting neurodegeneration. In this study, we generated curcuminoid submicron particle (CSP), which reduce the average size to ~60 nm in diameter. CSP had elevated the bioavailability in vivo and better neuroprotective effect against oligomeric Aß than un-nanosized curcuminoids in vitro. Two months of CSP consumption reversed spatial memory deficits and the loss of a calcium binding protein calbindin-D28k in the hippocampus of AD mouse model. In addition, CSP consumption lowered amyloid plaques and astrogliosis in vivo and enhanced microglial Aß phagocytosis in vitro, implying that the beneficial effects of CSP also mediated via modulating neuroinflammation and enhancing amyloid clearance. Taken together, our study demonstrated the protective effects of CSP toward ameliorating the memory impairment and pathological deficits in AD mouse model.

Amelioration of amyloid-ß-induced deficits by DcR3 in an Alzheimer's disease model.

BACKGROUND: Microglia mediate amyloid-beta peptide (Aß)-induced neuroinflammation, which is one of the key events in the pathogenesis of Alzheimer's disease (AD). Decoy receptor 3 (DcR3)/TNFRSF6B is a pleiotropic immunomodulator that promotes macrophage differentiation toward the M2 anti-inflammatory phenotype. Based on its role as an immunosupressor, we examined whether DcR3 could alleviate neuroinflammation and AD-like deficits in the central nervous system. METHOD: We crossed human APP transgenic mice (line J20) with human DcR3 transgenic mice to generate wild-type, APP, DcR3, and APP/DcR3 mice for pathological analysis. The Morris water maze, fear conditioning test, open-field, and elevated-plus maze were used to access their cognitive behavioral changes. Furthermore, the pathological and immune profiles were examined by immunostaining, ELISA, Q-PCR, and IP. In vitro assays were designed to examine DcR3-mediated innate cytokine profile alteration and the potential protective mechanism. RESULTS: We reported that DcR3 ameliorates hippocampus-dependent memory deficits and reduces amyloid plaque deposition in APP transgenic mouse. The protective mechanism of DcR3 mediates through interacting with heparan sulfate proteoglycans and activating IL-4+YM1+ M2a-like microglia that reduces Aß-induced proinflammatory cytokines and promotes phagocytosis ability of microglia. CONCLUSION: The neuroprotective effect of DcR3 is mediated via modulating microglia activation into anti-inflammatory M2a phenotype, and upregulating DcR3 expression in the brain may be a potential therapeutic approach for AD.