Nitric oxide releasing nanofiber stimulates revascularization in response to ischemia via cGMP-dependent protein kinase.

Nitric oxide (NO) promotes angiogenesis via various mechanisms; however, the effective transmission of NO in ischemic diseases is unclear. Herein, we tested whether NO-releasing nanofibers modulate therapeutic angiogenesis in an animal hindlimb ischemia model. Male wild-type C57BL/6 mice with surgically-induced hindlimb ischemia were treated with NO-releasing 3-methylaminopropyltrimethoxysilane (MAP3)-derived or control (i.e., non-NO-releasing) nanofibers, by applying them to the wound for 20 min, three times every two days. The amount of NO from the nanofiber into tissues was assessed by NO fluorometric assay. The activity of cGMP-dependent protein kinase (PKG) was determined by western blot analysis. Perfusion ratios were measured 2, 4, and 14 days after inducing ischemia using laser doppler imaging. On day 4, Immunohistochemistry (IHC) with F4/80 and gelatin zymography were performed. IHC with CD31 was performed on day 14. To determine the angiogenic potential of NO-releasing nanofibers, aorta-ring explants were treated with MAP3 or control fiber for 20 min, and the sprout lengths were examined after 6 days. As per either LDPI (Laser doppler perfusion image) ratio or CD31 capillary density measurement, angiogenesis in the ischemic hindlimb was improved in the MAP3 nanofiber group; further, the total nitrate/nitrite concentration in the adduct muscle increased. The number of macrophage infiltrations and matrix metalloproteinase-9 (MMP-9) activity decreased. Vasodilator-stimulated phosphoprotein (VASP), one of the major substrates for PKG, increased phosphorylation in the MAP3 group. MAP3 nanofiber or NO donor SNAP (s-nitroso-n-acetyl penicillamine)-treated aortic explants showed enhanced sprouting in an ex vivo aortic ring assay, which was partially abrogated by KT5823, a potent inhibitor of PKG. These findings suggest that the novel NO-releasing nanofiber, MAP3 activates PKG and promotes therapeutic angiogenesis in response to hindlimb ischemia.

One-Stop Plasmonic Nanocube-Excited SERS Immunoassay Platform of Multiple Cardiac Biomarkers for Rapid Screening and Progressive Tracing of Acute Myocardial Infarction.

Rapid and precise acute myocardial infarction (AMI) diagnosis is essential for preventing patient death. In addition, the complementary roles of creatine kinase muscle brain (CK-MB) and cardiac troponin I (cTnI) cardiac biomarkers in the early and late stages of AMI demand their simultaneous detection, which is difficult to implement using conventional fluorescence and electrochemical technologies. Here, a nanotechnology-based one-stop immuno-surface-enhanced Raman scattering (SERS) detection platform is reported for multiple cardiac indicators for the rapid screening and progressive tracing of AMI events. Optimal SERS is achieved using optical property-based, excitation wavelength-optimized, and high-yield anisotropic plasmonic gold nanocubes. Optimal immunoassay reaction efficiencies are achieved by increasing immobilized antibodies. Multiple simultaneous detection strategies are implemented by incorporating two different Raman reports with narrow wavenumbers corresponding to two indicators and by establishing a computational SERS mapping process to accurately detect their concentrations, irrespective of multiple enzymes in the human serum. The SERS platform precisely estimated AMI onset and progressive timing in human serum and made rapid AMI identification feasible using a portable Raman spectrometer. This integrated platform is hypothesized to significantly contribute to emergency medicine and forensic science by providing timely treatment and observation.

Regional classification of high PM10 concentrations in the Seoul metropolitan and Chungcheongnam-do areas, Republic of Korea.

Since the Seoul metropolitan area is a highly developed megacity, many people are often exposed to high concentrations of particulate matter (PM), with mean aerodynamic diameters equal to or less than 10 µm (PM10), in cold seasons. PM10 concentrations can be influenced by a combination of various factors, including meteorological conditions, anthropogenic emissions, atmospheric chemical reactions, transboundary transport, and geographic characteristics. However, the establishment of an efficient air quality management plan remains challenging due to the limited understanding of the regional PM concentration characteristics. Here, the Seoul metropolitan (Seoul, Gyeonggi-do, and Incheon) and Chungcheongnam-do (Chungcheongnam-do, Daejeon, and Sejong) areas were regionally classified to identify the spatiotemporal air quality in areas where megacities and emission sources are mixed. The four representative regions were determined using the K-means clustering method based on the temporal variations in the observed PM10 concentrations. The first cluster consisted of small cities in the southern and eastern parts of Gyeonggi-do and Chungcheongnam-do, respectively, while the second cluster consisted of Incheon, West Gyeonggi-do, and Seoul. In addition, the third and fourth clusters included West Chungcheongnam-do and East Gyeonggi-do, which are adjacent to the Yellow Sea and downstream area of the westerly wind, respectively. The characteristics of each cluster during the high PM10 concentration events are explainable by wind patterns and the local air pollutant emissions, including nitrogen oxides and sulfur oxides. The obtained regional classification was different from the provincial-level administrative division of South Korea. Therefore, the present study is expected to be a scientific basis for overcoming the limitations of air quality management in administrative districts.

Antiviral Mechanisms of Saucerneol from Saururus chinensis against Enterovirus A71, Coxsackievirus A16, and Coxsackievirus B3: Role of Mitochondrial ROS and the STING/TKB-1/IRF3 Pathway.

Enterovirus A71 (EV71), coxsackievirus A16 (CVA16), and coxsackievirus B3 (CVB3) are pathogenic members of the Picornaviridae family that cause a range of diseases, including severe central nervous system complications, myocarditis, and pancreatitis. Despite the considerable public health impact of these viruses, no approved antiviral treatments are currently available. In the present study, we confirmed the potential of saucerneol, a compound derived from Saururus chinensis, as an antiviral agent against EV71, CVA16, and CVB3. In the in vivo model, saucerneol effectively suppressed CVB3 replication in the pancreas and alleviated virus-induced pancreatitis. The antiviral activity of saucerneol is associated with increased mitochondrial ROS (mROS) production. In vitro inhibition of mROS generation diminishes the antiviral efficacy of saucerneol. Moreover, saucerneol treatment enhanced the phosphorylation of STING, TBK-1, and IRF3 in EV71- and CVA16-infected cells, indicating that its antiviral effects were mediated through the STING/TBK-1/IRF3 antiviral pathway, which was activated by increased mROS production. Saucerneol is a promising natural antiviral agent against EV71, CVA16, and CVB3 and has potential against virus-induced pancreatitis and myocarditis. Further studies are required to assess its safety and efficacy, which is essential for the development of effective antiviral strategies against these viruses.

Daeshiho-tang Attenuates Atherosclerosis by Regulating Cholesterol Metabolism and Inducing M2 Macrophage Polarization.

Dyslipidemia, the commonest cause of cardiovascular disease, leads to lipid deposits on the arterial wall, thereby aggravating atherosclerosis. DSHT (Daeshiho-tang) has long been used as an anti-dyslipidemia agent in oriental medicine. However, the anti-atherosclerotic effects of DSHT have not been fully investigated. Therefore, this study was designed to evaluate whether DSHT could exert beneficial anti-atherosclerotic effects. We fed apolipoprotein E-deficient (ApoE-/-) mice on a high-fat diet and treated them with atorvastatin (AT) or DSHT, or the combination of DSHT and AT for 12 weeks. To determine the role of DSHT, atherosclerotic lesions in the aorta, aortic root, and aortic arch; lipids and apolipoprotein levels in serum; and macrophage polarization markers in aorta tissues were examined. We show here that the DSHT decreased the atherosclerotic plaque ratio in the aortic arch, aorta, and aortic root. DSHT also regulated lipid levels by decreasing the ApoB level and increasing the ApoA1 level. Moreover, DSHT effectively regulated cholesterol metabolism by increasing the levels of PPARγ, ABCA1 and ABCG1, and the LDL receptor genes. We further found that DSHT promoted polarization to the M2 phenotype by increasing the levels of M2 macrophage (ARG1, CD163, and PPARγ) markers. Our data suggested that DSHT enhances the anti-atherosclerotic effect by regulating cholesterol metabolism through the activation of the PPARγ signaling pathway and by promoting anti-inflammatory M2 macrophage polarization.

The sGC-cGMP Signaling Pathway as a Potential Therapeutic Target in Doxorubicin-Induced Heart Failure: A Narrative Review.

The anti-cancer agent doxorubicin (DOX) has high cardiotoxicity that is linked to DOX-mediated increase in oxidative stress, mitochondrial iron overload, DNA damage, autophagy, necrosis, and apoptosis, all of which are also associated with secondary tumorigenicity. This limits the clinical application of DOX therapies. Previous studies have attributed DOX-mediated cardiotoxicity to mitochondrial iron accumulation and the production of reactive oxygen species (ROS), which seem to be independent of its anti-tumor DNA damaging effects. Chemo-sensitization of soluble guanylate cyclase (sGC) in the cyclic guanosine monophosphate (cGMP) pathway induces tumor cell death despite the cardiotoxicity associated with DOX treatment. However, sGC-cGMP signaling must be activated during heart failure to facilitate myocardial cell survival. The sGC pathway is dependent on nitric oxide and signal transduction via the nitric oxide-sGC-cGMP pathway and is attenuated in various cardiovascular diseases. Additionally, cGMP signaling is regulated by the action of certain phosphodiesterases (PDEs) that protect the heart by inhibiting PDE, an enzyme that hydrolyses cGMP to GMP activity. In this review, we discuss the studies describing the interactions between cGMP regulation and DOX-mediated cardiotoxicity and their application in improving DOX therapeutic outcomes. The results provide novel avenues for the reduction of DOX-induced secondary tumorigenicity and improve cellular autonomy during DOX-mediated cardiotoxicity.

Colorimetric determination of phenolic compounds using peroxidase mimics based on biomolecule-free hybrid nanoflowers consisting of graphitic carbon nitride and copper.

Hybrid nanoflowers consisting of graphitic carbon nitride (GCN) and copper were successfully constructed without the involvement of any biomolecule, by simply mixing them at room temperature to induce proper self-assembly to achieve a flower-like morphology. The resulting biomolecule-free GCN-copper hybrid nanoflowers (GCN-Cu NFs) exhibited an apparent peroxidase-mimicking activity, possibly owing to the synergistic effect from the coordination of GCN and copper, as well as their large surface area, which increased the number of catalytic reaction sites. The peroxidase-mimicking GCN-Cu NFs were then employed in the colorimetric determination of selected phenolic compounds hydroquinone (HQ), methylhydroquinone (MHQ), and catechol (CC). For samples without phenolic compounds, GCN-Cu NFs catalyzed the oxidation of the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2, producing an intense blue color signal. Conversely, in the presence of phenolic compounds, the oxidation of TMB was inhibited, resulting in a significant reduction of the color signal. Using this strategy, HQ, MHQ, and CC were selectively and sensitively determined in a linear range up to 100 µM with detection limits down to 0.82, 0.27, and 0.36 µM, respectively. The practical utility of this assay system was also validated by using it to detect phenolic compounds spiked in tap water, yielding a good recovery of 97.1-108.9% and coefficient of variation below 3.0%, demonstrating the excellent reliability and reproducibility of this strategy. Colorimetric determination of phenolic compounds using peroxidase mimics based on biomolecule-free hybrid nanoflowers consisting of graphitic carbon nitride and copper.

Potential neuron-autonomous Purkinje cell degeneration by 2',3'-cyclic nucleotide 3'-phosphodiesterase promoter/Cre-mediated autophagy impairments.

Studies of neuroglial interaction largely depend on cell-specific gene knockout (KO) experiments using Cre recombinase. However, genes known as glial-specific genes have recently been reported to be expressed in neuroglial stem cells, leading to the possibility that a glia-specific Cre driver results in unwanted gene deletion in neurons, which may affect sound interpretation. 2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) is generally considered to be an oligodendrocyte (OL) marker. Accordingly, Cnp promoter-controlled Cre recombinase has been used to create OL-specific gene targeting mice. However, in this study, using Rosa26-tdTomato-reporter/Cnp-Cre mice, we found that many forebrain neurons and cerebellar Purkinje neurons belong to the lineages of Cnp-expressing neuroglial stem cells. To answer whether gene targeting by Cnp-Cre can induce neuron-autonomous defects, we conditionally deleted an essential autophagy gene, Atg7, in Cnp-Cre mice. The Cnp-Cre-mediated Atg7 KO mice showed extensive p62 inclusion in neurons, including cerebellar Purkinje neurons with extensive neurodegeneration. Furthermore, neuronal areas showing p62 inclusion in Cnp-Cre-mediated Atg7 KO mice overlapped with the neuronal lineage of Cnp-expressing neuroglial stem cells. Moreover, Cnp-Cre-mediated Atg7-KO mice did not develop critical defects in myelination. Our results demonstrate that a large population of central neurons are derived from Cnp-expressing neuroglial stem cells; thus, conditional gene targeting using the Cnp promoter, which is known to be OL-specific, can induce neuron-autonomous phenotypes.

Tyrosine Phosphorylation of the Kv2.1 Channel Contributes to Injury in Brain Ischemia.

In brain ischemia, oxidative stress induces neuronal apoptosis, which is mediated by increased activity of the voltage-gated K+ channel Kv2.1 and results in an efflux of intracellular K+. The molecular mechanisms underlying the regulation of Kv2.1 and its activity during brain ischemia are not yet fully understood. Here this study provides evidence that oxidant-induced apoptosis resulting from brain ischemia promotes rapid tyrosine phosphorylation of Kv2.1. When the tyrosine phosphorylation sites Y124, Y686, and Y810 on the Kv2.1 channel are mutated to non-phosphorylatable residues, PARP-1 cleavage levels decrease, indicating suppression of neuronal cell death. The tyrosine residue Y810 on Kv2.1 was a major phosphorylation site. In fact, cells mutated Y810 were more viable in our study than were wild-type cells, suggesting an important role for this site during ischemic neuronal injury. In an animal model, tyrosine phosphorylation of Kv2.1 increased after ischemic brain injury, with an observable sustained increase for at least 2 h after reperfusion. These results demonstrate that tyrosine phosphorylation of the Kv2.1 channel in the brain may play a critical role in regulating neuronal ischemia and is therefore a potential therapeutic target in patients with brain ischemia.

BAY60-2770 attenuates doxorubicin-induced cardiotoxicity by decreased oxidative stress and enhanced autophagy.

BACKGROUND: Doxorubicin (DOX) administration decreases cardiac soluble guanylate cyclase (sGC) activity. We hypothesized that bypassing impaired NO-sGC-cGMP pathway resulting from the activation of oxidized and heme-free soluble guanylate cyclase (sGC) could be a therapeutic target for DOX-mediated cardiomyopathy (DOX-CM). The present study investigated the therapeutic roles and mechanism of BAY60-2770, an activator of oxidized sGC, in alleviating DOX-CM. METHODS: H9c2 cardiomyocytes were pretreated with BAY60-2770 followed by DOX. Cell viability and intracellular reactive oxygen species (ROS) were subsequently measured. To determine the role BAY60-2770 in mitochondrial ROS generation and mitochondrial membrane potential, we examined mitoSOX RED and TMRE fluorescence under DOX exposure. As animal experiments, rats were orally administered with 5 mg/kg of BAY60-2770 at 1 h prior to every DOX treatment and then assessed by echocardiography and apoptotic marker and autophagy. RESULTS: BAY60-2770 ameliorated cell viability and DOX-induced oxidative stress in H9c2 cells, which was mediated by PKG activation. Mitochondrial ROS and TMRE fluorescence were attenuated by BAY60-2770 in DOX-treated H9c2 cells. DOX-induced caspase-3 activation decreased after pretreatment with BAY60-2770 in vivo and in vitro. Echocardiography showed that BAY60-2770 significantly improved DOX-induced myocardial dysfunction. Autophagosome was increased by BAY60-2770 in vivo. CONCLUSIONS: BAY60-2770 appears to mitigate DOX-induced mitochondrial ROS, membrane potential loss, autophagy, and subsequent apoptosis, leading to protection of myocardial injury and dysfunction. These novel results highlighted the therapeutic potential of BAY60-2770 in preventing DOX-CM.

Boosting Visible-Light Photocatalytic Redox Reaction by Charge Separation in SnO2 /ZnSe(N2 H4 )0.5 Heterojunction Nanocatalysts.

In this work, environmentally friendly photocatalysts with attractive catalytic properties are reported that have been prepared by introducing SnO2 quantum dots (QDs) directly onto ZnSe(N2 H4 )0.5 substrates to induce advantageous charge separation. The SnO2 /ZnSe(N2 H4 )0.5 nanocomposites could be easily synthesized through a one-pot hydrothermal process. Owing to the absence of capping ligands, the attached SnO2 QDs displayed superior photocatalytic properties, generating many exposed reactive surfaces. Moreover, the addition of a specified amount of SnO2 boosted the visible-light photocatalytic activity; however, the presence of excess SnO2 QDs in the substrate resulted in aggregation and deteriorated the performance. The spectroscopic data revealed that the SnO2 QDs act as a photocatalytic mediator and enhance the charge separation within the type II band alignment system of the SnO2 /ZnSe(N2 H4 )0.5 heterojunction photocatalysts. The separated charges in the heterojunction nanocomposites promote radical generation and react with pollutants, resulting in enhanced photocatalytic performance.

Pi5 and Pii Paired NLRs Are Functionally Exchangeable and Confer Similar Disease Resistance Specificity.

Effector-triggered immunity (ETI) is an effective layer of plant defense initiated upon recognition of avirulence (Avr) effectors from pathogens by cognate plant disease resistance (R) proteins. In rice, a large number of R genes have been characterized from various cultivars and have greatly contributed to breeding programs to improve resistance against the rice blast pathogen Magnaporthe oryzae. The extreme diversity of R gene repertoires is thought to be a result of co-evolutionary history between rice and its pathogens including M. oryzae. Here we show that Pii is an allele of Pi5 by DNA sequence characterization and complementation analysis. Pii-1 and Pii-2 cDNAs were cloned by reverse transcription polymerase chain reaction from the Pii -carrying cultivar Fujisaka5 . The complementation test in susceptible rice cultivar Dongjin demonstrated that the rice blast resistance mediated by Pii , similar to Pi5 , requires the presence of two nucleotide-binding leucine-rich repeat genes, Pii-1 and Pii-2 . Consistent with our hypothesis that Pi5 and Pii are functionally indistinguishable, the replacement of Pii-1 by Pi5-1 and Pii-2 by Pi5-2 , respectively, does not change the level of disease resistance to M. oryzae carrying AVR-Pii. Surprisingly, Exo70F3, required for Pii-mediated resistance, is dispensable for Pi5-mediated resistance. Based on our results, despite similarities observed between Pi5 and Pii, we hypothesize that Pi5 and Pii pairs require partially distinct mechanisms to function.

Protein kinase A-induced phosphorylation at the Thr154 affects stability of DJ-1.

INTRODUCTION: Most cases of Parkinson's disease (PD) are sporadic, but genetic variations have been discovered in PD patients. PARK7/DJ-1 is a known cause of early-onset autosomal-recessive PD and is implicated in neuroprotection against oxidative stress. Although several post-translational modifications of DJ-1 have been proposed, phospho-modification of DJ-1 and its functional consequences have been less studied. METHODS: Putative phosphorylation sites of DJ-1 were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS analysis). Subsequently, phosphorylation site of DJ-1 was confirmed by in vitro kinase assay and cell-based pull-down assay. Impaired dimer formation of phospho-null mutant was measured using DSS crosslinking assay and immunoprecipitation assay. To evaluate physiological consequences of this event, protein stability of DJ-1 WT and DJ-1 phospho-null mutant were compared using cycloheximide chase assay and ubiquitination assay. RESULTS: Here, we showed that DJ-1 directly bound to the catalytic subunit of protein kinase A (PKAcα). We found that PKAcα is responsible for phosphorylation of DJ-1 at the T154 residue. Interestingly, dimerization of DJ-1 was not detected in a DJ-1 T154A mutant. Furthermore, stability of the DJ-1 T154A mutant was dramatically reduced compared with that of wild-type DJ-1. We found that DJ-1 T154A was prone to degradation by the ubiquitin proteasome system (UPS). CONCLUSION: We identified a novel phosphorylation site of DJ-1. Furthermore, we determined protein kinase A that is responsible for this posttranslational modification. Finally, we demonstrated physiological consequences of this event focusing on dimerization and protein stability of DJ-1.

p75 and neural cell adhesion molecule 1 can identify pathologic Schwann cells in peripheral neuropathies.

OBJECTIVE: Myelinated Schwann cells (SCs) in adult peripheral nerves dedifferentiate into immature cells in demyelinating neuropathies and Wallerian degeneration. This plastic SC change is actively involved in the myelin destruction and clearance as demyelinating SCs (DSCs). In inherited demyelinating neuropathy, pathologically differentiated and dysmyelinated SCs constitute the main nerve pathology. METHODS: We investigated whether this SC plastic status in human neuropathic nerves could be determined by patient sera to develop disease-relevant serum biomarkers. Based on proteomics analysis of the secreted exosomes from immature SCs, we traced p75 neurotrophin receptor (p75) and neural cell adhesion molecule 1 (NCAM) in the sera of patients with peripheral neuropathy. RESULTS: Enzyme-linked immunosorbent assay (ELISA) revealed that p75 and NCAM were subtype-specifically expressed in the sera of patients with peripheral neuropathy. In conjunction with these ELISA data, pathological analyses of animal models and human specimens suggested that the presence of DSCs in inflammatory neuropathy and of supernumerary nonmyelinating or dysmyelinating SCs in inherited neuropathy could potentially be distinguished by comparing the expression profiles of p75 and NCAM. INTERPRETATION: This study indicates that the identification of disease-specific pathological SC stages might be a valuable tool for differential diagnosis of peripheral neuropathies.

Removal of toluene using ozone at room temperature over mesoporous Mn/Al2O3 catalysts.

The catalytic oxidation of toluene with ozone at room temperature was carried out over hierarchically ordered mesoporous catalysts (CeO2 (meso), Mn2O3 (meso), ZrO2 (meso), and γ-Al2O3 (meso)) and Al2O3 with various textural properties and phases (γ-Al2O3 (meso), γ-Al2O3 (13 nm), and α-Al2O3) to examine the effects of the nature of the catalyst on the catalytic activity. The catalysts were characterized by N2-physisorption measurements, powder X-ray diffraction, temperature programmed reduction, X-ray photoelectron spectroscopy and scanning transmission electron microscopy with energy dispersive spectroscopy. Among the ordered mesoporous catalysts, γ-Al2O3 (meso) had the highest toluene removal efficiency because of its highest surface area and pore volume, which in turn was selected for further investigation. Manganese (Mn) was introduced to various Al2O3 to improve the toluene removal efficiency. Comparing the Mn-loaded catalysts supported on various Al2O3 with different crystalline phases or pore structures, Mn/γ-Al2O3 (meso), had the highest catalytic activity as well as the highest CO2/CO ratio. The higher activity was attributed to the larger surface area, weaker interaction between Mn and Al2O3, and larger portion of Mn2O3 phase. The increase in ozone concentration led to an improvement in the carbon balance but this enhancement was insufficient due to the deposition of by-products on the catalyst. After long term tests at room temperature, the reaction intermediates and carbonaceous deposits of the used catalysts were identified.

Acetaldehyde removal and increased H2/CO gas yield from biomass gasification over metal-loaded Kraft lignin char catalyst.

Acetaldehyde removal tests were performed to compare the catalytic activity of the Kraft lignin char (KC), KOH-treated Kraft lignin char (KKC), and activated carbon (AC) along with their impregnation with Mn in a plasma reactor. The gasification characteristics (syngas content, and H2/CO ratio) of yellow poplar were investigated using nickel catalysts supported on KC, KKC, AC, and γ-Al2O3 in a U-type quartz reactor. KKC and Mn/KKC improved significantly the surface area and contents of O and N functional groups over the raw char. In particular, Mn/KKC showed the highest acetaldehyde-removal efficiency. The catalytic activity of Ni-impregnated KC, KKC, AC, and γ-Al2O3 decreased in the order of Ni/KKC > Ni/AC > Ni/KC > Ni/γ-Al2O3 for the gas yield and Ni/γ-Al2O3 >Ni/KC > Ni/AC >Ni/KKC for the oil yield, respectively. The Ni/KKC provides a more conducive environment for gasification, resulting in larger amounts of syngas (H2 and CO) in the product gases. Moreover, Ni impregnated with char may be the most inexpensive and effective solution for achieving maximum tar reduction and syngas generation.

A Proteotranscriptomic-Based Computational Drug-Repositioning Method for Alzheimer's Disease.

Numerous clinical trials of drug candidates for Alzheimer's disease (AD) have failed, and computational drug repositioning approaches using omics data have been proposed as effective alternative approaches to the discovery of drug candidates. However, little multi-omics data is available for AD, due to limited availability of brain tissues. Even if omics data exist, systematic drug repurposing study for AD has suffered from lack of big data, insufficient clinical information, and difficulty in data integration on account of sample heterogeneity derived from poor diagnosis or shortage of qualified post-mortem tissue. In this study, we developed a proteotranscriptomic-based computational drug repositioning method named Drug Repositioning Perturbation Score/Class (DRPS/C) based on inverse associations between disease- and drug-induced gene and protein perturbation patterns, incorporating pharmacogenomic knowledge. We constructed a Drug-induced Gene Perturbation Signature Database (DGPSD) comprised of 61,019 gene signatures perturbed by 1,520 drugs from the Connectivity Map (CMap) and the L1000 CMap. Drugs were classified into three DRPCs (High, Intermediate, and Low) according to DRPSs that were calculated using drug- and disease-induced gene perturbation signatures from DGPSD and The Cancer Genome Atlas (TCGA), respectively. The DRPS/C method was evaluated using the area under the ROC curve, with a prescribed drug list from TCGA as the gold standard. Glioblastoma had the highest AUC. To predict anti-AD drugs, DRPS were calculated using DGPSD and AD-induced gene/protein perturbation signatures generated from RNA-seq, microarray and proteomic datasets in the Synapse database, and the drugs were classified into DRPCs. We predicted 31 potential anti-AD drug candidates commonly belonged to high DRPCs of transcriptomic and proteomic signatures. Of these, four drugs classified into the nervous system group of Anatomical Therapeutic Chemical (ATC) system are voltage-gated sodium channel blockers (bupivacaine, topiramate) and monamine oxidase inhibitors (selegiline, iproniazid), and their mechanism of action was inferred from a potential anti-AD drug perspective. Our approach suggests a shortcut to discover new efficacy of drugs for AD.

Cell-penetrating artificial mitochondria-targeting peptide-conjugated metallothionein 1A alleviates mitochondrial damage in Parkinson's disease models.

An excess of reactive oxygen species (ROS) relative to the antioxidant capacity causes oxidative stress, which plays a role in the development of Parkinson's disease (PD). Because mitochondria are both sites of ROS generation and targets of ROS damage, the delivery of antioxidants to mitochondria might prevent or alleviate PD. To transduce the antioxidant protein human metallothionein 1A (hMT1A) into mitochondria, we computationally designed a cell-penetrating artificial mitochondria-targeting peptide (CAMP). The recombinant CAMP-conjugated hMT1A fusion protein (CAMP-hMT1A) successfully localized to the mitochondria. Treating a cell culture model of PD with CAMP-hMT1A restored tyrosine hydroxylase expression and mitochondrial activity and reduced ROS production. Furthermore, injection of CAMP-hMT1A into the brain of a mouse model of PD rescued movement impairment and dopaminergic neuronal degeneration. CAMP-hMT1A delivery into mitochondria might be therapeutic against PD by alleviating mitochondrial damage, and we predict that CAMP could be used to deliver other cargo proteins to the mitochondria.

Identification and characterization of site-specific N-glycosylation in the potassium channel Kv3.1b.

The potassium ion channel Kv3.1b is a member of a family of voltage-gated ion channels that are glycosylated in their mature form. In the present study, we demonstrate the impact of N-glycosylation at specific asparagine residues on the trafficking of the Kv3.1b protein. Large quantities of asparagine 229 (N229)-glycosylated Kv3.1b reached the plasma membrane, whereas N220-glycosylated and unglycosylated Kv3.1b were mainly retained in the endoplasmic reticulum (ER). These ER-retained Kv3.1b proteins were susceptible to degradation, when co-expressed with calnexin, whereas Kv3.1b pools located at the plasma membrane were resistant. Mass spectrometry analysis revealed a complex type Hex3 HexNAc4 Fuc1 glycan as the major glycan component of the N229-glycosylated Kv3.1b protein, as opposed to a high-mannose type Man8 GlcNAc2 glycan for N220-glycosylated Kv3.1b. Taken together, these results suggest that trafficking-dependent roles of the Kv3.1b potassium channel are dependent on N229 site-specific glycosylation and N-glycan structure, and operate through a mechanism whereby specific N-glycan structures regulate cell surface expression.