Characterization of proteome profile data of chemicals based on data-independent acquisition MS with SWATH method.

Transcriptomic data of cultured cells treated with a chemical are widely recognized as useful numeric information that describes the effects of the chemical. This property is due to the high coverage and low arbitrariness of the transcriptomic data as profiles of chemicals. Considering the importance of posttranslational regulation, proteomic profiles could provide insights into the unrecognized aspects of the effects of chemicals. Therefore, this study aimed to address the question of how well the proteomic profiles obtained using data-independent acquisition (DIA) with the sequential window acquisition of all theoretical mass spectra, which can achieve comprehensive and arbitrariness-free protein quantification, can describe chemical effects. We demonstrated that the proteomic data obtained using DIA-MS exhibited favorable properties as profile data, such as being able to discriminate chemicals like the transcriptomic profiles. Furthermore, we revealed a new mode of action of a natural compound, harmine, through profile data analysis using the proteomic profile data. To our knowledge, this is the first study to investigate the properties of proteomic data obtained using DIA-MS as the profiles of chemicals. Our 54 (samples) × 2831 (proteins) data matrix would be an important source for further analyses to understand the effects of chemicals in a data-driven manner.

pH-Responsive Lipid Nanoparticles Achieve Efficient mRNA Transfection in Brain Capillary Endothelial Cells.

The blood-brain barrier (BBB), which is comprised of brain capillary endothelial cells, plays a pivotal role in the transport of drugs from the blood to the brain. Therefore, an analysis of proteins in the endothelial cells, such as transporters and tight junction proteins, which contribute to BBB function, is important for the development of therapeutics for the treatment of brain diseases. However, gene transfection into the vascular endothelial cells of the BBB is fraught with difficulties, even in vitro. We report herein on the development of lipid nanoparticles (LNPs), in which mRNA is encapsulated in a nano-sized capsule composed of a pH-activated and reductive environment-responsive lipid-like material (ssPalm). We evaluated the efficiency of mRNA delivery into non-polarized human brain capillary endothelial cells, hCMEC/D3 cells. The ssPalm LNPs permitted marker genes (GFP) to be transferred into nearly 100% of the cells, with low toxicity in higher concentration. A proteomic analysis indicated that the ssPalm-LNP had less effect on global cell signaling pathways than a Lipofectamine MessengerMAX/GFP-encoding mRNA complex (LFN), a commercially available transfection reagent, even at higher mRNA concentrations.

Upregulation of ribosome complexes at the blood-brain barrier in Alzheimer's disease patients.

The cerebrovascular-specific molecular mechanism in Alzheimer's disease (AD) was investigated by employing comprehensive and accurate quantitative proteomics. Highly purified brain capillaries were isolated from cerebral gray and white matter of four AD and three control donors, and examined by SWATH (sequential window acquisition of all theoretical fragment ion spectra) proteomics. Of the 29 ribosomal proteins that were quantified, 28 (RPLP0, RPL4, RPL6, RPL7A, RPL8, RPL10A, RPL11, RPL12, RPL14, RPL15, RPL18, RPL23, RPL27, RPL27A, RPL31, RPL35A, RPS2, RPS3, RPS3A, RPS4X, RPS7, RPS8, RPS14, RPS16, RPS20, RPS24, RPS25, and RPSA) were significantly upregulated in AD patients. This upregulation of ribosomal protein expression occurred only in brain capillaries and not in brain parenchyma. The protein expression of protein processing and N-glycosylation-related proteins in the endoplasmic reticulum (DDOST, STT3A, MOGS, GANAB, RPN1, RPN2, SEC61B, UGGT1, LMAN2, and SSR4) were also upregulated in AD brain capillaries and was correlated with the expression of ribosomal proteins. The findings reported herein indicate that the ribosome complex, the subsequent protein processing and N-glycosylation-related processes are significantly and specifically upregulated in the brain capillaries of AD patients.

Zinc transport via ZNT5-6 and ZNT7 is critical for cell surface glycosylphosphatidylinositol-anchored protein expression.

Glycosylphosphatidylinositol (GPI)-anchored proteins play crucial roles in various enzyme activities, cell signaling and adhesion, and immune responses. While the molecular mechanism underlying GPI-anchored protein biosynthesis has been well studied, the role of zinc transport in this process has not yet been elucidated. Zn transporter (ZNT) proteins mobilize cytosolic zinc to the extracellular space and to intracellular compartments. Here, we report that the early secretory pathway ZNTs (ZNT5-ZNT6 heterodimers [ZNT5-6] and ZNT7-ZNT7 homodimers [ZNT7]), which supply zinc to the lumen of the early secretory pathway compartments are essential for GPI-anchored protein expression on the cell surface. We show, using overexpression and gene disruption/re-expression strategies in cultured human cells, that loss of ZNT5-6 and ZNT7 zinc transport functions results in significant reduction in GPI-anchored protein levels similar to that in mutant cells lacking phosphatidylinositol glycan anchor biosynthesis (PIG) genes. Furthermore, medaka fish with disrupted Znt5 and Znt7 genes show touch-insensitive phenotypes similar to zebrafish Pig mutants. These findings provide a previously unappreciated insight into the regulation of GPI-anchored protein expression and protein quality control in the early secretory pathway.

Activation of Annexin A2 signaling at the blood-brain barrier in a mouse model of multiple sclerosis.

Blood-brain barrier (BBB) dysfunction is a fundamental cause of multiple sclerosis and identifying the molecules that are responsible is an urgent matter. Protein expression was comprehensively quantified at the BBB of experimental autoimmune encephalomyelitis (EAE) mice, a model of multiple sclerosis, using the SWATH method. Concerning tight junction molecules, the level of expression of Claudin-5, which, in a previous immunohistochemical analysis, was confirmed to be down-regulated by EAE, remained unchanged, but the expression of Claudin-11 and Occludin was decreased by 0.69- and 0.62-fold, respectively, in brain capillaries isolated from EAE mice. A number of other cell-cell junctional molecules including ESAM, CADM1, CADM2, CADM3, CADM4, and HEPACAM were also down-regulated. The levels of expression of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1), which directly mediate the infiltration of lymphocytes across the BBB, were increased in EAE mice by 3.3- and 2.6-fold, respectively. The expression of CXADR, which possibly facilitates the adhesion of migrating cells, was also increased by 3.5-fold. Interestingly, various members of the Annexin A (ANXA) family were also up-regulated in brain capillaries that were isolated from EAE mice. In a pathway associated with cell infiltration and tight junction disruption, a series of molecules that are involved in ANXA2 signaling (ANXA2, PTP1B, Ahnak, S100A11, CD44, Kindlin2, Integrin α5, Fibronectin, Fibrinogen) were up-regulated. ANXA2 is selectively and abundantly expressed in endothelial cells in the brain. The daily administration of an ANXA2 inhibitor (LCKLSL peptide) significantly suppressed the development of EAE in mice. In summary, the activation of ANXA2 signaling at the BBB appear to play an important role in the pathogenesis of EAE.

An Atlas of the Quantitative Protein Expression of Anti-Epileptic-Drug Transporters, Metabolizing Enzymes and Tight Junctions at the Blood-Brain Barrier in Epileptic Patients.

The purpose of the present study was to quantitatively elucidate the levels of protein expression of anti-epileptic-drug (AED) transporters, metabolizing enzymes and tight junction molecules at the blood-brain barrier (BBB) in the focal site of epilepsy patients using accurate SWATH (sequential window acquisition of all theoretical fragment ion spectra) proteomics. Brain capillaries were isolated from focal sites in six epilepsy patients and five normal brains; tryptic digests were produced and subjected to SWATH analysis. MDR1 and BCRP were significantly downregulated in the epilepsy group compared to the normal group. Out of 16 AED-metabolizing enzymes detected, the protein expression levels of GSTP1, GSTO1, CYP2E1, ALDH1A1, ALDH6A1, ALDH7A1, ALDH9A1 and ADH5 were significantly 2.13-, 6.23-, 2.16-, 2.80-, 1.73-, 1.67-, 2.47- and 2.23-fold greater in the brain capillaries of epileptic patients than those of normal brains, respectively. The protein expression levels of Claudin-5, ZO-1, Catenin alpha-1, beta-1 and delta-1 were significantly lower, 1.97-, 2.51-, 2.44-, 1.90- and 1.63-fold, in the brain capillaries of epileptic patients compared to those of normal brains, respectively. Consistent with these observations, leakage of blood proteins was also observed. These results provide for a better understanding of the therapeutic effect of AEDs and molecular mechanisms of AED resistance in epileptic patients.

Acute acalculous cholecystitis caused by gallbladder metastasis due to the peritoneal dissemination of gastric cancer: A case report.

INTRODUCTION AND IMPORTANCE: Acute acalculous cholecystitis (AAC) is associated with a high mortality rate. AAC caused by metastasis to the gallbladder is rare. We report a case of AAC caused by gallbladder metastasis due to the peritoneal dissemination of gastric cancer. CASE PRESENTATION: An 84-year-old male visited our hospital because of epigastric pain. Ultrasonography and computed tomography revealed swelling and thickening of the gallbladder wall, but stones were not observed in the gallbladder. We performed emergency surgery with a diagnosis of acute cholecystitis. Laparoscopy revealed the presence of many nodules around the abdominal cavity including the hepatoduodenal ligament. Inflammation of Calot's triangle was severe, so we performed subtotal cholecystectomy. We also resected one of the peritoneal nodules. Macroscopically, there were no stones in the gallbladder and histopathological examination revealed acute cholecystitis and existence of adenocarcinoma involving the subserosa of the gallbladder wall and the resected peritoneal nodule. After surgery, esophagogastroduodenoscopy revealed Borrmann type II lesions at the antrum and gastric biopsy showed adenocarcinoma. He was diagnosed with advanced gastric cancer with peritoneal dissemination. His postoperative course was good. CLINICAL DISCUSSION: The cases of AAC caused by gallbladder metastasis have been little reported in the literature. This case is advanced gastric cancer with peritoneal dissemination and AAC was thought to be caused by peritoneal dissemination from operative and histopathological findings. We successfully treated this rare case of AAC with laparoscopic surgery. CONCLUSION: Although metastasis to the gallbladder is rare, it is necessary to be aware of this possibility when treating AAC.

Lipid nanoparticles loaded with ribonucleoprotein-oligonucleotide complexes synthesized using a microfluidic device exhibit robust genome editing and hepatitis B virus inhibition.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has considerable therapeutic potential for use in treating a wide range of intractable genetic and infectious diseases including hepatitis B virus (HBV) infections. While non-viral delivery technologies for the CRISPR/Cas system are expected to have clinical applications, difficulties associated with the clinically relevant synthesis of formulations and the poor efficiency of delivery severely hinder therapeutic genome editing. We report herein on the production of a lipid nanoparticle (LNP)-based CRISPR/Cas ribonucleoprotein (RNP) delivery nanoplatform synthesized using a clinically relevant mixer-equipped microfluidic device. DNA cleavage activity and the aggregation of Cas enzymes was completely avoided under the optimized synthetic conditions. The optimized formulation, which was identified through 2 steps of design of experiments, exhibited excellent gene disruption (up to 97%) and base substitution (up to 23%) without any apparent cytotoxicity. The addition of negative charges to the RNPs by complexing single-stranded oligonucleotide (ssON) significantly enhanced the delivery of both Cas9 and Cpf1 RNPs. The optimized formulation significantly suppressed both HBV DNA and covalently closed circular DNA (cccDNA) in HBV-infected human liver cells compared to adeno-associated virus type 2 (AAV2). These findings represent a significant contribution to the development of CRISPR/Cas RNP delivery technology and its practical applications in genome editing therapy.

Successful surgical outcomes after 23-, 25- and 27-gauge vitrectomy without scleral encircling for giant retinal tear.

PURPOSE: Retinal detachment due to giant retinal tears (GRTs), tears larger than 90°, is rare and difficult to treat. Here, we show and compare surgical results of 23-, 25- and 27-gauge (G) micro-incision vitrectomy surgery (MIVS) for GRT. STUDY DESIGN: Retrospective and interventional case series. METHODS: Retrospective review of 41 eyes of 38 patients with GRT who underwent MIVS. Surgical outcomes after MIVS, including reattachment rates and postoperative complications, were compared between instrument gauges. All patients were followed for at least 6 months postoperatively. RESULTS: MIVS with 23G, 25G and 27G instruments was performed in 7, 19 and 15 eyes, respectively. Silicone oil (SO) was used in 34 of 41 eyes (83%) with a mean removal time of 43.8 days after first surgery. Best-corrected visual acuity (BCVA) was recovered or maintained in 39 eyes (95%). Reattachment was attained after initial surgery in 38 of 41 eyes (93%) (23G: 6/7 [86%]; 25G: 17/19 [89%]; 27G: 15/15 [100%]). Final reattachment was eventually achieved in all eyes (two eyes needed support from scleral encircling). Postoperative complications occurred in 16 eyes (39%) (23G: 3/7 [43%]; 25G: 8/19 [42%]; 27G: 5/15 [33%]), including macular pucker, cystoid macular edema, macular hole, subretinal perfluorocarbon liquid, retinal folds, vitreous hemorrhage and redetachment. There were no significant differences between the three groups in rate of high myopia, GRT size, operation time, phacovitrectomy rate, SO usage rate, initial reattachment rate, final reattachment rate, preoperative BCVA, final BCVA or rate of postoperative complications. CONCLUSION: Despite occasional postoperative complications, primary MIVS, regardless of gauge size, appears to be a safe and feasible option for GRT surgery.

Isotopic evidence of arbuscular mycorrhizal cheating in a grassland gentian species.

All orchids and pyroloids are mycoheterotrophic at least in the early stage. Many species are predisposed to mycoheterotrophic nutrition even in the adult stage, due to the initial mycoheterotrophy during germination. Although other green plants, such as gentian species, also produce numerous minute seeds, whose germination may depend on fungal associations to meet C demands, physiological evidence for partial mycoheterotrophy in the adult stage is lacking for most candidate taxa. Here, we compared the natural abundances of 13C and 15N isotopes in the AM-associated gentian species Pterygocalyx volubilis growing in high-light-intensity habitats with those of co-occurring autotrophic C3 and C4 plants and AM fungal spores. We found that P. volubilis was significantly enriched in 13C compared with the surrounding C3 plants, which suggests the transfer of some C from the surrounding autotrophic plants through shared AM networks. In addition, the intermediate δ15N values of P. volubilis, between those of autotrophic plants and AM fungal spores, provide further evidence for partial mycoheterotrophy in P. volubilis. Although it is often considered that light deficiency selects partial mycoheterotrophy, we show that partial mycoheterotrophy in AM-forming plants can evolve even under light-saturated conditions. The fact that there have been relatively few descriptions of partial mycoheterotrophy in AM plants may not necessarily reflect the rarity of such associations. In conclusion, partial mycoheterotrophy in AM plants may be more common than hitherto believed.

Quantitative analysis of the macula with optical coherence tomography angiography in normal Japanese subjects: The Taiwa Study.

This study evaluated age-related changes in the superficial and deep retinal capillary plexus (SCP and DCP, respectively) and in the foveal avascular zone (FAZ). SCP and DCP perfusion density (PD) were measured in optical coherence tomography angiography (OCTA) macular scans of 145 eyes of 145 healthy Japanese subjects, and findings were compared with SCP FAZ and clinical data. We found that age was negatively correlated with SCP and DCP PD (r = -0.17, P = 0.04 and r = -0.20, P = 0.02, respectively) and positively correlated with FAZ area (r = 0.18, P = 0.03). SCP and DCP PD were correlated with each other (r = 0.67, P < 0.001). FAZ area was negatively correlated with SCP PD, DCP PD and central macular thickness (CMT) (r = -0.18, P = 0.03; r = -0.25, P < 0.01; and r = -0.39, P < 0.001, respectively). FAZ was larger and CMT was lower (P = 0.01 and P < 0.001, respectively) in women than men. SCP and DCP PD were positively correlated with estimated glomerular filtration rate (r = 0.17, P = 0.03 and r = 0.24, P < 0.01, respectively). Multiple regression analysis confirmed that age independently affected DCP PD and FAZ (P = 0.02 and P < 0.01, respectively) and that CMT independently affected FAZ area (P < 0.001). Thus, normal subjects showed age-related decreases in macular PD and renal function. FAZ and CMT were related, suggesting that age-related changes in macular thickness also affect capillary vasculature.

Neutralization of negative charges of siRNA results in improved safety and efficient gene silencing activity of lipid nanoparticles loaded with high levels of siRNA.

Lipid nanoparticles (LNPs) are one of the leading technologies for the in vivo delivery of short interfering RNA (siRNA). Cationic lipids are an important component for efficient endosomal escape via membrane fusion followed by release of siRNAs in cytosol where the site of action is located. A high cationic lipid/siRNA charge ratio is usually necessary for maximizing the gene silencing activity of the siRNA-loaded LNPs. However, high levels of cationic lipids are known to cause cytotoxicity through interactions with negatively charged biocomponents. A strategy for solving this dilemma is important, in terms of producing clinically applicable LNPs with a wide therapeutic window. We herein report on the development of LNPs with a high gene silencing activity and a low cationic lipid/siRNA charge ratio, which we refer to as low lipid core-nanoparticles (LLC-NPs). The negative charges of the siRNAs were neutralized by protamines, cationic proteins, to reduce the net dose of cationic lipid, YSK05, which was developed in our laboratory, for endosomal escape, resulting in preserved fusogenic activity and gene silencing activity, both in vitro and in vivo factor VII mouse model. In addition, the LLC-NPs showed an improved hepatotoxicity compared to conventional LNPs, which have a relatively higher cationic lipid/siRNA charge ratio. The concept of the LLC-NPs helps to realize clinically applicable LNPs with a wide therapeutic window and has the potential for use in various applications and for the delivery of different classes of nucleic acid.

Highly specific delivery of siRNA to hepatocytes circumvents endothelial cell-mediated lipid nanoparticle-associated toxicity leading to the safe and efficacious decrease in the hepatitis B virus.

Lipid nanoparticles (LNPs) are the leading technology for delivering short interfering RNA (siRNA) in vivo. While numerous attempts to improve the efficiency of siRNA delivery have been reported, only a few studies of the mechanism of LNP-mediated toxicity and attempts to develop safe LNPs in vivo have been reported, in spite of the significance of such systems, in the light of treatment and clinical applications. We herein report on the elucidation of the mechanism of hepatotoxicity following the intravenous injection of a high dose of hepatotropic LNPs. The LNPs accumulated in liver sinusoidal endothelial cells (LSECs), resulting in their activation and the induction of several cytokines related to neutrophils, followed by neutrophilic inflammation. To circumvent this toxic cascade, the LNPs were modified with a hepatocyte-specific ligand, N-acetyl-d-galactosamine (GalNAc), which resulted in a substantial improvement of hepatocyte-specificity and in a dramatic reduction in toxicity. Moreover, modification of the GalNAc-LNPs with polyethyleneglycol abrogated the LNP-associated toxicity without any detectable loss of gene silencing activity in hepatocytes. Finally, we observed that a single injection of the LNPs resulted in a significant reduction of hepatitis B virus (HBV) genomic DNA and their antigens without any sign of toxicity in chimeric mice with humanized livers that had been persistently infected with HBV. These lines of the fact suggest that the newly designed siRNA-loaded LNPs promise to be a useful technology for the treatment of liver diseases.

Role of conserved F(alpha)-helix residues in the native fold and stability of the kinase-inhibited oxy state of the oxygen-sensing FixL protein from Sinorhizobium meliloti.

The oxygen-sensing FixL protein from Sinorhizobium meliloti is part of the heme-PAS family of gas sensors that regulate many important signal transduction pathways in a wide variety of organisms. We examined the role of the conserved F(alpha)-9 arginine 200 and several other conserved residues on the proximal F(alpha)-helix in the heme domain of SmFixL* using site-directed mutagenesis in conjunction with UV-visible, EPR, and resonance Raman spectroscopy. The F(alpha)-helix variants R200A, E, Q, H, Y197A, and D195A were expressed at reasonable levels and purified to homogeneity. The R200I and Y201A variants did not express in observable quantities. Tyrosine 201 is crucial for forming the native protein fold of SmFixL* while Y197 and R200 are important for stabilizing the kinase-inhibited oxy state. Our results show a clear correlation between H-bond donor ability of the F(alpha)-9 side chain and the rate of heme autoxidation. This trend in conjunction with crystal structures of liganded BjFixL heme domains, show that H-bonding between the conserved F(alpha)-9 arginine and the heme-6-propionate group contributes to the kinetic stability of the kinase-inactivated, oxy state of SmFixL*.