Dual CRISPR-Cas3 system for inducing multi-exon skipping in DMD patient-derived iPSCs.

To restore dystrophin protein in various mutation patterns of Duchenne muscular dystrophy (DMD), the multi-exon skipping (MES) approach has been investigated. However, only limited techniques are available to induce a large deletion to cover the target exons spread over several hundred kilobases. Here, we utilized the CRISPR-Cas3 system for MES induction and showed that dual crRNAs could induce a large deletion at the dystrophin exon 45-55 region (∼340 kb), which can be applied to various types of DMD patients. We developed a two-color SSA-based reporter system for Cas3 to enrich the genome-edited cell population and demonstrated that MES induction restored dystrophin protein in DMD-iPSCs with three distinct mutations. Whole-genome sequencing and distance analysis detected no significant off-target deletion near the putative crRNA binding sites. Altogether, dual CRISPR-Cas3 is a promising tool to induce a gigantic genomic deletion and restore dystrophin protein via MES induction.

Compartmentalization of soluble endocytic proteins in synaptic vesicle clusters by phase separation.

Synaptic vesicle (SV) clusters, which reportedly result from synapsin's capacity to undergo liquid-liquid phase separation (LLPS), constitute the structural basis for neurotransmission. Although these clusters contain various endocytic accessory proteins, how endocytic proteins accumulate in SV clusters remains unknown. Here, we report that endophilin A1 (EndoA1), the endocytic scaffold protein, undergoes LLPS under physiologically relevant concentrations at presynaptic terminals. On heterologous expression, EndoA1 facilitates the formation of synapsin condensates and accumulates in SV-like vesicle clusters via synapsin. Moreover, EndoA1 condensates recruit endocytic proteins such as dynamin 1, amphiphysin, and intersectin 1, none of which are recruited in vesicle clusters by synapsin. In cultured neurons, like synapsin, EndoA1 is compartmentalized in SV clusters through LLPS, exhibiting activity-dependent dispersion/reassembly cycles. Thus, beyond its essential function in SV endocytosis, EndoA1 serves an additional structural function by undergoing LLPS, thereby accumulating various endocytic proteins in dynamic SV clusters in concert with synapsin.

Electrochemical determination of emodin in acidic media by high-performance liquid chromatography and its application to Polygoni Multiflori Radix samples.

Electrochemical reduction of emodin under acidic media occurs at a less negative potential when compared with that under neutral media. When emodin is electrochemically detected at a less negative potential, a decrease in background noise and improvement in specificity benefit the development of high-performance liquid chromatography with electrochemical detection (HPLC-ECD) for its determination. HPLC-ECD was performed using an octadecyl silica column, acetonitrile-water (60:40, v/v) containing 5 mmol L-1 hydrochloric acid and 10 mmol L-1 lithium perchlorate, as a mobile phase, and an applied potential at - 0.4 V vs. Ag/AgCl. Under these optimal HPLC-ECD conditions, the detection limit (signal-to-noise ratio, S/N = 3) of emodin was 0.61 µg L-1. When this HPLC-ECD system was applied to the determination of emodin in Polygoni Multiflori Radix (PMR) samples, other peaks did not appear close to the emodin peak on a chromatogram. The emodin contents in PMR samples were determined with relative standard deviations (RSDs, n = 6) of less than 3.9%, and their recoveries ranged from 92 to 106%. We have shown that our HPLC-ECD system performed an accurate, precise, and specific determination of emodin in PMR samples.

Lurasidone-induced hyperosmolar hyperglycemic syndrome: A case report.

INTRODUCTION: Lurasidone has few metabolic adverse effects and is recommended as an alternative when other antipsychotic drugs considerably increase body weight or blood sugar concentrations. CASE PRESENTATION: An 81-year-old man with bipolar disorder developed hyperosmolar hyperglycemic syndrome as a side effect of lurasidone. Routine monitoring of blood glucose concentrations led to the early detection and treatment of this disease, preventing life-threatening complications. DISCUSSION AND CONCLUSION: We describe a rare case of lurasidone-induced hyperosmolar hyperglycemic syndrome. The mortality rate of this syndrome is estimated to be up to 20%. This rate is significantly higher than that of diabetic ketoacidosis (currently <2%). Although lurasidone is considered to have a low risk of raising blood glucose concentrations, symptoms of hyperglycemia must be evaluated and blood glucose concentrations should be monitored regularly.

Bromination of Carbon and Formation of PBDD/Fs by Copper Bromide in Oxidative Thermal Process.

Brominated aromatic compounds are unintentionally generated during various thermal processes, including municipal solid waste incineration, electric-waste open burning, and secondary copper smelting. Copper (Cu) plays an important role in the formation of brominated aromatic compounds. In the present study, the thermochemical behaviors of Cu and Br in model samples, including copper bromide (CuBr2) and activated carbon, were studied using in situ X-ray absorption near-edge structure (XANES) and thermogravimetry. Quantification of polybrominated dibenzo-p-dioxins/furans (PBDD/Fs) was also conducted by gas chromatograph-high resolution mass spectrometer. Three key reactions were identified: (i) the reduction of CuBr2 to CuBr (room temperature to 300 °C), (ii) the generation of Br bonded with aromatic carbon (150-350 °C), and (iii) the oxidation of copper (>350 °C). Maximum amounts of PBDD/Fs were found in residual solid phase after heating at 300 °C. The analytical results indicated the direct bromination of aromatic carbon by the debromination of copper bromides (I, II) and that CuBr and CuO acted as catalysts in the oxidation of the carbon matrix. The bromination mechanisms revealed in this study are essential to the de novo formation of PBDD/Fs and other brominated aromatic compounds.

Novel glucoamylase-resistant gluco-oligosaccharides with adjacent α-1, 6 branches at the non-reducing end discovered in Japanese rice wine, sake.

Sake, a traditional Japanese rice wine, contains various oligosaccharides (Sake oligosaccharides; SAOs) derived from rice starch. We previously found that SAOs reach a high degree of polymerization (DP). In this study, we developed a hydrophilic interaction liquid chromatography-time-of-flight/mass spectrometry (HILIC-TOF/MS) based analytical method to separate isomeric SAOs. Isomers of SAOs with DP = 6, 7, and 8, which were named DP6-1, DP7-1, DP8-1 and DP8-2, respectively, were purified from sake and their structures were determined by two-dimensional NMR spectroscopy. These were novel oligosaccharides containing two α-1, 6 bonded branches on an α-1, 4-linked glucose main chain. Interestingly, adjacent double α-1, 6 branches that have not been identified in starch, were found in DP6-1, DP7-1, and DP8-1, suggesting the presence of the branching pattern in starch. DP6-1 was poorly digested by fungal glucoamylase, and this may be attributed to its adjacent double branches at the non-reducing end.

Regulatory mechanism of chicken lysozyme gene expression in oviducts examined using transgenic technology.

The use of promoters that strongly express target genes in the chicken oviduct is beneficial for the production of proteinaceous materials into egg white by transgenic chickens. To examine the regulatory mechanisms of chicken lysozyme gene expression in vivo, genetically manipulated chickens that express human erythropoietin under the control of a lysozyme promoter-enhancer were established. By using several deletion mutants of the promoter-flanking region, we found that a -1.9 kb DNase I hypersensitive site (DHS) was essential for oviduct-specific expression in genetically manipulated chickens. The concentration of human erythropoietin in egg white was 14-75 µg/ml, suggesting that the chicken lysozyme promoter containing -1.9 kb DHS is sufficient for the production of pharmaceuticals using transgenic chickens.

Tandem Deubiquitination and Acetylation of SPRTN Promotes DNA-Protein Crosslink Repair and Protects against Aging.

DNA-protein crosslinks (DPCs) are highly toxic DNA lesions that threaten genomic integrity. Recent findings highlight that SPRTN, a specialized DNA-dependent metalloprotease, is a central player in proteolytic cleavage of DPCs. Previous studies suggest that SPRTN deubiquitination is important for its chromatin association and activation. However, the regulation and consequences of SPRTN deubiquitination remain unclear. Here we report that, in response to DPC induction, the deubiquitinase VCPIP1/VCIP135 is phosphorylated and activated by ATM/ATR. VCPIP1, in turn, deubiquitinates SPRTN and promotes its chromatin relocalization. Deubiquitination of SPRTN is required for its subsequent acetylation, which promotes SPRTN relocation to the site of chromatin damage. Furthermore, Vcpip1 knockout mice are prone to genomic instability and premature aging. We propose a model where two sequential post-translational modifications (PTMs) regulate SPRTN chromatin accessibility to repair DPCs and maintain genomic stability and a healthy lifespan.

DNA-protein crosslinks from environmental exposure: Mechanisms of formation and repair.

Many environmental carcinogens cause DNA damage, which can result in mutations and other alterations in genomic DNA if not repaired promptly. Because of the bulkiness of the lesions, DNA-protein crosslinks (DPCs) are one of the types of toxic DNA damage with potentially deleterious consequences. Despite the importance of DPCs, how cells remove these complex DNA adducts has been incompletely understood. However, major progress in the DPC repair field over the past 5 years now supports the view that cells are equipped with multiple mechanisms to cope with DPCs. Here, we first provide an overview of environmental substances that induce DPCs, describing the sources of exposure and mechanisms of DPC formation. We then review current models of DPC repair and discuss their significance for environmental carcinogens.

FAM111A protects replication forks from protein obstacles via its trypsin-like domain.

Persistent protein obstacles on genomic DNA, such as DNA-protein crosslinks (DPCs) and tight nucleoprotein complexes, can block replication forks. DPCs can be removed by the proteolytic activities of the metalloprotease SPRTN or the proteasome in a replication-coupled manner; however, additional proteolytic mechanisms may exist to cope with the diversity of protein obstacles. Here, we show that FAM111A, a PCNA-interacting protein, plays an important role in mitigating the effect of protein obstacles on replication forks. This function of FAM111A requires an intact trypsin-like protease domain, the PCNA interaction, and the DNA-binding domain that is necessary for protease activity in vivo. FAM111A, but not SPRTN, protects replication forks from stalling at poly(ADP-ribose) polymerase 1 (PARP1)-DNA complexes trapped by PARP inhibitors, thereby promoting cell survival after drug treatment. Altogether, our findings reveal a role of FAM111A in overcoming protein obstacles to replication forks, shedding light on cellular responses to anti-cancer therapies.

Transglycosylation Forms Novel Glycoside Ethyl α-Maltoside and Ethyl α-Isomaltoside in Sake during the Brewing Process by α-Glucosidase A of Aspergillus oryzae.

Sake, the Japanese rice wine, contains a variety of oligosaccharides and glucosides produced by fungal enzymes during the brewing process. This study investigates the effect of knocking out the Aspergillus oryzae α-glucosidase (agdA) gene on the transglycosylation products in brewed sake. In addition to α-ethyl glucoside and α-glyceryl glucoside, the amount of two compounds that have molecular mass values similar to that of ethyl maltose decreased by agdA gene knockout. Both compounds were synthesized, in vitro, from maltose and ethanol with purified agdA. Nuclear magnetic resonance analysis identified the two compounds as ethyl α-maltoside and ethyl α-isomaltoside, respectively, which are novel compounds in sake as well as in the natural environment. Quantitative analysis of 111 commercially available types of sake showed that these novel compounds were widely present at concentrations of several hundred mg/L, suggesting that both of them are ones of the common glycosides in sake.

Usefulness of our proposed olfactory scoring system during endoscopic sinus surgery in patients with chronic rhinosinusitis.

INTRODUCTION: The primary aim of the current study was to examine the usefulness of our proposed olfactory scoring system in chronic rhinosinusitis (CRS) patients with olfactory disorders (n = 213) receiving endoscopic sinus surgery (ESS). MATERIALS AND METHODS: Analyzed patients were divided into two groups: an eosinophilic CRS (ECRS) group (n = 153); and a non-ECRS group (n = 60). The T&T recognition threshold test was used to evaluate olfaction at baseline and at 3 and 12 months after ESS. Patients with mean recognition threshold < 2.0 at 3 or 12 months or with a decrease of ≥ 1.0 as compared with baseline were defined as showing clinical improvement. We scored mucosal conditions as normal (0 points), edema (1 point), and polyp (2 points) at the canopy of olfactory cleft (OC), middle and superior turbinates, superior nasal meatus, and sphenoethmoidal recess during ESS. The total score of OCs (SOCs) was calculated (range 0-20 points). We compared SOCs between ECRS and non-ECRS groups. Factors related to olfactory improvement were also investigated using uni- and multivariate analyses. RESULTS: SOCs in the ECRS and non-ECRS groups showed significant correlations with mean recognition thresholds at baseline and at 3 and 12 months. In the multivariate analysis for predicting improvement of mean recognition threshold, lower SOCs were significantly associated with olfactory improvement factors at 3 and 12 months postoperatively in the ECRS group. CONCLUSION: SOCs appears promising for estimating olfactory prognosis after ESS in CRS patients.

Spartan deficiency causes accumulation of Topoisomerase 1 cleavage complexes and tumorigenesis.

Germline mutations in SPRTN cause Ruijs-Aalfs syndrome (RJALS), a disorder characterized by genome instability, progeria and early onset hepatocellular carcinoma. Spartan, the protein encoded by SPRTN, is a nuclear metalloprotease that is involved in the repair of DNA-protein crosslinks (DPCs). Although Sprtn hypomorphic mice recapitulate key progeroid phenotypes of RJALS, whether this model expressing low amounts of Spartan is prone to DPC repair defects and spontaneous tumors is unknown. Here, we showed that the livers of Sprtn hypomorphic mice accumulate DPCs containing Topoisomerase 1 covalently linked to DNA. Furthermore, these mice exhibited DNA damage, aneuploidy and spontaneous tumorigenesis in the liver. Collectively, these findings provide evidence that partial loss of Spartan impairs DPC repair and tumor suppression.

Analyses of chicken sialyltransferases related to O-glycosylation.

The chicken ß-galactoside α2,3-sialyltransferase 1, 2, and 5 (ST3Gal1, 2, and 5) genes were cloned, and their enzymes were expressed in 293FT cells. ST3Gal1 and 2 exhibited enzymatic activities toward galactose-ß1,3-N-acetylgalactosamine and galactose-ß1,3-N-acetylglucosamine. ST3Gal5 only exhibited activity toward lactosylceramide. ST3Gal1 and 2 and previously cloned ST3Gal3 and 6 transferred CMP-sialic acid to asialofetuin. Reverse-transcription-quantitative PCR indicated that ST3Gal1 was expressed at higher levels in the trachea, lung, spleen, and magnum, and the strong expression of ST3Gal5 was observed in the spleen, magnum, and small and large intestines. ST3Gal1, 5, and 6 were also expressed in the tubular gland cells of the magnum, which secretes egg-white proteins. ST3Gal1, 5, and 6 were expressed in the egg chorioallantoic membrane, in which influenza viruses are propagated for the production of vaccines.

Clinical Features of Patients Treated with Endoscopic Sinus Surgery for Posttraumatic Paranasal Sinus Mucocele.

AIM: The purpose of this study was to analyze the clinical features of patients with posttraumatic paranasal sinus mucocele (PSM). SUBJECTS AND METHODS: Between 2009 and 2013, we performed endoscopic sinus surgery (ESS) on 68 patients with PSM at the Department of Otolaryngology - Head and Neck Surgery at Hyogo College of Medicine. Five male patients (age range, 45-76 years) with posttraumatic PSM were analyzed retrospectively. Diagnosis was based on the history of injury and radiological findings. RESULTS: Posttraumatic PSM was found in 7% (5/68) of patients. The mean interval from injury to diagnosis was 28.4 years. All patients had frontal sinus mucocele. Four patients had symptoms of headache, diplopia, visual field defect, and forehead swelling, and 1 patient was asymptomatic. ESS was performed under general anesthesia in all cases, and the symptoms improved postoperatively. Reoperation was required in 1 patient (20%) because headache developed with obstruction of the frontal drainage route 7 months after ESS. CONCLUSIONS: Posttraumatic PSM was the least frequent form of PSM and was located predominantly in the frontal sinus, causing symptoms long after the forehead injury. The important lessons to be learned for treating posttraumatic PSM are to obtain a detailed history and to enlarge the route to the cyst to avoid its recurrence.

Sonographic Optic Nerve Sheath Diameter: A Simple and Rapid Tool to Assess the Neurologic Prognosis After Cardiac Arrest.

BACKGROUND AND PURPOSE: The early prediction of hypoxic encephalopathy after cardiac arrest is challenging. Measurement of the optic nerve sheath diameter (ONSD) by using sonography is a straightforward, noninvasive technique to detect an increased intracranial pressure, which can even be conducted at the bedside. However, it remains unknown whether or not sonographic ONSD measurement is valuable as a prognostic indicator of hypoxic encephalopathy. METHODS: Seventeen patients after cardiac arrest were retrospectively enrolled in this study. ONSD measurements 3 mm behind the papilla were recorded. A Glasgow Outcome Scale score of 4 or above was considered to indicate a favorable prognosis. RESULTS: The mean ONSD associated with a favorable prognosis was 5.0 mm (4.4-6.1 mm). The ONSD associated with a poor prognosis was 6.1 mm (5.4-7.2 mm). ONSD less than or equal to 5.4 mm was an indicator of a favorable prognosis, with a sensitivity of 83%, specificity of 73%, positive likelihood ratio of 3.1, and negative likelihood ratio of .23. CONCLUSIONS: Sonographic ONSD measurement is a simple, rapid technique to assess the neurological prognosis after cardiac arrest.

Analyses of chicken sialyltransferases related to N-glycosylation.

Proteins exogenously expressed and deposited in the egg whites of transgenic chickens did not contain terminal sialic acid in their N-glycan. Since this sugar is important for the biological stability of therapeutic proteins, we examined chicken sialyltransferases (STs). Based on homologies in DNA sequences, we cloned and expressed several chicken STs, which appeared to be involved in N-glycosylation in mammals, in 293FT cells. Enzymatic activity was detected with ST3Gal3, ST3Gal6 and ST6Gal1 using galactose-ß1,4-N-acetylglucosamine (Galß1,4GlcNAc) as an acceptor. Using Golgi fractions from the cell-free extracts of chicken organs, α2,3- and/or α2,6-ST activities were detected in the liver and kidney, but were absent in the oviduct cells in which egg-white proteins were produced. This result suggested that the lack of ST activities in oviduct cells mainly caused the lack of sialic acid in the N-glycan of proteins exogenously expressed and deposited in egg white.

Novel endoscopic scoring system after sinus surgery.

OBJECTIVE: To propose a simple post-operative endoscopic scoring system for use after endoscopic sinus surgery (ESS) in patients with chronic rhinosinusitis (CRS), and to demonstrate the usefulness of this approach. METHODS: Subjects comprised 116 patients (84 men, 32 women; mean age, 54 years) with CRS who were analyzed endoscopically and radiologically after ESS between 2006 and 2012. The study was designed as a case series with planned data collection in the setting of university medical centers. Patients were followed-up for ≥ 6 months after ESS (mean, 13.1 months). Both pre- and post-operative computed tomography (CT) findings of each sinus and olfactory cleft (OC) were scored according to the Lund-Mackay scoring system: 0, normal; 1, partially; or 2, completely occupied. CT score represents the total score expressed as a percentage of the maximum possible score (12 points per side). Post-operative endoscopic score (E score, %) was calculated as the maximum score according to physical findings on each operated sinus and OC: 0, normal; 1, partially diseased; or 2, completely closed. Post-operative course using E score was verified by comparison with the Lund-Kennedy (L-K) scoring system. RESULTS: E score was easily and quickly determined. Interclass correlation coefficient among 10 otolaryngologists indicated high-level inter-rater reliability (0.922). E score correlated strongly with both CT score (n=116, p<0.0001, rs=0.755) and L-K score (n=79, p<0.0001, rs=0.723). CONCLUSION: Endoscopic evaluation using E score for sinuses and OCs after ESS is a useful method, together with L-K score for the nasal cavity and radiological study.

Galactosylation of human erythropoietin produced by chimeric chickens expressing galactosyltransferase.

Human erythropoietin produced in the egg white of chimeric chicken contains N-glycan with lower amounts of terminal galactose and sialic acid; therefore, the chicken galactosyltransferase gene was introduced together with the human erythropoietin gene by a retroviral vector. We found that erythropoietin accumulated in the egg white was partially galactosylated.