Effects of the matrix-bounded nanovesicles of high-hydrostatic pressure decellularized tissues on neural regeneration.

Decellularized tissues have been used as implantable materials for tissue regeneration because of their high biofunctionality. We have reported that high hydrostatic pressured (HHP) decellularized tissue developed in our laboratory exhibits good in vivo performance, but the details of the mechanism are still not known. Based on previous reports of bioactive factors called matrix bound nanovesicles (MBVs) within decellularized tissues, this study aims to investigate whether MBVs are also present in decellularized tissues prepared by HHP decellularization, which is different from the previously reported methods. In this study, we tried to extract bioactive factors from HHP decellularized brain and placenta, and evaluated their effects on nerves in vitro and in vivo, where its effects have been previously reported. The results confirmed that those factors can be extracted even if the decellularization method and tissue of origin differ, and that they have effects on a series of processes toward nerve regeneration, such as neurite outgrowth and nerve fiber repair.

In this study, we evaluated the neuroregenerative effects of matrix-bounded nanovesicles extracted from decellularized tissue using a high hydrostatic pressure method. The results indicate that bioactive factors, including matrix-bounded nanovesicles, can be extracted regardless of the decellularization method and tissue origin.

Dehydrogenative Coupling of Alkylamines with Primary Alcohols Forming α-Amino Ketones.

Acceptorless dehydrogenative coupling reactions between C-H bonds offer straightforward and atom-economical methods connecting readily available materials while liberating gaseous hydrogen as the sole byproduct. Despite the growing interest in such transformations, their realization still poses a significant challenge. Here we report a photoinduced dehydrogenative coupling reaction of alkylamines with primary alcohols. C-H bonds adjacent to nitrogen and oxygen are site-selectively cleaved, and a C-C bond is created between the carbon atoms in a cross-selective manner to produce α-amino ketones. Diverse polar functionalities such as esters, amides, and carboxylic acids survived, demonstrating the broad applicability of the present method.

Endoplasmic reticulum transporter OAT2 regulates drug metabolism and interaction.

Xenobiotic metabolic reactions in the hepatocyte endoplasmic reticulum (ER) including UDP-glucuronosyltransferase and carboxylesterase play central roles in the detoxification of medical agents with small- and medium-sized molecules. Although the catalytic sites of these enzymes exist inside of ER, the molecular mechanism for membrane permeation in the ER remains enigmatic. Here, we investigated that organic anion transporter 2 (OAT2) regulates the detoxification reactions of xenobiotic agents including anti-cancer capecitabine and antiviral zidovudine, via the permeation process across the ER membrane in the liver. Pharmacokinetic studies in patients with colorectal cancer revealed that the half-lives of capecitabine in rs2270860 (1324C > T) variants was 1.4 times higher than that in the C/C variants. Moreover, the hydrolysis of capecitabine to 5'-deoxy-5-fluorocytidine in primary cultured human hepatocytes was reduced by OAT2 inhibitor ketoprofen, whereas capecitabine hydrolysis directly assessed in human liver microsomes were not affected. The immunostaining of OAT2 was merged with ER marker calnexin in human liver periportal zone. These results suggested that OAT2 is involved in distribution of capecitabine into ER. Furthermore, we clarified that OAT2 plays an essential role in drug-drug interactions between zidovudine and valproic acid, leading to the alteration in zidovudine exposure to the body. Our findings contribute to mechanistically understanding medical agent detoxification, shedding light on the ER membrane permeation process as xenobiotic metabolic machinery to improve chemical changes in hydrophilic compounds.

Aspergillus oryzae α-l-rhamnosidase: Crystal structure and insight into the substrate specificity.

The subsequent biochemical and structural investigations of the purified recombinant α-l-rhamnosidase from Aspergillus oryzae expressed in Pichia pastoris, designated as rAoRhaA, were performed. The specific activity of the rAoRhaA wild-type was higher toward hesperidin and narirutin, where the l-rhamnose residue was α-1,6-linked to ß-d-glucoside, than toward neohesperidin and naringin with an α-1,2-linkage to ß-d-glucoside. However, no activity was detected toward quercitrin, myricitrin, and epimedin C. rAoRhaA kinetic analysis indicated that Km values for neohesperidin, naringin, and rutin were lower compared to those for hesperidin and narirutin. kcat values for hesperidin and narirutin were higher than those for neohesperidin, naringin, and rutin. High catalytic efficiency (kcat /Km ) toward hesperidin and narirutin was a result of a considerably high kcat value, while Km values for hesperidin and narirutin were higher than those for naringin, neohesperidin, and rutin. The crystal structure of rAoRhaA revealed that the catalytic domain was represented by an (α/α)6 -barrel with the active site located in a deep cleft and two ß-sheet domains were also present in the N- and C-terminal sites of the catalytic domain. Additionally, five asparagine-attached N-acetylglucosamine molecules were observed. The catalytic residues of AoRhaA were suggested to be Asp254 and Glu524, and their catalytic roles were confirmed by mutational studies of D254N and E524Q variants, which lost their activity completely. Notably, three aspartic acids (Asp117, Asp249, and Asp261) located at the catalytic pocket were replaced with asparagine. D117N variant showed reduced activity. D249N and D261N variants activities drastically decreased.

Photocatalytic 1,2-Phosphorus-Migrative [3 + 2] Cycloaddition of Tri(t-butyl)phosphine with Terminal Alkynes.

Tri(t-butyl)phosphine and terminal alkynes undergo 1,2-phosphorus-migrative [3 + 2] cycloaddition in the presence of a proton source under photocatalytic conditions. The reaction exhibits broad functional group tolerance and affords substituted cyclic phosphonium salts, which are amenable to further derivatization by Wittig olefination. Theoretical studies suggest that the phosphorus 1,2-migration of a ß-phosphonioalkyl radical proceeds through a phosphine radical cation-alkene complex as a pseudointermediate, and the two fragments in the intermediate are bound to each other through multiple noncovalent interactions.

Enhancement and maintenance of hepatic metabolic functions by controlling 3D aggregation of cryopreserved human iPS cell-derived hepatocyte-like cells.

Hepatocyte-like cells (HLCs) generated from human induced pluripotent stem cells are potent cells to study individual-specific hepatotoxicity for drug screening test. However, the functions of metabolic enzymes are practically low. Here, we reconstituted stable and compact 3D spheroids of commercially available cryopreserved HLCs by our original spheroid formation method with high viscous methylcellulose medium. 3D formation enhanced the hepatic functions and maintained the functions for 14 days. Especially, the expression of cytochrome P450s was 10- to 100-fold enhanced compared to conventional 2D culture, which is applicable to a typical drug-metabolizing test using liquid chromatograph-tandem mass spectrometer. In conclusion, we successfully formed human HLC spheroid from commercially available cryo-preserved cells, which realized remarkable hepatic maturation by prolonged 3D culture, especially in terms of drug-metabolizing enzymes. Our spheroid formation technology has the potential to make HLC spheroids a potent tool in aspects of pharmaceutical research, such as drug screening and pharmacokinetic studies.

Spontaneous beat synchronization in rats: Neural dynamics and motor entrainment.

Beat perception and synchronization within 120 to 140 beats/min (BPM) are common in humans and frequently used in music composition. Why beat synchronization is uncommon in some species and the mechanism determining the optimal tempo are unclear. Here, we examined physical movements and neural activities in rats to determine their beat sensitivity. Close inspection of head movements and neural recordings revealed that rats displayed prominent beat synchronization and activities in the auditory cortex within 120 to 140 BPM. Mathematical modeling suggests that short-term adaptation underlies this beat tuning. Our results support the hypothesis that the optimal tempo for beat synchronization is determined by the time constant of neural dynamics conserved across species, rather than the species-specific time constant of physical movements. Thus, latent neural propensity for auditory motor entrainment may provide a basis for human entrainment that is much more widespread than currently thought. Further studies comparing humans and animals will offer insights into the origins of music and dancing.

Mutational analysis of the effects of N-glycosylation sites on the activity and thermal stability of rutinosidase from Aspergillus oryzae.

Purified recombinant rutinosidase from Aspergillus oryzae expressed in Pichia pastoris (rAoRutM) exhibits increase in thermal stability after treatment with endo-ß-N-acetylglucosaminidase H (endo-H). In this study, the role of N-glycosylation in the activity and thermal stability of rAoRutM was analyzed via site-directed mutagenesis. Based on the crystal structure of AoRutM, five N-glycosylation sites (N32, N128, N176, N288, and N359) were identified in the AoRut protein. Among five single variants constructed for these sites, the N128D, N176D, and N359D variants exhibited similar mobility bands compared to that of the wild-type enzyme based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the N32D and N288D variants exhibited slightly and considerably increasing mobility bands, respectively. The N128D and N288D variants showed increasing and decreasing rutinosidase activity, respectively, compared to the case for the wild-type, without and with endo-H treatments. While the N128D and N176D variants had lower Km values, the N288D and N359D variants had higher Km values, compared to the wild-type, without and with endo-H treatments. Surprisingly, the N32D and N176D variants exhibited considerably greater thermal stability than the wild-type, without or with the endo-H treatments, whereas the N128D and N359D variants exhibited drastically decreased thermal stability. Circular dichroism (CD) spectra of the N128D and N359D variants showed a similar CD profile to that of the wild-type treated with endo-H; however, the molar ellipticity values of the peaks at 208 nm and 212 nm in the above variants varied from those of the intact wild-type and other variants.

Extraction and Biological Evaluation of Matrix-Bound Nanovesicles (MBVs) from High-Hydrostatic Pressure-Decellularized Tissues.

Decellularized tissues are widely used as promising materials in tissue engineering and regenerative medicine. Research on the microstructure and components of the extracellular matrix (ECM) was conducted to improve the current understanding of decellularized tissue functionality. The presence of matrix-bound nanovesicles (MBVs) embedded within the ECM was recently reported. Results of a previous experimental investigation revealed that decellularized tissues prepared using high hydrostatic pressure (HHP) exhibited good in vivo performance. In the current study, according to the hypothesis that MBVs are one of the functional components in HHP-decellularized tissue, we investigated the extraction of MBVs and the associated effects on vascular endothelial cells. Using nanoparticle tracking assay (NTA), transmission electron microscopy (TEM), and RNA analysis, nanosized (100-300 nm) and membranous particles containing small RNA were detected in MBVs derived from HHP-decellularized small intestinal submucosa (SIS), urinary bladder matrix (UBM), and liver. To evaluate the effect on the growth of vascular endothelial cells, which are important in the tissue regeneration process, isolated SIS-derived MBVs were exposed to vascular endothelial cells to induce cell proliferation. These results indicate that MBVs can be extracted from HHP-decellularized tissues and may play a significant role in tissue remodeling.

Comprehensive validation of a wearable foot sensor system for estimating spatiotemporal gait parameters by simultaneous three-dimensional optical motion analysis.

BACKGROUND: Use of a wearable gait analysis system (WGAS) is becoming common when conducting gait analysis studies due to its versatility. At the same time, its versatility raises a concern about its accuracy, because its calculations rely on assumptions embedded in its algorithms. The purpose of the present study was to validate twenty spatiotemporal gait parameters calculated by the WGAS by comparison with simultaneous measurements taken with an optical motion capture system (OMCS). METHODS: Ten young healthy volunteers wore two inertial sensors of the commercially available WGAS, Physilog®, on their feet and 23 markers for the OMCS on the lower part of the body. The participants performed at least three sets of 10-m walk tests at their self-paced speed in the laboratory equipped with 12 high-speed digital cameras with embedded force plates. To measure repeatability, all participants returned for a second day of testing within two weeks. RESULTS: Twenty gait parameters calculated by the WGAS had a significant correlation with the ones determined by the OMCS. Bland and Altman analysis showed that the between-device agreement for twenty gait parameters was within clinically acceptable limits. The validity of the gait parameters generated by the WGAS was found to be excellent except for two parameters, swing width and maximal heel clearance. The repeatability of the WGAS was excellent when measured between sessions. CONCLUSION: The present study showed that spatiotemporal gait parameters estimated by the WGAS were reasonably accurate and repeatable in healthy young adults, providing a scientific basis for applying this system to clinical studies.

Photoinduced Hydrophosphination of Terminal Alkynes with Tri(o-tolyl)phosphine: Synthesis of Alkenylphosphonium Salts.

Herein reported is a photoinduced hydrophosphination reaction of terminal alkynes with tri(o-tolyl)phosphine to form alkenylphosphonium salts. The reaction is more sustainable than conventional methods since it dispenses with the need for elaborated starting materials and precious transition metals. The o-methyl groups of tri(o-tolyl)phosphine play two important roles: (1) to guide the phosphorus radical to add onto the terminal sp carbon and (2) to donate a hydrogen atom onto the developing sp2 carbon radical intramolecularly.

Laparoscopic remnant cholecystectomy for calculi in the remnant gallbladder following subtotal-cholecystectomy: a report of two cases.

Two cases of laparoscopic remnant cholecystectomy using near-infrared fluorescence cholangiography (NIFC) for remnant gallbladder calculi following subtotal-cholecystectomy are reported. Case 1: a 36-year-old woman was referred to our hospital with acute abdomen. Computed tomography showed remnant gallbladder calculi, with detected no other findings as the cause of the abdominal pain. For intraoperative exploration of the biliary anatomy, 0.25 mg/kg of indocyanine green (ICG) was administered intravenously the day before the operation. NIFC clearly showed the common bile duct and enabled safe laparoscopic remnant cholecystectomy. She was free from symptoms after the operation. Case 2: a 40-year-old woman was referred to our hospital with epigastralgia due to remnant gallbladder calculi after open cholecystectomy. ICG was administered intravenously the day before the operation. Severe adhesions were observed in the upper abdominal cavity and there was tight adherence of the duodenum to the remnant gallbladder. NIFC showed a clear margin that appeared to be the margin between the duodenum and remnant gallbladder. However, dissection of the margin observed by NIFC caused perforation of the duodenum. The clear margin seen with NIFC was likely due to visualization of the gallbladder through the duodenum. Although NIFC is a useful modality for confirming the intraoperative biliary anatomy, it is important not to rely too heavily on NIFC alone, which may lead to misinterpretation of the anatomy.

Visible-Light-Driven Dehydrogenative Coupling of Primary Alcohols with Phenols Forming Aryl Carboxylates.

A preparative method for obtaining aryl esters from aliphatic primary alcohols and phenols was developed. The reaction proceeds under the irradiation of visible light at ambient temperature, dispensing with any oxidant or hydrogen acceptor. Primary alcohols having a variety of functional groups are successfully esterified with phenols. The produced esters can be utilized as the precursor of various carbonyl compounds.

A Kinetic Pump Integrated Microfluidic Plate (KIM-Plate) with High Usability for Cell Culture-Based Multiorgan Microphysiological Systems.

Microphysiological systems (MPSs), including organ-on-a-chip (OoC), have attracted attention as a novel method for estimating the effects and side effects of drugs in drug discovery. To reproduce the dynamic in vivo environment, previous MPSs were connected to pump systems to perfuse culture medium. Therefore, most MPSs are not user-friendly and have poor throughput. We aimed to develop a kinetic pump integrated microfluidic plate (KIM-Plate) by applying the stirrer-based micropump to an open access culture plate to improve the usability of MPSs. The KIM-Plate integrates six multiorgan MPS (MO-MPS) units and meets the ANSI/SBS microplate standards. We evaluated the perfusion function of the kinetic pump and found that the KIM-Plate had sufficient agitation effect. Coculture experiments with PXB cells and hiPS intestinal cells showed that the TEER of hiPS intestinal cells and gene expression levels related to the metabolism of PXB cells were increased. Hence, the KIM-Plate is an innovative tool for the easy coculture of highly conditioned cells that is expected to facilitate cell-based assays in the fields of drug discovery and biology because of its usability and high throughput nature.

Sustainable System for Hydrogenation Exploiting Energy Derived from Solar Light.

Herein described is a sustainable system for hydrogenation that uses solar light as the ultimate source of energy. The system consists of two steps. Solar energy is captured and chemically stored in the first step; exposure of a solution of azaxanthone in ethanol to solar light causes an energy storing dimerization of the ketone to produce a sterically strained 1,2-diol. In the second step, the chemical energy stored in the vicinal diol is released and used for hydrogenation; the diol offers hydrogen onto alkenes and splits back to azaxanthone, which is easily recovered and reused repeatedly for capturing solar energy.

Intra-articular Lipoma of the Knee Joint Located in the Lateral Recess: A Case Report.

INTRODUCTION: Lipomas are the most common type of soft-tissue tumor, but intra-articular lipomas are very rare. Most cases occur in the knee joint. This is a report of intra-articular lipoma in the knee joint, 8 months after removal with good post-operative outcome. CASE REPORT: A 56-year-old female who presented soft-tissue mass on her right knee. She felt uncomfortable but not in pain. The mass grew gradually and she wanted to be surgically removed. The mass was too large (5 cm × 4 cm × 2 cm) to be removed endoscopically, so arthrotomy was made. The mass was true lipoma histologically. She is living happily without recurrence after surgery. CONCLUSION: We have described an intra-articular synovial lipoma in the knee of a middle-aged female. Intra-articular lipomas are very rare. The tumor in this case was relatively large but painless, and the chief complaint was discomfort. It grew gradually, so the removal was performed. At present, the patient is living happy without recurrence after surgery.

Cleavage of Carbon-Carbon σ-Bonds of Four-Membered Rings.

This article reviews synthetic transformations involving cleavage of a carbon-carbon bond of a four-membered ring, with a particular focus on the examples reported during the period from 2011 to the end of 2019. Most significant is the progress of catalytic reactions involving oxidative addition of carbon-carbon bonds onto transition metals or ß-carbon elimination of transition metal alkoxides. When they are looked at from synthetic perspectives, they offer unique and efficient methods to build complex natural products and structures that are difficult to construct by conventional methods. On the other hand, ß-scission of radical intermediates has also attracted increasing attention as an alternative elementary step to cleave carbon-carbon bonds. Its site-selectivity is often complementary to that of transition metal-catalyzed reactions. In addition, Lewis acid-mediated and thermally induced ring-opening of cyclobutanone derivatives has garnered renewed attention. On the whole, these examples demonstrate unique synthetic potentials of structurally strained four-membered ring compounds for the construction of organic skeletons.

Photoinduced Specific Acylation of Phenolic Hydroxy Groups with Aldehydes.

A convenient method is reported to specifically acylate phenolic hydroxyl groups through a radical pathway. When a mixture of an aldehyde and a phenol in ethyl acetate is irradiated with blue light in the presence of iridium and nickel bromide catalysts at ambient temperature, phenoxyl and acyl radicals are transiently generated in situ and cross-couple to furnish an ester. Aliphatic hydroxy groups remain untouched under the reaction conditions.

Palliative Care of Malignant Fibrous Histiocytoma of Spine with Cord Compression and Multiple Bone Metastases Treated by Multidisciplinary Therapy: Case Report.

Malignant fibrous histiocytoma (MFH) of the spine is rare, with only a few dozen cases reported in the literature. A 60-year-old male was referred to us with symptoms of thoracic myelopathy. A solid tumor in the Th8 right costovertebral junction invading the spinal canal and compressing the spinal cord, and multiple bony metastases were discovered. Biopsy confirmed MFH. The thoracic spine tumor showed good response to irradiation followed by embolization and partial resection. The patient was followed until his death 22 months later. A good quality of life was sustained for more than 18 months. Despite a poor prognosis and an aggressive course of MFH of the spine, a good quality of life could be sustained for more than a year with palliative interventions.

Dehydrogenative Coupling of Benzylic and Aldehydic C-H Bonds.

A photoinduced dehydrogenative coupling reaction between benzylic and aldehydic C-H bonds is reported. When a solution of an alkylbenzene and an aldehyde in ethyl acetate is irradiated with visible light in the presence of iridium and nickel catalysts, a coupled α-aryl ketone is formed with evolution of dihydrogen. An analogous C-C bond forming reaction occurs between a C-H bond next to the nitrogen of an N-methylamide and an aldehydic C-H bond to produce an α-amino ketone. These reactions provide a straightforward pathway from readily available materials leading to valued structural motifs of pharmacological relevance.