Transferrin Receptor-Targeted Iduronate-2-sulfatase Penetrates the Blood-Retinal Barrier and Improves Retinopathy in Mucopolysaccharidosis II Mice.

Mucopolysaccharidoses (MPSs) make up a group of lysosomal storage diseases characterized by the aberrant accumulation of glycosaminoglycans throughout the body. Patients with MPSs display various signs and symptoms, such as retinopathy, which is also observed in patients with MPS II. Unfortunately, retinal disorders in MPS II are resistant to conventional intravenous enzyme-replacement therapy because the blood-retinal barrier (BRB) impedes drug penetration. In this study, we show that a fusion protein, designated pabinafusp alfa, consisting of an antihuman transferrin receptor antibody and iduronate-2-sulfatase (IDS), crosses the BRB and reaches the retina in a murine model of MPS II. We found that retinal function, as assessed by electroretinography (ERG) in MPS II mice, deteriorated with age. Early intervention with repeated intravenous treatment of pabinafusp alfa decreased heparan sulfate deposition in the retina, optic nerve, and visual cortex, thus preserving or even improving the ERG response in MPS II mice. Histological analysis further revealed that pabinafusp alfa mitigated the loss of the photoreceptor layer observed in diseased mice. In contrast, recombinant nonfused IDS failed to reach the retina and hardly affected the retinal disease. These results support the hypothesis that transferrin receptor-targeted IDS can penetrate the BRB, thereby ameliorating retinal dysfunction in MPS II.

One-pot enzymatic synthesis of 2-deoxy-scyllo-inosose from d-glucose and polyphosphate.

2-Deoxy-scyllo-inosose (2DOI, [2S,3R,4S,5R]-2,3,4,5-tetrahydroxycyclohexan-1-one) is a biosynthetic intermediate of 2-deoxystreptamine-containing aminoglycoside antibiotics, including butirosin, kanamycin, and neomycin. In producer microorganisms, 2DOI is constructed from d-glucose 6-phosphate (G6P) by 2-deoxy-scyllo-inosose synthase (DOIS) with the oxidized form of nicotinamide adenine dinucleotide (NAD+). 2DOI is also known as a sustainable biomaterial for production of aromatic compounds and a chiral cyclohexane synthon. In this study, a one-pot enzymatic synthesis of 2DOI from d-glucose and polyphosphate was investigated. First, 3 polyphosphate glucokinases (PPGKs) were examined to produce G6P from d-glucose and polyphosphate. A PPGK derived from Corynebacterium glutamicum (cgPPGK) was found to be suitable for G6P production under ordinary enzymatic conditions. Next, 7 DOISs were examined for the one-pot enzymatic reaction. As a result, cgPPGK and BtrC, the latter of which is a DOIS derived from the butirosin producer Bacillus circulans, achieved nearly full conversion of d-glucose to 2DOI in the presence of polyphosphate.

A single amino acid substitution converts a histidine decarboxylase to an imidazole acetaldehyde synthase.

Histidine decarboxylase (HDC; EC 4.1.1.22), an enzyme that catalyzes histamine synthesis with high substrate specificity, is a member of the group II pyridoxal 5'-phosphate (PLP) -dependent decarboxylase family. Tyrosine is a conserved residue among group II PLP-dependent decarboxylases. Human HDC has a Y334 located on a catalytically important loop at the active site. In this study, we demonstrated that a HDC Y334F mutant is capable of catalyzing the decarboxylation-dependent oxidative deamination of histidine to yield imidazole acetaldehyde. Replacement of the active-site Tyr with Phe in group II PLP-dependent decarboxylases, including mammalian aromatic amino acid decarboxylase, plant tyrosine/DOPA decarboxylase, and plant tryptophan decarboxylase, is expected to result in the same functional change, given that a Y-to-F substitution at the corresponding residue (number 260) in the HDC of Morganella morganii, another group II PLP-dependent decarboxylase, yielded the same effect. Thus, it was suggested that the loss of the OH moiety from the active-site Tyr residue of decarboxylase uniquely converts the enzyme to an aldehyde synthase.

Common carotid artery ligation at the proximal side before rupture in patients with ligation or occlusion of the external carotid artery at risk of carotid blowout syndrome.

BACKGROUND: Carotid blowout syndrome (CBS) is among the fatal complications in head and neck cancer treatment. However, the optimal treatment for CBS has not been established yet. This study aimed to describe our experience with two patients at high risk of CBS who underwent common carotid artery (CCA) ligation at the proximal side of the bleeding point under local anesthesia and before CCA rupture, and to review and compare the medical records of these two patients against 10 CBS cases treated in our department. METHODS: The institutional electronic medical record was searched, and clinical information was extracted for all patients who showed CBS from 2007 to 2017. Our treatment method was performed as follows. Ligation of the proximal side of the CCA was performed under local anesthesia. The CCA was identified and clamped with two bulldog forceps for 10 minutes to check for any adverse neurological symptoms. Subsequently, the CCA was ligated using 2-0 silk threads and sutured with an absorbable suture between the silk threads. However, ligation or occlusion of the external carotid artery by previous treatment is a prerequisite for this method. RESULTS: Eight patients received interventions, with six patients undergoing prophylactic interventions before rupture. Four patients who did not undergo treatment died owing to CBS. Two patients who underwent treatment with the novel method did not experience re-bleeding, but their conditions deteriorated owing to cancer progression. CONCLUSION: The present method is one of the treatment choices for CBS, especially in patients with an 'impending' risk of CBS.

Identification of three LRR-RKs involved in perception of root meristem growth factor in Arabidopsis.

A peptide hormone, root meristem growth factor (RGF), regulates root meristem development through the PLETHORA (PLT) stem cell transcription factor pathway, but it remains to be uncovered how extracellular RGF signals are transduced to the nucleus. Here we identified, using a combination of a custom-made receptor kinase (RK) expression library and exhaustive photoaffinity labeling, three leucine-rich repeat RKs (LRR-RKs) that directly interact with RGF peptides in Arabidopsis These three LRR-RKs, which we named RGFR1, RGFR2, and RGFR3, are expressed in root tissues including the proximal meristem, the elongation zone, and the differentiation zone. The triple rgfr mutant was insensitive to externally applied RGF peptide and displayed a short root phenotype accompanied by a considerable decrease in meristematic cell number. In addition, PLT1 and PLT2 protein gradients, observed as a gradual gradient decreasing toward the elongation zone from the stem cell area in wild type, steeply declined at the root tip in the triple mutant. Because RGF peptides have been shown to create a diffusion-based concentration gradient extending from the stem cell area, our results strongly suggest that RGFRs mediate the transformation of an RGF peptide gradient into a PLT protein gradient in the proximal meristem, thereby acting as key regulators of root meristem development.

Involvement of a cyclic adenosine monophosphate-dependent signal in the diet-induced canalicular trafficking of adenosine triphosphate-binding cassette transporter g5/g8.

UNLABELLED: The adenosine triphosphate-binding cassette (ABC) half-transporters Abcg5 and Abcg8 promote the secretion of neutral sterol into bile. Studies have demonstrated the diet-induced gene expression of these transporters, but the regulation of their trafficking when the nutritional status changes in the liver remains to be elucidated. Here, we generated a novel in vivo kinetic analysis that can monitor the intracellular trafficking of Abcg5/Abcg8 in living mouse liver by in vivo transfection of the genes of fluorescent protein-tagged transporters and investigated how hypernutrition affects the canalicular trafficking of these transporters. The kinetic analysis showed that lithogenic diet consumption accelerated the translocation of newly synthesized fluorescent-tagged transporters to intracellular pools in an endosomal compartment and enhanced the recruitment of these pooled gene products into the bile canalicular membrane in mouse liver. Because some ABC transporters are reported to be recruited from intracellular pools to the bile canaliculi by cyclic adenosine monophosphate (cAMP) signaling, we next evaluated the involvement of this machinery in a diet-induced event. Administration of a protein kinase A inhibitor, N-(2-{[3-(4-bromophenyl)-2-propenyl]amino}ethyl)-5-isoquinolinesulfonamide, decreased the canalicular expression of native Abcg5/Abcg8 in lithogenic diet-fed mice, and injection of a cAMP analog, dibutyryl cAMP, transiently increased their levels in standard diet-fed mice, indicating the involvement of cAMP signaling. Indeed, canalicular trafficking of the fluorescent-tagged Abcg5/Abcg8 was enhanced by dibutyryl cAMP administration. CONCLUSION: These observations suggest that diet-induced lipid loading into liver accelerates the trafficking of Abcg5/Abcg8 to the bile canalicular membrane through cAMP signaling machinery.

[Development of a Tool for Training and Evaluation of the Competencies in Occupational Mental Health Necessary for Labor and Social Security Attorneys].

Labor and Social Security Attorneys (LSSAs) advise their clients about occupational mental health, but the competencies necessary in this field are not clear to them. We standardized the necessary competencies as a counseling guide for LSSAs, and we also designed a related discussion training program. These competencies were summarized in a brainstorming session at a research conference comprised of physicians, an occupational health nurse, LSSAs, an instructional design expert, and a management consultant, and then a training program (lasting 9 hours 30 minutes) was developed. Nineteen trainees who were introduced by members of the research conference collectively completed a seven-question written test, both before and after the training, in order to assess its effectiveness. Sixteen trainees who completed the training were surveyed, with a recovery rate of 100%. The necessary competencies that they identified were: information about circular notices from the Ministry of Health, Labor and Welfare; behavior such as the gathering of information; and dealing with the reinstatement of employees. The scores were subjected to the Wilcoxon signed-rank test in order to evaluate the training, and the answers from the pre-training were compared with those from the post-training. A significant difference (P < 0.05) was seen for each question. These results show the effectiveness of the developed training program for the learning of the competencies necessary for LSSAs.

In Vivo Behavior of Hydrolyzable Tannins after Oral Administration of the Trapa bispinosa Extract to Rats.

The pericarp extract of Trapa bispinosa (TBPE), which is rich in hydrolyzable tannins, has been reported to inhibit α-glucosidase and glycation reactions. We investigated the in vivo behavior of hydrolyzable tannins and related metabolites after administration of TBPE to rats. Using high pressure liquid chromatography-electrospray ionization-tandem mass spectroscopy (HPLC-ESI-MS/MS), 12 ellagitannin metabolites, such as urolithins and 6 gallotannin metabolites, produced in the collected plasma and urine were quantified. Urolithins and gallic acid metabolites reached their maximum blood concentration after 24 and 1 h of administration, respectively. Conversely, the excretion of urolithins in urine required up to 72 h and followed a sigmoidal curve, whereas gallic acid metabolites were rapidly excreted earlier after administration. The results suggest that the metabolites gallotannin and ellagitannin are responsible for the antiglycation effect of TBPE, which proceeds via different mechanisms and times. Our findings provide basic data demonstrating the functionality of hydrolyzable tannins as well as Trapa ingredients.

A Case of Autoimmune Pulmonary Alveolar Proteinosis With Predominantly Peripheral Opacities Diagnosed by Transbronchial Lung Biopsy.

Although pulmonary alveolar proteinosis (PAP) showed various shadows, its shadows are usually distributed predominantly in the central lung area. We report a case of autoimmune PAP with localized subpleural ground-glass shadows in the bilateral upper lobes, which was diagnosed based on transbronchial lung biopsy (TBLB) specimen findings and anti-granulocyte macrophage colony PAP stimulating factor antibody positivity. PAP should be listed as a differential diagnosis for subpleural shadows. If subpleural shadows are observed, TBLB should be performed aggressively, and anti-granulocyte macrophage colony-stimulating factor (anti-GM-CSF) antibodies should be submitted.

Reddening of the Unicellular Green Alga Euglena gracilis by Dried Bonito Stock and Intense Red Light Irradiation.

This study confirms for the first time that the significant red coloration of Euglena gracilis is induced by bonito stock (BS), a traditional Japanese food, and intense red light exposure (605~660 nm, 1000~1300 µmol photons/m2/s). Under the condition, excessive photosynthetic activity destroyed many chloroplasts, while carotenoids were maintained, resulting in the formation of reddened cells. The HPLC analysis revealed that diadinoxanthin was the primary carotenoid present in reddened cells. Additionally, an undefined xanthophyll, not produced under normal culture conditions, was synthesized and suggested to contain a C=O bond. While it has been reported that strong light stress can increase the total carotenoid content of cells, this study did not verify this claim, and it should be investigated further in future research. Under white light irradiation conditions (90 µmol photons/m2/s) in BS medium, no reddening of cells was observed, and good growth was achieved (over four times the cell density in CM medium on the seventh day). This cell suspension is considered to have a high nutritional value because it is composed of functional food, BS and E. gracilis. The fact that this method does not involve genetic modification suggests the possibility of industrial applications, including food use, even in reddened cells.

Non-fibril form but not fibril form of human islet amyloid polypeptide 8-20 changes brain functions in mice.

Whether fibril formation increases or decreases cytotoxicity remains unclear. Aggregation of human islet amyloid polypeptide (hIAPP), a pivotal regulator of glucose homeostasis, impairs the function and viability of pancreatic ß cells. Evidence suggests that low-order oligomers of hIAPP are more toxic to ß cells than fibril. However, it remains unclear whether non-fibril form of hIAPP specifically alters brain functions. This study produced fibril and non-fibril forms from a single hIAPP 8-20 peptide. The non-fibril form-injected mice showed changes in spontaneous motor activities, preference for location in the open field and social behavior. In contrast, the fibril-injected mice showed no changes in these behavioral tests. In line with the behavioral changes, the non-fibril form led to impaired neurite outgrowth of cultured neuron-like cells and the loss of neurons in the mouse hippocampus. These findings suggest that non-fibril form but not fibril form of hIAPP changes brain functions.

Analysis of Unique Motility of the Unicellular Green Alga Chlamydomonas reinhardtii at Low Temperatures down to -8 °C.

Previous studies of motility at low temperatures in Chlamydomonas reinhardtii have been conducted at temperatures of up to 15 °C. In this study, we report that C. reinhardtii exhibits unique motility at a lower temperature range (-8.7 to 1.7 °C). Cell motility was recorded using four low-cost, easy-to-operate observation systems. Fast Fourier transform (FFT) analysis at room temperature (20-27 °C) showed that the main peak frequency of oscillations ranged from 44 to 61 Hz, which is consistent with the 60 Hz beat frequency of flagella. At lower temperatures, swimming velocity decreased with decreasing temperature. The results of the FFT analysis showed that the major peak shifted to the 5-18 Hz range, suggesting that the flagellar beat frequency was decreasing. The FFT spectra had distinct major peaks in both temperature ranges, indicating that the oscillations were regular. This was not affected by the wavelength of the observation light source (white, red, green or blue LED) or the environmental spatial scale of the cells. In contrast, cells in a highly viscous (3.5 mPa·s) culture at room temperature showed numerous peaks in the 0-200 Hz frequency band, indicating that the oscillations were irregular. These findings contribute to a better understanding of motility under lower-temperature conditions in C. reinhardtii.

Markers for obese and non-obese Type 2 diabetes identified using whole blood metabolomics.

Definitive differences in blood metabolite profiles between obese and non-obese Type 2 diabetes (T2D) have not been established. We performed an LC-MS-based non-targeted metabolomic analysis of whole blood samples collected from subjects classified into 4 types, based on the presence or absence of obesity and T2D. Of the 125 compounds identified, 20, comprising mainly nucleobases and glucose metabolites, showed significant increases or decreases in the T2D group. These included cytidine, UDP-glucuronate, UMP, 6-phosphogluconate, and pentose-phosphate. Among those 20 compounds, 11 enriched in red blood cells (RBCs) have rarely been studied in the context of diabetes, indicating that RBC metabolism is more extensively disrupted than previously known. Correlation analysis revealed that these T2D markers include 15 HbA1c-associated and 5 irrelevant compounds that may reflect diabetic conditions by a different mechanism than that of HbA1c. In the obese group, enhanced protein and fatty acid catabolism causes increases in 13 compounds, including methylated or acetylated amino acids and short-chain carnitines. Our study, which may be considered a pilot investigation, suggests that changes in blood metabolism due to obesity and diabetes are large, but essentially independent.

In fission yeast, 65 non-essential mitochondrial proteins related to respiration and stress become essential in low-glucose conditions.

Mitochondria perform critical functions, including respiration, ATP production, small molecule metabolism, and anti-oxidation, and they are involved in a number of human diseases. While the mitochondrial genome contains a small number of protein-coding genes, the vast majority of mitochondrial proteins are encoded by nuclear genes. In fission yeast Schizosaccharomyces pombe, we screened 457 deletion (del) mutants deficient in nuclear-encoded mitochondrial proteins, searching for those that fail to form colonies in culture medium containing low glucose (0.03-0.1%; low-glucose sensitive, lgs), but that proliferate in regular 2-3% glucose medium. Sixty-five (14%) of the 457 deletion mutants displayed the lgs phenotype. Thirty-three of them are defective either in dehydrogenases, subunits of respiratory complexes, the citric acid cycle, or in one of the nine steps of the CoQ10 biosynthetic pathway. The remaining 32 lgs mutants do not seem to be directly related to respiration. Fifteen are implicated in translation, and six encode transporters. The remaining 11 function in anti-oxidation, amino acid synthesis, repair of DNA damage, microtubule cytoskeleton, intracellular mitochondrial distribution or unknown functions. These 32 diverse lgs genes collectively maintain mitochondrial functions under low (1/20-1/60× normal) glucose concentrations. Interestingly, 30 of them have homologues associated with human diseases.

Enzyme replacement with transferrin receptor-targeted α-L-iduronidase rescues brain pathology in mucopolysaccharidosis I mice.

Mucopolysaccharidosis I (MPS I), a lysosomal storage disease caused by dysfunction of α-L-iduronidase (IDUA), is characterized by the deposition of dermatan sulfate (DS) and heparan sulfate (HS) throughout the body, which causes several somatic and central nervous symptoms. Although enzyme-replacement therapy (ERT) is currently available to treat MPS I, it does not alleviate central nervous disorders, as it cannot penetrate the blood-brain barrier. Here we evaluate the brain delivery, efficacy, and safety of JR-171, a fusion protein comprising humanized anti-human transferrin receptor antibody Fab and IDUA, using monkeys and MPS I mice. Intravenously administered JR-171 was distributed in major organs, including the brain, and reduced DS and HS concentrations in the central nervous system and peripheral tissues. JR-171 exerted similar effects on peripheral disorders similar to conventional ERT and further reversed brain pathology in MPS I mice. We found that JR-171 improved spatial learning ability, which was seen to deteriorate in the vehicle-treated mice. Further, no safety concerns were noted in repeat-dose toxicity studies in monkeys. This study provides nonclinical evidence that JR-171 might potentially prevent and even improve disease conditions in patients with neuronopathic MPS I without serious safety concerns.

Processing and stability of type IIc sodium-dependent phosphate cotransporter mutations in patients with hereditary hypophosphatemic rickets with hypercalciuria.

Mutations in the apically located Na(+)-dependent phosphate (NaPi) cotransporter, SLC34A3 (NaPi-IIc), are a cause of hereditary hypophosphatemic rickets with hypercalciuria (HHRH). We have characterized the impact of several HHRH mutations on the processing and stability of human NaPi-IIc. Mutations S138F, G196R, R468W, R564C, and c.228delC in human NaPi-IIc significantly decreased the levels of NaPi cotransport activities in Xenopus oocytes. In S138F and R564C mutant proteins, this reduction is a result of a decrease in the V(max) for P(i), but not the K(m). G196R, R468W, and c.228delC mutants were not localized to oocyte membranes. In opossum kidney (OK) cells, cell surface labeling, microscopic confocal imaging, and pulse-chase experiments showed that G196R and R468W mutations resulted in an absence of cell surface expression owing to endoplasmic reticulum (ER) retention. G196R and R468W mutants could be partially stabilized by low temperature. In blue native-polyacrylamide gel electrophoresis analysis, G196R and R468W mutants were either denatured or present in an aggregation complex. In contrast, S138F and R564C mutants were trafficked to the cell surface, but more rapidly degraded than WT protein. The c.228delC mutant did not affect endogenous NaPi uptake in OK cells. Thus, G196R and R468W mutations cause ER retention, while S138F and R564C mutations stimulate degradation of human NaPi-IIc in renal epithelial cells. Together, these data suggest that the NaPi-IIc mutants in HHRH show defective processing and stability.

Diabetes Mellitus, Elevated Hemoglobin A1c, and Glycated Albumin Are Associated with the Presence of All-Cause Dementia and Alzheimer's Disease: The JPSC-AD Study.

BACKGROUND: Glucose dysmetabolism is an important risk factor for dementia. OBJECTIVE: We investigated the associations of diabetes mellitus, the levels of glycemic measures, and insulin resistance and secretion measures with dementia and its subtypes in a cross-sectional study. METHODS: In this study, 10,214 community-dwelling participants were enrolled. Hemoglobin A1c (HbA1c), the homeostasis model assessment (HOMA) for insulin resistance (HOMA-IR), the HOMA of percent ß-cell function (HOMA-ß), and the glycated albumin (GA) was evaluated. The associations of each measure with Alzheimer's disease (AD) and vascular dementia (VaD) were investigated. RESULTS: The multivariable-adjusted odds ratios (ORs) of AD were significantly higher in participants with diabetes mellitus than in those without diabetes (1.46 [95% CI: 1.08-1.97]). Higher HbA1c levels were significantly associated with AD at diabetes (≥6.5%) and even at prediabetes (5.7 %-6.4 %) levels; multivariable-adjusted ORs for AD in participants at the diabetes level were 1.72 (95% CI: 1.19-2.49), and those in participants at the prediabetes level were 1.30 (95% CI: 1.00-1.68), compared with those in normal participants. Moreover, higher GA levels were associated with AD. No associations were observed between the diabetic status or the levels of glycemic measures and VaD. In addition, no significant relationships were observed between insulin resistance and secretion measurements and AD and VaD. CONCLUSION: Our findings indicate that diabetes mellitus and hyperglycemia are significantly associated with AD, even in individuals at the prediabetes level.

Absorbance spectroscopy of light scattering samples placed inside an integrating sphere for wide dynamic range absorbance measurement.

In the absorbance measurement of a sample that scatters light significantly, it is necessary to consider the effect of the attenuation of incident light due to scattering on the measured absorbance. Since the usual absorbance measurement with an integrating sphere (IS) cannot remove the influence of backscattering, we performed the absorbance measurement considering the light scattered to almost all solid angles by placing the sample inside the IS. Ni(NO3)2 and Co(NO3)2 aqueous solutions were used as non-scattering samples, and Ni(NO3)2 solutions mixed with submicrometer polystyrene spheres as scatterers were used as scattering samples. The sample-concentration dependence of the measured absorbance was investigated for the cell containing the sample placed at the entrance of or inside the IS. It was found that even inside the IS, the measured absorbance does not match the true absorbance because light is partially multiply transmitted through the sample or detected without being transmitted through the sample. Due to the latter reason, the saturated absorbance inside the IS was lower than that at the entrance. We derived the formula with three fitting parameters relating the measured and true absorbance taking these factors into account, which quantitatively reproduced the concentration dependence of the absorbance in the non-scattering sample. When the scattering samples were placed at the entrance and inside of the IS, the measured absorbance increased and decreased, respectively, compared to those without scatterers. This decrease in absorbance for the scattering samples inside the IS was also explained by the proposed formula slightly modified.