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Abstract It was reported by Jung and Day in 2011 that a cotton-like glass fiber pad made of borate glass 13-93B3 demonstrated a remarkable wound healing effect. It was approved for sale as a novel wound dressing in the management of acute and chronic wounds in 2016. However, the detailed mechanism of its wound healing effect has not been reported. In the present study, glass fibers of different composition in the system CaO-B2O3-SiO2 were prepared and their in vitro properties investigated to determine the role of the constituent components in wound healing. Fine glass fibers that were 0.6–2.0 μm in diameter were obtained by a melt blown method. However, these fibers were accompanied by small glass beads because of the low viscosity of the glass melts. 13-93B3 glass released an appreciable amount of borate and calcium ions into simulated body fluid (SBF). The amounts of these released ions decreased with partial replacement of the B2O3 in 13-93B3 with SiO2. The addition of large amounts of the borate and calcium ions into the culture medium decreased the viability of the L929 fibroblasts. Partial replacement of the B2O3 in 13-93B3 with SiO2 induced the formation of an apatite-like phase amenable to the adsorption of biological components on its surface in SBF. The wound healing effect of these glass fibers of different composition is worth examining in future animal experiments.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is an inherited metabolic disease caused by a defect in electron transfer flavoprotein alpha (ETFA), ETF beta (ETFB), or ETF dehydrogenase (ETFDH), and riboflavin metabolism disorders have recently been reported to present as mimicking MADD. MADD is roughly classified into neonatal (type 1 or 2) and later-onset (type 3) forms. To identify clinicogenetic characteristics in Japan, we investigated 37 Japanese patients with MADD diagnosed from 1997 to 2020. The causes of MADD were ETFDH deficiency in 26 patients, ETFA deficiency in four, ETFB deficiency in six, and riboflavin metabolism disorder in one. All 15 patients with the neonatal-onset type died by 2 years of age, while five of 22 patients with the later-onset form died by 3 years of age. Furthermore, 8 of 15 patients with the later-onset form of ETFDH deficiency treated with riboflavin were riboflavin non-responders. p.Y507D in ETFDH was identified as the most common variant (9 of 48 alleles, 18.8%). Of two patients with a homozygous p.Y507D variant, one experienced disease onset and died in the neonatal period, while the other experienced disease onset at two months of age and died at two years old, suggesting that the p.Y507D variant results in fatal outcomes. Our study concluded that more than half of Japanese patients with MADD died by three years old, and more than half of patients with the later-onset form had poor responsiveness to riboflavin, partly due to the unique Japanese p.Y507D variant in ETFDH.

Abstract Sol–gel-derived TiO2 coatings have been confirmed to effectively promote bone-bonding behavior on polyetheretherketone (PEEK) surfaces; however, the optimal layer thickness to maximize the osseointegration and adhesive performance has not been yet determined. In this study, we applied sol–gel-derived TiO2 coatings with different layer thicknesses (40 and 120 nm) on PEEK implants to determine the effects of layer thickness on the surface characteristics, adhesive strength, and bone bonding capabilities (including histological osseointegration). The surface analysis results of both coated implants indicated no significant differences concerning the water contact angle, layer adhesion strength, and apatite formation ability in a simulated body fluid. Additionally, the in vivo biomechanical tests revealed a higher bone-bonding strength for both coated PEEK implants (compared with that of the uncoated sample). It was thus concluded that the factor of layer thickness marginally influences the bioactive advantages attained by sol–gel-derived TiO2 coatings on PEEK surfaces, highlighting the significant versatility and clinical availability of this coating technology.

There is a paucity of data on nodular regenerative hyperplasia after liver transplant. We aim to define the clinical disease trajectory and identify predictors of outcome for this rare diagnosis. This is a retrospective review of postulated risk factors and outcome in patients with nodular regenerative hyperplasia. Patients were classified as having a late presentation if nodular regenerative hyperplasia was diagnosed > 48 months from transplant, and symptomatic if portal hypertensive symptoms were present. Forty-nine of 3,711 (1.3%) adult recipients developed nodular regenerative hyperplasia, and mortality was 32.7% with an average follow up of 84.6 months. The MELD-Na 6 months after diagnosis did not change significantly. Patients with symptomatic portal hypertension at the time of diagnosis had a significantly higher risk of mortality (51.8%) compared to patients with liver test abnormalities alone (10.5%). 44.9% of patients had no previously postulated risk factor. Anastomotic vascular complications do not appear to be the etiology in most patients. The results suggest the vast majority of patients presenting with liver test abnormalities alone have stable disease and excellent long term survival, in contrast to the 56.3% mortality seen in patients that present more than 48 months after LT with symptomatic portal hypertension at diagnosis.

The main unmet medical need of bone implants is multifunctional activity, including their ability to induce rapid and physiological osseointegration, counteract bacterial biofilm formation, and prevent in situ chronic inflammation at the same time. This research starts from an already developed c.p. titanium surface with proven bioactive (in vitro hydroxyl apatite precipitation) and antibacterial activities, due to a calcium titanate layer with nano- and micro-scale roughness and loaded with iodine ions. Here, antioxidant ability was added to prevent chronic inflammation by grafting polyphenols of a green tea extract onto the surface, without compromising the other functionalities of the surface. The surface was characterized before and after functionalization through XPS analysis, zeta potential titrations, ion release measurements, in vitro bioactivity tests, SEM and fluorescence microscopy, and Folin–Ciocalteu and biological tests. The presence of grafted polyphenols as a homogeneous layer was proven. The grafted polyphenols maintained their antioxidant ability and were anchored to the surface through the linking action of Ca2+ ions added to the functionalizing solution. Iodine ion release, cytocompatibility towards human mesenchymal stem cells (hMSC), and antibacterial activity were maintained even after functionalization. The antioxidant ability of the functionalized surface was effective in preserving hMSC viability in a chemically induced pro-inflammatory environment, thus showing a scavenger activity towards toxic active species responsible for inflammation.

Background: Scoring systems have been proposed to select donation after circulatory death (DCD) donors and recipients for liver transplantation (LT). We hypothesized that complex scoring systems derived in large datasets might not predict outcomes locally.Methods: Based on 1-year DCD-LT graft survival predictors in multivariate logistic regression models, we designed, validated, and compared a simple index using the University of California, San Francisco (UCSF) cohort (n = 136) and a universal-comprehensive (UC)-DCD score using the United Network for Organ Sharing (UNOS) cohort (n = 5,792) to previously published DCD scoring systems.Results: The total warm ischemia time (WIT)-index included donor WIT (dWIT) and hepatectomy time (dHep). The UC-DCD score included dWIT, dHep, recipient on mechanical ventilation, transjugular-intrahepatic-portosystemic-shunt, cause of liver disease, model for end-stage liver disease, body mass index, donor/recipient age, and cold ischemia time. In the UNOS cohort, the UC-score outperformed all previously published scores in predicting DCD-LT graft survival (AUC: 0.635 vs. ≤0.562). In the UCSF cohort, the total WIT index successfully stratified survival and biliary complications, whereas other scores did not.Conclusion: DCD risk scores generated in large cohorts provide general guidance for safe recipient/donor selection, but they must be tailored based on non-/partially-modifiable local circumstances to expand DCD utilization.

Abstract Purpose Current newborn screening (NBS) for mucopolysaccharidosis type I (MPSI) has very high false positive rates and low positive predictive values (PPVs). To improve the accuracy of presymptomatic prediction for MPSI, we propose an NBS tool based on known biomarkers, alpha‐L‐iduronidase enzyme activity (IDUA) and level of the glycosaminoglycan (GAG) heparan sulfate (HS). Methods We developed the NBS tool using measures from dried blood spots (DBS) of 5000 normal newborns from Gifu Prefecture, Japan. The tool's predictive accuracy was tested on the newborn DBS from these infants and from seven patients who were known to have early‐onset MPSI (Hurler's syndrome). Bivariate analyses of the standardized natural logarithms of IDUA and HS levels were employed to develop the tool. Results Every case of early‐onset MPSI was predicted correctly by the tool. No normal newborn was incorrectly identified as having early‐onset MPSI, whereas 12 normal newborns were so incorrectly identified by the Gifu NBS protocol. The PPV was estimated to be 99.9%. Conclusions Bivariate analysis of IDUA with HS in newborn DBS can accurately predict early MPSI symptoms, control false positive rates, and enhance presymptomatic treatment. This bivariate analysis‐based approach, which was developed for Krabbe disease, can be extended to additional screened disorders.

Robots able to coexist and interact with humans are key elements for Society 5.0. To produce the right expectations towards robots, it will be necessary to expose the true current capabilities of robots to the general public. In this context, Human–Robot Interaction (HRI) in the wild emerges as a relevant paradigm. In this article, we affront the challenge of bringing an industrial robot (NEXTAGE Open) outside factories and laboratories to be used in a public setting. We designed a multi-modal interactive scenario that integrates state-of-the-art sensory devices, deep learning methods for perception, and a human–machine graphical interface that monitors the system and provides useful information to participants. The main objective of the presented work is to build a robust and fully autonomous robotic system able to: (1) share the same space as humans, (2) work in a public and crowded space, and (3) provide an intuitive and engaging experience for a robotic exposition. In addition, we measured the attitudes, perceptions, expectations, and emotional reactions of volunteers. Results suggest that participants considered our proposed scenario as enjoyable, safe, interesting, and clear. Those points are also the main concerns of participants about sharing workspaces of daily environments with robots. However, we can point out some limitations with a biased population mainly composed of Japanese and males. In future work, we will improve our scenario with non-functional features or emotional expressions from the robot.

Background and Aim: Tocilizumab, a humanized anti-IL-6 receptor antibody, has been used to treat severely to critically ill patients with COVID-19. A living systematic review with meta-analysis of recent RCTs indicates that the combination therapy of corticosteroids and tocilizumab produce better outcomes, while previous observational studies suggest that tocilizumab monotherapy is beneficial for substantial numbers of patients. However, what patients could respond to tocilizumab monotherapy remained unknown.Methods: In this retrospective study we evaluated the effects of tocilizumab monotherapy on the clinical characteristics, serum biomediator levels, viral elimination, and specific IgG antibody induction in 13 severely to critically ill patients and compared with those of dexamethasone monotherapy and dexamethasone plus tocilizumab.Results: A single tocilizumab administration led to a rapid improvement in clinical characteristics, inflammatory findings, and oxygen supply in 7 of 11 patients with severe COVID-19, and could recover from mechanical ventilation management (MVM) in 2 patients with critically ill COVID-19. Four patients exhibited rapidly worsening even after tocilizumab administration and required MVM and additional methylprednisolone treatment. Tocilizumab did not delay viral elimination or inhibit IgG production specific for the virus, whereas dexamethasone inhibited IgG induction. A multiplex cytokine array system revealed a significant increase in the serum expression of 54 out of 80 biomediators in patients with COVID-19 compared with that in healthy controls. Compared with those who promptly recovered in response to tocilizumab, patients requiring MVM showed a significantly higher ratio of basal level of ferritin/CRP and a persistent increase in the levels of CRP and specific cytokines and chemokines including IL-6, IFN-γ, IP-10, and MCP-1. The basal high ratio of ferritin/CRP was also associated with clinical deterioration even in patients treated with dexamethasone and tocilizumab.Conclusion: Tocilizumab as monotherapy has substantial beneficial effects in some patients with severe COVID-19, who showed a relatively low level of the ratio of ferritin/CRP and prompt reduction in CRP, IL-6, IFN-γ, IP-10, and MCP-1. The high ratio of ferritin/CRP is associated with rapid worsening of pneumonia. Further evaluation is warranted to clarify whether tocilizumab monotherapy or its combination with corticosteroid is preferred for severely to critically ill patients with COVID-19.

Flow injection analysis–tandem mass spectrometry (FIA-TMS) has been applied in a first-tier test of newborn screening (NBS). Although isovalerylcarnitine (i-C5), which is a diagnostic indicator of isovaleric acidemia (IVA), is isobaric with pivaloylcarnitine (p-C5), 2-methylbutyrylcarnitine, and n-valerylcarnitine, these isomers cannot be distinguished by the FIA-TMS. There are many reports of false positives derived from p-C5 due to the use of pivalate-conjugated antibiotics. In this study, we developed a new FIA-TMS method to distinguish i-C5 and p-C5. We found that the intensity ratio of product ions for i-C5 and p-C5 was different in a certain range even under the same analytical conditions. The product ions with the most distinct differences in ionic intensity between the isomers and the collision energies that produce them were determined to be m/z 246.2 > 187.1 and −15 V, respectively. In addition to the quantification ion, a reference ion was defined, and the similarity of the i-C5 and p-C5 reference ion ratios (i-C5 score and p-C5 score, respectively) were used to estimate which isomer (i-C5 and p-C5) was responsible for elevated C5 acylcarnitine in dried blood spots (DBSs). As a result of analyses of 11 DBS samples derived from pivalate-conjugated antibiotics and four DBS samples from IVA patients using our method, it was found that our method was able to correctly determine the type of C5-acylcarnitine (i-C5 or p-C5) in the DBS samples. Implementation of this new FIA-TMS method into the current NBS protocol will allow for a reduction in false positives in IVA.

Abstract Background Urea cycle disorders (UCDs) are inherited metabolic disorders that present with hyperammonemia, and cause significant mortality and morbidity in infants and children. These disorders are not well reported in the Indian population, due to lack of a thorough study of the clinical and molecular profile. Results We present data from two major metabolic centres in India, including 123 cases of various UCDs. The majority of them (72/123, 58%) presented in the neonatal period (before 30 days of age) with 88% on or before day 7 of life (classical presentation), and had a high mortality (64/72, 88%). Citrullinemia type 1 was the most common UCD, observed in 61/123 patients. Ornithine transcarbamylase (OTC) deficiency was the next most common, seen in 24 cases. Argininosuccinic aciduria was diagnosed in 20 cases. Deficiencies of arginase, N-acetylglutamate synthase, carbamoyl phosphate synthetase, citrin, and lysinuric protein intolerance were also observed. Molecular genetic analysis revealed two common ASS1 mutations: c.470G > A (p.Arg157His) and c.1168G > A (p.Gly390Arg) (36 of 55 tested patients). In addition, few recurrent point mutations in ASL gene, and a deletion of the whole OTC gene were also noted. A total of 24 novel mutations were observed in the various genes studied. We observed a poor clinical outcome with an overall all time mortality of 63% (70/110 cases with a known follow-up), and disability in 70% (28/40) among the survivors. Prenatal diagnosis was performed in 30 pregnancies in 25 families, including one pre-implantation genetic diagnosis. Conclusions We report the occurrence of UCDs in India and the spectrum that may be different from the rest of the world. Citrullinemia type 1 was the most common UCD observed in the cohort. Increasing awareness amongst clinicians will improve outcomes through early diagnosis and timely treatment. Genetic diagnosis in the proband will enable prenatal/pre-implantation diagnosis in subsequent pregnancies.

In Japan, carnitine palmitoyltransferase II (CPTII) deficiency has been included as one of the primary target diseases in the expanded newborn mass screening program since 2018. However, many cases of the severe infantile hepatocardiomuscular form of CPTII deficiency showed severe neurodevelopmental delay or sudden death, which indicated that management of CPTII deficiency in the acute phase remains to be studied in detail. Herein, we discuss two cases diagnosed by newborn mass screening. Patient 1 was under strict clinical management from the neonatal period, with >20 admissions in 14 months, while Patient 2 was managed using a relatively relaxed approach, with only 2 admissions in the same period. Patient 1 showed normal development; however, Patient 2 expired at the age of 1 year 2 months. To develop strategies for preventing sudden deaths in patients with CPTII deficiency, this retrospective study focused on detailed clinical management practices and biochemical findings during the acute phase. We also investigated the correlation between conventional biomarkers (such as creatine kinase) and long-chain acylcarnitines. We propose that strict monitoring and immediate medical attention, even in case of slight fever or minor abdominal symptoms, can help prevent sudden death in patients with CPTII deficiency. Considering the higher morbidity rate of such patients, strict and acute management of CPTII deficiency cannot be overemphasized.

Mucopolysaccharidoses (MPS) are a family of lysosomal storage disorders which can lead to degenerative and irreversible skeletal, cardiovascular, pulmonary, and neurological damage. Current treatments, including hematopoietic stem cell transplantation and enzyme replacement therapy, have been found most effective if administered before clinical symptoms are present, highlighting the urgent need for the development of newborn screening.This study analyzed 18,222 dried blood spot samples from newborns for both enzyme activity and glycosaminoglycan (GAG) concentration levels. GAG levels were measured using liquid chromatography tandem mass spectrometry. Results were compared to our previously established cutoff values for three subtypes of GAGs: dermatan sulfate (DS) and heparan sulfate (HS0S and HSNS). Samples that were high for two of the three GAGs were identified and screened a second time. Samples were also measured for iduronate-2-sulfatase and alfa-L-iduronidase activity.A total of 300 samples were above the established cutoff values for at least two of the three GAGs after the first screening. One sample was determined through clinical and genetic testing to be a true positive for MPS II. The false positive rate after the first GAG screening was 1.64%. A Cochran's formula test showed that the samples available for the second screening were representative samples (p = .0000601). False positive rate after second GAG screening, extrapolated from the representative sample was 0.4%. False positive rate after enzyme activity assay by fluorimetry for IDUA and IDS enzymes was 0.21% and 0.18%. A combination of GAG and enzyme assays provided no false positive and false negative samples.Two-tier screening involving a combination of enzyme activity and multiple GAGs should be considered the gold standard for the diagnosis of MPS patients. Keywords: Newborn screening, Mucopolysaccharidosis, Fetal GAG contamination, Tandem mass spectrometry, Glycosaminoglycans

Background: Expanded newborn screening (ENBS) utilizing tandem mass spectrometry (MS/MS) for inborn metabolic diseases (IMDs), such as organic acidemias (OAs), fatty acid oxidation disorders, (FAODs), and amino acid disorders (AAs), is increasingly popular but has not yet been introduced in many Asian countries. This study aimed to determine the incidence rates of OAs, FAODs, and AAs in Asian countries and Germany using selective screening and ENBS records. Materials and methods: Selective screening for IMDs using gas chromatography–mass spectrometry and MS/MS was performed among patients suspected to be afflicted in Asian countries (including Japan, Vietnam, China, and India) between 2000 and 2015, and the results from different countries were compared. Similarly, ENBS results from Japan, South Korea, Taiwan, and Germany were compared. Additionally, the results of selective screening and ENBS in Japan were compared. Results: Among 39,270 patients who underwent selective screening, IMDs were detected in 1170. Methylmalonic acidemia was frequently identified in several countries, including Japan (81/377 diagnosed IMDs), China (94/216 IMDs), and India (72/293 IMDs). In Vietnam, however, β-ketothiolase deficiency was particularly frequent (33/250 IMDs). ENBS yielded differences in overall IMD rates by country: 1:8557 in Japan, 1:7030 in Taiwan, 1:13,205 in South Korea, and 1:2200 in Germany. Frequently discovered diseases included propionic acidemia (PPA) and phenylketonuria (PKU) in Japan, 3-methylcrotonyl-CoA carboxylase deficiency (MCCD) and PKU in Taiwan, MCCD and citrullinemia type I in South Korea, and PKU and medium-chain acyl-CoA dehydrogenase deficiency in Germany. Furthermore, in Japan, selective screening and ENBS yielded respective PPA frequencies of 14.7% and 49.4% among all organic acidemias. Conclusion: The incidence rates of IMDs vary by country. Moreover, the disease spectra of IMDs detected via selective screening differ from those detected via ENBS. Keywords: Organic acidemia, Fatty acid oxidation disorder, Amino acid disorder, Inherited metabolic disease, Expanded newborn screening, Incidence rate

Introduction: Various markers, such as C14:1 and the C14:1/C2 ratio, are used as diagnostic markers of very long-chain acyl-CoA dehydrogenase deficiency (VLCADD). However, the levels of these markers in patients with VLCADD overlap with those in heterozygous carriers and even healthy subjects. Materials and methods: In twenty-three affected patients and 15 heterozygous carriers with VLCADD, the accuracies of C14:1, C14:1/C12:1, C14:1/C2, and C14:1/C16 in dried blood spots (DBS) and serum were statistically estimated. Results: Among the serum markers, the sensitivity, specificity, positive predictive value, negative predictive value, false-positive rate, false-negative rate, and validity of C14:1/C12:1 were superior to those of C14:1, C14:1/C2, and C14:1/C16, but C14:1/C2 demonstrated a statistical advantage compared with only C14:1 and C14:1/C16. Elevation in serum C14:1/C12:1 was observed in only one heterozygous carrier, whereas almost half of the carriers displayed false positive results for the other markers. Among the DBS markers, although the accuracy of C14:1/C2 was ostensibly the best, no statistical significance was observed. Discussion: Serum C14:1/C12:1 might be useful for differentiating patients with VLCADD from heterozygous carriers. Although serum C14:1/C2 was significantly useful for the detection of VLCADD, this marker could not distinguish the affected patients from carriers. C14:1/C12:1 might be optimal compared with the other markers. Keywords: Very long-chain acyl-CoA dehydrogenase deficiency, Tetradecenoyl carnitine, Dodecenoyl carnitine, Heterozygous carrier, Newborn screening, Diagnostic markers

Introduction: Fatty acid oxidation disorders (FAODs) are rare diseases caused by defects in mitochondrial fatty acid oxidation (FAO) enzymes. While the efficacy of bezafibrate, a peroxisome proliferator-activated receptor agonist, on the in vitro FAO capacity has been reported, the in vivo efficacy remains controversial. Therefore, we conducted a clinical trial of bezafibrate in Japanese patients with FAODs. Materials and methods: This trial was an open-label, non-randomized, and multicenter study of bezafibrate treatment in 6 patients with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency and 2 patients with carnitine palmitoyltransferase-II (CPT-2) deficiency (median age, 8.2 years; ranging from 5.8 to 26.4 years). Bezafibrate was administered for 6 months following a 6-month observation period. The primary endpoint was the frequency of myopathic attacks, and the secondary endpoints were serum acylcarnitines (ACs, C14:1 or C16 + C18:1), creatine kinase (CK) levels, degree of muscle pain (VAS; visual analog scale) during myopathic attacks, and quality of life (QOL; evaluated using validated questionnaires). Results: The frequency of myopathic attacks after bezafibrate administration decreased in 3 patients, increased in 3, and did not change in 2. The CK, AC, and VAS values during attacks could be estimated in only three or four patients, but a half of the patients did not experience attacks before or after treatment. Changes in CK, AC, and VAS values varied across individuals. In contrast, three components of QOL, namely, physical functioning, role limitation due to physical problems (role physical), and social functioning, were significantly elevated. No adverse drug reactions were observed. Conclusion: In this study, the frequency of myopathic attacks and CK, AC, and VAS values during the attacks could not be evaluated due to several limitations, such as a small trial population. Our findings indicate that bezafibrate improves the QOL of patients with FAODs, but its efficacy must be examined in future investigations. Keywords: Bezafibrate, Fatty acid oxidation disorders (FAODs), Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, Carnitine palmitoyltransferase-II (CPT-2) deficiency, Clinical trial

Costello syndrome is a “RASopathy” that is characterized by growth retardation, dysmorphic facial appearance, hypertrophic cardiomyopathy and tumor predisposition. >80% of patients with Costello syndrome harbor a heterozygous germline G12S mutation in HRAS. Altered metabolic regulation has been suspected because patients with Costello syndrome exhibit hypoketotic hypoglycemia and increased resting energy expenditure, and their growth is severely retarded. To examine the mechanisms of energy reprogramming by HRAS activation in vivo, we generated knock-in mice expressing a heterozygous Hras G12S mutation (HrasG12S/+ mice) as a mouse model of Costello syndrome. On a high-fat diet, HrasG12S/+ mice developed a lean phenotype with microvesicular hepatic steatosis, resulting in early death compared with wild-type mice. Under starvation conditions, hypoketosis and elevated blood levels of long-chain fatty acylcarnitines were observed, suggesting impaired mitochondrial fatty acid oxidation. Our findings suggest that the oncogenic Hras mutation modulates energy homeostasis in vivo.

Jaw reconstruction using an additive-manufacturing titanium artificial bone (AMTAB) has recently attracted considerable attention. The synthesis of a titanium artificial bone is based on three-dimensional computed tomography images acquired before surgery. A histological evaluation of porous AMTAB (pAMTAB) embedded in rat calvarial bone defects was conducted. This study examined three groups: rats implanted with mixed-acid and heat-treated pAMTAB, rats implanted with untreated pAMTAB, and rats with no implant. In both pAMTAB groups, bone defects were created in rat calvarial bones using a 5-mm trephine bar, followed by pAMTAB implantation. The pAMTAB was fixed to the defect using the fitting force of the surrounding bones. The rats were sacrificed at 4, 8, and 16 weeks after implantation, and the skull was dissected. Undecalcified ground slides were prepared and stained with Villanueva Goldner. Compared with the no implant control group, both pAMTAB groups exhibited new bone formation inside the defect, with greater bone formation in the mixed-acid and heat-treated pAMTAB group than in the untreated pAMTAB group, but the difference was not significant. These data suggest that pAMTAB induces bone formation after implantation in bone defects. Bone formation appears to be enhanced by prior mixed-acid and heat-treated pAMTAB.

In the orthopedic and dental fields, simultaneously conferring titanium (Ti) and its alloy implants with antibacterial and bone-bonding capabilities is an outstanding challenge. In the present study, we developed a novel combined solution and heat treatment that controllably incorporates 0.7% to 10.5% of iodine into Ti and its alloys by ion exchange with calcium ions in a bioactive calcium titanate. The treated metals formed iodine-containing calcium-deficient calcium titanate with abundant Ti-OH groups on their surfaces. High-resolution XPS analysis revealed that the incorporated iodine ions were mainly positively charged. The surface treatment also induced a shift in the isoelectric point toward a higher pH, which indicated a prevalence of basic surface functionalities. The Ti loaded with 8.6% iodine slowly released 5.6 ppm of iodine over 90 days and exhibited strong antibacterial activity (reduction rate >99%) against methicillin-resistant Staphylococcus aureus (MRSA), S. aureus, Escherichia coli, and S. epidermidis. A long-term stability test of the antibacterial activity on MRSA showed that the treated Ti maintained a >99% reduction until 3 months, and then it gradually decreased after 6 months (to a 97.3% reduction). There was no cytotoxicity in MC3T3-E1 or L929 cells, whereas apatite formed on the treated metal in a simulated body fluid within 3 days. It is expected that the iodine-carrying Ti and its alloys will be particularly useful for orthopedic and dental implants since they reliably bond to bone and prevent infection owing to their apatite formation, cytocompatibility, and sustainable antibacterial activity.

Glycosaminoglycans (GAGs) are present in proteoglycans, which play critical physiological roles in various tissues. They are known to be elevated in mucopolysaccharidoses (MPS), a group of rare inherited metabolic diseases in which the lysosomal enzyme required to break down one or more GAG is deficient. In a previous study, we found elevation of GAGs in a subset of patients without MPS. In the current study, we aim to investigate serum GAG levels in patients with conditions beyond MPS. In our investigated samples, the largest group of patients had a clinical diagnosis of viral or non-viral encephalopathy. Clinical diagnoses and conditions also included epilepsy, fatty acid metabolism disorders, respiratory and renal disorders, liver disorders, hypoglycemia, developmental disorders, hyperCKemia, myopathy, acidosis, and vomiting disorders. While there was no conclusive evidence across all ages for any disease, serum GAG levels were elevated in patients with encephalopathy and some patients with other conditions. These preliminary findings suggest that serum GAGs are potential biomarkers in MPS and other disorders. In conclusion, we propose that GAGs elevated in blood can be used as biomarkers in the diagnosis and prognosis of various diseases in childhood; however, further designed experiments with larger sample sizes are required.

Mucopolysaccharidoses (MPS) and mucolipidosis (ML II/III) are a group of lysosomal storage disorders (LSDs) that occur due to a dysfunction of the lysosomal hydrolases responsible for the catabolism of glycosaminoglycans (GAGs). However, ML is caused by a deficiency of the enzyme uridine-diphosphate N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase, EC2.7.8.17), which tags lysosomal enzymes with a mannose 6-phosphate (M6P) marker for transport to the lysosome. A timely diagnosis of MPS and ML can lead to appropriate therapeutic options for patients. To improve the accuracy of diagnosis for MPS and ML in a high-risk population, we propose a combination method based on known biomarkers, enzyme activities, and specific GAGs. We measured five lysosomal enzymes (α-L-iduronidase (MPS I), iduronate-2-sulfatase (MPS II), α-N-acetylglucosaminidase (MPS IIIB), N-acetylglucosamine-6-sulfatase (MPS IVA), and N-acetylglucosamine-4-sulfatase (MPS VI)) and five GAGs (two kinds of heparan sulfate (HS), dermatan sulfate (DS), and two kinds of keratan sulfate (KS)) in dried blood samples (DBS) to diagnose suspected MPS patients by five-plex enzyme and simultaneous five GAGs assays. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) for both assays. These combined assays were tested for 43 patients with suspected MPS and 103 normal control subjects. We diagnosed two MPS I, thirteen MPS II, one MPS IIIB, three MPS IVA, two MPS VI, and six ML patients with this combined method, where enzymes, GAGs, and clinical manifestations were compatible. The remaining 16 patients were not diagnosed with MPS or ML. The five-plex enzyme assay successfully identified MPS patients from controls. Patients with MPS I, MPS II, and MPS IIIB had significantly elevated HS and DS levels in DBS. Compared to age-matched controls, patients with ML and MPS had significantly elevated mono-sulfated KS and di-sulfated KS levels. The results indicated that the combination method could distinguish these affected patients with MPS or ML from healthy controls. Overall, this study has shown that this combined method is effective and can be implemented in larger populations, including newborn screening.

Selective laser melting (SLM) is a promising technology capable of producing individual characteristics with a high degree of surface roughness for implants. These surfaces can be modified so as to increase their osseointegration, bone generation and biocompatibility, features which are critical to their clinical success. In this study, we evaluated the effects on preosteoblast proliferation and differentiation of titanium metal (Ti) with a high degree of roughness (Ra = 5.4266 ± 1.282 µm) prepared by SLM (SLM-Ti) that was also subjected to surface bioactive treatment by mixed acid and heat (MAH). The results showed that the MAH treatment further increased the surface roughness, wettability and apatite formation capacity of SLM-Ti, features which are useful for cell attachment and bone bonding. Quantitative measurement of osteogenic-related gene expression by RT-PCR indicated that the MC3T3-E1 cells on the SLM-Ti MAH surface presented a stronger tendency towards osteogenic differentiation at the genetic level through significantly increased expression of Alp, Ocn, Runx2 and Opn. We conclude that bio-activated SLM-Ti enhanced preosteoblast differentiation. These findings suggest that the mixed acid and heat treatment on SLM-Ti is promising method for preparing the next generation of orthopedic and dental implants because of its apatite formation and cell differentiation capability.

Japanese newborn screening (NBS) for phenylketonuria (PKU) was initiated in 1977. We surveyed the neurological outcomes of Japanese adult patients with PKU to investigate the long-term effects and of and issues with NBS. Eighty-five patients with PKU aged over 19 years who continued to be treated with a phenylalanine-free amino acid formula were investigated by administering questionnaires regarding clinical characteristics, such as mental ability, education status, and therapeutic condition. Of the 85 subjects, 68 patients were detected by NBS (NBS group), while the other 17 were clinically diagnosed before the initiation of NBS (pre-NBS group). Further, 10 of the 68 NBS patients presented intellectual and/or psychiatric disabilities, 5 of whom had a history of treatment discontinuation; in contrast, 12 of the 17 pre-NBS patients presented with neuropsychiatric symptoms. Regarding social outcomes, almost all patients in the NBS group could live an independent life, while over half of the patients in the pre-NBS group were not employed or lived in nursing-care facilities. Neurological outcomes are obviously improved by NBS in Japan. However, some patients, even those detected by NBS, developed neuropsychiatric symptoms due to treatment disruption. Lifelong and strict management is essential to maintain good neurological and social prognoses for patients with PKU.

We recently found that NFATc1, a member of the NFAT family and a key regulator of the immune response, could induce bladder carcinogenesis and cancer progression. In this study, we immunohistochemically stained for NFATc1 in upper urinary tract urothelial carcinoma (UUTUC) specimens and paired nonneoplastic urothelial tissues. NFATc1 was positive in 51 [52%; 40 (40%) weak (1+), 9 (9%) moderate (2+), and 2 (2%) strong (3+)] of 99 UUTUCs, which was significantly higher than in benign urothelium [30 (36%) of 83; 28 (34%) weak and 2 (2%) moderate] (0 vs 1+/2+/3+, P = .038; 0/1+ vs 2+/3+, P = .023). There were no significant associations between NFATc1 expression pattern and tumor grade or pT stage. However, the positive rates of NFATc1 expression tended to be higher in renal pelvic tumors (60%) than in ureteral tumors (42%; P = .080) as well as in pN+ tumors (75%) than in pN0 tumors (49%; P = .089). Kaplan-Meier and log-rank tests revealed that moderate (2+) to strong (3+) NFATc1 expression correlated with lower progression-free survival (P = .032) and cancer-specific survival (P = .005) rates in the 99 cases. Patients with high (2+/3+) NFATc1 muscle-invasive tumor (n = 9) also had a significantly higher risk of cancer-specific mortality (P = .021) compared to those with low (0/1+) NFATc1 muscle-invasive tumor (n = 53). Thus, compared with nonneoplastic urothelium, a significant increase in the expression of NFATc1 in UUTUC was seen, implying the involvement of NFATc1 signals in the development of UUTUC. The current results further suggest that NFATc1 overexpression serves as a predictor of poor prognosis in patients with UUTUC.

Introduction: Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is an autosomal recessive mitochondrial fatty acid oxidation disorder and presents as hypoketotic hypoglycemia or rhabdomyolysis during childhood. l-Carnitine supplementation for patients with VLCAD deficiency is controversial. Herein, we describe two siblings with VLCAD deficiency who experienced more frequent episodes of rhabdomyolysis after l-carnitine supplementation. Case presentation: Case 1 involved a 6-year-old boy who was diagnosed with VLCAD deficiency after repeated episodes of hypoketotic hypoglycemia at 3 years of age. He developed rhabdomyolysis more frequently after starting l-carnitine supplementation. Case 2 involved an 8-year-old boy, the elder brother of case 1, who was also diagnosed with VLCAD deficiency by sibling screening at the age of 5 years. He first developed rhabdomyolysis during a common cold after treatment with l-carnitine. Both patients had fewer rhabdomyolysis episodes after the cessation of l-carnitine supplementation. Conclusion: Our cases suggest that l-carnitine supplementation can increase rhabdomyolysis attacks in patients with VLCAD deficiency. Keywords: Very long-chain acyl-CoA dehydrogenase deficiency, l-Carnitine, Supplementation, Rhabdomyolysis, Side effect

Introduction: Fatty acid oxidation disorders (FAODs) are rare diseases caused by a defective mitochondrial fatty acid oxidation (FAO) enzyme. We recently reported that bezafibrate improved patient quality of life (QOL) based on the SF-36 questionnaire score in patients with FAODs during a 50-week, open-label, clinical trial. Herein we conducted further survey assessments of the trial patients to define the long-term efficacy and safety of bezafibrate. Materials and methods: This trial was an open-label, non-randomized, and multicenter study of bezafibrate treatment in five patients with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency and one patient with carnitine palmitoyltransferase-II (CPT-2) deficiency (median age, 15.9 years; range, 5.8–26.4 years). The bezafibrate administration was continued for a further 102–174 weeks after the 24-week treatment described in our previous study. QOL was quantitated using the 36-Item Short Form Health Survey (SF-36) questionnaire, which constitutes eight components: physical functioning (PF), role limitation due to physical problems, bodily pain, general health perception, vitality, social functioning, role limitation due to emotional problems, and mental health. Results: PF was elevated in all patients and continued to rise during the study, with the total QOL scores increased from baseline in five of the six cases. In particular, three patients older than 20 years showed treatment efficacy, and all subcategories of QOL were elevated in two of these cases. Conclusion: Our findings supported one of the stated benefits of bezafibrate in improving QOL for patients with FAODs. Keywords: Bezafibrate, Fatty acid oxidation disorders (FAODs), Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, Carnitine palmitoyltransferase-II (CPT-2) deficiency, Clinical trial, Quality of life

Glycosaminoglycans (GAGs), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS), are the primary biomarkers in patients with mucopolysaccharidoses (MPS); however, little is known about other biomarkers. To explore potential biomarkers and their correlation with GAGs, blood samples were collected from 46 MPS II patients, 34 MPS IVA patients, and 5 MPS IVB patients. We evaluated the levels of 8 pro-inflammatory factors (EGF, IL-1β, IL-6, MIP-1α, TNF-α, MMP-1, MMP-2, and MMP-9), collagen type II, and DS, HS (HS0S, HSNS), and KS (mono-sulfated, di-sulfated) in blood.Eight biomarkers measured were significantly elevated in untreated MPS II patients, compared with those in normal controls: EGF, IL-1β, IL-6, HS0S, HSNS, DS, mono-sulfated KS, and di-sulfated KS. The same eight biomarkers remained elevated in ERT-treated patients. However, only three biomarkers remained elevated in post-HSCT MPS II patients: EGF, mono-sulfated KS, and di-sulfated KS. Post-HSCT patients with MPS II showed that IL-1β and IL-6 were normalized as HS and DS levels decreased. Eight biomarkers were significantly elevated in untreated MPS IVA patients: EGF, IL-1β, IL-6, MIP-1α, MMP-9, HSNS, mono-sulfated KS, and di-sulfated KS, and four biomarkers were elevated in MPS IVA patients under ERT: IL-6, TNF-α, mono-sulfated KS, and di-sulfated KS. There was no reduction of KS in the ERT-treated MPS IVA patient, compared with untreated patients. Two biomarkers were significantly elevated in untreated MPS IVB patients: IL-6 and TNF-α.Reversely, collagen type II level was significantly decreased in untreated and ERT-treated MPS II patients and untreated MPS IVA patients.In conclusion, selected pro-inflammatory factors can be potential biomarkers in patients with MPS II and IV as well as GAGs levels. Keywords: Morquio syndrome, Hunter syndrome, Glycosaminoglycans, Cytokines, Inflammation

The additive manufacturing (AM) technique has attracted attention as one of the fully customizable medical material technologies. In addition, the development of new surface treatments has been investigated to improve the osteogenic ability of the AM titanium (Ti) plate. The purpose of this study was to evaluate the osteogenic activity of the AM Ti with mixed-acid and heat (MAH) treatment. Fully customized AM Ti plates were created with a curvature suitable for rat calvarial bone, and they were examined in a group implanted with the MAH-treated Ti in comparison with the untreated (UN) group. The AM Ti plates were fixed to the surface of rat calvarial bone, followed by extraction of the calvarial bone 1, 4, 8, and 12 weeks after implantation. The bonding between the bone and Ti was evaluated mechanically. In addition, AM Ti plates removed from the bone were examined histologically by electron microscopy and Villanueva–Goldner stain. The mechanical evaluation showed significantly stronger bone-bonding in the MAH group than in the UN group. In addition, active bone formation was seen histologically in the MAH group. Therefore, these findings indicate that MAH resulted in rapid and strong bonding between cortical bone and Ti.

Some special implantable materials are defined as “bioactive” if they can bond to living bone, forming a tight and chemically-stable interface. This property, which is inherent to some glass compositions, or can be induced by applying appropriate surface treatments on otherwise bio-inert metals, can be evaluated in vitro by immersion studies in simulated body fluid (SBF), mimicking the composition of human plasma. As a result, apatite coating may form on the material surface, and the presence of this bone-like “biomimetic skin” is considered predictive of bone-bonding ability in vivo. This review article summarizes the story and evolution of in vitro bioactivity testing methods using SBF, highlighting the influence of testing parameters (e.g., formulation and circulation of the solution) and material-related parameters (e.g., composition, geometry, texture). Suggestions for future methodological refinements are also provided at the end of the paper.

Mitochondrial trifunctional protein (TFP) is a multienzyme complex that catalyzes the last three steps of the β-oxidation cycle of long-chain fatty acids. In the prenatal diagnosis of TFP deficiency, acylcarnitine (AC) analysis has been considered difficult because of limited excretion of long-chain ACs into the fetal urine and hence into the amniotic fluid. Here, we report our experience with prenatally diagnosing TFP deficiency using AC analysis of amniotic fluid. The index case was a boy born at 38 weeks gestation and weighing 2588 g. He suddenly became unconscious and hypoglycemic and died on day 6 of life. Postmortem blood AC analysis and gene sequencing revealed TFP deficiency. Therefore, the parents underwent prenatal diagnoses for their subsequent 2 pregnancies. Mutation analysis suggested that one (Case 1) was affected and the other (Case 2) was not. AC analysis also demonstrated identical results, with significantly elevated 3-hydroxy-AC levels in the amniotic fluid of the affected pregnancy compared with those of heterozygotes and normal controls (n = 2 for heterozygotes and n = 8 for normal controls). Our findings suggest that AC analysis can functionally confirm results even in families with unidentified mutations, without raising issues related to maternal cell contamination. During prenatal diagnosis, misdiagnosis has to be avoided, and combining AC analysis with gene sequencing may result in more accurate prenatal diagnosis of TFP deficiency.

The recent introduction of metabolic autopsy in the field of forensic science has made it possible to detect hidden inherited metabolic diseases. Since the next generation sequencing (NGS) has recently become available for use in postmortem examinations, we used NGS to perform metabolic autopsy in 15 sudden unexpected death in infancy cases. Diagnostic results revealed a case of carnitine palmitoyltransferase II deficiency and some cases of fatty acid oxidation-related gene variants. Metabolic autopsy performed with NGS is a useful method, especially when postmortem biochemical testing is not available.

Carnitine palmitoyltransferase II (CPT-2) deficiency, an autosomal recessive disorder of fatty acid oxidation, can be detected by newborn screening using tandem mass spectrometry (TMS). Our case was a boy born at 38 weeks and 6 days of gestation via normal vaginal delivery; his elder sister was affected with CPT-2 deficiency. Acylcarnitine (AC) was analyzed in both dried blood spots (DBS) and serum 2 h after birth to determine whether the boy was also affected. His C16 and C18:1 AC levels in DBS were in the normal range, while his serum long-chain AC levels were marginally increased but lower than those of his sister. After the samples were taken, he was treated with glucose infusion to prevent any catabolism for 2 days. On day 4, the long-chain AC levels in both DBS and serum obtained were higher than those on day 0 and were equivalent to those of his sister. Genetic testing confirmed the presence of the same mutation found in his sister, a homozygous F383Y mutation in the CPT2 gene, thus leading to the diagnosis of CPT-2 deficiency. The sample for TMS should be taken between days 1 and 7. If the sample is not obtained at an appropriate time, correct diagnosis may not be made, as in our case. Although early diagnosis is required, samples taken within 24 h after birth should not be used for TMS.

Background: Homocystinuria (HCU) is a rare inherited metabolic disease. In Japan, newborn screening (NBS) for HCU (cystathionine β-synthase deficiency) was initiated in 1977. We compared the outcomes between patients detected by NBS (NBS group) and clinically detected patients (non-NBS group). Methods: We administered questionnaires about clinical symptoms and social conditions to 16 attending physicians of 19 adult HCU patients treated with methionine-free formula. Results: Eighteen patients (nine patients each in the NBS and non-NBS groups) participated. The frequency of patients with ocular, vascular, central nervous system, and skeletal symptoms in the NBS group was lower than that in the non-NBS group. Intellectual disability was observed in one and eight patients in the NBS and non-NBS groups, respectively. Concerning their social conditions, all patients in the NBS group were employed or still attending school, while only two patients in the non-NBS group were employed. Three of the four patients who discontinued treatment presented some symptoms, even in the NBS group. Conclusion: The social and intellectual outcomes of adult Japanese patients with HCU detected by NBS were favorable. However, even in the patients in the NBS group, some symptoms might not be preventable without continuous treatment.

Enfortumab vedotin is a novel antibody–drug conjugate targeting Nectin-4, which is highly expressed in urothelial carcinoma. However, the expression status of Nectin-4 in upper tract urothelial carcinoma (UTUC) remains unclear. The relationship between Nectin-4 and Programmed Death Ligand 1 (PD-L1) in UTUC is also ambiguous. We performed immunohistochemical analysis of 99 UTUC tissue microarray to assess the expression of Nectin-4 and PD-L1 in UTUC. Nectin-4-positivity was detected in 65 (65.7%) samples, and PD-L1 was detected in 24 (24.2%) samples. There was no correlation between the expression of Nectin-4 and PD-L1. Patients with strong Nectin-4-expressing tumors had a significantly higher risk of progression (p = 0.031) and cancer-specific mortality (p = 0.036). Strong Nectin-4 expression was also an independent predictor of disease progression in the high-risk group (pT3 ≤ or presence of lymphovascular invasion or lymph node metastasis) (Hazard ratio, 3.32 [95% confidence interval, 1.20–7.98; p = 0.027]). In conclusion, we demonstrated that Nectin-4 expression rate in UTUC was 65.7% and independent of PD-L1 expression. Strong Nectin-4 expression was associated with worse progression-free survival in high-risk UTUC. These findings suggested that enfortumab vedotin may be effective in a broad range of patients with UTUC, regardless of PD-L1 expression.

Spinal muscular atrophy (SMA) is a common neuromuscular disease with autosomal recessive inheritance. The disease gene, SMN1, is homozygously deleted in 95% of SMA patients. Although SMA has been an incurable disease, treatment in infancy with newly developed drugs has dramatically improved the disease severity. Thus, there is a strong rationale for newborn and carrier screening for SMA, although implementing SMA carrier screening in the general population is controversial. We previously developed a simple, accurate newborn SMA screening system to detect homozygous SMN1 deletions using dried blood spots (DBS) on filter paper. Here, we modified our previous system to detect the heterozygous deletions of SMN1, which indicates SMA carrier status. The system involves a calibrator-normalized relative quantification method using quantitative nested PCR technology. Our system clearly separated the DBS samples with one SMN1 copy (carrier status with a heterozygous deletion of SMN1) from the DBS samples with two SMN1 copies (non-carrier status with no deletion of SMN1). We also analyzed DBS samples from SMA families, confirmed SMA in the affected children, and determined the carrier status of their parents based on the SMN1 copy number. In conclusion, our system will provide essential information for risk assessment and genetic counseling, at least for SMA families.

Mucopolysaccharidoses (MPSs) are rare lysosomal storage diseases caused by the accumulation of undegraded glycosaminoglycans in cells and tissues. The effectiveness of early intervention for MPS has been reported. Multiple-assay formats using tandem mass spectrometry have been developed. Here, we developed a method for simultaneous preparation and better measurement of the activities of five enzymes involved in MPSs, i.e., MPS I, MPS II, MPS IIIB, MPS IVA, and MPS VI, which were validated using 672 dried blood spot samples obtained from healthy newborns and 23 patients with MPS. The mean values of the enzyme activities and standard deviations in controls were as follows: α-iduronidase (IDUA), 4.19 ± 1.53 µM/h; iduronate-2-sulfatase (I2S), 8.39 ± 2.82 µM/h; N-acetyl-α-glucosaminidase (NAGLU), 1.96 ± 0.57 µM/h; N-acetylgalactosamine-6-sulfatase (GALNS), 0.50 ± 0.20 µM/h; and N-acetylgalactosamine-4-sulfatase (ARSB), 2.64 ± 1.01 µM/h. All patients displayed absent or low enzyme activity. In MPS I, IIIB, and VI, each patient group was clearly separated from controls, whereas there was some overlap between the control and patient groups in MPS II and IVA, suggesting the occurrence of pseudo-deficiencies. Thus, we established a multiplex assay for newborn screening using liquid chromatography tandem mass spectrometry, allowing simultaneous pretreatment and measurement of five enzymes relevant to MPSs.

Nowadays, there is a large amount of research aimed at improving the multifunctional behavior of the biomaterials for bone contact, including the concomitant ability to induce apatite formation (bioactivity), fast and effective osteoblasts colonization, and antibacterial activity. The aim of this study is to develop antibacterial and bioactive surfaces (Ti6Al4V alloy and a silica-based bioactive glass) by chemical doping with strontium and/or silver ions. The surfaces were characterized by Scanning Electron Microscopy equipped with Energy Dispersive X ray Spectroscopy (SEM-EDS), X-ray photoelectron spectroscopy (XPS), and Transmission Electron Microscopy (TEM). To better focus on the cells−bacteria competition for the implant surface, in addition to the standard assays for the evaluation of the bacteria adhesion (ISO22196) and for single-cell cultures or biofilm formation, an innovative set of co-cultures of cells and bacteria is here proposed to simulate a competitive surface colonization. The results suggest that all the bioactive tested materials were cytocompatible toward the bone progenitor cells representative for the self-healing process, and that the doped ones were effective in reducing the surface colonization from a pathogenic drug-resistant strain of Staphylococcus aureus. The co-cultures experiments demonstrated that the doped surfaces were able to protect the adhered osteoblasts from the bacteria colonization as well as prevent the infection prior to the surface colonization by the osteoblasts.

The leading reason for implant revision surgery globally is lack of implant integration with surrounding bone. A new titanium alloy GUMMETAL ® (Ti 59 Nb 36 Ta 2 Zr 3 O 0.3 ) is currently used in biomedical devices and has a Young’s modulus that is better matched to bone. The surface was subject to NaOH, CaCl 2 , heat and water treatment (BioGum) after which the surfaces were evaluated using atomic force microscope, scanning electron microscope, X-ray diffractometer and elemental analysis using energy dispersive X-ray. To demonstrate enhanced bone bonding ability and cytocompatibility, apatite formation in simulated body fluid and in vitro stem cell attachment, proliferation and cytoskeleton organisation were examined. The formation of a ~200 nm nanoscale needle-like calcium titanate network on the surface following treatment was revealed and upon soaking in simulated body fluid, the formation of a ~5 µm layer of apatite. Metabolic activity of rat bone marrow stem cells on BioGum was increased in comparison to control and the cell number appeared greater, with more elongated morphology as early as 2 h post-seeding. This positions the modification as a simple and potentially universal technology for the improvement of implant integration.

Titanium (Ti)-6Al-4V alloy, which is widely used in spinal instrumentation with a pedicle screw (PS) system. However, significant clinical problems, including loosening and back-out of PSs, persist. During the last decade, a novel technology that produces bioactive Ti from chemical and heat treatments has been reported that induces the spontaneous formation of a hydroxyapatite (HA) layer on the surface of Ti materials. The purpose of this study was to study the effect of bioactivation of Ti-6Al-4V PSs on the ability of HA formation in vitro and its biocompatibility and bone-bonding ability in vivo.Ti-6V-4Al alloy PSs were prepared and bioactivated by NaOH-CaCl2-heat-water treatments. The HA-forming ability of bioactive PSs in simulated body fluid (SBF) was evaluated by field emission scanning electron microscopy (FE-SEM) and energy dispersive X-ray analysis (EDX). Six 11-month-old female beagle dogs were used for the in vivo study. Bioactive and control (without bioactivation) PSs were left and right randomly placed from L1 to L6. One and three months after surgery, lumbar spines were removed for biomechanical and histological analyses.In vitro: The surface analysis of bioactive PSs by FE-SEM and EDX showed substantial HA deposits over the entire surface. In vivo: The mean extraction torque was significantly higher for bioactive PSs compared to controls PSs (P

Signal transducer and activator of transcription 3 (STAT3) plays a prominent role in the growth and invasion of several types of solid tumors. In this study, to assess the expression status and prognostic significance of the STAT3 pathway in upper urinary tract urothelial carcinoma (UTUC), we immunohistochemically stained for STAT3 and STAT3 pathway proteins, sphingosine-1-phosphate receptor 1 (S1PR1) and interleukin-6 (IL-6), in a tissue microarray containing 99 UTUC specimens. There were no significant associations between STAT3, S1PR1, or IL-6 expression pattern and tumor grade or pT stage. However, the patients with high STAT3 tumor had a significantly higher risk of both disease progression (p = 0.009) and cancer-specific mortality (p = 0.009), but not with tumors expressing S1PR1 or IL-6. High STAT3 expression in the nucleus was also associated with a significantly higher risk of both disease progression (p = 0.003) and cancer-specific mortality (p = 0.034). Multivariate analysis revealed that high STAT3 expression in the nucleus was significantly associated with cancer-specific survival after adjustment for pathological stage, lymph node involvement, lymphovascular invasion, and tumor grade (HR = 2.136, 95% CI = 1.009-4.767, p = 0.047). Our findings indicated that STAT3 could be a cancer-promoting factor and potentially a significant prognostic factor in UTUC.

Polyetheretherketone (PEEK) is a widely accepted biomaterial, especially in the field of spinal surgery. However, PEEK is not able to directly integrate with bone tissue, due to its bioinertness. To overcome this drawback, various studies have described surface coating approaches aimed at increasing the bioactivity of PEEK surfaces. Among those, it has been shown that the recently developed sol-gel TiO2 coating could provide PEEK with the ability to bond with bone tissue in vivo without the use of a bone graft.This in vivo experimental study using a canine model determined the efficacy of bioactive TiO2-coated PEEK for anterior cervical fusion.Sol-gel-derived TiO2 coating, which involves sandblasting and acid treatment, was used to give PEEK bone-bonding ability. The cervical interbody spacer, which was designed to fit the disc space of a beagle, was fabricated using bioactive TiO2-coated PEEK. Both uncoated PEEK (control) and TiO2-coated PEEK spacers were implanted into the cervical intervertebral space of beagles (n = 5 for each type). After the 3-month survival period, interbody fusion success was evaluated based on μ-CT imaging, histology, and manual palpation analyses.Manual palpation analyses indicated a 60% (3/5 cases) fusion (no gap between bone and implants) rate for the TiO2-coated PEEK group, indicating clear advantage over the 0% (0/5 cases) fusion rate for the uncoated PEEK group. The bony fusion rate of the TiO2-coated PEEK group was 40% according to μCT imaging; however, it was 0% of for the uncoated PEEK group. Additionally, the bone-implant contact ratio calculated using histomorphometry demonstrated a better contact ratio for the TiO2-coated PEEK group than for the uncoated PEEK group (mean, 32.6% vs 3.2%; p = 0.017).The TiO2-coated bioactive PEEK implant demonstrated better fusion rates and bone-bonding ability than did the uncoated PEEK implant in the canine anterior cervical fusion model. Bioactive PEEK, which has bone-bonding ability, could contribute to further improvements in clinical outcomes for spinal interbody fusion.

Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by deficiency of the lysosomal enzymes essential for catabolism of glycosaminoglycans (GAGs). Accumulation of undegraded GAGs results in dysfunction of multiple organs, resulting in distinct clinical manifestations. A range of methods have been developed to measure specific GAGs in various human samples to investigate diagnosis, prognosis, pathogenesis, GAG interaction with other molecules, and monitoring therapeutic efficacy. We established ELISA, liquid chromatography tandem mass spectrometry (LC-MS/MS), and an automated high-throughput mass spectrometry (HT-MS/MS) system (RapidFire) to identify epitopes (ELISA) or disaccharides (MS/MS) derived from different GAGs (dermatan sulfate, heparan sulfate, keratan sulfate, and/or chondroitin sulfate). These methods have a high sensitivity and specificity in GAG analysis, applicable to the analysis of blood, urine, tissues, and cells. ELISA is feasible, sensitive, and reproducible with the standard equipment. HT-MS/MS yields higher throughput than conventional LC-MS/MS-based methods while the HT-MS/MS system does not have a chromatographic step and cannot distinguish GAGs with identical molecular weights, leading to a limitation of measurements for some specific GAGs. Here we review the advantages and disadvantages of these methods for measuring GAG levels in biological specimens. We also describe an unexpected secondary elevation of keratan sulfate in patients with MPS that is an indirect consequence of disruption of catabolism of other GAGs.

3-Hydroxyisobutyryl-CoA hydrolase (HIBCH) deficiency is an autosomal recessive disorder characterized by episodes of ketoacidosis and a Leigh-like basal ganglia disease, without high concentrations of pyruvate and lactate in the cerebrospinal fluid. Only 4 cases of HIBCH deficiency have been reported. However, clinical–biochemical correlation in HIBCH deficiency by determining the detailed residual enzyme activities has not yet been elucidated. Here, we report a case of two Japanese siblings with HIBCH deficiency carrying a new homozygous missense mutation (c.287C > A, [p.A96D]) at the substrate-binding site. A transfection study using HIBCH expression vectors harboring wild type or 4 reported mutations, including the newly identified mutation (p.A96D, p.Y122C, p.G317E, and p.K74Lfs*13), revealed a correlation between residual HIBCH activities and the severity of the disease. All HIBCH mutants, except p.K74Lfs*13, showed residual enzyme activity and only the patient with p.K74Lfs*13 had congenital anomalies. p.G317E showed only low enzyme activity (~3%) of that of wild-type HIBCH. Although p.A96D had approximately 7 times higher enzyme activity than p.G317E, patients with p.A96D died during childhood. These findings are essential for clinical management, genetic counseling, and specific meal and concomitant drug considerations as part of the treatment for patients with HIBCH deficiency.

Internal tandem duplication (ITD) mutations in the Fms-related tyrosine kinase 3 (FLT3) gene (FLT3-ITD) are associated with poor prognosis in patients with acute myeloid leukemia (AML). Due to the development of drug resistance, few FLT3-ITD inhibitors are effective against FLT3-ITD+ AML. In this study, we show that FLT3-ITD activates a novel pathway involving p21Cdkn1a (p21) and pre-B cell leukemia transcription factor 1 (Pbx1) that attenuates FLT3-ITD cell proliferation and is involved in the development of drug resistance. FLT3-ITD up-regulated p21 expression in both mouse bone marrow c-kit+-Sca-1+-Lin- (KSL) cells and Ba/F3 cells. The loss of p21 expression enhanced growth factor-independent proliferation and sensitivity to cytarabine as a consequence of concomitantly enriching the S+G2/M phase population and significantly increasing the expression of Pbx1, but not Evi-1, in FLT3-ITD+ cells. This enhanced cell proliferation following the loss of p21 was partially abrogated when Pbx1 expression was silenced in FLT3-ITD+ primary bone marrow colony-forming cells and Ba/F3 cells. When FLT3-ITD was antagonized with AC220, a selective inhibitor of FLT3-ITD, p21 expression was decreased coincident with Pbx1 mRNA up-regulation and a rapid decline in the number of viable FLT3-ITD+ Ba/F3 cells; however, the cells eventually became refractory to AC220. Overexpressing p21 in FLT3-ITD+ Ba/F3 cells delayed the emergence of cells that were refractory to AC220, whereas p21 silencing accelerated their development. These data indicate that FLT3-ITD is capable of inhibiting FLT3-ITD+ cell proliferation through the p21/Pbx1 axis and that treatments that antagonize FLT3-ITD contribute to the subsequent development of cells that are refractory to a FLT3-ITD inhibitor by disrupting p21 expression.

Patients with severe hypophosphatasia (HPP) develop osteogenic impairment with extremely low alkaline phosphatase (ALP) activity, resulting in a fatal course during infancy. Mesenchymal stem cells (MSCs) differentiate into various mesenchymal lineages, including bone and cartilage. The efficacy of allogeneic hematopoietic stem cell transplantation for congenital skeletal and storage disorders is limited, and therefore we focused on MSCs for the treatment of HPP. To determine the effect of MSCs on osteogenesis, we performed multiple infusions of ex vivo expanded allogeneic MSCs for two patients with severe HPP who had undergone bone marrow transplantation (BMT) from asymptomatic relatives harboring the heterozygous mutation. There were improvements in not only bone mineralization but also muscle mass, respiratory function, and mental development, resulting in the patients being alive at the age of 3. After the infusion of MSCs, chimerism analysis of the mesenchymal cell fraction isolated from bone marrow in the patients demonstrated that donor-derived DNA sequences existed. Adverse events of BMT were tolerated, whereas those of MSC infusion did not occur. However, restoration of ALP activity was limited, and normal bony architecture could not be achieved. Our data suggest that multiple MSC infusions, following BMT, were effective and brought about clinical benefits for patients with lethal HPP. Allogeneic MSC-based therapy would be useful for patients with other congenital bone diseases and tissue disorders if the curative strategy to restore clinically normal features, including bony architecture, can be established.

Using preclinical models, we have recently found that ELK1, a transcriptional factor that activates downstream targets, including c-fos proto-oncogene, induces bladder cancer outgrowth. Here, we immunohistochemically determined the expression status of phospho-ELK1, an activated form of ELK1, in upper urinary tract urothelial carcinoma (UUTUC). Overall, phospho-ELK1 was positive in 47 (47.5%; 37 weak (1+) and 10 moderate (2+)) of 99 UUTUCs, which was significantly (P = 0.002) higher than in benign urothelium (21 (25.3%) of 83; 17 1+ and 4 2+) and was also associated with androgen receptor expression (P = 0.001). Thirteen (35.1%) of 37 non-muscle-invasive versus 34 (54.8%) of 62 muscle-invasive UUTUCs (P = 0.065) were immunoreactive for phospho-ELK1. Lymphovascular invasion was significantly (P = 0.014) more often seen in phospho-ELK1(2+) tumors (80.0%) than in phospho-ELK1(0/1+) tumors (36.0%). There were no statistically significant associations between phospho-ELK1 expression and tumor grade, presence of concurrent carcinoma in situ or hydronephrosis, or pN status. Kaplan-Meier and log-rank tests revealed that patients with phospho-ELK1(2+) tumor had marginally and significantly higher risks of disease progression (P = 0.055) and cancer-specific mortality (P = 0.008), respectively, compared to those with phospho-ELK1(0/1+) tumor. The current results thus support our previous observations in bladder cancer and further suggest that phospho-ELK1 overexpression serves as a predictor of poor prognosis in patients with UUTUC.

Sodium titanate formed on Ti metal by NaOH and heat treatments induces apatite formation on its surface in a body environment and bonds to living bone. These treatments have been applied to porous Ti metal in artificial hip joints, and have been used clinically in Japan since 2007. Calcium titanate formed on Ti-15Zr-4Nb-4Ta alloy by NaOH, CaCl2, heat, and water treatments induces apatite formation on its surface in a body environment. Titanium oxide formed on porous Ti metal by NaOH, HCl, and heat treatments exhibits osteoinductivity as well as osteoconductivity. This is now under clinical tests for application to a spinal fusion device.

Rotavirus and norovirus are common pathogens associated with gastroenteritis in children. Although rotavirus occasionally induces central nervous system disease, only 3 cases with rotavirus-induced acute myositis have been reported in the English literature. We recently treated a female patient with acute myositis associated with gastroenteritis induced by concurrent infection with rotavirus and norovirus. Having suffered from gastroenteritis for 3 days, she suddenly developed myositis affecting her lower extremities with concomitant creatine kinase elevation. Herein, we present our patient and review the previous cases including those reported in the Japanese literature.

Titanium metal (Ti) and its alloys are widely used in orthopedic and dental fields. We have previously shown that acid and heat treatment was effective to introduce bone bonding, osteoconduction and osteoinduction on pure Ti. In the present study, acid and heat treatment with or without initial NaOH treatment was performed on typical Ti-based alloys used in orthopedic and dental fields. Dynamic movements of alloying elements were developed, which depended on the kind of treatment and type of alloy. It was found that the simple acid and heat treatment enriched/remained the alloying elements on Ti–6Al–4V, Ti–15Mo–5Zr–3Al and Ti–15Zr–4Nb–4Ta, resulting in neutral surface charges. Thus, the treated alloys did not form apatite in a simulated body fluid (SBF) within 3 days. In contrast, when the alloys were subjected to a NaOH treatment prior to an acid and heat treatment, alloying elements were selectively removed from the alloy surfaces. As a result, the treated alloys became positively charged, and formed apatite in SBF within 3 days. Thus, the treated alloys would be useful in orthopedic and dental fields since they form apatite even in a living body and bond to bone.