Scoring system for diagnosis and pretreatment risk assessment of neuroblastoma using urinary biomarker combinations.

The urinary catecholamine metabolites, homovanillic acid (HVA) and vanillylmandelic acid (VMA), are used for the adjunctive diagnosis of neuroblastomas. We aimed to develop a scoring system for the diagnosis and pretreatment risk assessment of neuroblastoma, incorporating age and other urinary catecholamine metabolite combinations. Urine samples from 227 controls (227 samples) and 68 patients with neuroblastoma (228 samples) were evaluated. First, the catecholamine metabolites vanillactic acid (VLA) and 3-methoxytyramine sulfate (MTS) were identified as urinary marker candidates through comprehensive analysis using liquid chromatography-mass spectrometry. The concentrations of these marker candidates and conventional markers were then compared among controls, patients, and numerous risk groups to develop a scoring system. Participants were classified into four groups: control, low risk, intermediate risk, and high risk, and the proportional odds model was fitted using the L2-penalized maximum likelihood method, incorporating age on a monthly scale for adjustment. This scoring model using the novel urine catecholamine metabolite combinations, VLA and MTS, had greater area under the curve values than the model using HVA and VMA for diagnosis (0.978 vs. 0.964), pretreatment risk assessment (low and intermediate risk vs. high risk: 0.866 vs. 0.724; low risk vs. intermediate and high risk: 0.871 vs. 0.680), and prognostic factors (MYCN status: 0.741 vs. 0.369, histology: 0.932 vs. 0.747). The new system also had greater accuracy in detecting missing high-risk neuroblastomas, and in predicting the pretreatment risk at the time of screening. The new scoring system employing VLA and MTS has the potential to replace the conventional adjunctive diagnostic method using HVA and VMA.

Synergy between Laminin-Derived Elastin-like Polypeptides (LELPs) Optimizes Cell Spreading.

A major component of the extracellular matrix (ECM), laminins, modulates cells via diverse receptors. Their fragments have emerging utility as components of "ECM-mimetics" optimized to promote cell-based therapies. Recently, we reported that a bioactive laminin peptide known as A99 enhanced cell binding and spreading via fusion to an elastin-like polypeptide (ELP). The ELP "handle" serves as a rapid, noncovalent strategy to concentrate bioactive peptide mixtures onto a surface. We now report that this strategy can be further generalized across an expanded panel of additional laminin-derived elastin-like polypeptides (LELPs). A99 (AGTFALRGDNPQG), A2G80 (VQLRNGFPYFSY), AG73 (RKRLQVQLSIRT), and EF1m (LQLQEGRLHFMFD) all promote cell spreading while showing morphologically distinct F-actin formation. Equimolar mixtures of A99:A2G80-LELPs have synergistic effects on adhesion and spreading. Finally, three of these ECM-mimetics promote the neurite outgrowth of PC-12 cells. The evidence presented here demonstrates the potential of ELPs to deposit ECM-mimetics with applications in regenerative medicine, cell therapy, and tissue engineering.

Benzoxaborole Catalyst Embedded with a Lewis Base: A Highly Active and Selective Catalyst for cis-1,2-diol Modification.

The regioselective modification of polyols allows rapid access to their derivatives, thereby accelerating the polyol-related biology and drug discovery. We previously reported that benzoxaborole is a potent catalyst for the regioselective modification of polyols containing a cis-1,2-diol structure. In this study, we developed a bifunctional benzoxaborole catalyst embedded with a Lewis base. Benzoxaborole and Lewis base groups were designed to cooperatively activate a substrate (cis-1,2-diol) and reactant (electrophile), respectively, hence lowering the reaction barrier for the cis-1,2-diol moiety. The bifunctional catalyst indeed exhibited a significantly higher catalytic activity and selectivity for cis-1,2-diol modifications rather than a benzoxaborole catalyst without a Lewis base group. Mechanistic analyses, using both experimental and theoretical methods, supported the design of our catalyst. The bifunctional catalyst reported herein would be a new tool for the straightforward synthesis of polyol derivatives.

Characterization of Novel Cell-Adhesive Peptides for Biomaterial Development.

In previous studies, my group developed cell-adhesive peptide-polysaccharide complexes as biomaterials for tissue engineering. Having a wide variety of cell-adhesive peptides is important as the biological functions of peptide-polysaccharide complexes are highly dependent on the biological activity of peptides. This paper reviews the biological activities of two types of recently characterized cell-adhesive peptides. The first is peptides rich in basic amino acids originating from octaarginine. We analyzed the relationships between the amino acid composition of basic peptides and cell adhesion, elongation, and proliferation and identified the most suitable peptide for cell culture. The second was arginine-glycine-aspartic acid (RGD)-containing peptides that promote the adhesion of induced pluripotent stem cells (iPSCs). We identified the RGD-surrounding sequences necessary for iPSC adhesion, clarified the underlying mechanism, and improved cell adhesion by modifying the structure-activity relationships. The novel cell-adhesive peptides identified in our previous studies may aid in the development of novel peptide-based biomaterials.

Data resource profile of an online database system for forensic mental health services.

This paper introduces a forensic psychiatry database established in Japan and discusses its significance and future issues. The purpose of this Database, created under the Medical Treatment and Supervision Act (MTSA) Database Project, is to improve the quality of forensic psychiatry treatment. It can collect monthly data on "basic information," "Orders and hospitalizations under the MTSA," "Treatment process," "Criminal and medical treatment history," and "problematic behavior in the unit." The online system has accumulated data on more than 8,000 items in 24 broad categories. Medical data are exported from the medical care assisting system of 32 designated inpatient facilities in XML format and then saved on USB memory sticks. The files are imported into the Database system client, which sends the data to the Database server via a virtual private network. This system minimizes errors and efficiently imports patient data. However, there is a limitation that it is difficult to set items that need to be analyzed to solve everyday clinical problems into the database system because they tend to change over time. By evaluating the effectiveness of the Database, and collecting appropriate data, it is expected to disseminate a wide range of knowledge that will contribute to the future development of mental health and welfare care.

RGDX1 X2 motif regulates integrin αvß5 binding for pluripotent stem cell adhesion.

The arginine-glycine-aspartic acid (RGD) motif is a cell adhesion sequence that binds to integrins. Some RGD-containing peptides promote adhesion of both embryonic stem cells and induced pluripotent stem cells (iPSCs); however, not all such RGD-containing peptides are active. In this study, we elucidated the role of RGD-neighboring sequences on iPSC adhesion using diverse synthetic peptides and recombinant proteins. Our results indicate that iPSC adhesion requires RGDX1 X2  sequences, such as RGDVF and RGDNY, and that the X1 X2 residues are essential for the adhesion via integrin αvß5 but not αvß3. iPSCs express integrin αvß5 but not αvß3; therefore, iPSC adhesion requires the RGDX1 X2 -containing sequences. The importance of the X1 X2 residues was confirmed with both HeLa and A549 cells, which express integrin αvß5 but not αvß3. Analysis of RGD-neighboring sequences provides important insights into ligand-binding specificity of integrins. Identification of integrin αvß5-binding motifs is potentially useful in drug development, drug delivery, cell culture, and tissue engineering.

Isomer Stability Dependence of Hydrogen-Bonded Benzoxazole Clusters on Solvent Molecules.

The structures of hydrogen-bonded benzoxazole clusters with methanol and ammonia, BO-(CH3OH)n (n = 1-3) and BO-(NH3)n (n = 1, 2), in a supersonic jet have been investigated by measuring the S1-S0 electronic spectra and isomer-selected vibrational spectra with the aid of quantum chemical calculations. Similar to BO-(H2O)1, two isomers of BO-(NH3)1 were observed, which form two types of hydrogen bond networks starting from the CH bond at the 2-/7-position to the nitrogen atom of BO (C2HN/C7HN). The relative stability of these isomers strongly depends on solvent molecules. Natural bond orbital analysis reveals that the OH···N hydrogen bond is dominant in BO-(H2O)1 and that intermolecular interaction between the CH group and the nitrogen atom of ammonia, especially C2H···N, is significantly enhanced, resulting in a more stable C2HN isomer. Symmetry-adapted perturbation theory calculations indicate that the dispersion interaction between the methyl group of methanol and π electron cloud on the BO ring is responsible for the extreme stability of the C7HN of BO-(CH3OH)1. Furthermore, using time-dependent density functional theory calculations, the isomer tendency of the electronic transition shifts from the monomer origin is reproduced and it is proposed that the significant blue shift in C2HN is due to the shortened C2H bond length upon electronic excitation.

Spectroscopic and Theoretical Studies on Conformational Stability of Benzyl Methyl Ether.

Conformer-selected electronic and vibrational spectra of benzyl methyl ether and its terminal methyl group-substituted derivative in a supersonic jet have been measured using ultraviolet (UV)-UV hole burning and fluorescence-detected infrared spectroscopy to investigate the conformational stability of flexible molecules. Various quantum chemical calculations as well as experimental observations reveal the coexistence of three conformers with different CCOC dihedral angles and side-chain orientations relative to the benzene ring plane. Vibrational analysis in the excited state with time-dependent density functional theory and IR simulations containing anharmonic coupling sufficiently reproduce the experimental results, suggesting that these three conformers can be distinguished into one gauche-conformer and two trans-ones with respect to the CCOC dihedral angle. We also observe that the gauche conformer exhibits higher-frequency CH2 modes. The natural bond orbital analysis indicates that this phenomenon is attributed to the electron delocalization from the non-bonding orbitals and the C-O orbitals associated with the neighboring oxygen atom, which leads to a conformer dependence of the methylene C-H bond strength.

Quantitative assessment of copy number alterations by liquid biopsy for neuroblastoma.

Liquid biopsy, a method of detecting genomic alterations using blood specimens, has recently attracted attention as a noninvasive alternative to surgical tissue biopsy. We attempted quantitative analysis to detect amplification of MYCN (MYCNamp) and loss of heterozygosity at 11q (11qLOH), which are clinical requisites as prognostic factors of neuroblastoma (NB). In this study, cell-free DNA (cfDNA) was extracted from plasma samples from 24 NB patients at diagnosis. Copy numbers of MYCN and NAGK genes were quantitatively analyzed by droplet digital PCR (ddPCR). 11qLOH was also assessed by detecting allelic imbalances of heterozygous single nucleotide polymorphisms in the 11q region. The results obtained were compared to those of specimens from tumor tissues. The correlation coefficient of MYCN copy number of cfDNA and tumor DNA was 0.88 (p < 0.00001). 11qLOH was also accurately detected from cfDNA, except for one case with localized NB. Given the high accuracy of liquid biopsy, to investigate components of cfDNA, the proportion of tumor-derived DNA was estimated by examining the variant allele frequency of tumor-specific mutations in cfDNA. The proportion of tumor-derived DNA in cfDNA was 42.5% (range, 16.9%-55.9%), suggesting sufficient sensitivity of liquid biopsy for NB. In conclusion, MYCN copy number and 11qLOH could be quantitatively analyzed in plasma cfDNA by ddPCR assay. These results suggest that plasma cfDNA can be substituted for tumor DNA and can also be applied for comprehensive genomic profiling analysis.

[Peritoneal Recurrence of Gastric Cancer Treated with Chemoradiotherapy-A Case Report].

A 76-year-old male patient underwent a distal gastrectomy for advanced gastric cancer. As the postoperative serum CA19-9 level was elevated, chemotherapy was initiated. Computed tomography(CT)detected a solitary peritoneal recurrence in the left subhepatic space 17 months later. Consequently, chemoradiotherapy(CRT)at a total dose of 60 Gy, combined with S-1 therapy, was administered for local tumor control. After CRT, CT scans revealed a remarkable reduction in the peritoneal recurrence. Presently, 8 months after CRT, the patient remains alive with no indications of regrowth. CRT could prove efficacious as a treatment for gastric cancer patients with localized peritoneal recurrences.

Low NUDT15 expression levels due to biallelic NUDT15 variants and 6-mercaptopurine intolerance.

6-Mercaptopurine (6-MP) is widely used for the treatment of paediatric leukaemia and lymphoma. Recently, germline variants in the NUDT15 gene have been identified as one of the major genetic causes for 6-MP-associated adverse effects such as myelosuppression. Patients with hypomorphic NUDT15 variants accumulate excessive levels of DNA-incorporated thioguanine in white blood cells, resulting in severe myelosuppression. Although preclinical studies suggest that these variants may influence the protein stability of NUDT15, this has not been directly characterised in patients. In this study, we report the development of a series of novel monoclonal antibodies against NUDT15, using which we quantitatively assessed NUDT15 protein levels in 37 patients with acute lymphoblastic leukaemia treated with 6-MP, using sandwich enzyme-linked immunosorbent assay (ELISA). The NUDT15 genotype was highly correlated with its protein levels (p < 0.0001), with homozygous and compound heterozygous patients showing exceedingly low NUDT15 expression. There was a positive correlation between NUDT15 protein level and 6-MP tolerance (r = 0.631, p < 0.0001). In conclusion, our results point to low NUDT15 protein abundance as the biochemical basis for NUDT15-mediated 6-MP intolerance, thus providing a phenotypic readout of inherited NUDT15 deficiency.

HLA loci predisposing to immune TTP in Japanese: potential role of the shared ADAMTS13 peptide bound to different HLA-DR.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare autoimmune disorder caused by neutralizing anti-ADAMTS13 autoantibodies. In white individuals, HLA allele DRB1*11 is a predisposing factor for iTTP, whereas DRB1*04 is a protective factor. However, the role of HLA in Asians is unclear. In this study, we analyzed 10 HLA loci using next-generation sequencing in 52 Japanese patients with iTTP, and the allele frequency in the iTTP group was compared with that in a Japanese control group. We identified the following HLA alleles as predisposing factors for iTTP in the Japanese population: DRB1*08:03 (odds ratio [OR], 3.06; corrected P [Pc] = .005), DRB3/4/5*blank (OR, 2.3; Pc = .007), DQA1*01:03 (OR, 2.25; Pc = .006), and DQB1*06:01 (OR,: 2.41; Pc = .003). The estimated haplotype consisting of these 4 alleles was significantly more frequent in the iTTP group than in the control group (30.8% vs 6.0%; Pc < .001). DRB1*15:01 and DRB5*01:01 were weak protective factors for iTTP (OR, 0.23; Pc = .076; and OR, 0.23, Pc = .034, respectively). On the other hand, DRB1*11 and DRB1*04 were not associated with iTTP in the Japanese. These findings indicated that predisposing and protective factors for iTTP differ between Japanese and white individuals. HLA-DR molecules encoded by DRB1*08:03 and DRB1*11:01 have different peptide-binding motifs, but interestingly, bound to the shared ADAMTS13 peptide in an in silico prediction model.

Effects of laminin-111 peptide coatings on rat neural stem/progenitor cell culture.

Neurons require adhesive scaffolds for their growth and differentiation. Laminins are a major cell adhesive component of basement membranes and have various biological activities in the peripheral and central nervous systems. Here, we evaluated the biological activities of 5 peptides derived from laminin-111 as a scaffold for mouse neuroblastoma Neuro2a cells and rat neural stem/progenitor cells (NPCs). The 5 peptides showed Neuro2a cell attachment activity similar to that of poly-d-lysine. However, when NPCs were cultured on the peptides, 2 syndecan-binding peptides, AG73 (RKRLQVQLSIRT, mouse laminin α1 chain 2719-2730) and C16 (KAFDITYVRLKF, laminin γ1 chain 139-150), demonstrated significantly higher cell attachment and neurite extension activities than other peptides including integrin-binding ones. Long-term cell culture experiments showed that both AG73 and C16 supported the growth of neurons and astrocytes that had differentiated from NPCs. Furthermore, C16 markedly promoted the expression of neuronal markers such as synaptosomal-associated protein-25 and syntaxin 1A. These results indicate that AG73 and C16 are useful for NPC cultures and that C16 can be applied to specialized research on synapses in differentiated neurons. These peptides have the potential for use as valuable biomaterials for NPC research.

Gemtuzumab Ozogamicin Followed by Unrelated Cord Blood Transplantation With Reduced-intensity Conditioning for a Child With Refractory Acute Promyelocytic Leukemia.

There is no established treatment for patients with acute promyelocytic leukemia (APL) refractory to targeted therapies with all-trans retinoic acid (ATRA) and/or arsenic trioxide (ATO). We report here a case of an 8-month-old girl with APL who failed standard ATRA-combined chemotherapy. Although molecular remission was achieved after introducing ATRA/ATO combination therapy, molecular relapse occurred during the ATO consolidation courses. Subsequent molecular remission was rapidly achieved after administering 2 doses of gemtuzumab ozogamicin. She was successfully treated with unrelated cord blood transplantation using reduced-intensity conditioning. Gemtuzumab ozogamicin might be a preferable choice for patients with APL refractory to standard therapy.

Octa-arginine and Octa-lysine Promote Cell Adhesion through Heparan Sulfate Proteoglycans and Integrins.

Octa-arginine (R8) has been extensively studied as a cell-penetrating peptide. R8 binds to diverse transmembrane heparan sulfate proteoglycans (HSPGs), including syndecans, and is internalized by cells. R8 is also reported to bind to integrin ß1. In this study, we evaluated the biological activities of R8 and octa-lysine (K8), a peptide similar to R8, with a focus on cell adhesion. R8 and K8 were immobilized on aldehyde-agarose matrices via covalent conjugation, and the effect of these peptides on cell attachment, spreading, and proliferation was examined using human dermal fibroblasts. The results indicated that R8- and K8-matrices mediate cell adhesion mainly via HSPGs. Moreover, R8- and K8-matrices interacted with integrin ß1 and promote cell spreading and proliferation. These results are useful for further understanding of the R8-membrane interactions and the cellular uptake mechanisms. In addition, the R8- and K8-matrices may potentially be used as a multi-functional biomaterial to promote cell adhesion, spreading, and proliferation.

Effect of Amino Acid Substitution on Cell Adhesion Properties of Octa-arginine.

Octa-arginine (R8) is a cell-permeable peptide with excellent cell adhesion properties. Surface-immobilized R8 mediates cell attachment via cell surface receptors, such as heparan sulfate proteoglycans and integrin ß1, and promotes cell spreading and proliferation. However, it is not clear how these properties are affected by specific peptide composition and if they could be improved. Here, we synthesized XR8 peptides, in which half of the original R8 arginine residues were replaced with another amino acid (X). We then aimed to investigate the effect of the substitution on cell adhesion and proliferation on XR8-conjugated agarose matrices. The XR8-matrix showed slightly better cell attachment when X was a hydrophobic or aromatic amino acid. However, hydrophobic XR8-matrices tended to promote cell proliferation to a less extent. Eventually, YR8-matrix most efficiently promoted cell adhesion, spreading, and proliferation among the XR8-matrices tested. Collectively, these observations indicate that the properties of residue X play a major role in the biological activity of XR8-matrices and shed light on the interaction between small peptides and the cell membrane. Further, YR8 is a promising cell-adhesive peptide for the development of cell culture substrates and biomaterials.

Structural Requirement of hA5G18 Peptide (DDFVFYVGGYPS) from Laminin α5 Chain for Amyloid-like Fibril Formation and Cell Adhesion.

The hA5G18 peptide (DDFVFYVGGYPS) identified from the human laminin α5 chain G domain promotes cell attachment and spreading when directly coated on a plastic plate, but does not show activity when it is conjugated on a chitosan matrix. Here, we focused on the structural requirement of hA5G18 for activity. hA5G18 was stained with Congo red and formed amyloid-like fibrils. A deletion analysis of hA5G18 revealed that FVFYV was a minimum active sequence for the formation of amyloid-like fibrils, but FVFYV did not promote cell attachment. Next, we designed functional fibrils using FVFYV as a template for amyloid-like fibrils. When we conjugated an integrin binding sequence Arg-Gly-Asp (RGD) to the FVFYV peptide with Gly-Gly (GG) as a spacer, FVFYVGGRGD promoted cell attachment in a plate coat assay, but a negative control sequence RGE conjugated peptide, FVFYVGGRGE, also showed activity. However, when the peptides were conjugated to Sepharose beads, the FVFYVGGRGD beads showed cell attachment activity, but the FVFYVGGRGE beads did not. These results suggest that RGD and RGE similarly contribute to cell attachment activity in amyloid-like fibrils, but only RGD contributes the activity on the Sepharose beads. Further, we conjugated a basic amino acid (Arg, Lys, and His) to the FVFYV peptide. Arg or Lys-conjugated FVFYV peptides, FVFYVGGR and FVFYVGGK, showed cell attachment activity when they were coated on a plate, but a His-conjugated FVFYV peptide FVFYVGGH did not show activity. None of the basic amino acid-conjugated peptides showed cell attachment in a Sepharose bead assay. The cell attachment and spreading on FVFYVGGR and FVFYVGGK were inhibited by an anti-integrin ß1 antibody. These results suggest that the Arg and Lys residues play critical roles in the interaction with integrins in amyloid-like fibrils. FVFYV is useful to use as a template for amyloid-like fibrils and to develop multi-functional biomaterials.

Evaluation of extracellular matrix mimetic laminin bioactive peptide and elastin-like polypeptide.

The extracellular matrix (ECM) is comprised of a large network of proteins that are essential for tissue development and repair. A bioactive RGD-containing peptide from laminin α1 chain, A99 (AGTFALRGDNPQG), promotes strong cell attachment and has demonstrated utility in cell culture and tissue engineering. Various materials can be utilized as a scaffold for bioactive peptides; however, it may be advantageous to design materials that use bioconjugation strategies that do not affect bioactivity, generate homogenous products, and can be produced at scale. This report is the first to compare the methods for preparing chemically conjugated and recombinant A99 to elastin-like polypeptides (ELPs) as the scaffold and characterize the biological and cell attachment activity using human dermal fibroblasts (HDFs). ELPs are biocompatible protein-polymers that are also thermo-responsive. Below a lower critical solution temperature (LCST), they are highly soluble. Above the LCST, ELPs phase separate into a polymer-rich liquid, known as a coacervate. Both chemically conjugated and recombinant fusion between A99 and an ELP (A99-ELP-R) show dose-dependent cell attachment. In addition, coating above the LCST provides better cell spreading compared to coating at 4°C. ELPs provide an excellent structural framework for deposition of bioactive peptides of the ECM, and their intrinsic biophysical properties make laminin peptide-ELPs promising biomaterials for cell culture and tissue engineering.

A case of primary CNS embryonal rhabdomyosarcoma with PAX3-NCOA2 fusion and systematic meta-review.

PURPOSE: Primary central nervous system (CNS) rhabdomyosarcoma is a rare mesenchymal tumor predominantly seen in children and associated with a poor outcome. We report a case of primary CNS rhabdomyosarcoma with PAX3-NCOA2 fusion and present a systematic meta-review of primary CNS rhabdomyosarcoma to characterize this rare tumor. METHODS: We present the case of a 6-year-old boy with primary CNS rhabdomyosarcoma in the posterior fossa. In a systematic meta-review, we compare the demographic data of primary CNS rhabdomyosarcoma with data of rhabdomyosarcoma at all sites from the SEER database and analyze clinical factors associated with survival outcome. RESULTS: Our patient underwent gross total resection and received vincristine, actinomycin-D, cyclophosphamide with early introduction of concurrent focal radiation and remained alive with no evidence of disease for 2 years after the end of therapy. Histopathological review revealed embryonal-type rhabdomyosarcoma, and whole-transcriptome analysis revealed PAX3 (EX6)-NCOA2 (EX12) fusion. In all, 77 cases of primary CNS rhabdomyosarcoma were identified through the meta-review. The demographic data of primary CNS rhabdomyosarcoma were similar to data of rhabdomyosarcoma at all sites. Overall and event-free survival outcomes were available for 64 and 56 patients, respectively, with a 3-year OS of 29.0% and a 3-year EFS of 25.7%. The group that received trimodal treatment exhibited better survival outcomes, with a 3-year OS of 57.4% and a 3-year EFS of 46.3%. CONCLUSIONS: Primary CNS rhabdomyosarcoma shares common histological, molecular, and demographic features with non-CNS rhabdomyosarcoma. A trimodal treatment approach with early introduction of radiation therapy may result in favorable survival outcomes.