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Abstract Craniometaphyseal dysplasia (CMD), a rare craniotubular disorder, occurs in an autosomal dominant (AD) or autosomal recessive (AR) form. CMD is characterized by hyperostosis of craniofacial bones and metaphyseal flaring of long bones. Many patients with CMD suffer from neurological symptoms. The pathogenesis of CMD is not fully understood. Treatment is limited to craniofacial surgery. Here, we report a knock in (KI) mouse model for AR CMD carrying a Cx43R239Q mutation. Cx43 KI/KI mice replicate typical features of AR CMD, including thickening of craniofacial bones, club-shaped femurs, and widened diaphyseal cortical bones. Female Cx43 KI/KI mice display remarkably more bone overgrowth than male Cx43 KI/KI mice as they age. In contrast to Cx43 +/+ littermates, Cx43 KI/KI mice exhibit periosteal bone deposition and increased osteoclast (OC) numbers in the endosteum of long bones. Although formation of resting OCs in Cx43 +/+ and Cx43 KI/KI mice is comparable, the actively resorbing Cx43 KI/KI OCs have reduced resorption on bone chips. Cx43 KI/KI mice display reduced osteocyte dendrites. RNA from Cx43 KI/KI femoral cortical bones show reduced expression levels of Sost, Tnf-α, IL-1β, Esr1, Esr2, and a lower Rankl/Opg ratio. Moreover, the Cx43R239Q mutation results in altered spatial expression of Cx43 protein and mild reduction of gap junction and hemichannel activity. The distinct phenotype seen in Cx43 KI/KI mice but not in Cx43 ablation models suggests that Cx43 loss-of-function is unlikely the main cause of AR CMD. Additional studies are required to investigate new roles of CMD-mutant Cx43.

Dental pulp stem cells (DPSCs) demonstrate high proliferative and multilineage differentiation potential. As previously reported, the helioxanthin derivative 4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH) has been demonstrated to induce the osteogenic differentiation of DPSCs. However, the mechanism of osteogenesis induced by TH in DPSCs remains unknown. The objective of this study was to identify functional extracellular vesicle (EV) microRNAs (miRNAs), and the principal genes involved in the TH-induced osteogenesis of DPSCs. DPSCs were derived from dental pulp extracted from the third molars of three healthy subjects, and were cultured with or without TH. miRNAs were extracted from DPSC-derived EVs. The gene expression patterns of mRNA and miRNA were compared using RNA-Seq and miRNA-Seq. To investigate miRNA/mRNA interacting networks, functional analyses were performed by Ingenuity Pathway Analysis. Alkaline phosphatase (ALP) staining demonstrated that treatment with TH resulted in enhanced ALP activity in DPSCs after 7 days. The expression levels of ALP and type 1 collagen alpha 1 were significantly higher in TH-induced DPSCs on day 7. RNA-Seq and miRNA-Seq analyses identified 869 differentially expressed genes (DEGs) and 18 miRNA-DEGs. Gene Ontology analysis of the mRNA-Seq results showed that TH induced several biological activities associated with signal transduction, cell adhesion, and cell differentiation. Integrated miRNA-mRNA analyses showed that these miRNAs contain the targeting information of 277 mRNAs of the DEGs. Among them, 17 target genes known to be involved in the differentiation of osteoblasts, and 24 target genes known to be involved in the differentiation of bone cells were identified. Quantitative real-time PCR showed that WNT5a expression in DPSCs was upregulated by 48 h of TH treatment. Upstream regulator analysis indicated that WNT3a, FOS, and RAC1 may be responsible for gene expression changes in DPSCs after TH treatment. EV miRNA regulatory networks might play crucial roles in TH-induced osteogenic differentiation of DPSCs. Our results presented herein offer valuable insights that will facilitate further research into the mechanism of osteogenesis of DPSCs, which is expected to lead to the clinical application of TH-induced DPSCs for bone regeneration. Furthermore, EVs derived from TH-induced DPSCs might be useful as therapeutic tools for bone defects.

Abstract In clinical studies, the next-generation anti-tumor necrosis factor-alpha (TNF-α) single domain antibody ozoralizumab showed high clinical efficacy shortly after the subcutaneous injection. To elucidate the mechanism underlying the rapid onset of the effects of ozoralizumab, we compared the biodistribution kinetics of ozoralizumab and adalimumab after subcutaneous injection in an animal model of arthritis. Alexa Fluor 680-labeled ozoralizumab and adalimumab were administered by subcutaneous injection once (2 mg/kg) at five weeks after induction of collagen-induced arthritis (CIA) in an animal arthritis model. The time-course of changes in the fluorescence intensities of the two compounds in the paws and serum were evaluated. The paws of the CIA mice were harvested at four and eight hours after the injection for fluorescence microscopy. Biofluorescence imaging revealed better distribution of ozoralizumab to the joint tissues than of adalimumab, as early as at four hours after the injection. Fluorescence microscopy revealed a greater fluorescence intensity of ozoralizumab in the joint tissues than that of adalimumab at eight hours after the injection. Ozoralizumab showed a significantly higher absorption rate constant as compared with adalimumab. These results indicate that ozoralizumab enters the systemic circulation more rapidly and is distributed to the target tissues earlier and at higher levels than conventional IgG antibodies. Our investigation provides new insight into the mechanism underlying the rapid onset of the effects of ozoralizumab in clinical practice.

Recent studies have demonstrated that the interaction of dietary constituents with taste and olfactory receptors and nociceptors expressed in the oral cavity, nasal cavity and gastrointestinal tract regulate homeostasis through activation of the neuroendocrine system. Polyphenols, of which 8000 have been identified to date, represent the greatest diversity of secondary metabolites in plants, most of which are bitter and some of them astringent. Epidemiological studies have shown that polyphenol intake contributes to maintaining and improving cardiovascular, cognitive and sensory health. However, because polyphenols have very low bioavailability, the mechanisms of their beneficial effects are unknown. In this review, we focused on the taste of polyphenols from the perspective of sensory nutrition, summarized the results of previous studies on their relationship with bioregulation and discussed their future potential.

Biologics have become an important component of treatment strategies for a variety of diseases, but the immunogenicity of large immune complexes (ICs) and aggregates of biologics may increase risk of adverse events is a concern for biologics and it remains unclear whether large ICs consisting of intrinsic antigen and therapeutic antibodies are actually involved in acute local inflammation such as injection site reaction (ISR). Ozoralizumab is a trivalent, bispecific NANOBODY® compound that differs structurally from IgGs. Treatment with ozoralizumab has been shown to provide beneficial effects in the treatment of rheumatoid arthritis (RA) comparable to those obtained with other TNFα inhibitors. Very few ISRs (2%) have been reported after ozoralizumab administration, and the drug has been shown to have acceptable safety and tolerability. In this study, in order to elucidate the mechanism underlying the reduced incidence of ISRs associated with ozoralizumab administration, we investigated the stoichiometry of two TNFα inhibitors (ozoralizumab and adalimumab, an anti-TNFα IgG) ICs and the induction by these drugs of Fcγ receptor (FcγR)-mediated immune responses on neutrophils. Ozoralizumab-TNFα ICs are smaller than adalimumab-TNFα ICs and lack an Fc portion, thus mitigating FcγR-mediated immune responses on neutrophils. We also developed a model of anti-TNFα antibody-TNFα IC-induced subcutaneous inflammation and found that ozoralizumab-TNFα ICs do not induce any significant inflammation at injection sites. The results of our studies suggest that ozoralizumab is a promising candidate for the treatment of RA that entails a lower risk of the IC-mediated immune cell activation that leads to unwanted immune responses.

Sagittal split ramus osteotomy (SSRO) sometimes induces an irregular split pattern referred to as a bad split. We investigated the risk factors for bad splits in the buccal plate of the ramus during SSRO. Ramus morphology and bad splits in the buccal plate of the ramus were assessed using preoperative and postoperative computed tomography images. Of the 53 rami analyzed, 45 had a successful split, and 8 had a bad split in the buccal plate. Horizontal images at the height of the mandibular foramen showed that there were significant differences in the ratio of the forward thickness to the backward thickness of the ramus between patients with a successful split and those with a bad split. In addition, the distal region of the cortical bone tended to be thicker and the curve of the lateral region of the cortical bone tended to be smaller in the bad split group than in the good split group. These results indicated that a ramus shape in which the width becomes thinner towards the back frequently induces bad splits in the buccal plate of the ramus during SSRO, and more attention should be paid to patients who have rami of these shapes in future surgeries.

In the oral and maxillofacial region, the treatment of severe bone defects, caused by fractures, cancers, congenital abnormalities, etc., remains a great challenge. In addition, neurological disorders are frequently accompanied by these bone defects or the treatments for them. Therefore, novel bone regenerative techniques and methods to repair nerve injury are eagerly sought. Among them, strategies using dental pulp stem cells (DPSCs) are promising options. Human DPSCs can be collected easily from extracted teeth and are now considered a type of mesenchymal stem cell with higher clonogenic and proliferative potential. DPSCs have been getting attention as a cell source for bone and nerve regeneration. In this article, we reviewed the latest studies on osteogenic or neural differentiation of DPSCs as well as bone or neural regeneration methods using DPSCs and discussed the potential of DPSCs for bone and nerve tissue regeneration.

Human dental pulp stem cells (DPSCs) exhibit multilineage differentiation capabilities and superior clonogenic and proliferative properties. However, the use of animal-derived components such as FBS raises concerns regarding the clinical application of stem-cell-based therapies. Platelet-rich fibrin (PRF) derived from human blood is rich in fibrin, platelets, and growth factors and acts as a bioactive scaffold for grafting with biomaterials. In this study, we assessed the efficacy of PRF-conditioned medium (CM) in promoting DPSCs proliferation and osteogenic differentiation compared with the standard culture medium supplemented with FBS. A comparison of DPSCs cultured in FBS and PRF-CM revealed no differences in characteristics or morphology. However, cells cultured with PRF-CM exhibited inferior proliferation rates and cell numbers during passage in comparison with those cultured with FBS. In contrast, DPSCs cultured in PRF-CM showed significantly higher levels of calcification, and RT-PCR confirmed that the gene expression levels of markers associated with osteoblast differentiation were significantly increased. The PRF-CM approach offers a convenient, straightforward, and advantageous method for culturing DPSCs, without relying on animal-derived components. In summary, this study introduces a novel application of PRF-CM for enhancing the osteogenesis of DPSCs, which provides an alternative to FBS culture medium and addresses concerns associated with the use of animal-derived components in clinical settings.

B-type procyanidins, a series of catechin oligomers, are among the most ingested polyphenols in the human diet. Results of meta-analyses have suggested that intake of B-type procyanidins reduces cardiovascular disease risk. Another recent focus has been on the effects of B-type procyanidins on central nervous system (CNS) function. Although long-term B-type procyanidin ingestion is linked to health benefits, a single oral intake has been reported to cause physiological alterations in circulation, metabolism, and the CNS. Comprehensive analyses of previous reports indicate an optimal mid-range dose for the hemodynamic effects of B-type procyanidins, with null responses at lower or higher doses, suggesting hormesis. Indeed, polyphenols, including B-type procyanidins, elicit hormetic responses in vitro, but animal and clinical studies are limited. Hormesis of hemodynamic and metabolic responses to B-type procyanidins was recently confirmed in animal studies, however, and our work has linked these effects to the CNS. Here, we evaluate the hormetic response elicited by B-type procyanidins, recontextualizing the results of intervention trials. In addition, we discuss the possibility that this hormetic response to B-type procyanidins arises via CNS neurotransmitter receptors. We have verified the direction of future research for B-type procyanidins in this review.

Introduction: Cell-based therapies require an emerging alternative treatment using easily harvested cell sources. Neural stem cells derived from various tissues, including brain, bone marrow, skin and retina can give rise to both neurons and glial cells. Recently, human dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHED) were demonstrated to have mesenchymal stem cell-like abilities such as self-renewal and multi-lineage differentiation, including neuron and glial cells. Moreover, DPSCs and SHED show a higher proliferation rate and a higher number of population doublings compared with adult bone marrow stromal stem cells. Therefore, DPSCs are a useful source that can be applied in cell replacement therapy for various neurological disorders. Generally, the conventional culture methods for DPSCs have used serum, therefore the undefined components in culture medium may complicate investigations of the molecular mechanisms that control the self-renewal and differentiation of DPSCs. However, neural stem cells proliferate to form ‘neurospheres’ in suspension in vitro in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). No study to date has obtained neurospheres from DPSCs in serum-free conditions in primary culture. Thus, the aim of this study was to establish a method for the proliferation and neural differentiation of DPSCs in xeno- and serum-free conditions in primary culture. Methods: DPSCs were obtained from the dental pulp of wisdom teeth from healthy individuals (18–41 years old) and cultured in conventional medium containing 15% fetal bovine serum and xeno-/serum-free medium. We evaluated the proliferation of DPSCs, neurosphere generation, and neural differentiation under xeno-/serum-free conditions by flow cytometry, immunohistochemistry, and real-time polymerase chain reaction. Results: In proliferation medium without xeno/serum, DPSCs can proliferate and generate neurospheres, however, the neurospheres had limited self-renewal ability. Under differentiation conditions, class III β-tubulin (TUBB3) and microtubule-associated protein (MAP2) were more significantly expressed in neurospheres derived from DPSCs in xeno-/serum-free culture conditions than in DPSCs in conventional culture conditions. Conclusions: Our result demonstrated that neurosphere generation from DPSCs in xeno-/serum-free culture may be an accessible source for clinical cell replacement therapies for neuronal degenerative diseases.

Although the introduction of tumor necrosis factor (TNF) inhibitors represented a significant advance in the treatment of rheumatoid arthritis (RA), traditional anti-TNFα antibodies are somewhat immunogenic, and their use results in the formation of anti-drug antibodies (ADAs) and loss of efficacy (secondary failure). Ozoralizumab is a trivalent, bispecific NANOBODY® compound that differs structurally from IgGs. In this study we investigated the suppressant effect of ozoralizumab and adalimumab, an anti-TNFα IgG, on arthritis and induction of ADAs in human TNF transgenic mice. Ozoralizumab markedly suppressed arthritis progression and did not induce ADAs during long-term administration. We also developed an animal model of secondary failure by repeatedly administering adalimumab and found that switching from adalimumab to ozoralizumab was followed by superior anti-arthritis efficacy in the secondary-failure animal model. Moreover, ozoralizumab did not form large immune complexes that might lead to ADA formation. The results of our studies suggest that ozoralizumab, which exhibited low immunogenicity in the animal model used and has a different antibody structure from that of IgGs, is a promising candidate for the treatment of RA patients not only at the onset of RA but also during secondary failure of anti-TNFα treatment.

ABSTRACT Cherubism (CBM), characterized by expansile jawbones with multilocular fibrocystic lesions, is caused by gain‐of‐function mutations in SH3 domain‐binding protein 2 (SH3BP2; mouse orthologue Sh3bp2). Loss of jawbone and dental integrity significantly decrease the quality of life for affected children. Treatment for CBM is limited to multiple surgeries to correct facial deformities. Despite significant advances made with CBM knockin (KI) mouse models (Sh3bp2KI/KI), the activation mechanisms of CBM lesions remain unknown because mutant mice do not spontaneously develop expansile jawbones. We hypothesize that bony inflammation of an unknown cause triggers jawbone expansion in CBM. To introduce jawbone inflammation in a spatiotemporally controlled manner, we exposed pulp of the first right mandibular molar of 6‐week‐old Sh3bp2+/+, Sh3bp2KI/+, and Sh3bp2KI/KI mice. Bacterial invasion from the exposed pulp into root canals led to apical periodontitis in wild‐type and mutant mice. The pathogen‐associated molecular patterns (PAMPs)‐induced inflammation of alveolar bone resulted in jawbone expansion in Sh3bp2KI/+ and Sh3bp2KI/KI mice. CBM‐like lesions developed exacerbated inflammation with increased neutrophil, macrophage, and osteoclast numbers. These lesions displayed excessive neutrophil extracellular traps (NETs) compared to Sh3bp2+/+ mice. Expression levels of IL‐1β, IL‐6, and TNF‐α were increased in periapical lesions of Sh3bp2+/+, Sh3bp2KI/+, and Sh3bp2KI/KI mice and also in plasma and the left untreated mandibles (with no pulp exposure) of Sh3bp2KI/KI mice, suggesting a systemic upregulation. Ablation of Tlr2/4 signaling or depletion of neutrophils by Ly6G antibodies ameliorated jawbone expansion induced by PAMPs in Sh3bp2KI/KI mice. In summary, successful induction of CBM‐like lesions in jaws of CBM mice is important for studying initiating mechanisms of CBM and for testing potential therapies. Our findings further emphasize a critical role of host immunity in the development of apical periodontitis and the importance of maintaining oral health in CBM patients. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Abstract Background Human dental pulp stem cells (DPSCs), which have the ability to differentiate into multiple lineages, were recently identified. DPSCs can be collected readily from extracted teeth and are now considered to be a type of mesenchymal stem cell with higher clonogenic and proliferative potential than bone marrow stem cells (BMSCs). Meanwhile, the treatment of severe bone defects, such as fractures, cancers, and congenital abnormalities, remains a great challenge, and novel bone regenerative techniques are highly anticipated. Several studies have previously shown that 4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH), a helioxanthin derivative, induces osteogenic differentiation of preosteoblastic and mesenchymal cells. However, the osteogenic differentiation activities of TH have only been confirmed in some mouse cell lines. Therefore, in this study, toward the clinical use of TH in humans, we analyzed the effect of TH on the osteogenic differentiation of DPSCs, and the in-vivo osteogenesis ability of TH-induced DPSCs, taking advantage of the simple transplantation system using cell-sheet technology. Methods DPSCs were obtained from dental pulp of the wisdom teeth of five healthy patients (18–22 years old) and cultured in regular medium and osteogenic medium with or without TH. To evaluate osteogenesis of TH-induced DPSCs in vivo, we transplanted DPSC sheets into mouse calvaria defects. Results We demonstrated that osteogenic conditions with TH induce the osteogenic differentiation of DPSCs more efficiently than those without TH and those with bone morphogenetic protein-2. However, regular medium with TH did not induce the osteogenic differentiation of DPSCs. TH induced osteogenesis in both DPSCs and BMSCs, although the gene expression pattern in DPSCs differed from that in BMSCs up to 14 days after induction with TH. Furthermore, we succeeded in bone regeneration in vivo using DPSC sheets with TH treatment, without using any scaffolds or growth factors. Conclusions Our results demonstrate that TH-induced DPSCs are a useful cell source for bone regenerative medicine, and the transplantation of DPSC sheets treated with TH is a convenient scaffold-free method of bone healing.

OBJECTIVES:We previously found that elevated energy expenditure following a single oral dose of flavan 3-ols (FL), a mixture of catechins and B type procyanidins, is caused by sympathetic nerve activation. In the present study, we compared the activity of the FL components (-)-epicatechin (EC; monomer), procyanidin B2 (B2; dimer), procyanidin C1 (C1; trimer), cinnamtannin A2 (A2; tetramer), and more than pentamer fraction (P5). METHODS:Male ICR mice were treated with a single oral dose of FL, EC, B2, C1, A2, or P5. The animals were sacrificed and blood and brown adipose tissue (BAT) sampled. The plasma catecholamine (CA) levels and BAT uncoupling protein (UCP)-1 mRNA expression were determined. RESULTS:A single dose of 10 mg/kg FL significantly increased plasma CA and UCP-1 mRNA levels. B2, C1, and A2, but not EC and P5 (all at 1 mg/kg), significantly increased plasma adrenaline levels. Plasma noradrenaline was significantly elevated by B2 and A2, but not by EC, C1, or P5. UCP-1 mRNA levels were significantly increased by C1 and P5. In the dose response study of A2, 10-3 mg/kg A2 increased UCP-1 mRNA levels significantly, but not 10-2 and 10-1 mg/kg A2. In addition, combination treatment with 10-1 mg/kg A2 and yohimbine, an α2 adrenalin blocker, remarkably increased UCP-1 mRNA levels. CONCLUSION:These results suggest that FL and its components, except EC, increase UCP-1 mRNA and plasma CA with varying efficacy.

Flavan-3-ols (FLs), specifically catechin and its oligomer B-type procyanidins, are suggested to potently bind to bovine serum albumin (BSA). We examined the interaction between BSA and FLs by fluorescence quenching and found the following order of binding activities to BSA: cinnamtannin A2 (A2; tetramer) > procyanidin C1 (C1; trimer) ≈ procyanidin B2 (B2, dimer) > (−)epicatechin (EC, monomer). Docking simulations between BSA and each compound at the binding site showed that the calculated binding energies were consistent with the results of our experimental assay. FLs exerted cytotoxicity at 1000 μg/mL in F11 cell culture with fetal bovine serum containing BSA. In culture containing serum-free medium, FLs exhibited significant cell proliferation at 10−4 μg/mL and cytotoxicity was observed at concentrations greater than 10 μg/mL. Results of this study suggest that interactions between polyphenols and BSA should be taken into account when evaluating procyanidin in an in vitro cell culture system.

ABSTRACT We sequenced the genome of Theileria orientalis, a tick-borne apicomplexan protozoan parasite of cattle. The focus of this study was a comparative genome analysis of T. orientalis relative to other highly pathogenic Theileria species, T. parva and T. annulata. T. parva and T. annulata induce transformation of infected cells of lymphocyte or macrophage/monocyte lineages; in contrast, T. orientalis does not induce uncontrolled proliferation of infected leukocytes and multiplies predominantly within infected erythrocytes. While synteny across homologous chromosomes of the three Theileria species was found to be well conserved overall, subtelomeric structures were found to differ substantially, as T. orientalis lacks the large tandemly arrayed subtelomere-encoded variable secreted protein-encoding gene family. Moreover, expansion of particular gene families by gene duplication was found in the genomes of the two transforming Theileria species, most notably, the TashAT/TpHN and Tar/Tpr gene families. Gene families that are present only in T. parva and T. annulata and not in T. orientalis, Babesia bovis, or Plasmodium were also identified. Identification of differences between the genome sequences of Theileria species with different abilities to transform and immortalize bovine leukocytes will provide insight into proteins and mechanisms that have evolved to induce and regulate this process. The T. orientalis genome database is available at http://totdb.czc.hokudai.ac.jp/. IMPORTANCE Cancer-like growth of leukocytes infected with malignant Theileria parasites is a unique cellular event, as it involves the transformation and immortalization of one eukaryotic cell by another. In this study, we sequenced the whole genome of a nontransforming Theileria species, Theileria orientalis, and compared it to the published sequences representative of two malignant, transforming species, T. parva and T. annulata. The genome-wide comparison of these parasite species highlights significant genetic diversity that may be associated with evolution of the mechanism(s) deployed by an intracellular eukaryotic parasite to transform its host cell.

The relationship between sequence polymorphisms and human disease has been studied mostly in terms of effects of single nucleotide polymorphisms (SNPs) leading to single amino acid substitutions that change protein structure and function. However, less attention has been paid to more drastic sequence polymorphisms which cause premature termination of a protein's sequence or large changes, insertions, or deletions in the sequence. We have analyzed a large set (n = 512) of insertions and deletions (indels) and single nucleotide polymorphisms causing premature termination of translation in disease-related genes. Prediction of protein-destabilization effects was performed by graphical presentation of the locations of polymorphisms in the protein structure, using the Genomes TO Protein (GTOP) database, and manual annotation with a set of specific criteria. Protein-destabilization was predicted for 44.4% of the nonsense SNPs, 32.4% of the frameshifting indels, and 9.1% of the non-frameshifting indels. A prediction of nonsense-mediated decay allowed to infer which truncated proteins would actually be translated as defective proteins. These cases included the proteins linked to diseases inherited dominantly, suggesting a relation between these diseases and toxic aggregation. Our approach would be useful in identifying potentially aggregation-inducing polymorphisms that may have pathological effects.

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology.