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Background: Group B Streptococci (GBS) are common vaginal bacteria found in 20–30% of pregnant women and a significant cause of invasive infections in newborns. Recently, attention has been focused on the efficacy of probiotics during the perinatal period. However, the effect of probiotic intake on the mother-to-child transmission (MTCT) of GBS remains unknown. Methods: Pregnant women with positive GBS results from vaginal and rectal swab cultures at 35–37 weeks of gestation were randomly assigned to the probiotic group or the control group in an open-label manner at the Department of Obstetrics and Gynecology, San-ikukai Hospital, Tokyo, Japan. The probiotic group received Lactobacillus reuteri during antenatal checkups from 35 to 37-week gestation to 1 month after delivery. Rectal swabs were obtained from the newborns at 5 days and at 1 month of age. Whole-genome sequencing was performed to test for GBS strains in the mother, whose newborn carried GBS at the 1-month checkup. Multi-locus sequence typing and single nucleotide polymorphism analyses were performed to identify MTCT. Results: Overall, 67 mother-infant pairs were included, with 31 in the probiotic group and 36 in the control group. The positivity rate of GBS in newborns at 1 month of age was 10% (n = 3) in the probiotic group and 28% (n = 10) in the control group. In newborns carrying GBS at 1 month of age, genetic analysis revealed that the MTCT rate was 6% in the probiotic group and 22% in the control group, although the difference was not statistically significant (p = 0.0927). Conclusion: No statistically significant difference was found; however, the consumption of L. reuteri by women with GBS-positive pregnancies may inhibit the MTCT of GBS.

Summary: Ferroptosis is a type of regulated cell death characterized by iron-dependent lipid peroxidation. A model cell system is constructed to induce ferroptosis by re-expressing the transcription factor BACH1, a potent ferroptosis inducer, in immortalized mouse embryonic fibroblasts (iMEFs). The transfer of the culture supernatant from ferroptotic iMEFs activates the proliferation of hepatoma cells and other fibroblasts and suppresses cellular senescence-like features. The BACH1-dependent secretion of the longevity factor FGF21 is increased in ferroptotic iMEFs. The anti-senescent effects of the culture supernatant from these iMEFs are abrogated by Fgf21 knockout. BACH1 activates the transcription of Fgf21 by promoting ferroptotic stress and increases FGF21 protein expression by suppressing its autophagic degradation through transcriptional Sqstm1 and Lamp2 repression. The BACH1-induced ferroptotic FGF21 secretion suppresses obesity in high-fat diet-fed mice and the short lifespan of progeria mice. The inhibition of these aging-related phenotypes can be physiologically significant regarding ferroptosis.

Abstract Background Surgical options for patients vary with age and comorbidities, advances in medical technology and patients’ wishes. This complexity can make it difficult for surgeons to determine appropriate treatment plans independently. At our institution, final decisions regarding treatment for patients are made at multidisciplinary meetings, termed High-Risk Conferences, led by the Patient Safety Committee. Methods In this retrospective study, we assessed the reasons for convening High-Risk Conferences, the final decisions made and treatment outcomes using conference records and patient medical records for conferences conducted at our institution from April 2010 to March 2018. Results A total of 410 High-Risk Conferences were conducted for 406 patients during the study period. The department with the most conferences was cardiovascular surgery (24%), and the reasons for convening conferences included the presence of severe comorbidities (51%), highly difficult surgeries (41%) and nonmedical/personal issues (8%). Treatment changes were made for 49 patients (12%), including surgical modifications for 20 patients and surgery cancellation for 29. The most common surgical modification was procedure reduction (16 patients); 4 deaths were reported. Follow-up was available for 21 patients for whom surgery was cancelled, with 11 deaths reported. Conclusions Given that some change to the treatment plan was made for 12% of the patients discussed at the High-Risk Conferences, we conclude that participants of these conferences did not always agree with the original surgical plan and that the multidisciplinary decision-making process of the conferences served to allow for modifications. Many of the modifications involved reductions in procedures to reflect a more conservative approach, which might have decreased perioperative mortality and the incidence of complications as well as unnecessary surgeries. High-risk patients have complex issues, and it is difficult to verify statistically whether outcomes are associated with changes in course of treatment. Nevertheless, these conferences might be useful from a patient safety perspective and minimize the potential for legal disputes.

Abstract This is the first report of large presacral Tarlov cysts (cerebrospinal fluid‐filled perineural cysts) diagnosed during pregnancy in which a cesarean delivery mode was selected to avoid the risk associated with vaginal delivery.

The assessment of airborne viruses in air is a critical step in the design of appropriate prevention and control measures. Hence, herein, we developed a novel wet-type electrostatic air sampler using a viral dissolution buffer containing a radical scavenging agent, and verified the concentration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in the air of hospital rooms inhabiting coronavirus disease 2019 (COVID-19) patients and public areas. RNA damage caused by corona discharge was negligible when Buffer AVL was used as the collecting electrode. The viral RNA concentration in the air of the room varied by patient: 3.9 × 103 copy/m3 on the 10th day after onset in a mild case and 1.3 × 103 copy/m3 on the 18th day in a severe case. Viral RNA levels were 7.8 × 102 and 1.9 × 102 copy/m3 in the air of the office and food court, respectively, where people removed their masks when eating and talking, but it remained undetected in the station corridor where all the people were wearing masks. The assessment of airborne SARS-CoV-2 RNA using the proposed sampler can serve as a basis for the safe discontinuation of COVID-19 isolation precautions to identify exposure hotspots and alert individuals at increased infection risks.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the most lethal cancer, mainly because of its invasive and metastatic characteristics. Pancreatic intraepithelial neoplasia (PanIN) is one of the major precursor lesions of PDAC. Although epithelial‐to‐mesenchymal transition (EMT) is known to play an important role for these malignant behaviors, the association between PanIN and EMT has not been clearly understood. Therefore, we explored possible molecules for regulation of EMT immunohistochemically. Using surgically resected specimens from 71 PDAC patients, expressions of SNAIL, SLUG, TWIST1, and ZEB1 were investigated in high‐grade PanIN (HG‐PanIN) and PDAC. Results demonstrated that PDAC accompanied by SNAIL‐positive HG‐PanIN showed a significantly better relapse‐free survival (RFS) (median survival time (MST) of 11.3 months vs 4.4 months, P

Hydrogen sulfide (H 2 S) has been reported to exacerbate stroke outcome in experimental models. Cystathionine β-synthase (CBS) has been implicated as the predominant H 2 S-producing enzyme in central nervous system. When SH-SY5Y cells were transfected to overexpress CBS, these cells were able to synthesize H 2 S when exposed to high levels of enzyme substrates but not substrate concentrations that may reflect normal physiological conditions. At the same time, these cells demonstrated exacerbated cell death when subjected to oxygen and glucose deprivation (OGD) together with high substrate concentrations, indicating that H 2 S production has a detrimental effect on cell survival. This effect could be abolished by CBS inhibition. The same effect was observed with primary astrocytes exposed to OGD and high substrates or sodium hydrosulfide. In addition, CBS was upregulated and activated by truncation in primary astrocytes subjected to OGD. When rats were subjected to permanent middle cerebral artery occlusion, CBS activation was also observed. These results imply that in acute ischemic conditions, CBS is upregulated and activated by truncation causing an increased production of H 2 S, which exacerbate the ischemic injuries. Therefore, CBS inhibition may be a viable approach to stroke treatment.

Skeletal muscle degeneration is a complication arising from a variety of chronic diseases including advanced cancer. Pro-inflammatory cytokine TNF-α plays a pivotal role in mediating cancer-related skeletal muscle degeneration. Here, we show a novel function for retinoblastoma protein (Rb), where Rb causes sarcomeric disorganization. In human skeletal muscle myotubes (HSMMs), up-regulation of cyclin-dependent kinase 4 (CDK4) and concomitant phosphorylation of Rb was induced by TNF-α treatment, resulting in the translocation of phosphorylated Rb to the cytoplasm. Moreover, induced expression of the nuclear exporting signal (NES)-fused form of Rb caused disruption of sarcomeric organization. We identified mammalian diaphanous-related formin 1 (mDia1), a potent actin nucleation factor, as a binding partner of cytoplasmic Rb and found that mDia1 helps maintain the structural integrity of the sarcomere. These results reveal a novel non-nuclear function for Rb and suggest a potential mechanism of TNF-α-induced disruption of sarcomeric organization.

Competing Interests: The authors declare that they have no competing interests.

Phylogenetic analyses over the last two decades have united a few small, and previously orphan clades, the nematodermatids, acoels and xenoturbelids, into the phylum Xenacoelomorpha. Some phylogenetic analyses support a sister relationship between Xenacoelomorpha and Ambulacraria (Xenambulacraria), while others suggest that Xenacoelomorpha may be sister to the rest of the Bilateria (Nephrozoa). An understanding of the cell type complements of Xenacoelomorphs is essential to assessing these alternatives as well as to our broader understanding of bilaterian cell type evolution. Employing whole organism single-cell RNA-seq in the marine xenacoelomorph worm Xenoturbella bocki, we show that Xenambulacrarian nerve nets share regulatory features and a peptidergic identity with those found in cnidarians and protostomes and more broadly share muscle and gland cell similarities with other metazoans. Taken together, these data are consistent with broad homologies of animal gland, muscle, and neurons as well as more specific affinities between Xenoturbella and acoel gut and epidermal tissues, consistent with the monophyly of Xenacoelomorpha., (© 2024. The Author(s).)

Schizophrenia (SZ) is a heterogeneous and debilitating psychiatric disorder with a strong genetic component. To elucidate functional networks perturbed in schizophrenia, we analysed a large dataset of whole-genome studies that identified SNVs, CNVs, and a multi-stage schizophrenia genome-wide association study. Our analysis identified three subclusters that are interrelated and with small overlaps: GO:0007017~Microtubule-Based Process, GO:00015629~Actin Cytoskeleton, and GO:0007268~SynapticTransmission. We next analysed three distinct trio cohorts of 75 SZ Algerian, 45 SZ French, and 61 SZ Japanese patients. We performed Illumina HiSeq whole-exome sequencing and identified de novo mutations using a Bayesian approach. We validated 88 de novo mutations by Sanger sequencing: 35 in French, 21 in Algerian, and 32 in Japanese SZ patients. These 88 de novo mutations exhibited an enrichment in genes encoding proteins related to GO:0051015~actin filament binding ( p = 0.0011) using David, and enrichments in GO: 0003774~transport ( p = 0.019) and GO:0003729~mRNA binding ( p = 0.010) using Amigo. One of these de novo variant was found in CORO1C coding sequence. We studied Coro1c haploinsufficiency in a Coro1c +/- mouse and found defects in the corpus callosum. These results could motivate future studies of the mechanisms surrounding genes encoding proteins involved in transport and the cytoskeleton, with the goal of developing therapeutic intervention strategies for a subset of SZ cases.

Anti-CD19 chimeric antigen receptor (CAR) T cell therapy represents a breakthrough for the treatment of B cell malignancies. Yet, it can lead to severe adverse events, including cytokine release syndrome (CRS), which may require urgent clinical management. Whether interpatient variability in CAR T cell subsets contributes to CRS is unclear. Here, we show that CD4 + CAR T cells are the main drivers of CRS. Using an immunocompetent model of anti-CD19 CAR T cell therapy, we report that CD4 + , but not CD8 + , CAR T cells elicit physiological CRS-like manifestations associated with the release of inflammatory cytokines. In CAR T cell-treated patients, CRS occurrence and severity are significantly associated with high absolute values of CD4 + CAR T cells in the blood. CRS in mice occurs independently of CAR T cell-derived interferon γ (IFN-γ) but requires elevated tumor burden. Thus, adjusting the CD4:CD8 CAR T cell ratio to patient tumor load may help mitigate CAR T cell-associated toxicities., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent advances in synthetic embryology have opened new avenues for understanding the complex events controlling mammalian peri-implantation development. Here, we show that mouse embryonic stem cells (ESCs) solely exposed to chemical inhibition of SUMOylation generate embryo-like structures comprising anterior neural and trunk-associated regions. HypoSUMOylation-instructed ESCs give rise to spheroids that self-organize into gastrulating structures containing cell types spatially and functionally related to embryonic and extraembryonic compartments. Alternatively, spheroids cultured in a droplet microfluidic device form elongated structures that undergo axial organization reminiscent of natural embryo morphogenesis. Single-cell transcriptomics reveals various cellular lineages, including properly positioned anterior neuronal cell types and paraxial mesoderm segmented into somite-like structures. Transient SUMOylation suppression gradually increases DNA methylation genome wide and repressive mark deposition at Nanog. Interestingly, cell-to-cell variations in SUMOylation levels occur during early embryogenesis. Our approach provides a proof of principle for potentially powerful strategies to explore early embryogenesis by targeting chromatin roadblocks of cell fate change., Competing Interests: Declaration of interests J.-C.C., T.T., S.S., C.N.B., and A.D. are designated as inventors of the patent application WO 2023/002057 A2 covering the aspects of the in vitro generation of organized 3D cell structures and the microfluidic device described in the manuscript., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The coordinated differentiation of progenitor cells into specialized cell types and their spatial organization into distinct domains is central to embryogenesis. Here, we developed and applied an unbiased spatially resolved single-cell transcriptomics method to identify the genetic programs underlying the emergence of specialized cell types during mouse limb development and their spatial integration. We identify multiple transcription factors whose expression patterns are predominantly associated with cell type specification or spatial position, suggesting two parallel yet highly interconnected regulatory systems. We demonstrate that the embryonic limb undergoes a complex multiscale reorganization upon perturbation of one of its spatial organizing centers, including the loss of specific cell populations, alterations of preexisting cell states' molecular identities, and changes in their relative spatial distribution. Our study shows how multidimensional single-cell, spatially resolved molecular atlases can allow the deconvolution of spatial identity and cell fate and reveal the interconnected genetic networks that regulate organogenesis and its reorganization upon genetic alterations.

Down syndrome (DS) is caused by human chromosome 21 (HSA21) trisomy. It is characterized by a poorly understood intellectual disability (ID). We studied two mouse models of DS, one with an extra copy of the <i>Dyrk1A</i> gene (189N3) and the other with an extra copy of the mouse Chr16 syntenic region (Dp(16)1Yey). RNA-seq analysis of the transcripts deregulated in the embryonic hippocampus revealed an enrichment in genes associated with chromatin for the 189N3 model, and synapses for the Dp(16)1Yey model. A large-scale yeast two-hybrid screen (82 different screens, including 72 HSA21 baits and 10 rebounds) of a human brain library containing at least 10<sup>7</sup> independent fragments identified 1,949 novel protein-protein interactions. The direct interactors of HSA21 baits and rebounds were significantly enriched in ID-related genes (<i>P</i>-value < 2.29 × 10<sup>-8</sup>). Proximity ligation assays showed that some of the proteins encoded by HSA21 were located at the dendritic spine postsynaptic density, in a protein network at the dendritic spine postsynapse. We located HSA21 DYRK1A and DSCAM, mutations of which increase the risk of autism spectrum disorder (ASD) 20-fold, in this postsynaptic network. We found that an intracellular domain of DSCAM bound either DLGs, which are multimeric scaffolds comprising receptors, ion channels and associated signaling proteins, or DYRK1A. The DYRK1A-DSCAM interaction domain is conserved in <i>Drosophila</i> and humans. The postsynaptic network was found to be enriched in proteins associated with ARC-related synaptic plasticity, ASD, and late-onset Alzheimer's disease. These results highlight links between DS and brain diseases with a complex genetic basis., (© 2022 Viard et al.)

A nanoporous gold (NPG) electrode prepared through a facile anodization technique was employed in the electrochemical reductive amination of biomass-derivable α-keto acids in the presence of a nitrogen source to produce the corresponding amino acids. NPG showed a clear reductive current in the presence of α-keto acid and NH 2 OH, and the electrolysis experiments confirmed the production of L-amino acid. A reductive voltammetric signal at the NPG electrode appeared at a more positive potential by 0.18-0.79 V, compared with those at the planar-gold electrode without anodization and other previously reported electrode systems, indicating the high activity of the prepared nanostructure for the electrochemical reaction. Maximum Faradaic efficiencies (FEs) of 74-93% in the reductive molecular conversion to amino acids of Ala, Asp, Glu, Gly, and Leu were obtained under the optimized conditions. The FE values were strongly dependent on the applied potential in the electrolysis, suggesting that the hydrogen evolution reaction at the electrode surface was more significant as the applied potential became more negative. The effect of potential at the NPG was lower than that at the planar-gold electrode. These results indicate that nanostructurization decreases the overpotential for the electrochemical reductive amination, resulting in high FE.

Chimeric antigen receptor (CAR) T cell therapy relies on the activity of a large pool of tumor-targeting cytotoxic effectors. Whether CAR T cells act autonomously or require interactions with the tumor microenvironment (TME) remains incompletely understood. Here, we report an essential cross-talk between CAR T cell subsets and the TME for tumor control in an immunocompetent mouse B cell lymphoma model of anti-CD19 CAR T cell therapy. Using single-cell RNA sequencing, we revealed substantial modification of the TME during CAR T cell therapy. Interferon-γ (IFN-γ) produced by CAR T cells not only enhanced endogenous T and natural killer cell activity but was also essential for sustaining CAR T cell cytotoxicity, as revealed by intravital imaging. CAR T cell-derived IFN-γ facilitated host interleukin-12 production that supported host immune and CAR T cell responses. Compared with CD8 + CAR T cells, CD4 + CAR T cells were more efficient at host immune activation but less capable of direct tumor killing. In summary, CAR T cells do not act independently in vivo but rely instead on cytokine-mediated cross-talk with the TME for optimal activity. Invigorating CAR T cell interplay with the host represents an attractive strategy to prevent relapses after therapy., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Non-enzymatic saccharide sensors are of great interest in diagnostics, but their non-selectivity limits their practical diagnostic abilities. In this study, we investigated the electrochemical oxidation of monosaccharides at nanoporous gold (NPG) catalysts with different contributions of surface crystallographic orientations. Fructose elicited no clear electrochemical response, but glucose, galactose, and mannose produced clear oxidative current. The onset potentials for oxidation of these saccharides depended on the surface atomic structure of the NPG. The oxidation potential was approximately 100 mV less positive at the Au(100)-enhanced NPG than at the Au(111)-enhanced NPG. Furthermore, the voltammetric responses significantly differed among the saccharides. Galactose was oxidized at less positive potential and exhibited a higher current response than the other saccharides. This tendency was enhanced in the presence of chloride ions. These features enabled the selective and sensitive detection of galactose at an NPG electrode without enzymes under physiological conditions. A linear range of 10 μM to 1.8 mM was obtained in the calibration plot, which was comparable to those in previously reported enzymatic galactose sensors. Thus, we demonstrated that controlling the crystallographic orientation on the nanostructured electrode surface is useful in developing electrochemical sensors.

The direct electron transfer between human cytoglobin (Cygb) and the electrode surface, which would allow manipulating the oxidation states of the heme iron in Cygb, was first observed by immobilizing Cygb on a nanoporous gold (NPG) electrode via a carboxy-terminated alkanethiol. The voltammetric performances of the wild type and mutated Cygb-immobilized NPG electrodes were evaluated in the absence or presence of potential substrates. The obtained results demonstrated that the usefulness of the proposed method in understanding the function of Cygb in molecular basis.

Alzheimer's disease and other types of dementia are the top cause for disabilities in later life and various types of experiments have been performed to understand the underlying mechanisms of the disease with the aim of coming up with potential drug targets. These experiments have been carried out by scientists working in different domains such as proteomics, molecular biology, clinical diagnostics and genomics. The results of such experiments are stored in the databases designed for collecting data of similar types. However, in order to get a systematic view of the disease from these independent but complementary data sets, it is necessary to combine them. In this study we describe a heterogeneous network-based data set for Alzheimer's disease (HENA). Additionally, we demonstrate the application of state-of-the-art graph convolutional networks, i.e. deep learning methods for the analysis of such large heterogeneous biological data sets. We expect HENA to allow scientists to explore and analyze their own results in the broader context of Alzheimer's disease research.

The emergence and diversification of cell types is a leading factor in animal evolution. So far, systematic characterization of the gene regulatory programs associated with cell type specificity was limited to few cell types and few species. Here, we perform whole-organism single-cell transcriptomics to map adult and larval cell types in the cnidarian Nematostella vectensis, a non-bilaterian animal with complex tissue-level body-plan organization. We uncover eight broad cell classes in Nematostella, including neurons, cnidocytes, and digestive cells. Each class comprises different subtypes defined by the expression of multiple specific markers. In particular, we characterize a surprisingly diverse repertoire of neurons, which comparative analysis suggests are the result of lineage-specific diversification. By integrating transcription factor expression, chromatin profiling, and sequence motif analysis, we identify the regulatory codes that underlie Nematostella cell-specific expression. Our study reveals cnidarian cell type complexity and provides insights into the evolution of animal cell-specific genomic regulation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

MicroRNA (miRNA)-guided argonaute (Ago) controls gene expression upon binding to the 3' UTR of mRNA. The miRNA function can be competitively inhibited by single-stranded anti-miRNA oligonucleotides (AMOs). In this study, we constructed a novel type of AMO flanked by interstrand cross-linked 2'-O-methylated RNA duplexes (CLs) that confer a stable helical conformation. Compared with other structured AMOs, AMO flanked by CLs at the 5' and 3' termini exhibited much higher inhibitory activity in cells. Anti-miRNA activity, nuclease resistance, and miRNA modification pattern distinctly differed according to the CL-connected positions in AMOs. Moreover, we found that the 3'-side CL improves nuclease resistance, whereas the 5'-side CL contributes to stable binding with miRNA in Ago upon interaction with the 3' part of miRNA. These structure-function relationship analyses of AMOs provide important insights into the function control of Ago-miRNA complexes, which will be useful for basic miRNA research as well as for determining therapeutic applications of AMO., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Imaging and chromosome conformation capture studies have revealed several layers of chromosome organization, including segregation into megabase-sized active and inactive compartments, and partitioning into sub-megabase domains (TADs). It remains unclear, however, how these layers of organization form, interact with one another and influence genome function. Here we show that deletion of the cohesin-loading factor Nipbl in mouse liver leads to a marked reorganization of chromosomal folding. TADs and associated Hi-C peaks vanish globally, even in the absence of transcriptional changes. By contrast, compartmental segregation is preserved and even reinforced. Strikingly, the disappearance of TADs unmasks a finer compartment structure that accurately reflects the underlying epigenetic landscape. These observations demonstrate that the three-dimensional organization of the genome results from the interplay of two independent mechanisms: cohesin-independent segregation of the genome into fine-scale compartments, defined by chromatin state; and cohesin-dependent formation of TADs, possibly by loop extrusion, which helps to guide distant enhancers to their target genes.

The neuropeptide somatostatin (SOM) is widely expressed in rodent brain and somatostatin-IRES-Cre (SOM-cre) mouse strains are increasingly used to unravel the physiology of SOM-containing neurons. However, while knock-in targeting strategy greatly improves Cre-Lox system accuracy, recent reports have shown that genomic insertion of Cre construct per se can markedly affect physiological function. We show that Cre transgene insertion into the 3'UTR of the somatostatin gene leads to the selective and massive depletion of endogenous SOM in all tested brain regions. It also strongly impacts SOM-related neuroendocrine responses in a similar manner to what has been reported for SST KO mice: increased corticosterone levels after 30-min restraint stress, decreased amplitude and regularity of ultradian growth hormone secretory patterns accompanied by changes in sexually dimorphic liver gene expression ( serpina1, Cyp2b9, Cyp2a4, Cyp2d9, and Cyp7b1 ). In addition to demonstrating the need for examination of the consequences of Cre transgenesis, these results also reveal how this SOM-cre strain may be a useful tool in studying the functional consequences of moderate to low SOM levels as reported in neurological and psychiatric disorders.

DNA molecules have attracted considerable attention as functional materials in various fields such as electrochemical sensors with redox-labeled DNA. However, the recently developed interstrand cross-link (ICL) technique for double-stranded DNA can adequately modify the electronic properties inside the duplex. Hence, the electrochemical investigation of ICL-DNA helps us to understand the electron transfer of redox-labeled DNA at an electrode surface, which would develop useful sensors. In this study, the first insight into this matter is presented. We prepared 17-mer DNA duplexes incorporating Nile blue (NB-DNA) at one end as a redox marker and a disulfide tether at the other end for immobilization onto an electrode. The duplexes were covalently cross-linked by bifunctional cross-linkers that utilize either a propyl or naphthalene residue to replace a base pair. Their electrochemical responses at the electrode surface were compared to evaluate the effect of the ICL on the electron-transfer reactions of the redox-labeled DNA duplexes. A direct transfer of electrons between NB and the electrode was observed for a standard DNA, as previously reported, whereas interstrand cross-linked DNA (CL-DNA) strands showed a decrease in the direct electron-transfer pathway. This is expected to result from constraining the elastic bending/flexibility of the duplex caused by the covalent cross-links. Interestingly, the CL-DNA incorporating naphthalene residues exhibited additional voltammetric peaks derived from DNA-mediated electron transfer (through base π stacking), which was not observed in the mismatched CL-DNA. The present results indicate that the ICL significantly affects electron transfer in the redox-labeled DNA at the electrode and can be an important determinant for electrochemical signaling in addition to its role in stabilizing the duplex structure.

The SMARCA2 gene, which encodes BRM in the SWI/SNF chromatin-remodeling complex, was recently identified as being associated with schizophrenia (SZ) in a genome-wide approach. Polymorphisms in SMARCA2, associated with the disease, produce changes in the expression of the gene and/or in the encoded amino acid sequence. We show here that an SWI/SNF-centered network including the Smarca2 gene is modified by the down-regulation of REST/NRSF in a mouse neuronal cell line. REST/NRSF down-regulation also modifies the levels of Smarce1, Smarcd3 and SWI/SNF interactors (Hdac1, RcoR1 and Mecp2). Smarca2 down-regulation generates an abnormal dendritic spine morphology that is an intermediate phenotype of SZ. We further found that 8 (CSF2RA, HIST1H2BJ, NOTCH4, NRGN, SHOX, SMARCA2, TCF4 and ZNF804A) out of 10 genome-wide supported SZ-associated genes are part of an interacting network (including SMARCA2), 5 members of which encode transcription regulators. The expression of 3 (TCF4, SMARCA2 and CSF2RA) of the 10 genome-wide supported SZ-associated genes is modified when the REST/NRSF-SWI/SNF chromatin-remodeling complex is experimentally manipulated in mouse cell lines and in transgenic mouse models. The REST/NRSF-SWI/SNF deregulation also results in the differential expression of genes that are clustered in chromosomes suggesting the induction of genome-wide epigenetic changes. Finally, we found that SMARCA2 interactors and the genome-wide supported SZ-associated genes are considerably enriched in genes displaying positive selection in primates and in the human lineage which suggests the occurrence of novel protein interactions in primates. Altogether, these data identify the SWI/SNF chromatin-remodeling complex as a key component of the genetic architecture of SZ.

We have succeeded to immobilize fluorescent proteins selectively using a micro-structured organosilane self-assembled monolayer as a template. An organosilane layer with amino terminal group was formed on a thermally oxidized Si wafer by liquid-phase method and then was pattern-etched by vacuum ultraviolet light (VUV). The second organosilane layer with thiol terminal group was deposited on the etched area by chemical vapor surface modification method (CVSM). These micro-structured organosilane layer containing two reactive terminal groups were chemically modified using bi-functional linkers. Two kinds of fluorescent protein, Enhanced Cyan Fluorescent Protein (ECFP) and R-phycoerythrin were selectively immobilized on the chemically modified surface.

Cytochrome c-552 from Pseudomonas alcaliphila AL15-21(T), which is a small cytochrome c(5) from Pseudomonas spp., was first purified and characterized in our previous study. Although it has been found that cytochrome c-552 is induced at a high pH under air-limited condition, the physiological role of this cytochrome c has not been clarified yet. Therefore, to understand its physiological role, further characterization of this cytochrome expressed in Escherichia coli was performed. The yield of the recombinant protein reached 2.8 mg/l of culture, which was 76.4-fold larger than that of native cells. Analytical data of the recombinant protein exactly agreed with that of native cytochrome c-552. The recombinant cytochrome c-552 was oxidized by partially purified cb-type cytochrome c oxidase from P. alcaliphila AL15-21(T) at a rate of 9.6 mu mol min(-1) mg oxidase(-1). Unlike reported cytochromes c from other Pseudomonas spp., the E degrees ' values between pHs 5.0 and 10.0 were nearly unchanged. Cytochrome c-552 oxidized very slowly at pHs 8.0 (6.1 x 10(-4) h(-1)), 9.0 (1.4 x 10(-3) h(-1)) and 10.0 (1.6 x 10(-3) h(-1)), whereas it oxidized more rapidly at pH 7.0 (2.5 x 10(-3) h(-1)). On the other hand, horse heart cytochrome c showed higher oxidation rates at pHs 6.0-10.0 than cytochrome c-552. It is considered that the high electron-retaining ability of cytochrome c-552 at high pHs is important for its physiological function in the environmental adaptation of this bacteria for superior growth at high pHs under air-limited conditions.

Rapid analyses of drug metabolism reactions by human cytochrome P450s (CYPs) that metabolize 95% of all current drugs are very important in drug development processes and effective therapies. Since CYPs need electrons to metabolize drugs, electrons supplied from electrodes to activate CYP molecules are expected to be very useful to develop rapid assay methods for CYP reactions. Although several studies on the direct electrochemistry of isolated and purified human CYPs have been reported, the use of microsomes (membranes) containing CYP is more suitable, because they are frequently used in drug research due to their easy preparation and low cost. Herein, we demonstrate electrons supplied from an electrode to microsomes containing CYP and CYP-reductase (CPR) on an electrode coated with hydrophobic thin films and observe electrochemically driven drug metabolisms by voltammetry for the first time. We tested the immobilization of microsomes on gold electrodes coated with several thiolates and found that microsomes immobilized on thin hydrophobic surfaces of aromatic compounds showed well-defined redox peaks. Furthermore, electrochemically driven drug metabolism reactions of CYP3A4 were clearly observed by voltammetry, and these reactions were inhibited by a CYP3A4 inhibitor, ketoconazole. We also found that metabolism reactions were facilitated in the presence of CPR.

A membrane-anchored cytochrome c-550, which is highly expressed in obligately alkaliphilic Bacillus clarkii K24-1U, was purified and characterized. The protein contained a conspicuous sequence of Gly(22)-Asn(34), in comparison with the other Bacillus small cytochromes c. Analytical data indicated that the original and lipase-treated intermediate forms of cytochrome c-550 bind to fatty acids of C(15), C(16) and C(17) chain lengths and C(15) chain length, respectively, and it was considered that these fatty acids are bound to glycerol-Cys(18). Since there was a possibility that the presence of a diacylglycerol anchor contributed to the formation of dimeric states of this protein (20 and 17 kDa in SDS-PAGE), a C18M (Cys(18) --> Met)-cytochrome c-550 was constructed. The molecular mass of the C18M-cytochrome c-550 was determined as 15 and 10 kDa in SDS-PAGE and 23 kDa in blue native PAGE. The C18M-cytochrome c-550 bound with or without Triton X-100 formed a tetramer as the original cytochrome c-550 bound with Triton X-100, as determined by gel filtration. The midpoint redox potential of cytochrome c-550 as determined by redox titration was +83 mV, while that determined by cyclic voltammetric measurement was +7 mV. The above results indicate that cytochrome c-550 is a novel cytochrome c.

The molecular mechanisms that lead to the cognitive defects characteristic of Down syndrome (DS), the most frequent cause of mental retardation, have remained elusive. Here we use a transgenic DS mouse model (152F7 line) to show that DYRK1A gene dosage imbalance deregulates chromosomal clusters of genes located near neuron-restrictive silencer factor (REST/NRSF) binding sites. We found that Dyrk1a binds the SWI/SNF complex known to interact with REST/NRSF. The mutation of a REST/NRSF binding site in the promoter of the REST/NRSF target gene L1cam modifies the transcriptional effect of Dyrk1a-dosage imbalance on L1cam. Dyrk1a dosage imbalance perturbs Rest/Nrsf levels with decreased Rest/Nrsf expression in embryonic neurons and increased expression in adult neurons. Using transcriptome analysis of embryonic brain subregions of transgenic 152F7 mouse line, we identified a coordinated deregulation of multiple genes that are responsible for dendritic growth impairment present in DS. Similarly, Dyrk1a overexpression in primary mouse cortical neurons induced severe reduction of the dendritic growth and dendritic complexity. We propose that DYRK1A overexpression-related neuronal gene deregulation via disturbance of REST/NRSF levels, and the REST/NRSF-SWI/SNF chromatin remodelling complex, significantly contributes to the neural phenotypic changes that characterize DS.

Several redox enzymes were examined for enzymatic/electrochemical-recycling systems in order to measure p-aminophenol (PAP) with high sensitivity. Glucose oxidase (GOD) and diaphorase (DI) worked well as catalysts for recycling electrode systems: these enzymes effectively reduced p-iminoquinone (PIQ), the electrochemically-oxidized form of PAP, and caused an enhancement in the electrochemical signals (anodic currents in the voltammogram and amperogram) by approximately 100 fold. The lower detection limits for PAP were estimated to be 50 nM with the GOD system and 2 nM with the DI system. We combined the enzymatic-recycling electrode using DI with an enzyme immunoassay system to measure atrial natriuretic peptide (ANP), an important marker peptide hormone involved in heart diseases. ANPs from serum samples at ppt-levels were determined appropriately using the present assay system.

The rapid and reversible electron transfer reaction of cytochrome b562 was observed at an In2O3 electrode. The estimated heterogeneous electron transfer rate constant (k0') was k0' > or = 5.0 x 10(-3) cm s(-1) at pH 6.5. When the methionine-7 (Met-7) residue, which coordinates to the heme iron as an axial ligand, of the wild-type cytochrome b562 was replaced by an Ala or Gly residue, a water molecule bound to the heme iron and the electron transfer rate constants decreased to 1.3 x 10(-3) and 1.8 x 10(-3) cm s(-1), respectively. This decrease in the electron transfer rate would be due to the larger reorganization energy for the structural change at the redox site. The midpoint potential of cytochrome b562 was shifted negatively by approximately 135 mV by replacing Met-7 with Ala or Gly. Similar dissociation kinetics of cyanide for the mutated molecules as compared to native myoglobin was obtained.

The electron transfer reaction of wild-type myoglobin at an electrode was significantly facilitated in a D2O buffer as compared with that in an H2O buffer, with k(0)'(H2O)/k(0)'(D2O)= 0.13, while a minimal deuterium kinetic isotope effect on the myoglobin with modification at distal histidine (His-64) was observed.

We replaced protoheme-IX in native myoglobin with the symmetric protohemes-III and -XIII, in order to investigate the role of heme vinyl-globin contacts on Mb function. The UV-visible spectra and the resonance Raman spectra in the high-frequency region (containing oxidation, spin, and coordination state marker lines) of the two reconstituted Mbs were very similar. However, the signal intensity of the Soret band in the CD spectra and the resonance Raman lines for vinyl bending modes in the low-frequency region notably differed, thereby reflecting altered heme peripheral contacts. The redox potentials, formal heterogeneous electron-transfer rates, and thermal denaturation temperatures of the two reconstituted Mbs were also indistinguishable. In addition, the oxygen binding properties of the ferrous deoxy Mbs were comparable. These results demonstrate that altered heme vinyl-globin interactions only slightly affect the physical properties of Mb. It is therefore likely that the orientation of protoheme-IX about the alpha,gamma-axis in the heme pocket is not necessarily a crucial factor for oxygen binding to native Mb.