Endothelial CLEC-1b plays a protective role against cancer hematogenous metastasis.

Metastasis, which is the spread of cancer cells into distant organs, is a critical determinant of prognosis in patients with cancer, and blood vessels are the major route for cancer cells to spread systemically. Extravasation is a critical process for the hematogenous metastasis; however, its underlying molecular mechanisms remain poorly understood. Here, we identified that senescent ECs highly express C-type lectin domain family 1 member B (CLEC-1b), and that endothelial CLEC-1b inhibits the hematogenous metastasis of a certain type of cancer. CLEC-1b expression was enhanced in ECs isolated from aged mice, senescent cultured human ECs, and ECs of aged human. CLEC-1b overexpression in ECs prevented the disruption of endothelial integrity, and inhibited the transendothelial migration of cancer cells expressing podoplanin (PDPN), a ligand for CLEC-1b. Notably, target activation of CLEC-1b in ECs decreased the hematogenous metastasis in the lungs by cancer cells expressing PDPN in mice. Our data reveal the protective role of endothelial CLEC-1b against cancer hematogenous metastasis. Considering the high CLEC-1b expression in senescent ECs, EC senescence may play a beneficial role with respect to the cancer hematogenous metastasis.

A single nucleotide polymorphism (-250 A/C) of the GFAP gene is associated with brain structures and cerebral blood flow.

AIM: Glial fibrillary acidic protein (GFAP), the intermediate filament protein expressed in astrocytes, plays a key role in many aspects of brain function through communication with neurons or blood vessels. A common single nucleotide polymorphism (SNP), GFAP -250 C/A (rs2070935), is associated with the transcriptional regulation of GFAP, which can potentially result in the genotype-specific brain structure. This study aimed to verify the biological effects of the GFAP variants on brain structure and function. METHODS: We investigated the associations between the GFAP variants and magnetic resonance imaging findings, including gray and white matter volumes, white matter integrity, and resting arterial blood flow, from 1212 healthy Japanese subjects. RESULTS: The GFAP -250 C/A genotype was significantly associated with total gray matter volume, total white matter volume, average mean diffusivity, and mean cerebral blood flow. In voxel-by-voxel analyses, the GFAP genotype showed significant associations with the regional gray and white matter volumes in the inferior frontal lobe and corpus callosum, the regional mean diffusivity in the left posterior region, and the regional cerebral blood flow throughout the brain. CONCLUSION: This study revealed a common SNP that is significantly associated with multiple global brain structure parameters.

Polymorphisms in the microglial marker molecule CX3CR1 affect the blood volume of the human brain.

AIM: CX3CR1, a G-protein-coupled receptor, is involved in various inflammatory processes. Two non-synonymous single nucleotide polymorphisms, V249I (rs3732379) and T280M (rs3732378), are located in the sixth and seventh transmembrane domains of the CX3CR1 protein, respectively. Previous studies have indicated significant associations between T280M and leukocyte functional characteristics, including adhesion, signaling, and chemotaxis, while the function of V249I is unclear. In the brain, microglia are the only proven and widely accepted CX3CR1-expressing cells. This study aimed to specify whether there were specific brain regions on which these two single nucleotide polymorphisms exert their biological impacts through their functional effects on microglia. METHODS: Associations between the single nucleotide polymorphisms and brain characteristics, including gray and white matter volumes, white matter integrity, resting arterial blood volume, and cerebral blood flow, were evaluated among 1300 healthy Japanese individuals. RESULTS: The major allele carriers (V249 and T280) were significantly associated with an increased total arterial blood volume of the whole brain, especially around the bilateral precuneus, left posterior cingulate cortex, and left posterior parietal cortex. There were no significant associations between the genotypes and other brain structural indicators. CONCLUSION: This finding suggests that the CX3CR1 variants may affect arterial structures in the brain, possibly via interactions between microglia and brain microvascular endothelial cells.

The Application of a Modified d-ROMs Test for Measurement of Oxidative Stress and Oxidized High-Density Lipoprotein.

Reactive oxygen species (ROS) are involved in the initiation and progression of atherosclerosis. ROS-derived hydroperoxides, as an indicator of ROS production, have been measured by using the diacron reactive oxygen metabolites (d-ROMs) test, which requires iron-containing transferrin in the reaction mixture. In this study we developed a modified d-ROMs test, termed the Fe-ROMs test, where iron ions were exogenously added to the reaction mixture. This modification is expected to exclude the assay variation that comes from different blood iron levels in individuals. In addition, this Fe-ROMs test was helpful for determining the class of plasma lipoproteins that are hydroperoxidized. Low-density lipoprotein/very low-density lipoprotein (LDL/VLDL) and high-density lipoprotein (HDL) were purified by use of an LDL/VLDL purification kit and the dextran sulfate-Mg2+ precipitation method, respectively; their hydroperoxide contents were assessed by performing the Fe-ROMs test. The majority of the hydroperoxides were detected only in the HDL fraction, not in the LDL/VLDL. Further detailed analysis of HDLs by size-exclusion high-performance liquid chromatography revealed that the hydroperoxide-containing molecules were small-sized HDLs. Because HDL was shown to be the principal vehicle for the plasma hydroperoxides, this Fe-ROMs test is a beneficial method for the assessment of oxidized-HDL levels. Indeed, Fe-ROMs levels were strongly associated with the levels of oxidized HDL, which were determined by performing the malondialdehyde-modified HDL enzyme immunoassay. In conclusion, the Fe-ROMs test using plasma itself or the HDL fraction after dextran sulfate-Mg2+ precipitation is useful to assess the functionality of HDL, because the oxidation of HDL impairs its antiatherogenic capacity.

DNA cytosine and methylcytosine deamination by APOBEC3B: enhancing methylcytosine deamination by engineering APOBEC3B.

APOBEC (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like) is a family of enzymes that deaminates cytosine (C) to uracil (U) on nucleic acid. APOBEC3B (A3B) functions in innate immunity against intrinsic and invading retroelements and viruses. A3B can also induce genomic DNA mutations to cause cancer. A3B contains two cytosine deaminase domains (CD1, CD2), and there are conflicting reports about whether both domains are active. Here we demonstrate that only CD2 of A3B (A3BCD2) has C deamination activity. We also reveal that both A3B and A3BCD2 can deaminate methylcytosine (mC). Guided by structural and functional analysis, we successfully engineered A3BCD2 to gain over two orders of magnitude higher activity for mC deamination. Important determinants that contribute to the activity and selectivity for mC deamination have been identified, which reveals that multiple elements, rather than single ones, contribute to the mC deamination activity and selectivity in A3BCD2 and possibly other APOBECs.

Engineering the allosteric properties of archaeal non-phosphorylating glyceraldehyde-3-phosphate dehydrogenases.

The archaeal non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN, EC 1.2.1.9) is a highly allosteric enzyme activated by glucose 1-phosphate (Glc1P). Recent kinetic analyses of two GAPN homologs from Sulfolobales show different allosteric behaviors toward the substrate glyceraldehyde-3-phosphate (GAP) and the allosteric effector Glc1P. In GAPN from Sulfolobus tokodaii (Sto-GAPN), Glc1P-induced activation follows an increase in affinity for GAP rather than an increase in maximum velocity, whereas in GAPN from Sulfolobus solfataricus (Sso-GAPN), Glc1P-induced activation follows an increase in maximum velocity rather than in affinity for GAP. To explore the molecular basis of this difference between Sto-GAPN and Sso-GAPN, we generated 14 mutants and 2 chimeras. The analyses of chimeric GAPNs generated from regions of Sto-GAPN and Sso-GAPN indicated that a 57-residue module located in the subunit interface was clearly involved in their allosteric behavior. Among the point mutations in this modular region, the Y139R variant of Sto-GAPN no longer displayed a sigmoidal K-type-like allostery, but instead had apparent V-type allostery similar to that of Sso-GAPN, suggesting that the residue located in the center of the homotetramer critically contributes to the allosteric behavior.

Effect of concomitant use of valproic acid during clozapine initiation on clozapine-induced inflammation among Japanese patients with schizophrenia.

During clozapine initiation, titration speed and concomitant valproate administration have been reported as risk factors for clozapine-induced fever and myocarditis. We tested the risk of concomitant valproate administration by stratifying patients according to titration rate. Concomitant valproate use was only associated with increased inflammatory adverse events in the slower titration group. The frequency of inflammatory adverse events was approximately 30 % during faster titration, regardless of concomitant valproate administration. However, the faster titration group with valproate had a higher frequency of severe adverse effects such as myocarditis. Clinicians should avoid concomitant valproate administration during clozapine initiation, regardless of titration rate.

Molecular mechanism for regulating APOBEC3G DNA editing function by the non-catalytic domain.

APOBEC3G (A3G) belongs to the AID/APOBEC cytidine deaminase family and is essential for antiviral immunity. It contains two zinc-coordinated cytidine-deaminase (CD) domains. The N-terminal CD1 domain is non-catalytic but has a strong affinity for nucleic acids, whereas the C-terminal CD2 domain catalyzes C-to-U editing in single-stranded DNA. The interplay between the two domains in DNA binding and editing is not fully understood. Here, our studies on rhesus macaque A3G (rA3G) show that the DNA editing function in linear and hairpin loop DNA is greatly enhanced by AA or GA dinucleotide motifs present downstream (in the 3'-direction) but not upstream (in the 5'-direction) of the target-C editing sites. The effective distance between AA/GA and the target-C sites depends on the local DNA secondary structure. We present two co-crystal structures of rA3G bound to ssDNA containing AA and GA, revealing the contribution of the non-catalytic CD1 domain in capturing AA/GA DNA and explaining our biochemical observations. Our structural and biochemical findings elucidate the molecular mechanism underlying the cooperative function between the non-catalytic and the catalytic domains of A3G, which is critical for its antiviral role and its contribution to genome mutations in cancer.

Molecular mechanism for regulating APOBEC3G DNA editing function by the non-catalytic domain.

APOBEC3G, part of the AID/APOBEC cytidine deaminase family, is crucial for antiviral immunity. It has two zinc-coordinated cytidine-deaminase domains. The non-catalytic N-terminal domain strongly binds to nucleic acids, whereas the C-terminal domain catalyzes C-to-U editing in single-stranded DNA. The interplay between the two domains is not fully understood. Here, we show that DNA editing function of rhesus macaque APOBEC3G on linear and hairpin loop DNA is enhanced by AA or GA dinucleotide motifs present downstream in the 3'-direction of the target-C editing sites. The effective distance between AA/GA and the target-C sites is contingent on the local DNA secondary structure. We present two co-crystal structures of rhesus macaque APOBEC3G bound to ssDNA containing AA and GA, revealing the contribution of the non-catalytic domain in capturing AA/GA DNA. Our findings elucidate the molecular mechanism of APOBEC3G's cooperative function, which is critical for its antiviral role and its contribution to mutations in cancer genomes.

Structural Basis for Polθ-Helicase DNA Binding and Microhomology-Mediated End-Joining.

DNA double-strand breaks (DSBs) present a critical threat to genomic integrity, often precipitating genomic instability and oncogenesis. Repair of DSBs predominantly occurs through homologous recombination (HR) and non-homologous end joining (NHEJ). In HR-deficient cells, DNA polymerase theta (Polθ) becomes critical for DSB repair via microhomology-mediated end joining (MMEJ), also termed theta-mediated end joining (TMEJ). Thus, Polθ is synthetically lethal with BRCA1/2 and other HR factors, underscoring its potential as a therapeutic target in HR-deficient cancers. However, the molecular mechanisms governing Polθ-mediated MMEJ remain poorly understood. Here we present a series of cryo-electron microscopy structures of the Polθ helicase domain (Polθ-hel) in complex with DNA containing 3'-overhang. The structures reveal the sequential conformations adopted by Polθ-hel during the critical phases of DNA binding, microhomology searching, and microhomology annealing. The stepwise conformational changes within the Polθ-hel subdomains and its functional dimeric state are pivotal for aligning the 3'-overhangs, facilitating the microhomology search and subsequent annealing necessary for DSB repair via MMEJ. Our findings illustrate the essential molecular switches within Polθ-hel that orchestrate the MMEJ process in DSB repair, laying the groundwork for the development of targeted therapies against the Polθ-hel.

SKGQA, a Peptide Derived from the ANA/BTG3 Protein, Cleaves Amyloid-ß with Proteolytic Activity.

Despite the extensive research conducted on Alzheimer's disease (AD) over the years, no effective drug for AD treatment has been found. Therefore, the development of new drugs for the treatment of AD is of the utmost importance. We recently reported the proteolytic activities of JAL-TA9 (YKGSGFRMI) and ANA-TA9 (SKGQAYRMA), synthetic peptides of nine amino acids each, derived from the Box A region of Tob1 and ANA/BTG3 proteins, respectively. Furthermore, two components of ANA-TA9, ANA-YA4 (YRMI) at the C-terminus end and ANA-SA5 (SKGQA) at the N-terminus end of ANA-TA9, exhibited proteolytic activity against amyloid-ß (Aß) fragment peptides. In this study, we identified the active center of ANA-SA5 using AEBSF, a serine protease inhibitor, and a peptide in which the Ser residue of ANA-SA5 was replaced with Leu. In addition, we demonstrate the proteolytic activity of ANA-SA5 against the soluble form Aß42 (a-Aß42) and solid insoluble form s-Aß42. Furthermore, ANA-SA5 was not cytotoxic to A549 cells. These results indicate that ANA-SA5 is a promising Catalytide and a potential candidate for the development of new peptide drugs targeting Aß42 for AD treatment.

Demonstration of shape analysis of neutron resonance transmission spectrum measured with a laser-driven neutron source.

Laser-driven neutron sources (LDNSs) can generate strong short-pulse neutron beams, which are valuable for scientific studies and engineering applications. Neutron resonance transmission analysis (NRTA) is a nondestructive technique used for determining the areal density of each nuclide in a material sample using pulsed thermal and epithermal neutrons. Herein, we report the first successful NRTA performed using an LDNS driven by the Laser for Fast Ignition Experiment at the Institute of Laser Engineering, Osaka University. The key challenge was achieving a well-resolved resonance transmission spectrum for material analysis using an LDNS with a limited number of laser shots in the presence of strong background noise. We addressed this by employing a time-gated 6 Li -glass scintillation neutron detector to measure the transmission spectra, reducing the impact of electromagnetic noise and neutron and gamma-ray flashes. Output waveforms were recorded for each laser shot and analyzed offline using a counting method. This approach yielded a spectrum with distinct resonances, which were attributed to 115 In and 109 Ag , as confirmed through neutron transmission simulation. The spectrum was analyzed using the least-square nuclear-resonance fitting program, REFIT, demonstrating the possibility of using an LDNS for nondestructive areal-density material characterization.

Structural basis for a Polθ helicase small-molecule inhibitor revealed by cryo-EM.

DNA polymerase theta (Polθ) is a DNA helicase-polymerase protein that facilitates DNA repair and is synthetic lethal with homology-directed repair (HDR) factors. Thus, Polθ is a promising precision oncology drug-target in HDR-deficient cancers. Here, we characterize the binding and mechanism of action of a Polθ helicase (Polθ-hel) small-molecule inhibitor (AB25583) using cryo-EM. AB25583 exhibits 6 nM IC50 against Polθ-hel, selectively kills BRCA1/2-deficient cells, and acts synergistically with olaparib in cancer cells harboring pathogenic BRCA1/2 mutations. Cryo-EM uncovers predominantly dimeric Polθ-hel:AB25583 complex structures at 3.0-3.2 Å. The structures reveal a binding-pocket deep inside the helicase central-channel, which underscores the high specificity and potency of AB25583. The cryo-EM structures in conjunction with biochemical data indicate that AB25583 inhibits the ATPase activity of Polθ-hel helicase via an allosteric mechanism. These detailed structural data and insights about AB25583 inhibition pave the way for accelerating drug development targeting Polθ-hel in HDR-deficient cancers.

RecQ5 protein translocation into the nucleus by a nuclear localization signal.

RecQ5, a member of the RecQ helicase family, maintains genome stability via participation in many DNA metabolic processes including DNA repair, DNA resolution, and RNA transcription, processes occurring in the nucleus. Previously, we reported that RecQ5 and Rad51, also involved in DNA repair, become co-localized in nuclei when co-expressed in cultured cells. Nuclear localization of RecQ5 appears to be important for cellular function along with Rad51. However, little is known about the nuclear localization of RecQ5. Here, we generated enhanced green fluorescent protein (EGFP)-tagged RecQ5 transgenic flies and analyzed localization of this protein in early embryos by live imaging. In syncytial embryos, RecQ5 was localized synchronously in interphase nuclei, and spread repeatedly over the embryos in mitosis. Thus, RecQ5 was transported into nuclei at the early interphase. Furthermore, we examined the subcellular localization of a series of truncated forms of Drosophila RecQ5 in cultured cells to determine the nuclear localization signal (NLS). Entire coding or deleted RecQ5 sequences of various sizes were ligated into EGFP vectors, which were then used to transfect cultured Drosophila cells. The region responsible for nuclear localization of Drosophila RecQ5 contained a short stretch of positively charged basic amino acids, 2 of which were particularly important for the nuclear localization. This stretch was sufficient for nuclear localization when fused with EGFP. Although the NLS of Drosophila RecQ5 was distinct from that of human RECQL5 in terms of position and amino acid sequence, this fly RecQ5 protein was translocated into the nucleus by an NLS.

Archaeal aldehyde dehydrogenase ST0064 from Sulfolobus tokodaii, a paralog of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase, is a succinate semialdehyde dehydrogenase.

Aldehyde dehydrogenase ST0064, the closest paralog of previously characterized allosteric non-phosphorylating glyceraldehyde-3-phosphate (GAP) dehydrogenase (GAPN, ST2477) from a thermoacidophilic archaeon, Sulfolobus tokodaii, was expressed heterologously and characterized in detail. ST0064 showed remarkable activity toward succinate semialdehyde (SSA) (Km of 0.0029 mM and kcat of 30.0 s(-1)) with no allosteric regulation. Activity toward GAP was lower (Km of 4.6 mM and kcat of 4.77 s(-1)), and previously predicted succinyl-CoA reductase activity was not detected, suggesting that the enzyme functions practically as succinate semialdehyde dehydrogenase (SSADH). Phylogenetic analysis indicated that archaeal SSADHs and GAPNs are closely related within the aldehyde dehydrogenase superfamily, suggesting that they are of the same origin.

Emotional processing during speech communication and positive symptoms in schizophrenia.

AIMS: The recognition of emotion is often impaired in patients with schizophrenia. The relationship of this deficit with symptoms of psychosis remains unclear. In the current study, we investigated the relationship between emotional processing and positive psychotic symptoms in schizophrenia. METHODS: Twenty-eight patients with schizophrenia and 37 healthy participants were included in the study. They were instructed to listen to a set of sentences and judge whether the emotional valence expressed verbally and that expressed by affective prosody were congruous or incongruous. RESULTS: Overall, the patients with schizophrenia had more inaccurate responses than the healthy participants and the poor performance was prominent when the patients processed affectively negative scenarios. The percentage of accurate responses negatively correlated with the severity of positive symptoms when the scenarios and/or the affective prosody had a negative valence. CONCLUSION: Patients with schizophrenia appear to have impaired function in the processing of negative verbal information. Impaired processing of negative verbal and prosodic information seems to be associated with positive symptoms in schizophrenia.

Structural basis for polyuridine tract recognition by SARS-CoV-2 Nsp15.

SARS-CoV-2 non-structural protein 15 (Nsp15) is critical for productive viral replication and evasion of host immunity. The uridine-specific endoribonuclease activity of Nsp15 mediates the cleavage of the polyuridine [poly(U)] tract of the negative-strand coronavirus genome to minimize the formation of dsRNA that activates the host antiviral interferon signaling. However, the molecular basis for the recognition and cleavage of the poly(U) tract by Nsp15 is incompletely understood. Here, we present cryogenic electron microscopy (cryoEM) structures of SARS-CoV-2 Nsp15 bound to viral replication intermediate dsRNA containing poly(U) tract at 2.7-3.3 Å resolution. The structures reveal one copy of dsRNA binds to the sidewall of an Nsp15 homohexamer, spanning three subunits in two distinct binding states. The target uracil is dislodged from the base-pairing of the dsRNA by amino acid residues W332 and M330 of Nsp15, and the dislodged base is entrapped at the endonuclease active site center. Up to 20 A/U base pairs are anchored on the Nsp15 hexamer, which explains the basis for a substantially shortened poly(U) sequence in the negative strand coronavirus genome compared to the long poly(A) tail in its positive strand. Our results provide mechanistic insights into the unique immune evasion strategy employed by coronavirus Nsp15.

Structural basis of HIV-1 Vif-mediated E3 ligase targeting of host APOBEC3H.

Human APOBEC3 (A3) cytidine deaminases are antiviral factors that are particularly potent against retroviruses. As a countermeasure, HIV-1 uses a viral infectivity factor (Vif) to target specific human A3s for proteasomal degradation. Vif recruits cellular transcription cofactor CBF-ß and Cullin-5 (CUL5) RING E3 ubiquitin ligase to bind different A3s distinctively, but how this is accomplished remains unclear in the absence of the atomic structure of the complex. Here, we present the cryo-EM structures of HIV-1 Vif in complex with human A3H, CBF-ß and components of CUL5 ubiquitin ligase (CUL5, ELOB, and ELOC). Vif nucleates the entire complex by directly binding four human proteins, A3H, CBF-ß, CUL5, and ELOC. The structures reveal a large interface area between A3H and Vif, primarily mediated by an α-helical side of A3H and a five-stranded ß-sheet of Vif. This A3H-Vif interface unveils the basis for sensitivity-modulating polymorphism of both proteins, including a previously reported gain-of-function mutation in Vif isolated from HIV/AIDS patients. Our structural and functional results provide insights into the remarkable interplay between HIV and humans and would inform development efforts for anti-HIV therapeutics.