CRISPR-Cas locus architecture in <i>H</i>. <i>cinaedi</i> PAGU597^T strain.

<p>CRISPR loci in <i>H</i>. <i>cinaedi</i>: (A) CRISPR1, (B) CRISPR2. CRISPR1 and CRISPR2 loci were present in <i>H</i>. <i>cinaedi</i> genomes (AP012492) in 1362505–1364592 and 1889862–1890162, respectively. The signature gene for each type is shown in red (<i>cas</i>9 and RAMP for Type II and III, respectively). The universal <i>cas</i>1 and <i>cas</i>2 genes are blue. Accessory genes are white. CRISPR loci are shown in green. The arrows indicate the directions of the coding sequences.</p

Diversity and microevolution of CRISPR loci in <i>Helicobacter cinaedi</i>

<div><p><i>Helicobacter cinaedi</i> is associated with nosocomial infections. The CRISPR-Cas system provides adaptive immunity against foreign genetic elements. We investigated the CRISPR-Cas system in <i>H</i>. <i>cinaedi</i> to assess the potential of the CRISPR-based microevolution of <i>H</i>. <i>cinaedi</i> strains. A genotyping method based on CRISPR spacer organization was carried out using 42 <i>H</i>. <i>cinaedi</i> strains. The results of sequence analysis showed that the <i>H</i>. <i>cinaedi</i> strains used in this study had two CRISPR loci (CRISPR1 and CRISPR2). The lengths of the consensus direct repeat sequences in CRISPR1 and CRISPR2 were both 36 bp-long, and 224 spacers were found in the 42 <i>H</i>. <i>cinaedi</i> strains. Analysis of the organization and sequence similarity of the spacers of the <i>H</i>. <i>cinaedi</i> strains showed that CRISPR arrays could be divided into 7 different genotypes. Each genotype had a different ancestral spacer, and spacer acquisition/deletion events occurred while isolates were spreading. Spacer polymorphisms of conserved arrays across the strains were instrumental for differentiating closely-related strains collected from the same hospital. MLST had little variability, while the CRISPR sequences showed remarkable diversity. Our data revealed the structural features of <i>H</i>. <i>cinaedi</i> CRISPR loci for the first time. CRISPR sequences constitute a valuable basis for genotyping, provide insights into the divergence and relatedness between closely-related strains, and reflect the microevolutionary process of <i>H</i>. <i>cinaedi</i>.</p></div

MLST analysis of 42 <i>H</i>. <i>cinaedi</i> isolates.

<p>MLST analysis of 42 <i>H</i>. <i>cinaedi</i> isolates.</p

Unweighted Pair Group Method with Arithmetic Averages (UPGMA) dendrogram derived by comparing the spacer patterns for CRISPR1 and CRISPR2 profiles from 42 <i>H</i>. <i>cinaedi</i> strains.

<p>The scale indicates the genetic distances calculated by UPGMA method. All sequences are labeled by strain number, hospital, and year of isolation in parentheses. CRISPR genotypes, CRISPR1 and CRISPR2 patterns, MLST sequence type, and MLST clonal complexes are indicated. PAGU1922 strain was assigned to an unknown ST, indicated in quotes. PAGU1930, PAGU1931, and PAGU1932 were also not assigned to any clonal complexes. Strains assigned to ST-3, ST-4, ST-8, and ST-16, which had identical sequences at all seven loci in each ST, had different spacer distributions in CRISPR analysis. In some clinically relevant strains (PAGU 611, 1294, 1703, 1708 and 1811), the spacer distribution of CRISPR1 differed, even though that of CRISPR2 was same, and vice versa.</p

CRISPR spacer content and polymorphisms in CRISPR2.

<p>The CRISPR2 arrays from 42 <i>H. cinaedi</i> strains are represented graphically. Identical spacers are shown as squares representing the same combination of numerals and letters, and are aligned so that they have same number (apart from duplicate spacers). Spacer numbering is initiated at the ancestral (right) end towards the most recently acquired spacers per strain (left).</p

Phylogenetic tree of 11 STs of <i>H</i>. <i>cinaedi</i> isolates using MLST analysis.

<p>Phylogenetic analysis was generated with the neighbor-joining method. Numbers at the nodes represent bootstrap values > 50% (obtained from 100 resamplings). All sequences are labeled by strain number, hospital, and year of isolation in parentheses. The colors represent the different hospitals. Bars: 0.001 substitutions per nucleotide position.</p

Structural Determination of the Nanocomplex of Borate with Styrene–Maleic Acid Copolymer-Conjugated Glucosamine Used as a Multifunctional Anticancer Drug

The development of effective anticancer drugs is essential for chemotherapy that specifically targets cancer tissues. We recently synthesized a multifunctional water-soluble anticancer polymer drug consisting of styrene–maleic acid copolymer (SMA) conjugated with glucosamine and boric acid (BA) (SGB complex). It demonstrated about 10 times higher tumor-selective accumulation compared with accumulation in normal tissues because of the enhanced permeability and retention effect, and it inhibited tumor growth via glycolysis inhibition, mitochondrial damage, and thermal neutron irradiation. Gaining insight into the anticancer effects of this SGB complex requires a determination of its structure. We therefore investigated the chemical structure of the SGB complex by means of nuclear magnetic resonance, infrared (IR) spectroscopy, and liquid chromatography–mass spectrometry. To establish the chemical structure of the SGB complex, we synthesized a simple model compoundmaleic acid–glucosamine (MAG) conjugateby using a maleic anhydride (MA) monomer unit instead of the SMA polymer. We obtained two MAG–BA complexes (MAGB) with molecular weights of 325 and 343 after the MAG reaction with BA. We confirmed, by using IR spectroscopy, that MAGB formed a stable complex via an amide bond between MA and glucosamine and that BA bound to glucosamine via a diol bond. As a result of this chemical design, identified via analysis of MAGB, the SGB complex can release BA and demonstrate toxicity to cancer cells through inhibition of lactate secretion in mild hypoxia that mimics the tumor microenvironment. For clinical application of the SGB complex, we confirmed that this complex is stable in the presence of serum. These findings confirm that our design of the SGB complex has various advantages in targeting solid cancers and exerting therapeutic effects when combined with neutron irradiation

Reactive Sulfur Species-Mediated Activation of the Keap1–Nrf2 Pathway by 1,2-Naphthoquinone through Sulfenic Acids Formation under Oxidative Stress

Sulfhydration by a hydrogen sulfide anion and electrophile thiolation by reactive sulfur species (RSS) such as persulfides/polysulfides (e.g., R-S-SH/R-S-S_n-H­(R)) are unique reactions in electrophilic signaling. Using 1,2-dihydroxynaphthalene-4-thioacetate (1,2-NQH₂-SAc) as a precursor to 1,2-dihydroxynaphthalene-4-thiol (1,2-NQH₂-SH) and a generator of reactive oxygen species (ROS), we demonstrate that protein thiols can be modified by a reactive sulfenic acid to form disulfide adducts that undergo rapid cleavage in the presence of glutathione (GSH). As expected, 1,2-NQH₂-SAc is rapidly hydrolyzed and partially oxidized to yield 1,2-NQ-SH, resulting in a redox cycling reaction that produces ROS through a chemical disproportionation reaction. The sulfenic acid forms of 1,2-NQ-SH and 1,2-NQH₂-SH were detected by derivatization experiments with dimedone. 1,2-NQH₂-SOH modified Keap1 at Cys171 to produce a Keap1-S-S-1,2-NQH₂ adduct. Subsequent exposure of A431 cells to 1,2-NQ or 1,2-NQH₂-SAc caused an extensive chemical modification of cellular proteins in both cases. Protein adduction by 1,2-NQ through a thio ether (C–S–C) bond slowly declined through a GSH-dependent S-transarylation reaction, whereas that originating from 1,2-NQH₂-SAc through a disulfide (C–S–S–C) bond was rapidly restored to the free protein thiol in the cells. Under these conditions, 1,2-NQH₂-SAc activated Nrf2 and upregulated its target genes, which were enhanced by pretreatment with buthionine sulfoximine (BSO), to deplete cellular GSH. Pretreatment of catalase conjugated with poly­(ethylene glycol) suppressed Nrf2 activation by 1,2-NQH₂-SAc. These results suggest that RSS-mediated reversible electrophilic signaling takes place through sulfenic acids formation under oxidative stress

8‑Nitro-cGMP Enhances SNARE Complex Formation through S‑Guanylation of Cys90 in SNAP25

Nitrated guanine nucleotide 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) generated by reactive oxygen/nitrogen species causes protein S-guanylation. However, the mechanism of 8-nitro-cGMP formation and its protein targets in the normal brain have not been identified. Here, we investigated 8-nitro-cGMP generation and protein S-guanylation in the rodent brain. Immunohistochemistry indicated that 8-nitro-cGMP was produced by neurons, such as pyramidal cells and interneurons. Using liquid chromatography-tandem mass spectrometry, we determined endogenous 8-nitro-cGMP levels in the brain as 2.92 ± 0.10 pmol/mg protein. Based on S-guanylation proteomics, we identified several S-guanylated neuronal proteins, including SNAP25 which is a core member of the soluble <i>N</i>-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex. SNAP25 post-translational modification including palmitoylation, phosphorylation, and oxidation, are known to regulate neurotransmission. Our results demonstrate that S-guanylation of SNAP25 enhanced the stability of the SNARE complex, which was further promoted by Ca²⁺-dependent activation of neuronal nitric oxide synthase. Using site-directed mutagenesis, we identified SNAP25 cysteine 90 as the main target of S-guanylation which enhanced the stability of the SNARE complex. The present study revealed a novel target of redox signaling via protein S-guanylation in the nervous system and provided the first substantial evidence of 8-nitro-cGMP function in the nervous system

Persistent Activation of cGMP-Dependent Protein Kinase by a Nitrated Cyclic Nucleotide via Site Specific Protein <i>S</i>‑Guanylation

8-Nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) is a nitrated derivative of guanosine 3′,5′-cyclic monophosphate (cGMP) formed endogenously under conditions associated with production of both reactive oxygen species and nitric oxide. It acts as an electrophilic second messenger in the regulation of cellular signaling by inducing a post-translational modification of redox-sensitive protein thiols via covalent adduction of cGMP moieties to protein thiols (protein <i>S</i>-guanylation). Here, we demonstrate that 8-nitro-cGMP potentially <i>S</i>-guanylates thiol groups of cGMP-dependent protein kinase (PKG), the enzyme that serves as one of the major receptor proteins for intracellular cGMP and controls a variety of cellular responses. <i>S</i>-Guanylation of PKG was found to occur in a site specific manner; Cys42 and Cys195 were the susceptible residues among 11 Cys residues. Importantly, <i>S</i>-guanylation at Cys195, which is located in the high-affinity cGMP binding domain of PKG, causes persistent enzyme activation as determined by <i>in vitro</i> kinase assay as well as by an organ bath assay. <i>In vivo</i>, <i>S</i>-guanylation of PKG was demonstrated to occur in mice without any specific treatment and was significantly enhanced by lipopolysaccharide administration. These findings warrant further investigation in terms of the physiological and pathophysiological roles of <i>S</i>-guanylation-dependent persistent PKG activation