Transcriptome and metabolome analyses of Streptococcus gordonii DL1 under acidic conditions.

OBJECTIVES: Streptococcus gordonii is associated with the formation of biofilms, especially those that comprise dental plaque. Notably, S. gordonii DL1 causes infective endocarditis (IE). Colonization of this bacterium requires a mechanism that can tolerate a drop in environmental pH by producing acid via its own sugar metabolism. The ability to survive acidic environmental conditions might allow the bacterium to establish vegetative colonization even in the endocardium due to inflammation-induced lowering of pH, increasing the risk of IE. At present, the mechanism by which S. gordonii DL1 survives under acidic conditions is not thoroughly elucidated. The present study was thus conducted to elucidate the mechanism(s) by which S. gordonii DL1 survives under acidic conditions. METHODS: We analyzed dynamic changes in gene transcription and intracellular metabolites in S. gordonii DL1 exposed to acidic conditions, using transcriptome and metabolome analyses. RESULTS: Transcriptome analysis revealed upregulation of genes involved in heat shock response and glycolysis, and down regulation of genes involved in phosphotransferase systems and biosynthesis of amino acids. The most upregulated genes were a beta-strand repeat protein of unknown function (SGO_RS06325), followed by copper-translocating P-type ATPase (SGO_RS09470) and malic enzyme (SGO_RS01850). The latter two of these contribute to cytoplasmic alkalinization. S. gordonii mutant strains lacking each of these genes showed significantly reduced survival under acidic conditions. Metabolome analysis revealed that cytoplasmic levels of several amino acids were reduced. CONCLUSIONS: S. gordonii survives the acidic conditions by recovering the acidic cytoplasm using the various activities, which are regulated at the transcriptional level.

Phylloquinone is preferable over menadione as a growth factor for Porphyromonas gingivalis.

OBJECTIVES: Porphyromonas gingivalis is the etiological agent of chronic periodontitis. Menadione (vitamin K3) and phylloquinone (vitamin K1) are well-known growth factors for P. gingivalis, while menadione is widely used in growth experiments. Here we attempted to determine the differences in phylloquinone and menadione in P. gingivalis growth experiments, which have not been well studied to date. METHODS: We investigated the effects of menadione and phylloquinone on the growth of two W83 strains and seven ATCC 33277 strains of P. gingivalis. RESULTS: The ATCC 33277 strains grew well with phylloquinone at 2.9 µM in a complex medium (nutrient medium) and at 29 µM in two minimal media. In contrast, the W83 strains grew well without menadione or phylloquinone in three different culture media. Menadione at 2.9 µM, the conventionally used concentration for culturing P. gingivalis, supported the growth of most ATCC 33277 strains but inhibited the growth of some W83 and ATCC 33277 strains. Furthermore, menadione at 14.5 µM frequently inhibited cell growth, while phylloquinone at 145 µM promoted cell growth. CONCLUSIONS: These results indicate that menadione and phylloquinone act as growth factors for ATCC 33277 but that menadione also can inhibit P. gingivalis growth. Thus, we propose that phylloquinone be used instead of menadione in P. gingivalis growth experiments requiring vitamin K.

Relationship Between Nonsmoothness in Adversarial Training, Constraints of Attacks, and Flatness in the Input Space.

Adversarial training (AT) is a promising method to improve the robustness against adversarial attacks. However, its performance is not still satisfactory in practice compared with standard training. To reveal the cause of the difficulty of AT, we analyze the smoothness of the loss function in AT, which determines the training performance. We reveal that nonsmoothness is caused by the constraint of adversarial attacks and depends on the type of constraint. Specifically, the L∞ constraint can cause nonsmoothness more than the L2 constraint. In addition, we found an interesting property for AT: the flatter loss surface in the input space tends to have the less smooth adversarial loss surface in the parameter space. To confirm that the nonsmoothness causes the poor performance of AT, we theoretically and experimentally show that smooth adversarial loss by EntropySGD (EnSGD) improves the performance of AT.

Intraplaque wiring enables drug-coated balloons to be utilized for percutaneous recanalization of chronically occluded coronary arteries.

OBJECTIVES: This study aimed to determine whether drug-coated balloon (DCB) angioplasty following intraplaque wiring and the use of modified balloons is safe and effective in the percutaneous treatment of coronary chronic total occlusions (CTOs). BACKGROUND: DCB is an alternative therapeutic option without the limitations of permanent vascular implants. However, its efficacy in CTOs has yet to be confirmed. The combination of modified balloons and DCB can be effectively applied when the intraplaque passage of the guidewire is achieved in CTOs. METHODS: Data from 124 consecutive CTO lesions (105 patients) treated at our hospital between February 2016 and December 2020 were screened for inclusion and retrospectively analyzed. Among the 118 lesions successfully recanalized, intraplaque wiring was achieved in 108, and 85 were treated by the DCB-only approach following cutting/scoring balloon dilatation. RESULTS: Follow-up data were available for 82 lesions (71 patients). The median occlusion length was 18.5 mm, and the J-CTO score was 1.7 ± 0.9. No in-hospital major adverse cardiac events occurred, including abrupt vessel closure. During the median 29-month follow-up period, target lesion revascularization was performed for 10 lesions. Follow-up coronary angiography (8.7 ± 3.9 months after the index procedure) was performed for 64 lesions, demonstrating late lumen loss of -0.15 mm (interquartile range -0.4 to 0.23 mm), binary restenosis (diameter stenosis ≥50%) in 12 lesions (18.8%), and late lumen enlargement in 37 (57.8%). CONCLUSION: The DCB-only approach following the use of modified balloons is a promising strategy for coronary CTOs when intraplaque wiring is achieved.

Analysis of sagittal thoracolumbar spine and hip joint movements during sit-to-stand using a 2D image analysis freeware.

[Purpose] This study aimed to verify the utility of an image analysis freeware in the evaluation of thoracolumbar spine and hip joint movements during sit-to-stand movement and show the importance of separately analyzing the movements of the thoracolumbar spine and the hip joint. [Participants and Methods] We used a two-dimensional image analysis freeware to analyze the kinematics of the thoracolumbar spine and the hip joint during sit-to-stand movements in seven healthy young males. We further examined the usefulness of the freeware by verifying the concordance of its angle measurements with those of a three-dimensional motion analysis device. Moreover, we evaluated joint coordination of the thoracolumbar spine with hip joint movements in pregnant female before and after delivery by measuring the relative phase angle. [Results] The trunk angle and relative phase angle between the thoracolumbar spine and the hip joint obtained using the two different analytical methods were fairly consistent. In the analysis of the pregnant female, the degree of thoracolumbar flexion prior to hip flexion tended to decrease. Similarly, the degree of hip extension tended to decrease during pregnancy. [Conclusion] This study shows that a two-dimensional image analysis freeware could be useful and meaningful in the calculation of thoracolumbar spine and hip joint movements and in the detection of synergistic patterns of these entities during sit-to-stand movement.

Calcium ions and vitamin B12 are growth factors for Porphyromonas gingivalis.

BACKGROUND: Porphyromonas gingivalis is a causative agent of chronic periodontitis. Standard strains of P. gingivalis, such as W83 and ATCC 33277, proliferate in minimal medium when protein is added as the energy source and hemin and menadione are added as growth factors. Nevertheless, minimal medium containing bovine serum albumin sometimes fails to support growth. HIGHLIGHTS: The proliferation of two W83 strains and seven ATCC 33277 strains in various minimal media was investigated. Previously, we determined that calcium chloride (CaCl2) was a growth factor for W83NM, a W83 strain. In this study, we found that vitamin B12 enhanced the proliferation of W83NM in a minimal medium with cultures from the fourth passage but not from the first to the third passage. Therefore, using fourth-passage cultures, we assessed the proliferation of two W83 and seven ATCC 33277 strains in minimal media and the effects of CaCl2 and vitamin B12. Surprisingly, the nine P. gingivalis strains all differed with respect to their proliferation in minimal media, and protein products used as energy sources showed product-to-product and lot-to-lot heterogeneity. Even though strains or protein products were different, we found CaCl2-dependent growth in nine strains and vitamin B12-dependent growth in seven strains. CONCLUSION: These results suggest that calcium ions and vitamin B12 are novel growth factors for P. gingivalis.

Regulation of constant cell elongation and Sfm pili synthesis in Escherichia coli via two active forms of FimZ orphan response regulator.

Escherichia coli (E. coli) has multiple copies of the chaperone-usher (CU) pili operon in five fimbria groups: CU pili, curli, type IV pili, type III secretion pili, and type IV secretion pili. Commensal E. coli K-12 contains 12 CU pili operons. Among these operons, Sfm is expressed by the sfmACDHF operon. Transcriptome analyses, reporter assays, and chromatin immunoprecipitation PCR analyses reported that FimZ directly binds to and activates the sfmA promoter, transcribing sfmACDHF. In addition, FimZ regularly induces constant cell elongation in E. coli, which is required for F-type ATPase function. The bacterial two-hybrid system showed a specific interaction between FimZ and the α subunit of the cytoplasmic F1 domain of F-type ATPase. Studies performed using mutated FimZs have revealed two active forms, I and II. Active form I is required for constant cell elongation involving amino acid residues K106 and D109. Active form II additionally required D56, a putative phosphorylation site, to activate the sfmA promoter. The chromosomal fimZ was hardly expressed in parent strain but functioned in phoB and phoP double-gene knockout strains. These insights may help to understand bacterial invasion restricted host environments by the sfm γ-type pili.

Functional analysis of human olfactory receptors with a high basal activity using LNCaP cell line.

Humans use a family of more than 400 olfactory receptors (ORs) to detect odorants. However, deorphanization of ORs is a critical issue because the functional properties of more than 80% of ORs remain unknown, thus, hampering our understanding of the relationship between receptor function and perception. HEK293 cells are the most commonly used heterologous expression system to determine the function of a given OR; however, they cannot functionally express a majority of ORs probably due to a lack of factor(s) required in cells in which ORs function endogenously. Interestingly, ORs have been known to be expressed in a variety of cells outside the nose and play critical physiological roles. These findings prompted us to test the capacity of cells to functionally express a specific repertoire of ORs. In this study, we selected three cell lines that endogenously express functional ORs. We demonstrated that human prostate carcinoma (LNCaP) cell lines successfully identified novel ligands for ORs that were not recognized when expressed in HEK293 cells. Further experiments suggested that the LNCaP cell line was effective for functional expression of ORs, especially with a high basal activity, which impeded the sensitive detection of ligand-mediated activity of ORs. This report provides an efficient functional assay system for a specific repertoire of ORs that cannot be characterized in current cell systems.

Hemagglutinating properties of a Streptococcus gordonii strain expressing sialic acid-binding adhesin homolog with low binding site similarity to that of strain DL1.

OBJECTIVES: The Hsa adhesin of Streptococcus gordonii strain DL1 was previously identified as a hemagglutinin that binds specifically to sialoglycoconjugates. We recently found that among oral streptococcal species, S. gordonii strains most frequently express Hsa homologs on the bacterial cell surface. However, the effect of amino acid sequence diversity of nonrepetitive region 2 (NR2), a putative binding site of Hsa, on antigenicity and hemagglutinating (HA) properties is unclear due to difficulties in DNA sequencing the NR2 coding region. The aim of this study was to elucidate the similarity of the low NR2 antigenicity Hsa homolog of strain NDU1118 to that of strain DL1 and the association of the homolog with HA properties of the strain. METHODS: The hsa homolog of NDU1118 was sequenced using a long-read next-generation sequencer, and the Hsa homolog was assessed by alignment analysis of the deduced amino acid sequences. The hsa mutant of NDU1118 was generated by insertion of the erythromycin resistance gene. The HA properties of the wild type and the hsa mutant were assessed with human erythrocytes. RESULTS: The NR2 amino acid sequence of the NDU1118 Hsa homolog was almost identical to that of the S. gordonii M99 Hsa homolog, also known as GspB, and less similar to that of DL1 Hsa. The hsa mutation of NDU1118 induced reduction of HA activity in untreated erythrocytes, but surprisingly increased lactose-inhibitable HA activity in neuraminidase-treated erythrocytes. CONCLUSIONS: The results suggest the existence of an adhesin other than the Hsa homolog on the cell surface of NDU1118.

Laser vaporization of atherothrombotic burden before drug-coated balloon application in ST-segment elevation myocardial infarction: Two-year outcomes of the laser-DCB trial.

OBJECTIVES: This study aimed to examine whether the combination of excimer laser coronary atherectomy (ELCA) and drug-coated balloon (DCB) angioplasty can provide feasible clinical outcome in patients with ST-segment elevation myocardial infarction (STEMI) with 8-month and 2-year scheduled follow-up angiography. BACKGROUND: Intracoronary thrombus elevates the risk of interventional treatment in patients with STEMI and hampers drug absorption into the vasculature released from DCB. METHODS: Sixty-two patients with STEMI within 24 h after the onset of symptoms were enrolled in this prospective, single-center, single-arm study. RESULTS: The laser catheter was successfully crossed distal to the culprit lesion in all cases. No ELCA-related adverse events occurred. Bail-out stenting was required in two patients (3.2%) after adjunctive ballooning; thus, the remaining 60 patients were completed with DCB angioplasty without stenting. Scheduled angiography at 8 months and 2 years was completed in 100% and 85.2%, respectively, and minimal lumen diameters were 3.4 ± 0.5, 3.4 ± 0.6, and 3.4 ± 0.5 mm after the procedure, at 8 months and at 2 years, respectively. Binary restenosis was observed in five patients (8.1%) in whom target lesion revascularization was performed. The duration of dual antiplatelet therapy was 2.3 ± 2.2 months, and neither abrupt vessel closure, reinfarction, cardiac death nor major bleeding was observed. CONCLUSION: A combination of DCB angioplasty with ELCA is a feasible therapeutic option for STEMI.

Expression and diversity of the sialic acid-binding adhesin and its homologs associated with oral streptococcal infection.

Streptococcus gordonii, one of the early colonizers of oral biofilms, is involved in the development of dental caries, periodontal disease, and infective endocarditis. The Hsa adhesin of S. gordonii DL1 has the ability to bind strongly to the terminal sialic acid groups of host glycoproteins via the binding region, nonrepetitive region 2 (NR2), which is important for the pathogenicity of S. gordonii DL1. Low similarity with the NR2 of Hsa homologs among other streptococcal species has been reported. However, the reports have been limited to certain strains. This study attempted to assess frequency of the expression on the bacterial cell surface and to analyze the diversity of Hsa homologs among different wild strains of oral streptococci. We isolated 186 wild-type strains of oral streptococci from healthy volunteers and analyzed their hemagglutinating (HA) activity on human erythrocytes and their Hsa homologs and NR2 homologous regions by dot immunoblotting using anti-Hsa and anti-NR2 antisera, respectively. We found 30 strains reacted with anti-NR2 antiserum (NR2 positive) and determined the sequence of the NR2 regions. Many strains with high HA activity were also NR2 positive, suggesting that the NR2 region may be associated with HA activity. Among the NR2-positive strains, four different amino acid sequence patterns were observed, demonstrating diversity in the NR2 region. Notably, S. gordonii strains frequently possessed Hsa homologs and NR2-like antigens compared with other streptococci. It is speculated that the possessing frequency of Hsa homologs and the amino acid sequence of NR2 region may vary among streptococcal species.

The hdeD Gene Represses the Expression of Flagellum Biosynthesis via LrhA in Escherichia coli K-12.

Escherichia coli survives under acid stress conditions by the glutamic acid-dependent acid resistance (GAD) system, which enzymatically decreases intracellular protons. We found a linkage between GAD and flagellar systems in E. coli. The hdeD gene, one of the GAD cluster genes, encodes an uncharacterized membrane protein. A reporter assay showed that the hdeD promoter was induced in a GadE-dependent manner when grown in the M9 glycerol medium. Transcriptome analysis revealed that most of the transcripts were from genes involved in flagellum synthesis, and cell motility increased not only in the hdeD-deficient mutant but also in the gadE-deficient mutant. Defects in both the hdeD and gadE increased the intracellular level of FliA, an alternative sigma factor for flagellum synthesis, activated by the master regulator FlhDC. The promoter activity of the lrhA gene, which encodes repressor for the flhDC operon, was found to decrease in both the hdeD- and gadE-deficient mutants. Transmission electron microscopy showed that the number of flagellar filaments on the hdeD-, gadE-, and lrhA-deficient cells increased, and all three mutants showed higher motility than the parent strain. Thus, HdeD in the GAD system activates the lrhA promoter, resulting in a decrease in flagellar filaments in E. coli cells. We speculated that the synthesis of HdeD, stimulated in E. coli exposed to acid stress, could control the flagellum biosynthesis by sensing slight changes in pH at the cytoplasmic membrane. This could help in saving energy through termination of flagellum biosynthesis and improve bacterial survival efficiency within the animal digestive system. IMPORTANCE E. coli cells encounter various environments from the mouth down to the intestines within the host animals. The pH of gastric juice is lower than 2.0, and the bacterial must quickly respond and adapt to the following environmental changes before reaching the intestines. The quick response plays a role in cellular survival in the population, whereas adaptation may contribute to species survival. The GAD and flagellar systems are important for response to low pH in E. coli. Here, we identified the novel inner membrane regulator HdeD, encoding in the GAD cluster, to repress the synthesis of flagella. These insights provide a deeper understanding of how the bacteria enter the animal digestive system, survive, and form colonies in the intestines.

Streptococcus gordonii DL1 evades polymorphonuclear leukocyte-mediated killing via resistance to lysozyme.

Streptococcus gordonii is an etiological bacterial agent of infective endocarditis. Although the pathogenesis mechanisms are not well understood, the interaction between streptococci and phagocytes is considered important for the development of infective endocarditis. Previous studies show that some S. gordonii strains, including DL1, survive in polymorphonuclear leukocytes (PMNs), whereas other strains such as SK12 are sensitive to PMN-dependent killing. In this study, we assessed the differences between the sensitivity of S. gordonii DL1 and S. gordonii SK12 to PMN-dependent killing. S. gordonii DL1 showed a higher survival when treated with PMNs than SK12. Both S. gordonii DL1 and S. gordonii SK12 showed high resistance to low pH condition. Compared to S. gordonii SK12, S. gordonii DL1 was sensitive to hydrogen peroxide. However, the resistance of S. gordonii DL1 to the tested bactericidal agents, especially lysozyme, was higher than that of SK12. Furthermore, we performed a bactericidal assay by treating a mixture of S. gordonii DL1 and SK12 with PMNs. S. gordonii DL1 did not enhance the survival of S. gordonii SK12 exposed to PMNs. These results indicated that S. gordonii DL1 is resistant to bactericidal agents that degrade bacteria in phagolysosomes. In addition, there was no secretory factor involved in the resistance to bactericidal agents. The findings of this study may help develop treatments for infective endocarditis caused by S. gordonii.

Laser Vaporization of Intracoronary Thrombus and Identifying Plaque Morphology in ST-Segment Elevation Myocardial Infarction as Assessed by Optical Coherence Tomography.

OBJECTIVES: We evaluated the thrombus-vaporizing effect of excimer laser coronary angioplasty (ELCA) in patients with ST-segment elevation myocardial infarction (STEMI) by optical coherence tomography (OCT). BACKGROUND: Larger intracoronary thrombus elevates the risk of interventional treatment and mortality in patients with STEMI. METHODS: A total of 92 patients with STEMI who presented within 24 hours from the onset and underwent ELCA following manual aspiration thrombectomy (MT) were analyzed. RESULTS: The mean baseline thrombolysis in myocardial infarction flow grade was 0.4 ± 0.6, which subsequently improved to 2.3 ± 0.7 after MT (p < 0.0001) and 2.7 ± 0.5 after ELCA (p=0.0001). The median residual thrombus volume after MT was 65.7 mm3, which significantly reduced to 47.5 mm3 after ELCA (p < 0.0001). Plaque rupture was identified by OCT in only 22 cases (23.9%) after MT, but was distinguishable in 36 additional cases after ELCA (total: 58 cases; 63.0%). Ruptured lesions contained a higher proportion of red thrombus than nonruptured lesions (75.9% vs. 43.3%, p=0.001). Significantly larger thrombus burden after MT (69.6 mm3 vs. 56.3 mm3, p < 0.05) and greater thrombus reduction by ELCA (21.2 mm3 vs. 11.8 mm3, p < 0.01) were observed in ruptured lesions than nonruptured lesions. CONCLUSIONS: ELCA effectively vaporized intracoronary thrombus in patients with STEMI even after MT. Lesions with plaque rupture contained larger thrombus burden that was frequently characterized by red thrombus and more effectively reduced by ELCA.

Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors.

Type three secretion systems enable bacterial pathogens to inject effectors into the cytosol of eukaryotic hosts to reprogram cellular functions. It is technically challenging to label effectors and the secretion machinery without disrupting their structure/function. Herein, we present a new approach for labeling and visualization of previously intractable targets. Using genetic code expansion, we site-specifically labeled SsaP, the substrate specificity switch, and SifA, a here-to-fore unlabeled secreted effector. SsaP was secreted at later infection times; SsaP labeling demonstrated the stochasticity of injectisome and effector expression. SifA was labeled after secretion into host cells via fluorescent unnatural amino acids or non-fluorescent labels and a subsequent click reaction. We demonstrate the superiority of imaging after genetic code expansion compared to small molecule tags. It provides an alternative for labeling proteins that do not tolerate N- or C-terminal tags or fluorophores and thus is widely applicable to other secreted effectors and small proteins.

Role of Streptococcus intermedius phosphoglucosamine mutase in bacterial growth, cell morphology, and resistance to polymorphonuclear leukocyte killing.

OBJECTIVES: Streptococcus intermedius is a member of the anginosus group of streptococci, an oral commensal bacterium found in infected root canals, and the causative agent of deep-seated abscesses. This organism has slow clearance when phagocytosed within neutrophils. Here, we investigated the role of its phosphoglucosamine mutase (GlmM), an enzyme associated with peptidoglycan synthesis, in bacterial growth, cell morphology, and resistance to polymorphonuclear leukocyte killing. METHODS: The glmM-deletion (ΔglmM) mutant and the plasmid-borne complementation (ΔglmM/glmM) strain of S. intermedius were generated. The wild type, the ΔglmM mutant, and the ΔglmM/glmM strain were phagocytosed with human polymorphonuclear leukocytes (PMNs), and bacterial viability in PMNs was determined by LIVE/DEAD staining. Additionally, bacterial growth and cell morphology were also compared. RESULTS: The survival rate of the ΔglmM mutant was significant lower than that of the wild type. Although the difference in the survival rate of the ΔglmM/glmM strain compared to that of the wild type or the ΔglmM mutant was not significant, the rate appeared to be restored to the middle level. Compared to the wild type and the ΔglmM/glmM strain, the ΔglmM mutant showed reduced growth potential, a significant increase in the number of bacterial chains, and heterogeneous bacteria. CONCLUSIONS: GlmM is one of the factors responsible for the stable resistance of S. intermedius to clearance by PMNs.

Measurement of the Promoter Activity in Escherichia coli by Using a Luciferase Reporter.

The reporter system is widely used technique for measuring promoter activity in bacterial cells. Until now, a number of reporter system have been developed, but the bioluminescent reporter constructed from the bacterial luciferase genes is one of the useful systems for measuring in vivo dynamics of gene expression. The introduced bioluciferase lux reporter enables easy, fast, and sensitive measurement of the promoter activity without cell lysis because the substrates of bioluminescent reaction are synthesized inside the bacterial cell, thereby allowing low-cost experiments. This protocol describes a high throughput technique to measure the promoter activity in Escherichia coli K-12 using the lux reporter system.

Efficacy of Cutting Balloon Angioplasty for Chronic Total Occlusion of Femoropopliteal Arteries.

BACKGROUND: Chronic total occlusion (CTO) of femoropopliteal artery (FP) continues to be a lesion subset where maintaining long-term patency after endovascular treatment is challenging. We evaluated the efficacy of cutting balloon angioplasty (CBA) for de novo FP-CTOs in patients with symptomatic lower limb ischemia. METHODS: Seventy-three limbs of 67 symptomatic patients with de novo FP-CTOs successfully recanalized using CBA alone were enrolled in this study. Primary patency was defined as the absence of recurrent symptoms and no deterioration of the ankle-brachial index (ABI) >0.10 from the immediate postinterventional value. RESULTS: The mean age was 73.5 ± 7.3 years, and 59.7% of patients had diabetes mellitus. Most lesions were classified as Trans-Atlantic Inter-Society Consensus II type C (n = 18; 24.7%) or type D (n = 44; 60.3%), with mean lesion and occluded lengths of 24.8 ± 11.4 and 17.8 ± 11.2 cm, respectively. No procedure-related adverse events occurred, except one distal embolization. The ABI significantly increased after intervention from 0.52 ± 0.12 to 0.80 ± 0.15 (P < 0.0001), with marked improvement in clinical symptoms (Rutherford stage: 2.7 ± 1.0 to 1.1 ± 1.2, P < 0.0001). The mean follow-up period was 31.2 ± 18.0 months, and the primary patency rates at 12 and 24 months were 75.3% and 60.6%, respectively. The independent predictive factors of failed patency were baseline hemoglobin A1c (P = 0.031, hazard radio [HR] 1.51 per 1%), occluded length ≥15 cm (P = 0.036, HR 2.90), and severe dissection (P = 0.033, HR 2.85). Vessel calcification and diameter did not affect primary patency. CONCLUSIONS: CBA is a feasible option for endovascular treatment of FP-CTOs. Diabetic status, occlusion length, and severe dissection after CBA are independent negative predictors of long-term patency.

Dimerization site 2 of the bacterial DNA-binding protein H-NS is required for gene silencing and stiffened nucleoprotein filament formation.

The bacterial nucleoid-associated protein H-NS is a DNA-binding protein, playing a major role in gene regulation. To regulate transcription, H-NS silences genes, including horizontally acquired foreign genes. Escherichia coli H-NS is 137 residues long and consists of two discrete and independent structural domains: an N-terminal oligomerization domain and a C-terminal DNA-binding domain, joined by a flexible linker. The N-terminal oligomerization domain is composed of two dimerization sites, dimerization sites 1 and 2, which are both required for H-NS oligomerization, but the exact role of dimerization site 2 in gene silencing is unclear. To this end, we constructed a whole set of single amino acid substitution variants spanning residues 2 to 137. Using a well-characterized H-NS target, the slp promoter of the glutamic acid-dependent acid resistance (GAD) cluster promoters, we screened for any variants defective in gene silencing. Focusing on the function of dimerization site 2, we analyzed four variants, I70C/I70A and L75C/L75A, which all could actively bind DNA but are defective in gene silencing. Atomic force microscopy analysis of DNA-H-NS complexes revealed that all of these four variants formed condensed complexes on DNA, whereas WT H-NS formed rigid and extended nucleoprotein filaments, a conformation required for gene silencing. Single-molecule stretching experiments confirmed that the four variants had lost the ability to form stiffened filaments. We conclude that dimerization site 2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing.

Altered Distribution of RNA Polymerase Lacking the Omega Subunit within the Prophages along the Escherichia coli K-12 Genome.

The RNA polymerase (RNAP) of Escherichia coli K-12 is a complex enzyme consisting of the core enzyme with the subunit structure α2ßß'ω and one of the σ subunits with promoter recognition properties. The smallest subunit, omega (the rpoZ gene product), participates in subunit assembly by supporting the folding of the largest subunit, ß', but its functional role remains unsolved except for its involvement in ppGpp binding and stringent response. As an initial approach for elucidation of its functional role, we performed in this study ChIP-chip (chromatin immunoprecipitation with microarray technology) analysis of wild-type and rpoZ-defective mutant strains. The altered distribution of RpoZ-defective RNAP was identified mostly within open reading frames, in particular, of the genes inside prophages. For the genes that exhibited increased or decreased distribution of RpoZ-defective RNAP, the level of transcripts increased or decreased, respectively, as detected by reverse transcription-quantitative PCR (qRT-PCR). In parallel, we analyzed, using genomic SELEX (systemic evolution of ligands by exponential enrichment), the distribution of constitutive promoters that are recognized by RNAP RpoD holoenzyme alone and of general silencer H-NS within prophages. Since all 10 prophages in E. coli K-12 carry only a small number of promoters, the altered occupancy of RpoZ-defective RNAP and of transcripts might represent transcription initiated from as-yet-unidentified host promoters. The genes that exhibited transcription enhanced by RpoZ-defective RNAP are located in the regions of low-level H-NS binding. By using phenotype microarray (PM) assay, alterations of some phenotypes were detected for the rpoZ-deleted mutant, indicating the involvement of RpoZ in regulation of some genes. Possible mechanisms of altered distribution of RNAP inside prophages are discussed. IMPORTANCE The 91-amino-acid-residue small-subunit omega (the rpoZ gene product) of Escherichia coli RNA polymerase plays a structural role in the formation of RNA polymerase (RNAP) as a chaperone in folding the largest subunit (ß', of 1,407 residues in length), but except for binding of the stringent signal ppGpp, little is known of its role in the control of RNAP function. After analysis of genomewide distribution of wild-type and RpoZ-defective RNAP by the ChIP-chip method, we found alteration of the RpoZ-defective RNAP inside open reading frames, in particular, of the genes within prophages. For a set of the genes that exhibited altered occupancy of the RpoZ-defective RNAP, transcription was found to be altered as observed by qRT-PCR assay. All the observations here described indicate the involvement of RpoZ in recognition of some of the prophage genes. This study advances understanding of not only the regulatory role of omega subunit in the functions of RNAP but also the regulatory interplay between prophages and the host E. coli for adjustment of cellular physiology to a variety of environments in nature.