Genomic SELEX Screening of Regulatory Targets of Transcription Factors.

The genome of Escherichia coli K-12 is transcribed by a single species of RNA polymerase. The selectivity of transcriptional targets is determined via interaction with one of seven species of the sigma subunit and a total of approximately 300 species of transcription factor (TFs). For comprehensive identification of the regulatory targets of these two groups of regulatory proteins on the genome, we developed an in vitro approach, "Genomic SELEX" (gSELEX) screening. Here we describe a detailed protocol of the gSELEX screening system, which uses purified regulatory proteins and fragments of genomic DNA from E. coli. Moreover, we describe methods and examples of results using cell-free synthetic proteins.

CsgI (YccT) Is a Novel Inhibitor of Curli Fimbriae Formation in Escherichia coli Preventing CsgA Polymerization and Curli Gene Expression.

Curli fimbriae are amyloids-found in bacteria (Escherichia coli)-that are involved in solid-surface adhesion and bacterial aggregation during biofilm formation. The curli protein CsgA is coded by a csgBAC operon gene, and the transcription factor CsgD is essential to induce its curli protein expression. However, the complete mechanism underlying curli fimbriae formation requires elucidation. Herein, we noted that curli fimbriae formation was inhibited by yccT-i.e., a gene that encodes a periplasmic protein of unknown function regulated by CsgD. Furthermore, curli fimbriae formation was strongly repressed by CsgD overexpression caused by a multicopy plasmid in BW25113-the non-cellulose-producing strain. YccT deficiency prevented these CsgD effects. YccT overexpression led to intracellular YccT accumulation and reduced CsgA expression. These effects were addressed by deleting the N-terminal signal peptide of YccT. Localization, gene expression, and phenotypic analyses revealed that YccT-dependent inhibition of curli fimbriae formation and curli protein expression was mediated by the two-component regulatory system EnvZ/OmpR. Purified YccT inhibited CsgA polymerization; however, no intracytoplasmic interaction between YccT and CsgA was detected. Thus, YccT-renamed CsgI (curli synthesis inhibitor)-is a novel inhibitor of curli fimbriae formation and has a dual role as an OmpR phosphorylation modulator and CsgA polymerization inhibitor.

Roles of methionine and cysteine residues of the Escherichia coli sensor kinase HprS in reactive chlorine species sensing.

Sensor histidine kinase HprS, an oxidative stress sensor of Escherichia coli, senses reactive oxygen species (ROS) and reactive chlorine species (RCS), and is involved in the induction of oxidatively damaged protein repair periplasmic enzymes. We reinvestigated the roles of six methionine and four cysteine residues of HprS in the response to HClO, an RCS. The results of site-directed mutagenesis revealed that methionine residues in periplasmic and cytoplasmic regions (Met225) are involved in HprS activation. Interestingly, the Cys165Ser substitution reduced HprS activity, which was recovered by an additional Glu22Cys substitution. Our results demonstrate that the position of the inner membrane cysteine residues influences the extent of HprS activation in HClO sensing.

Case Report: A Very Low Birth Weight Female Infant With Congenital Bilateral Periventricular Leukomalacia, Born to a Mother With Coronavirus Disease 2019.

A 26-year-old primipara woman with COVID-19 performed an emergency Cesarean section due to further hypoxemia at 28 weeks 5/7 days gestation. The female neonate was born weighing 1,347 gram with an Apgar score of four at 1 min, three at 5 min, and eight at 10 min. RT-PCR from nasopharyngeal swabs for COVID-19 were performed at birth, 24 h, and 48 h after birth, all of which were negative. On head ultrasound bilateral cystic lesions were found in the anterior horn of the lateral ventricles at birth. A brain magnetic resonance imaging (MRI) test at 56 days of life (corrected 36 weeks and 6/7 days) revealed cystic lesions with T1 low signal, T2 high signal, and T2 Flair high signal around the anterior horn of the lateral ventricle and We diagnose it as Grade 2 periventricular leukomalacia (PVL). She was discharged on day 64 of life, with no abnormality on exam. While the majority of neonates born to women with COVID-19 during pregnancy have favorable outcome, we report a case of a neonate with Grade 2 periventricular leukomalacia and this should prompt clinicians to monitor fetal cerebral function and structure shortly after birth.

Single-Target Regulators Constitute the Minority Group of Transcription Factors in Escherichia coli K-12.

The identification of regulatory targets of all transcription factors (TFs) is critical for understanding the entire network of genome regulation. A total of approximately 300 TFs exist in the model prokaryote Escherichia coli K-12, but the identification of whole sets of their direct targets is impossible with use of in vivo approaches. For this end, the most direct and quick approach is to identify the TF-binding sites in vitro on the genome. We then developed and utilized the gSELEX screening system in vitro for identification of more than 150 E. coli TF-binding sites along the E. coli genome. Based on the number of predicted regulatory targets, we classified E. coli K-12 TFs into four groups, altogether forming a hierarchy ranging from a single-target TF (ST-TF) to local TFs, global TFs, and nucleoid-associated TFs controlling as many as 1,000 targets. Using the collection of purified TFs and a library of genome DNA segments from a single and the same E. coli K-12, we identified here a total of 11 novel ST-TFs, CsqR, CusR, HprR, NorR, PepA, PutA, QseA, RspR, UvrY, ZraR, and YqhC. The regulation of single-target promoters was analyzed in details for the hitherto uncharacterized QseA and RspR. In most cases, the ST-TF gene and its regulatory target genes are adjacently located on the E. coli K-12 genome, implying their simultaneous transfer in the course of genome evolution. The newly identified 11 ST-TFs and the total of 13 hitherto identified altogether constitute the minority group of TFs in E. coli K-12.

[A Case of Immediately-Treated Internal Hernia Occurring during Chemotherapy for Liver Metastasis after Gastric GIST Resection].

A 71-year-old man underwent total gastrectomy with Roux-en-Y reconstruction for gastric GIST in October 2017. Liver metastasis was identified in June 2019, and chemotherapy with imatinib was started in July. In December, the patient presented with acute upper abdominal pain and back pain. Abdominal contrast-enhanced CT showed that the jejunum extending from the duodenal stump was dilated. In addition, part of the jejunum had a poor wall contrast effect, with ascites also found surrounding it. We suspected a strangulated ileus and immediately performed emergency surgery. We found an internal hernia with incarceration of the afferent loop at the Petersen's defect. The time from the onset of symptoms to the surgery was relatively short, and the surgery was completed with hernial repair and closure of the hernial orifice without the development of bowel necrosis; the patient's postoperative course was good. Although the frequency of internal hernia after gastrectomy is relatively low, there is a risk that it may be severe if it occurs. Therefore, care should be taken to not cause internal hernias during surgery, and an internal hernia should be considered in the event of sudden abdominal pain after gastric surgery.

Novel regulators of the csgD gene encoding the master regulator of biofilm formation in Escherichia coli K-12.

Under stressful conditions, Escherichia coli forms biofilm for survival by sensing a variety of environmental conditions. CsgD, the master regulator of biofilm formation, controls cell aggregation by directly regulating the synthesis of Curli fimbriae. In agreement of its regulatory role, as many as 14 transcription factors (TFs) have so far been identified to participate in regulation of the csgD promoter, each monitoring a specific environmental condition or factor. In order to identify the whole set of TFs involved in this typical multi-factor promoter, we performed in this study 'promoter-specific transcription-factor' (PS-TF) screening in vitro using a set of 198 purified TFs (145 TFs with known functions and 53 hitherto uncharacterized TFs). A total of 48 TFs with strong binding to the csgD promoter probe were identified, including 35 known TFs and 13 uncharacterized TFs, referred to as Y-TFs. As an attempt to search for novel regulators, in this study we first analysed a total of seven Y-TFs, including YbiH, YdcI, YhjC, YiaJ, YiaU, YjgJ and YjiR. After analysis of curli fimbriae formation, LacZ-reporter assay, Northern-blot analysis and biofilm formation assay, we identified at least two novel regulators, repressor YiaJ (renamed PlaR) and activator YhjC (renamed RcdB), of the csgD promoter.

Cellulose acetate/multi-wall carbon nanotube/Ag nanofiber composite for antibacterial applications.

Herein we propose cellulose acetate/carbon nanotube/silver nanoparticles (CA/CNT/Ag) nanofiber composite for antibacterial applications. The nanofiber composite are expected to avoid harmful effects of silver (i.e. argyria and argyrosis) owing to anchoring of silver nanoparticles on carbon nanotubes (CNTs) and embedding of the composite inside cellulose acetate (CA) matrix. The carbon nanotubes/silver nanoparticles (CNT/Ag) nanocomposite localized inside the CA polymer matrix allow minimal/no direct contact of silver nanoparticles with human cells and are expected to show reduced silver leaching. The cellulose acetate (CA) nanofibers loaded with silver nanoparticles anchored multiwall carbon nanotubes (CNT/Ag) were fabricated by electrospinning. The samples were studied with scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), energy dispersive X-ray spectroscopy (EDS), transmission electron microscopy (TEM), Fourier transform infra-red spectroscopy (FTIR), tensile strength tests and antibacterial assays. Synthesis of the CNT/Ag nanocomposite was confirmed with XPS, XRD, EDS and TEM analysis. SEM images showed regular morphology of the CA/CNT/Ag nanofiber composites. TEM images depicted anchoring of silver nanoparticles on CNTs and embedding of CNT/Ag in the CA nanofiber matrix. The antibacterial test results demonstrated excellent antibacterial performance of the CA/CNT/Ag. The CA/CNT/Ag samples ensured effective bacterial growth inhibition on agar plates, in liquid medium (optical density, OD590nm) (for 48 h) and in bactericidal assay (relative cell viability, %). Our results suggested CA/CNT/Ag composite nanofibers as potential candidate for safer antibacterial applications.

Regulatory role of pyruvate-sensing BtsSR in biofilm formation by Escherichia coli K-12.

Pyruvate, the key regulator in connection of a variety of metabolic pathways, influences transcription of the Escherichia coli genome through controlling the activity of two pyruvate-sensing two-component systems (TCSs), BtsSR and PyrSR. Previously, we identified the whole set of regulatory targets of PyrSR with low-affinity to pyruvate. Using gSELEX screening system, we found here that BtsSR with high-affinity to pyruvate regulates more than 100 genes including as many as 13 transcription factors genes including the csgD gene encoding the master regulator of biofilm formation. CsgD regulates more than 20 target genes including the csg operons encoding the Curli fimbriae. In addition, we identified the csgBAC as one of the regulatory targets of BtsR, thus indicating the involvement of two pyruvate-dependent regulatory pathways of the curli formation: indirect regulation by CsgD; and direct regulation by BtsR. Based on the findings of the whole set of regulatory targets by two pyruvate-sensing BtsR and PyrR, we further propose an innovative concept that the pyruvate level-dependent regulation of different gene sets takes place through two pyruvate-sensing TCS systems, high-affinity BtsSR and low-affinity PyrSR to pyruvate.

Characterizations and application of CA/ZnO/AgNP composite nanofibers for sustained antibacterial properties.

Although silver based nanofibers possess excellent bactericidal and bacteriostatic characteristics. However, excess release/contact with silver may induce harmful side-effects including carcinoma, argyria, argyrosis and allergies. Similarly, silver depletion may limit prolonged antibacterial activities as well. Thus present research proposes electrospun CA/ZnO/AgNPs composite nanofibers for biologically safer and sustained antibacterial applications. The ZnO/AgNPs were synthesized using dopamine hydrochloride (Dopa) as reducing agent to immobilize AgNPs on ZnO nanoparticles. A simple solution-mixing procedure effectively generated AgNPs on ZnO nanoparticles. Strong adhesive characteristics of Dopa initiate adsorption of silver ions on ZnO nanoparticle surfaces and its metal ion reducing properties generate AgNPs. Additionally, the Dopa mediation generates strongly adhered AgNPs. The ZnO/AgNPs were used to fabricate CA/ZnO/AgNPs nanofibers. Characterization techniques, XRD, XPS, TEM, FTIR and SEM confirmed synthesis of nanocomposites. Crystallite sizes of ZnO and AgNPs calculated by Debye-Scherrer equation were 17.85 nm and 11.68 nm respectively. Antibacterial assays confirmed CA/ZnO/AgNP's effectiveness in growth inhibition of E. coli and S. aureus strains on agar plate and in liquid medium. The nanofiber composites demonstrated 100% bactericidal properties against both the test strains. Bacterial growth inhibition in LB medium for 108 h indicated suitability of CA/ZnO/AgNPs composite nanofibers in sustained antibacterial applications such as antibacterial wound dressings and other applications demanding sustained antimicrobial properties.

Genomic SELEX Screening of Regulatory Targets of Escherichia coli Transcription Factors.

The genome of Escherichia coli K-12 is transcribed by a single species of RNA polymerase. The selectivity of its transcriptional targets is modulated via two-steps of protein-protein interaction: at the first step, seven species of the sigma subunit are involved, at the second step, a total of approximately 300 species of transcription factor (TFs). For the identification of the regulatory targets of these two groups of regulatory proteins, we developed two in vitro approaches, "Genomic SELEX" (currently designated as gSELEX) and "PS (promoter-specific)-TF" screenings. Here, we describe a detailed protocol of the genomic SELEX screening system which uses purified regulatory proteins and fragments of genomic DNA from E. coli.

Single-target regulators form a minor group of transcription factors in Escherichia coli K-12.

The identification of regulatory targets of all TFs is critical for understanding the entire network of the genome regulation. The lac regulon of Escherichia coli K-12 W3110 is composed of the lacZYA operon and its repressor lacI gene, and has long been recognized as the seminal model of transcription regulation in bacteria with only one highly preferred target. After the Genomic SELEX screening in vitro of more than 200 transcription factors (TFs) from E. coli K-12, however, we found that most TFs regulate multiple target genes. With respect to the number of regulatory targets, a total of these 200 E. coli TFs form a hierarchy ranging from a single target to as many as 1000 targets. Here we focus a total of 13 single-target TFs, 9 known TFs (BetI, KdpE, LacI, MarR, NanR, RpiR, TorR, UlaR and UxuR) and 4 uncharacterized TFs (YagI, YbaO, YbiH and YeaM), altogether forming only a minor group of TFs in E. coli. These single-target TFs were classified into three groups based on their functional regulation.

Digestion of peptidoglycan near the cross-link is necessary for the growth of Bacillus subtilis.

Bacterial cells are covered with peptidoglycan (PG) layer(s), serving as the cellular exoskeleton. The PG sacculus changes its shape during cell growth, and thus both the synthesis and disassembly of PG are important for cell proliferation. In Bacillus subtilis, four dl-endopeptidases (DLEPases; LytE, LytF, CwlO and CwlS) are involved in the maintenance of cell morphology. The lytE cwlO double mutant exhibits synthetic lethality and defective cell elongation, while the lytE lytF cwlS triple mutant exhibits defective cell separation, albeit with septum formation. LytE is involved in both cell separation and elongation. We propose that DLEPases have varied roles in cell separation and elongation. To determine these roles, the catalytic domain of LytE was substituted with another catalytic domain that digests the other bonds in PG. By using the chimeric enzymes, we assessed the suppression of the synthetic lethality by the cell elongation defect and the disruption of chain morphology by the cell separation defect. All the constructed chimeric enzymes suppressed the cell separation defect, restoring the chain morphology. Digestion at any position of PG broke the linkage between two daughter cells, releasing them from each other. However, only d,d-endopeptidases suppressed the lack of DLEPase in the lytE cwlO double mutant. This indicated that the release of tension on the expanding PG sacculus is not the sole essential function of DLEPases. Considering that the structure of the digested PG is important for cell elongation, the digested product might be reused in the growth process in some way.

[The Impact of Neutrophil-Lymphocyte Ratio on the Prognosis in Patients Who Underwent Esophagectomy for Esophageal Cancer].

BACKGROUND: Thene utrophil-lymphocyteratio (NLR)reflects a patient's systemic inflammatory response. Several studies have revealed that the NLR is associated with a poor prognosis in several types of malignant tumors such as colorectal and lung cancer. The aim of this study was to evaluate the impact of preoperative NLR on the prognosis of patients with esophageal cancer. METHODS: The NLR was calculated for 93 consecutive patients with clinical Stage II or III esophageal cancer, who underwent curative esophagectomy following neoadjuvant chemotherapy between 2011 and 2013. The impact of preoperativeNLR on overall survival(OS)after esophagectomy was evaluated. The NLR cut off value was set to 2. RESULTS: The 3-year OS of patients with NLR≥2 was significantly shorter than patients with NLR<2(40.5% vs 67.9%, p=0.005). In a multivariateCox model, NLR≥2(HR: 2.342, 95%CI: 1.095-5.007, p=0.028), pathological depth of tumor(HR: 3.207, 95%CI: 1.114- 9.233, p=0.031), and an ageove r 60(HR: 2.342, 95%CI: 1.117-6.501, p=0.027)were identified as independent prognostic factors for OS after esophagectomy. CONCLUSIONS: The preoperative NLR was significantly associated with a poor prognosis in esophageal cancer patients who underwent curative esophagectomy following neoadjuvant chemotherapy.

A structural sketch of RcdA, a transcription factor controlling the master regulator of biofilm formation.

RcdA is a regulator of curlin subunit gene D, the master regulator of biofilm formation in Escherichia coli. Here, we determined the X-ray structure of RcdA at 2.55 Å resolution. RcdA consists of an N-terminal DNA-binding domain (DBD) containing a helix-turn-helix (HTH) motif and a C-terminal dimerization domain, and forms a homodimer in crystals. A computational docking model of the RcdA-DNA complex allowed prediction of the candidate residues responsible for DNA binding. Our structure-guided mutagenesis, in combination with gel shift assay, atomic force microscopic observation, and reporter assay, indicate that R32 in α2 of the HTH motif plays an essential role in the recognition and binding of target DNA while T46 in α3 influences the mode of oligomerization. These results provide insights into the DNA-binding mode of RcdA.

Cross-regulation between two common ancestral response regulators, HprR and CusR, in Escherichia coli.

The uncharacterized two-component system YedVW of Escherichia coli is involved in stress response to hydrogen peroxide. To identify the H2O2-sensing role of YedV, a set of single Cys-to-Ala substitution mutants were constructed. One particular mutant with C165A substitution in the membrane domain rendered YedV inactive in H2O2-dependent transcription of its regulatory target hiuH. We then proposed to rename YedVW to HprSR (hydrogen peroxide response sensor/regulator). One unique characteristic of HprR is the overlapping of its recognition sequence with that of the Cu(II)-response two-component system regulator CusR. Towards understanding this unique regulation system, in this study we analysed the interplay between HprR and CusR with respect to transcription of hiuH, a regulatory target of HprR, and cusC, a target of CusR. Under low protein concentrations in vitro and in vivo, two regulators recognize and transcribe both hiuH and cusC promoters, albeit at different efficiency, apparently in a collaborative fashion. This is a new type of transcription regulation of the common target genes in response to different external signals. Upon increase in protein concentrations, however, HprR and CusR compete with each other in transcription of the common targets, thereby exhibiting a competitive interplay.

[Laparoscopic Lateral Lymph Node Dissection for Rectal Cancer with Lateral Lymph Node Metastasis - A Case Report].

BACKGROUND: Lateral lymph node dissection(LLND)for locally advanced lower rectal cancer is the standard treatment procedure in Japan. We performed LLND with an extraperitoneal approach. Recently, we introduced laparoscopic surgery for locally advanced rectal cancer and laparoscopic LLND. We performed laparoscopic LLND in a patient havinglower rectal cancer with lateral lymph node metastasis that was detected via preoperative imaging. CASE PRESENTATION: The patient was a woman in her 50s who experienced melena and visited a physician. Colonoscopy revealed a tumor in the lower rectum and computed tomography showed lateral lymph node swelling and liver metastasis. The patient was referred to our institution and she was diagnosed with lower rectal cancer having lateral lymph node and synchronous liver metastases. We performed laparoscopic abdominoperineal resection and laparoscopic LLND. The operatingtime was 260 min, and the blood loss was 60g. CONCLUSION: The magnification of laparoscopy enables precision in the surgical operation of the narrow pelvis during lymph node dissection, allowingautonomic nerve preservation. Therefore, laparoscopic LLND is a helpful procedure in the treatment of locally advanced rectal cancer with a lateral lymph node metastasis.

Role of transcription factor NimR (YeaM) in sensitivity control of Escherichia coli to 2-nitroimidazole.

The binding site(s) on the Escherichia coli genome was determined for an uncharacterized AraC/XylS superfamily transcription factor YeaM by using the in vitro genomic SELEX screening system. The only one clear binding target of YeaM was found to locate in the spacer between the divergently transcribed yeaM and yeaN genes. After the phenotype microarray analysis, the major facilitator superfamily transporter YeaN was found to confer E. coli the resistance to 2-nitroimidazole, the antibacterial and antifungal antibiotic, suggesting that YeaN plays a role in 2-nitromidazole efflux. Purified YeaM bound to three sites within this yeaM-yeaN spacer region. Several lines of in vitro and in vivo evidence indicate that YeaM regulates transcription of both the yeaM gene itself and the yeaNO operon. Taken together we propose to rename yeaN to nimT (nitroimidazole transporter) and yeaM to nimR (regulator of nimT).

Cooperative regulation of the common target genes between H2O2-sensing YedVW and Cu²âº-sensing CusSR in Escherichia coli.

YedVW is one of the uncharacterized two-component systems (TCSs) of Escherichia coli. In order to identify the regulation targets of YedVW, we performed genomic SELEX (systematic evolution of ligands by exponential enrichment) screening using phosphorylated YedW and an E. coli DNA library, and identified YedW-binding sites within three intergenic spacers, yedW-hiuH, cyoA-ampG and cusR-cusC, along the E. coli genome. Using a reporter assay system, we found that transcription of hiuH, encoding 5-hydroxyisourate hydrolase, was induced at high concentrations of either Cu(2+) or H2O2. Cu(2+)-dependent expression of hiuH was observed in the yedWV knockout mutant, but was reduced markedly in the cusRS-null mutant. However, H2O2-induced hiuH expression was observed in the cusRS-null mutant, but not in the yedWV-null mutant. Gel mobility shift and DNase I footprinting analyses showed binding of both YedW and CusR to essentially the same sequence within the hiuH promoter region. Taken together, we concluded that YedVW and CusSR formed a unique cooperative TCS pair by recognizing and regulating the same targets, but under different environmental conditions - YedVW played a role in H2O2 response regulation, whilst CusSR played a role in Cu(2+) response regulation.

Ex vivo application of endocytoscopy for determining the longitudinal extent of bile duct cancer: a pathological analysis of 28 resected cases.

BACKGROUND: The aim was to examine the diagnostic ability of an endocytoscope system (ECS) for detecting the longitudinal extent of bile duct cancer. METHODS: We observed the surfaces of the bile duct of 28 consecutive patients undergoing biliary surgery both directly and under methylene blue staining by using an ECS. Based on the change in the microvascular caliber, abnormal vascularity, structural atypia, and cellular atypia, we created an ECS score. RESULTS: The ECS score produced a median value of 1 (range 0-3) point in normal bile ducts, 3 (0-6) points in cholangitis, 9 (1-10) points in carcinoma in situ (CIS), and 8 (3-11) points in invasive cancer. A significant difference in the scores between cancerous and non-cancerous lesions was found (P < 0.001). The ECS score was able to distinguish non-cancerous lesions from cancerous lesions using the cut-off value of a score less than 5, the accuracy being 92.1%, with a sensitivity of 91.1% and specificity of 93.5%. CONCLUSIONS: With ECS observation of bile duct specimens, the newly created ECS score was able to clearly distinguish non-cancerous lesions from cancerous lesions. This ECS score method should be validated in the differentiation of various BilIN lesions in the future.