Photorhabdus temperata strain M1021 is an entomopathogenic bacterium belonging to the family Enterobacteriaceae and is symbiotically associated with nematodes. The draft genome sequence of P. temperata strain M1021 consists of 5,598,253 bp with a G+C content of 43.7%, and it has 6,120 protein-coding genes.
Hematopoietic cells arise from spatiotemporally restricted domains in the developing embryo. Although studies of non-mammalian animal and in vitro embryonic stem cell models suggest a close relationship among cardiac, endocardial, and hematopoietic lineages, it remains unknown whether the mammalian heart tube serves as a hemogenic organ akin to the dorsal aorta. Here we examine the hemogenic activity of the developing endocardium. Mouse heart explants generate myeloid and erythroid colonies in the absence of circulation. Hemogenic activity arises from a subset of endocardial cells in the outflow cushion and atria earlier than in the aorta-gonad-mesonephros region, and is transient and definitive in nature. Interestingly, key cardiac transcription factors, Nkx2-5 and Isl1, are expressed in and required for the hemogenic population of the endocardium. Together, these data suggest that a subset of endocardial/endothelial cells expressing cardiac markers serve as a de novo source for transient definitive hematopoietic progenitors.
The preparation and characterization of nitric oxide (NO)-releasing silica particles formed following the synthesis of N-diazeniumdiolate-modified aminoalkoxysilanes are reported. Briefly, an aminoalkoxysilane solution was prepared by dissolving an appropriate amount of aminoalkoxysilane in a mixture of ethanol, methanol, and sodium methoxide (NaOMe) base. The silane solution was reacted with NO (5 atm) to form N-diazeniumdiolate NO donor moieties on the amino-alkoxysilanes. Tetraethoxy- or tetramethoxysilane (TEOS or TMOS) was then mixed with different ratios of N-diazeniumdiolate-modified aminoalkoxysilane (10 – 75 mol%, balance TEOS or TMOS). Finally, the silane mixture was added into ethanol in the presence of an ammonia catalyst to form NO donor silica nanoparticles via a sol–gel process. This synthetic approach allows for the preparation of NO delivery silica scaffolds with remarkably improved NO storage and release properties, surpassing all macromolecular NO donor systems reported to date with respect to NO payload (11.26μmol·mg−1), maximum NO release amount (357000 ppb·mg−1), NO release half-life (253 min), and NO release duration (101 h). The N-diazeniumdiolate-modified silane monomers and the resulting silica nanoparticles were characterized by 29Si nuclear magnetic resonance (NMR) spectroscopy, UV-visible spectroscopy, chemiluminescence, atomic force microscopy (AFM), gas adsorption-desorption isotherms, and elemental analysis.
The serum level of carcinoembryonic antigen (CEA) is a clinical prognostic factor in the follow-up evaluation of patients with colon cancer. We aimed to evaluate the prognostic significance of the rate of decrease of the perioperative serum CEA level in patients with colon cancer after a curative resection.
A total of 605 patients who underwent a curative resection for colon cancer between January 2000 and December 2007 were enrolled retrospectively. The rate of decrease was calculated using the following equation: ([preoperative CEA - postoperative CEA]/[preoperative CEA] ×100).
In the group with a preoperative serum CEA level of >5 ng/mL, the normalized group with a postoperative serum CEA level of ≤5 ng/mL showed a better overall survival (OS) rate and disease-free survival (DFS) rate than those of the non-normalized group (P ≤ 0.0001). The "cutoff values" of the rate of decrease in the perioperative serum CEA that determined the OS and the DFS were 48.9% and 50.8%, respectively. In the multivariate analysis of preoperative serum CEA levels >5 ng/mL, the prognostic factors for the OS and the DFS were the cutoff value (P < 0.0001) and the pN stage (P < 0.0001).
A rate of decrease of more than 50% in the perioperative serum CEA level, as well as the normalization of the postoperative serum CEA level, may be useful factors for determining a prognosis for colon cancer patients with high preoperative CEA levels.
Colon neoplasms; Carcinoembryonic antigen; Prognostic factor
The utility of nitric oxide (NO)-releasing silica nanoparticles as a novel antibacterial is demonstrated against Pseudomonas aeruginosa. Nitric oxide-releasing nanoparticles were prepared via co-condensation of tetraalkoxysilane with aminoalkoxysilane modified with diazeniumdiolate NO donors, allowing for the storage of large NO payloads. Comparison of the bactericidal efficacy of the NO-releasing nanoparticles to 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (PROLI/NO), a small molecule NO donor, demonstrated enhanced bactericidal efficacy of nanoparticle-derived NO and reduced cytotoxicity to healthy cells (mammalian fibroblasts). Confocal microscopy revealed that fluorescently-labeled NO-releasing nanoparticles associated with the bacteria, providing rationale for the enhanced bactericidal efficacy of the nanoparticles. Intracellular NO concentrations were measurable when the NO was delivered from nanoparticles as opposed to PROLI/NO. Collectively, these results demonstrate the advantage of delivering NO via nanoparticles for antimicrobial applications.
nitric oxide; silica nanoparticle; antibacterial; bactericidal; cytotoxicity; reactive nitrogen species; reactive oxygen species
Although the potent anti-tumor activity of nitric oxide (NO) supports its promise as an anti-neoplastic agent, effective and selective delivery and action on tumor and not normal cells remains a limiting factor. Nanoparticle-based delivery of NO has been considered as one approach to overcome these limitations. Therefore, we determined the utility of NO delivery using silica nanoparticles and evaluated their anti-tumor efficacy against human ovarian tumor and nontumor cells. The NO-releasing nanoparticles exhibited enhanced growth inhibition of ovarian tumor cells when compared to both control nanoparticles and a previously reported small molecule NO donor, PYRRO/NO. In addition, the NO-releasing nanoparticles showed greater inhibition of the anchorage-independent growth of tumor-derived and Ras-transformed ovarian cells. Confocal microscopy analysis revealed that fluorescently-labeled NO-releasing nanoparticles entered the cytosol of the cell and localized to late endosomes and lysosomes. Furthermore, we observed a nanoparticle size dependency on efficacy against normal versus transformed ovarian cells. Our study provides the first application of nanoparticle-derived NO as an antitumor therapy and supports the merit for future studies examining nanoparticle formulation for in vivo applications.
Nanoparticle; silica; nitric oxide; ovarian cancer; Ras
The important biological roles of nitric oxide (NO) have prompted the development of analytical techniques capable of sensitive and selective detection of NO. Electrochemical sensing, more than any other NO-detection method, embodies the parameters necessary for quantifying NO in challenging physiological environments such as blood and the brain. Herein, we provide a broad overview of the field of electrochemical NO sensors, including design, fabrication, and analytical performance characteristics. Both electrochemical sensors and biological applications are detailed.
Disease progression of ankylosing spondylitis has been considered irreversible. However, we report a case of spontaneous regression of syndesmophyte development following allogeneic peripheral blood stem cell transplantation in a patient with acute myeloid leukemia, who was also diagnosed as having ankylosing spondylitis. To the best of our knowledge, this is the first case report presenting the partial radiologic regression of syndesmophytes.
A 39-year-old man with active ankylosing spondylitis achieved clinical remission and partial radiological regression of cervical spine syndesmophytes following a peripheral blood stem cell transplantation for acute myeloid leukemia. Our patient received an allogeneic peripheral blood stem cell transplantation following a pre-transplantation conditioning regimen of total body irradiation and cyclophosphamide. The donor was a human leukocyte antigen-matched 29-year-old man. Our patient has remained asymptomatic and has received no medication for ankylosing spondylitis for nearly three years.
Several explanations are proposed for the regression of syndesmophytes and clinical improvement in active ankylosing spondylitis observed in our patient, including changes in bone remodeling and immune reconstitution following stem cell transplantation, the effect of immunosuppressive agents, or fluctuation in the natural course of ankylosing spondylitis although further studies are required. The regression of syndesmophytes in ankylosing spondylitis in this case raises the possibility that stem cell transplantation might contribute to the development of a novel therapeutic strategy for treatment of the disease.
A superhydrophobic xerogel coating synthesized from a mixture of nanostructured fluorinated silica colloids, fluoroalkoxysilane, and a backbone silane is reported. The resulting fluorinated surface was characterized using contact angle goniometry, SEM, and AFM. Quantitative bacterial adhesion studies performed using a parallel plate flow cell demonstrated that the adhesion of Staphylococcus aureus and Pseudomonas aeruginosa were reduced by 2.08 ± 0.25 and 1.76 ± 0.12 log over controls, respectively. This simple superhydrophobic coating synthesis may be applied to any surface regardless of geometry and does not require harsh synthesis or processing conditions, making it an ideal candidate as a biopassivation strategy.
This experimental study verified the effect of adipose-tissue-derived stem cells (ASCs) on the healing of ischemic colonic anastomoses in rats.
ASCs were isolated from the subcutaneous fat tissue of rats and identified as mesenchymal stem cells by identification of different potentials. An animal model of colonic ischemic anastomosis was induced by modifying Nagahata's method. Sixty male Sprague-Dawley rats (10-week-old, 370 ± 50 g) were divided into two groups (n = 30 each): a control group in which the anastomosis was sutured in a single layer with 6-0 polypropylene without any treatment and an ASCtreated group (ASC group) in which the anastomosis was sutured as in the control group, but then ASCs were locally transplanted into the bowel wall around the anastomosis. The rats were sacrificed on postoperative day 7. Healing of the anastomoses was assessed by measuring loss of body weight, wound infection, anastomotic leakage, mortality, adhesion formation, ileus, anastomotic stricture, anastomotic bursting pressure, histopathological features, and microvascular density.
No differences in wound infection, anastomotic leakage, or mortality between the two groups were observed. The ASC group had significantly more favorable anastomotic healing, including less body weight lost, less ileus, and fewer ulcers and strictures, than the control group. ASCs augmented bursting pressure and collagen deposition. The histopathological features were significantly more favorable in the ASC group, and microvascular density was significantly higher than it was in the control group.
Locally-transplanted ASCs enhanced healing of ischemic colonic anastomoses by increasing angiogenesis. ASCs could be a novel strategy for accelerating healing of colonic ischemic risk anastomoses.
Colonic anastomosis; Ischemia; Anastomotic healing; Adipose-tissue-derived stem cell; Angiogenesis
To report the clinical data and visual outcomes after treatment of patients with dysthyroid optic neuropathy (DON).
We retrospectively reviewed the medical records and orbital images of 40 patients (65 eyes) with DON and analyzed the visual outcomes after treatment with intravenous steroids pulse therapy, radiotherapy and orbital decompression.
The study included 21 men and 19 women, with 10 (25%) being diabetic patients. Visual field test results revealed defects in 88.7% of DON eyes; afferent pupillary defects in 63.2%; reduced color vision in 78.5%; and abnormal visual evoked potentials in 84%. Orbital imaging showed moderate to severe apical crowding in 95% of the orbits and intracranial fat prolapse in 24.2%. Median best corrected visual acuity improved from 0.4 to 1.0 after one year of treatment (p < 0.001). We noted more improvement in vision with the use of decompressive surgery than with non-surgical methods (p < 0.05). Recurrences occurred in 7 patients who had not received orbital radiotherapy.
Visual field defects and apical crowding seen on orbital imaging were the most sensitive indicators for the detection of DON. Treatment with intravenous steroids pulse therapy, radiotherapy and orbital decompression effectively improved visual outcomes in cases of DON.
Decompression; Optic neuropathy; Radiation; Steroid; Thyroid eye diseases
The aim of this study was to analyze the oncologic outcomes and the risk factors for recurrence after a tumor-specific mesorectal excision (TSME) of resectable rectal cancer in a single institution.
A total of 782 patients who underwent a TSME for resectable rectal cancer between February 1995 and December 2005 were enrolled retrospectively. Oncologic outcomes included 5-year cancer-specific survival and its affecting factors, as well as risk factors for local and systemic recurrence.
The 5-year cancer-specific survival rate was 77.53% with a mean follow-up period of 61 ± 31 months. The overall local and systemic recurrence rates were 9.2% and 21.1%, respectively. The risk factors for local recurrence were pN stage (P = 0.015), positive distal resection margin, and positive circumferential resection margin (P < 0.001). The risk factors for systemic recurrence were pN stage (P < 0.001) and preoperative carcinoembryonic antigen level (P = 0.005). The prognostic factors for cancer-specific survival were pT stage (P < 0.001), pN stage (P < 0.001), positive distal resection margin (P = 0.005), and positive circumferential resection margin (P = 0.016).
The oncologic outcomes in our institution after a TSME for patients with resectable rectal cancer were similar to those reported in other recent studies, and we established the risk factors that could be crucial for the planning of treatment and follow-up.
Rectal neoplasms; Colorectal cancer recurrence; Oncologic outcome; Tumor-specific mesorectal excision
The purpose of this study is to examine the impact of prenatal ultrasonography (US) on the birth of babies with diseases listed on the Korean pediatric surgery index diseases (IDs).
Depending the ease of diagnosis using prenatal US, [diagnostic facility if prenatal US] IDs were divided into easily diagnosed (ED), not easily diagnosed (NED) and detected with difficulty (DD) groups. Five-year data were obtained for the total live birth number (TBN) from the Korean Statistical Information Service, and the actual birth number of IDs (ABNID) from the Korean Health Insurance Review and Assessment Service. The certified incidences of IDs (I) were obtained from a prestigious textbook of pediatric surgery. The estimated abortion rate (AR) of fetus in each group was obtained using the following formula: AR (%) = [1 - (ABNID)/(TBN × I)] × 100.
The AR with all IDs was 38 to 77%. The AR was 78 to 93% for ED group, 38 to 66% for NED group and 0% for DD group.
In spite of high survival rates after treatment, the AR of each group depends on the ease of diagnosis using prenatal US in Korea. A recommendatory policy for the fetus with IDs should be urgently established after general consensus within the related medical societies.
Prenatal ultrasonography; Birth rate; Induced abortion; Congenital; Fetus
Patients with biliary atresia (BA) treated with Kasai portoenterostomy may later develop intractable cholangitis (IC) that is unresponsive to routine conservative treatment. It may cause biliary cirrhosis and eventually hepatic failure with portal hypertension. Control of IC requires prolonged hospitalization for the administration of intravenous antibiotics. To reduce the hospitalization period, we designed a home intravenous antibiotic treatment (HIVA) which can be administered after initial inpatient treatment. In this study, we reviewed the effects of this treatment.
We reviewed medical records of 10 patients treated with HIVA for IC after successful Kasai portoenterostomies performed for BA between July 1997 and June 2009.
The duration of HIVA ranged from 8 to 39 months (median, 13.5 months). The median length of hospital stay was 5.7 days per month for conventional treatments to manage IC before HIVA and, 1.5 days per month (P = 0.012) after HIVA. The median amount of medical expenses per month was reduced by about one tenth with HIVA. One patient underwent liver transplantation due to uncontrolled esophageal variceal bleeding, but the other nine patients had acceptable hepatic function with native livers.
HIVA may be an effective primary treatment for IC after Kasai portoenterostomies for BA, and reduce length of hospital stay and medical expense.
Biliary atresia; Intractable cholangitis; Home intravenous antibiotics treatment
The ability of nitric oxide (NO)-releasing silica nanoparticles to kill biofilm-based microbial cells is reported. Biofilms of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans were formed in vitro and exposed to NO-releasing silica nanoparticles. Replicative viability experiments revealed that ≥ 99% of cells from each type of biofilm were killed via NO release, with the greatest efficacy (≥ 99.999% killing) against gram-negative P. aeruginosa and E. coli biofilms. Cytotoxicity testing demonstrated that the highest dose of NO-releasing silica nanoparticles inhibited fibroblast proliferation to a lesser extent than clinical concentrations of currently-administered antiseptics (e.g., chlorhexidine) with proven wound-healing benefits. This study demonstrates the promise of employing nanoparticles for delivering an antimicrobial agent to microbial biofilms.
Nitric oxide; nanoparticle; biofilm; antimicrobial; cytotoxicity
An amperometric fluorinated xerogel-derived nitric oxide (NO) microelectrode is described. A range of fluorine-modified xerogel polymers were synthesized via the co-hydrolysis and condensation of alkylalkoxy- and fluoroalkoxysilanes. Such polymers were evaluated as NO sensor membranes to identify the optimum composition for maximizing NO permeability while providing sufficient selectivity for NO in the presence of common interfering species. By taking advantage of both the versatility of sol–gel chemistry and the “poly(tetrafluoroethylene) (PTFE)-like” high NO permselective properties of the xerogels, the performance of the fluorinated xerogel-derived sensors was excellent, surpassing all miniaturized NO sensors reported to date. In contrast to previous electrochemical NO sensor designs, xerogel-based NO microsensors were fabricated using a simple, reliable dip-coating procedure. An optimal permselective membrane was achieved by synthesizing xerogels of methyltrimethoxysilane (MTMOS) and 20% (heptadecafluoro-1,1,2,2-tetrahydrodecyl)trimethoxysilane (17FTMS, balance MTMOS) under acid-catalyzed conditions. The resulting NO microelectrode had a conical tip of ~20 μm in diameter and ~55 mm in length, and exhibited sensitivities of 7.91 pA·nM−1 from 0.2 to 3.0 nM (R2 = 0.9947) and 7.60 nA·mM−1 from 0.5 to 4.0 μM (R2 = 0.9999), detection limit of 83 pM (S/N = 3), response time (t95%) of <3 sec, and selectivity (logKNO,jamp) of −5.74, <−6, <−6, <−6, <−6, −5.84, and −1.33 for j = nitrite, ascorbic acid, uric acid, acetaminophen, dopamine, ammonia/ammonium, and carbon monoxide. In addition, the sensor proved functional up to 20 d, maintaining ≥90% of the sensor's initial sensitivity without serious deterioration in selectivity.
The mini-chromosome maintenance (MCM) proteins serve as the replicative helicases in archaea and eukaryotes. Interestingly, an MCM homolog was identified, by BLAST analysis, within a phage integrated in the bacterium Bacillus cereus (Bc). BcMCM is only related to the AAA region of MCM-helicases; the typical amino-terminus is missing and is replaced by a segment with weak homology to primases. We show that BcMCM displays 3′→5′ helicase and ssDNA-stimulated ATPase activity, properties that arise from its conserved AAA domain. Isolated BcMCM is a monomer in solution but likely forms the functional oligomer in vivo. We found that the BcMCM amino-terminus can bind ssDNA and harbors a zinc atom, both hallmarks of the typical MCM amino-terminus. No BcMCM-catalyzed primase activity could be detected. We propose that the divergent amino-terminus of BcMCM is a paralog of the corresponding region of MCM-helicases. A divergent amino terminus makes BcMCM a useful model for typical MCM-helicases since it accomplishes the same function using an apparently unrelated structure.
The Cdc6-1 and -2 proteins from the archaeon Methanothermobacter thermautotrophicus were previously shown to bind the minichromosome maintenance (MCM) helicase. It is shown here that Cdc6-2 protein dissociates the MCM complex. This observation supports the hypothesis that the Cdc6-2 protein functions as a helicase loader.
Helicases play essential roles in many cellular processes including replication, transcription and translation. Most helicases translocate along one strand of the duplex while displacing the complementary strand (of either DNA or RNA). Thus, helicases have directionality. They move along nucleic acids in either the 3'→ 5' or 5'→ 3' direction. The directionality of helicases with low activity or of those that cannot initiate duplex unwinding from a substrate that contains only one single-stranded overhang region is difficult to determine.
An improved assay to determine helicase directionality was developed that uses a substrate containing biotinylated oligonucleotides. As a proof of concept, it was shown that the substrates substantially improve helicase activity and directionality determination for several DNA helicases in comparison to more traditional substrates. In addition, a universal substrate that can be used to determine the directionality of both 3'→ 5' and 5'→ 3' helicases was developed.
It is shown here that the use of a biotin-streptavidin complex as a helicase substrate improves helicase activity and the determination of helicase directionality. The method described is simpler that the currently available techniques.
Replicative DNA helicases are ring-shaped hexamers that play an essential role in chromosomal DNA replication. They unwind the two strands of the duplex DNA and provide the single-stranded (ss) DNA substrate for the polymerase. The minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in eukarya and archaea. The proteins of only a few archaeal organisms have been studied and revealed that although all have similar amino acid sequences and overall structures they differ in their biochemical properties. In this report the biochemical properties of the MCM protein from the archaeon Thermoplasma acidophilum is described. The enzyme has weak helicase activity on a substrate containing only a 3′-ssDNA overhang region and the protein requires a forked DNA structure for efficient helicase activity. It was also found that the helicase activity is stimulated by one of the two T.acidophilum Cdc6 homologues. This is an interesting observation as it is in sharp contrast to observations made with MCM and Cdc6 homologues from other archaea in which the helicase activity is inhibited when bound to Cdc6.
The Cdc6 proteins from the archaeon Methanothermobacter thermautotrophicus were previously shown to bind double-stranded DNA. It is shown here that the proteins also bind single-stranded DNA. Using minichromosome maintenance (MCM) helicase mutant proteins unable to bind DNA, it was found that the interaction of MCM with Cdc6 inhibits the DNA binding activity of Cdc6.
The origin recognition complex, Cdc6 and the minichromosome maintenance (MCM) complex play essential roles in the initiation of eukaryotic DNA replication. Homologs of these proteins may play similar roles in archaeal replication initiation. While the interactions among the eukaryotic initiation proteins are well documented, the protein–protein interactions between the archaeal proteins have not yet been determined. Here, an extensive structural and functional analysis of the interactions between the Methanothermobacter thermautotrophicus MCM and the two Cdc6 proteins (Cdc6-1 and -2) identified in the organism is described. The main contact between Cdc6 and MCM occurs via the N-terminal portion of the MCM protein. It was found that Cdc6–MCM interaction, but not Cdc6–DNA binding, plays the predominant role in regulating MCM helicase activity. In addition, the data showed that the interactions with MCM modulate the autophosphorylation of Cdc6-1 and -2. The results also suggest that MCM and DNA may compete for Cdc6-1 protein binding. The implications of these observations for the initiation of archaeal DNA replication are discussed.
DNA helicases play essential roles in many cellular processes. The currently available techniques to generate substrates for helicase assays are fairly complicated and need some expertise not available in all laboratories. Here, a PCR-based method to generate a substrate for a helicase assay is described, and its application for several archaeal, bacterial and viral enzymes is demonstrated.