Naked-eye detection of specific DNA sequences amplified by the polymerase chain reaction with nanocomposite beads.

We developed a novel nanocomposite bead system for detection by the naked eye of specific DNA sequences amplified by the polymerase chain reaction (PCR). The DNA probes, which were complementary to the target DNA, are conjugated with the nanocomposite beads. If the amplified products contained sequences complementary to the probes, the beads aggregated through sandwich hybridization. The aggregation was detectable as precipitation of the nanocomposite beads. The results were determined visually and did not require instrumental detection. The assay was sensitive enough to detect PCR products with a detection limit of 10 copies/tube for DNA templates. This technique is that all needed components are included within the initial cap, so that the risk of carryover contamination is very low. The nanocomposite bead system has broad application prospects for the detection of specific DNA sequences in biological and biomedical research.

MicroRNA biomarkers for chemical hazard screening identified by RNA deep sequencing analysis in mouse embryonic stem cells.

We investigated the responses of microRNAs (miRNAs) using mouse embryonic stem cells (mESCs) exposed to nine chemicals (bis(2-ethylhexyl)phthalate, p-cresol, p-dichlorobenzene, phenol, pyrocatecol, chloroform, tri-n-butyl phosphate, trichloroethylene, and benzene), which are listed as "Class I Designated Chemical Substances" from the Japan Pollutant Release and Transfer Register. Using deep sequencing analysis (RNA-seq), several miRNAs were identified that show a substantial response to general chemical toxicity (i.e., to these nine chemicals considered as a group) and several miRNA biomarkers that show a substantial and specific response to benzene. The functions of the identified miRNAs were investigated in accordance with Gene Ontology terms of their predicted target genes, indicating regulation of cellular processes. We compared the results with those for the long non-coding RNAs (ncRNAs) and mRNAs reported in our previous studies in addition to previously identified miRNAs that are either up- or down-regulated in response to the benzene as stimuli. We also observed that the changes in expression of miRNAs were smaller than those for long ncRNAs and mRNAs. Taken together the current and previous results revealed that toxic chemical stimuli regulate the expression of miRNAs. We believe that the use of miRNAs, including the thus identified miRNAs, as biomarkers contribute to predicting the potential toxicity of particular chemicals or identifying human individuals that have been exposed to chemical hazards.

Improving attainment of the critical view of safety during laparoscopic cholecystectomy.

INTRODUCTION: We hypothesized that practicing surgeons would successfully achieve a better and more frequent Critical View of Safety (CVS) during laparoscopic cholecystectomy (LC) after participation in a structured Safe CVS Curriculum. METHODS: All surgeons performing LC at a regional health system had four LC cases recorded: twice before and twice after a curriculum focused on the CVS, which was led by a member of the Society of American Gastrointestinal and Endoscopic Surgeons (SAGES) Safe LC Task Force. Videos were then de-identified and randomly ordered for grading on a 6-point CVS assessment tool by two expert surgeons, who were blinded to the operator and whether the surgeries were performed before or after the curriculum. Confidence surveys and performance on a CVS identification video quiz were also compared pre- and post-curriculum. RESULTS: Twelve surgeons (five general, four acute care, and three minimally invasive) with an average experience of 17.9 ± 6.3 years participated in the study. After the curriculum, surgeons achieved all three CVS criteria in more cases (1/24 (4%) versus 10/24 (42%), p < 0.004). There was also significant improvement in correctly identifying whether the CVS was achieved in 10 video clips from the Internet (7.9 ± 1.5 vs. 9.3 ± 0.8, p = 0.006) and increased confidence on a 5-point Likert scale in accurately identifying the CVS (4.5 ± 0.5 vs. 4.9 ± 0.3, p = 0.017). CONCLUSION: A structured curriculum on achieving a quality CVS for practicing, experienced surgeons improved their confidence and frequency of obtaining the Critical View of Safety during LC. We recommend that the Safe CVS Curriculum be considered for widespread use in order to increase the quality and frequency of attaining the Critical View of Safety.

Vascular Anatomy of Mesopancreas in Pancreatoduodenectomy Using an Intestinal Derotation Procedure.

BACKGROUND: Mesopancreas excision in pancreatoduodenectomy is technically complicated because of the anatomical complexity of the mesopancreas with the inferior peripancreatic blood vessels which is caused by intestinal rotation in fetal life. We have developed a novel artery-first approach (the intestinal derotation procedure) for facilitating mesopancreas excision. The aim of this study was to clarify the vascular anatomy of the mesopancreas after derotation. METHODS: The right-sided colon and small intestine are mobilized from the retroperitoneum, and the intestinal loop is then derotated. In 136 cases of pancreatoduodenectomy employing the derotation procedure, we analyzed the vascular anatomy of the mesopancreas. RESULTS: After derotation, the anatomy was simplified. The mesopancreas extended from the right aspect of the superior mesenteric artery (SMA), forming a horizontal plane. The first jejunal trunk (FJT) was situated in parallel with the second jejunal artery and was anterior (91%) or posterior (9%) to the SMA. The inferior pancreaticoduodenal vein (IPDV) entered the right side of the FJT (83%) or the superior mesenteric vein (17%). Besides the IPDV, 1-4 tributaries entered the right wall of the FJT, in 89% of cases. The inferior pancreaticoduodenal artery was observed to originate from the right wall of the SMA, sharing a common stem with the first jejunal artery (70%) or branching directly from the SMA (29%). CONCLUSIONS: Intestinal derotation simplifies the mesopancreas anatomy and reveals the anatomical details of the inferior peripancreatic blood vessels in pancreatoduodenectomy.

Identification of RNA biomarkers for chemical safety screening in neural cells derived from mouse embryonic stem cells using RNA deep sequencing analysis.

Chemical safety screening requires the development of more efficient assays that do not involve testing in animals. In vitro cell-based assays are among the most appropriate alternatives to animal testing for screening of chemical toxicity. Most studies performed to date made use of mRNAs as biomarkers. Recent studies have however indicated the presence of many unannotated non-coding RNAs (ncRNAs) in the transcriptome that do appear to encode proteins. In the present study, we performed whole-transcriptome sequencing analysis (RNA-Seq) to identify novel RNA biomarkers, including ncRNAs, which showed marked responses to the toxicity of nine chemicals. Chemical safety screening was performed in cell-based assays using mouse embryonic stem cell (mESC)-derived neural cells. Marked responses in the expression of some ncRNAs to the chemical compounds were observed. The results of the present study suggested that ncRNAs may be useful in chemical safety screening as novel RNA biomarkers.

Relation of dietary inorganic arsenic exposure and urinary inorganic arsenic metabolites excretion in Japanese subjects.

Inorganic arsenic (InAs) is a ubiquitous metalloid that has been shown to exert multiple adverse health outcomes. Urinary InAs and its metabolite concentration has been used as a biomarker of arsenic (As) exposure in some epidemiological studies, however, quantitative relationship between daily InAs exposure and urinary InAs metabolites concentration has not been well characterized. We collected a set of 24-h duplicated diet and spot urine sample of the next morning of diet sampling from 20 male and 19 female subjects in Japan from August 2011 to October 2012. Concentrations of As species in duplicated diet and urine samples were determined by using liquid chromatography-ICP mass spectrometry with a hydride generation system. Sum of the concentrations of urinary InAs and methylarsonic acid (MMA) was used as a measure of InAs exposure. Daily dietary InAs exposure was estimated to be 0.087 µg kg-1 day-1 (Geometric mean, GM), and GM of urinary InAs+MMA concentrations was 3.5 ng mL-1. Analysis of covariance did not find gender-difference in regression coefficients as significant (P > 0.05). Regression equation Log 10 [urinary InAs+MMA concentration] = 0.570× Log 10 [dietary InAs exposure level per body weight] + 1.15 was obtained for whole data set. This equation would be valuable in converting urinary InAs concentration to daily InAs exposure, which will be important information in risk assessment.

Pancreatic Duct Holder and Mucosa Squeeze-out Technique for Duct-to-Mucosa Pancreatojejunostomy After Pancreatoduodenectomy: Propensity Score Matching Analysis.

BACKGROUND: Duct-to-mucosa pancreatojejunostomy after pancreatoduodenectomy can be technically difficult, particularly in cases with a non-dilated pancreatic duct. We devised a novel procedure employing a pancreatic duct holder and mucosa squeeze-out technique facilitating duct-to-mucosa anastomosis. We compared the perioperative outcomes of pancreatoduodenectomy with duct-to-mucosa pancreatojejunostomy between the novel and conventional procedures. METHODS: Our pancreatic holder has a cone-shaped tip with a slit. The holder can expand the pancreatic duct and provides a good surgical field for anastomosis. A small incision for anastomosis is made on the jejunum, while the jejunum is grasped around the incision. Then, the jejunal mucosa becomes squeezed-out and everted. This mucosa squeeze-out technique facilitates suturing the full thickness of the jejunum. Propensity score matching yielded 113 cases each undergoing the novel and the conventional procedure, among 308 cases receiving pancreatoduodenectomy with duct-to-mucosa pancreatojejunostomy. RESULTS: The overall morbidity rate was significantly lower in the novel procedure group. The pancreatic fistula (ISGPF grade B/C) rate was significantly lower in the novel (5 %) than in the conventional (13 %) procedure group. For cases with a non-dilated pancreatic duct (≤3 mm), the rate was significantly lower in the novel (10 %) than in the conventional procedure group (24 %). Multivariate analysis identified a non-dilated pancreatic duct, soft pancreas, and the conventional procedure as factors independently predicting the complication of pancreatic fistula formation. CONCLUSIONS: Our novel procedure facilitates duct-to-mucosa pancreatojejunostomy and decreases the pancreatic fistula rate. This procedure is simple, rational, and useful for achieving anastomosis, particularly in cases with a non-dilated pancreatic duct.

Long-term retention of pristine multi-walled carbon nanotubes in rat lungs after intratracheal instillation.

As a result of the growing potential industrial and medical applications of multi-walled carbon nanotubes (MWCNTs), people working in or residing near facilities that manufacture them may be exposed to airborne MWCNTs in the future. Because of concerns regarding their toxicity, quantitative data on the long-term clearance of pristine MWCNTs from the lungs are required. We administered pristine MWCNTs well dispersed in 0.5 mg ml(-1) Triton-X solution to rats at doses of 0.20 or 0.55 mg via intratracheal instillation and investigated clearance over a 12-month observation period. The pristine MWCNTs pulmonary burden was determined 1, 3, 7, 28, 91, 175 and 364 days after instillation using a method involving combustive oxidation and infrared analysis, combined with acid digestion and heat pretreatment. As 0.15- and 0.38-mg MWCNTs were detected 1 day after administration of 0.20 and 0.55 mg MWCNTs, respectively, approximately 30% of administrated MWCNTs may have been cleared by bronchial ciliary motion within 24 h of administration. After that, the pulmonary MWCNT burden did not decrease significantly over time for up to 364 days after instillation, suggesting that MWCNTs were not readily cleared from the lung. Transmission electron microscopy (TEM) showed that alveolar macrophages internalized the MWCNTs and retained in the lung for at least 364 days after instillation. MWCNTs were not detected in the liver or brain within the 364-day study period (<0.04 mg per liver, < 0.006 mg per brain).

Insights from the ganglionic acetylcholine receptor autoantibodies in patients with Sjögren's syndrome.

OBJECTIVE: It is not known whether autonomic neuropathy is a feature of Sjögren's syndrome (SS) or whether it is related to circulating antiganglionic acetylcholine receptor (gAChR) antibodies. The goal of the present study was to investigate the autonomic dysfunction in patients with SS and the associations between autonomic dysfunction, anti-gAChR antibodies, and clinical features of SS. METHODS: (1) The first observational study tested for the presence of gAChR antibodies in the serum samples from 39 patients with SS (absent information regarding autonomic symptoms) and healthy volunteers. (2) In the second study, serological and clinical data from 10 Japanese patients diagnosed with SS were reviewed. These patients showed autonomic dysfunction, and luciferase immunoprecipitation systems (LIPS) test was conducted to detect anti-α3 and anti-ß4 gAChR antibodies. (3) In the final analysis, we combined the data of seropositive SS patients with autonomic symptom from the first study with all of the patients from the second study, and analyzed the clinical features. RESULTS: (1) The LIPS assay revealed that anti-gAChRα3 and anti-gAChRß4 antibodies were detected in the sera from patients with SS (23.1%, 9/39). Five of nine SS patients had autonomic symptoms. (2) Anti-α3 and anti-ß4 gAChR antibodies were also detected in 80.0% (8/10) of patients with SS with autonomic symptoms. Six of the ten patients were diagnosed as having SS after neurological symptoms developed. These seropositive patients had predominant and severe autonomic symptoms and were diagnosed with autonomic neuropathy. (3) Thirteen of fifteen SS patients with autonomic symptoms (86.7%) were seropositive for anti-gAChR antibodies, and we confirmed sicca complex, orthostatic hypotension, upper and lower gastrointestinal (GI) symptoms, and bladder dysfunction at high rates. CONCLUSION: The present results suggest the possibility of anti-gAChR antibodies aiding the diagnostics of SS with autonomic dysfunction.

Inorganic arsenic in the Japanese diet: daily intake and source.

The concentrations of arsenic (As) species in 19 food composites prepared from 159 food items purchased in Shizuoka city, Japan, were determined (1) to estimate total daily intake of inorganic As (InAs) and some organic As species and (2) to determine food contributing to total daily InAs intake. As analysis included extraction of As species with a synthetic gastric juice (0.07 mol/L HCl + 0.01 % pepsin) from food composite and high-performance liquid chromatography-high efficiency photo-oxidation-hydride generation-inductively coupled plasma mass spectrometry. InAs was detected in 9 of 19 food composites at a concentration of 0.423-450 ng As/g fresh-weight. Daily intake of InAs from cereals was greatest (13 µg/person/day) followed by algae (5.7 µg/person/day), and the intake from the two categories constituted 90 % of the total daily InAs intake of adults (21 µg/person/day on a bioaccessible-fraction basis and 24 µg/person/day on a content basis). Analysis of individual food items showed that rice and hijiki contributed virtually 100 % of InAs from cereals and algae, respectively. The present survey indicated that InAs from rice and hijiki consumption contributed to total daily InAs intake and consequently to significant cancer risk of the general Japanese population. Daily intake of some organic forms of As and their contributing food categories was also estimated.

[Pancreatic ductal carcinoma concomitant with intraductal papillaly mucinous neoplasm].

IPMN is a slow-growing tumor and has a good prognosis, but is very often associated with a high incidence of pancreatic ductalcarcinoma(DC). Unlike IPMN, DC progresses rapidly, and has a poor prognosis. However, DC concomitant with IPMN has a better prognosis than DC without IPMN. The reason for the good prognosis of the former is undetermined, but perhaps it is the early detection of DC or its not so malignant behavior. It is important to thoroughly examine the entire pancreas for the potentialco -occurrence of DC in patients with IPMN.

Biopersistence of inhaled MWCNT in rat lungs in a 4-week well-characterized exposure.

It is important to conduct a risk assessment that includes hazard assessment and exposure assessment for the safe production and handling of newly developed nanomaterials. We conducted an inhalation study of a multi-wall carbon nanotube (MWCNT) as a hazard assessment. Male Wistar rats were exposed to well-dispersed MWCNT for 4 weeks by whole body inhalation. The exposure concentration in the chamber was 0.37 ± 0.18 mg/m³. About 70% of the MWCNTs in the chamber were single fiber. The geometric mean diameter (geometric standard deviation, GSD) and geometric mean length (GSD) of the aerosolized MWCNTs in the chamber were 63 nm (1.5) and 1.1 µm (2.7), respectively. The amounts of MWCNT deposited in the rat lungs were determined by the X-ray diffraction method and elemental carbon analysis. The average deposited amounts at 3 days after the inhalation were 68 µg/lung by the X-ray diffraction method and 76 µg/lung by elemental carbon analysis. The calculated deposition fractions were 18% and 20% in each analysis. The amount of retained MWCNT in the lungs until 3 months after the inhalation decreased exponentially and the calculated biological half times of MWCNT were 51 days and 54 days, respectively. The clearance was not delayed, but a slight increase in lung weight at 3 days after the inhalation was observed.

Optimal Lymphadenectomy of the Mesopancreas Based on Fluorescence Imaging During Pancreaticoduodenectomy.

BACKGROUND: Excision of the mesopancreas with lymphadenectomy is an important component of pancreatoduodenectomy. However, the optimal extent of lymphadenectomy remains unclear. Furthermore, accurate description of the mesopancreatic lymphatic pathways is difficult, probably because of the complex anatomy. Intestinal derotation simplifies the anatomy and facilitates both examination of lymphatic flow and the surgical procedure. The aim of this study was to evaluate lymphatic flow in the mesopancreas using indocyanine green fluorescence imaging with an intestinal derotation technique, and to clarify the optimal extent of mesopancreas excision and lymphadenectomy in pancreatoduodenectomy. METHODS: Indocyanine green solution (2.5 × 10-3 mg) was injected into the pancreatic head parenchyma. After intestinal derotation, the spread of indocyanine green was observed using near-infrared imaging. RESULTS: Participants comprised 10 patients who underwent pancreatoduodenectomy for periampullary neoplasms. With indocyanine green fluorescence imaging, 9 of the 10 patients showed lymphatic flow from the pancreatic head to the superior mesenteric artery via the inferior pancreaticoduodenal artery and first jejunal artery (but not via the second and more distant arteries), with eventual drainage into the paraaortic region. CONCLUSIONS: Lymphatic pathways from the pancreatic head were connected to the superior mesenteric artery via the inferior pancreaticoduodenal artery and first jejunal artery. Excision of the mesopancreas with the inferior pancreaticoduodenal artery and first jejunal artery while preserving the second or more distant arteries appears optimal in pancreatoduodenectomy for periampullary malignancies.

Restrictive ventilatory impairment and thrombosis due to a giant liver cyst.

Most patients with liver cysts are asymptomatic and require no treatment. In this patient with symptoms and restrictive ventilatory impairment, percutaneous needle aspiration with injection of minocycline hydrochloride was effective.

Gene expression profiles in rat lung after inhalation exposure to C60 fullerene particles.

Concern over the influence of nanoparticles on human health has risen due to advances in the development of nanotechnology. We are interested in the influence of nanoparticles on the pulmonary system at a molecular level. In this study, gene expression profiling of the rat lung after whole-body inhalation exposure to C(60) fullerene (0.12mg/m(3); 4.1x10(4) particles/cm(3), 96nm diameter) and ultrafine nickel oxide (Uf-NiO) particles (0.2mg/m(3); 9.2x10(4) particles/cm(3), 59nm diameter) as a positive control were employed to gain insights into these molecular events. In response to C(60) fullerene exposure for 6h a day, for 4 weeks (5 days a week), C(60) fullerene particles were located in alveolar epithelial cells at 3 days post-exposure and engulfed by macrophages at both 3 days and 1 month post-exposures. Gene expression profiles revealed that few genes involved in the inflammatory response, oxidative stress, apoptosis, and metalloendopeptidase activity were up-regulated at both 3 days and 1 month post-exposure. Only some genes associated with the immune system process, including major histocompatibility complex (MHC)-mediated immunity were up-regulated. These results were significantly different from those of Uf-NiO particles which induced high expression of genes associated with chemokines, oxidative stress, and matrix metalloproteinase 12 (Mmp12), suggesting that Uf-NiO particles lead to acute inflammation for the inhalation exposure period, and the damaged tissues were repaired in the post-exposure period. We suggest that C(60) fullerene might not have a severe pulmonary toxicity under the inhalation exposure condition.

384-Channel electrochemical sensor array chips based on hybridization-triggered switching for simultaneous oligonucleotide detection.

We investigated the feasibility of simultaneous detection of multiple environmentally- and biomedically-relevant RNA biomarker target sequences on a single newly fabricated 384-ch sensor array chip aiming at practical application. The individual sensor is composed of a photolithographically-fabricated Au/Cr-based electrode modified with peptide nucleic acid (PNA) probes. The sensor array chips showed sequence-specific responses upon hybridization of the probes with target sequences complementary to the probes in contrast to mismatch versions. The target oligonucleotides have 15-22 mer sequences from messenger RNAs for estrogen-responsive genes and microRNAs for lung cancer biomarkers. The dependence on target concentrations of sensor responses was observed by using a single chip on which experiments for detection of several target concentrations proceeded simultaneously, with the detection limit of 7.33 × 10-8 M. As more realistic samples, oligonucleotide samples amplified by PCR from a synthesized template sequence were applied to the chip. They showed sequence-specific responses, revealing the potential for fabricated sensor array chips to be utilized to analyze PCR samples. Unlike complicated and expensive chips that require nanofabrication, our sensor array chips based on glass coated with gold thin films are simple and can be fabricated from inexpensive and readily available materials.