Negative Effects of Endoscopists' Fatigue on Colonoscopy Quality on 34,022 Screening Colonoscopies.

BACKGROUND AND AIMS: There is still considerable controversy surrounding the relationship between fatigue of endoscopists and the quality of colonoscopy. The aim of this study is to comprehensively explore the association between fatigue and adenoma detection rate (ADR) and cecal intubation rate (CIR). METHODS: The mixed effects logistic regression model was used to explore the relationship between fatigue- related factors including procedure order, session of procedures and the day of week and ADR as well as CIR. RESULTS: When controlling for confounders, the day of week (Monday as reference, Friday, p=0.022; weekends, p=0.015) and session of procedures (P<0.001) were significantly associated with ADR while procedure order (<5 as reference, 6-10, p<0.001; >10, p=0.001) and session of procedures (p=0.004) were independent predictors for CIR. Additionally, there was a significant downward trend on ADR and CIR with the approaching of weekends (p=0.005) and increasing procedure orders (p<0.001), respectively. In the subgroup analysis stratified by gender, age and workload intensity, significant lower ADR was found in the afternoon in all subgroups (male, p<0.001; female, p=0.005; <40 years, p<0.001; ≥40 years, p=0.020; intensity<50 per month, p=0.017; intensity≥50 per month, p<0.001) but the downward trend on ADR as the week progressed was only found in endoscopists with male gender (p=0.011), age<40 (p=0.027) and high workload intensity (p=0.003). Moreover, a significant downward trend on CIR as the procedure order increased was found in all subgroups except endoscopists with age≥40 (male, p=0.005; female, p<0.001; <40 years, p<0.001; intensity<50 per month, p=0.001; intensity≥50 per month, p<0.001). CONCLUSIONS: Colonoscopies in the afternoon will affect ADR negatively while increasing procedure order will cause a lower CIR. Importantly, the significant negative influence of Friday and weekends on ADR was first discovered in this study. Moreover, endoscopists with female gender and advanced age (≥40) but not high workload intensity showed superiority in resistance of fatigue caused by the end of the week and increasing daily procedures.

Comprehensive evaluation of the learning curve to achieve satisfactory adenoma detection rate.

BACKGROUND AND AIM: The number of colonoscopies required to reach satisfactory adenoma detection rate (ADR) is not well established. The aim of this study was to identify the appropriate number of procedures required to attain satisfactory ADR for those well-trained endoscopists who have a cecal intubation rate (CIR) ≥ 90% and start to perform colonoscopy independently. METHODS: All endoscopists with compelete independent colonoscopy data during career in our database were enrolled. The number of procedures required to achieve ADR ≥ 20% was identified by cumulative summation (Cusum), learning curve Cusum (LC-Cusum), and moving average method. Mixed effect logistic regression model was developed to determine the relationship between endoscopist as well as patient-related factors and adenoma detection. RESULTS: A total of 24 943 procedures and 14 endoscopists were enrolled. By Cusum analysis, the interest point was at 207 procedures. By LC-Cusum analysis, 71% (10/14) and 86% (12/14) of endoscopists had attained satisfactory ADR after 200 and 300 procedures, respectively. By moving average method, endoscopists reached a mean ADR of 20% at 216 and 261 procedures over blocks of 50 and 100 procedures, respectively. The total number of procedures, number of daily procedures, patient age and gender, bowel preparation, sedation, and diverticulosis were significantly associated with adenoma detection. CONCLUSIONS: This is the first study to investigate the learning curve of ADR for those well-trained endoscopists who have a CIR ≥ 90% and start to perform colonoscopy independently. Two hundred procedures might be an optimal number required to reach an ADR ≥ 20%.

Comparison of droplet digital PCR and direct Sanger sequencing for the detection of the BRAFV600E mutation in papillary thyroid carcinoma.

BACKGROUND: The BRAFV600E mutation status is a useful diagnostic and prognostic marker for papillary thyroid carcinoma (PTC). Although it is a commonly used method, Sanger sequencing has several limitations in detecting the BRAFV600E mutation. The aim of this study was to evaluate the efficiency of droplet digital PCR (ddPCR) as an alternative method for the detection of the BRAFV600E mutation in PTC patients. METHODS: Samples from a total of 120 patients with PTC and 30 patients with benign nodular thyroid disease who underwent thyroid surgery were collected. The BRAFV600E mutation status of the PTC patients was tested by Sanger sequencing and ddPCR. RESULTS: The BRAFV600E mutation was detected in 67 samples (44.67%) by Sanger sequencing and 92 samples (61.33%) by ddPCR. The detection of the mutation by the two methods was inconsistent in twenty-five samples (16.67%). The sensitivity and specificity of the ddPCR method were 100% and 69.88%, respectively, and the positive predictive and negative predictive values were 72.83% and 100%, respectively. The concordance rate between the two methods in detecting the BRAFV600E mutation was 83.33%. Neither Sanger sequencing nor ddPCR detected BRAFV600E in 30 patients with benign nodular thyroid disease. The 92 samples with the BRAFV600E mutation were detected by ddPCR at a fractional abundance from 0.28% to 45.40% as follows: ≥10% (59 samples, 64.13%), 5%-10% (8 samples, 8.70%), and ≤5% (25 samples, 27.17%). The BRAFV600E mutation was detected in all 59 samples at a fractional abundance ≥10% and in four samples at a fractional abundance from 5% to 10%, and no BRAFV600E mutation was detected at a fractional abundance ≤5% by Sanger sequencing. CONCLUSIONS: ddPCR was a reliable, highly sensitive alternative method for the detection of the BRAFV600E mutation in PTC patients.

Next­generation sequencing­based detection of EGFR, KRAS, BRAF, NRAS, PIK3CA, Her­2 and TP53 mutations in patients with non­small cell lung cancer.

In recent years, the incidence of non­small cell lung cancer (NSCLC) has become the highest lethal rate of cancer worldwide. Molecular assays of EGFR, KRAS, BRAF, NRAS, PIK3CA and Her­2 are widely used to guide individualized treatment in NSCLC patients. Somatic mutations in 112 NSCLC patients, including 7 oncogenic driver genes, were detected by Iontorrent personal genome machine (PGM). Sanger sequencing was used to test and verify the results of PGM. Apart from uncommon mutations of EGFR, 101 NSCLC specimens were tested by droplet digital PCR (ddPCR). According to NGS results, mutations were detected in EGFR (58/112, 51.79% of tumors), KRAS (10/112, 8.93%), BRAF (2/112, 1.79%), NRAS (2/112, 1.79%), Her­2 (2/112, 1.79%), PIK3CA (6/112, 5.36%) and TP53 (31/112, 27.69%). There were 27 samples without any somatic mutations in all genes while 24 samples harboured mutations in two or more genes. A total of 61 samples had one or more mutations in a single gene. All alterations of 7 genes were presented and the overall detection rate of NGS and Sanger sequencing was determined to be 51.79% (58/112) and 37.50% (42/112), respectively (χ2=5.88, P=0.015). Compared with Sanger sequencing, the total sensitivity and specificity of NGS assays was 95.24% (40/42) and 77.14% (54/70), respectively. The overall detection rate of NGS and ddPCR was 45.54% (46/101) and 47.52% (48/101), respectively (χ2=0.000598, P=0.98). Compared with ddPCR, the overall sensitivity and specificity of NGS assays was 95.83% (46/48) and 98.11% (52/53), respectively. The findings indicated that the positive mutation rate of EGFR tested by NGS was significantly lower than that by Sanger sequencing, but the difference between ddPCR and NGS was not statistically significant. The high degree of agreement of reportable variants is proposed in both NGS and ddPCR analysis, suggesting the performance of NGS assays in routine clinical detection may be useful in determining the treatment decisions in NSCLC patients.

A novel bioremediation strategy for petroleum hydrocarbon pollutants using salt tolerant Corynebacterium variabile HRJ4 and biochar.

The present work aimed to develop a novel strategy to bioremediate the petroleum hydrocarbon contaminants in the environment. Salt tolerant bacterium was isolated from Dagang oilfield, China and identified as Corynebacterium variabile HRJ4 based on 16S rRNA gene sequence analysis. The bacterium had a high salt tolerant capability and biochar was developed as carrier for the bacterium. The bacteria with biochar were most effective in degradation of n-alkanes (C16, C18, C19, C26, C28) and polycyclic aromatic hydrocarbons (NAP, PYR) mixture. The result demonstrated that immobilization of C. variabile HRJ4 with biochar showed higher degradation of total petroleum hydrocarbons (THPs) up to 78.9% after 7-day of incubation as compared to the free leaving bacteria. The approach of this study will be helpful in clean-up of petroleum-contamination in the environments through bioremediation process using eco-friendly and cost effective materials like biochar.

Characterization of potassium hydroxide (KOH) modified hydrochars from different feedstocks for enhanced removal of heavy metals from water.

Hydrochars produced from different feedstocks (sawdust, wheat straw, and corn stalk) via hydrothermal carbonization (HTC) and KOH modification were used as alternative adsorbents for aqueous heavy metals remediation. The chemical and physical properties of the hydrochars and KOH-treated hydrochars were characterized, and the ability of hydrochars for removal of heavy metals from aqueous solutions as a function of reaction time, pH, and initial contaminant concentration was tested. The results showed that KOH modification of hydrochars might have increased the aromatic and oxygen-containing functional groups, such as carboxyl groups, resulting in about 2-3 times increase of cadmium sorption capacity (30.40-40.78 mg/g) compared to that of unmodified hydrochars (13.92-14.52 mg/g). The sorption ability among different feedstocks after modification was as the following: sawdust > wheat straw > corn stack. Cadmium sorption kinetics on modified hydrochars could be interpreted with a pseudo-second order, and sorption isotherm was simulated with Langmuir adsorption model. High cadmium uptake on modified hydrochars was observed over the pH range of 4.0-8.0, while for other heavy metals (Pb(2+), Cu(2+), and Zn(2+)) the range was 4.0-6.0. In a multi-metal system, the sorption capacity of heavy metals by modified hydrochars was also higher than that by unmodified ones and followed the order of Pb(II) > Cu(II) > Cd(II) > Zn(II). The results suggest that KOH-modified hydrochars can be used as a low cost, environmental-friendly, and effective adsorbent for heavy metal removal from aqueous solutions.

Hoechst 33342-induced autophagy protected HeLa cells from caspase-independent cell death with the participation of ROS.

Autophagy, an evolutionarily-conserved intracellular organelle and protein degradation process, may exhibit drastically different effects on cell survival depending on the particular environmental and culturing conditions. Hoechst 33342 (HO), a fluorescent dye widely used for staining DNA, has been reported to induce apoptosis in mammalian cells. Here we showed that, in addition to caspase-independent cell death, HO also induced autophagy in HeLa cells, as evidenced by the accumulation of autophagosomes, LC3 form conversion and LC3 puncta formation in a cell line stably expressing GFP-LC3. HO treatment led to generation of reactive oxygen species (ROS), and inhibition of ROS with N-acetyl-l-cysteine (NAC) abrogated both autophagy and caspase-independent cell death. Finally, autophagy played a protective role against caspase-independent cell death, as cell death induced by HO was enhanced under pharmacological and siRNA-mediated genetic inhibition of autophagy.

Improved efficiency of female germline stem cell purification using fragilis-based magnetic bead sorting.

The enrichment of female germline stem cells (FGSCs) and the establishment of cell lines are influenced by the efficiency of cell purification. A previous study using mouse vasa homolog (MVH)-magnetic bead sorting for the isolation and purification of mouse FGSCs showed a relatively low efficiency. In this study, we tested 3 further proteins with the aim of improving the efficiency of FGSC purification. Immunofluorescence assays and magnetic sorting were performed using short-type pituitary gland and brain-cadherin (Stpb-c), CD9, and interferon-inducible transmembrane protein 3 (Iftm3, Fragilis), all of which are expressed in germ cells. Although all 3 proteins were expressed in FGSCs, CD9 was unsuitable because of its lack of germline specificity, and Stpb-c was also unsuitable because of the unavailability of an appropriate primary antibody. The efficiency of FGSC purification was remarkably enhanced using the germline-specific protein Fragilis, compared with that using MVH. This new method for the purification of FGSCs may have extensive applications in stem cell studies and clinical research.

Production of transgenic mice by random recombination of targeted genes in female germline stem cells.

Oocyte production in most mammalian species is believed to cease before birth. However, this idea has been challenged with the finding that postnatal mouse ovaries possess mitotically active germ cells. A recent study showed that female germline stem cells (FGSCs) from adult mice were isolated, cultured long term and produced oocytes and progeny after transplantation into infertile mice. Here, we demonstrate the successful generation of transgenic or gene knock-down mice using FGSCs. The FGSCs from ovaries of 5-day-old and adult mice were isolated and either infected with recombinant viruses carrying green fluorescent protein, Oocyte-G1 or the mouse dynein axonemal intermediate chain 2 gene, or transfected with the Oocyte-G1 specific shRNA expression vector (pRS shOocyte-G1 vector), and then transplanted into infertile mice. Transplanted cells in the ovaries underwent oogenesis and produced heterozygous offspring after mating with wild-type male mice. The offspring were genetically characterized and the biological functions of the transferred or knock-down genes were investigated. Efficiency of gene-transfer or gene knock-down was 29%-37% and it took 2 months to produce transgenic offspring. Gene manipulation of FGSCs is a rapid and efficient method of animal transgenesis and may serve as a powerful tool for biomedical science and biotechnology.

Autophagy-mediated chemosensitization in cancer cells by fullerene C60 nanocrystal.

Autophagy may represent a common cellular response to nanomaterials, and modulation of autophagy holds great promise for improving the efficacy of cancer therapy. Fullerene C60 possesses potent anti-cancer activities, but its considerable toxicity towards normal cells may hinder its practical applications. It has been reported that fullerene C60 induces certain hallmarks of autophagy in cancer cells. Here we show that the water-dispersed nanocrystal of underivatized fullerene C60 (Nano-C60) at noncytotoxic concentrations caused authentic autophagy and sensitized chemotherapeutic killing of both normal and drug-resistant cancer cells in a reactive oxygen species (ROS)-dependent and photo-enhanced fashion. We further demonstrated that the chemosensitization effect of Nano-C60 was autophagy-mediated and required a functional Atg5, a key gene in the autophagy signaling pathway. Our results revealed a novel biological function for Nano-C60 in enhancing the cytotoxic action of chemotherapeutic agents through autophagy modulation and may point to the potential application of Nano-C60 in adjunct chemotherapy.

Production of offspring from a germline stem cell line derived from neonatal ovaries.

The idea that females of most mammalian species have lost the capacity for oocyte production at birth has been challenged recently by the finding that juvenile and adult mouse ovaries possess mitotically active germ cells. However, the existence of female germline stem cells (FGSCs) in postnatal mammalian ovaries still remains a controversial issue among reproductive biologists and stem cell researchers. We have now established a neonatal mouse FGSC line, with normal karyotype and high telomerase activity, by immunomagnetic isolation and culture for more than 15 months. FGSCs from adult mice were isolated and cultured for more than 6 months. These FGSCs were infected with GFP virus and transplanted into ovaries of infertile mice. Transplanted cells underwent oogenesis and the mice produced offspring that had the GFP transgene. These findings contribute to basic research into oogenesis and stem cell self-renewal and open up new possibilities for use of FGSCs in biotechnology and medicine.

Functional analysis of three AHL autoinducer synthase genes in Mesorhizobium loti reveals the important role of quorum sensing in symbiotic nodulation.

One of the most important signal transduction pathways in bacteria, quorum sensing, is involved in many regulatory circuits in rhizobia, especially in the control of communication between rhizobia and their plant hosts. In this study, we identified 3 autoinducer synthase genes - mrlI1, mrlI2, and mrlI3 - in Mesorhizobium loti NZP 2213. We found that MrlI1 and MrlI2 could synthesize distinct N-acyl homoserine lactone (AHL) autoinducers in rich medium cultures, and the expression of mrlI1 was shown to be growth-phase-dependent. MrlI3 did not produce any detectable AHL molecules under the culture conditions tested. To investigate whether these AHL synthases affect nodulation, we examined the nodulation of AHL-deficient mutants on their native plant host Lotus corniculatus and found that the efficiency of nodulation of bacteria with mutations of any of these 3 synthase genes was reduced, suggesting that quorum sensing systems in M. loti may play an important role in successful establishment of rhizobium-legume symbiosis.