Six-well plate-based colony-forming efficacy assay and Co-Culture application to assess toxicity of metal oxide nanoparticles.

The development of a universal, label-free, and reliable in vitro toxicity testing method for nanoparticles is urgent because most nanoparticles can interfere with toxicity assays. In this regard, the colony-forming efficacy (CFE) assay has been suggested as a suitable in vitro toxicity assay for testing nanoparticles without such interference. Recently, the Organisation for Economic Co-operation and Development (OECD) developed a 60 × 15 mm Petri dish-based CFE assay for testing nanoparticles in MDCK-1 cells. However, further investigations are needed, including testing with other cell types, at a smaller scale for greater efficiency, and the application of the co-culture technique. In this study, we selected TiO2, CuO, CeO2, and SiO2 as test nanoparticles and successfully developed a 6-well plate-based CFE assay using HepG2 and A549 cells and a co-culture assay for combinations of HepG2 cells and THP-1 macrophages or A549 cells and THP-1 monocytes. The results suggest that the 6-wellplate-based CFE assay for HepG2 and A549 cells can be applied to nanoparticles, but the co-culture CFE assay has limitations in that it is not different from the single culture study, and it inhibits colony-formation by A549 cells in the presence of macrophages; this warrant further study.

New Bone Formation in the Whole Decellularized Cortical Bone Scaffold Using the Model of Revitalizing a Haversian System.

ABSTRACT: Decellularized allogeneic bone chips act as scaffolds for bone tissue regeneration. Owing to their lack of osteogenic potentials compared to autologous bone graft, decellularized bone scaffolds (DBSs) have applied only to small partial bone defects in clinical settings. Furthermore, only decellularized cancellous bone chips have been limitedly used for the purpose of bone regeneration. The cortical bone has less porosity and less osteogenic materials such as bone morphogenetic proteins in comparison with cancellous bone. In this study, we tried to accelerate new bone formation within the decellularized cortical bone scaffold using a vascular pedicle as an in vivo bioreactor.Forty DBSs were divided into 4 groups with different conditionings (DBS+ demineralized bone matrix [DBM], DBS+DBM+me+mesenchymal stem cells, DBS+DBM+vascular pedicle, and DBS+DBM+vascular pedicle+mesenchymal stem cells) and implanted into the back of 5 rabbits. Half of the DBSs were examined at 8 weeks and the other half at 16 weeks to determine vascularization level and osteogenesis within each group. New bone formation and bone-forming cells related to osteogenesis were observed via histological staining. Inclusion of the vascular pedicle resulted in larger areas of bone regeneration. With time, osteon structures became more prominent in groups containing the vascular pedicle.In summary, vascularized DBSs combined with a vascular pedicle have shown promising results for bone regeneration, thereby representing potential therapeutic alternatives for autologous bone grafts or bone tissue free transfer in large or segmental bone defects. In addition, demineralized whole cortical bone matrix along with vascular pedicle and various bone inductive materials, such as DBM and recombinant human bone morphogenetic protein-2, may be an additional new option of an ideal osteoinductive system.

Prevention of Breast Implant Displacement Using the Acellular Dermal Matrix Garter Belt.

BACKGROUND: Since the issue of breast implant-associated anaplastic large cell lymphoma, smooth breast implants tend to be the more preferred option in implant-based breast reconstructions, compared to its use previously. The most unfavorable aspect of smooth implants is lateral and inferior displacements, which are more common in patients who undergo breast reconstruction compared to augmentation mammoplasty. Hence, we introduce a prevention method for implant displacement using an acellular dermal matrix garter belt. METHODS: This study is a retrospective review of patients who had undergone implant-based breast reconstruction between April 2019 and December 2020. Some patients who have highly possibility of implant displacement, had undergone the application of an ADM garter belt to prevent lateral or superior displacement. Implant displacement was assessed before and at least 6 months postoperatively. RESULTS: A total of 155 IBR cases were recorded. ADM garter belts were applied in 27 patients (17.4%) who had a high tendency of implant displacement for several reasons, which could be classified into two categories: wide breast pocket (56%) and tight inferomedial breast pocket (44%). The intraoperative average distance of lateral slipping on patients' reconstructed breasts from the chest wall midline in supine position was 3.02 ± 0.81 cm and corrected to1.54 ± 0.69 cm at least 6 months postoperative follow-up. CONCLUSIONS: We utilized an ADM strap as an internalized garter belt to minimize implant displacement. This ADM garter belt combined with capsuloplasty might be an effective way to prevent the displacement of smooth implants in the patients with a greater risk of implant displacement. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors   www.springer.com/00266.

Nipple Reconstruction Using the Semilunar Flap and Omega-shaped Acellular Dermal Matrix Strut.

BACKGROUND: Various operative methods exist for nipple reconstruction. Selection of an appropriate skin flap and core strut material is imperative in achieving a satisfactory outcome in nipple reconstruction. Long-term maintenance of nipple projection requires further investigation by surgeons. We propose a new technique that uses a semilunar flap and omega-shaped acellular dermal matrix (ADM). METHODS: Total 53 nipples were reconstructed by this method. An omega-shaped ADM strut was inserted into the barrel made by a semilunar flap. The footplates of omega-shaped ADM struts were spread out under the subcutaneous tissue of the donor site of the semilunar flap to support the dome of the omega strut. RESULTS: The mean maintenance rate of nipple projection was 95.12 ± 6.30% at 3 weeks, 80.60 ± 8.93% at 3 months, and 71.70 ± 8.67% at 6 months postoperatively when compared to the projection observed in the immediate postoperative period. Thirty-five patients (66.0%) showed a maintenance rate over 70% at 6 months post operation, with most patients (94.3%) demonstrating a maintenance rate greater than 60%. CONCLUSIONS: Our study with the omega-shaped ADM strut showed superior maintenance rates of projection when compared to other studies on that used AlloDerm® as a core strut for nipple reconstruction. Omega-shaped struts, when made with cross-linked thick ADM, supported the skin flap quite well. We propose that our method combining the semilunar flap with an omega-shaped ADM may be a good option for nipple reconstruction. LEVEL OF EVIDENCE IV: "This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors   www.springer.com/00266 ."

Prediction of Postoperative Mortality in Patients with Organ Failure After Gastric Cancer Surgery.

BACKGROUND: Scarce data are available on the characteristics of postoperative organ failure (POF) and mortality after gastrectomy. We aimed to describe the causes of organ failure and mortality related to gastrectomy for gastric cancer and to identify patients with POF who are at a risk of failure to rescue (FTR). METHODS: The study examined patients with POF or in-hospital mortality in Seoul National University Hospital between 2005 and 2014. We identified patients at a high risk of FTR by analyzing laboratory findings, complication data, intensive care unit records, and risk scoring including Acute Physiology and Chronic Health Evaluation (APACHE) IV, Sequential Organ Failure Assessment (SOFA) score, and Simplified Acute Physiology Score (SAPS) 3 at ICU admission. RESULTS: Among the 7304 patients who underwent gastrectomy, 80 (1.1%) were identified with Clavien-Dindo classification (CDC) grade ≥ IVa. The numbers of patients with CDC grade IVa, IVb, and V were 48 (0.66%), 11 (0.15%), and 21 (0.29%), respectively. Pulmonary failure (43.8%), surgical site complication (27.5%), and cardiac failure (13.8%) were the most common causes of POF and mortality. Cancer progression (100%) and cardiac events (45.5%) showed high FTR rates. In univariate analysis, acidosis, hypoalbuminemia, SOFA, APACHE IV, and SAPS 3 were identified as risk factors for FTR (P < 0.05). Finally, SAPS 3 was identified as an independent predictive factor for FTR. CONCLUSIONS: Cancer progression and acute cardiac failure were the most lethal causes of FTR. SAPS 3 is an independent predictor of FTR among POF patients after gastrectomy.

The Effect of Hyaluronidase on the Fat Graft.

BACKGROUND: Fat grafting has been widely used for facial rejuvenation and soft tissue reconstruction. However, it is associated with a lower retention rate than expected and complications such as fat necrosis or calcification. Several techniques that may increase the survival rate of fat grafts have been proposed. The techniques that promote the recipient sites vascularity to increase the survival rate of fat grafts include administration of growth factors, platelet- rich plasma, and adipose derived-stem cells or preconditioning of the recipient fat graft site. METHODS: In this study, the authors evaluated the effect of hyaluronidase on autologous fat graft survival by pretreatment with hyaluronidase at the recipient site by using an animal model. In the experimental group, the recipient site of the fat graft was pretreated with hyaluronidase before fat grafting, whereas the control group was pretreated with normal saline. RESULTS: After 8 weeks of fat grafting, the average volume retention was 78.2% in the experimental group and 68.6% in control group. Considerable fibrosis between the fat globules in the control group was confirmed with Masson trichrome staining. CD31 immunofluorescence staining was performed and stained vessels were counted. Counted vessel number was significantly greater in the experimental group than in the control group. CONCLUSIONS: Pretreatment of hyaluronidase on the fat graft recipient site is a good option to enhance the outcome of the fat graft in the clinical setting.

Risk Factors for Recurrence of Right Colonic Diverticulitis.

BACKGROUND: Right colonic diverticulitis (RCD) is more common in Asian countries than in Western countries, and the risk factors for recurrence of RCD are not fully understood. The objective of this study was to assess the risk factors for recurrence of RCD. METHODS: We analyzed 296 patients admitted for treatment of RCD in the Gachon University Gil Medical Center from December 2001 to October 2014. Gender, age, BMI, obesity, hypertension, diabetes mellitus, alcohol consumption, smoking, Hinchey classification, and hospital stay were investigated as risk factors for recurrence. RESULTS: Of the 296 patients with RCD, 31 patients recurred after conservative treatment. The median time interval between the initial episode and recurrence of diverticulitis was 10.4 months. In the univariate analysis, a high recurrence rate was observed in patients with a history of alcohol consumption, smoking, and long hospital stay. In the multivariate analysis, the recurrence rate was much higher (p < 0.001) in patients who stayed in the hospital for more than 10 days after the first attack. Smoking also elevated the recurrence rate (p = 0.011). CONCLUSION: Factors associated with recurrence of RCD may include smoking and the long hospital stay due to complexity when first diverticulitis occurs. Further prospective large-scale studies are needed to draw a definite conclusion.

The comprehensive complication index (CCI) is a more sensitive complication index than the conventional Clavien-Dindo classification in radical gastric cancer surgery.

BACKGROUND: The comprehensive complication index (CCI) integrates all complications of the Clavien-Dindo classification (CDC) and offers a metric approach to measure morbidity. The aim of this study was to evaluate the CCI at a high-volume center for gastric cancer surgery and to compare the CCI to the conventional CDC. METHODS: Clinical factors were collected from the prospective complication data of gastric cancer patients who underwent radical gastrectomy at Seoul National University Hospital from 2013 to 2014. CDC and CCI were calculated, and risk factors were investigated. Correlations and generalized linear models of hospital stay were compared between the CCI and CDC. The complication monitoring model with cumulative sum control-CCI (CUSUM-CCI) was displayed for individual surgeons, for comparisons between surgeons, and for the institution. RESULTS: From 1660 patients, 583 complications in 424 patients (25.5%) were identified. The rate of CDC grade IIIa or greater was 9.7%, and the overall CCI was 5.8 ± 11.7. Age, gender, Charlson score, combined resection, open method, and total gastrectomy were associated with increased CCI (p < 0.05). The CCI demonstrated a stronger relationship with hospital stay (ρ = 0.721, p < 0.001) than did the CDC (ρ = 0.634, p < 0.001). For prolonged hospital stays (≥30 days), only the CCI showed a moderate correlation (ρ = 0.544, p = 0.024), although the CDC did not. The CUSUM-CCI model displayed dynamic time-event differences in individual and comparison monitoring models. In the institution monitoring model, a gradual decrease in the CCI was observed. CONCLUSIONS: The CCI is more strongly correlated with postoperative hospital stay than is the conventional CDC. The CUSUM-CCI model can be used for the continuous monitoring of surgical quality.

WNT5A Correlates with Clinicopathological Characteristics in Gastric Cancer: a Meta-Analysis.

BACKGROUND/AIMS: Gastric cancer (GC), the third-leading cause of cancer death in the world, is typically diagnosed only in its advanced stages. WNT signaling has been associated with clinicopathological characteristics in diverse cancer types. But the systematic analysis of WNT5A, a member in the signaling, has not been inspected. Thus, our study used a meta-analysis to statistically associate WNT5A expression with GC clinicopathological characteristics. METHODS: For a systematic literature review of GC in combination with the WNT signaling molecule WNT5A, we searched for PubMed, Cochrane Library, and Web of Science. It led to the five cohorts, in four eligible studies, consisting of 1,034 patients (617 WNT5A-positive and 417 WNT5A-negative patients). These patients were inspected by the library "meta" in R software for our meta-analysis. RESULTS: Our meta-analysis, revealed a statistically significant associations of WNT5A-positivity with lymph node metastasis (p=0.0047), some types of Lauren diffuse subtype GCs (p<0.0001), advanced tumor depth (p<0.0001), and advanced UICC stages (p=0.0461) with no observation of bias or confounding factors. CONCLUSIONS: These results support the feasibility of targeting this embryonic signaling pathway, both for therapy, and as a biomarker to "guide" various individual interventions (i.e., "personalized medicine").

Short- and Long-Term Outcomes After Gastrectomy in Elderly Gastric Cancer Patients.

BACKGROUND: The number of elderly patients undergoing gastric cancer surgery has recently increased. We therefore evaluated the short- and long-term outcomes of elderly patients after curative gastrectomy. METHODS: Overall, 824 patients were included in this retrospective study, which comprised of a non-elderly group (60-64 years; n = 558), an early-elderly group (75-79 years; n = 198), and a late-elderly group (≥80 years; n = 68) who underwent curative gastrectomy for gastric cancer between 2005 and 2009. Postoperative complications, according to the Clavien-Dindo classification, and survival of both elderly groups were compared with the non-elderly group. Postoperative life expectancy of the late-elderly group was compared with the corresponding aged general population. RESULTS: Overall and severe (grade III or higher) complications in the early-elderly group were comparable with the non-elderly group; however, those in the late-elderly group were significantly more common than in the non-elderly group (p = 0.013 and p = 0.043, respectively). Multivariable analysis revealed that age ≥80 years was an independent risk factor for severe complications (hazard ratio 3.02, 95 % confidence interval 1.12-8.17; p = 0.029), and the disease-specific survivals of both elderly groups were comparable with the non-elderly group in all TNM stages. Postoperative life expectancy of late-elderly patients eliminating death from recurrence was comparable with the corresponding aged general population eliminating death from gastric cancer. CONCLUSIONS: Gastric cancer surgery in elderly patients aged ≥80 years achieves reasonable long-term survival despite the increased risk of severe complications.

Identification of Candidates for Early Discharge After Gastrectomy.

OBJECTIVE: The aim of this study was to analyze clinical and laboratory variables associated with complications after gastrectomy for gastric cancer to predict candidates for successful early discharge. METHODS: Consecutive patients undergoing gastrectomy at Seoul National University Hospital from January through December 2013 were identified from a prospective complications database. Clinicopathologic and postoperative laboratory parameters were analyzed to determine variables associated with complications. An additional validation study was performed from March through May 2014. RESULTS: Overall, complications occurred in 180/855 patients (21.1 %). Age >68 years (odds ratio [OR] 1.64), use of an open approach (OR 1.9), and use of combined resection (OR 1.67) were significant independent risk factors for complications (p < 0.05). The postoperative day (POD) 5 to preoperative white blood cell count (WBC) ratio (risk ratio [RR] 2.01), C-reactive protein (CRP) level on POD 5 (RR 1.1), and maximum body temperature on POD 4 (RR 2.36) independently predicted complications in a multivariate analysis (p < 0.05). After establishing an early discharge profile (EDP) based on these six variables, 152/855 patients (17.8 %) were predicted to have an uncomplicated course. Of these, 8/152 (5.3 %) experienced complications. In a validation study of 217 patients, 43/217 (19.8 %) were candidates for early discharge on POD 5, and 3 (7.0 %) had a false-positive EDP. CONCLUSIONS: Patients younger than 68 years of age who underwent laparoscopic gastrectomy without combined resection might be candidates for early discharge on POD 5 if the POD 5 to preoperative WBC ratio is ≤1.2, POD 5 CRP level is ≤5.38 g/mL, and POD 4 body temperature is ≤37.4 °C.

The role of surface functionalization on the pulmonary inflammogenicity and translocation into mediastinal lymph nodes of graphene nanoplatelets in rats.

Graphene, a two-dimensional monocrystalline layer of carbon atoms, has potential in many applications not only in material sciences, but also in the biomedical fields, but there is little information about the role of surface modification on the toxicity of graphene-based nanomaterials. Here, we evaluated the role of surface functionalization of the graphene nanoplatelets (GNPs) on the pulmonary inflammogenicity and translocation into mediastinal lymph nodes using a rat intratracheal instillation model. Six types of GNPs were used: All types of GNPs were based on the pristine GNPs (GNPdot), and different functional groups were conjugated onto them including a COOH (GNPCOOH), COH [Formula: see text], N-H [Formula: see text], F x (GNPF), and N=H [Formula: see text]. All types of GNPs showed very high potential for the generation of reactive oxygen species (ROS) in a dose-dependent manner when measured by a 2'7'-dichlorofluorescin diacetate assay. GNPs were instilled into the lungs of rats at 0.3 and 1 mg/rat for the evaluation of acute (24 h) inflammation and at 3 mg/rat for chronic (1 and 4 weeks) inflammation. At 24 h after instillation, all types of GNPs showed good dose-dependent increases in polymorphonuclear leukocytes with a clear dose-dependency although significant increases compared to vehicle control were found only in positively charged GNPs [Formula: see text]. While the acute inflammation in all treatment groups was returned to control levels at 1 and 4 weeks after instillation, GNPs showed similar patterns of translocation into the mediastinal lymph nodes with a higher degree over time. This study implies that the main factors of GNPs for producing lung inflammation are the potential for ROS generation and surface charge. In addition, functional groups on the GNPs might not play an important role in the extrapulmonary translocation into the mediastinal lymph nodes.

Is There Any Role of Adjuvant Chemotherapy for T3N0M0 or T1N2M0 Gastric Cancer Patients in Stage II in the 7th TNM but Stage I in the 6th TNM System?

BACKGROUND: Controversy surrounds adjuvant chemotherapy (CTx) for T3N0M0 and T1N2M0 in the American Joint Committee on Cancer (AJCC) 7th edition stage IIA gastric cancer patients. The purpose of this study was to evaluate the benefit of adjuvant CTx for stage IIA cancer, including T3N0M0 and T1N2M0. METHODS: A total of 630 patients with stage IIA cancer who underwent a radical gastrectomy between January 1999 and December 2009 at Seoul National University Hospital were retrospectively analyzed. We compared the outcomes of 434 patients who did not receive CTx (the non-CTx group) with those of 196 patients who received CTx comprising of 5-fluorouracil-based regimens (the CTx group). RESULTS: The 5-year overall survival (OS) rates of the non-CTx and CTx groups were 86.4 and 89.3 %, respectively (p = 0.047). In the subgroup analysis of T2N1M0 (6th II/7th IIA), there was a significant difference in OS between the non-CTx and CTx groups (p = 0.003), but no differences were observed in T3N0M0 and T1N2M0 (6th IB/7th IIA) (p = 0.574 and p = 0.934). The multivariate analysis showed that a tumor size greater than 5 cm in T3N0M0 [odds ratio (OR) 1.929; p = 0.030], no adjuvant CTx in T2N1M0 (OR 4.853; p = 0.025), and no factors in T1N2M0 were found to be risk factors for recurrence-free survival. CONCLUSIONS: Adjuvant CTx may be associated with an improved outcome of patients with T2N1M0 (6th II/7th IIA), but not T3N0M0 or T1N2M0 (6th IB/7th IIA), gastric cancer. To confirm these results, further studies are needed.

Portomesenteric vein thrombosis after gastric surgery.

BACKGROUND: Postoperative portomesenteric venous thrombosis (PMVT) is a rare but potentially serious complication of gastric surgery. This study analyzed the incidence, characteristics, risk factors, and outcomes of PMVT following gastric surgery. METHODS: Medical records of patients who underwent gastric surgery between January 2007 and December 2012 were reviewed retrospectively. The risk factors of PMVT were analyzed by a logistic regression analysis with control group matched 1:4 by age, sex, and cancer stage and by a Poisson regression analysis with unmatched control group. The resolution rate of PMVT in 12 months was compared between the treatment group and the nontreatment group. RESULTS: The total incidence of PMVT after gastric surgery was 0.67 % (31/4611). Most (54.84 %) PMVT cases were detected within 1 month postoperatively. No accompanying deep vein thrombosis (DVT) was noted. Multivariate comparison with 1:4 matched control showed that combined splenectomy, synchronous malignancy, and intra-abdominal complication were independent risk factors. Advanced stage, combined splenectomy, and synchronous malignancy were independent risk factors in Poisson regression analysis using unmatched controls. The resolution rate of PMVT was not different from patients treated with anticoagulation (n = 6) or antiplatelet therapy (n = 1) and were not significantly different with those of the untreated group [85.7 % (6/7) vs. 82.3 % (14/17), p = 0.935] during 1-year follow up. CONCLUSIONS: PMVT after gastric surgery was associated with advanced cancer stage, combined splenectomy, and synchronous malignancy, but it was not related to laparoscopy or DVT. Significant differences in the natural course of PMVT were not found between the treatment group and observation group.

Is preoperative staging enough to guide lymph node dissection in clinically early gastric cancer?

BACKGROUND: Limited by the accuracy of preoperative staging, some cases of gastric cancer invading the muscularis propria (pT2) are underestimated as early gastric cancer (EGC) in the preoperative assessment. The aim of this present study was to determine prognostic factors and to propose indications for limited lymph node dissection in patients with clinically EGC (cEGC). METHODS: Patients of cEGC (n = 2072) who were postoperatively diagnosed as pT1 (cT1pT1, n = 1858) and pT2 (cT1pT2, n = 214) from 2005 to 2009 at Seoul National University Hospital were retrospectively analyzed. RESULTS: There was no difference in 5-year survival rate between the cT1pT1 and cT1pT2 group (95.5 % vs. 92.5 %, P = 0.059), and both groups had better overall survival than pT2 patients who were preoperatively diagnosed as locally advanced gastric cancer (cT2-4pT2), whose 5-year survival rate was 78.0 % (P < 0.001). Multivariate analysis indicated lymph node metastasis (LNM) was the independent prognostic factor for cEGC (P < 0.001). In cEGC patients, three preoperative factors, including N stage by multidetector-row computed tomography (MDCT) (P < 0.001), preoperative histological type (P < 0.001), and tumor size (P < 0.001), were associated with LNM by multivariate analysis. Regarding the possibility of LNM, low-risk (4.4 %) and high-risk (17.3 %) groups were developed based on weighted scores of the aforementioned independent three variables. Among 52 patients in the low-risk group, the extension of LNM was limited to the perigastric area. CONCLUSIONS: Comprehensive evaluation based on MDCT, preoperative histological type, and tumor size is an effective method to predict LNM and guide tailored LN dissection for cEGC.

Prognostic factors for reoperation of recurrent retroperitoneal sarcoma: The role of clinicopathological factors other than histologic grade.

BACKGROUND AND OBJECTIVES: Reoperation is recommended for resectable retroperitoneal sarcoma (RS) recurrence; however, the long-term overall survival (OS) benefit varies. Although histologic grade is an important OS predictor after primary tumor resection, its prognostic value tends to diminish with subsequent reoperations. The objective of this study was to identify prognostic factors of OS after reoperation for recurrent RS. METHODS: The medical records of 95 patients who underwent resection for RS at Seoul National University Hospital between January 1999 and July 2011 were retrospectively reviewed. Of the 95 patients, 50 patients underwent second resection for recurrence, and 26 of these patients underwent third resection. Prognostic factors were analyzed at each reoperation. RESULTS: Higher histologic grade and gross residual disease were poor prognostic factors of OS after first resection. After second resection, higher histologic grade and time since previous operation of within 1 year were poor prognostic factors. After third resection, only contiguous organ resection was a significant independent prognostic factor. CONCLUSIONS: The significance of prognostic factors changes with repetitive reoperations for RS recurrence. The prognostic value of histologic grade diminishes after the third resection, whereas other clinical factors such as time since previous operation and contiguous organ resection achieve significance.

Simple high-throughput analytical method using ultra-performance liquid chromatography coupled with tandem mass spectrometry to quantify total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in urine.

BACKGROUND: Since the urinary concentration of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a reliable biomarker of exposure to tobacco smoke, we developed a relatively simple high-throughput chromatographic method to quantify total urinary NNAL concentrations in the general population. METHODS: The high-throughput analytical method was developed using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) to identify and quantify total urinary NNAL concentrations in 10 non-smokers and 15 otherwise healthy smokers. RESULTS: Loss of nitric oxide at m/z 30 was found to be the predominant mass transitioned, and therefore was used as the SIM transition to quantify both NNAL and NNAL-methyl-d3 in urine. The analytical method did not require sample derivatization. Standard curves for total NNAL concentrations were linear between 20 and 1500 pg/mL, with coefficients of determination >0.95. Precision and accuracy ranged from 2.2% to 8.6% (CV) and from -5.6% to 10.9% (percent error), respectively. The lowest limit of quantification was 6.7 pg/mL, and 2.0 pg/mL the lowest limit of detection (LLOD). Total urinary NNAL concentrations in non-smoker subjects were

Evaluation of developmental toxicity using undifferentiated human embryonic stem cells.

An embryonic stem cell test (EST) has been developed to evaluate the embryotoxic potential of chemicals with an in vitro system. In the present study, novel methods to screen toxic chemicals during the developmental process were evaluated using undifferentiated human embryonic stem (hES) cells. By using surface marker antigens (SSEA-4, TRA-1-60 and TRA-1-81), we confirmed undifferentiated conditions of the used hES cells by immunocytochemistry. We assessed the developmental toxicity of embryotoxic chemicals, 5-fluorouracil, indomethacin and non-embryotoxic penicillin G in different concentrations for up to 7 days. While expressions of the surface markers were not significantly affected, the embryotoxic chemicals influenced their response to pluripotent ES cell markers, such as OCT-4, NANOG, endothelin receptor type B (EDNRB), secreted frizzled related protein 2 (SFRP2), teratocarcinoma-derived growth factor 1 (TDGF1), and phosphatase and tensin homolog (PTEN). Most of the pluripotent ES cell markers were down-regulated in a dose-dependent manner after treatment with embryotoxic chemicals. After treatment with 5-fluorouracil, indomethacin and penicillin G, we observed a remarkable convergence in the degree of up-regulation of development, cell cycle and apoptosis-related genes by gene expression profiles using an Affymetrix GeneChips. Taken together, these results suggest that embryotoxic chemicals have cytotoxic effects, and modulate the expression of ES cell markers as well as development-, cell cycle- and apoptosis-related genes that have pivotal roles in undifferentiated hES cells. Therefore, we suggest that hES cells may be useful for testing the toxic effects of chemicals that could impact the embryonic developmental stage.

Role of Recurrence Pattern Multiplicity in Predicting Post-recurrence Survival in Patients Who Underwent Curative Gastrectomy for Gastric Cancer.

PURPOSE: This study aimed to investigate the recurrence patterns in patients who underwent curative surgery for gastric cancer (GC) and analyze their prognostic value for post-recurrence survival (PRS). MATERIALS AND METHODS: We retrospectively reviewed the medical records of 204 patients who experienced GC recurrence following curative gastrectomy for GC at a single institution between January 2012 and December 2017. Specific recurrence patterns (lymph node, peritoneal, and hematogenous) and their multiplicity were analyzed as prognostic factors of PRS. RESULTS: The median PRS of the 204 patients was 8.3 months (interquartile range [IQR]: 3.2-17.4). For patients with a single recurrence pattern (n=164), the difference in each recurrence pattern did not show a significant prognostic value for PRS (lymph node vs. peritoneal, P=0.343; peritoneal vs. hematogenous, P=0.660; lymph node vs. hematogenous, P=0.822). However, the patients with a single recurrence pattern had significantly longer PRS than those with multiple recurrence patterns (median PRS: 10.2 months [IQR: 3.7-18.7] vs. 3.9 months [IQR: 1.8-10.4]; P=0.037). In the multivariate analysis, multiple recurrence patterns emerged as independent prognostic factors for poor PRS (hazard ratio, 1.553; 95% confidence interval, 1.092-2.208; P=0.014) along with serosal invasion, recurrence within 1 year after gastrectomy, and the absence of post-recurrence chemotherapy. CONCLUSIONS: Regardless of the specific recurrence pattern, multiple recurrence patterns emerged as independent prognostic factors for poor PRS compared with a single recurrence pattern.

Trust your gut: Establishing confidence in gastrointestinal models - An overview of the state of the science and contexts of use.

The webinar series and workshop titled Trust Your Gut: Establishing Confidence in Gastrointestinal Models - An Overview of the State of the Science and Contexts of Use was co-organized by NICEATM, NIEHS, FDA, EPA, CPSC, DoD, and the Johns Hopkins Center for Alternatives to Animal Testing (CAAT) and hosted at the National Institutes of Health in Bethesda, MD, USA on October 11-12, 2023. New approach methods (NAMs) for assessing issues of gastrointestinal tract (GIT)-related toxicity offer promise in addressing some of the limitations associated with animal-based assessments. GIT NAMs vary in complexity, from two-dimensional monolayer cell line-based systems to sophisticated 3-dimensional organoid systems derived from human primary cells. Despite advances in GIT NAMs, challenges remain in fully replicating the complex interactions and processes occurring within the human GIT. Presentations and discussions addressed regulatory needs, challenges, and innovations in incorporating NAMs into risk assessment frameworks; explored the state of the science in using NAMs for evaluating systemic toxicity, understanding absorption and pharmacokinetics, evaluating GIT toxicity, and assessing potential allergenicity; and discussed strengths, limitations, and data gaps of GIT NAMs as well as steps needed to establish confidence in these models for use in the regulatory setting.

Non-animal methods to assess whether chemicals may be toxic to the human digestive tract promise to complement or improve on animal-based methods. These approaches, which are based on human or animal cells and/or computer models, are faced with their own technical challenges and need to be shown to predict adverse effects in humans. Regulators are tasked with evaluating submitted data to best protect human health and the environment. A webinar series and workshop brought together scientists from academia, industry, military, and regulatory authorities from different countries to discuss how non-animal methods can be integrated into the risk assessment of drugs, food additives, dietary supplements, pesticides, and industrial chemicals for gastrointestinal toxicity.