Thiopurine is metabolized to 6-thio-(deoxy) guanosine triphosphate (6-thio-(d) GTP), which is then incorporated into DNA or RNA and causes cytotoxicity. Nudix hydrolase 15 (NUDT15) reduces the cytotoxic effects of thiopurine by converting 6-thio-(d) GTP to 6-thio-(d) guanosine monophosphate (6-thio-(d) GMP). NUDT15 polymorphisms like the Arg139Cys variant are strongly linked to thiopurine-induced severe leukocytopenia and alopecia. Therefore, measurement of NUDT15 enzymatic activity in individual patients can help predict thiopurine tolerability and adjust the dosage. We aimed to develop a quantitative assay for NUDT15 enzymatic activity in human blood samples. Blood samples were collected from donors whose NUDT15 genetic status was determined. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to assess the 6-thio-GTP metabolic activity in cell extracts. Because 6-thio-guanosine diphosphate (6-thio-GDP) and 6-thio-GMP were generated upon incubation of 6-thio-GTP with human blood cell extracts, the method detecting 6-thio-GTP, 6thio-GDP, and 6-thio-GMP was validated. All three metabolites were linearly detected, and the lower limit of quantification (LLOQ) of 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 5 mu M, 1 mu M, and 2 mu M, respectively. Matrix effects of human blood cell extracts to detect 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 99.0 %, 100.5 %, and 101.4 %, respectively, relative to the signals in the absence of blood cell extracts. The accuracy and precision of the method and the stability of the samples were also assessed. Using this established method, the genotype-dependent differences in NUDT15 activities were successfully determined using cell extracts derived from human blood cells with NUDT15 wild-type (WT) or Arg139Cys variant and 6-thio-GTP (100 mu M) as a substrate (18.1, 14.9, and 6.43 mu M/h/106 cells for WT, Arg139Cys heterozygous, and homozygous variant, respectively). We developed a method for quantifying intracellular NUDT15 activity in peripheral blood mononuclear cells (PBMCs), which we defined as the conversion of 6-thio-GTP to 6-thio-GMP. Although PBMCs preparation takes some time, its reproducibility in experiments makes it a promising candidate for clinical application. This method can tell the difference between WT and Arg139Cys homozygous blood samples. Even in

INTRODUCTION:Cancer genome analysis using next-generation sequencing requires adequate and high-quality DNA samples. Genomic analyses were conventionally performed using formalin-fixed paraffin-embedded sections rather than cytology samples such as cell block or smear specimens. Specimens collected from liquid-based cytology (LBC) have the potential to be sources of high-quality DNA suitable for genetic analysis even after long-term storage.METHODS:We collected breast tumor/lesion fractions from 92 residual LBC specimens using fine-needle aspiration (FNA) biopsy, including breast carcinoma (1 invasive carcinoma and 4 ductal carcinomas in situ), papillomatous lesion (5 intraductal papillomas), and fibroepithelial lesion (19 phyllodes tumors and 53 fibroadenomas) samples, and others (1 ductal adenoma, 1 hamartoma, 1 fibrocystic disease, and 7 unknown). DNA was extracted from all samples and subjected to DNA integrity number (DIN) score analysis.RESULTS:Average DIN score collected from 92 LBC specimens was significantly higher score. In addition, high-quality DNA with high DIN values (7.39 ± 0.80) was successfully extracted more than 12 months after storage of residual LBC specimens.CONCLUSION:Residual LBC specimens collected from FNA of the breast were verified to carry high-quality DNA and could serve as an alternate source for genetic analysis.

A list of sgRNAs in a small-scale custom library

Figure S1. Overview of the CRISPR/Cas9 screen performed in EGFR mutation positive NSCLC PC9 cells. Figure S2. Cell viability assay of cytotoxic agents or ALK-TKI in SHOC2-depleted cells. Figure S3. Supplementary data for domain analysis. Figure S4. Supplementary data for phospho-proteomics analysis. Figure S5. Cell viability assay of celastrol in PC9 and H1975 cells. Figure S6. Supplementary data for xenograft model. Supplemental Table Legends 1-3

Background: Clarifying the presence of viable severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rather than SARS-CoV-2 viral RNA in inpatient rooms is important for infection control of coronavirus disease 2019 (COVID-19). In this study, we investigated levels of viral RNA and viable virus on environmental surfaces and in patient saliva. Methods: Environmental samples from 23 sites in hospital rooms were collected every other day until patient discharge. Saliva specimens and samples from the inner surface of patient masks were also collected. Additionally, environmental samples were collected from 46 sites in hospital rooms on discharge day. The samples were examined using quantitative reverse transcription polymerase chain reaction (RT-qPCR) and plaque assays. Results: The 10 enrolled cases were classified as mild COVID-19, and patients were discharged after 6-9 days. The viral RNA was detected in 12.4% (105/849) of serially collected environmental samples during hospitalization, whereas viable virus was detected only in 0.47% (4/849), which were from sinks and tap levers. Although all patients recovered, three cases retained viable virus in the last saliva specimen collected. In the 15 discharged rooms, viral RNA was detected in 6.6% (45/682) of the samples, and viable virus was detected in only one sample from the sink. Conclusions: Although environmental surfaces surrounding patients with COVID-19 were frequently contaminated with viral RNA, the presence of viable virus was rare and limited only to areas around sinks. These results suggest that contact infection risk via fomites in hospital rooms is extremely rare.

A list of genes would enhance the cytotoxic effect of erlotinib when they are depleted ranked by STARS algorithm.

The combination of osimertinib and celastrol demonstrated a synergistic anti-proliferative effect in PC9 cells and H1975 cells.

Although many therapeutic options are available for inflammatory bowel disease (IBD), 5-aminosalicylic acid (5-ASA) is still the key medication, particularly for ulcerative colitis (UC). However, the mechanism of action of 5-ASA remains unclear. The intestinal microbiota plays an important role in the pathophysiology of IBD, and we hypothesized that 5-ASA alters the intestinal microbiota, which promotes the anti-inflammatory effect of 5-ASA. Because intestinal inflammation affects the gut microbiota and 5-ASA can change the severity of inflammation, assessing the impact of inflammation and 5-ASA on the gut microbiota is not feasible in a clinical study of patients with UC. Therefore, we undertook a translational study to demonstrate a causal link between 5-ASA administration and alterations of the intestinal microbiota. Furthermore, by rigorously controlling environmental confounders and excluding the effect of 5-ASA itself with a vertical transmission model, we observed that the gut microbiota altered by 5-ASA affected host mucosal immunity and decreased susceptibility to dextran sulfate sodium-induce colitis. Although the potential intergenerational transmission of epigenetic changes needs to be considered in this study, these findings suggested that alterations in the intestinal microbiota induced by 5-ASA directed the host immune system towards an anti-inflammatory state, which underlies the mechanism of 5-ASA efficacy.

Thiopurines are often used in combination with mesalazine for the treatment of ulcerative colitis (UC). Mesalazine formulations are delivered to the digestive tract by various delivery systems and absorbed as 5‐aminosalicylic acid (5‐ASA). 5‐ASA is known to inhibit thiopurine S‐methyltransferase (TPMT) activity and to affect thiopurine metabolism. There have been no studies comparing TPMT inhibition by multimatrix mesalazine (MMX) with other formulations. We investigated the difference in TPMT inhibition by different mesalazine formulations and prospectively confirmed the clinical relevance.

Polymorphisms in the nucleotide diphosphate-linked moiety X-type motif 15 (NUDT15) gene are associated with thiopurine-induced leukopenia in patients with inflammatory bowel disease (IBD). NUDT15-associated subcellular thiopurine metabolism has not been investigated in primary lymphocytes. We hypothesized that NUDT15 mutation increases DNA-incorporated deoxythioguanosine (dTG) and induces apoptosis in lymphocytes. DNA-incorporated dTG in peripheral blood mononuclear cells (PBMCs) and 6-thioguanine nucleotides (6-TGN) in red blood cells were measured in patients with IBD undergoing thiopurine treatment. The association of a single nucleotide polymorphism for NUDT15 (rs116855232) with dTGPBMC was examined. The pro-apoptotic effect of DNA-incorporated dTG was examined ex vivo in association with NUDT15 genotypes by co-culturing patient-derived peripheral CD4+ T lymphocytes with 6-thioguanine (6-TG). dTGPBMC was significantly higher in NUDT15 variants than in non-variants. dTGPBMC, but not 6-TGNRBC, negatively correlated with peripheral lymphocyte counts (r =  – 0.31 and  – 0.12, p = 0.012 and 0.173, respectively). DNA-incorporated dTG significantly accumulated to a greater extent in lymphocytes from NUDT15 variants when co-cultured with 6-TG ex vivo than in those from non-variants and was associated with decreased proliferation and increased apoptosis. Increased DNA-incorporated dTG may be responsible for thiopurine-induced leukocytopenia through cell apoptosis in IBD patients with NUDT15 mutation.

Abstract EGFR mutation-positive patients with non–small cell lung cancer (NSCLC) respond well to treatment with EGFR–tyrosine kinase inhibitors (EGFR–TKI); however, treatment with EGFR–TKIs is not curative, owing to the presence of residual cancer cells with intrinsic or acquired resistance to this class of drugs. Additional treatment targets that may enhance the efficacy of EGFR–TKIs remain elusive. Using a CRISPR/Cas9-based screen, we identified the leucine-rich repeat scaffold protein SHOC2 as a key modulator of sensitivity to EGFR–TKI treatment. On the basis of in vitro assays, we demonstrated that SHOC2 expression levels strongly correlate with the sensitivity to EGFR–TKIs and that SHOC2 affects the sensitivity to EGFR–TKIs in NSCLC cells via SHOC2/MRAS/PP1c and SHOC2/SCRIB signaling. The potential SHOC2 inhibitor celastrol phenocopied SHOC2 depletion. In addition, we confirmed that SHOC2 expression levels were important for the sensitivity to EGFR–TKIs in vivo. Furthermore, IHC showed the accumulation of cancer cells that express high levels of SHOC2 in lung cancer tissues obtained from patients with NSCLC who experienced acquired resistance to EGFR–TKIs. These data indicate that SHOC2 may be a therapeutic target for patients with NSCLC or a biomarker to predict sensitivity to EGFR–TKI therapy in EGFR mutation-positive patients with NSCLC. Our findings may help improve treatment strategies for patients with NSCLC harboring EGFR mutations. Implications: This study showed that SHOC2 works as a modulator of sensitivity to EGFR–TKIs and the expression levels of SHOC2 can be used as a biomarker for sensitivity to EGFR–TKIs.

Background] Fecal calprotectin (FC) and fecal immunochemical tests (FIT) have the disadvantage of requiring fecal samples.It has been reported that prostaglandin E-major urinary metabolite (PGE-MUM) values correlate with Mayo endoscopic scores (MESs) for ulcerative colitis (UC).However, there has been no report that PGE-MUM can determine endoscopic remission under remission phase UC, nor comparative study of PGE-MUM with FC and FIT.Thus, we aimed to examine the association between PGE-MUM values and the colonoscopy (CS) results of patients in the remission phase of UC, and to compare the accuracy of using PGE-MUM versus that of using FC or FIT for determining endoscopic remission.[Method] UC Patients who were under clinical remission (Simple Clinical Colitis Activity Index < 2) and had planned to undergo CS from August 2017 to March 2019 were enrolled.FC levels were measured and FITs were performed on the day of CS; PGE-MUM was measured either the day before or the day after CS.Three physicians independently scored the CS findings (MES, Modified Mayo Endoscopic Score [MMES], and UC endoscopic index of severity [UCEIS]) while blinded from clinical information.We used the Wilcoxin ranksum test to analyze the differences in PGE-MUM values between two groups, which were divided between those that did achieve and those that did not achieve the following scores: 1) MES 0 point, 2) MES £ 1 point, 3) modified MES 0 point, 4) modified MES £1 point, 5) UCEIS 0 point, 6) UCEIS £ 2 points.In addition, the accuracy of PGE-MUM, FC, and FIT with respect to determining the achievement of 1) through 6) were compared by using areas under the receiver operating characteristics curves.Patients with altered UC activity between the day of PGE-MUM measurement and CS, and those who received NSAIDs on the day of PGE-MUM measurement, were excluded from the analysis.[Results] Of the 125 enrolled subjects, 30 patients were excluded (urine specimens not submitted, 11; poor stool specimens, 6; NSAIDs users, 10; clearly altered UC activity, 3).The remaining 95 patients (average age 48.2 years, 57 males, and 54 patients with total colitis type), were eligible for analysis.The median PGE-MUM values (in µg/gCr) for groups that did or did not achieve 1) through 6) were as follows: 1) 14.6/17.2,p = 0.106; 2) 14.9/20.5,p = 0.039; 3) 14.5/ 17.4, p = 0.059; 4) 14.1/21.8,p < 0.001; 5) 14.5/17.4,p = 0.059; 6) 14.7/22.2,p = 0.003.The areas under the receiver operating characteristics curves for PGE-MUM/FC/FIT used for determining the achievement of 1) through 6) were as follows: 1) 0.597/0.664/0.682,2) 0.692/0.74/0.825,3) 0.613/0.686/0.692,4) 0.794/0.82/0.786,5) 0.613/0.686/0.692and 6) 0.778/0.824/0.825.[Conclusion] PGE-MUM, which is measured using urine samples, is equally as effective as FC and FIT for determining the achievement of endoscopic remission. Su1933

Background/Aims: The pharmacokinetics of tacrolimus (TAC) is known to be largely influenced by single-nucleotide polymorphisms (SNPs) in CYP3A5. Patients starting TAC require careful dose adjustment, owing to the wide range of optimal dosages, depending on their CYP3A5 expression status. Here, we evaluated whether individualization of TAC dosages based on CYP3A5 SNPs would improve its therapeutic efficacy in ulcerative colitis. Methods: Twenty-one patients were prospectively treated, with their initial dosage adjusted according to their CYP3A5 status (0.1, 0.15, and 0.2 mg/kg/day for CYP3A5*3/*3, CYP3A5*1/*3, and CYP3A5*1/*1, respectively). Their clinical outcomes were compared with those of patients treated with a fixed dose (0.1 mg/kg/day). Results: The first blood trough level of CYP3A5 expressors, CYP3A5*1/*3 or CYP3A5*1/*1, and the overall rate in achieving the target blood trough level within a week in the individualized-dose group were significantly higher than those in the fixed-dose group (5.15 +/- 2.33 ng/mL vs. 9.63 +/- 0.79 ng/mL, P =0.035 and 12.5% vs. 66.7%, P =0.01). The remission rate at 2 weeks in the expressors was as high as that in the nonexpressors, CYP3A5*3/*3, in the individualized-dose group. Conclusions: Individualized TAC treatment is effective against ulcerative colitis regardless of the CYP3A5 genotype.

BackgroundSecond-generation colon capsule endoscopy (CCE-2) has been reported as a potential tool for monitoring ulcerative colitis (UC). However, its excretion rate is still unsatisfactory, and the bowel preparation regimen is not well tolerated. Furthermore, a standard bowel preparation regimen validated for UC has not been established. The aim of this study was to develop a simple 1-day CCE-2 procedure while evaluating its excretion rate and acceptability in UC. Factors associated with the colonic transit time and acceptability of CCE-2 were evaluated.MethodsThirty-three patients were prospectively evaluated. Five hundred milliliters of hypertonic polyethylene glycol solution, followed by 250 mL of water, was ingested 2.5 hours before, then 1, 3, and 6 hours after capsule ingestion until its excretion, with castor oil added to the second ingestion. Mayo endoscopic subscore (MES) and Ulcerative Colitis Endoscopic Index of Severity (UCEIS) were graded, and their correlations with fecal calprotectin (FC) were assessed. A questionnaire comparing CCE-2 with previous colonoscopy (CS) was conducted.ResultsThe excretion rate was 93.9% (31/33). The acceptability of CCE-2 was superior to CS (CCE-2 42.4% vs CS 27.3%). The median colonic transit time was 119 minutes and showed a positive correlation with MES (P = 0.010), UCEIS (P = 0.010), and FC (P = 0.041). CCE-2 was not favored by patients whose colonic transit times were longer.ConclusionsA novel bowel preparation regimen of CCE-2 was well tolerated, with a high excretion rate, by UC patients. Patients with active disease required longer colonic transit time, which may have resulted in the lower acceptability of CCE-2.

Introduction: It is well accepted that fecal calprotectin (fCal) is a non-invasive biomarker, which reflects mucosal inflammation in ulcerative colitis (UC). However, its value as a marker of response during the early phase of induction treatment has not been well studied. Therefore, in the present study, fCal levels were monitored in combination with clinical activity assessment indices during the induction treatment to evaluate its role as a predictor for early response to treatment for UC. In addition, within-stool and within-day variability were assessed to evaluate reliability of fCal during the acute phase of UC. Methods: 1) Sixteen consecutive patients diagnosed with active UC undergoing induction treatment (prednisolone 8, anti-TNF 2, tacrolimus 6) were included in the study. fCal and clinical activity scores (Lichtiger index, LI and partial Mayo score; pMayo) were prospectively followed up for 12 weeks. Clinical response was defined based on LI at week 12. fCal was measured by fluoro enzyme immunoassay (FEIA) using Phadia 250. 2) Within-stool and within-day variability were assessed by collecting fecal samples 4 times a day (n=10). A coefficient of variation was calculated for each stool sample and sampling day. Data are shown as mean±SD%. Results: 1) A significant decrease of fCal levels was observed after 4 weeks of treatment, however LI and pMayo were decreased much earlier in patients who demonstrated clinical response at week 12 (n=9). There was no significant decrease of fCal levels in non-responders at week 12 (n=7). 2) Within-stool variability was 17.4±17.0% and within-day variability was 55.6±18.6%. Conclusion: fCal levels decreased with ongoing induction therapy and corresponded to response to treatment, but was less sensitive than clinical activity indices in detecting response during the early phase of treatment. Significant within-day variability in active UC patients might make sensitive detection of early change difficult. In conclusion, fCal is a useful non-invasive biomarker to help predict response to induction therapy in UC, however, it is not sensitive enough to detect the early change within a few weeks.

specificity of 89.29%, PPV 89.66% and NPV 86.21% in detecting DL.DSC had a sensitivity of 66.67%, specificity of 88.57%, PPV 50%, NPV 93.94% and EVC had a sensitivity of 100%, specificity of 83.3%, PPV 64.71% and NPV 100%.Conclusions: DL are frequently detected in long-standing IBD.Our Results indicate that DSC is not more accurate than either HD or VEC in detecting DL.In fact, the majority of dysplastic lesions were detected in the HD group, suggesting that advances in high definition technology may favour this technique as the surveillance method of choice for IBD in future.