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PDF file, 779K, Figure S1. Expressionofp-KIT,p-AKT,andp-ERK increased according to the increased SCF concentrationin two KIT-expressingcelllines. Figure S2. Expression of KIT, p-KIT, p-AKT, and p-ERK after SCF treatment in two tumor cell lines with KIT mutation. FigureS3.Quantification of Westernblotting results for Fig.1E. FigureS4. Membranous KITexpression of DLD-1 was determined by FACS. Figure S5. Mutant KIT is not degraded after SCF treatment. Figure S6. FACS analysis of p-AKT and p-ERK expressing colon cancer cells treated with SCF and PMA. Figure S7. FACS analysis of KIT expression in the DLD-1 cells transfectedwith wild-type (Rab11-WT) or dominant-negative mutant form of Rab11 (Rab11-S25N). Figure S8. Effects of imatinibon CRC cells expressing KIT. FigureS9. DLD-1 cell number and invasion were increased after SCF and PMA treatment. FigureS10. Expression of SCF mRNA expression in CRC cell lines. Figure S11.Colon cancers expressing WT-KIT constantly generated activated KIT-SCF signaling as a result of KIT recycling in a condition of endogenous PKC-delta activation.

Supplementary Information - PDF file 587K, Figure S1. Semiquantitative RT-PCR analysis of the selected genes in 10 MSI-H and 2 MSS cell lines. Figure S2. Endogenous levels of wild type TTK, TCF7L2, and MARCKS proteins decrease according to the mutation status. Figure S3. Generation of an antibody specific for neopeptides of mutant MARCKS. Figure S4. Endogenous mutant TTK, TCF7L2, and MARCKS are detected after proteasome inhibition. Figure S5. Inhibition of proteasome degradation by MG132 increased the expression of the mutants derived from the cDNA expression constructs. Figure S6. Generation of neopeptides from frameshift mutations in 29 genes that displayed frequent insertion/deletion mutations. Table S1: Primers used for RT-PCR and construction of expression vectors. Table S2. List of genes containing coding mononucleotide repeats longer than 9 nt in the last exon

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PDF file, 160K, Supplementary Table 1. Primer and siRNA sequences Supplementary Table 2. Clinicopathological characteristics of 250 colorectal cancers according to KIT expression.

Purpose: Abnormal signaling through receptor tyrosine kinase (RTK) moieties is important in tumorigenesis and drug targeting of colorectal cancers. Wild-type KIT (WT-KIT), a RTK that is activated upon binding with stem cell factor (SCF), is highly expressed in some colon cancers; however, little is known about the functional role of SCF-dependent KIT activation in colon cancer pathogenesis. We aimed to elucidate the conditions and roles of WT-KIT activation in colon cancer tumorigenesis.Experimental Design: Colorectal cancers with KIT expression were characterized by immunoblotting and immunohistochemistry. The biologic alterations after KIT-SCF binding were analyzed with or without protein kinase C (PKC) activation.Results: We found that WT-KIT was expressed in a subset of colon cancer cell lines and was activated by SCF, leading to activation of downstream AKT and extracellular signal-regulated kinase (ERK) signaling pathways. We also showed that KIT expression gradually decreased, after prolonged SCF stimulation, due to lysosomal degradation. Degradation of WT-KIT after SCF binding was significantly rescued when PKC was activated. We also showed the involvement of activated PKC-δ in the recycling of WT-KIT. We further showed that a subset of colorectal cancers exhibit expressions of both WT-KIT and activated PKC-δ and that expression of KIT is correlated with poor patient survival (P = 0.004).Conclusions: Continuous downstream signal activation after KIT-SCF binding is accomplished through PKC-δ–mediated recycling of KIT. This sustained KIT activation may contribute to tumor progression in a subset of colon cancers with KIT expression and might provide the rationale for a therapeutic approach targeting KIT. Clin Cancer Res; 19(18); 4961–71. ©2013 AACR.

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Purpose: Frameshift mutations in coding mononucleotide repeats (cMNR) are common in tumors with high microsatellite instability (MSI-H). These mutations generate mRNAs containing abnormal coding sequences and premature termination codons (PTC). Normally, mRNAs containing PTCs are degraded by nonsense-mediated mRNA decay (NMD). However, mRNAs containing PTCs located in the last exon are not subject to degradation by NMD (NMD-irrelevant). This study aimed to discover whether genes with frameshift mutations in the last exon generate truncated mutant proteins.Experimental Design: We identified 66 genes containing cMNRs in the last exon by bioinformatic analysis. We found frequent insertion/deletion mutations in the cMNRs of 29 genes in 10 MSI-H cancer cell lines and in the cMNRs of 3 genes in 19 MSI-H cancer tissues. We selected 7 genes (TTK, TCF7L2, MARCKS, ASTE1, INO80E, CYHR1, and EBPL) for mutant mRNA expression analysis and 3 genes (TTK, TCF7L2, and MARCKS) for mutant protein expression analysis.Results: The PTC-containing NMD-irrelevant mRNAs from mutated genes were not degraded. However, only faint amounts of endogenous mutant TTK and TCF7L2 were detected, and we failed to detect endogenous mutant MARCKS. By polysome analysis, we showed that mRNAs from genomic mutant MARCKS constructs are normally translated. After inhibiting 3 protein degradation pathways, we found that only inhibition of the proteasomal pathway facilitated the rescue of endogenous mutant TTK, TCF7L2, and MARCKS.Conclusions: Our findings indicate that cancer cells scavenge potentially harmful neopeptide-containing mutant proteins derived from NMD-irrelevant abnormal mRNAs via the ubiquitin–proteasome system, and these mutant proteins may be important substrates for tumor-specific antigens. Clin Cancer Res; 19(13); 3369–82. ©2013 AACR.

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Purpose: Gain-of-function mutations and KIT overexpression are well-known tumorigenesis mechanisms in gastrointestinal stromal tumors (GIST). This study aimed to discover microRNAs (miRNA) that target KIT and reveal the relationship between the discovered miRNAs and KIT expression in GISTs.Experimental Design: Fresh-frozen GISTs from 31 patients were used to confirm the relationship between miR-494 and KIT expression using quantitative reverse transcription-PCR to assess miR-494 expression levels and Western blotting to assess KIT protein expression levels. A luciferase assay was conducted for the target evaluation. The functional effects of miR-494 on GIST882 cells (GIST cell line with activating KIT mutation) were validated by a cell proliferation assay and fluoresce-activated cell sorting analysis.Results: An inverse relationship was found between the expression levels of miR-494 and KIT in GISTs (r = −0.490, P = 0.005). The direct targeting of KIT by miR-494 was shown by the reduction in KIT expression after miR-494 overexpression and the increase in KIT expression after inhibiting endogenous miR-494 expression. We showed that miR-494 regulates KIT by binding two different seed match sites. Induced miR-494 overexpression in GIST882 reduced the expression of downstream molecules in KIT signaling transduction pathways, including phospho-AKT and phospho-STAT3. Finally, miR-494 overexpression provoked apoptosis and inhibited GIST cell growth, which were accompanied by changes in G1 and S phase content.Conclusion: Our findings indicate that miR-494 is a negative regulator of KIT in GISTs and overexpressing miR-494 in GISTs may be a promising approach to GIST treatment. Clin Cancer Res; 17(24); 7584–94. ©2011 AACR.

In addition to the absence of a domestic definition of bioelectronic medicine, there are cases in which digital medicine and bioelectronic medicine are confused and used because the technology that stimulates the nerves or the clear mechanism has not been identified. It is necessary to suggest a direction for R&D that can induce continuous growth and lead to industrial performance through analysis of domestic technological competitiveness and market ecosystem through domestic and international status surveys. In addition, due to the lack of research on bioelectronic medicine, information on development and research trends is provided and future research directions are set.

BackgroundWe describe two unrelated patients who display similar clinical features including telangiectasia, ectodermal dysplasia, brachydactyly and congenital heart disease.MethodsWe performed trio whole exome sequencing and functional analysis using in vitro kinase assays with recombinant proteins.ResultsWe identified two different de novo mutations in protein kinase D1 (PRKD1, NM_002742.2): c.1774G>C, p.(Gly592Arg) and c.1808G>A, p.(Arg603His), one in each patient. PRKD1 (PKD1, HGNC:9407) encodes a kinase that is a member of the protein kinase D (PKD) family of serine/threonine protein kinases involved in diverse cellular processes such as cell differentiation and proliferation and cell migration as well as vesicle transport and angiogenesis. Functional analysis using in vitro kinase assays with recombinant proteins showed that the mutation c.1808G>A, p.(Arg603His) represents a gain-of-function mutation encoding an enzyme with a constitutive, lipid-independent catalytic activity. The mutation c.1774G>C, p.(Gly592Arg) in contrast shows a defect in substrate phosphorylation representing a loss-of-function mutation.ConclusionThe present cases represent a syndrome, which associates symptoms from several different organ systems: skin, teeth, bones and heart, caused by heterozygous de novo mutations in PRKD1 and expands the clinical spectrum of PRKD1 mutations, which have hitherto been linked to syndromic congenital heart disease and limb abnormalities.

Background We investigated the clinical course of patients with prolonged disorders of consciousness (PDoC), predictors of emergence from PDoC (EDoC), and the temporal dynamics of six neurobehavior domains based on the JFK Coma Recovery Scale-Revised (CRS-R) during the recovery. Methods A total of 50 traumatic and non-traumatic patients with PDoC were enrolled between October 2014 and February 2017. A retrospective analysis of the clinical findings and neurobehavioral signs was conducted using standardized methodology such as CRS-R. The findings were used to investigate the incidence and predictors of EDoC and determine the cumulative pattern of neurobehavioral recovery at 6 months, 1 year, and 2 years post-injury. Results The results showed that 46% of the subjects emerged from PDoC after 200 median days (64–1197 days) of injury onset. The significant predictors of EDoC included minimally conscious state (MCS) (vs. vegetative state), higher auditory, communication, arousal, total CRS-R scores, shorter lag time post-injury, and the absence of intra-axial lesions. In terms of cumulative recovery of motor and communication signs in patients who emerged from PDoC, 39 and 32% showed EDoC at 6 months post-injury, and 88 and 93% exhibited EDoC at 2 years post-injury, respectively. Conclusions Nearly half of the patients with PDoC recovered consciousness during inpatient rehabilitation. MCS, shorter lag time, the absence of intra-axial lesions, higher auditory, communication, arousal, and total CRS-R scores were important predictors for EDoC. Motor scores in the early stage of recovery and communication scores after prolonged intervals contributed to the higher levels of cumulative EDoC.

Optimal treatments for multiple myeloma (MM) patients with high-risk cytogenetics must be determined, but subgroup features are not well-defined. We used real-world data from the Korean Myeloma Registry (KMR) to analyze the characteristics and clinical outcomes of newly diagnosed MM patients with ≥ 1 high-risk cytogenetic abnormality: Group 1: t(4;14) or t(14;16); Group 2: del(17p); Group 3: t(4;14)/del(17p) or t(14;16)/del(17p). Overall, 347 high-risk patients were identified (males, 48.7%; median age, 63 years). Median progression-free survival (PFS) and overall survival (OS) were 19.0 months (95% CI 17.0-20.0) and 50.0 months (95% CI 37.0-61.0), respectively. PFS (p = 0.047) and OS (p = 0.020) differed significantly between groups. After stratification by transplant eligibility, PFS and OS were significantly poorer in Group 3 among transplant-eligible patients, and even poorer in those with gain(1q). Patients stratified by cytogenetic abnormality and revised International Staging System (R-ISS) had significantly different PFS (p 0.001) and OS (p = 0.003), with the worst survival in R-ISS III/Group 3 (median OS 21.0 months). Higher number of high-risk cytogenetic abnormalities was a negative prognostic marker for PFS and OS (p 0.001). Real-world KMR data showed that risk factors for poor prognosis of MM patients included del(17p), R-ISS stage, and number of cytogenetic abnormalities.

Preclinical evaluation models have been developed for precision medicine, with patient-derived xenograft models (PDXs) and patient-derived organoids (PDOs) attracting increasing attention. However, each of these models has application limitations. In this study, an advanced xenograft model was established and used for drug screening. PDO and endothelial colony-forming cells (ECFCs) were cotransplanted in NRGA mice (PDOXwE) to prepare the model, which could also be subcultured in Balb/c nude mice. Our DNA sequencing analysis and immunohistochemistry results indicated that PDOXwE maintained patient genetic information and tumor heterogeneity. Moreover, the model enhanced tumor growth more than the PDO-bearing xenograft model (PDOX). The PDO, PDOXwE, and clinical data were also compared in the liver metastasis of a colorectal cancer patient, demonstrating that the chemosensitivity of PDO and PDOXwE coincided with the clinical data. These results suggest that PDOXwE is an improvement of PDOX and is suitable as an evaluation model for precision medicine.

Radiotherapy is a leading treatment for various types of cancer. However, exposure to high-dose ionizing radiation causes acute gastrointestinal injury and gastrointestinal syndrome. This has significant implications for human health, and therefore, radioprotection is a major area of research. Radiation induces the loss of intestinal stem cells; hence, the protection of stem cells expressing LGR5 (a marker of intestinal epithelial stem cells) is a key strategy for the prevention of radiation-induced injury. In this study, we identified valproic acid (VPA) as a potent radioprotector using an intestinal organoid culture system. VPA treatment increased the number of LGR5+ stem cells and organoid regeneration after irradiation. N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT, an inhibitor of NOTCH signaling) blocked the radioprotective effects of VPA, indicating that NOTCH signaling is a likely mechanism underlying the observed effects of VPA. In addition, VPA acted as a radiosensitizer via the inhibition of histone deacetylase (HDAC) in a colorectal cancer organoid. These results demonstrate that VPA exerts strong protective effects on LGR5+ stem cells via NOTCH signaling and that the inhibition of NOTCH signaling reduces these protective effects, providing a basis for the improved management of radiation injury.

Background: Behçet's disease (BD) is a chronic inflammatory systemic disease that affects multiple organs. The causes of BD are still unknown, but it is primarily characterized by autoimmune reaction in the blood vessels. Current research focuses on treatments that can reduce the non-typical inflammatory responses of BD. Nevertheless, studies on improving the inflammatory effect of BD using inflammation mechanisms are still insufficient. Therefore, we conducted the integrated treatments related to inflammation modulation and achieved alleviation of symptoms in BD mice. Methods: To understand the complex etiology of BD and compare its management, the herpes simplex virus (HSV)-induced BD mouse model was used. In order to alleviate the inflammatory response in BD mice, Taraxaci Herba (TH, herbal medicine), R7050-a TNFα inhibitor, and a mixture of TH and R7050 were injected for 2 weeks repetitively. The SCORAD index was examined to evaluate the cutaneous inflammations. In addition, histological changes and inflammatory factors were analyzed. Results: Repetitive injection of TH and/or R7050 reduced the symptoms of BD and significantly decreased IL-6, IL-1β, and TNFα in blood sera. Moreover, this treatment reduced the ulcers and the deterioration of skin. Conclusions: The results of our study showed that the down-regulation of inflammatory factors is related to the control of immune responses in BD models, suggesting that a mixed drug treatment may be more effective in improving the condition of BD.

Additional file 2. Summary of our bimaxillary orthognathic surgery protocol.

Additional file 3. Comparison of perioperative inflammatory and coagulation findings between the two groups.

Additional file 1. Consolidated Standards of Reporting Trials (CONSORT) guidelines.

For sustainable soil management, there is an increasing demand for soil quality, resilience, and health assessment. After remediation of petroleum hydrocarbon (PHC)-contaminated soils, changes in the physicochemical and ecological characteristics of the soil were investigated. Two kinds of remediation technologies were applied to contaminated soils: land farming (LF) and high temperature thermal desorption (HTTD). As a result of total petroleum hydrocarbons (TPH), PHC-contaminated soils were efficiently remediated by LF and HTTD. The soil health could not be completely recovered after the removal of pollutants due to adverse changes in the soil properties, especially in soil enzyme activities. Therefore, monitoring is necessary for accurate estimation of soil ecotoxicity and effective remediation, and additional soil management, such as fertilizer application or organic amendments, is needed to restore soil heath. In the case of HTTD, soil ecological properties are severely changed during the remediation process. The decision to reuse or recycle remediated soils should reflect changes in soil quality. HTTD is a harsh remediation method that results in deterioration of soil fertility and ecological functions. Alternatives, such as low-temperature thermal desorption or additional soil management using fertilizer or organic amendments, for example, are needed.

Background Previous studies have reported the efficacy and safety of intravenous (IV) iron therapy during the perioperative period as an alternative and adjunct to allogeneic blood transfusion. Preemptive IV iron therapy provides noninferior hemoglobin levels on postoperative day (POD) 1 compared to autologous whole blood therapy (AWBT) in healthy patients who had undergone bimaxillary orthognathic surgery. Methods This was a prospective, patient-randomized, noninferiority trial. After excluding 2 patients, 64 patients were divided into two groups: the IV iron therapy group (patients received IV iron infusion 4 weeks before surgery; n = 32) and the AWBT group (2 units of autologous whole blood were collected 4 and 2 weeks before surgery; n = 32). The primary outcome was hemoglobin level on POD 1 and the prespecified noninferiority limit was − 1 g/dL. Results Baseline data were comparable, including hemoglobin and iron levels, between the two groups. Immediately before surgery, the levels of hemoglobin, iron, and ferritin were higher in the IV iron group than in the AWBT group. The mean treatment difference (iron group—whole blood group) in hemoglobin level on POD 1 between the two groups was 0.09 (95% CI = − 0.83 to 1.0). As the lower limit of the 95% CI (− 0.83) was higher than the prespecified noninferiority margin (δ = − 1), noninferiority was established. On POD 2, the hemoglobin level became lower in the iron group, which eventually led to greater requirement of allogeneic blood transfusion compared to the whole blood group. However, the iron group did not require allogeneic blood transfusion during or early after surgery, and the whole blood group showed continuously higher incidence of overt iron deficiency compared to the iron group. Conclusion As collection of autologous whole blood caused overt iron loss and anemia before surgery and intraoperative transfusion of whole blood was not able to prevent the occurrence of persistent iron deficiency after surgery, IV iron therapy was found to have potential benefits for iron homeostasis and subsequent erythropoiesis in healthy patients early after bimaxillary orthognathic surgery. Trial registration: Clinical Research Information Service, Republic of Korea, approval number: KCT0003680 on March 27, 2019. https://cris.nih.go.kr/cris/search/search_result_st01_kren.jsp?seq=15769&sLeft=2&ltype=my&rtype=my.

Background This study analyzed remnant kidney function recovery in living donors after laparoscopic nephrectomy to establish a risk stratification model for delayed recovery and further investigated clinically modifiable factors. Patients and methods This retrospective study included 366 adult living donors who underwent elective donation surgery between January 2017 and November 2019 at our hospital. ITMB was included as an analgesic component in the living donor strategy for early postoperative pain relief from November 2018 to November 2019 (n = 116). Kidney function was quantified based on the estimated glomerular filtration rate (eGFR), and delayed functional recovery of remnant kidney was defined as eGFR

Enhancing the radioresponsiveness of colorectal cancer (CRC) is essential for local control and prognosis. However, consequent damage to surrounding healthy cells can lead to treatment failure. We hypothesized that short‑chain fatty acids (SCFAs) could act as radiosensitizers for cancer cells, allowing the administration of a lower and safer dose of radiation. To test this hypothesis, the responses of three‑dimensional‑cultured organoids, derived from CRC patients, to radiotherapy, as well as the effects of combined radiotherapy with the SCFAs butyrate, propionate and acetate as candidate radiosensitizers, were evaluated via reverse transcription‑quantitative polymerase chain reaction, immunohistochemistry and organoid viability assay. Of the three SCFAs tested, only butyrate suppressed the proliferation of the organoids. Moreover, butyrate significantly enhanced radiation‑induced cell death and enhanced treatment effects compared with administration of radiation alone. The radiation‑butyrate combination reduced the proportion of Ki‑67 (proliferation marker)‑positive cells and decreased the number of S phase cells via FOXO3A. Meanwhile, 3/8 CRC organoids were found to be non‑responsive to butyrate with lower expression levels of FOXO3A compared with the responsive cases. Notably, butyrate did not increase radiation‑induced cell death and improved regeneration capacity after irradiation in normal organoids. These results suggest that butyrate could enhance the efficacy of radiotherapy while protecting the normal mucosa, thus highlighting a potential strategy for minimizing the associated toxicity of radiotherapy.

Background The aim of this study was to investigate the effect of the steep Trendelenburg position (STP) with pneumoperitoneum on whole-blood viscosity (WBV) in patients undergoing robot-assisted laparoscopic prostatectomy (RALP). The study also analyzed the associations of clinical patient-specific and time-dependent variables with WBV and recorded postoperative outcomes. Methods Fifty-eight adult male patients (ASA physical status of I or II) undergoing elective RALP were prospectively analyzed in this study. WBV was intraoperatively measured three times: at the beginning of surgery in the supine position without pneumoperitoneum; after 30 min in the STP with pneumoperitoneum; and at the end of surgery in the supine position without pneumoperitoneum. The WBV at a high shear rate (300 s− 1) was recorded as systolic blood viscosity (SBV) and that at a low shear rate (5 s− 1) was recorded as diastolic blood viscosity (DBV). Systolic blood hyperviscosity was defined as > 13.0 cP at 300 s− 1 and diastolic blood hyperviscosity was defined as > 4.1 cP at 5 s− 1. Results The WBV and incidences of systolic and diastolic blood hyperviscosity significantly increased from the supine position without pneumoperitoneum to the STP with pneumoperitoneum. When RALP was performed in the STP with pneumoperitoneum, 12 patients (27.3%) who had normal SBV at the beginning of surgery and 11 patients (26.8%) who had normal DBV at the beginning of surgery developed new systolic and diastolic blood hyperviscosity, respectively. The degree of increase in WBV after positioning with the STP and pneumoperitoneum was higher in the patients with hyperviscosity than in those without hyperviscosity at the beginning of surgery. Higher preoperative body mass index (BMI) and hematocrit level were associated with the development of both systolic and diastolic blood hyperviscosity in the STP with pneumoperitoneum. All patients were postoperatively discharged without fatal complications. Conclusions Changes in surgical position may influence WBV, and higher preoperative BMI and hematocrit level are independent factors associated with the risk of hyperviscosity during RALP in the STP with pneumoperitoneum. Trial registration Clinical Research Information Service, Republic of Korea, approval number: KCT0003295 on October 25, 2018.