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XLS file - 59K, Differentially regulated proteins for proteomic analysis comparing 344SQ/393P tumor lysates.

XLS file - 18K, Downregulated genes with predicted binding sites for Zeb1.

PDF file 1293K, Localization of cytochrome c by immunofluorescence

PDF file 60KB, Cell viability in H1299 and A549

XLS file - 12K, Enrichment for predicted transcription factor DNA binding domains in 344SQ/393P and 344SQ_miR-200/344SQ_control data sets.

XLS file - 20K, Complete Gene Ontology tables for the 344SQ_mir200/344SQ_vector proteomic comparison.

SIRT1 is an NAD+-dependent protein deacetylase induced by metabolic stresses, such as nutrition or oxygen deprivation. Although SIRT1 contributes to aging and metabolic disorders, its role in cancer progression and therapeutic responses remains controversial. Because hypoxia occurs widely in solid tumors, where it provokes drug resistance, we investigated the involvement of SIRT1 in hypoxia-induced chemoresistance. SIRT1 was downregulated in a panel of non–small cell lung carcinoma (NSCLC) cells exposed to hypoxia for 48 hours. The master metabolic kinase AMP-activated protein kinase (AMPK) was inactivated under the same conditions, likely due to attenuation of the SIRT1/LKB1-mediated AMPK activation process. Notably, hypoxic inactivation of this SIRT1–AMPK pathway led to cisplatin and doxorubicin resistance. Mechanistic investigations suggested that this pathway supported the cytotoxic response to cisplatin and doxorubicin by licensing an apoptotic process controlled by mitochondria. We confirmed the involvement of this pathway in a mouse xenograft model of human NSCLC. Furthermore, we demonstrated that a SIRT1 activator SRT1720 augmented the antitumor effects of cisplatin, and these effects could be blocked by administration of an AMPK inhibitor compound C. Taken together, our results offer preclinical proof-of-concept to target the SIRT1–AMPK pathway as a strategy to overcome hypoxia-induced chemoresistance in NSCLC. Cancer Res; 74(1); 298–308. ©2013 AACR.

PDF file 57K, Localization of cytochrome c by Western blotting

PDF file - 57K

PDF file 1207K, DNA fragmentation (TUNEL) and active caspase-9 expression

PDF file - 256K, The most significant networks TGFB containing networks from Ingenuity Pathway Analysis for cellular compartments.

Supplementary Figure 3 PDF file 158K, Cell viability in H1299 and A549

XLS file - 153K, Differentially regulated proteins for proteomic analysis comparing 344SQ_miR-200/344SQ_control cells.

Kirsten rat sarcoma viral oncogene homologue (KRAS) is a frequent oncogenic driver of solid tumors, including non-small cell lung cancer (NSCLC). Its treatment and outcomes have been dramatically revolutionized by direct targeted therapies because of the lack of deep binding pockets for specific small -molecule inhibitors. Here, we demonstrated that the class III histone deacetylase SIRT1 mRNA and protein levels were upregulated by the KRASMut-Raf-MEK-c-Myc axis in KRASMut lung cancer cells and lung tumors of a spontaneous KrasG12D expression mouse model. KRASMut-induced SIRT1 bound to KRASMut again and stably deacetylased KRASMut lysine 104, which increased KRASMut activity. SIRT1 knock down (K/D) or SIRT1H363Y increased KRASMut acetylation, which decreased KRASMut activity, which sensitized the anti-cancer effect of cisplatin and erlotinib. Furthermore, KrasG12D/+;Sirt1co/co mice treated with cisplatin and erlotinib robustly reduced tumor burden and increased survival rates compared with these in spontaneous LSL-KrasG12D/+;Sirt1+/+ mice and each drug treatment group. By extension, we found p300 as a KRASMut acetyltransferase, which reinforced KRASMut lysine 104 acetylation and robustly decreased KRASMut activity. KRASMut lysine 104 acetylation by p300 and deacetylation by SIRT1 was confirmed by LC-MS/MS. Consistent with this finding, SIRT1 inhibitor, EX527 repressed KRASMut acetylation and activity, which abolished synergistically cell proliferation, colony formation, and tumor burden of KRASMut combined treatment with cisplatin or erlotinib. Our data reveals a novel pathway critical for the regulation of KRASMut lung cancer activity and provides important evidence for the potential application of SIRT1 inhibitor and p300 activator for the combination regimen of KRASMut lung cancer patients. Significance KRASMut lung cancer activity dependents on acetylation or deacetylation status at lysine 104 by SIRT1 and p300 and could be effective combined regimen to sensitize KRASMut NSCLC.

Background Clear cell Renal cell carcinoma (ccRCC) is an immunogenic tumor. B7 family members, such as CTLA-4, PD-1, and PD-L1, are the main components of immune checkpoints that regulate various immune responses. Specifically, B7-H3 regulates T cell-mediated immune responses against cancer. This study aimed to analyze the association between B7-H3 and CTLA-4 expression and the prognostic factors of ccRCC to provide a basis for their potential use as predictive factors and in immunotherapy. Methods Formalin-fixed paraffin-embedded specimens were obtained from 244 ccRCC patients, and B7-H3, CTLA-4, and PD-L1 expressions were evaluated using immunohistochemical staining. Results B7-H3 and CTLA-4 were positive in 73 (29.9%) and 57 (23.4%) of the 244 patients, respectively. B7-H3 expression was significantly associated with PD-L1 expression (P P = 0.842). Kaplan–Meier analysis showed that positive B7-H3 expression was associated with poor progression-free survival (PFS) (P P = 0.457). Multivariate analysis revealed that B7-H3 was correlated with poor PFS (P = 0.031), whereas CTLA-4 was not (P = 0.173). Conclusions To the best of our knowledge, this study is the first to investigate B7-H3 and PD-L1 expression and survival in ccRCC. B7-H3 expression is an independent prognostic factor for ccRCC. Furthermore, multiple immune cell inhibitory targets, such as B7-H3 and PD-L1, can be used for therapeutic tumor regression in a clinical setting.

Radiofrequencyablation (RFA) is one of the treatment options for lung nodules; however, the need for exact delivery of the rigid metal electrode into the center of the target mass often leads to complications or suboptimal results. To overcome these limitations, we report a novel bronchoscopy-guided atraumatic no-touch RFA procedure that uses a specially designed radiofrequency (RF) conductor balloon catheter (CAROL catheter) and an injectable bronchial electrode based on a medical grade liquid metal (E-GaIn) in porcine lungs. The bronchial electrode injected from the CAROL catheter was able to turn the target-site bronchial air pipe into a temporally multi-tined RF electrode. The mean volume of E-GaIn (bronchial electrode) for each effective CAROL was 0.46 ± 0.47 ml. The ablation results showed highly efficacious and consistent results especially in the peripheral lung. Most bronchial electrodes were also retrieved by either bronchoscopic suction immediately after the procedure or by natural expectoration thereafter. The residual bronchial electrode did not cause any significant safety issues. Our computer simulation model also supported preclinical data.

Laparoscopic liver donor surgery is a technically difficult and demanding procedure. Our aim was toevaluate its feasibility at an established transplant center. Although our hospital is a small-volume center with20 liver transplants per year, laparoscopic donor surgery has been performed regularly. In this study, we have reported our experiences with laparoscopic donor right hepa-tectomy and its outcomes.Between May 2014 and March 2021, 26 deceased donor liver transplants and 37 living donor liver transplants, approved by the Korean Network for Organ Sharing, were performed at out center. From these, we reviewed the medical records, including clinical and demographic characteristics and operative outcomes, of 3 living donors who under-went pure laparoscopic donor right hepatectomy and their recipients.Each of the 3 laparoscopic donor right hepatectomies took over 10 hours with the prolonged Pringle maneuver time and warm ischemic time. However, there were no significant events during surgery or critical postoperative complications. In the recipients, posttransplant complications included middle hepatic vein obstruction, postoperative bleeding, bile leak, septic shock, and primary nonfunction of the graft. We managed and resolved these complications using various approaches, including retransplant, and all 3 recipients recovered and survived.Laparoscopic donor right hepatectomy had a relatively long operative time at our small-volume center. We believe that successful laparoscopic donor hepatectomy is possible if the donor is selected appropriately according to the center's experiences and there are constant efforts to overcome the learning curve.

The early rectal cancer (ERC) has increased with the national cancer screening project for early detection of colorectal cancer. The gold standard treatment for low rectal cancer is low anterior resection (LAR) based on total mesorectal excision. However, radical resection results in a high mortality and complication rate, and the deterioration of quality of life due to LAR syndrome, genitourinary dysfunction, and possible permanent stoma. Local excision with chemoradiation therapy could be one of the alternative therapeutic strategies for the organ preservation and a cure of cancer. To decide the treatment strategies, it is important to establish sophisticated indications that can maximize the therapeutic effect. ERC has heterogeneous pathological features, including aggressive behavior and occult lymph node metastasis, with different responses to chemoradiotherapy. Therefore, radiologic, endoscopic and pathologic evaluation to predict the risk of lymph node metastasis and local recurrence has been evolving to determine the optimal treatment strategy in the patient-tailored medicine. Recently, the long-term outcomes of prospective randomized clinical trials provide new hope for organ preservation in patients with ERC. In this paper, we aim to review various risk factors related to local recurrence and discuss the optimal treatment strategy for ERC.

Background: The level of surgical difficulty in laparoscopic cholecystectomy might be predictable based on preoperative imaging and intraoperative findings indicative of cholecystitis severity. Several scales for laparoscopic cholecystectomy have been developed, but most are complex, unverified, and not widely adopted. This study evaluated the association of the cystic duct fibrosis score (range, 0–3) with surgical difficulty in laparoscopic cholecystectomy. Methods: Between July 2018 and November 2018, 163 laparoscopic cholecystectomy cases were retrospectively reviewed at a single center. Patients’ demographics, preoperative laboratory data, operation time, complications, hospital stay, and cholecystitis severity grade were investigated. We also evaluated the associations of the Tokyo Guidelines 2018 and the Parkland grading scale with the cystic fibrosis score. Results: The cystic duct fibrosis score was associated with preoperative white blood cells (pp=0.046), preoperative total bilirubin (pppp=0.002), estimated blood loss (pp=0.004), open conversion (pp=0.010). The cystic duct fibrosis score was also correlated with the Tokyo Guidelines 2018 and the Parkland grading scale (p

External bile stents may be used to prevent biliary complications. However, the external biliary stent itself has a risk of complications. This study evaluated the frequency and treatment of complications associated with external bile stent.From May 2015 to September 2019, 18 deceased donor liver transplantations (DDLTs) and 25 living donor liver transplantations (LDLTs) were performed. We retrospectively reviewed these patients' demographic profiles, type of transplantation and presence of biliary complications, external bile stent-related complications, and treatment results.Overall biliary complications occurred in 12 patients (27.9%): 3 strictures (6.9%), 2 leakages (4.6%), and 7 external bile stent-related complications (16.2%). Among the 7, 4 were self-removal or stent fractures at home, and 2 occurred after removal by a physician. One patient had ileus with peritonitis. Local peritonitis was controlled by antibiotics and fluid therapy, but 1 patient needed an operation because of intestinal obstruction with recurrent local peritonitis. All biliary complications occurred in LDLT, and external biliary stent-related complications also occurred only in LDLT, not in DDLT (P = .014). Interestingly, only 1 of 7 external bile stent-related complications occurred after we adopted the stent buried suture technique on the duodenum (P = .062).External bile stent-related complications were higher in LDLT than in DDLT. When performing external bile stent implantation, the stent buried suture technique will help reduce stent-related complications, especially in LDLT.

Although 2D materials hold great potential for next-generation pressure sensors, recent studies revealed that gases permeate along the membrane-surface interface that is only weakly bound by van der Waals interactions, necessitating additional sealing procedures. In this work, we demonstrate the use of free-standing complex oxides as self-sealing membranes that allow the reference cavity of pressure sensors to be sealed by a simple anneal. To test the hermeticity, we study the gas permeation time constants in nano-mechanical resonators made from SrRuO3 and SrTiO3 membranes suspended over SiO2/Si cavities which show an improvement up to 4 orders of magnitude in the permeation time constant after annealing the devices for 15 minutes. Similar devices fabricated on Si3N4/Si do not show such improvements, suggesting that the adhesion increase over SiO2 is mediated by oxygen bonds that are formed at the SiO2/complex oxide interface during the self-sealing anneal. We confirm the enhancement of adhesion by picosecond ultrasonics measurements which show an increase in the interfacial stiffness by 70% after annealing. Since it is straigthforward to apply, the presented self-sealing method is thus a promising route toward realizing ultrathin hermetic pressure sensors., 7 pages, 5 figures

The inherent properties of 2D materials—light mass, high out-of-plane flexibility, and large surface area—promise great potential for precise and accurate nanomechanical mass sensing, but their application is often hampered by surface contamination. Here we demonstrate a tri-layer graphene nanomechanical resonant mass sensor with sub-attogram resolution at room temperature, fabricated by a bottom-up process. We found that Joule-heating is effective in cleaning the graphene membrane surface, which results in a large improvement in the stability of the resonance frequency. We characterized the sensor by depositing Cr metal using a stencil mask and found a mass-resolution that is sufficient to weigh very small particles, like large proteins and protein complexes, with potential applications in the fields of nanobiology and medicine.

Metagonimiasis, commonly seen in East Asian countries, is a parasitic disorder caused by definitive hosts’ ingestion of undercooked freshwater fishes. Recently, genetic analysis has proved 28S rRNA and cytochrome c oxidase subunit I (COI) mtDNA gene to be a successful marker differentiating species of the genus Metagonimus. In the present study, using specimens from the newly discovered Joseon Dynasty human remains of Goryeong, we obtained updated genetic data on genus Metagonimus, which was also prevalent during the Joseon period. The ancient DNA (aDNA) was retrieved from the coprolite sample of the seventeenth century, half-mummified individual discovered at Goryeong Country, South Korea. Cloning and sequencing were performed on PCR-amplified amplicons for M. yokogawai 28S rRNA and COI mtDNA gene. The consensus sequences were used for species identification and phylogenetic analysis using NCBI/BLAST and MEGA X software. Based on the COI mtDNA gene region, the Goryeong sequence was confirmed as belonging to M. yokogawai, as it was shown to form a separate cluster with other M. yokogawai taxa that are distinct also from M. takahashii and M. miyatai. In a series of our genetic analyses on genus Metagonimus using samples retrieved from Joseon-period cases, aDNA sequences of genus Metagonimus revealed in South Korea thus far are those of M. yokogawai, but not of M. miyatai or M. takahashii yet.

Testicular carcinoid tumors are very rare, accounting for less than 1% of all testicular tumors. We report a rare case of a testicular carcinoid tumor with extensive lymphatic invasion. A 42-year-old man presented with a painless, enlarged right testicular mass. There was no history of injury or discomfort in this region. Right radical orchiectomy was performed, which showed a well-defined, non-encapsulated solid white mass with calcification (7.0 × 4.5 × 3.5 cm) and absence of cystic components. Microscopic examination using hematoxylin and eosin staining of the tumor sections identified organoid, trabecular, and solid patterns with rosette formation. Extensive multifocal lymphatic invasion was observed. Immunohistochemistry was positive for synaptophysin, chromogranin, and CD56. Testicular carcinoid tumors usually show good prognoses; however, there was extensive lymphovascular invasion in this case. Thus, in the case of unusual presentation of the disease, close follow-up is necessary.

Background: Because Korea is the fastest aging country, the stroke incidence is increasing rapidly. We investigate the trend of the number of patients with acute stroke in recent years and estimate the burden of the neurologist to treat the acute stroke patient visited the emergency department.Methods: We requested a questionnaire survey to all teaching hospitals on the number of hospital beds, the number of stroke patients who visited the emergency department, the number of stroke patients in charge of the neurologist, and the number of days on duty of residents from 2016 to 2019.Results: Of 69 teaching hospitals, 41 hospitals answered the survey. The average hospital beds per hospital were increased annually from 909 to 916. The average patients who visited the emergency department with stroke and were in charged to neurologists were rapidly increased from 799 to 867 per hospital. In particular, the number of patients with hyperacute cerebral infarction requiring the thrombolytic administration or mechanical thrombectomy were rapidly increased from 105 to 131. On the other hand, the average number of residents per hospital was decreased from 5.1 to 4.8. Therefore, the days on duty per resident were increased from 74 to 77.Conclusions: The number of acute stroke patients, especially, hyperacute stroke required the rapid cooperation and high labor were increasing rapidly in recent years. However, because the number of residents were decreased, the burden was increasing. To improve the quality of acute stroke treatment, it is necessary to increase the number of residents.

Hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels function as pacemaker channels in spontaneously active cells. We studied the existence of HCN channels and their functional roles in the interstitial cells of Cajal (ICC) from the mouse colon using electrophysiological, immunohistochemical and molecular techniques. HCN1 and HCN3 channels were detected in anoctamin‐1 (Ca2+‐activated Cl− channel; ANO1)‐positive cells within the muscular and myenteric layers in colonic tissues. The mRNA transcripts of HCN1 and HCN3 channels were expressed in ANO1‐positive ICC. In the deletion of HCN1 and HCN3 channels in colonic ICC, the pacemaking potential frequency was reduced. Basal cellular adenylate cyclase activity was decreased by adenylate cyclase inhibitor in colonic ICC, whereas cAMP‐specific phosphodiesterase inhibitors increased it. 8‐Bromo‐cyclic AMP and rolipram increased spontaneous intracellular Ca2+ oscillations. In addition, Ca2+‐dependent adenylate cyclase 1 (AC1) mRNA was detected in colonic ICC. Sulprostone, a PGE2‐EP3 agonist, increased the pacemaking potential frequency, maximum rate of rise of resting membrane in pacemaker potentials and basal cellular adenylate cyclase activity in colonic ICC. These results indicate that HCN channels exist in colonic ICC and participate in generating pacemaking potentials. Thus, HCN channels may be therapeutic targets in disturbed colonic motility disorders.

The efficacy of programmed death ligand (PD-L)-1/PD-1 checkpoint blockade in renal cell carcinoma (RCC) remains unknown. The effects of mTOR inhibitors are uncertain, and patients may develop resistance to them. The limited understanding of cancer cell-intrinsic mTOR-mediated pathways remains a challenge in developing effective treatments. Whether transcription factor (TF)-E3 regulates PD-L1 expression and the tumor microenvironment was investigated, and the effects of an mammalian target of rapamycin (mTOR) inhibitor on translocation RCC were explored. TFE3 was overexpressed in clear cell RCC cell lines, and PD-L1 expression was analyzed by Western blot analysis. PD-L1 activity in translocation RCC was analyzed in relation to TFE3 expression via TFE3 knockdown and treatment with an mTOR inhibitor. The results were correlated with the gene expression profile, evaluated using digital multiplex analysis. TFE3 and PD-L1 expression were positively correlated in RCC cells. TFE3 overexpression was associated with the expression of PD-L1 in RCC. Furthermore, mTOR inhibition was associated with enhanced PD-L1 expression via TFE3 activation in translocation RCC. These data support the feasibility of combination therapy based on mTOR inhibition and PD-L1 blockade as a novel strategy for the treatment of patients with translocation RCC.

In adoptive T cell therapy (ACT), the transfer of tumor-specific T cells is paralleled by the conditioning regimen to increase therapeutic efficacy. Pre-conditioning depletes immune-suppressive cells and post-conditioning increases homeostatic signals to improve the persistence of administered T cells. Identifying the favorable immunological factors involved in a conditioning regimen is important to design effective strategies in ACT. Here, by using an ACT model of murine melanoma, we evaluate the effect of the total body irradiation (TBI) and interleukin-2 (IL-2) treatment combination. The use of a Rag1 knock-out strain, which lacks endogenous T cells, enables the identification of factors in a way that focuses more on transferred T cells. We demonstrate that the TBI/IL-2 combination has no additive effect in ACT, although each conditioning improves the therapeutic outcome. While the combination increases the frequency of transferred T cells in lymphoid and tumor tissues, the activation intensity of the cells is reduced compared to that of the sole TBI treatment. Notably, we show that in the presence of TBI, the IL-2 treatment reduces the frequency of intra-tumoral dendritic cells, which are crucial for T cell activation. The current study provides insights into the immunological events involved in the TBI/IL-2 combination in ACT.

Background Although COVID-19 is a viral infection, it is known that antibiotics are often prescribed due to concerns about combined bacterial infection. Therefore, we aimed to analyze how many patients with COVID-19 received the antibiotic prescription as well as what kinds of factors contributed to it using the National Health Insurance database. Methods We retrospectively reviewed claims data for adults 19 years of age and older hospitalized for COVID-19 from December 1, 2019 to December 31, 2020. According to severity classification of the National Institutes of Health guidelines, we calculated not only the proportion of patients receiving antibiotics but also days of treatment per 1000 patient days. In addition, we investigated the factors contributing to antibiotic use by linear regression analysis. Results Of the 55,228 patients, 47% were male, 55% were older than 50 years of age, and most patients (89%) had no underlying diseases. The majority (84%, 46,576) were classified as having mild to moderate illness, with 11% (6,168) and 5% (2,484) having severe and critical, respectively. Antibiotics were prescribed in a total of 27% (15,081). While 74% of patients with severe illness and 88% of those with critical illness received antibiotic treatment, even 18% of mild to moderate cases were prescribed antibiotics. Fluoroquinolones were the most commonly prescribed antibiotics (8,348), followed by third generation cephalosporins (5,729) and beta-lactam/beta-lactamase inhibitors (3,822) as shown in Figure 1. Older age, severity of disease and underlying medical conditions contributed to overall prescription rates as well as days of antibiotic use significantly (Table 1). Conclusion Although most of COVID-19 patients had mild to moderate illness, more than a quarter were prescribed antibiotics. Judicious use of broad-spectrum antibiotics is necessary for COVID-19 patients, considering the severity of disease and the risk of bacterial co-infection. Disclosures All Authors: No reported disclosures.

A high-throughput, accurate screening is crucial for the prevention and control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current methods, which involve sampling from the nasopharyngeal (NP) area by medical staffs, constitute a fundamental bottleneck in expanding the testing capacity. To meet the scales required for population-level surveillance, self-collectable specimens can be used; however, its low viral load has hindered its clinical adoption. Here, we describe a magnetic nanoparticle functionalized with synthetic apolipoprotein H (ApoH) peptides to capture, concentrate, and purify viruses. The ApoH assay demonstrates a viral enrichment efficiency of >90% for both SARS-CoV-2 and its variants, leading to an order of magnitude improvement in analytical sensitivity. For validation, we apply the assay to a total of 84 clinical specimens including nasal, oral, and mouth gargles obtained from COVID-19 patients. As a result, a 100% positivity rate is achieved from the patient-collected nasal and gargle samples, which exceeds that of the traditional NP swab method. The simple 12 min pre-enrichment assay enabling the use of self-collectable samples will be a practical solution to overcome the overwhelming diagnostic capacity. Furthermore, the methodology can easily be built on various clinical protocols, allowing its broad applicability to various disease diagnoses.