Complexation of drug and hapten-conjugated aptamer with universal hapten antibody for pancreatic cancer treatment.

Owing to a lack of reliable markers and therapeutic targets, pancreatic ductal adenocarcinoma (PDAC) remains the most lethal malignant tumor despite numerous therapeutic advances. In this study, we utilized cell-SELEX to isolate a DNA aptamer recognizing the natural conformation of the target on the cell surface. PAp7T8, an aptamer optimized by size and chemical modification, exhibited specific targeting to pancreatic cancer cells and orthotopic xenograft pancreatic tumors. To confer therapeutic functions to the aptamer, we adopted a drug-conjugated oligobody (DOligobody) strategy. Monomethyl auristatin E was used as a cytotoxic drug, digoxigenin acted as a hapten, and the humanized anti-digoxigenin antibody served as a universal carrier of the aptamer. The resulting PAp7T8-DOligobody showed extended in vivo half-life and markedly inhibited tumor growth in an orthotopic pancreatic cancer xenograft model without causing significant toxicity. Therefore, PAp7T8-DOligobody represents a promising novel therapeutic delivery platform for PDAC.

Standardized cotton swab sampling with nested quantitative polymerase chain reaction is effective for diagnosing ordinary scabies.

BACKGROUND: Low sensitivity of the PCR assay for diagnosing scabies has been noted because of the difficulty in obtaining tissue containing Sarcoptes scabiei DNA. AIM: To evaluate nested real-time quantitative PCR (nRT-qPCR) with nonexpert-dependent standardized cotton swab sampling (CSW) as a tool for diagnosing scabies. METHODS: All patients underwent dermoscopic and microscopic examination (MS) with scraped sampling (Sc). Patient samples were acquired with a single, dry swab rubbed across the flexor areas of both wrists as well as the eight interdigital spaces and on any suspected scabies lesions. nRT-qPCRs were performed with Sc and CSW samples. RESULTS: Out of 125 patients with suspected scabies, 120 patients were sampled, and 57 were positive (positive with: MS n = 53; nRT-qPCR with Sc n = 52; nRT-qPCR with CSW n = 46) and 63 were negative for scabies. The sensitivities of these tests were 93.0%, 91.2% and 80.7%, respectively, which were not different statistically (P > 0.05). However, upon subsequent monitoring after treatment, the sensitivity of nRT-qPCR with CSW was only 36.6%, which was significantly lower than 83.0% for MS and 92.7% for nRT-qPCR with Sc (P < 0.001). The obtained sequences showed 97%-100% homology with scabies sequences deposited in GenBank. CONCLUSION: CSW with nRT-qPCR shows sensitivity close to MS with scraping performed by experts for diagnosing scabies in an outpatient setting, but not for post-treatment monitoring. CSW with nRT-qPCR may be useful for physicians unfamiliar with a traditional diagnostic method, and for screening an outbreak in community facilities.

Establishment of Patient-derived Preclinical Models for Invasive Papillary Cholangiocarcinoma.

BACKGROUND/AIM: Invasive papillary cholangio-carcinoma (IPC) is a minor subtype of extrahepatic cholangiocarcinoma. However, its etiology and characteristics remain unknown because of the unavailability of in vitro and in vivo models. We aimed to establish a novel preclinical model for translational research of IPC. MATERIALS AND METHODS: A patient-derived xenograft (PDX) was engrafted in NOG mice and the cell line National Cancer Center human IPC (NCChIPC) was subsequently established from the PDX tumors. Immunohistochemistry and RNA-sequencing were used to determine the retention of original characteristics of patient tissues. RESULTS: PDX tumors showed successful amplification, and the NCChIPC-derived xenograft largely retained the histopathological features of the original tumor with CK19, MUC1 and MUC5AC expression. Transcriptome analysis showed a high correlation between patient and preclinical models. Additionally, anticancer drugs response was analyzed in the NCChIPC PDX. CONCLUSION: These novel preclinical models here will help elucidate IPC etiology and facilitate translational research.

Cohnella cholangitidis sp. nov., a novel species of the genus Cohnella isolated from a clinical specimen in Korea.

A Gram-positive, aerobic, rod-shaped bacterium, designated as strain 1605-214T, was isolated from the blood sample of a patient with cholangitis. Based on its 16S rRNA gene sequence, the strain 1605-214T belonged to the genus Cohnella and exhibited 97.9% sequence identity with Cohnella luojiensis DSM 24270T (GQ214052). DNA-DNA hybridization, digital DNA-DNA hybridization, and average nucleotide identity values between the two species were 23% ± 1.9, 21.1%, and 77.2%, respectively. The cellular fatty acids of strain 1605-214T were mainly comprised of anteiso-C15:0 (36.1%), iso-C16:0 (16.5%), and C16:0 (15.1%). The predominant quinone was menaquinone-7; predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, and aminophospholipid-1. The cell wall peptidoglycan of strain 1605-214T contained meso-diaminopimelic acid. DNA G + C content of strain 1605-214T was 50.6 mol%. 5187 genes out of a total of 5413 (94.6%) were assigned putative functions using eggNOG v5.0. Based on genotypic characteristics and genomic sequence analysis results, strain 1605-214T was confirmed to represent a novel species of genus Cohnella, for which the name Cohnella cholangitidis sp. nov., was proposed.

Intradiploic epidermoid cyst in a dog.

CASE DESCRIPTION: A 10-year-old 7-kg (15.4-lb) neutered male Shih Tzu was referred for evaluation because of a sudden onset of dullness and intermittent vomiting of 1 to 2 weeks' duration. Two days prior to evaluation, clinical signs had worsened and 1 seizure was reported. CLINICAL FINDINGS: A 3.5 × 2.5-cm soft, dome-shaped mass of the right occipital region of the head was noted on physical examination. Radiography and CT confirmed the presence of the mass and indicated its intra- and extracranial expansion. The MRI images showed compression of the cerebellum by the mass, with distinct margins of hypointensity on both T1- and T2-weighted images. TREATMENT AND OUTCOME: Surgery was performed, and the mass was completely excised. The dog had mild ataxia, hypermetria, and head tremors after surgery. Histologic examination of the mass yielded a diagnosis of intradiploic epidermoid cyst. On examination 3 weeks after surgery, the previous neurologic signs had resolved. On examination 25 months after surgery, the dog remained free of clinical signs. CLINICAL RELEVANCE: The present report described the clinical signs, diagnostic imaging results, and successful surgical removal of an intradiploic epidermoid cyst in a dog. Long-term prognosis may be good with complete removal of intradiploic epidermoid cysts in dogs.

A Variant of SLC1A5 Is a Mitochondrial Glutamine Transporter for Metabolic Reprogramming in Cancer Cells.

Glutamine is an essential nutrient that regulates energy production, redox homeostasis, and signaling in cancer cells. Despite the importance of glutamine in mitochondrial metabolism, the mitochondrial glutamine transporter has long been unknown. Here, we show that the SLC1A5 variant plays a critical role in cancer metabolic reprogramming by transporting glutamine into mitochondria. The SLC1A5 variant has an N-terminal targeting signal for mitochondrial localization. Hypoxia-induced gene expression of the SLC1A5 variant is mediated by HIF-2α. Overexpression of the SLC1A5 variant mediates glutamine-induced ATP production and glutathione synthesis and confers gemcitabine resistance to pancreatic cancer cells. SLC1A5 variant knockdown and overexpression alter cancer cell and tumor growth, supporting an oncogenic role. This work demonstrates that the SLC1A5 variant is a mitochondrial glutamine transporter for cancer metabolic reprogramming.

Detection of Gram-negative bacterial outer membrane vesicles using DNA aptamers.

Infection of various pathogenic bacteria causes severe illness to human beings. Despite the research advances, current identification tools still exhibit limitations in detecting Gram-negative bacteria with high accuracy. In this study, we isolated single-stranded DNA aptamers against multiple Gram-negative bacterial species using Toggle-cell-SELEX (systemic evolution of ligands by exponential enrichment) and constructed an aptamer-based detection tool towards bacterial secretory cargo released from outer membranes of Gram-negative bacteria. Three Gram-negative bacteria, Escherichia coli DH5α, E. coli K12, and Serratia marcescens, were sequentially incubated with the pool of random DNA sequences at each SELEX loop. Two aptamers selected, GN6 and GN12, were 4.2-times and 3.6-times higher binding to 108 cells of Gram-negative bacteria than to Gram-positive bacteria tested, respectively. Using GN6 aptamer, we constructed an Enzyme-linked aptamer assay (ELAA) to detect bacterial outer membrane vesicles (OMVs) of Gram-negative bacteria, which contain several outer membrane proteins with potent immunostimulatory effects. The GN6-ELAA showed high sensitivity to detect as low as 25 ng/mL bacterial OMVs. Aptamers developed in this study show a great potential to facilitate medical diagnosis and early detection of bacterial terrorism, based on the ability to detect bacterial OMVs of multiple Gram-negative bacteria.

Measurement of Vibrating Tympanic Membrane in an In Vivo Mouse Model Using Doppler Optical Coherence Tomography.

Optical coherence tomography (OCT) has a micro-resolution with a penetration depth of about 2 mm and field of view of about 10 mm. This makes OCT well suited for analyzing the anatomical and internal structural assessment of the middle ear. To study the vibratory motion of the tympanic membrane (TM) and its internal structure, we developed a phase-resolved Doppler OCT system using Kasai's autocorrelation algorithm. Doppler optical coherence tomography is a powerful imaging tool which can offer the micro-vibratory measurement of the tympanic membrane and obtain the micrometer-resolved cross-sectional images of the sample in real-time. To observe the relative vibratory motion of individual sections (malleus, thick regions, and the thin regions of the tympanic membrane) of the tympanic membrane in respect to auditory signals, we designed an experimental study for measuring the difference in Doppler phase shift for frequencies varying from 1 to 8 kHz which were given as external stimuli to the middle ear of a small animal model. Malleus is the very first interconnecting region between the TM and cochlea. In our proposed study, we observed that the maximum change in Doppler phase shift was seen for the 4 kHz acoustic stimulus in the malleus, the thick regions, and in the thin regions of the tympanic membrane. In particular, the vibration signals were higher in the malleus in comparison to the tympanic membrane.

Cell-SELEX Based Identification of an RNA Aptamer for Escherichia coli and Its Use in Various Detection Formats.

Escherichia coli are important indicator organisms, used routinely for the monitoring of water and food safety. For quick, sensitive and real-time detection of E. coli we developed a 2'F modified RNA aptamer Ec3, by Cell-SELEX. The 31 nucleotide truncated Ec3 demonstrated improved binding and low nano-molar affinity to E. coli. The aptamer developed by us out-performs the commercial antibody and aptamer used for E. coli detection. Ec3(31) aptamer based E. coli detection was done using three different detection formats and the assay sensitivities were determined. Conventional Ec3(31)-biotin-streptavidin magnetic separation could detect E. coli with a limit of detection of 1.3 × 106 CFU/ml. Although, optical analytic technique, biolayer interferometry, did not improve the sensitivity of detection for whole cells, a very significant improvement in the detection was seen with the E. coli cell lysate (5 × 104 CFU/ml). Finally we developed Electrochemical Impedance Spectroscopy (EIS) gap capacitance biosensor that has detection limits of 2 × 104 CFU/mL of E. coli cells, without any labeling and signal amplification techniques. We believe that our developed method can step towards more complex and real sample application.

Endoplasmic reticulum stress induces 5-fluorouracil resistance in human colon cancer cells.

Colon cancer can be treated with 5-fluorouracil (5-FU), but 5-FU resistance frequently occurs. We determined whether 5-FU resistance arises as a result of endoplasmic reticulum (ER) stress. 5-FU-resistant SNUC5 colon cancer cells (SNUC5/FUR cells) expressed higher levels of ER stress-related proteins than drug-sensitive SNUC5 cells. SNUC5/FUR cells also exhibited more intense ER staining and higher level of mitochondrial Ca(2+) overload. SNUC5/FUR cells transfected with siRNA against GRP78, ATF6, ERK, or AKT were more sensitive to 5-FU than siControl RNA-transfected cells. These results suggested that 5-FU resistance was associated with ER stress in colon cancer.

Induction of Endoplasmic Reticulum Stress via Reactive Oxygen Species Mediated by Luteolin in Melanoma Cells.

BACKGROUND: This study aimed to investigate whether luteolin, a flavonoid, induces apoptosis in human melanoma cells via endoplasmic reticulum (ER) stress. MATERIALS AND METHODS: To investigate the effects of luteolin in human melanoma cells, the anti-proliferation, apoptosis, ER stress induction and reactive oxygen species (ROS) generation were evaluated using MTT, Hoechst 33342, ER-tracker Blue White DPX and DCF-DA staining assays, respectively. RESULTS: Luteolin inhibited cell proliferation and increased apoptotic body formation. Luteolin induced ER stress, as shown by ER staining and mitochondrial Ca(2+) overloading. Luteolin increased expression of the ER stress-related proteins; protein kinase RNA-like ER kinase, phospho eukaryotic translation initiation factor 2α, activating transcription factor (ATF) 6, CCAAT/enhancer-binding protein-homologous protein (CHOP), and cleaved caspase 12. Furthermore, luteolin increased the level of intracellular ROS, leading to ROS-mediated apoptosis and ER stress. However, N-acetyl cysteine, a ROS scavenger, decreased ROS levels, apoptosis, and ER stress induced by luteolin treatment. In addition, knockdown of CHOP and ATF6 by small-interfering RNA inhibited luteolin-induced cell death. CONCLUSION: Luteolin induces apoptosis by ER stress via increasing ROS levels.

Spatial regression analysis of traffic crashes in Seoul.

Traffic crashes can be spatially correlated events and the analysis of the distribution of traffic crash frequency requires evaluation of parameters that reflect spatial properties and correlation. Typically this spatial aspect of crash data is not used in everyday practice by planning agencies and this contributes to a gap between research and practice. A database of traffic crashes in Seoul, Korea, in 2010 was developed at the traffic analysis zone (TAZ) level with a number of GIS developed spatial variables. Practical spatial models using available software were estimated. The spatial error model was determined to be better than the spatial lag model and an ordinary least squares baseline regression. A geographically weighted regression model provided useful insights about localization of effects. The results found that an increased length of roads with speed limit below 30 km/h and a higher ratio of residents below age of 15 were correlated with lower traffic crash frequency, while a higher ratio of residents who moved to the TAZ, more vehicle-kilometers traveled, and a greater number of access points with speed limit difference between side roads and mainline above 30 km/h all increased the number of traffic crashes. This suggests, for example, that better control or design for merging lower speed roads with higher speed roads is important. A key result is that the length of bus-only center lanes had the largest effect on increasing traffic crashes. This is important as bus-only center lanes with bus stop islands have been increasingly used to improve transit times. Hence the potential negative safety impacts of such systems need to be studied further and mitigated through improved design of pedestrian access to center bus stop islands.

Generation of Reactive Oxygen Species and Endoplasmic Reticulum Stress by Dictyopteris undulata Extract Leads to Apoptosis in Human Melanoma Cells.

In this study, we evaluated the hypothesis that a marine brown algae, Dictyopteris undulata ethanol extract (DUE), provokes apoptosis in a human melanoma cell line, A2058, via reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. DUE inhibited A2058 cell proliferation and increased apoptotic body formation, as indicated by the presence of fragmented nuclei and the activation of caspase-3. Moreover, DUE-treated cells showed elevated ER staining, mitochondrial calcium cation (Ca2+) overloading, augmented levels of ER stress-related and cell death modulatory proteins, including RNA-dependent protein kinase-related ER kinase, phospho-inositol-requiring enzyme 1α, phospho-eukaryotic translation initiation factor 2α, and CCAAT/enhancer-binding protein-homologous protein, as well as increased intracellular ROS levels. However, the antioxidant N-acetyl cysteine reversed the elevated ROS levels, decreased apoptosis, and mitigated ER stress in A2058 cells following DUE treatment. These findings suggest that DUE treatment triggers apoptosis in human melanoma cells through a mechanism involving ER stress and ROS.

Dictyopteris undulata Extract Induces Apoptosis via Induction of Endoplasmic Reticulum Stress in Human Colon Cancer Cells.

BACKGROUND: Induction of endoplasmic reticulum (ER) stress-mediated apoptosis in cancer cells represents an alternative approach for cancer therapy. The objective of this study was to elucidate whether ethanol extract of the marine brown alga Dictyopteris undulata can induce apoptosis, via ER stress, in human colon adenocarcinoma cells. METHODS: Anti-proliferative activity was evaluated by the colony forming assay. ER stress response was evaluated using flow cytometry and confocal imaging after Rhod2 and ER tracker staining. The expression of ER stress-related proteins was assessed by Western blotting. RESULTS: D. undulata extract (DUE) inhibited colony forming ability in SW480 cells. Furthermore, DUE induced characteristic signs of ER stress: mitochondrial Ca(2+) overloading, ER staining, expression of ER stress-related proteins, phosphorylation of RNA-dependent protein kinase-like ER kinase and inositol requiring enzyme 1, cleavage of activating transcription factor 6, and induction of the pro-apoptotic factors, CCAAT/enhancer-binding protein-homologous protein (CHOP) and caspase-12. Moreover, down-regulation of CHOP by siCHOP RNA attenuated DUE-induced apoptosis. CONCLUSIONS: The ER stress response plays an important role in DUE-induced apoptosis in human colon cancer cells.