Here, we report a high-sensitivity dual immunoassay using Lumulus amebocyte lysate (LAL) and blood coagulation cascade reactions with redox cycling in a nanoscale-gap electrode. Endotoxin and factor ...XIa were used as the label molecules for the immunoassay of two types of analytes to induce the LAL and coagulation cascade reactions, respectively, when each corresponding analyte existed in the sample solution. In addition to the signal amplification by the cascade reactions, we employed redox cycling in a nanoscale gap to achieve a highly sensitive assay. The nanoscale-gap electrode amplifies the amperometric signals from p-aminophenol liberated from artificial substrates in the final steps of the cascade reactions. First, the cross reaction between the LAL and coagulation cascade reactions was investigated. The results indicated that these cascade reactions did not efficiently proceed in a single solution owing to the cross reaction. Therefore, we selected to induce two cascade reactions in different solutions by bisecting the beads after the immunocomplex formation on the beads. The cross reactions of factor XIa with the LAL cascade reaction and of endotoxin with the coagulation cascade reaction were investigated. The effects of these cross reactions were revealed to be negligible by bisecting the beads before inducing the cascade reactions. Finally, a dual immunoassay for goat and human immunoglobulin G was performed, for which the limits of detection were 70 pg/mL (470 fmol/L) and 1.0 ng/mL (6.6 pmol/L), respectively. Thus, our dual immunoassay provides a sensitive platform for clinical diagnosis requiring detection of multiple analytes.
Circulating microRNAs (miRNAs) are attracting major interest as potential non-invasive biomarkers for colorectal cancer (CRC). This study aimed to identify a novel serum miRNA biomarker for the early ...detection and/or evaluating prognosis of CRC patients.
Comprehensive miRNA array analysis was carried out using serum samples from patients with colorectal neoplasia and healthy controls. Next, to verify whether the candidate miRNA possessed a secretory potential, we screened miRNA expression levels in culture medium from 2 CRC cell lines, followed by serum analysis from 12 stage IV CRC, 12 adenoma, and 12 control subjects. Thereafter, we validated expression of candidate miRNAs in 179 primary CRC tissues, as well as serum samples from an independent cohort of 211 CRCs, 56 adenomas, and 57 control subjects.
Through microarray analysis, we identified significantly higher levels of miRNA-1290 (miR-1290) in serum from patients with colorectal adenomas and cancers. We verified miR-1290 overexpression in serum of CRC patients in a training cohort. In the validation cohort, serum miR-1290 levels were significantly up-regulated in patients with colorectal adenomas (P < 0.0001) and cancers (P < 0.0001). Serum miR-1290 levels could robustly distinguish adenoma area under the curve (AUC) = 0.718 and CRC patients (AUC = 0.830) from normal subjects. High miR-1290 expression in serum and tissue was significantly associated with tumor aggressiveness and poor prognosis. Moreover, serum miR-1290 levels were an independent prognostic factor hazard ratio (HR) = 4.51; 95% confidence interval (CI) = 1.23–23.69; P = 0.0096 and an independent predictor for tumor recurrence (hazard ratio = 3.92; 95% confidence interval = 1.11–25.14; P = 0.032) in CRC.
Serum miR-1290 is a novel biomarker for early detection, recurrence, and prognosis in CRC.
Aims/hypothesis
The small G-protein ras-related C3 botulinum toxin substrate 1 (RAC1) plays various roles in mammalian cells, such as in the regulation of cytoskeletal organisation, cell adhesion, ...migration and morphological changes. The present study examines the effects of RAC1 ablation on pancreatic beta cell function.
Methods
Isolated islets from pancreatic beta cell-specific
Rac1
-knockout (beta
Rac1
−/−
) mice and RAC1 knockdown INS-1 insulinoma cells treated with small interfering RNA were used to investigate insulin secretion and cytoskeletal organisation in pancreatic beta cells.
Results
Beta
Rac1
−/−
mice showed decreased glucose-stimulated insulin secretion, while there were no apparent differences in islet morphology. Isolated islets from the mice had blunted insulin secretion in response to high glucose levels. In RAC1 knockdown INS-1 cells, insulin secretion was also decreased in response to high glucose levels, consistent with the phenotype of beta
Rac1
−/−
mice. Even under high glucose levels, RAC1 knockdown INS-1 cells remained intact with F-actin, which inhibits the recruitment of the insulin granules, resulting in an inhibition of insulin secretion.
Conclusions/interpretation
In RAC1-deficient pancreatic beta cells, F-actin acts as a barrier for insulin granules and reduces glucose-stimulated insulin secretion.
In this study, a carbon paste filling method was proposed as a simple strategy for fabricating high-density bipolar electrode (BPE) arrays for bipolar electrochemical microscopy (BEM). High ...spatiotemporal resolution imaging was achieved using the fabricated BPE array. BEM, which is an emerging microscopic system in recent years, achieves label-free and high spatiotemporal resolution imaging of molecular distributions using high-density BPE arrays and electrochemiluminescence (ECL) signals. We devised a simple method to fabricate a BPE array by filling a porous plate with carbon paste and succeeded in fabricating a high-density BPE array (15 μm pitch). After a detailed observation of the surface of the BPE array using a scanning electron microscope, the basic electrochemical and ECL emission characteristics were evaluated using potassium ferricyanide solution as a sample solution. Moreover, inflow imaging of the sample molecules was conducted to evaluate the imaging ability of the prepared BPE array. In addition, Prussian Blue containing carbon ink was applied to the sample solution side of the BPE array to provide catalytic activity to hydrogen peroxide, and the quantification and inflow imaging of hydrogen peroxide by ECL signals was achieved. This simple fabrication method of the BPE array can accelerate the research and development of BEM. Furthermore, hydrogen peroxide imaging by BEM is an important milestone for achieving bioimaging with high spatiotemporal resolution such as biomolecule imaging using enzymes.
Abstract
We report the systematic analysis of knots, hotspots, and lobes in 57 active galactic nuclei (AGNs) to investigate the variation of the magnetic field along the jet from the sub-parsec base ...to the terminus on kiloparsec-to-megaparsec scales. Expanding the number of radio/X-ray samples in the work of Kataoka & Stawarz, we analyzed the data in 12 FR
i
and 30 FR
ii
radio galaxies, 12 quasars, and three BL Lac objects, which contained 76 knots, 42 hotspots, and 29 radio lobes. We first derived the equipartition magnetic fields in the cores and then estimated those in various jet components by assuming
B
est
∝
d
−1
, where
d
is the distance from the jet base. On the other hand, the magnetic field in large-scale jets (knots, hotspots, and lobes),
B
eq
, can be estimated from the observed flux and spatial extent under the equipartition hypothesis. We show that the magnetic field decreases as the distance along the jet increases, but generally in a more gentle way than ∝
d
−1
. The increase in
B
eq
/
B
est
at large
d
may suggest the deceleration of the jet downstream, but there is no difference between FR
i
and FR
ii
jets. Moreover, the magnetic fields in the hotspots are systematically larger than those in knots and lobes. Finally, we applied the same analysis to knots and lobes in Centaurus A to check whether the above discussion will hold even in a
single
jet source.
Centaurus A (Cen A) is the nearest active radio galaxy, which has kiloparsec-scale jets and giant lobes detected by various instruments in radio and X-ray frequency ranges. The Fermi-Large Area ...Telescope and High Energy Stereoscopic System (HESS) confirmed that Cen A is a very high-energy (VHE; >0.1 TeV) γ-ray emitter with a known spectral softening in the energy range from a few GeV to TeV. In this work, we consider a synchrotron self-Compton model in the nucleus for the broadband spectrum below the break energy and an external Compton model in kiloparsec-scale jets for the γ-ray excess. Our results show that the observed γ-ray excess can be suitably described by the inverse Compton scattering of the starlight photons in the kiloparsec-scale jets, which is consistent with the recent tentative report by HESS on the spatial extension of the TeV emission along the jets. Considering the spectral fitting results, the excess can only be seen in Cen A, which is probably due to two factors: (1) the host galaxy is approximately 50 times more luminous than other typical radio galaxies and (2) the core γ-ray spectrum quickly decays above a few MeV due to the low maximum electron Lorentz factor of γc = 2.8 × 103 resulting from the large magnetic field of 3.8 G in the core. By the comparison with other γ-ray detected radio galaxies, we found that the magnetic field strength of relativistic jets scales with the distance from the central black holes d with B(d) ∝ d−0.88 0.14.
The prognosis of gastric cancer (GC) patients with peritoneal dissemination remains poor, and a better understanding of the underlying mechanisms is critical for the development of new treatments ...that will improve survival in these patients. This study aimed to clarify the clinical and biological role of two key metastasis-associated long non-coding RNAs (lncRNAs) in GC. We analyzed the expression levels of two lncRNAs-Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) and HOX-Antisense Intergenic RNA (HOTAIR)-by real-time reverse transcription PCR in 300 gastric tissues (150 GC and 150 adjacent normal mucosa), and in seven GC cell lines. Functional characterization for the role of HOTAIR in GC was performed by small interfering RNA (siRNA) knockdown, followed by series of in-vitro and in-vivo experiments. Expression of both lncRNAs was significantly higher in cancerous tissues than in corresponding normal mucosa, and higher expression of these lncRNAs significantly correlated with peritoneal metastasis in GC patients. In addition, elevated HOTAIR expression emerged both as an independent prognostic and risk factor for peritoneal dissemination. SiRNA knockdown of HOTAIR in GC cells significantly inhibited cell proliferation, migration and invasion, but concurrently enhanced the anoikis rate in transfected cells. In an in vivo assay, HOTAIR siRNA-transfected MKN45 cells injected into nude mice inhibited the growth of xenograft tumors and peritoneal metastasis compared with controls. Our data provide novel evidence for the biological and clinical significance of HOTAIR expression as a potential biomarker for identifying patients with peritoneal metastasis, and as a novel therapeutic target in patients with gastric neoplasia.
Here, we report a highly sensitive immunoassay for human immunoglobulin G (IgG) that uses signal amplification of the coagulation cascade. Z-Phe-Pro-Lys-p-nitroaniline (FPK-pNA) was used as a ...substrate for thrombin activation in the last step of the coagulation cascade. During the coagulation cascade, pNA is liberated from FPK-pNA and can be detected electrochemically. Using square wave voltammetry with a glassy carbon electrode, we demonstrated that pNA can be quantified in a solution modeling the coagulation cascade prepared by mixing FPK-pNA and pNA. Characterization of the reactivity of thrombin toward FPK-pNA revealed that thrombin efficiently reacted with FPK-pNA. Subsequent characterization of factor XIa activity of factor XIa-labeled antibody revealed that factor XIa was not inactivated during labeling. Finally, a coagulation cascade-based immunoassay for human IgG was performed using a factor XIa-labeled antibody on magnetic beads. The limit of detection for human IgG was 5.0 pg/mL (33 fM) indicating that the coagulation cascade can amplify the immunoassay sensitivity compared to immunoassay using a thrombin-labeled antibody as a condition without a coagulation cascade. Coagulation cascade-based immunoassay was also highly selective. In the near future, we will report a highly sensitive immunoassay for the simultaneous detection of multiple analytes using a coagulation cascade-based immunoassay and Limulus amebocyte lysate reaction-based immunoassay we previously reported.
REASON FOR PERFORMING STUDY: Diagnostic techniques for oviductal obstruction in the mare are limited and development of a more reliable and direct method to assess oviductal patency is needed. ...OBJECTIVE: To evaluate the feasibility of hysteroscopic selective hydrotubation of the equine oviduct via videoendoscopy in standing mares. METHODS: Using a catheter inserted under endoscopic guidance into the uterotubal junctions of 10 mares, 5 ml of indigo carmine solution (4 mg/ml) was flushed into the oviduct. After introduction of the dye, peritoneal fluid was obtained via abdominocentesis. A colpotomy was also performed to allow introduction of a videoendoscope into the abdominal cavity to assess the presence of dye visually at the ovarian end of the oviduct. RESULTS AND CONCLUSIONS: In 15 of 20 (75.0%) attempts, the catheter was successfully inserted into the uterotubal junction, and dye was observed at the ampulla, fimbria, overlying the ovary or surrounding tissue. In 2 mares, the videoendoscope could not be manipulated to identify the uterotubal junction. Only one of 2 oviducts was flushed in an additional mare because insufflation of the uterus could not be maintained. The colour of the dye was evident macroscopically and spectrophotometrically in 4 of 8 mares from which peritoneal fluid was successfully collected. POTENTIAL RELEVANCE: The equine oviduct can be evaluated by hysteroscopic selective hydrotubation.
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