Resected Intrahepatic Cholangiocarcinoma with Anaphylactic Shock from a Preoperative Liver Function Test before Hepatectomy

The indocyanine green test is a reliable liver function examination before major hepatectomy, and anaphylaxis is rarely a concern. A 65-year-old male patient without epigastralgia was diagnosed with a 2.2-cm intrahepatic cholangiocarcinoma. He had no history of allergic reactions. Some liver dysfunction was indicated by the laboratory data; however, there was no marked obstructive jaundice and the liver functional reserve was maintained by technetium-99m galactosyl serum albumin. The indocyanine green test was routinely performed, but the patient immediately demonstrated severe anaphylaxis due to indocyanine green administration. He had cardiorespiratory arrest, but recovered after immediate resuscitation. Although acute renal and respiratory failure was significant, the patient recovered at day 10 after the event, and his liver function and other organ functions were improved. Then, the scheduled left hepatectomy with caudate and extrahepatic duct resection was successfully performed without issues. The patient exhibited no allergic response against the administration of antibiotics or other drugs and the postoperative course was uneventful. The patient was discharged on day 17. The tumor was diagnosed as stage III intrahepatic cholangiocarcinoma and R0 resection was accomplished. Preoperative management, including the liver functional loading test, should be carefully carried out before major hepatectomy

In Vivo Imaging of Hierarchical Spatiotemporal Activation of Caspase-8 during Apoptosis.

[Background]: Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the dynamics of CASP8 activation are not fully understood. [Methodology/Principal Findings]: We have established a biosensor based on fluorescence resonance energy transfer (FRET) for visualizing apoptotic signals associated with CASP8 activation at the single-cell level. Our dual FRET (dual-FRET) system, comprising a triple fusion fluorescent protein, enabled us to simultaneously monitor the activation of CASP8 and its downstream effector, caspase-3 (CASP3) in single live cells. With the dual-FRET-based biosensor, we detected distinct activation patterns of CASP8 and CASP3 in response to various apoptotic stimuli in mammalian cells, resulting in the positive feedback amplification of CASP8 activation. We reproduced these observations by in vitro reconstitution of the cascade, with a recombinant protein mixture that included procaspases. Furthermore, using a plasma membrane-bound FRET-based biosensor, we captured the spatiotemporal dynamics of CASP8 activation by the diffusion process, suggesting the focal activation of CASP8 is sufficient to propagate apoptotic signals through death receptors. [Conclusions]: Our new FRET-based system visualized the activation process of both initiator and effector caspases in a single apoptotic cell and also elucidated the necessity of an amplification loop for full activation of CASP8

Angiotensin‐converting enzyme 2 deficiency accelerates and angiotensin 1‐7 restores age‐related muscle weakness in mice

Abstract Background A pharmacologic strategy for age‐related muscle weakness is desired to improve mortality and disability in the elderly. Angiotensin‐converting enzyme 2 (ACE2) cleaves angiotensin II into angiotensin 1‐7, a peptide known to protect against acute and chronic skeletal muscle injury in rodents. Since physiological aging induces muscle weakness via mechanisms distinct from other muscle disorders, the role of ACE2‐angiotensin 1‐7 in age‐related muscle weakness remains undetermined. Here, we investigated whether deletion of ACE2 alters the development of muscle weakness by aging and whether angiotensin 1‐7 reverses muscle weakness in older mice. Methods After periodic measurement of grip strength and running distance in male ACE2KO and wild‐type mice until 24 months of age, we infused angiotensin 1‐7 or vehicle for 4 weeks, and measured grip strength, and excised tissues. Tissues were also excised from younger (3‐month‐old) and middle‐aged (15‐month‐old) mice. Microarray analysis of RNA was performed using tibialis anterior (TA) muscles from middle‐aged mice, and some genes were further tested using RT‐PCR. Results Grip strength of ACE2KO mice was reduced at 6 months and was persistently lower than that of wild‐type mice (p < 0.01 at 6, 12, 18, and 24‐month‐old). Running distance of ACE2KO mice was shorter than that of wild‐type mice only at 24 months of age [371 ± 26 vs. 479 ± 24 (m), p < 0.01]. Angiotensin 1‐7 improved grip strength in both types of older mice, with larger effects observed in ACE2KO mice (% increase, 3.8 ± 1.5 and 13.3 ± 3.1 in wild type and ACE2KO mice, respectively). Older, but not middle‐aged ACE2KO mice had higher oxygen consumption assessed by a metabolic cage than age‐matched wild‐type mice. Angiotensin 1‐7 infusion modestly increased oxygen consumption in older mice. There was no difference in a wheel‐running activity or glucose tolerance between ACE2KO and wild‐type mice and between mice with vehicle and angiotensin 1‐7 infusion. Analysis of TA muscles revealed that p16INK4a, a senescence‐associated gene, and central nuclei of myofibers increased in middle‐aged, but not younger ACE2KO mice. p16INK4a and central nuclei increased in TA muscles of older wild‐type mice, but the differences between ACE2KO and wild‐type mice remained significant (p < 0.01). Angiotensin 1‐7 did not alter the expression of p16INK4a or central nuclei in TA muscles of both types of mice. Muscle ACE2 expression of wild‐type mice was the lowest at middle age (2.6 times lower than younger age, p < 0.05). Conclusions Deletion of ACE2 induced the early manifestation of muscle weakness with signatures of muscle senescence. Angiotensin 1‐7 improved muscle function in older mice, supporting future application of the peptide or its analogues in the treatment of muscle weakness in the elderly population

Estimation of the diffusion coefficient of CASP8CS/mKikGR.

<p>Estimation of the diffusion coefficient of CASP8CS/mKikGR.</p

Monitoring of caspase activation with the CYR83 in single cells.

<p>(<b>A</b>) A schematic structure of CYR83 and its variants. In CYR83(IETA) variant, the IETD sequence was replaced by IETA; in the CYR83(DEVA) variant, DEVD was replaced by DEVA. (<b>B</b>) The graphic pattern of the emission ratio based on the fluorescence intensity of the CYR83 in single cells undergoing apoptosis. The CYR83-expressing HeLa cells were induced to undergo apoptosis with an agonistic anti-Fas antibody and monitored by dual-FRET. As shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050218#pone.0050218.s003" target="_blank">Figure S3</a>, time course was set up before and after 1 h of cell shrinkage. The temporal fluctuations of the emission ratio on Venus/seCFP and mRFP1/Venus in single cells are plotted as red and blue lines, respectively. The IETDase and DEVDase activities are inversely proportional to graphic data. The arrow indicates a rebound detected by monitoring the fluorescence. (<b>C</b>) The CYR83-expressing HeLa cells were induced to undergo apoptosis by UV-irradiation and monitored by dual-FRET. A time course of the emission ratio is indicated. (<b>D</b>, <b>E</b>) HeLa cells expressing CYR83 variants were monitored for fluorescence. Transfected cells expressing CYR83(IETA) (D) or CYR83(DEVA) (E) were treated with an anti-Fas antibody and monitored for fluorescence. <b>(F</b>, <b>G)</b> Fluorescence image analyses on the proteolytic processing profiles of CYR83 and its variants. HeLa cells expressing CYR83 or its variants were subjected to extrinsic (F) and intrinsic (G) apoptotic stimuli at indicated times. Cell extracts prepared from those cells were resolved by SDS-PAGE and scanned for fluorescence in the gel using the imaging analyzer. Among fluorescence bands detected in panels of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050218#pone.0050218.s005" target="_blank">Figure S5A–C and S5E–G</a>, three bands corresponding to each seCFP, Venus and mRFP1 peptide fragments were chosen and represented as (F) and (G).</p

A mathematical model on the propagation of CASP8 activation.

<p>(<b>A</b>–<b>C</b>) The propagation of CASP8 activation was simulated by an one-dimensional diffusion model. The vertical axis (<i>u</i>) indicates the concentration of the activated CASP8. The horizontal axis (<i>x</i>) indicates the distance from the input source of the apoptotic signal and time zero means the starting point of the cell-cell interaction. CASP8 activation propagates from the left to the right in the graph. Each colored line in the figures gives the spatial distribution of activated CASP8 for different time, where time zero is the starting point of the cell-cell interaction. A blue line indicates the start time when the apoptotic signal is inputted. A yellow line indicates 2000 unit time that has passed from the start time. The duration (<i>τ</i>) of the input signal and was set to 100 (A), 500 (B), and 1000 (C) and <i>f</i>₀ = 10. Here, diffusion coefficient is set to <i>D</i> = 1 and the cell size is set to <i>L</i> = 200.</p

Effects of the downregulation of CASP8 on the processing of the SCAT8.1 biosensor.

<p>(<b>A</b>) Immunoblot analyses of CASP8 proteins downregulated by RNA interference. Cell extracts from HeLa cells carrying either pCSIIU6Tet-Neo or pCSIIU6Tet-sh<i>CASP8</i>-Neo were prepared and analyzed by SDS-PAGE, followed by immunoblotting with anti-CASP8 and anti-actin antibodies, respectively. The numbers indicate the ratio of endogenous CASP8 between two stable lines by calculating the measurements of CASP8 and actin using an image analyzer. (<b>B</b>) Immunoblot analyses of CASP8-knockdown cells subjected to intrinsic apoptotic stimuli. Cell extracts from parental HeLa cells (lanes 1–4) or HeLa/CASP8-KD cells (lanes 5–8) expressing SCAT8.1 were prepared at the indicated times after UV-irradiation. Endogenous CASP3, PARP, actin and exogenous SCAT8.1 were examined with indicated specific antibodies. A representative of four independent experiments is shown. Arrows indicate intact proteins while arrowheads identify the processed peptide fragments. (<b>C</b>) The static analysis of the immunoblot data. The relative ratio of processed peptide fragments to total SCAT8.1 proteins was estimated by measuring the intensity of immunoblot bands in four independent experiments using an image analyzer. The ratio of the processed form relative to total CASP3 proteins was also shown as percentage. The both graphs indicate the means and standard deviations of the estimated ratios. Significant differences between the two groups were evaluated by Student’s <i>t</i>-test. An asterisk shows p < 0.05. (<b>D</b>) Immunoblot analyses of HeLa cells overexpressing CASP6 or its mutant. HeLa cells were transiently transfected either with plasmids carrying CASP6 (lanes 5–8), CASP6CS (lanes 9–12) or control vector (lanes 1–4), and cell extracts were prepared at indicated times after UV-irradiation. Endogenous CASP3, PARP, actin and exogenous CASP6 and SCAT8.1 were examined with indicated specific antibodies. A representative of four independent experiments is shown. The arrow and arrowhead indicate the full-length and the cleaved form of proteins examined, respectively. (<b>E</b>) The static analysis of the immunoblot data. Both ratio and percentage of processed fragments to total SCAT8.1 and processed CASP3 to total CASP3 were estimated and shown as the bar graph. Statistical validation was performed by Student’s <i>t</i>-test. *p < 0.05, **p < 0.01.</p