Participation of c-FLIP in NLRP3 and AIM2 inflammasome activation.

Cellular FLICE-inhibitory protein (c-FLIP) is an inhibitor of caspase-8 and is required for macrophage survival. Recent studies have revealed a selective role of caspase-8 in noncanonical IL-1ß production that is independent of caspase-1 or inflammasome. Here we demonstrated that c-FLIP(L) is an unexpected contributor to canonical inflammasome activation for the generation of caspase-1 and active IL-1ß. Hemizygotic deletion of c-FLIP impaired ATP- and monosodium uric acid (MSU)-induced IL-1ß production in macrophages primed through Toll-like receptors (TLRs). Decreased IL-1ß expression was attributed to a reduced activation of caspase-1 in c-FLIP hemizygotic cells. In contrast, the production of TNF-α was not affected by downregulation in c-FLIP. c-FLIP(L) interacted with NLRP3 or procaspase-1. c-FLIP is required for the full NLRP3 inflammasome assembly and NLRP3 mitochondrial localization, and c-FLIP is associated with NLRP3 inflammasome. c-FLIP downregulation also reduced AIM2 inflammasome activation. In contrast, c-FLIP inhibited SMAC mimetic-, FasL-, or Dectin-1-induced IL-1ß generation that is caspase-8-mediated. Our results demonstrate a prominent role of c-FLIP(L) in the optimal activation of the NLRP3 and AIM2 inflammasomes, and suggest that c-FLIP could be a valid target for treatment of inflammatory diseases caused by over-activation of inflammasomes.

Identification of the forensically important beetles Nicrophorus japonicus, Ptomascopus plagiatus and Silpha carinata (Coleoptera: Silphidae) based on 16S rRNA gene in China.

Sarcophagous beetles play an important role in estimating postmortem interval time (PMI) in the later stages decomposition of carcasses. However, the morphological similarity of beetles usually poses a challenge for forensic scientists within their routine work. As a supplementary to traditional morphological method, molecular genetics identification is simple and time-saving. A molecular identification method involving a 288-bp segment of the 16S ribosomal RNA (16S rRNA) gene from 15 beetles of Silphidae (Coleoptera), collected from 5 locations in 4 Chinese provinces, was evaluated. Phenogram analysis of the sequenced segments by the unweighted pairgroup method analysis (UPGMA) method showed that all specimens were properly assigned into four species with strong similarity, which indicated the possibility of separation congeneric species with the short 16S rRNA fragment. These results will be instrumental for implementation of the Chinese database of forensically relevant beetles.

A program to interest medical students in Changhua, Taiwan in the incorporation of visual arts in medicine.

INTRODUCTION: Visual arts have been used to facilitate the teaching of the United States Accreditation Council for Graduate Medical Education (ACGME) competencies used in some countries. Some medical students may not appreciate the usefulness of incorporating arts in medical education. Therefore, arts programs that can interest medical students are necessary. We initiated and evaluated a visual arts program at the Changhua Christian Hospital in Changhua, Taiwan, with an aim to give the students a short review of visual arts and to interest them in the incorporation of arts in medicine. METHODS: A total of 110 students in clerkship or internship participated in a visual arts program with emphasis on medicine-related visual arts. Content analysis of the data from the notes made by the instructor from direct observation of students; descriptions during discussions and the written feedback from students at the end of the program was used to evaluate the effect of the program. Anonymous questionnaires were also used for self-assessment of students. RESULTS: Qualitative analysis of the data revealed that the course was interesting to students. Themes emerged including its helpfulness to students in interpreting paintings, enhanced empathy, increased cultural awareness, enhanced observational skills, better team work, listening and communication skills and reduced stress. Ratings on the questionnaire showed similar results. Moreover, students had an increase in their confidence and desire to interpret paintings. CONCLUSION: The structured visual arts program, with emphasis on medicine-related visual arts and other humanities subjects, was able to attract the attention of medical students. It might be helpful to improve the required skills of ACGME competencies, but further studies are needed to support these conclusions.

Ezrin is a negative regulator of death receptor-induced apoptosis.

Ezrin links cortical actin filaments with the cell membrane, and has a critical role in many membrane-initiated events. Fas is directly associated with ezrin, but conflicting results have been reported for the involvement of ezrin in Fas-induced cell death. In this study we show that ezrin was associated with Fas in T cells before stimulation and was released shortly after Fas ligand (FasL) engagement. The knockdown of ezrin moderately increased Fas-triggered or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-triggered cell death in normal T lymphocytes and in H9 cells, but had no effect on death receptor-induced apoptosis in type II cells, such as Jurkat and CEM. Expression of a dominant-negative form of ezrin also led to an increased Fas-induced apoptosis in H9 cells. Ezrin deficiency did not affect the internalization of Fas after Fas ligation. Instead, an enhanced formation of death-inducing signaling complex (DISC) was observed in H9 cells with ezrin knockdown, leading to accelerated caspase-8 activation. Together, our results suggest that ezrin has a negative role in the recruitment of Fas into signaling complexes in type I T cells. Loss of ezrin likely removes the constraint imposed by ezrin and facilitates the assembly of death receptor complex in T cells.

Transcripts of enriched germ cells responding to heat shock as potential markers for porcine semen quality.

A cDNA microarray-assisted experiment was conducted to survey genes that respond early to heat shock in enriched immature porcine germ cells; the 5'-UTR flanking the highest upregulated gene, heat shock 105/110 kDa protein 1 (Hsph1 or Hsp105), in response to heat shock was also investigated. We established a porcine testis cDNA microarray with 9944 transcripts from two libraries constructed from the testes of mature boars, with or without heat shock. After a mild heat shock treatment (39 degrees C for 1h and recovered at 34 degrees C for 2h), 380 transcripts demonstrated significant gene expression in enriched immature germ cells; 326 were upregulated and 54 were downregulated. Ten transcripts of interest exhibiting significance analysis of microarrays (SAM) scores higher than the median were subjected to quantitative real-time PCR; three (Hsp105, Hspa4l and Thap4) were upregulated >1.5-fold. The sequence of the 5'-UTR of Hsp105, the highest upregulated transcript, was cloned and analyzed. A single nucleotide polymorphism (SNP) was found at position -762 (C or T) upstream of the translational start site (ATG codon). Only two genotypes (CC or TC) were found in the mature boars that were studied (n=31). A heterozygous genotype (TC) at this SNP site revealed an elevated percentage of morphologically normal sperm during hot and cold seasons; this SNP may be a useful marker for semen quality in boars. Furthermore, the cell-model established from enriched primitive germ cells has potential for the study of reproduction in mature animals.

Expressed transcripts associated with high rates of egg production in chicken ovarian follicles.

The purpose of this study was to characterize differentially expressed transcripts associated with varying rates of egg production in Taiwan country chickens. Ovarian follicles were isolated from two strains of chicken which showed low (B) or high (L2) rates of egg production, then processed for RNA extraction and cDNA library construction. Three thousand and eight forty clones were randomly selected from the cDNA library and amplified by PCR, then used in microarray analysis. Differentially expressed transcripts (P<0.05, log(2)> or = 1.75) were sequenced, and aligned using GenBank. This analysis revealed 20 non-redundant sequences which corresponded to known transcripts. Eight transcripts were expressed at a higher level in ovarian tissue prepared from chicken strain B, and 12 transcripts were expressed at a higher level in L2 birds. These differential patterns of expression were confirmed by semi-quantitative RT-PCR. We show that transcripts of cyclin B2 (cycB2), ferritin heavy polypeptide 1 (FTH1), Gag-Pol polyprotein, thymosin beta4 (TB4) and elongation factor 1 alpha1 (EEF1A1) were enriched in B strain ovarian follicles. In contrast, thioredoxin (TXN), acetyl-CoA dehydrogenase long chain (ACADL), inhibitor of growth family member 4 (ING4) and annexin II (ANXA2) were expressed in at higher levels in the L2 strain. We suggest that our approach may lead to the isolation of effective molecular markers that can be used in selection programs in Taiwan country chickens.

AFLP fingerprinting for paternity testing in ducks.

1. The accuracy and reproducibility of AFLP fingerprinting was investigated in the duck (Anas Platyrhynchos), using a multicolour fluorescent labeling technique. The fluorescent labelling fragments were separated on a capillary electrophoresis-base ABI PRISM 3100 Genetic Analyzer. 2. A total of 337 AFLP peaks with 103 of them being polymorphic markers were generated by 16 sets consisting of EcoRI/TaqI primer pair combinations. The number and size range of AFLP polymorphisms detected per primer pair varied from 3 to 11 and 58 to 290 bp, respectively. About 30.6% (103/337) of AFLP peaks were detected polymorphisms, with an average of 6.4 polymorphic markers per primer pair. 3. The clear polymorphic peaks were amplified with EcoR+AC/Taq+AC primer combinations. The AFLP peaks showed high reproducibility. From the family testing, we found that the fingerprints of all the offspring were derived from one or other parent. Therefore, we conclude that AFLP fingerprinting might be a suitable method for duck paternity testing.

Evaluation of activity of epiphyseal plates in growing males and females.

To investigate the age-related activity of the epiphyseal plates, a retrospective study of (99m)Tc-methylene diphosphonate bone scans was undertaken. The study comprised 81 males and 46 females aged 2 weeks to 24 years. The total percentage (%) whole-body (ratio of total physis activity to whole-body activity) and the regional % whole-body (ratio of physis activity of one region to whole-body activity) were derived. The ratio of physis activity of one region to the total physis activity was defined as % physis. Before age 12, total physis activity was found to contribute about 10% to whole-body activity. All total and regional % whole-body activities followed sigmoid curves with age. The differences of the parameters (transition centers and widths) suggested that there might be a later and longer period for the disappearance of physis activity in males than in females. For all the regions, % physis changed little with age until after puberty. At age <1, the proportion of bone activity in the body was about 30-35% for skull, 20-25% for lower limbs, and 5-15% for the rest of the regions. The maximal changes during growth occurred in the skull and the lower limbs. The age-related changes of physis activity during growth reflect a combination of the potential of bone to grow and the processes of bone growth and bone turnover. Bone scintigraphy is useful in understanding the changes of physis activity during growth.

Activation of the transient receptor potential M2 channel and poly(ADP-ribose) polymerase is involved in oxidative stress-induced cardiomyocyte death.

Overproduction of reactive oxygen species is one of the major causes of cell death in ischemic-reperfusion (I/R) injury. In I/R animal models, electron microscopy (EM) has shown mixed apoptotic and necrotic characteristics in the same cardiomyocyte. The present study shows that H(2)O(2) activates both apoptotic and necrotic machineries in the same myocyte and that the ultrastructure seen using EM is very similar to that in I/R animal studies. The apoptotic component is caused by the activation of clotrimazole-sensitive, NAD(+)/ADP ribose/poly(ADP-ribose) polymerase (PARP)-dependent transient receptor potential M2 (TRPM2) channels, which induces mitochondrial [Na(+)](m) (and [Ca(2+)](m)) overload, resulting in mitochondrial membrane disruption, cytochrome c release, and caspase 3-dependent chromatin condensation/fragmentation. The necrotic component is caspase 3-independent and is caused by PARP-induced [ATP](i)/NAD(+) depletion, resulting in membrane permeabilization. Inhibition of either TRPM2 or PARP activity only partially inhibits cell death, while inhibition of both completely prevents the ultrastructural changes and myocyte death.

Objective and noninvasive detection of sub-clinical lung injury in breast cancer patients after radiotherapy.

AIMS: We employed technetium-99m hexamethylpropylene amine oxime (Tc-99m HMPAO) lung scan to detect sub-clinical lung injury after radiation therapy of 60 female patients diagnosed with right breast cancer. METHODS: The degree of pulmonary vascular endothelium damage was represented as lung/liver uptake ratios (L/L ratios) calculated on Tc-99m HMPAO lung scan. All patients underwent simple mastectomy and post-operative radiotherapy of approximately 50 Gy. We divided the patients into three groups according to the interval between radiotherapy and lung Tc-99m HMPAO lung scan: Group 1 included 20 patients who received the lung scan within 1-3 months after radiotherapy, group 2 included 20 patients were within 3-9 months after radiotherapy, and group 3 included 20 patients were more than 9 months after radiotherapy. In addition, 20 age-matched normal women were included as the control group. RESULTS: The L/L ratios were 0.32+/-0.04 for normal controls, 0.59+/-0.10 for group 1, 0.55+/-0.07 for group 2, and 0.34+/-0.04 for group 3, respectively. Based on our preliminary results, we found that sub-clinical lung injury and significantly increased L/L ratio in breast cancer patients received radiotherapy may occur within the first 6 months after radiotherapy. However, the L/L ratio is markedly decreased after 9 months. CONCLUSION: Our findings concluded that the degree of pulmonary vascular endothelium damage represented as the L/L ratio on Tc-99m HMPAO lung scan has the potential to be a sensitive, objective and noninvasive method to detect sub-clinical lung injury in breast cancer patients received radiotherapy.

Arachidonic acid-induced H+ and Ca2+ increases in both the cytoplasm and nucleoplasm of rat cerebellar granule cells.

1. Arachidonic acid (AA) exerts multiple physiological and pathophysiological effects in the brain. By continuously measuring the intracellular pH (pH(i)) and Ca2+ levels ([Ca2+]i) in primary cultured rat cerebellar granule cells, we have found, for the first time, that 20 min treatment with 10 microM AA resulted in marked increases in Ca2+ and H+ levels in both the cytosol and nucleus. 2. A much higher concentration (40 mM) of another weak acid, propionic acid, was needed to induce a similar change in pH(i). The [Ca2+]i increase was probably caused by AA-induced activation of Ni2+-sensitive cationic channels, but did not involve NMDA channels or the Na+-Ca2+ exchanger. 3. AA-induced acidosis occurs by a different mechanism involving predominantly the passive diffusion of the un-ionized form of AA, rather than a protein carrier, as proposed by Kamp & Hamilton for fatty acids (FAs) in artificial phospholipid bilayers (the 'flip-flop' model). The following results, which are similar to those observed in lipid bilayers, support this conclusion: (1) FAs containing a -COOH group (AA, linoleic acid, alpha-linolenic acid, and docosahexaenoic acid) induced intracellular acidosis, whereas a FA with a -COOCH3 group (AA methyl ester) had little effect on pH(i), (2) a FA amine, tetradecylamine, induced intracellular alkalosis, and (3) the AA-/FA-induced pH(i) changes were reversed by bovine serum albumin. 4. Further evidence in support of a passive diffusion model, rather than a membrane protein carrier, is that: (1) there was a linear relationship between the initial rate of acid flux and the concentration of AA (2-100 microM), (2) acidosis was not inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid, a potent inhibitor of the plasma membrane FA carrier protein, and (3) the involvement of most known H+-related membrane carriers and H+ conductance has been ruled out. 5. Since AA can be released under both physiological and pathophysiological conditions, the possible significance of the AA-evoked increases in H+ and Ca2+ in both the cytoplasm and nucleoplasm is discussed.

Correlation of vasomotor- and respiratory-controlling mechanisms around the caudal ventrolateral medulla in cats.

We examined the involvement of caudal ventrolateral medulla (CVLM) in respiratory control. Microinjection of glutamate (Glu) into CVLM decreased systemic arterial blood pressure (SAP) and altered phrenic nerve activities (PNA). Among 143 depressor sites, 55% (78/143) increased respiratory frequency (Rf), while 72% altered PNA amplitude (36% increased and 36% decreased). A small but significant positive correlation was observed between the magnitudes of depressor responses and inhibition of PNA amplitude (r = 0.1718, n = 143), indicating a substantial cross-talk between depressor and PNA inhibitory neurons. Furthermore, microinjections of acetylcholine (ACh) mimicked the Glu-induced depressor responses. However, ACh did not alter Rf, but still reduced PNA amplitude. Our findings suggest that Rf-regulating and depressor neurons are two separate neuronal populations, coexisting in CVLM. The PNA inhibitory and depressor neurons, in contrast, could have stronger correlation.

Fragile X premutation is a significant risk factor for premature ovarian failure: the International Collaborative POF in Fragile X study--preliminary data.

The preliminary results of an international collaborative study examining premature menopause in fragile X carriers are presented. A total of 760 women from fragile X families was surveyed about their fragile X carrier status and their menstrual and reproductive histories. Among the subjects, 395 carried a premutation, 128 carried a full mutation, and 237 were noncarriers. Sixty-three (16%) of the premutation carriers had experienced menopause prior to the age of 40 compared with none of the full mutation carriers and one (0.4%) of the controls. Based on these preliminary data, there is a significant association between fragile X premutation carrier status and premature menopause.

Evaluation of global and regional left ventricular function using technetium-99m sestamibi ECG-gated single-photon emission tomography.

We evaluated a method for the assessment of left ventricular (LV) function with technetium-99m sestamibi ECG-gated single-photon emission tomography (GSPET). GSPET was performed at rest in 21 patients. Images were reconstructed to obtain end-diastolic (ED) and end-systolic (ES) images. Endocardial and epicardial edges of the left ventricle for the ED and ES images were defined using the gradient images and the algorithm developed. LV wall thickness was measured for the mid-ventricular slices of ED and ES images at 10 degrees intervals. The systolic thickening (ST) and the LV ejection fraction [LVEF(GSPECT)] could be determined. These values were compared with the LV ejection fraction (LVEF) estimated using the gated blood pool method. There was a linear correlation between LVEF and ST (r=0.79), and LVEF and LVEF(GSPET) (r=0.87). Histograms of LV thicknesses were generated. Agreement for evaluation of regional wall motion between the method using histograms of LV thicknesses and the gated blood pool study was 92.8% (kappa=0.75). It is concluded that with an appropriate method for LV edge detection, GSPET with 99mTc-labelled perfusion agents is of use for simultaneous evaluation of myocardial perfusion and assessment of LV function.

A semi-automated method for edge detection in the evaluation of left ventricular function using ECG-gated single-photon emission tomography.

Thallium-201 gated single-photon emission tomography (GSPET) of myocardium was performed at rest in 18 patients. Images were reconstructed to obtain end-diastolic (ED) and end-systolic (ES) images. The endocardial and epicardial edges of the left ventricle (LV) for the ED and ES images were defined for the mid-ventricular images of the short-axis slices, using a semi-automated method. LV wall thickness was measured for ED and ES images at 10 degrees intervals. Mean LV thickness was derived as the mean of the LV thickness for the three mid-ventricular slices. The systolic thickening (ST) was derived as: mean LV thickness (ES)--mean LV thickness (ED). The systolic thickening ratio (STR) was defined as: ST/mean LV thickness (ED). LV cavity area was measured. The dilation ratio (DR) was defined as: [mean cavity area (ED)--mean cavity area (ES)]/mean cavity area (ED). LV ejection fraction (LVEF) was estimated using technetium-99m gated blood pool study. There was a linear correlation between LVEF and ST (r = 0.85), LVEF and STR (r = 0.77) and LVEF and DR (r = 0.81). There was a strong correlation (r = 0.85) between regional STR and regional percent count increase in 52 segments which did not have perfusion defects. As well as for the evaluation of myocardial perfusion, GSPET images can be of use for the assessment of LV function using an appropriate method for LV edge detection.

Radionuclide hysterosalpingography with technetium-99m-pertechnetate: application and radiation dose to the ovaries.

Although radionuclide hysterosalpingography (RNHSG) has been suggested as an efficient procedure for assessing function of fallopian tubes, the radiation dose to the ovaries was addressed as an important issue to be taken into consideration. We describe a modified method of RNHSG, calculating the radiation dose to the ovaries. A small dose of approximately 18.5 MBq (0.5 mCi) of [99mTc]pertechnetate was administered directly into the uterine cavity without overpressure. The accuracy of the method was 84.5% as compared with the contrast hysterosalpingography. The estimated average dose to the ovaries was 0.057 mGy/MBq (0.21 rad/mCi) or 1.08 mGy (108 mrad) per study. RNHSG is an accurate method for functional study of fallopian tube patency with low radiation dose.

Usefulness of Tl-201 SPECT imaging in evaluation of left ventricular hypertrophy and dilation in patients with previous myocardial infarction.

Using short axis slices of Tl-201 myocardial SPECT images, total left ventricular (LV) mass, perfused LV mass, defect size, defect mass, ratio of total LV area to cavity area (T/C), ratio of radius of total LV area to radius of cavity area (TR/CR), and ratio of wall thickness to radius of cavity area (W/R) were obtained. The left ventricular ejection fraction (LVEF) was calculated using blood pool imaging studies. A total of 33 patients with old myocardial infarctions were studied. The patients were divided into group 1 (n = 16) with no history of heart failure, and group 2 (n = 17) with a history of heart failure. The LVEF (P less than 0.001), T/C (P less than 0.005), TR/CR (P less than 0.005) and W/R (P less than 0.001) were significantly lower, while the total LV mass (P less than 0.001) and perfused LV mass (P less than 0.01) were significantly higher in group 2. In the whole group of 33 patients, there was inverse linear correlation between the total LV mass and LVEF (r = -0.82), perfused LV mass and LVEF (r = -0.67), and defect mass and LVEF (r = -0.59). There was linear correlation between W/R and LVEF (r = 0.64). Nonlinear correlations were found between T/C and LVEF (r = 0.81) and TR/CR and LVEF (r = 0.80). Tl-201 myocardial SPECT imaging is useful for the evaluation of LV hypertrophy and cavity dilation in patients with previous myocardial infarction.