[Consistency of ALK Ventana-D5F3 immunohistochemistry interpretation in lung adenocarcinoma among Chinese histopathologists].

Objective: To understand the consistency of ALK Ventana-D5F3 immunohistochemistry (IHC) interpretation in Chinese lung adenocarcinoma among histopathologists from different hospitals, and to recommend solution for the problems found during the interpretation of ALK IHC in real world, with the aim of the precise selection of patients who can benefit from ALK targeted therapy. Methods: This was a multicenter and retrospective study. A total of 109 lung adenocarcinoma cases with ALK Ventana-D5F3 IHC staining were collected from 31 lung cancer centers in RATICAL research group from January to June in 2018. All cases were scanned into digital imaging with Ventana iSCANcoreo Digital Slide Scanning System and scored by 31 histopathologists from different centers according to ALK binary (positive or negative) interpretation based on its manufacturer's protocol. The cases with high inconsistency rate were further analyzed using FISH/RT-PCR/NGS. Results: There were 49 ALK positive cases and 60 ALK negative cases, confirmed by re-evaluation by the specialist panel. Two cases (No. 2302 and No.2701) scored as positive by local hospitals were rescored as negative, and were confirmed to be negative by RT-PCR/FISH/NGS. The false interpretation rate of these two cases was 58.1% (18/31) and 48.4% (15/31), respectively. Six out of 31 (19.4%) pathologists got 100% accuracy. The minimum consistency between every two pathologists was 75.8%.At least one pathologist gave negative judgement (false negative) or positive judgement (false positive) in the 49 positive or 60 negative cases, accounted for 26.5% (13/49), 41.7% (25/60), respectively, with at least one uncertainty interpretation accounted for 31.2% (34/109). Conclusion: There are certain heterogeneities and misclassifications in the real world interpretation of ALK-D5F3 IHC test, which need to be guided by the oncoming expert consensus based on the real world data.

[Role of electrocardiogram in predicting cardiac resynchronization therapy response].

OBJECTIVE: To explore the role of electrocardiogram(ECG)in predicting cardiac resynchronization therapy (CRT) response. METHODS: This study retrospectively analyzed ECG of 92 CRT patients, who received CRT therapy from 2001 to 2013 in our center and were followed up for 6 months. The patients were divided into responder group (n=64) and non-responder group (n=28). The baseline and 6-month data including QRS width, heart rhythm and axis variation were analyzed. The definition of responder is left ventricular end systolic volume (LVESV) reduction ≥15% within 6 months after CRT. After CRT therapy, the ventricular activation was changed as left to right (frontal plane), posterior to anterior and axis changed in a clockwise direction. The change in more than two directions was defined as prominent axis change. Logistic analysis was performed to analyze the role of ECG in predicting CRT response. RESULTS: (1) Baseline parameter comparison between the two groups: the proportion of female and LBBB is significantly higher (P<0.01; P=0.04), while the proportion of atrial fibrillation/flutter (Af/AF) is significantly lower (P<0.01) in responder group than in non-responder group. The pre-CRT average QRS duration is much wider in responder group than in non-responder group (P=0.01). (2) Comparison of follow-up with baseline results in two groups: NYHA heart function level, 6 minutes walking distance, QRS duration, LVEF, LVESV improved significantly (P<0.01) post-CRT in responder group. In non-responder group, the QRS duration and LVESV deteriorated significantly (P=0.02, P<0.01), while post-CRT NYHA heart function level improved significantly. In responder group, pre-CRT ECG axis of 53 patients (82.8%) pointed to left and 58 patients (90.6%) pointed to posterior; post-CRT ECG axis of 49 patients (76.6%) pointed to right and 30 patients (40.6%) pointed to anterior. In non-responder group, pre-CRT ECG axis of 25 patients (89.3%) pointed to left and 24 patients (85.7%) pointed to posterior; post-CRT ECG axis of 17 patients (60.7%) pointed to right and 12 patients (42.9%) pointed to anterior. Post-CRT, the proportion of ECG axis prominent change was significantly higher in responder than in non-responder group (62.5%(40/64) vs. 32.1%(9/28), P=0.007). (3)Predicting value: pre-CRT QRS width ≥140 ms (OR=4.97, 95% CI 1.53 to 16.13, P=0.008)and post-CRT prominent axis change (OR=5.1, 95% CI 1.67 to 15.5, P=0.004)were found to be independent predictors of CRT responders. Af/AF pre-CRT was associated with reduced CRT response (OR=0.25, 95% CI 0.08 to 0.80, P=0.02). CONCLUSIONS: ECG may play a role in predicting CRT response. QRS width and Af/AF before CRT and ECG axis change post-CRT could be used to predict CRT response.

Liquid-gated interface superconductivity on an atomically flat film.

Liquid/solid interfaces are attracting growing interest not only for applications in catalytic activities and energy storage, but also for their new electronic functions in electric double-layer transistors (EDLTs) exemplified by high-performance organic electronics, field-induced electronic phase transitions, as well as superconductivity in SrTiO(3) (ref. 12). Broadening EDLTs to induce superconductivity within other materials is highly demanded for enriching the materials science of superconductors. However, it is severely hampered by inadequate choice of materials and processing techniques. Here we introduce an easy method using ionic liquids as gate dielectrics, mechanical micro-cleavage techniques for surface preparation, and report the observation of field-induced superconductivity showing a transition temperature T(c)=15.2 K on an atomically flat film of layered nitride compound, ZrNCl. The present result reveals that the EDLT is an extremely versatile tool to induce electronic phase transitions by electrostatic charge accumulation and provides new routes in the search for superconductors beyond those synthesized by traditional chemical methods.

Differential patterns of antioxidant enzyme mRNA expression in guinea pig lung and liver during development.

cDNA clones for guinea pig antioxidant enzymes, copper-zinc (Cu-Zn) and manganese (Mn-) superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were isolated by reverse transcription (RT)-polymerase chain reaction (PCR) cloning, to explore the mechanism regulating the differential expression of antioxidant enzymes (AOEs) in guinea pig lung and liver, during development. Increases in MnSOD, CAT and GPx mRNA expression in lung and, MnSOD mRNA in liver, were seen during the final period of gestation, whereas CuZnSOD and CAT mRNA expression in liver, which was constant during gestation, increased in the postnatal period. In lung, CuZnSOD mRNA level decreased just prior to birth while in liver, GPx mRNA expression declined markedly over the last third of gestation. In lung, while the mRNA levels of MnSOD, CAT, and GPx increased pre-natally, they declined following birth. In contrast, the postnatal increase in mRNA for CuZnSOD and CAT and the prenatal increase in MnSOD mRNA expression in liver remained at least to adolescence. In adolescent guinea pigs, CuZnSOD and CAT mRNA were most abundantly expressed in liver, while MnSOD and GPx mRNA were most abundant in heart and spleen, respectively. These results demonstrate markedly different developmental patterns of AOEs expression in guinea pig lung and liver during both the pre- and post-natal period. The short-lasting, late-gestational increases of MnSOD, CAT, and GPx mRNA expression in lung, may be responsible for the temporary increases in the activity of these antioxidants in the late gestational period, whereas the steady increases of CuZnSOD, CAT mRNA following birth, and also the prenatal increases in MnSOD mRNA expression, are probably responsible for the higher postnatal activity of these antioxidants in liver.

Effects of des-Asp-angiotensin I on the contractile action of angiotensin II and angiotensin III.

Nanomolar concentrations of des-Asp-angiotensin I potentiated the contractile action of angiotensin II on the rabbit aortic ring but attenuated the contractile action of angiotensin III in the same tissue. Indomethacin had no effect on the potentiation of angiotensin II but inhibited the attenuation of angiotensin III. The action of angiotensin II, angiotensin III and des-Asp-angiotensin I was not inhibited by (S)-1-}[4-(dimethylamino)-3-methylphenyl]methyl}-5-(diphenylacetyl )-4,5,6, 7-tetrahydro-1H-imidazo-[4,5-c]pyridine-6-carboxylic acid, ditrifluoroacetate, dihydrate (PD123319), an angiotensin AT2 receptor antagonist. The data show that angiotensin II and angiotensin III act on different subclasses of angiotensin receptors and that their actions are differentially modulated by des-Asp-angiotensin I. The data also indicate the possibility that des-Asp-angiotensin I is a functional peptide that modulates the contractile action of the two angiotensins at sub-nanomolar concentrations.

Electrically induced ferromagnetism at room temperature in cobalt-doped titanium dioxide.

The electric field effect in ferromagnetic semiconductors enables switching of the magnetization, which is a key technology for spintronic applications. We demonstrated electric field-induced ferromagnetism at room temperature in a magnetic oxide semiconductor, (Ti,Co)O(2), by means of electric double-layer gating with high-density electron accumulation (>10(14) per square centimeter). By applying a gate voltage of a few volts, a low-carrier paramagnetic state was transformed into a high-carrier ferromagnetic state, thereby revealing the considerable role of electron carriers in high-temperature ferromagnetism and demonstrating a route to room-temperature semiconductor spintronics.

Discovery of superconductivity in KTaO3 by electrostatic carrier doping.

Superconductivity at interfaces has been investigated since the first demonstration of electric-field-tunable superconductivity in ultrathin films in 1960(1). So far, research on interface superconductivity has focused on materials that are known to be superconductors in bulk. Here, we show that electrostatic carrier doping can induce superconductivity in KTaO(3), a material in which superconductivity has not been observed before. Taking advantage of the large capacitance of the self-organized electric double layer that forms at the interface between an ionic liquid and KTaO(3) (ref. 12), we achieve a charge carrier density that is an order of magnitude larger than the density that can be achieved with conventional chemical doping. Superconductivity emerges in KTaO(3) at 50 mK for two-dimensional carrier densities in the range 2.3 × 10(14) to 3.7 × 10(14) cm(-2). The present result clearly shows that electrostatic carrier doping can lead to new states of matter at nanoscale interfaces.

Angiopoietin growth factors and Tie receptor tyrosine kinases in renal vascular development.

Angiopoietin-1 (Ang-1) is a secreted growth factor which binds to and activates the Tie-2 receptor tyrosine kinase. The factor enhances endothelial cell survival and capillary morphogenesis, and also limits capillary permeability. Ang-2 binds the same receptor but fails to activate it: hence, it is a natural inhibitor of Ang-1. Ang-2 destabilises capillary integrity, facilitating sprouting when ambient vascular endothelial growth factor (VEGF) levels are high, but causing vessel regression when VEGF levels are low. Tie-1 is a Tie-2 homologue but its ligands are unknown. Angiopoietin and Tie genes are expressed in the mammalian metanephros, the precursor of the adult kidney, where they may play a role in endothelial precursor growth. Tie-1-expressing cells can be detected in the metanephros when it first forms and, based on transplantation experiments, these precursors contribute to the generation of glomerular capillaries. During glomerular maturation, podocyte-derived Ang-1 and mesangial-cell-derived Ang-2 may affect growth of nascent capillaries. After birth, vasa rectae acquire their mature configuration and Ang-2 expressed by descending limbs of loops of Henle would be well placed to affect the growth of this medullary microcirculation. Finally, preliminary data implicate angiopoietins in deregulated vessel growth in Wilms' kidney tumours and in vascular remodelling after nephrotoxicity.

Hypoxia up-regulates angiopoietin-2, a Tie-2 ligand, in mouse mesangial cells.

BACKGROUND: Angiopoietins are secreted factors modulating endothelial survival and morphogenesis. Our previous studies demonstrated angiopoietin-2 (Ang-2) promoter activity in vivo in maturing kidney vascular smooth muscle and mesangial cells, with Tie-2 expressed by adjacent endothelia, including glomerular capillaries. METHODS: In this study we investigated Ang-2 expression in immortalized mouse mesangial cell lines and studied the response to hypoxia. RESULTS: Using reverse transcription-polymerase chain reaction, Ang-2 and Ang-3 mRNA were detected but Ang-1 and Tie-2 transcripts were absent. As assessed by Northern and slot blotting, 8 to 24 hours hypoxia (3% O(2)) significantly increased Ang-2 mRNA levels versus normoxic (21% O(2)) cells and the rate of Ang-2 mRNA degradation was similar in both conditions, consistent with increased transcription. Hypoxia also increased immunoreactive Ang-2 in cell lysates. Hypoxic stimulation of Ang-2 mRNA was significantly reduced by inhibitors of tyrosine kinase (genistein) and protein kinase C (GF109203X), but not by a mitogen-activated protein kinase 1 inhibitor (PD98059). Furthermore, hypoxia coincidentally up-regulated levels of vascular endothelial growth factor (VEGF) mRNA in these cells. Finally, in vivo, immunoreactive Ang-2 was observed in the cores of immature glomeruli of neonatal mice, but immunostaining in this location was absent in four-week postnatal mice. CONCLUSION: This is the first demonstration that isolated mesangial cells express Ang-2 mRNA and protein and up-regulate Ang-2 in response to hypoxia. We speculate that hypoxia-induced, mesangial-derived Ang-2 and VEGF may have synergistic paracrine roles in the growth of glomerular endothelia during normal development and diseases.

Effects of oxygen on vascular patterning in Tie1/LacZ metanephric kidneys in vitro.

Tie1 is an endothelial lineage-specific receptor. Using Tie1/LacZ mice we previously demonstrated in situ differentiation of glomerular capillaries after transplantation of renal precursors into the neonatal nephrogenic kidney cortex. We now report studies with Tie1/LacZ metanephric kidneys explanted in vitro at a stage when Tie1/LacZ-expressing cells surround nephron precursors but glomeruli are unformed. After 4 days of serum-free organ culture in 21% O2, transgene-expressing vessels regressed. In contrast, in 3% O2, transgene was expressed between epithelial tubules by cellular masses containing poorly defined lumens. The normal branching of Tie1/LacZ-expressing vessels which occurred in vivo was absent in vitro and glomeruli forming in culture lacked capillaries. Similar observations were made in wild-type metanephroi using vascular endothelial growth factor receptor 2 (Flk1) as an endothelial marker. We speculate that the metanephros is hypoxic in vivo to permit endothelial growth but other cues must be required for construction of the microcirculation since hypoxia failed to elicit normal patterning in vitro.

Expression and potential role of angiopoietins and Tie-2 in early development of the mouse metanephros.

Angiopoietins (Ang) are secreted factors which bind the Tie-2 receptor and modulate endothelial growth. This signalling system is known to be expressed in later stages of maturation of the mouse metanephros, the adult kidney precursor. In this study, by using reverse transcription polymerase chain reaction and Northern and Western blotting, we demonstrated that Ang-1, Ang-2, and Tie-2 were expressed during early metanephrogenesis when interstitial and glomerular capillaries begin to form. By using immunohistochemistry, embryonic kidney capillaries in the interstitium and glomeruli expressed Tie-2 at a later stage of differentiation compared with vascular endothelial growth factor receptor-2 and platelet-endothelial cell adhesion molecule. Addition of 200 ng/ml Ang-1 to explanted embryonic day (E) 12.5 metanephroi increased the proportion of vascular glomeruli that formed during 1 week in culture. These results are consistent with the hypotheses that Tie-2 has a role in vascular growth in the early stages of mammalian nephrogenesis and that Tie-2 activation may maintain the integrity of recently formed interstitial and glomerular vessels.

Effect of endothelin-1 on the vascular smooth muscle cell cycle.

Rabbit vascular smooth muscle cells were cultured in various concentrations of endothelin-1 (ET-1) at 37 degrees C for 24 h. The vascular smooth muscle cell cycle distribution was determined by flow cytometry according to DNA content. In the control group, the mitotically active phase (S + G2 + M phase) of vascular smooth muscle cells was 11.94%. In ET-1-cultured smooth muscle cells, the mitotically active phase was 11.69% at a concentration of 1 pM ET-1, whereas the mitotically active phases were 18.98 and 26.91% at concentrations of 0.1 and 10 nM ET-1, respectively. The findings show that ET-1 significantly increased mitotic activity of cultured vascular smooth muscle cells.

Cloning of guinea pig surfactant protein A defines a distinct cellular distribution pattern within the lung.

A full-length cDNA to guinea pig pulmonary surfactant protein (SP) A was cloned by screening a newborn guinea pig lung cDNA library with a human SP-A cDNA probe. The full-length guinea pig SP-A cDNA consists of 1,839 bp and is highly conserved at both nucleotide and amino acid sequence levels with those from other species. As expected, guinea pig SP-A mRNA is abundantly expressed in adolescent lung tissue and is undetectable in nonpulmonary tissues. In situ hybridization studies clearly show a unique cellular distribution pattern of SP-A mRNA within the guinea pig lung. SP-A mRNA expression is confined to cells of the alveolar epithelium with no expression in the bronchiolar epithelial cells, whereas SP-B mRNA is expressed in both alveolar and bronchiolar epithelial cell populations. This distinct expression pattern suggests that the guinea pig lung will be a useful model in which to study expression of transcription factors implicated in the regulation of SP genes.

Expression of angiopoietin-1, angiopoietin-2, and the Tie-2 receptor tyrosine kinase during mouse kidney maturation.

The Tie-2 receptor tyrosine kinase transduces embryonic endothelial differentiation, with Angiopoietin-1 (Ang-1) acting as a stimulatory ligand and Ang-2 postulated to be a naturally occurring inhibitor. Expression of these genes was sought during mouse kidney maturation from the onset of glomerulogenesis (embryonic day 14 [E14]) to the end of nephron formation (2 wk postnatal [P2]), and during medullary maturation into adulthood (P8). Using Northern and slot blotting of RNA extracted from whole organs, these three genes were expressed throughout the experimental period with peak levels at P2 to P3. By in situ hybridization analysis at E18, P1, and P3, Ang-1 mRNA was found to localize to condensing renal mesenchymal cells, proximal tubules, and glomeruli in addition to maturing tubules of the outer medulla. In contrast, Ang-2 transcripts were more spatially restricted, being detected only in differentiating outer medullary tubules and the vasa recta bundle area. Using in situ hybridization and immunohistochemistry, Tie-2 was detected in capillaries of the nephrogenic cortex, glomerular tufts, cortical interstitium, and medulla including vessels in the vasa recta. Using Western blotting of protein extracted from whole organs, Tie-2 protein was detected between E14 and P8 with tyrosine phosphorylated Tie-2 evident from E18. These data are consistent with the hypothesis that Tie-2 has roles in maturation of both glomeruli and vasa rectae.

Proliferation and remodeling of the peritubular microcirculation after nephron reduction: association with the progression of renal lesions.

Little is known about the serial changes that might occur in renal capillaries after reduction of renal mass. In the current study, our aim was to document potential alterations in the morphology and proliferation of the renal cortical peritubular microcirculation at specific time points (7 and 60 days) after experimental 75% surgical nephron reduction using two strains of mice that we here demonstrate react differently to the same initial insult: one strain (C57BL6xDBA2/F1 mice) undergoes compensatory growth alone, whereas the other (FVB/N mice) additionally develops severe tubulo-interstitial lesions. Our data demonstrate that significant remodeling and proliferation occur in renal cortical peritubular capillaries after experimental nephron reduction, as assessed by microangiography using infusion of fluorescein isothiocyanate-labeled dextran, expression of the endothelial markers CD34 and Tie-2, and co-expression of CD34 and proliferating cell nuclear antigen, a surrogate marker of cell proliferation. This was accompanied by an increase of renal vascular endothelial growth factor protein levels and a change in distribution of this protein within the kidney itself. Moreover, most of these responses were accentuated in FVB/N mice in the presence of progressive renal disease and positively correlated with tubular epithelial cell proliferation. Hence, we have made three significant novel observations that illuminate the complex pathophysiology of chronic kidney damage after nephron reduction: 1) cortical peritubular capillaries grow by proliferation and remodeling, 2) vascular endothelial growth factor expression is altered, and 3) the development of tubulo-interstitial disease is genetically determined.

The influence of mode of delivery, hormonal status and postnatal O2 environment on epithelial sodium channel (ENaC) expression in perinatal guinea-pig lung.

We have studied factors that potentially modulate the expression of mRNA coding for subunits of the amiloride-sensitive sodium channel, alphaENaC and betaENaC, in lungs of vaginally and Caesarean (CS)-delivered late gestation fetal guinea-pigs. Expression of alphaENaC and betaENaC mRNAs was developmentally regulated in the late gestation fetus, reaching peak levels at term (68 days post conception, PC) and postnatally, respectively. In animals delivered by CS at 65 days PC and term, alphaENaC mRNA expression was significantly increased by day 1 post partum, reaching levels greater than those normally achieved in vaginally delivered animals at term. In contrast, betaENaC mRNA levels remained significantly lower postnatally in animals delivered by CS at 65 days PC compared with those in vaginally and CS-delivered animals at term. Plasma cortisol and total triiodothyronine (T3) levels increased towards term, were higher 1 day after vaginal delivery but declined towards pre-term levels by day 3. Cortisol levels also increased rapidly in the CS-delivered animals, reaching levels similar to those in vaginally delivered animals at day 1. Plasma T3 levels at days 1 and 3 were significantly lower in animals delivered by CS at 65 days PC. The increase in alphaENaC mRNA paralleled the increase in plasma cortisol after delivery, but not T3, and inhibition of cortisol synthesis with 2-methyl-1,2-di-3-pyridyl-1-propanone (metyrapone) after CS delivery suppressed the increase in alphaENaC mRNA expression. Concomitant with the increase in alphaENaC mRNA expression after CS delivery at 65 days PC was an increase in the amiloride-blockable component of lung fluid clearance by day 3 postnatally. We conclude that in late gestation guinea-pigs delivered by CS there is a significant increase in lung alphaENaC expression postnatally, which is mediated, in part, by the postnatal rise in cortisol at delivery. This in turn leads to an increase in amiloride-sensitive lung fluid clearance, which is unrelated to labour.

Potential biological role of transforming growth factor-beta1 in human congenital kidney malformations.

Transformations between epithelial and mesenchymal cells are widespread during normal development and adult disease, and transforming growth factor-beta1 (TGF-beta1) has been implicated in some of these phenotypic switches. Dysplastic kidneys are a common cause of chronic kidney failure in young children and result from perturbed epithelial-mesenchymal interactions. In this study, we found that components of the TGF-beta1 axis were expressed in these malformations: TGF-beta1 mRNA and protein were up-regulated in dysplastic epithelia and surrounding mesenchymal cells, whereas TGF-beta receptors I and II were expressed in aberrant epithelia. We generated a dysplastic kidney epithelial-like cell line that expressed cytokeratin, ZO1, and MET, and found that exogenous TGF-beta1 inhibited proliferation and decreased expression of PAX2 and BCL2, molecules characterizing dysplastic tubules in vivo. Furthermore, addition of TGF-beta1 specifically induced morphological changes compatible with a shift to a mesenchymal phenotype, accompanied by loss of ZO1 at cell borders and up-regulation of the mesenchymal markers alpha-smooth muscle actin and fibronectin. The descriptive and functional data presented in this report potentially implicate TGF-beta1 in the pathobiology of dysplastic kidneys and our results provide preliminary evidence that an epithelial-to-mesenchymal phenotypic switch may be implicated in a clinically important developmental aberration.