The deposition was carried out using a TiC-NiCr-Eu2O3 electrode and 5140 steel substrates. Structural, elemental, and phase compositions of the deposited coatings were investigated by scanning electron microscopy, energy-dispersive spectrometry, and X-ray diffraction. The mechanical properties of the coatings were measured by nanoindentation using a 4 mN load. The tribological properties of the coatings were measured using the pin-on-disc setup in air and in distilled water at a 5 N load. The experimental data suggest that VESA coatings are characterized by surface defects, a hardness of 12.2 GPa, and a friction coefficient of 0.4. To ensure good adhesion between the VESA coating and the upper layer containing diamond-like carbon (DLC), an intermediate layer was deposited by PCAE in the Ar atmosphere. The intermediate layer had a hardness of up to 31 GPa. The upper layer of the coating ensured a low and stable friction coefficient of 0.2 and high wear resistance due to the formation of an sp2–sp3 bound carbon phase. Multilayer TiC-based coating with the upper DLC layer, in addition to high tribological properties, was characterized by the lowest corrosion current density (12 μA/cm2).A novel two-stage technology combining vacuum electro-spark alloying (VESA) and pulsed cathodic arc evaporation (PCAE) was approbated for the deposition of TiC-based coatings in inert (Ar) and reactive (C The electro-spark alloying (ESA) method is a quite widespread technology for the enhancement of wear resistance and lifespan of critical parts in the industry ,2. ESA iNevertheless, the ESA method has a number of disadvantages, such as a high content of structural defects  , high leThe ESA process can be optimized in multiple ways, including the application of protecting gas  or liquid  environments, which allow one to improve the quality of the coatings ,12,13. V2, Al2O3, NbC, WC, and other additives into TiC-Ni and TiC-NiCr electrodes allows one to increase the coating growth rate, hardness, and heat resistance as well as to decrease the coefficient of friction [2O3 into the electrode composition leads to the increase in their strength [The application of TiC-Ni electrodes is quite widespread in ESA technology ,29,30. Tstrength , coatingstrength , mechanistrength ,35. The strength . Sputterstrength .2O3 electrodes and steel 5140 as the substrate. The upper layer was formed by PCAE in the ethylene environment in order to increase the tribological performance of the whole coating due to the formation of the DLC-based layer.The current study is aimed at research on coatings deposited by a combination of VESA and PCAE technologies using TiC-NiCr-Eu2O3 electrodes of 4 × 4 × 50 and 10 × 10 × 75 mm size were manufactured for the VESA and PCAE processes using the powder metallurgy route. Mixing and mechanical activation of the powder mixture  were performed in a planetary ball mill “Aktivator-2S” with a jar radius of 0.040 m and carrier radius of 0.104 m. Milling was performed for 5 min at the velocity of 694 rotations per minute using steel milling bodies  with a weight of 0.8 g and a radius of 0.003 m in the Ar environment. As-treated powder mixtures were compacted on a hydraulic press with a pressure of 200 MPa. Sintering was performed at 1450 °C for 60 min in the Al2O3 filler in a vacuum oven VE-3-16 . Coatings were deposited on Ø30 × 5 mm polished discs of 5140 steel. Before the deposition, substrates were ultrasonicated in isopropanol for 5 min using the UZDN-2T device .TiC-NiCr-EuAutomated VESA processing and PCAE deposition were performed on a custom device based on the UVN-2M vacuum system  and two different blocks for deposition processing. Electro-spark coatings were fabricated using a three-coordinate VESA module a, designVESA processing was carried out using the following parameters: Residual pressure of 10 Pa, normal polarity (electrode–anode), voltage of 100 V, pulse duration of 20 μs, pulse frequency of 100 Hz, 20 iterations, and rotational velocity of 1000 rounds/min.2H4 (99.95%) in a chamber. The pressure of working gases was 0.4 Pa, and residual pressure was 4 × 10−3 Pa.For the PCAE process, we used a module with an originally-designed evaporator b. The ig2O3 ball with a diameter of 6 mm at room temperature in air and in liquid (distilled water) with a linear velocity of 10 cm/s and perpendicular load of 5 N. In order to examine the wear tracks and determine the surface roughness, an optical profiler “WYKO NT 110”  was used. The wear of the counter-body and of the coatings was calculated by the formula mentioned in [2SO4 solution using the Ag/AgCl reference electrode and auxiliary Pt electrode. All potentials were recounted in relation to the standard hydrogen electrode. The density of the corrosion current (icor) was calculated using the Tafel formula.The morphological, elemental, and phase compositions of coatings were investigated by scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS) using a Hitachi S-3400N microscope with a Noran 7 Thermo add-on. X-ray diffraction analysis (XRD) was performed on the AXS D8 AD-VANCE device  using Cu–Ka radiation. Raman spectroscopy was performed using the NTEGRA NT-MDT  installation equipped with a red laser (633 nm wavelength). Mechanical properties of coatings, such as hardness (H), elastic modulus (E), and elastic recovery (W), were measured on a Nano Hardness Tester  equipped with a Berkovich diamond indenter. Transversal cross-cuts of the VESA samples were tested at a 10 mN load, whereas the PCAE coatings were examined by indentation from the top with a 4 mN load. Tribological testing was performed using a pin-on-disc scheme on the Tribometer CSM Instruments device. The samples were tested in contact with an Alioned in . The eleThe elemental compositions of the substrates and of each of the layers in the multi-layered coatings are presented in A total of 21.5 wt. % of Fe  is present in the VESA coatings. The Ti to C ratio is close to the stoichiometric composition (1:1), whereas the Ni to Cr ratio is 1:2. The Eu concentration is 3.9 wt. %. The PCAE coating deposited in argon features reduced iron content (1.6 wt. %) due to the specifics of the deposition process. The contents of the main elements, Ti, C, Ni, and Cr, as compared to the VESA coating increased by up to 2.2, 1.4, 0.6, and 2.9 times, respectively.2H4, and the coating fabricated by the VESA–PCAE technique.2O3 grains, which are present both on the substrate–coating interface and in the coating’s volume.On the surface of the VESA coating, small quantities of microcracks, droplets, and condensed metallic spatter can be observed. The transverse fractures of single- and multilayered coatings  demonstr2O3 grains were in the 0.2–1.5 μm and 0.2–2.0 μm ranges, respectively. In the multilayer coatings, the sizes of the TiC grains were similar, but the Eu2O3 grains were finer (0.1–0.5 μm). The VESA–PCAE sample consisted of a lower VESA layer with a thickness of 28 μm, middle PCAE layer deposited in Ar (0.8 μm), and upper PCAE layer (deposited in C2H4) with a similar thickness of 0.8 μm.In the case of the single-layer coatings, the sizes of TiC and Eu2H4), and multilayer VESA–PCAE coatings.2H4 featured TiC peaks ) corresponding to planes (111), (200), (220), and (311). In the samples obtained by VESA and VESA–PCAE, the peaks of a solid solution of Ni and Cr in Fe (Fe (NiCr)) (ICCD 52-0513) are presented on the XRD patterns. Samples 3 and 4 demonstrate that upon the deposition, Cr3C2 (ICCD 65-0897) grains are formed, corresponding to planes (211), (301), (121), and (221). The peak at 2Θ = 32.1 is specific to Eu2O3 (ICCD 65-3182).All coatings featured peaks corresponding to (110), (200), (220), (311), (222), and (400) lines of the TiC-based face centered cubic phase. The PCAE coatings deposited in Ar and C2H4) coating, the observed TiC sizes further decrease to 4–7 nm, accompanied by the formation of Cr3C2 crystals with sizes of 25–40 nm. The peaks for VESA–PCAE contain all of the peaks observed for single-layer coatings.The crystallite size was determined using the Scherrer formula. The sizes of the TiC crystals calculated from the broadening of lines (111), (200), and (220) for the VESA sample were 22, 23, and 16 nm, respectively. For the PCAE (Ar) coating, the sizes of the crystals are lower: 9, 11, and 7 nm, respectively. For the PCAE  phase [The Raman spectra for all coatings contained peaks corresponding to TiC . The−1 [−1 ,41. The The mechanical characteristics of the coatings are shown in 2H4) lead to a decrease in H and E values by 4.5 and 4 times, respectively, along with a decrease of W to 68%.The VESA coating was characterized by a hardness of H = 12.2 GPa, Young’s modulus of E = 273 GPa, and elastic recovery of W = 37%. The VESA coatings provided a threefold increase in the hardness and elastic recovery of the substrate. The intermediate layer of PCAE (Ar) had the highest mechanical properties: H = 30.4 GPa, E = 286 GPa, and W = 79%. Reactive deposition (in Cf) for PCAE (Ar) smoothly grew in the 0–70 m range with an average f value of 0.64. After 70 m, f grew to 0.8, which is the coefficient of friction of the substrate. The PCAE (C2H4) demonstrated a stable f = 0.36 in the range of 0–46 m. Afterward, the value jumps up due to the wearing of the coating and contact between the counter-body and the substrate [According to the results of tribological testing at room temperature, a coefficient of friction  during the whole distance of 100 m. The average coefficient of friction value was 0.41. The multilayer coating had a stable coefficient of friction equal to 0.33. Interestingly, the VESA–PCAE coatings demonstrated a higher initial friction coefficient as compared to the VESA samples  can be observed in the tribocontact zone, but the coating was not fully worn out. Despite its lower coefficient of friction, the PCAE layer of a multi-layered coating was fully worn out by the end of the test. DLC-containing wear products from the upper layer were present in the tribocontact zone and decreased the coefficient of friction and counter-body wear to 3.5 × 10–5 mm3/N/m.In the case of the single-layer coatings, the wear of the AlThe tribological characteristics were also examined in distilled water .f = 0.55, which then decreased to 0.38. In the 400–1000 m distance, the friction coefficient stabilized at a value of 0.5. The VESA–PCAE coating had a low initial f = 0.18. At the distance of 200 m, the friction coefficient experienced a sharp increase, but then it decreased again and remained stable in the distance of 250–1000 m. The average f value for the VESA–PCAE coating was 0.40, which is 15% lower than the friction coefficient of the single-layer coating (0.47). The wear zones of the counter-bodies after the contact with VESA and VESA–PCAE coatings are shown in The VESA coating had a relatively high initial −6 mm3/N/m. In contact with the multilayer coating, the wear of the counter-body was much lower (0.54 × 10−6 mm3/N/m). The VESA coating experienced little to no wear, although some wear products (FexOx) were observed in the tribocontact zone. Similarly to the results of friction in the air, the upper layer of the VESA–PCAE coating was worn down, and the wear products decreased the friction coefficient. The examination of the tribocontact zone revealed the presence of carbon, Fe, and Al oxides along with small shards of VESA and PCAE coatings.In the case of the VESA sample, the specific wear of the alumina ball was 4.67 × 10The data pertaining to the electrochemical properties of single-layer and multi-layer coatings are provided in 3C2, with the subsequent isolation of the surface from the electrolyte by the as-formed oxide layers. At potentials above 1.75 V, all coatings experienced the electrical breakdown of the passivating film. The free corrosion potential φ and density of corrosion current icor are provided in All coatings were passivated in sulphuric acid. Under anode polarization in the 0.5–1.5 V diapason, all coatings demonstrated an activational current density peak, related to the oxidation of nickel, TiC, and Cr2H4, and VESA–PCAE, respectively. The density of corrosion current for the VESA coating was 3500 μA/cm2, which is close to the iron corrosion current density (4500 μA/cm2). The icor value for the PCAE (Ar) coating was 54 μA/cm2, and for the PCAE (C2H4) coating, it was 12 μA/cm2. Thus, the PCAE deposition of the upper DLC layer reduced the current density of the corrosion by 4.5 times. The multilayered VESA–PCAE coatings demonstrated the current density at the level of 150 μA/cm2, which is 23 times smaller than the mono-layer VESA samples.The stable free corrosion potentials were –170, –200, –230, and –190 mV for the coatings of VESA, PCAE deposited in Ar and C2H4 featured the highest carbon content of 87.2 wt. %, probably due to the additional influx of C atoms from reactive C2H4. The surplus of carbon atoms should lead to the formation of amorphous diamond-like carbon in addition to TiC. This coating is also characterized by the reduced concentration of metallic elements.The VESA coating contains up to 22 wt. % of Fe . The Ti to C ratio is close to the stoichiometric composition (1:1), whereas the Ni to Cr ratio is 1:2. Eu concentration is 3.9 wt. %. The PCAE coating deposited in argon features a reduced iron content (1.6 wt. %) due to the specifics of the deposition process. The contents of the main elements Ti, C, Ni, and Cr, as compared to the VESA coating, increased by up to 2.2, 1.4, 0.6, and 2.9 times, respectively. The PCAE coating produced in CThe VESA coating had a characteristic microstructure with the presence of microcracks, droplets, and condensed metallic spatter . All of 2O3 coatings is in the range of 17–21 GPa [3 bonds and the prevalence of sp2 bonds [3 bonds and the hardness of the phase. We cannot rule out the influence of the formation of new phases, such as Cr3C2 (which was confirmed by XRD).It is known that VESA coatings based on TiC-Ni have relatively low hardness in the range of 13.9–15 GPa ,42. PCAE7–21 GPa . Howeverp2 bonds . This miThe results of tribological tests suggest that the deposition of VESA–PCAE coatings results in the reduction of the friction coefficient during the wear of the base layer, both in air and in distilled water. In multilayer coatings, the upper PCAE layer provides a low friction coefficient and the lower VESA layer has a high wear resistance.2) was achieved for the coating sputtered in ethylene, which might be related to the positive influence of chromium carbide and increased carbon content [cor of the multi-layered coating decreased by at least 20 times in comparison with the basic VESA coating. This indicates an improvement in the corrosion resistance of VESA coatings due to the deposition of the upper layer with high carbon content. The decrease of the corrosion current density might also result from the healing of cracks and other defects of the spark-deposited layer, as was demonstrated in [The corrosion potentials of all single-layer coatings deposited on the steel substrates are equal to the free corrosion potential of iron (190 V). This is related to the spread of the electrolyte through the cracks and defects of the coating towards the substrate with the subsequent active dissolution of the iron. The lowest corrosion current density  in the case of VESA and VESA–PCAE deposition and of NiCr in the case of PCAE, Cr3C2 (for PCAE in C2H4 and VESA–PCAE), and Eu2O3. The VESA coating was characterized by relatively low hardness (H = 12.2 GPa); however, this property could be fortified by the deposition of an intermediate PCAE layer in Ar (H = 30 GPa). The deposition of the DLC-containing upper layer of PCAE reduced the friction coefficient by at least 50% in air and 20% in distilled water. The upper PCAE layer, deposited in C2H4, provided a lower corrosion current density in the 1 N solution of H2SO4 (150 versus 3500 μA/cm2).TiCNiCr-Eu2H4 was somewhat lower, some multilayered coatings demonstrated a lower coefficient of friction in air and distilled water due to the formation of the DLC phase in the upper layer. Due to the tight sealing of the defects in the base layer by the upper PCAE layer, the corrosion current decreases. Therefore, the VESA–PCAE technology can be applied for the increase of wear and corrosion resistance for parts which are used in the air or in corrosive environments.For the first time, the two-stage VESA–PCAE technology was applied for the deposition of multilayered coatings based on TiC with an NiCr binder. Such coatings surpass the single-layered VESA samples by a large margin when it comes to tribological characteristics and corrosion resistance. Although the strength of the upper layer deposited in C2O3-devised coatings deposited via the VESA–PCAE technique are the automotive and aerospace industries. The electrodes and VESA–PCAE technique which were approbated in this work can be applied for the increase of the performance and lifespan of parts which are usually treated by the single-stage ESA technique, such as crankshafts, camshafts, valves and pistons of internal combustion engines, edges and roots of turbine blades, parts of pumps, etc.The potential areas of application of multi-layered TiC-NiCr-Eu


Few studies examined the effect of long-acting nitrates on renal function in chronic heart failure (CHF). Thus, we aimed to investigate the effect of long-acting nitrate on the expression of adrenoceptors (AR) and angiotensin II receptor (ATR) subtypes of the renal cortex, in rats with myocardial infarction-induced CHF.1R, and AT2R were measured using reverse transcription-polymerase chain reaction and western blot analysis. Histopathology of the kidney was determined on hematoxylin and eosin-stained sections.Rats were randomly divided into the following groups: control, sham-operated, CHF, low- and high-dose nitrate, positive drug control (olmesartan), and high-dose of long-acting nitrate + olmesartan. Ultrasound echocardiography markers were compared, and the levels of AR subtypes, AT1R expression and downregulated α1A-, β1-, β2-AR, and AT2R expression compared to the sham control. High-dose nitrate or olmesartan alone, and especially in combination, decreased the levels of PRA and angiotensin II and downregulated the CHF-induced expression of AT1R, α1A-, β1-, and β2-AR, and AT2R. CHF resulted in significant impairment of the renal tissue, including inflammatory cells infiltration to the tubular interstitium and surrounding the renal glomerulus, and tubular necrosis, which was alleviated in all treatment groups to different degrees.CHF significantly increased plasma renin activity (PRA) and angiotensin II levels, upregulated ATLong-acting nitrates could reverse CHF-induced changes in AR and ATR subtypes in the kidney, and improve cardiac function to protect renal function. Compared with monotherapy, the combination of nitrates and olmesartan shows more significant benefits in regulating AR and ATR subtypes. Chronic heart failure (CHF) is a complex clinical syndrome in which the heart’s function cannot meet the demand of the body’s healthy blood circulation. In CHF, the useful circulating blood volume is reduced to decrease the rate of renal perfusion, which in turn activates the sympathetic nervous system (SNS), leading to increased blood catecholamine levels, which stimulates the juxtaglomerular apparatus cells to secrete and release more renin to ultimately activate the renin-angiotensin system (RAS) –4. Activ1-AR subtypes in the kidney tissue, within which α1A-AR and α1D-AR mainly regulate renal vasoconstriction, while α1B-AR mediates the proliferation and hypertrophy of vascular smooth muscle cells, and together with α1A-AR promotes sodium and water reabsorption in the proximal tubules [Moreover, SNS activation in CHF will have direct effects on kidney function. Elevated catecholamine acts on the adrenergic receptor (AR) of the kidney tissue, contributing to a series of conditions, including renal interstitial inflammatory cell infiltration, renal fibrosis, and renal tubular necrosis, ultimately resulting in altered renal hemodynamics. When the RAS is activated after heart failure develops, the plasma level of angiotensin II (Ang II) also increases by mediating renal vasomotor and sodium retention, as well as the proliferation, hypertrophy, and profibroblast effect of kidney mesangial cells and the extracellular matrix. There are three kinds of α tubules –7.Nitrates have beneficial effects on CHF by expanding the vein, coronary artery, and small peripheral arteries to reduce blood reflow and thus the cardiac preload, thereby improving myocardial blood supply and the cardiac afterload, respectively. However, few studies have examined whether long-acting nitrates can inhibit excessive activation of the SNS and RAS by regulating the expression of AR and angiotensin receptor (ATR) in the kidneys under a condition of CHF. Therefore, in this study, we examined the influence of the administration of long-acting nitrates to a rat model of heart failure induced by myocardial infarction on the expression of AR and ATR in the kidney. These results can lay the foundation for a renal protective effect of long-acting nitrate as a treatment or preventive strategy in patients with CHF.Clean inbred male Wistar rats  were obtained from Beijing Vital River Laboratory Animal Technology Co. Ltd. . All rats were barrier-housed in the clean animal room with the temperature maintained at 22 ± 3 °C and relative humidity of 50 ± 20% at Capital Medical University affiliated Beijing Anzhen Hospital. The experimental protocol was approved by the institutional ethics committee (permission license: SCXK-2012-0001), and the rats were fed with standardized rat chow and water throughout the experiment.n = 9), sham-operated group , and CHF model rats (n = 73). The CHF model was induced by ligation of the left anterior descending (LAD) artery. In brief, the rats were anesthetized by intra-abdominal injection of 1% pentobarbital sodium and set on a ventilator for small animals . Anterior myocardial infarction was created by ligation of the LAD artery near the main pulmonary artery. Four weeks later, echocardiography was performed to evaluate left ventricular (LV) function based on the left ventricular ejection fraction (LVEF); rats with an LVEF ≤45% were considered to have CHF and were included in subsequent experiments [A total of 90 Wistar rats were randomly divided into three main groups: healthy control group , in which the rats received intragastric administration of saline once a day; low-dose nitrate group (n = 9), receiving intragastric administration of 3.6 mg/kg isosorbide-5-mononitrate  in 2 ml saline once a day; high-dose nitrate group (n = 9), receiving intragastric administration of 7.2 mg/kg IS-5-MN in 2 ml saline once a day; positive drug control group , receiving intragastric administration of 3 mg/kg olmesartan  in 2 ml saline once a day; and high-dose nitrate combined with olmesartan group , receiving intragastric administration with 7.2 mg/kg IS-5-MN and 3 mg/kg olmesartan in 2 ml saline once a day. Sham-operated rats were subject to the same procedure as those in the CHF-induced model but without ligation of the LAD artery. Both the CTL and sham groups received intragastric administration with saline once a day. The treatment lasted for 6 weeks. Rats were anesthetized by intraperitoneal (i.p) injection with sodium pentobarbital (40 mg/kg). And then all blood samples were taken from the abdominal aorta of rats. After blood sampling procedures the animals were euthanized by an injection of sodium pentobarbital (150 mg/kg) administered through the abdominal aorta.These CHF rats were then randomly divided into the following five treatment groups: CHF model group . The rats were anesthetized, and B-mode measurement in the LV short-axis view (papillary muscle level) was performed with a 12-MHz phased array transducer . LVEF was measured and averaged for three consecutive cardiac cycles., expressed as nanograms per milliliter per hour.At the end of the treatment period, blood samples were collected from the abdominal aorta, and plasma was separated immediately and stored at − 80°C until analysis. For renin activity determination, the plasma was incubated with rabbit angiotensinogen at 37 °C for 60 min and the renin concentration was measured according to the standard protocol of the radioimmunoassay (RIA) kit 125I-labeled Ang II for 18 h at 4 °C. Antibody-bound Ang II was separated from the free Ang II using donkey anti-rabbit-coated cellulose. After incubation for 30 min at room temperature and centrifugation at 5000 rpm for 15 min at 4 °C, the concentration of Ang II in each sample was read with the RIA kit according to a prepared standard curve.For Ang II determination, the plasma was incubated with antiserum (anti-rabbit) for 6 h, and then with The kidneys were removed after blood collection, and specimens of the renal cortex were obtained and frozen in liquid nitrogen at − 80°C until analysis.Gapdh was used as an internal standard, and the target mRNA  levels were normalized to that of Gapdh. The target genes and Gapdh were amplified according to the parameters shown in Table Total RNA was extracted from the rat renal cortex by Trizol reagent, and an ultraviolet spectrophotometer was used to detect the concentration of total RNA for each sample. cDNA was synthesized and amplified with a Promega RT-PCR Kit  according to the manufacturer instructions using sense and antisense oligonucleotide primers synthesized by Bejing SBS Genetech . To quantify the transcripts obtained by RT-PCR amplification, RT-PCR products were resolved by electrophoresis on a 1.5% agarose gel , and stained with ethidium bromide for visualization on a Bio-rad scanner. Densitometry was used for relative semi-quantitative assessment of expression levels.1-AR (1:200), anti-β2-AR (1:200), anti-β3-AR (1:200), anti-α1A-AR (1:200), anti-α1B-AR (1:200), anti-α1D-AR (1:200), anti-AT1R (1:200), anti-AT2R (1:200), and anti-GAPDH (1:200)  at 4 °C overnight, followed by incubation with goat anti-rabbit fluorescent (IRDye-conjugated) secondary antibodies  for 2 h at room temperature. The images were quantified by the Odyssey infrared imaging system . Levels of proteins were normalized to that of GAPDH.Total protein was extracted after homogenizing the rat renal cortex, and the concentration was determined with a bicinchoninic acid protein assay kit. The proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes, which were incubated with the primary antibodies rabbit anti-βThe kidneys were perfused with formalin, collected, and fixed in paraffin. Fixed specimens were then cut into 4-μm sections and stained with hematoxylin and eosin following standard procedures. Images were captured with a Nikon Labophot 2 microscope equipped with a Sony CCD-Iris/RGB color video camera attached to a computerized imaging system .t-test or one-way analysis of variance, followed by the Newman-Keuls test in GraphPad Prism 5.0 . P < 0.05 was considered statistically significant.Data are expressed as mean ± standard error of the mean. Differences between groups were analyzed by Student’s P < 0.01), with no significant differences among the different CHF model groups.The echocardiography data are summarized in Table After treatment, the LVEF of the high-dose nitrate, olmesartan, and nitrate + olmesartan groups significantly increased compared to that of the CHF group, whereas there was no significant effect of treatment with low-dose nitrate. Although the LVEF of the olmesartan group was slightly increased compared to that of the high-dose nitrate group, the difference was not statistically significant. However, the combination of high-dose nitrate and olmesartan had a significant effect on improving the LVEF compared to either treatment alone.P < 0.01). However, this increase was significantly alleviated by treatment with high-dose nitrate or olmesartan (P < 0.05) and was more strongly improved with their combination (P < 0.01); however, low-dose nitrate did not effectively increase the reduction in PRA or Ang II plasma levels. Although olmesartan had a slightly stronger effect on improving PRA and Ang II plasma levels than high-dose nitrate, the difference was not statistically significant. However, their combination had a significant effect on reducing these levels compared to either treatment alone.As shown in Table The CTL and sham groups both showed a clear structure of the renal glomerulus and tubules with organized glomerular epithelial cells, an intact basement membrane, and no interstitial inflammatory cell infiltration. By contrast, the renal tissue showed significant impairment in the CHF group, including glomerular mesangial area expansion, interstitial inflammatory cell infiltration, partial glomerulus necrosis, and corresponding renal tubular atrophy. However, this inflammatory cell infiltration and tubular necrosis were reduced in all treatment groups to different extents, demonstrating a renal protective effect Fig. .Fig. 1a1, β-AR, and ATR subtypes between the sham and CTL groups. Compared with the sham group, the expression of AT1R was significantly up-regulated, whereas the expression of α1A-AR, β1-AR, β2-AR, and AT2R was down-regulated in the CHF model. Low-dose nitrate treatment did not affect the CHF-induced changes to the levels of α1, β-AR, and ATR subtypes, whereas the other treatments significantly reduced the level of AT1R and increased the levels of α1A-AR, β1-AR, β2-AR, and AT2R. Consistent with the other findings, olmesartan had a more significant effect than high-dose nitrate treatment, but the difference was not statistically significant. Furthermore, the combination treatment had a much greater effect than either treatment alone. There was no significant difference in the expression levels of α1B-AR, α1D-AR, and β3-AR among groups.As shown in Figs. Kidneys are vital organs to maintain the body’s blood pressure, as well as the water and electrolyte balance, and the SNS and RAS regulate their physiological functions. After heart failure develops, SNS is activated and the release of catecholamine increases.In this experiment, the renal tissue showed significant impairment in the CHF group. Inflammatory cell infiltration and tubular necrosis were reduced in all treatment groups to different extents, demonstrating a renal protective effect. However, due to the original design of the study, no quantitative analysis of the histology was performed. We only described the pathological changes under the optical microscope. As the study was completed some time ago and some pathological specimen had been damaged, quantitative analysis of histology could not be performed retrospectively.In our study, a CHF model was established by ligation of the LV, resulting in a decrease of the LVEF, and an increase of plasma PRA and Ang II levels, indicating excessive activation of the RAS and confirming the successful establishment of the model . However1A-AR, but did not affect the expression of the other subtypes α1B-AR and α1D-AR. The result is consistent with previous findings [1A-AR internalization resulting from persistent activation of the SNS in a condition of heart failure, and further weakens its regulatory effect on renal hemodynamics, while RAS activation inhibits the down-regulation of α1D-AR and maintains α1B-AR at a relatively low baseline expression level. Such receptor-mediated regulation has been shown to play an essential role in maintaining renal perfusion and normal renal function [1A-AR expression induced by long-acting nitrate treatment may indicate that nitrate improved cardiac function and inhibited excessive activation of the local renal SNS, which then normalized AR expression under the pathological condition to restore its renal protective effect.Moreover, we found that CHF significantly downregulated αfindings , 6, suggfunction , 11. The1-, β2-, and β3-AR. Stimulating β1-AR and β2-AR could mediate dilation of the renal arteries and glomerular mesangial cells to regulate blood flow and the glomerular filtration rate (GFR), increase sodium and chloride reabsorption, and increase the secretion of renin and erythropoietin. Besides, β1-AR plays a dominant role in the regulation of renal function [3-AR can activate nitric oxide synthase (NOS) and promote NO release, and thus cause dilation of the glomerular afferent arteries to adjust the renal perfusion rate [There are three β-AR subtypes in the kidney tissue: βfunction –14. β3-Aion rate .1- and β2-AR expression in the heart failure model group was significantly down-regulated, which was consistent with the findings of Fung et al. [1- and β2-AR expression in the kidney was reversed after nitrate therapy, suggesting that local SNS activity decreases in the kidney after heart function improves so that the receptor expression reverts to the average level under the pathological condition [3-AR in the kidney tissue showed a relatively low baseline expression level, and no significant changes were found among the groups.Our results showed that βg et al. . One reaondition . By cont1R and AT2R are the two ATR subtypes expressed in the renal vasculature and renal tubules [1R causes renal vasoconstriction, promotes renal sodium and water reabsorption, and enhances cell proliferation, whereas binding of Ang II to AT2R can increase NO release, which further contributes to renal vessel dilation, increasing sodium and water excretion, and inhibiting cell proliferation and hypertrophy [AT tubules . Bindingertrophy , 17.1R expression was markedly up-regulated in the heart failure group, while AT2R expression was significantly down-regulated, suggesting changes of ATR expression levels in the kidney during CHF, which could contribute to renal interstitial edema, inflammatory cell infiltration, glomerular fibrosis, and tubular necrosis, ultimately affecting renal perfusion and reserve function [1R expression was down-regulated while AT2R was up-regulated after nitrate therapy, which may be related to the fact that nitrates can cause renal vascular relaxation, increase renal perfusion, and inhibit RAS activation. Up-regulation of AT2R can suppress the expression of AT1R, thereby the receptor adjustment protected against impaired renal function during CHF [In this study, ATfunction , 19. AT1ring CHF , 21.1R antagonist, olmesartan can inhibit RAS activation, and reduce plasma PRA and Ang II levels as well as the renal vascular sensitivity to catecholamines [1R antagonists, olmesartan has an inverse activating effect when binding to AT1R, and thus inactivate AT1R and alleviate damage to the kidney induced by Ang II [2R plays a role in physiological renal protection [As a selective ATolamines . In conty Ang II , 23. Morotection .1R and increasing the expression levels of α1A-, β1-, β2-AR, and AT2R. These results may relate to the fact that olmesartan blocks AT1R and promotes the rise of the tissue endothelial NOS/inducible NOS level and NO synthesis, and by inducing the up-regulation of AT2R expression. Thus, olmesartan elevates endothelial NOS levels and increases the bioavailability of NO, which further enhances the vasodilating effect of nitrates [Compared with monotherapy, high doses of nitrates combined with olmesartan had a more significant effect on improving cardiac function, reducing PRA and Ang II levels, and normalized the receptor expression level, namely by lowering the expression level of ATnitrates , 26. In 1R to down-regulate the expression of α1-AR, and also phosphorylates and desensitizes β1-AR through the PLC/PKC/c-src/PI3K pathway, eventually deteriorating renal function in heart failure [3-AR promotes the synthesis of NO by activating NOS, thus mediating renal vasodilation, resulting in reduced AT1R expression [During CHF, the plasma Ang II level and catecholamine secretion rise, and the sympathetic nerve is activated so that the RAS and SNS interaction increases, resulting in changes of AR and ATR expression in the kidney. Previous studies have shown that Ang II acts on AT failure , 14. Aftpression , 28. ThiOverall, our results demonstrate that application of long-acting nitrates can, through the interaction between SNS and RAS, inversely regulate the expression of AR and ATR subtypes in the CHF kidney to normal levels, thus playing a beneficial role in protecting renal function. Compared with monotherapy, the combination of nitrates and olmesartan has a more significant effect in regulating the expression of AR and ATR subtypes, showing that long-term nitrates and olmesartan have a synergistic effect on protecting renal function.


The use of religious bells as symbolism and ritual is prevalent in many faiths worldwide. However, the sound of bells emanating from churches is by nature not exclusive to the church, as these sounds can effectively become part of the “public domain.” The value of church bell ringing can therefore be attributed to the church community and clergy as well as the wider community. Cessation of these sounds affects not only the soundscape of the area, but the people who place value on these sounds or soundscapes. Data are presented from a previous survey from 2018 investigating church bell practices in New South Wales  and compared to the current practice of bell ringing, which has been heavily influenced by regulations introduced due to the COVID-19 pandemic. I.The emergence of COVID-19, the coronavirus disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) , in JanuII.We undertook a survey in 2018 to investigate past and current trends of church bell ringing practices across NSW, looking at the six most populous denominations of the Christian faith in 2016 : Roman CA large proportion of Orthodox (91%), Catholic (78%), and Anglican churches (76%) reported bells on church premises,III.Following the first positive case of COVID-19 in NSW on 25 January 2020 and the first case of community transmission on 2 March 2020, the NSW State Government began to implement staged, community-wide public health control plans, placing restrictions on gathering and movement. On 16 March the NSW Minister for Health and Medical Research introduced the first restrictions on gatherings of people to 500 under the Public Health Act. Two days later, the order was amended to restrict public gatherings, including church services, to 500 people outdoors and 100 people indoors. By 23 March, all churches were closed.IV.The soundscape generated by church bells depends on the nature of ringing, whether bells are rung as part of liturgical practice, such as calls to prayer or mass, or whether bells are rung as peals, a form of bell music generated by change ringing. While the former requires a single individual to ring the tenor bell, the latter requires a group of bellringers acting in unison, which has obvious implications in times of social distancing rules during a pandemic.In order to ascertain the impact of the COVID-19 pandemic on church bell ringing, we first sourced denomination-specific directives for NSW churches regarding bell ringing. We then sent a brief targeted questionnaire to church individuals who expressed particular interest from our original 2018 survey, soliciting information pertaining to bell ringing practices during the COVID-19 pandemic, and how ringing patterns have deviated from the norm. We combined these data with information from publicly available sources  to present a narrative on the effects (to date) of the pandemic on church bell ringing in NSW.A.In anticipation of the Stage 3 lockdown, the Catholic Archdiocese of Sydney activated its own response plan aimed at ensuring that liturgical services could be provided during lockdown . The dioIn the early stages of incremental restrictions, the Anglican Cathedrals of Parramatta (population 257 197), Sydney [population 2 085 000 (area of diocese only)] and Wollongong (population 295 669) rang their bells not only on Sunday, but also on Mondays and Thursdays to call for public services and prayer , with thElsewhere too, bells were used to reaffirm the presence of the church in the community and to provide audible and public emotional support in smaller outer regional communities such as Mudgee (population 10 923 in 2016) , Glen InIn some instances, this occurred daily, as in the case of the Anglican Churches in Moree  and GlenOther congregations, however, fell silent during lockdown. Some Catholic churches, such as Albury  and LeetOnce the church services gradually resumed, bell ringing was no longer mentioned in the pastoral letters ,d,b, howB.Imposed regulations also affected the convention of change ringing in many churches. Rapid change of events is clearly evident in St Andrew's Cathedral, Sydney. With cathedral services suspended on 20 March 2020, the usual bell ringing associated with the Sunday services was no longer required, and a 5 min ringing was undertaken from midday for bi-weekly outdoor prayer services instead. Regular weekly evening bell practices were suspended two days later, with training limited to a maximum number of six ringers in the tower, until the cessation of all bell ringing activities on 30 March 2020 . Other c2 per person. In essence, as most ringing rooms are too small and the ropes are usually less than 1.5 m apart, ringing is either completely suspended  maintain a 1.5 m separation between individuals and (b) that any room needs to provide space at a ratio of 4 muspended  or limituspended . At the uspended .V.The changes in church environment soundscapes as a result of the COVID-19 pandemic are not homogenous across regions or denominations. Some churches increased their frequency of bell ringing in communal support for the medical staff and humanity in general, almost in defiance against the difficulties at hand. In cases where churches increased bell ringing, soundscapes here would be richer in this sense, especially in comparison with the expected reduced occurrence of other urban environmental sounds as a result of movement restrictions. Such an example would be the soundscape of Glen Innes, with a seven-fold increase in bell ringing at the Anglican church, coinciding with regular ringing of the Catholic church (twice daily) and the Uniting church (weekly)  Fig. 2)2). On thThere was a distinct lack of bells rung for weddings during the COVID-19 pandemic, and this correlates with the lack of church-based weddings over this time. Whilst this is expected to partially correlate with imposed restrictions on the numbers permitted at weddings in NSW, many churches reported that the period of June-August (winter) was also generally poorly represented in 2019. It would be interesting to see how bells are represented for this activity and the resultant soundscape in NSW as Australia approaches spring and into summer.VI.The COVID-19 pandemic has demonstrated how a stochastic disruptive event can dramatically alter community soundscapes. With the reduction in ambient transportation noises due to the reduction in human activity during the lockdown period, the ringing of church bells created a stronger audible presence than otherwise. Likewise, the absence of church bells sounds made the COVID-19 silence even more pronounced.Unlike soundscapes that are a by-product of human activity and thus sensitive to changes in that activity , church bell ringing creates a soundscape that is directly influenced by purposive human action. We have shown elsewhere the myriad of values imparted by society on church bell ringing in NSW, being both religious and secular in origin .The COVID-19 induced changes to urban soundscapes have brought to the fore the role and power of the individual. Some clergy acquiesced to the situation and embraced the physical shutting down of services as new reality, pausing all church bell sounds in the process. For others, however, COVID-19 enkindled a sense of defiance, whereby the ringing of the bells was construed as a public signaling system to alert the community of the continued presence of the church.It remains to be seen to what extent the pandemic engenders a “new normal,” with durably altered soundscapes, or whether old patterns reemerge once the community has put the pandemic behind it.


Gastric cancer (GC) remains one of the most common digestive malignancies worldwide and ranked third causes of cancer-related death. Mounting evidence has revealed that miRNAs exert critical regulatory roles in GC development.vivo. Luciferase reporter assay, western blot and qRT-qPCR were employed to identify the miRNAs which directly targeted NRP1. Furthermore, Bioinformatics analysis and experimental verification were used to explore the potential molecular mechanism and signalling pathway.Immunohistochemistry (IHC) and western blot assay were performed to determine the protein expression levels of neuropilin-1 (NRP1) and mRNA levels were confirmed by quantitative RT-PCR (qRT-PCR) in GC tissues. Kaplan–Meier analysis was performed to evaluate the prognostic value of NRP1 in GC. Knockdown of NRP1 was conducted to analyse its function in vitro and In the current study, we revealed that NRP1 was highly expressed in GC tumor tissues and was associated with poor prognosis in GC patients. NRP1 knockdown inhibited GC cell growth, migration and invasion in vitro, while suppressed GC xenograft tumor development in vivo. Bioinformatics analysis predicted that miR-19b-3p down-regulated NRP1 expression by targeting its 3′-UTR. Functional assay demonstrated that miR-19b-3p inhibited GC cell growth, migration and invasion via negatively regulating NRP1. Overexpression NRP1 partially reversed the regulatory effect of miR-19b-3p. Moreover, we showed that miR-19b-3p/NRP1 axis regulated the epithelial-to-mesenchymal transition and focal adhesion in GC, which might contribute the GC development and progression.Taken together, our findings suggest a regulatory network of miR-19b-3p/NRP1 in GC development. The miR-19b-3p/NRP1 axis might be further explored as a potential diagnostic and therapeutic target in GC. Gastric cancer (GC) remains one of the most common digestive malignancies worldwide and ranked third causes of cancer-related death . TechnicNeuropilin-1 (NRP1) is a trans-membrane glycoprotein that function as a co-receptor for multiple extracellular ligands such as semaphorins, hepatic growth factor, FGF and TGF-β , 6. MounMicroRNAs (miRNAs) are small non-coding RNAs participating in various biological processes including tumorigenesis –16. MiRNIn the current study, we found that NRP1 expression was upregulated in GC patients and was associated with poor prognosis. Knockdown of NRP1 inhibited GC cell growth, migration and invasion in vivo, and suppressed xenograft tumor development in vivo. Bioinformatics analysis demonstrated that miR-19b-3p specifically dampened NRP1 expression by binding to its 3′-UTR. MiR-19b-3p exerted its tumor suppressor function via negatively regulating NRP1 and EMT/focal adhesion process. Taken together, our findings reveal a novel regulatory network of miR-19b-3p/NRP1 in GC, which might provide novel diagnostic and therapeutic targets in GC.102 paired GC tissues and adjacent nontumorous tissues were collected from patients undergoing surgery at Ganzhou People’s Hospital and the First Affiliated Hospital of Zhengzhou University (GZPH GC cohort) between year 2010 and 2018. Tissue microarray (TMA) were constructed with GC tissue specimen. All patients signed the written informed consent. The Ethics Review Committee of Ganzhou People’s Hospital approved the study.GC cell lines  and control gastric epithelial cell line GES-1 were purchased from the Cell Bank, Chinese Academy of Science . HEK293 cells were from ATCC and maintained in the lab. Cells were cultured in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal bovine serum , 100 U/ml Penicillin, and 100 μg/ml Streptomycin at 37 °C under 5% CO2 in a humidified incubator.SGC-7901 or MGC-823 cells were transiently transfected with si-NRP1 or negative control (NC), miR-19b-3p mimics or NC, miR-19b-3p inhibitor or NC, or pcDNA3.1-NRP1 using lipofectamine 3000 . SGC-7901 cells were transduced with sh-NRP1 or NC and stably NRP1 knockdown cells were selected using neomycin before cell implantation.Complementary DNA was prepared from total RNA using First strand cDNA synthesis Kit . NRP1 and miR-19b-3p expression were analyzed by quantitative PCR using SYBR Green master mix . GAPDH and U6 were used as control. The primers used in the study were listed as following: NRP1, 5′-ACCCAAGTGAAAAATGCGAATG -3′ and 5′-CCTCCAAATCGAAGTGAGGGTT-3′; miR-19b-3p, 5′-AACAGAAGTTTTGCAGGTTTGCATC-3′ and 5′-CAGTGCAGGGTCCGAGGT-3′; U6, 5′-CCAGUUUACCUAACGCAAUTT-3′ and 5′-TTCACGAATTTGCGTGTCAT-3′; GAPDH, 5′-CTGGGCTACACTGAGCACC-3′ and 5′-AAGTGGTCGTTGAGGGCAATG-3′.Western blot was performed as previously described . The priCell growth was analyzed by CCK-8 assay, EdU staining assay and Colony formation assay as previously described .Cell invasion was evaluated with transwell chamber . Briefly, 1 × 10  transfecSGC-7901 or MGC-823 cells were cultured to form monolayer in 6-well plate. An artificial wound was created using a sterile 200 μL pipette tip. Floating cells were washed awat with PBS and rest cells were cultured in serum-free medium for 48 h. The wound was recorded at 0 h and 48 h to calculate the migration distance.Luciferase vector containing wild type or mutated 3′-UTR sequence of NRP1 was cloned using pGL3-luc vector . HEK393 cells were seeded into 24-well plates and transfected with luciferase vector, together with miR-19b-3p mimics or negative control. Relative luciferase activity was analyzed using the Dual-Glo luciferase reporter assay kit  48 h later.Briefly, 5 μm paraffin tumor section slides were de-paraffined and blocked with normal rat serum, and then incubated with primary antibodies overnight at 4 °C. Then, slides were incubated with HRP conjugated secondary antibodies, and the specific protein expression was detected using a DAB kit . The primary antibodies used in the study were listed as following: Ki-67 , NRP1 , E-cadherin , vimentin , N-cadherin , BMP4 , ICAM1 , VCAM1 .6 stable transfected SGC-7901 cells with luciferase reporter were subcutaneously inoculated into the nude mice. Tumor growth was monitored every week and tumor bioluminescence was photographed using an IVIS Lumina II system  following the manual. Tumor volume was calculated (length × width2/2). Tumor were extracted and weighed at 5 weeks. The animal experiment was approved by the Institutional Animal Care and Use Committee of Ganzhou People’s Hospital.BALB/C nude mice  were obtained from Vital River Laboratory . 3 × 10t test and one-way ANOVA was conducted to calculate the difference between two or more groups. A *p < 0.05 is considered to be statistically significant.Results were displayed as mean ± SD and analyzed with GraphPad Prism V6 . Student To explore the expression of NRP1, we examined the NRP1 expression in 30-paired GC and adjacent nontumorous tissues. The mRNA levels of NRP1 were significantly upregulated in GC tissues Fig. a–d. ThesTo illustrate the function of NRP1 in GC, RNA silencing was performed to knockdown the expression of NRP1 in GC cells by using siRNA Fig. a. FunctiWe further investigated the role of NRP1 in xenograft GC tumor model. SGC-7901 cells stably knockdown NRP1 or control cells were subcutaneously inoculated into nude mice to establish the xenograft tumor. As shown in Fig. To further understand how NRP1 is regulated in GC development, bioinformatics analysis was performed using online database miRBase to predict the potential miRNAs interacting with NRP1. MiR-19b-3p had the putative binding sequences targeting 3′-URT of WT NRP1 Fig. a. In addRescue experiment was conducted to further verify the regulation of NRP1 by miR-19b-3p. Overexpression of miR-19b-3p notably suppressed NRP1 expression while the protein expression of NRP1 was partially restored by NRP1 overexpression in SGC-7901 or MGC-823 cells Fig. a. FunctiGene set variation analysis (GSVA) using TCGA GC datasets was performed to further understand the functional mechanism of NPR1 in GC development. As shown in Fig. Accumulating studies suggest that miRNA/mRNA loop exerts critical function in various human diseases including cancers . In thisGC can be classified into different subtypes according to the different anatomic locations, development stages, or major morphologic components . Based oStudies have shown that NRP1 was regulated by miRNAs in different tumors , 32. MiRMiR-19b-3p has been investigated in various cancers including breast cancer, pancreatic cancer, colon cancer and so on –37. MicrIn summary, our findings verified miR-19b-3p/NRP1 axis plays a critical role in the development of GC. MiR-19b-3p inhibited GC cell growth, migration and invasion via negatively regulating NRP1 and EMT/cell adhesion process. Thus, the results suggest a novel potential diagnostic and therapeutic value of miR-19b-3p/NRP1 in GC.


On rhinoscopy, vigorous PA was significantly associated with watery rhinorrhea  but not pale mucosa. Total immunoglobulin E (IgE) and three allergen-specific IgE were not significantly elevated in participants who performed PA. Therefore, regular vigorous PA is associated with subjective and objective aggravation of AR symptoms, which may not necessarily manifest as increased serum IgE levels.Evidence regarding the association between allergic rhinitis (AR) and physical activity (PA) is conflicting. Previous studies have mostly relied only on self-reported symptoms to define AR, did not classify AR by severity or persistence, and included only children or athletes. The present cross-sectional study evaluated the association between PA and objectively-defined AR and its subtypes in the general adult population using data for 1932 eligible participants aged 19 years or older in the 2010 Korea National Health and Nutrition Examination Survey. Multivariable logistic regression analyses were performed to evaluate the relationship between three types of PA and overall AR, AR subtypes, and rhinoscopy findings showed that moderate-severe AR was positively associated with vigorous (odds ratio [OR] = 3.392,  Allergic rhinitis (AR) is one of the most common health conditions worldwide and is estimated to affect more than 500 million people; while the prevalence varies based on age and region, the prevalence is rising in most countries . The symPA is associated with a wide range of health benefits and is strongly recommended to improve health; however, in some instances, it can elicit dysregulated inflammatory responses and exacerbate pre-existing illnesses such as asthma, cystic fibrosis, and systemic lupus erythematosus . RegardiAlmost all previous studies on the association between AR and PA have used self-reported symptoms of rhinitis to define AR. However, this definition may include non-allergic rhinitis (NAR) or occupational rhinitis. Thus, allergen-specific immunoglobulin E (IgE) reactivity must be present to confirm a diagnosis of AR and clinical examination may improve the accuracy of the diagnosis . These sThe KNHANES is a nationwide, population-based, cross-sectional health examination and survey conducted to understand the health and nutrition status of the Korean population since 1998. The KNHANES is conducted annually by randomly sampling 23 households in each of the 192 regions and includes 10,000 household members over one year of age. Foreigners, people enlisted in the army, people in jail, and people in nursing homes are excluded from the survey. The survey consists of a health interview , nutritional survey , and clinical examinations .The present study used data collected by the KNHANES V-1 conducted in 2010, which was the only year in which data for serum IgE levels were collected. In particular, an otorhinolaryngology survey and endoscopic examinations were completed by 150 residents from the otorhinolaryngology departments of 47 institutes. Before participating in the survey, the residents were educated on the following: the overview of the survey; purpose of, and caution related to, the investigation of otolaryngology diseases; detailed guidance on the diagnosis and severity classification of diseases; and simulation training with the actual examination equipment. Among the 8313 subjects who participated in the otorhinolaryngology survey, 1932 were 19 years of age or older and underwent IgE testing, and thus, were included in this study . All parDermatophagoides farinae, dog, cockroach) were measured using ImmunCAP 100  and were logarithmically transformed (base 10) for statistical analyses [AR was defined as having replied “yes” to the question “In the past year, have you experienced rhinitis symptoms such as sneezing, rhinorrhea, nasal obstruction, and nasal itching that were unrelated to the common cold ?” as well as positivity for at least one allergen-specific IgE (>0.35 kU/L). NAR was defined as having replied “yes” to the above question but being negative for any of the specific IgE. AR was further classified according to the Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines based on severity (mild/moderate-severe) or persistence (intermittent/persistent) . Rhinoscanalyses .In the KNHANES V, the participants are asked three questions regarding PA: (1) “In the past week, on how many days have you performed more than 10 min of vigorous physical activity that made you extremely tired or more out of breath than usual , hiking, cycling at high speeds, fast swimming, soccer, basketball, jump rope, squash, singles tennis, lifting heavy weights, etc.)?”; (2) “In the past week, on how many days have you performed more than 10 min of moderate physical activity that made you extremely tired or more out of breath than usual ?”; and (3) “In the past week, on how many days have you walked more than 10 min?” If subjects performed any of the three PAs on even a single day, they were also asked for how many minutes they performed the activity per day, on average. Then, we determined whether the participants had satisfied the recommendations of the American College of Sports Medicine for each PA: minimum 20 min of vigorous PA a day, at least three times a week; minimum 30 min of moderate PA a day, at least five times a week; or minimum 30 min of walking a day, at least five times a week .2 were categorized as normal and obese, respectively. In response to the statement “Usual perception of stress”, participants who replied “extremely stressed” or “quite stressed” were categorized as high-stress, while those who replied “slightly stressed” or “rarely stressed” were categorized as low-stress. Sleep hours were divided into ≥8 and <8 h. Participants who replied “yes” to the statement “I have experienced asthma” were categorized as having asthma and those who replied “yes” to the statement “I have experienced atopic dermatitis” were categorized as having atopic dermatitis.The health interview collected information on educational level, marital status, household number, household income, occupation type, residential location, smoking habits, alcohol consumption, body mass index (BMI), sleep hours, stress level, asthma, and atopic dermatitis. Participants who had graduated high school and above were categorized as having a high educational level. Households with income in the upper two quartiles were categorized as having a high household income. Current smokers were defined as those who had smoked at least five packs of cigarettes in a lifetime and who reported smoking “every day” or “frequently.” Alcohol consumption was determined by asking the participants whether they consumed alcohol at least once per month in the past year. Participants with a BMI <25 and ≥25 kg/mt-test for weighted means, while categorical variables were analyzed using Rao–Scott chi-square tests. Logistic regression analysis was performed to calculate the odds ratios (ORs) between PA and overall AR in the total study population and the associations between PA and AR subtypes or rhinoscopy findings in the AR population. Multiple logistic regression model was used to adjust for confounding factors including age, sex, and other variables with p-values less than 0.2 in p-values less than 0.05 were considered statistically significant. All statistical analyses were performed using IBM SPSS Statistics for Windows, version 24.0 , SAS software, version 9.4 , or R software, version 3.6.0 Complex sample analysis was performed using the stratification, cluster, and sample weight variables provided in the KNHANES dataset. Weighted numbers and percentages were calculated by multiplying the sample weight variable by unweighted sample values. For the differences between the baseline characteristics of AR and control groups, continuous variables were analyzed using Student’s p < 0.001). Compared to the control group, the participants in the overall AR group were more likely to be unmarried, have graduated high school, and have a higher level of stress. However, they did not differ in sex, location of residence, household income, household number, and occupation. Additionally, the overall AR group reported a higher prevalence of asthma and atopic dermatitis but not alcohol use, smoking, obesity, and sleep.Among the 1932 participants, 276 (15.1%) belonged to the AR group . The AR p = 0.696), moderate PA , and walking   ).p = 0.002) and moderate  PA, as did persistent AR with vigorous  and moderate  PA. In comparison, participants who had NAR  did not show positive correlations between the severity or persistence of rhinitis symptoms and PA performance  reported having moderate-severe AR and 78 (26.8%) reported having persistent AR . Univariformance .p = 0.048). While pale mucosa was more commonly found in participants performing vigorous or moderate PA, these associations were not significant in univariate or multivariate logistic regression. Total IgE and the three allergen-specific IgE levels were not significantly associated with any of the three types of PA. Differences in allergen-specific IgE levels (divided into six grades) between participants who performed PA and those who did not are shown in These significant associations between PA and self-reported symptoms of AR were further explored by analyzing the association between PA and rhinoscopy findings  or serumThe main finding of this study was that increased AR severity or persistence was related to higher rates of PA performance in adults. To our knowledge, this is the first detailed analysis of the association between AR and PA to classify AR by severity or persistence and PA by intensity. By diagnosing AR not only from clinical symptoms but also using allergen-specific IgE levels and by supplementing this diagnosis with rhinoscopy findings, we improved the reliability and the robustness of our results and reduced the risk of including non-AR patients, who accounted for approximately 45% of the participants complaining of rhinitis symptoms in our survey. By using a cross-sectional survey, our study is representative of the general adult population, unlike previous studies that have mostly focused on children or athletes.While no apparent association was found between overall AR and PA, classifying AR according to the ARIA (Allergic Rhinitis and its Impact on Asthma) guidelines revealed significant associations. In our study, AR patients who performed vigorous PA were more likely to have more severe or persistent symptoms and watery rhinorrhea on rhinoscopy. A previous study in children also showed that the association between AR and PA differed significantly after dividing patients by symptom severity, suggesting that subgroup analyses of AR patients can provide useful insights . FurtherThe pathophysiology behind exercise-induced AR remains unclear. One possible hypothesis is the T helper 2 (Th2) allergic response, which involves the release of cytokines such as interleukin (IL)-4, IL-5, and IL-13 . This shIn addition to the allergen-specific IgE data, the rhinoscopy findings add to the robustness of our results. We observed that vigorous PA was positively correlated with watery rhinorrhea, but not pale mucosa. Vigorous PA and the resulting inhalation of cold and dry air may stimulate cholinergic receptors in the airways and the enhanced cholinergic glandular secretory activity may be responsible for eliciting rhinorrhea . While tAnother possible mechanism is the exercise-induced neutrophilic infiltration of the nasal mucosa. Athletes may have an increased number of neutrophils in their nasal mucosa after exercise, which is accompanied by increased AR symptoms, especially rhinorrhea ,28. In vIt is worth comparing the association between AR symptoms and PA with exercised-induced bronchoconstriction (EIB), which occurs in approximately 90% of asthmatic individuals . AR is sDermatophagoides farinae, dog, and cockroach), it could not capture AR patients who were sensitized to other allergens. In particular, pollen is an important outdoor allergen that could cause AR symptoms in participants who perform PA outdoors; however, specific IgE against pollen was not measured in the KNHANES. Third, it was not possible to differentiate whether patients performed PA indoors or outdoors; thus, it is not clear whether the AR symptoms were dependent on exposure to specific allergens or the intensity of PA. Finally, the data used in this study were collected in 2010; therefore, the context and relationship between analyzed parameters may have changed since then. Despite these limitations, the strength of this study is that objective and robust data were used to diagnose and analyze AR and that classifying AR and PA provided a deeper understanding of the associations.The present study has several limitations. First, based on the cross-sectional study design, the causal relationship between PA and AR is difficult to determine due to the simultaneous collection of information from the survey. Second, because the KNHANES data only included sensitization to three indoor allergens . While further studies are needed to validate this relationship, clinicians and AR patients may consider vigorous PA as a potential risk factor for the aggravation of AR symptoms and modify the patients’ behavior to avoid aggravating symptoms such as performing less vigorous PA or improving adherence to environmental exposure to allergens.


Triple negative breast cancer (TNBC), usually presenting with a very aggressive phenotype, is a heterogeneous entity. We aim to discuss new biomarkers, suitable for prognostic and predictive purposes. We retrospectively collected clinical variables and immunohistochemical characteristics of early TNBCs, specifically focusing on the prognostic and predictive significance of tumor infiltrating lymphocytes (TILs) and androgen receptor (AR) expression, assessing their correlation with clinical variables. Among 159 patients, TILs were significantly higher in younger patients and with lower BMI, and in tumors with higher ki-67 and greater nodal involvement; conversely, AR was significantly higher in older patients and in tumors with lower ki-67. Interestingly and in line with literature, both TILs level and ARs expression were lower within metastatic sites, in patients who developed distant metastases, compared to those found in the primary site. Small (pT1) and node negative tumors were highly represented and no correlation of either TILs or AR with prognosis could be observed. Our findings support the use of stromal TILs to identify a more aggressive, but chemo-sensitive phenotype, mostly represented in younger women, while AR may identify a less aggressive, slow-growing luminal TNBC subtype, more common among older patients. TILs and AR are worth implementing in routine clinical practice to refine prognosis even if, in our case series, we couldn’t identify a significant correlation of the two variables with either disease-free and overall survival. In routine clinical practice, BC can be classified into prognostic and predictive subtypes based on the immunohistochemical (IHC) expression of the estrogen receptor (ER), the progesterone receptor (PgR) and the human epidermal growth factor receptor type 2 (HER2), with or without in situ hybridization of the latter, in equivocal 2 + tests2.Breast cancer (BC) is the most common cancer in women and the second cause of cancer female mortality6.Triple negative (TN) BC accounts for 15–20% of BCs and it is characterized by the absence of ER, PgR and HER2 overexpression; it usually presents with a very aggressive phenotype, frequently exhibiting high Ki-67 index and high histological grade. Due to its intrinsic aggressive clinical behavior and the lack of recognized predictive biomarkers to be used as targets for therapy, patients with TNBC have a poorer outcome compared with those presenting with other BC subtypes. Chemotherapy is still the standard of care for TNBC, in both early (neo-adjuvant and adjuvant) and advanced-stages of the disease, with anthracycline and taxanes based regimens routinely used in the early settings and sequential single-agent chemotherapy, with a predominant role for platinum-derived agents, to be used in the metastatic setting7, retain an important prognostic significance, with an improvement in both disease-free survival (DFS) and overall survival (OS)11. Moreover, TILs have been shown to predict pathological complete response (pCR) in early TNBC following neoadjuvant chemotherapy14. If TILs have been proved to be more represented in the more chemo-sensitive TNBC subtype, the androgen receptor (AR) seems to identify a “luminal”, less aggressive phenotype. AR has been detected in 25–35% of TNBC and AR negativity has been associated with a shorter DFS and OS as compared to AR-positive TNBCs19. Indeed, the prognostic role of AR in TNBC has been considered controversial, as some authors reported AR expression to be associated to worse outcome22 and the lack of prospective data concerning the interaction between AR and OS. Nevertheless, three recent large meta-analyses confirmed longer DFS in AR-positive versus AR-negative BC patients25 and multiple studies (both single and multicenter case series and prospective studies) have shown AR expression to be significantly associated with lower mitotic index and lower tumor grade at diagnosis28.It is widely accepted that TNBC itself is a heterogeneous entity and many efforts have been made to better refine the understanding of its features and to identify potential biomarkers for targeted therapies to be effectively used in this challenging BC subtype. Stromal tumor infiltrating lymphocytes (TILs) in TNBC, evaluated as a continuous variable, following the recommendations of the International TILs working group29. Nevertheless, these gene-expression based subtypes, although carrying prognostic and predictive information, due to their complexity, cannot be routinely integrated into clinical practice. Our study manly focuses on exploring the potential role in daily clinical practice of TNBC treatment for TILs and AR expression, and their correlation with clinical variables.In their pivotal paper, Lehmann et al. defined different TNBC subtypes on the basis of gene-expression profiles and among them they could distinguish an immunomodulatory subgroup and a luminal AR-expressing groupWe retrospectively collected, in an ad hoc data base, the clinical-pathologic characteristics of early TNBC, consecutively treated at our Institution between 2006 and 2014. Information on clinical history and follow-up was collected from patients’ medical charts. All patients received both the local  and systemic treatment with neo-adjuvant and/or adjuvant chemotherapy. Patients were eligible for the study if they had confirmed TNBC at the time of surgery and if data on surgical, radiation and medical treatment with follow-up or last contact information were available.2.Body mass index (BMI) was assessed for all patients based on weight and height at the time of primary surgery and calculated as the weight (in kilograms) divided by the square of the body height (in meters), expressed in units of kg/mNeutrophils-to-lymphocytes ratio (NLR) was calculated as the absolute neutrophil count divided by the absolute lymphocyte count based on preoperative blood values (5 to 1 day before surgery).All procedures were conducted in accordance with the Declaration of Helsinki and have been approved by the local ethics committee . Written informed consent to the use of clinical data for scientific purposes had been provided by all patients at the time of access for surgery.30 by one investigator (BF) who was blinded to clinical data. AR expression was measured by IHC, using an anti-androgen receptor  and the percentage of AR-positive nuclei was quantified. A cutoff of 10% or greater was used to define an AR-positive tumor31. The IHC threshold (= 0 + or < 10% or ≥ 1 + and ≥ 10%) was used for AR.Immunohistochemical analysis on BC specimens were performed at our Pathology Department and tumors were defined as TNBC if ER  and PgR  IHC staining was ≤ 1%, together with 0/1 + score HER2 on IHC  and/or non-amplified on fluorescent in situ hybridization (FISH) for 2 + score HER2 cases. For each study patient, formalin-fixed paraffin-embedded (FFPE) tumor samples were retrieved from Institutional Pathology archives. Stromal TILs were assessed according to consensus guidelinesAccording to the recommendations of the International TILs working group 2014, we analyzed TILs as a continuous variable. Correlation analysis between TILs, ARs and clinic-pathological data was performed by Spearman test. Differences between groups were calculated by Kruskal–Wallis test.Comparison of TILs and AR within the primary tumor and their respective metastatic sites was performed by Wilcoxon matched pairs test.p-values are two-sided, and significance level was set at p < 0.05.Disease-Free Survival (DFS) was defined as the the time from diagnosis to relapse at any site . Overall survival (OS) was defined as the time from diagnosis to death from any cause. The Kaplan–Meier method was used to estimate survival curves, the log-rank test was used to test difference between groups. All reported Statistical analyses were performed using STATA software (version 15).All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors.Informed consent was obtained from all individual participants included in the study.Between 2006 and 2014, 159 patients with TNBC were treated at our Institution; patients’ clinical-pathologic characteristics are summarized in Table Among 159 tumor specimens analyzed, TILs and AR were evaluable on 151 (94.9%). Median TILs score was 10% (interquartile range 3%-20%). When using 10%, the median stromal TILs score, as our internal cut-off, we could distinguish “low”  to “high”  cases and among the latter category, we were able to identify 5 (3.3%) so called lymphocyte predominant breast cancers (LPBCs), where mononuclear immune cells were more abundant than cancer cells in the stroma inside the tumor burden. Concerning AR IHC staining, we identified 77 (51%) tumor specimens with positive AR staining and 74 (49%) cases with a negative AR staining.We performed a correlation analysis between both TILs and AR and other routinely used pathologic features, such as tumor size, nodal status, histological grade, lympho-vascular invasion (LVI) and Ki-67, and clinical characteristics, such as age, BMI and NLR. TILs were significantly higher in younger patients  and in patients with lower BMI , in tumors with higher ki-67  and greater nodal involvement , and in tumors with lower ki-67 . There was no evidence of association between ARs and size, NLR, BMI, grading, pT, pN, histology, and LVI. In Fig. With a median follow-up of 6.16 years (range 0.89–13.12 years), we observed 38 (24%) distant recurrences, 4 (3%) contralateral breast cancers, and 2 (1%) ipsilateral breast cancers. Median time to metastasis was 15 months. Median number of metastatic sites was 3 (range 1–6). Thirteen out of 38 (34.2%) patients had visceral metastases, one (2.6%) patient recurred with bone-only disease and 27 (71.1%) developed central nervous system disease. For 17 (44.7%) patients, for whom biopsies of metastatic disease were available, we reviewed specimens and confirmed a TNBC phenotype. Our analysis showed that both TILs level and AR expression were lower within metastatic sites, in patients who developed distant metastases, compared to those found in the primary site, with a trend toward statistical significance . Kaplan-Meyer curves for the entire population are shown in Fig. 7 we were able to correlate immune features with well-established pathological data. In line with literature data on high stromal TILs predicting benefit from chemotherapy, we confirmed that the higher stromal TILs scoring, the higher the aggressiveness of pathologic profile of the tumor. In our case-series, around half of the population showed AR positivity, in a mixed literature context, where values of AR positivity in TNBCs can range from 7 to 75%34. Interestingly, low Ki-67 came up to be the only pathologic feature significantly correlating with high AR expression, clearly linking lower biological aggressiveness with the luminal TNBC phenotype. These findings endorse AR as a biomarker to be used in clinical practice, together with Ki-67, to refine TNBC prognosis and help clinicians to better identify “low-proliferation” TNBC, for whom a tailored strategy, taking into account AR-targeted agents, may be proposed. Nevertheless, wide variability exists in the literature on AR + TNBC specific prognosis, with some studies reporting a correlation of the luminal TNBC subtype with increased lymph node metastasis, increased mortality, and poor disease free survival36. Besides, early phase trials testing different androgen-blockade strategies, have shown only limited clinical benefit rate (CBR) in AR + TNBCs  and a phase III trial of paclitaxel plus enzalutamide versus placebo or enzalutamide monotherapy followed by paclitaxel was withdrawn . Moreover, when evaluating impact of AR in refining TNBC prognosis, probably a long-term follow-up, similar to the one routinely considered for luminal-like BCs, might be more suitable. Thus, many efforts still have to be made in defining AR+ TNBCs phenotype and clinical behavior, and possibly different biomarkers would need to be considered to refine luminal TNBCs prognosis, such as basal markers EGFR and CK 5/6, with EGFR−/AR + TNBCs exhibiting better clinical outcome compared to both EGFR+/AR+ and EGFR+/AR− disease39. Additionally, in line with the complexity of “luminal” TNBC, AR antagonists are being tested in combination with different targeted agents (such as CDK4/6 inhibitors and PI3K/AKT inhibitors) or immunotherapeutic strategies. The results of this research effort will hopefully help us identifying additional predictive biomarkers and understanding which proportion of AR+ TNBCs display a less aggressive behavior and fare prognostically better than other TNBCs.In our monocentric, real life, retrospective case series, assessment of stromal TILs and AR was feasible and following international consensus guidelines40. In addition, due to the retrospective nature of the data and the relatively small sample size, we could not do an a-priori power analysis for interaction of AR and TILs with OS and DFS. These considerations are to be kept in mind when analyzing our Kaplan-Meyer curves, because we have to acknowledge that the effect of TILs and AR in modifying prognosis may be masked by the stage of presentation. Moreover, we think this finding to be specifically relevant nowadays, when due to widespread use of screening procedures, a very high percentage of tumors presents in stage I–II.In our case series most of the patients presented with stage I–II, with a low disease burden; specifically, around 35% of them had pT1N0 tumors, which harbor an intrinsic very good prognosis regardless of tumor biology45, with this trend being more frequently observed in more aggressive tumors, such as TNBC and HER2-enriched cases. Moreover, in our case series, biopsies of the metastatic site were taken at first appearance of metastatic disease, before the initiation of any chemotherapeutic line, thus no impairment of the immune system was induced by pre-treatment. These observation are of paramount interest because we now face the introduction in standard first-line therapy of immune check-point blockade for PD-L1 positive TNBC, where the amount of stromal reactive immune cells has been proved to be crucial for treatment benefit46. Thus further efforts have to be made to render the tumor microenvironment more sensitive to immune therapies; on this regard the recently published results of the TONIC trial indicate that short-term administration of chemotherapy, mainly doxorubicin and cisplatin, may induce a more favorable tumor microenvironment and increase the likelihood of response to immune check-point blockade in TNBC47.Our data on the extent of lymphocytic infiltration in metastasis biopsies are in line with recent literature, reporting a lower TILs score in metastatic tissue compared to the matched primary specimen50. Some authors have linked the more advanced and aggressive disease in young women to the increased potential for a delayed diagnosis, due to difficulties in detecting tumors because of the density of the mammary glands52. Nevertheless, it seems apparent that the prognostic impact of age is mainly guided by a more aggressive biological phenotype. Compared to older women, the young counterpart presents at diagnosis with higher percentage of ER and PgR negativity, vascular or lymphatic invasion and pathologic grade 3 tumors56. Thus, patient’s age has been endorsed to be considered as a prognostic factor by the National Comprehensive Cancer Network (NCCN), St. Gallen, and European Society of Medical Oncology (EMSO) guidelines57. Here, in line with the tight relationship between age and prognosis, we observed young women to present with a more aggressive phenotype than the older counterpart.Interestingly we found that age remained a statistically significant factor associated with both TILs and AR, with younger patients presenting with a more prominent stromal TILs infiltration and older patients expressing higher levels of AR. Age has been long considered a confounding factor in breast cancer prognosis definition, younger age being historically seen as a predictor for worse prognosis, independently of clinical and treatment-related variables62. While in luminal/ER-positive BC subtypes numerous studies have identified an increased mortality risk in obese women66, BMI in TNBC is considered a confounding factor, as no direct relation has been established between BMI itself and recurrence-free survival (RFS) or OS in TNBC69. Here we propose a rationale for using BMI in refining TNBC prognosis in clinical practice, considering non-obese women to be at major risk for more aggressive, but chemo-sensitive, TN disease.We also found a statistically significant association between high stromal TILs infiltration and low BMI, which is not surprising if we take into account data showing higher pCR rates after neo-adjuvant chemotherapy in lower mean BMI patientsIn conclusion, stromal TILs can be used to identify a more aggressive, but chemo-sensitive phenotype, mostly represented in younger women, while AR expression characterizes a less aggressive, slow-growing TNBC subtype, more common among older patients. Furthermore, we confirm that metastatic sites tend to be less rich in TILs, supporting a role for induction treatments prior to check-point blockade treatments. In our opinion, these findings are very interesting in managing daily clinical practice therapeutic decisions and prognosis speculations because, outside clinical trials, we face many pathological reports interpretation difficulties and refining prognosis with more biological parameters is of crucial importance.