The nanobubbles produce the hydroxyl radical by ultrasonic waves, changing the pH and catalyst and so on, while the nanobubble is very reactive to scavenge free radicals. In this research especially hydrogen (4% H2 in argon) and CO2 nanobubbles have been blown into hydrogen peroxide (H2O2) added pure water, ethanol, and ethylene glycol aqueous solution through a porous ceramic sparger from the gas cylinder. The aqueous solutions with H2O2 are irradiated by ultraviolet (UV) light and the produced hydroxyl radical amount is measured with spin trapping reagent and electron spin resonance (ESR). The CO2 nanobubble blowing extremely has reduced the hydroxyl radical in water, ethanol, and ethylene glycol aqueous solution. On the other hand, when H2 nanobubbles are brown after CO2 nanobubble blowing, the hydroxyl radical amount has increased. For the disinfection test, the increase of hydroxyl radicals is useful to reduce the bacteria by the observation in the agar medium. Next, when the superoxide anion solution is mixed with nanobubble containing water, ethanol, and ethylene glycol aqueous solution, H2 nanobubble has reduced the superoxide anion slightly. The water containing both CO2 and H2 nanobubble reduces the superoxide anion. The less than 20% ethanol and the 30% ethylene glycol aqueous solution containing CO2 nanobubbles generated after H2 nanobubble blowing can diminish the superoxide anion much more. While the H2 nanobubble blowing after CO2 nanobubble blowing scavenges the superoxide anion slightly. The experimental results have been considered using a chemical reaction formula.The main findings are the hydroxyl radical scavenging and the superoxide anion diminishing by mixing the carbon dioxide (CO This report is not the free radical production by nanobubble but describes the free radical degradation in aqueous solution by adding nanobubbles. Various kinds of materials have been reported to scavenge free radicals in medical and pharmacy fields in the paper such as FREE RADICAL RESEARCH and so on. However, there are a few reports for free radical degradation by adding and mixing nanobubbles, while, there are several reports for the detection and production of free radicals by nanobubbles. The aim of this research is how to reduce or increase the existing free radical by nanobubble, therefore, in the beginning, the reports of free radical production conditions by nanobubble are investigated. Takahashi et al. reported that the hydroxyl radical generation with ESR measurement using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin-trapping reagent has been observed after collapsing of high concentration air microbubbles in water produced by a pump through a gas-dissolution tank and a microbubble-generating nozzle1. Next, his group reported that the ∙OH producing in both cases of air (oxygen microbubbles and nitrogen microbubbles) in the acidic condition at pH 2 and 32 and the generation of hydroxyl radicals from the collapse of oxygen or air microbubbles was markedly enhanced by the existence of copper ion catalyst by the copper wire under pH2.2 acidic HCl solution3. Also, the microbubbles on ozonized water indicated the presence of hydroxyl radical by the collapse of microbubble4. Recent papers are referred the above papers5 and the oxygen nanobubble stability was confirmed to be pH-dependent and the collapsed oxygen nanobubble generate the free radical that induced the photodegradation of oxytetracycline6. When the absolute value of zeta potential of nanobubble is low, there is a possibility to collapse of nanobubble by Brownian motion and produce the free radical and also the existence of catalysts enhance the radical generation. Ozone addition can produce the hydroxyl radical. On the other hand, the free radical could not generate by the self-collapse of air micro-nano bubbles in pure water produced by fiber membrane filter, and the hydroxyl radical peak was observed with weak supersonic wave7. The radical production by ultrasonic wave irradiation becomes more important to produce the radicals, especially hydroxyl radicals in water by comparing no irradiation and irradiation8 and the ultrasonic waves collapse the nanobubble (hydrogen) and increase the temperature of water9. Therefore, the pure water and alcohol mixed water that does not change the pH and not irradiated an ultrasonic wave is utilized in this report. As the application of produced radicals, there are reports for environmental cleaning related to wastewater treatment13 and the medical application by reducing oxygen molecules in a chaotic manner within the tumour14. In this paper, the bacteria reduction is also examined when the hydroxyl radical enhances in the aqueous solution.Reactive oxygen species are hydroxyl radical (∙OH), superoxide anion  such as ∙OH, ClO–, ONOO–, and∙ O2− both in vivo and in vitro18. Cancer cell growth was inhibited in the hydrogen nanobubble-containing medium compared to the non-containing medium (in vitro)19. They have reported mainly the scavenging of ROS by using hydrogen nanobubbles.On the other hand, the nanobubble effects to degrade the existing free radicals have been investigated for recent 10 years. In 2010 the nano-bubble hydrogen-dissolved water, which was prepared using a microporous-filter hydrogen-jetting device, scavenged reactive oxygen species (ROS) indispensable to slightly exist as a signal for tumor cell growth2 + Ar, CO2, N2, O2) and mixture nanobubbles (H2 + Ar and CO2) in water and alcohol aqueous solution. The hydroxyl radical in free radical included aqueous solution  is prepared by H2O2 addition followed by blowing various nanobubble into the liquid, and the hydroxyl radical scavenging is investigated for single nanobubbles and mixture nanobubbles. The superoxide anion in free radical-induced aqueous solution is prepared by a hypoxanthine (HX) and xanthine oxidase (XO) system and mixed with nanobubble included aqueous solution. The superoxide anion scavenging is examined using the single nanobubbles and mixture nanobubbles. The free radical concentration is measured with ESR by using G-CYPMPO as a spin trapping reagent. The degradation of free radicals is effective for healthy beverage and the increase of hydroxyl radical eliminates the bacteria concentration.In this experiment, the degradation of free radicals for hydroxyl radical and superoxide anion have been compared with single nanobubbles (H2 (4% in Ar), CO2, O2, and N2 nanobubble in water is between 100 and 200 nm. The mean diameter of H2 (4% in Ar) in ethanol aqueous solution is between 150 and 250 nm, while in the ethylene glycol aqueous solution the nanobubble diameter becomes larger between 500 and 1000 nm. The mean diameter of CO2 nanobubble in ethanol solution is between 250 and 300 nm and in the ethylene glycol aqueous solution the nanobubble diameter becomes larger between 400 and 1000 nm similar to H2 (4% in Ar) nanobubble in ethylene glycol. The mean diameter of nanobubble by both blowing H2 (4% in Ar) gas and next CO2 gas in ethanol aqueous solution is about 200 nm, however, the mean diameter of nanobubble by CO2 gas and next H2 (4% in Ar) gas becomes larger between 200 and 500 nm. H2 (4% in Ar) nanobubble size in water is gradually increased and stable about 400 nm after 10 days and exists more than 100 days. While the CO2 nanobubble is gradually increased and disappeared after several days. In Table 20. The CO2 gas solubility is one order larger comparing other gas like H2, Ar, O2, and N2.The mean diameter of produced nanobubble in various aqueous solutions are listed in Table The ESR spin adducts of sc-5--5 methyl-1-pyrroline N-oxide (G-CYPMPO) for hydrogen peroxide aqueous solution (0.1 wt%) under 10 s UV-illumination (A) and hypoxanthine/xanthine oxidase (HX/XO) system (B) is shown in Fig. 2, O2, H2 (4% in Ar), and CO2 are measured by ESR and shown in Fig. 2 nanobubble has decreased compared with the control water and the water containing other nanobubbles. Also, the 4th peak of the water containing H2 (4% in Ar) nanobubble has decreased. Therefore, the water containing CO2 nanobubble is used for the hydroxyl radical degradation experiment in other aqueous solutions. On the other hand, the degradation by nanobubble combination of CO2 after H2 (4% in Ar) nanobubble in water and combination of H2 (4% in Ar) after CO2 nanobubble in water show the similar to the degradation by CO2 bubbling. Degradation of superoxide anion radical in the water containing different kinds of nanobubbles N2, O2, H2 (4% in Ar), and CO2 are measured by ESR and shown in Fig. 2 (4% in Ar) nanobubble shows larger degradation peaks comparing with the control water. Therefore, the water bubbled H2 (4% in Ar) nanobubble is used for the superoxide anion radical degradation in the following experiment. Both degradation by nanobubble combination of CO2 after H2 (4% in Ar) nanobubble and combination of H2 (4% in Ar) after CO2 nanobubble show a little bit larger degradation of H2 (4% in Ar).Degradation of hydroxyl radical with ultraviolet in water blown different kinds of nanobubbles N2 and H2 (4% in Ar) are blown into 50%, 20%, and 10% of ethanol in water with 0.1% H2O2. The peaks of hydroxyl radical with ESR are shown in Fig. 2 (4% in Ar) gas bubbling reduced hydroxyl radical small. While the CO2 gas bubbling reduced hydroxyl radical almost completely in the used concentration of ethanol aqueous solution. The CO2 nanobubble in ethanol aqueous solution can eliminate the hydroxyl radical. The CO2 and H2 (4% in Ar) have been blown into 50% and 30% of ethylene glycol in water with 0.1% H2O2 as a dihydric alcohol aqueous solution and the peaks are shown in Fig. 2 (4% in Ar) gas bubbling can reduce the hydroxyl radical a little bit small, whereas the CO2 gas bubbling eliminates hydroxyl radical almost completely like the result of ethanol aqueous solution. Next, the hydroxyl radical degradation is investigated with the blowing order of CO2 and H2 (4% in Ar) gas. Peaks of hydroxyl radical with ESR by blowing CO2 after H2 (4% in Ar) gas and H2 (4% in Ar) after CO2 gas nanobubble into 50, 20, and 10% ethanol with 0.1% H2O2 aqueous solution are shown in Fig. 2 (4% in Ar) blowing cannot decrease the hydroxyl radical peak as shown in Fig. 2 blowing after H2 (4% in Ar) can disappear the hydroxyl radical peaks as shown in Fig. 2 blowing can decrease the hydroxyl radical peaks as shown in Fig. 2 (4% in Ar) blowing increased the hydroxyl radical peaks again as shown in Fig. 2 after H2 (4% in Ar) gas nanobubble and H2 (4% in Ar) after CO2 gas nanobubble into 50 and 30% ethylene glycol with 0.1% H2O2 aqueous solution is shown in Fig. 2 (4% in Ar) blowing after CO2 increased the hydroxyl radical peaks again.The CO2 and H2 % in Ar a2 and H2 % in Ar a2 and H2 % in Ar a2O2 aqueous solution H2 (4% in Ar) nanobubble injection after CO2 gas nanobubble blowing showed no living bacteria after 48 h incubation. The hydroxyl radical in the solution as shown in Fig. Photos of the plate are compared with the control and the solution containing nanobubbles treatment after 48 h incubation is shown in Fig. 2 and H2 (4% in Ar) nanobubbles. Then the peaks of superoxide anion radical with ESR using spin trapping reagent of G-CYPMPO are shown in Fig. 2 (4% in Ar) gas and CO2 nanobubble included 10% and 20% ethanol aqueous solution reduced superoxide anion radical slightly. However, radical degradation cannot be seen in the 50% ethanol aqueous solution. The peaks of superoxide anion radical with ESR in 30 and 50% ethylene glycol aqueous solution containing CO2 and H2 (4% in Ar) nanobubble are shown in Fig. 2 (4% in Ar) nanobubble shows much smaller peaks, however, the CO2 peaks do not decrease. Next, the superoxide anion radical degradation is investigated with the blowing order of CO2 and H2 (4% in Ar) gas nanobubble in ethanol aqueous solution. The peaks of superoxide anion radical with ESR by blowing CO2 after H2 (4% in Ar) gas nanobubble and H2 (4% in Ar) after CO2 gas nanobubble into 50, 20, and 10% ethanol aqueous solution are shown in Fig. 2 after H2 (4% in Ar) for 20 and 10% ethanol aqueous solution, the superoxide anion decreased comparing the peaks by only CO2 and H2 (4% in Ar) in Fig. 2 after H2 (4% in Ar) gas nanobubble and H2 (4% in Ar) after CO2 gas nanobubble into 50 and 30% ethylene glycol aqueous solution are shown in Fig. 2 gas after H2 (4% in Ar) gas decreased larger than the H2 (4% in Ar) gas after CO2 gas for 50 and 30% ethylene glycol aqueous solution. This is a similar phenomenon to the 20 and 10% ethanol aqueous solution.The hypoxanthine (HX)/xanthine oxidase (XOD) solution is added and mixed well with 50%, 20%, and 10% of ethanol in water containing CO18 and reduced especially hydroxyl radical17. Oxygen nanobubble caused tissue damage16. In this experiment, the used solvents are pure water, ethanol aqueous solution as monohydric alcohol, and ethylene glycol aqueous solution as a dihydric alcohol mixture. The hydroxyl radical (∙HO) is produced by adding H2O2 in an aqueous solution and irradiation of UV as shown in the Eq. (2−) was produced with an addition of hypoxanthine (HX) and xanthine oxidase (XO) in an aqueous solution. In the measurement of hydroxyl radical by ESR with UV, a large amount of CO2 and H2 nanobubbles are supplied into H2O2 added aqueous solution before measurement. While the superoxide anion was measured by ESR after the superoxide anion was mixed with O2 and H2 nanobubble containing aqueous solution. Some parts of CO2 and H2 nanobubbles are collapsed and the appeared gas seems to react to the radicals. There is a report that N2, O2 and CO2 nanobubbles produced by piston-type generator have gradually changed the size for 48 h21. After nanobubbles were produced, in a few days nanobubble size of N2, O2, and H2 + Ar changes and becomes almost constant in a week and stable. However, the CO2 gas bubble size constantly changes larger and disappeared in several days. The nanobubble gas can supply the gas reacts to the radicals for several days. Several chemical formula22 of hydroxyl radical and superoxide anion radical reactions with hydrogen and oxygen are as follows,There are some reports for the diminishment of free radicals by hydrogen nanobubble as shown in the introduction session. The hydrogen nanobubble scavenged various ROS2 nanobubble is shown in the reaction (2 (4% in Ar), O2, and N2 nanobubble it is difficult to react directly to ∙HO in an aqueous solution. As shown in the ESR peaks of Fig. 2 nanobubble can reduce the ∙HO in water. Also, the CO2 nanobubble can reduce ∙OH in the other alcohol aqueous solution as shown in Figs. 2 blowing after H2 (4% in Ar) gas nanobubble showed a clear decrease in the peak of ∙HO similar to only CO2 blowing. On the other hand, the opposite mixing of H2 (4% in Ar) gas nanobubble after CO2 blowing did not decrease the peak of ∙HO and H2 (4% in Ar) nanobubble increased the peak of ∙HO. The produced ∙O2− shown in the Eqs. (2 and ∙HO is produced again by the reaction shown in Eq. (2− in aqueous solution with H2 (4% in Ar) nanobubble is shown in the reaction  nanobubble. The produced ∙HO in (6) can be reduced by the CO2 nanobubble in the reaction of (2 (4% in Ar) nanobubble is blowing into ethanol and ethylene glycol with water, ∙HO is produced by the reaction (2− has mixed with both CO2 and H2 (4% in Ar) nanobubble mixed solution, the peaks of.The decrease of the ESR spectrum of ∙HO in aqueous solution with COreaction  → 4) →  → 2 nanoreaction , therefothe Eqs.  and 8)  2 nanobun in Eq. . The reareaction  and the ction of  → (4) → ction of . When H2reaction  and 13)2 nanobub2− has decreased clearly in the ethanol aqueous solution as shown in Fig. 2− have decreased slightly in the ethylene glycol aqueous solution as shown in Fig. 2 nanobubble amount by H2 (4% in Ar) blowing after CO2 gas nanobubble might contribute to the reaction  →  → 2− hasreaction  and incrreaction  → 4). N. N2− hasreaction . When etshown in  and 13)2− has de2 in argon (Ar), CO2, O2, and N2) from the gas cylinder into distilled water, ethanol, and ethylene glycol aqueous solution through a porous ceramic sparger of 500 nm mean pore diameter for 30 min23. Next, the aqueous solutions containing H2 (4% in Ar) gas are blown CO2 gas again and the solution containing CO2 nanobubbles is blown H2 (4% in Ar) gas again to investigate the effect of nanobubble gas mixture. The nanobubble size distributions are measured by the dynamic light scattering (DLS) method . The prepared nanobubble solution is supplied in the radical experiment within 24 h. In this experiment, a 4% hydrogen in argon is used to produce the nanobubble for the security as the 4 vol % hydrogen is the lower explosive limit. The carbon dioxide from the gas cylinder is more than 99.5 vol %. The existing nanobubble percentage in water is about 0.1 vol% after blowing 30 min. The nanobubble is blown in the water, 50%, 20%, and 10% ethanol as monohydric alcohol in water and 50%, 30% ethylene glycol as dihydric alcohol in water.Nanobubbles are generated by blowing the different gas (4% H2O2 addition in aqueous solution and 10-s ultraviolet irradiation. The superoxide anion was produced with a hypoxanthine (HX) and xanthine oxidase (XO) system. A mixture of 3.6 μL of 10.97 units/ml XO, 20 μl of 20 μM HX, 156.4 μL of the nanobubble sample solution, and 20 μl spin trapping reagent solution are mixed.The hydroxyl radical is produced with 0.1 wt% H24 has been used. CYPMPO can spin-trap superoxide and hydroxyl radicals and the half-life for the superoxide adduct of CYPMPO produced in UV-illuminated hydrogen peroxide solution and HX/XO solution are about 15 min and 50 min, respectively25. The kinetic evaluation of spin trapping rate constants of CYPMPO was reported26. The 0.1 wt% H2O2 was added to the aqueous solution before blowing gas bubbles. While the superoxide anion was added after blowing the gas bubbles.As spin-trapping reagent, sc-5--5 methyl-1-pyrroline N-oxide (G-CYPMPO)2O2 added aqueous solution, and the spectra of hydroxyl radicals by ESR is measured. While to decompose the superoxide anion, the superoxide anion is added into the various nanobubbles included aqueous solution and the spectra of superoxide anion have been measured by ESR.JEOL JES-TE25X ESR spectrometer is used to obtain ESR spectra of free radicals of hydroxyl radical and superoxide anion. To investigate the decomposition of hydroxyl radicals by nanobubbles, the various gas has been blown into 0.1 wt% H27 is used to measure the total number of living bacteria on hand. The hand is put into an aqueous solution containing viable bacteria and touch the plate. The plate is incubated for 48 h as control. The other plate is soaked with ethylene glycol aqueous solution containing nanobubble and after removing the solution the plate is incubated for 48 h. After 48 h the photos of the plate are compared with the control and the solution containing nanobubbles treatment.Hand Petancheck II Tryptone Soya Agar Medium


MPST deficiency was found in 1960s among rare hereditary mercaptolactate-cysteine disulfiduria patients. Mpst-knockout (KO) mice with enhanced liver Tst expression were recently generated as its model; however, the physiological roles/significances of Mpst remain largely unknown. Here we generated three independent germ lines of Mpst-KO mice by CRISPR/Cas9 technology, all of which maintained normal hepatic Tst expression/activity. Mpst/Cth-double knockout (DKO) mice were generated via crossbreeding with our previously generated Cth-KO mice. Mpst-KO mice were born at the expected frequency and developed normally like Cth-KO mice, but displayed increased urinary 3-mercaptolactate excretion and enhanced passive systemic anaphylactic responses when compared to wild-type or Cth-KO mice. Mpst/Cth-DKO mice were also born at the expected frequency and developed normally, but excreted slightly more 3-mercaptolactate in urine compared to Mpst-KO or Cth-KO mice. Our Mpst-KO, Cth-KO, and Mpst/Cth-DKO mice, unlike semi-lethal Cbs-KO mice and lethal Cars2-KO mice, are useful tools for analyzing the unknown physiological roles of endogenous H2S/RSS production.Mercaptopyruvate sulfurtransferase (Mpst) and its homolog thiosulfate sulfurtransferase (Tst = rhodanese) detoxify cyanide to thiocyanate. Mpst is attracting attention as one of the four endogenous hydrogen sulfide (H R)-2-amino-3-[(2-carboxy-2-hydroxyethyl) disulfanyl] propanoic acid; C(C(C=O)O)N)SSCC(C(=O)O)O) in his urine ) ,20,21,22In the present study, we examined Mpst-KO mice produced using CRISPR/Cas9 technology. We preserved normal TST expression by minimizing the size of Mpst gene deletion and found that our mice display increased urinary excretion of 3-mercaptolactate and enhanced passive systemic anaphylaxis (PSA), while maintaining hepatic Tst expression/activity. Moreover, we also generated Mpst/Cth-double knockout (DKO) mice as a useful tool to investigate their mutually compensatory roles in mice.Mpst which encodes 67% of the Mpst open reading frame  to greater or lesser degrees. This is in stark contrast to the other two H and Cth A. In all and Cth B. Theref and Cth  likely d and Cth . Using t and Cth B. Furthe and Cth C. Mpst a and Cth A,B. Howe and Cth A,B. At t and Cth A,B.Serum amino acid/thiol compound levels for all lines of Mpst-KO mice were indistinguishable from those of WT mice, which was in marked contrast to Cth mice; however, all Mpst-KO mice excreted 5.5–7.3 times the normal amount of 3-mercaptolactate (3-ML) in urine . Though We have previously generated Cth-KO mice  that req2S has been shown to regulate various inflammatory responses [Hesponses ,31, but esponses ,33,” remMCDU has historically been attributed to MPST deficiency, but studies containing direct experimental evidence are lacking. We provide here the first direct evidence that Mpst deficiency in mice causes enhanced secretion of 3-ML in urine, a hallmark trait of MCDU . CysteinMpst gene deletion strategies as the mouse Tst gene is located near the Mpst gene (Previously generated Mpst-KO mice exhibited increased anxiety-like behaviors associated with increased levels of serotonin and its major metabolite (5-hydroxyindoleacetic acid) in the prefrontal cortex and decreased levels of dopamine in the hippocampus . Furtherpst gene A and the2S/RSS in general thermoregulation. Further studies are necessary to delineate the roles of Mpst/Cth and produced H2S/RSS in the thermoregulation.In this study, we analyzed, for the first time, the systemic anaphylactic responses in Cth-KO and Mpst-KO mice and found greater PSA (but not PCA) responses in Mpst-KO mice . In anapMpst mutations, further studies including the examination of Mpst/Cth-DKO mice for PSA/PCA alterations are needed.Furthermore, we generated Mpst/Cth-DKO mice that develop normally  may provide new insight into the sulfur amino acid metabolism.In conclusion, we generated Mpst-KO mice as an animal model of MCDU and also Mpst/Cth-DKO mice as a useful tool for analyzing functions of HMpst gene: Mpst crRNA upstream (u) (5′-ACGACCGCGGGCGCCAGGAG-3′) and Mpst crRNA downstream (d) (5′-GCTGGGGAAACGGACTAATG-3′). Electroporated zygotes were transferred into the oviducts of pseudopregnant female ICR mice , and a total of 14 mice were born on E19.5. Correct targeting/deletion was confirmed by PCR using forwarding (f) (5′-TGCTGGGCAAGGAATAGAGTG-3′) and reverse (r) (5′-GCATATAAGGCCAGCACACG-3′) primers that are located outside the targeted locus (http://card.medic.kumamoto-u.ac.jp/card/english/index.html). All animal procedures conformed to the Guide for the Care and Use of Laboratory Animals, 8th Edition published by the US National Research Council and were approved by the Animal Care Committees of Showa Pharmaceutical University .Mpst-KO mice were generated using CRISPR/Cas9 technology from Setsurotech Inc.  on a C57BL/6J  background as described previously  with somed locus B. We seqed locus . Routineed locus C. Adult g, 4 °C, 20 min). Mpst and Tst recombinant proteins were affinity-purified with the TALON Metal Affinity Resins (Clontech) and a linear gradient of 10–100 mM imidazole. The pooled fraction was dialyzed twice against buffer E at 4 °C overnight using a Slide-A-Lyzer G2 Dialysis Cassette–20kDa cutoff (Thermo Fisher Scientific). The recombinant proteins were stored at −80 °C until use.His-tagged murine Mpst ) and Tst ) expression plasmids were purchased from GeneCopoeia . The plasmids were transformed to Rosetta (DE3) pLysS Competent Cells (Novagen (Merck)). Single colonies were picked up from LB agar plates containing 100 µg/mL Ampicillin (Amp) and 34 µg/mL Chloramphenicol (CP), and placed into 75 mL LB media (100 µg/mL Amp and 34 µg/mL CP) for growth overnight at 37 °C. 50-mL of the overnight culture was seeded into 1-L LB media (100 µg/mL Amp) and grown at 30 °C for 1.5 h and at 20 °C for 2 h. Following, isopropyl β-D-galactopyranoside (IPTG) was added  and the culture was further grown at 20 °C for 24 h. Cells were then centrifuged and resuspended in 40 mL buffer E (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM Tris(2-carboxyethyl)phosphine (TCEP) hydrochloride, and 10% (v/v) glycerol) containing a Complete EDTA-free Protease Inhibitor Cocktail . Cells were then lysed by sonication and the soluble fraction was isolated by centrifugation  was obtained. Efficient antibody titers  were confirmed by ELISA (data not shown).A single Japanese white rabbit ) was immunized with full-length mouse Mpst recombinant proteins using Freund’s complete adjuvant four times (day 0 (0.3 mg), 28 (0.2 mg), 35 (0.2 mg), and 42 (0.2 mg)) and whole blood was collected at day 49. The blood was allowed to clot by leaving undisturbed at room temperature for 20 min, and the clot was removed by centrifugation at 2000× per 100 mg; 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1.0 mM EDTA, 1% (w/v) Triton X-100, 0.1% (w/v) phenylmethylsulfonyl fluoride, and Complete EDTA-free Protease Inhibitor Cocktail) using a MicroSmash-100R homogenizing system  and zirconia beads . Homogenates were centrifuged at 15,300× g, at 4 °C for 20 min. Supernatants were recentrifuged at 15,300× g, at 4 °C for 30 min. After heating  with the sample buffer, samples (5 µg/lane) were resolved on 10% or 12% pre-cast PAGE gels, and then transferred to the Immobilon PVDF membrane (Millipore) and subjected to western blotting. Mpst (and Tst as a cross-reactive protein) were detected with the above polyclonal antibody ) and anti-rabbit IgG, HRP-linked whole Ab donkey antibody . Tst was detected with anti-human TST rabbit monoclonal antibody  and anti-rabbit IgG, HRP-linked whole Ab donkey antibody . Cbs was detected with anti-rat CBS rabbit polyclonal antibody ([Each mouse tissue sample (~100 mg) was homogenized in lysis buffer 3)3 + HNO3 + H2O; [–) formation was determined by the measurement of absorbance at 460 nm using the Epoch 2 microplate spectrophotometer . Tst enzyme assays were performed using the substrate thiosulfate as described previously [2PO4, and 71.4 mM KCN at 25 °C for 30 min and processed similarly. Specific Mpst and Tst activities were obtained at substrate concentrations of 5.95 mM (for 3MP) and 25 mM (for thiosulfate), respectively.Mpst enzyme assays were performed using the substrate 3-MP as described previously ,44, with3 + H2O; ). Thiocyeviously  with somSerum samples were analyzed for their amino acid concentrations using amino acid and thiol-derivatization fluorescent reagents (4-fluoro-7-nitrobenzofurazan (NBD-F) and 4-fluoro-7-sulfobenzofurazan (SBD-F), respectively; Dojindo, Kumamoto, Japan) via a high-performance liquid chromatography (HPLC) as previously described  Supplem. Urine sPSA and PCA assays were performed as previously described . For PSAt-test was used in PCA assays . Statistical comparison was performed using Prizm 5 software . Two-way repeated-measured ANOVA with Bonferroni’s multiple comparison test was used in PSA assays A and ratA assays B. One-wa


Lysine (K)-specific demethylase 6B (KDM6B), a stress-inducible H3K27me3 demethylase, plays oncogenic or antitumoral roles in malignant tumors depending on the type of tumor cell. However, how this histone modifier affects the progression of prostate cancer (PCa) is still unknown. Here we analyzed sequenced gene expression data and tissue microarray to explore the expression features and prognostic value of KDM6B in PCa. Further, we performed in vitro cell biological experiments and in vivo nude mouse models to reveal the biological function, upstream and downstream regulation mechanism of KDM6B. In addition, we investigated the effects of a KDM6B inhibitor, GSK-J4, on PCa cells. We showed that KDM6B overexpression was observed in PCa, and elevated KDM6B expression was associated with high Gleason Score, low serum prostate-specific antigen level and shorted recurrence-free survival. Moreover, KDM6B prompted proliferation, migration, invasion and cell cycle progression and suppressed apoptosis in PCa cells. GSK-J4 administration could significantly suppress the biological function of KDM6B in PCa cells. KDM6B is involved in the development of castration-resistant prostate cancer (CRPC), and combination of MDV3100 plus GSK-J4 is effective for CRPC and MDV3100-resistant CRPC. Mechanism exploration revealed that androgen receptor can decrease the transcription of KDM6B and that KDM6B demethylates H3K27me3 at the cyclin D1 promoter and cooperates with smad2/3 to prompt the expression of cyclin D1. In conclusion, our study demonstrates that KDM6B is an androgen receptor regulated gene and plays oncogenic roles by promoting cyclin D1 transcription in PCa and GSK-J4 has the potential to be a promising agent for the treatment of PCa. Histone lysine residues can be mono-, di-, or trimethylated, and the different degrees of methylation on a particular site could be linked to different functional outcomes2. H3K27 trimethylation (H3K27me3) is one of the most important repressive histone modifications and is mediated by the histone methyltransferase enhancer of zeste homolog 2 (EZH2)3. EZH2 is the catalytic subunit of the polycomb repressive complex (PRC2) and H3K27me3 contributes to the recruitment of the PRC1 complex. PRC1 and PRC2 repress the expression of various developmental genes4.Histone methylation can change the steric configuration of DNA, leading to gene expression or repression6. It is involved in multiple cellular processes, including differentiation, proliferation, senescence, and apoptosis, which tend to effect responses, such as vertebrate development, cancer, inflammatory diseases, and neurodegenerative diseases9. KDM6B expression could be induced by certain normal developmental cues and by stressful or pathogenic factors, such as inflammatory cytokines, cancerogenic factors, and mitochondrial stress inducers11. This is different from ubiquitously transcribed X chromosome tetratricopeptide repeat protein , which is a constitutively expressed H3K27 demethylase in various tissue types13.Lysine (K)-specific demethylase 6B (KDM6B), also called jumonji domain-containing protein D3 (JMJD3), could counter the effect of EZH2. It is well known that KDM6B, a member of the Jumonji C (JmjC) histone demethylase family, contains a JmjC domain and can remove all three mono-, di-, or trimethyl groups from methylated H3K27KDM6B in the development of malignant hematopoiesis has been widely detailed. Previous studies have reported the association between the abnormal elevation of KDM6B and the overactivated nuclear factor-κb (NF-κb)/innate immunity pathway in human CD34+ hematopoietic stem cells of myelodysplastic syndrome14, the oncogenic role of KDM6B in regulating immune cell differentiation and the immunological responses in lymphoid malignancies17, and the oncogenic activity of KDM6B in NOTCH1-driven human T-cell acute lymphocytic leukemia (T-ALL)18. Moreover, the abnormal expression or activity of KDM6B was observed in solid tumors, including cervical carcinoma19, pancreatic carcinoma21, glioma23, and renal cancer24. KDM6B expression is also upregulated in prostate cancer (PCa) and is further increased during metastasis25. Moreover, it has also been reported that global histone modification patterns can predict the risk of PCa occurrence and recurrence27, which indicates the great importance of histone modifications for the generation and development of PCa. However, the detailed effect and mechanism of KDM6B in PCa are still unclear.The vital role of KDM6B expression and its effect on PCa. The up- and downstream signaling pathways are also explored in this report. Further, the effects of a KDM6B inhibitor, GSK-J4, on PCa cells were also investigated. Considering the complexity of the internal environment in organisms, a subcutaneous PCa nude mouse model was applied to reveal the effects of KDM6B on PCa progression in vivo.Thus, we designed this study to observe alterations in https://genome-cancer.ucsc.edu) and five sets of data were downloaded from the GEO database . The Universal Expression Code approach was used to calculate the normalized expression as previously reported28. RNA sequencing of our Dingtianlidi program was conducted by Novogene Biotechnology , and the detailed sequencing procedures and data analysis methods are presented in the Supplemental Materials.The PCa gene expression data in The Cancer Genome Atlas (TCGA) database were manually retrieved from the UCSC Cancer Genomics Browser  and the date of BCR was assigned as the first date of total PSA levels >0.2 ng ml−1.This project was approved by the Clinical Research Ethics Committee of Shanghai Changhai Hospital of Naval Medical University. All of the clinical samples were obtained from Shanghai Changhai Hospital . Written informed consents were obtained from the participants before sampling. Construction of the tissue microarray was based on PCa and benign prostate hyperplasia histological specimens retrieved from the archives of the Department of Pathology at Changhai Hospital. Our pathology team identified the most representative area of each sample, and both the tumor tissue and the para-cancerous tissue were identified for the prostatectomy samples. The patients’ clinical and pathological data were obtained from the medical records. Different methods were applied to obtain the follow-up information as follows: reviewing the outpatient electronic medical record system and the PCa database of our department, phoning the patients or their family members, and sending letters or emails. A sustained elevation of the serum total prostate-specific antigen (PSA) level above 0.2 ng mlKDM6B-3xFLAG or the control vector to construct KDM6B overexpression or negative control cell lines. The cells were seeded into 96-well plates and cell proliferation was detected using a CCK-8 kit  according to the manufacturer’s protocol.The PC3 (RRID: CVCL_0035), C42B (RRID: CVCL_4784), and LNCaP (RRID: CVCL_0395) cell lines were bought from American Tissue Culture Collection and stored in the cell lines bank of our laboratory. Materials of cell authentication was uploaded as Supplemental Data. The human PCa cell line PC3 was maintained with F12 medium  supplemented with 10% fetal bovine serum, and the LNCaP cell line and C42B cell line was cultured with 1640 medium  supplemented with 10% fetal bovine serum. The small interfering RNA (siRNA) transfection was facilitated by Lipofectamine RNAiMAX reagent  according to the manufacturer’s instructions. The details of the siRNA sequence are presented in the Supplemental Materials. LNCaP cells were transfected with either pSLenti-EF1-Puro-CMV-4 cells were seeded on a transwell insert  in 100 μl culture medium  and were incubated for 1–2 days at 37 °C. The cells that adhered to the lower surface of the transwell insert were fixed with methanol, stained with a crystal violet solution, and counted under a microscope.After siRNA transfection for 24 h, 5–8 × 106 PCa cells per mouse were applied to construct the subcutaneous models in 36 mice. No blind method was applied in the research and random number method was applied to randomly divide all mice into the following six groups (n = 6 mice per group): (1) PC3 cell line + negative control siRNA, (2) PC3 cell line + KDM6B siRNA, (3) C42B cell line + negative control siRNA, (4) C42B cell line + KDM6B siRNA, (5) C42B cell line + dimethylsulfoxide (DMSO) (negative control), and (6) C42B cell line + GSK-J4 (50 mg/kg). For group 2 and group 4, the in vivo-jetPEI Delivery Kit  and the KDM6B siRNA were used to knock down the expression of KDM6B by an intratumor injection according to the manufacturer’s protocol. Intratumor injection of in vivo-jetPEI Delivery Kit and negative control siRNA was conducted in group 1 and group 3. GSK-J4 was administrated through intraperitoneal injection with the concentration of 50 mg/kg and DMSO was taken as the control. The siRNA and drug delivery were conducted every 3 days. Tumor volume was measured using a Vernier calliper every 3 days and the tumor volume was calculated with the formula: 0.5 × length × width2. Nasal anesthesia with isoflurane was introduced to alleviate pain to the mice throughout experimental studies. At the end of the animal experiments, subcutaneous tumors were collected from the killed mice for further analysis.All animal experiments were approved by the Laboratory Animal Ethics Committee of the Second Military Medical University with the Guidelines for Animal Health and Use  and performed inside a biosafety cabinet during the animal’s light time cycle on the fourth floor of the Experimental Animal Room at Navy Medical University. Four-week-old male Nu/nu athymic nude mice (RRID: MGI:5649767) were bought from Sippr-BK laboratory animal Co. Ltd  and raised under specific pathogen free (SPF) room conditions for feeding and observation. A total of 5 × 10KDM6B KD cell line was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNex® UltraTMRNA Library Prep Kit for Illumina® , following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. Clustering of the indexed samples was performed on a cBot Cluster Generation System using a HiSeq X PE Cluster Kit V2.5 (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated. We then selected Hisat2 as the mapping tool for which Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools. Differential expression analysis between C42B and C42B KDM6B KD  was performed using the DESeq2 R package (1.16.1). Genes with an adjusted P-value < 0.05 found by DESeq2 were assigned as differentially expressed. RNA-sequencing data were deposited into CNGB Sequence Archive (https://db.cngb.org/cnsa/) of CNGBdb with accession number CNP0001447.A total amount of 3 µg RNA of C42B or C42B The cells were collected 48 h after siRNA transfection or drug treatment. The samples for the cell cycle analysis were stained with propidium iodide and the samples for the apoptosis analysis were stained with Annexin V-fluorescein isothiocyanate and propidium iodide. Then, the samples were analyzed using the MACS Quant Analyzer 10 flow cytometer . The data were analyzed using FlowJo software .−ΔΔCT formula was then utilized to calculate the relative expression of the genes and β-actin was used as an internal control. The primers information is listed in the Supplemental Materials.The total RNA was extracted using the RNeasy Mini Kit  and reverse-transcription PCR (RT-PCR) was performed with the ReverTra Ace qPCR RT Kit  according to the manufacturer’s instructions. A real-time PCR assay was then conducted with SYBR green dye  on a StepOne Sequence Detection System . The 2The total protein content was isolated with an appropriate volume of a radio immunoprecipitation assay (RIPA) lysis buffer and protease inhibitor cocktail, which was then mixed with loading buffer and heated at 100 °C. Western blotting was performed according to the following classic procedures: electrophoresis on SDS-polyacrylamide gel electrophoresis, transfer onto polyvinylidene difluoride membranes , antibody incubation, and color development. The antibodies used in this study are as follows: anti-KDM6B , anti-β-actin , anti-cyclin D1 (CCND1) , and anti-smad2/3 .Immunohistochemistry was conducted to assess the protein expression of KDM6B , ARs , and CCND1 . The expression levels of KDM6B, ARs, and CCND1 were quantified using the Pannoramic Viewer and Quant Center image analysis software  to calculate the H-score. The H-score was calculated as follows: percent of weak staining  × 1 + percent of moderate staining  × 2 + percent of strong staining  × 3.0. Then, the H-score was applied to assist pathologists in categorizing the KDM6B expression into four levels: negative, low, moderate, and high.Chromatin immunoprecipitation (ChIP) assays were conducted by the Simple ChIP Plus Enzymatic Chromatin IP Kit  according to the manufacturer’s instructions. Chromatin was immunoprecipitated using anti-AR , anti-KDM6B , and anti-H3K27me3  antibodies. An anti-histone H3 antibody  and a normal rabbit IgG antibody  were used as the positive control and the negative control, respectively. The ChIP-derived DNA was quantified using quantitative RT-PCR and the related primers are listed in the Supplemental Materials.androgen receptor (AR) plasmid and its control plasmid. Then, these cells were cotransfected with pGiro dual-luciferase reporter and pGL4.10-KDM6B promoter (wild type (WT) or mutation (MUT)) with Lipofectamine 2000 (Invitrogen). Six times were repeated in each group. The luciferase activity was detected by Dual-Luciferase Reporter Assay System (Promega) after 48 h of transfection. Renilla luciferase activity was normalized against firefly luciferase activity.293T cells were first transfected with A protein lysate (1 mg) was incubated for 2 h with protein G-conjugated magnetic beads  to reduce nonspecific binding and was then centrifuged to remove the protein G-conjugated magnetic beads. The supernatant was incubated with the primary antibody overnight followed by a 2 incubation with the protein G-conjugated magnetic beads. And then the compounds of protein and magnetic beads were centrifuged and collected. The immunoprecipitated proteins were assessed by western blotting.The mass spectrometric analysis was conducted at AIMS Scientific Co., Ltd . First, the total protein was isolated from the PC3 cell line and KDM6B-targeted immunoprecipitation was conducted. Then, the peptide samples were analyzed with a Thermo Fisher LTQ Obitrap ETD mass spectrometer. The details of the mass spectrometry and bioinformatic analyses are presented in the Supplemental Materials.t-test or one-way ANOVA for data with normal distribution and homoscedasticity, and the Wilcoxon’s rank-sum test was used for other data. Spearman’s correlation analysis was utilized to explore the correlation between KDM6B expression and AR/CCND1 expression. The survival analysis was assessed by the log-rank test. p < 0.05 was considered statistically significant (two-sided).Statistical analysis was performed with SPSS 13.0 . Comparisons between the groups were calculated with a KDM6B mRNA was expressed in all samples of normal prostate tissue, PCa tissue, and metastatic PCa tissue. Further, GEO:GSE35988 dataset indicated that the KDM6B mRNA expression rates in normal control, PCa, and metastatic PCa were 25%, 57%, and 70%, respectively. The tissue microarray data showed that KDM6B protein expression rates in normal control and PCa were 87% and 93%, respectively. At the mRNA level, GEO:GSE6919, GEO:GSE35988, and GEO:GSE6752 datasets indicated that KDM6B expression was upregulated in PCa compared to the expression in the normal control, and the expression was even higher in metastatic PCa  efficiency was tested , we tested KDM6B mRNA and protein expression in hormone-sensitive LNCaP cells and castration-resistant C42B cells. Even though no significant difference of KDM6B mRNA expression was calculated between LNCaP and C42B cells, elevated KDM6B protein expression was observed in C42B cells  and MUT in the AR-binding sites at the 3′-untranslated region of Rluc. We found that AR reduced the luciferase activities of the WT reporter vector but not mutant reporter vector  had lower KDM6B protein expression levels than those in patients with low serum PSA (<20 ng/ml) , and the tissue microarray data indicated that there was a positive correlation trend between KDM6B and CCND1 expression, and the statistical significance was not calculated for the GEO:GSE46602 dataset (p = 0.05); this may be due to the limited sample size  is higher than that in the high PSA group (≥20), which causes us to wonder whether there is a negative correlation between ARs and KDM6B. Furthermore, a univariate and multivariate survival analysis showed that overexpressed KDM6B was correlated with the early recurrence of PCa, but no correlation was found between KDM6B expression and overall survival. KDM6B has the potential to independently predict the early recurrence of PCa.A previous study searched the Oncomine database and one set of data showed that the 30. On the other hand, it was reported that KDM6B-induced INK4a/P16 expression benefited the survival of cervical carcinoma19. Another study showed that a KDM6B insufficiency could promote the progression of pancreatic carcinoma by decreasing C/EBPα expression21. Moreover, a KDM6B deficiency with a subsequent H3K27me3 enrichment promoted the dedifferentiation of hypoxicV600E BRAF melanoma cells and then led to BRAF inhibitor resistance31. These observations implicate the role of KDM6B in the progression of malignant tumors depending on tumor cell types. In this study, we found that the siRNA-mediated depletion of KDM6B inhibited the proliferation, migration, and invasion abilities of the C42B and PC3 cell lines. After the depletion of KDM6B, a very large proportion of the C42B and PC3 cells was blocked at the G1 phase and more apoptosis was observed. In addition, the KD of KDM6B inhibited the growth of PCa in vivo. These evidences indicated that KDM6B prompted the development and progression of PCa.Previous studies have reported the oncogenic role of KDM6B in various malignant tumors32. Applying GSK-J4 to inhibit KDM6B could result in the growth inhibition of different cancer cell lines, including T-ALL and glioma29. In PCa, GSK-J4 could inhibit the proliferation of the C42B and PC3 cell lines, and the inhibition was demonstrated to be dose-dependent with 50% growth inhibition occurring at concentrations of 1.213 µM and 0.7166 µM for the PC3 and C42B cell lines, respectively. An analysis of the cell cycle distribution revealed that the G1 phase was the most increased cell cycle stage. In addition, GSK-J4 could also lead to greater levels of apoptosis in the C42B and PC3 cell lines, and could strongly inhibit PCa growth in vivo. Previous researches and our study, which illuminate the antitumoral effect of GSK-J4 by inhibiting KDM6B activity, indicate that a KDM6B-based target therapy is emerging as a promising cancer therapy for various tumors, including PCa.At present, GSK-J4 was reported to be the most useful tool for the inhibition of KDM6B demethylase activityNext, we focused on whether KDM6B promotes the development of CRPC. Compared to hormone-sensitive LNCaP cells, elevated KDM6B protein expression was found in castration-resistant C42B cells. In addition, we observed gradual increase of KDM6B expression from benign prostate tissue to PCa to CRPC. Of note, the KDM6B expression rise from PCa to CRPC was much greater than that from benign prostate tissue to PCa, which indicates that KDM6B might play a vital role in the development of CRPC. Further, MDV3100 administration failed to suppress the proliferation of LNCaP cells with KDM6B overexpression, whereas the combination of MDV3100 and GSK-J4 potently inhibited the growth of these cells. The above evidence indicates that KDM6B might involve in promoting PCa development after AR pathway was inhibited and the interaction between KDM6B and AR pathway need to be revealed. Compared to single MDV3100 or GAK-J4 treatment, the proliferation of castration-resistant C42B cells and MDV3100-resistant MDVR cell was significantly inhibited. It is reasonably believed that MDV3100 and GSK-J4 play a synergistic role in treating CRPC. Co-target AR and KDM6B could be a promising treatment for CRPC and MDV3100-resistant CRPC.34. As mentioned above, we found a negative association between KDM6B and PSA. Then, we speculated whether KDM6B expression could be induced or repressed by ARs. Therefore, we searched the GEO database and two sets of data (GSE46602 and GSE62872), and our Dingtianlidi sequencing data showed a negative correlation between KDM6B and ARs at the mRNA level. Although no statistically significant correlation between KDM6B protein expression and AR protein expression was calculated from the tissue microarray, a negative trend was observed. In reality, the H-score, applied to assess the immunohistochemistry results of the study, cannot provide enough precision to assess the protein expression levels. This may be the reason why no statistical significance was calculated when we observed a negative correlation between KDM6B and ARs at the protein level. To verify this finding, DHT and MDV3100 were applied to treat LNCaP cells. DHT decreased the expression of KDM6B mRNA and protein, whereas KDM6B mRNA and protein expression was increased by MDV3100 treatment. Further, a ChIP analysis revealed that DHT treatment increased the direct binding of ARs to the KDM6B promoter, and that MDV3100 reduced the direct binding of ARs to the KDM6B promoter. Dual-luciferase reporter assay also verified that ARs could decrease KDM6B transcription by directly binding its promotor. These evidence indicates that ARs play an inhibitory role in the transcription of KDM6B36. As AR activity loss is a common phenomenon in CRPC36, this relieves the repression of ARs on KDM6B transcription. Then, the accumulation of KDM6B facilitates the transcription of downstream oncogenic molecules in PCa; in turn, this prompts the conversion of PCa to CRPC.KDM6B is an inducible histone demethylase and its expression was shown to be mediated by NF-κB and signal transducer and activator of transcription 3 in previous reportsKDM6B has great effects on the biological function of PCa, but the downstream mechanism is still unknown. To solve this issue, RNA sequencing was applied to compare the differences in gene expression between the C42B WT sample and the C42B KDM6B depletion sample. Compared to the C42B WT sample, a series of genes was upregulated or downregulated in the C42B KDM6B depletion sample, such as the upregulation of CCND1, PTCH1, SH2B3, and FGFR1, and the downregulation of MET, TFRC, and NT5C2. As CCND1 was reported to have the functions of cell growth regulation, cell migration control, DNA repair, and mitochondrial activity modulation38, we chose CCND1 as the focus of our downstream research. Two sets of GEO data, our Dingtianlidi sequencing data, and the tissue microarray data verified the positive correlation between KDM6B and CCND1. Furthermore, the CCND1 mRNA and protein expression level reduction was observed after the depletion and inhibition of KDM6B. ChIP-PCR demonstrated that the depletion and inhibition of KDM6B could increase histone H3K27me3 on the CCND1 promoter. Additionally, an immunoprecipitation-based mass spectrometry analysis revealed an interaction between KDM6B and smad2/3. We also observed that the amount of the interaction between smad2/3 and KDM6B was reduced after the depletion and inhibition of KDM6B. Further, ChIP-PCR analysis showed that the depletion and inhibition of KDM6B reduced the binding of KDM6B and smad2/3 to the CCND1 promoter. Together, these data reveal that KDM6B binds to the promoter regions of the CCND1 gene, lessens H3K27me3 occupancy, and increases CCND1 expression; in turn, this causes the progression of PCa.The study revealed that KDM6B in PCa. KDM6B could serve as a predictor for the early recurrence of PCa. In addition, GSK-J4, an inhibitor of KDM6B, could suppress the vitality and progression of PCa and cooperate with MDV3100 to suppress CRPC and MDV3100-resistant CRPC, it can serve as a promising therapeutic for the treatment of PCa. Furthermore, our study reveals the upstream and downstream mechanisms of KDM6B in PCa. We first report that ARs decrease the transcription of KDM6B and we report that KDM6B demethylates H3K27me3 on the promoter of CCND1 and cooperates with smad2/3 to prompt the expression of CCND1.In summary, we systemically used clinical data, in vitro cellular biological studies, and in vivo mouse models to reveal the oncogenic role of Supplemental Figure LegendsSupplemental Fig.1Supplemental Fig.2.tifSupplemental Fig.3Supplemental Fig.4Supplemental TablessiRNA and Primers used in the studymass spectrometric analysisThe details of RNA sequencing and data processingDual-luciferase reporter assay reportC4-2B authenticationLncap authenticationPC-3 authentication


The kidney is innervated by afferent sensory and efferent sympathetic nerve fibers. Norepinephrine (NE) is the primary neurotransmitter for post-ganglionic sympathetic adrenergic nerves, and its signaling, regulated through adrenergic receptors (AR), modulates renal function and pathophysiology under disease conditions. Renal sympathetic overactivity and increased NE level are commonly seen in chronic kidney disease (CKD) and are critical factors in the progression of renal disease. Blockade of sympathetic nerve-derived signaling by renal denervation or AR blockade in clinical and experimental studies demonstrates that renal nerves and its downstream signaling contribute to progression of acute kidney injury (AKI) to CKD and fibrogenesis. This review summarizes our current knowledge of the role of renal sympathetic nerve and adrenergic receptors in AKI, AKI to CKD transition and CKDand provides new insights into the therapeutic potential of intervening in its signaling pathways. Acute kidney injury (AKI) is associated with increased mortality and risk of development of chronic kidney disease (CKD) in the long term ,2. CKD i1-, α2-, or β-AR [The kidneys are abundantly innervated by both renal afferent sensory and efferent sympathetic nerves and communicate with the central nervous system via the sensory afferent nerves ,7. Incre or β-AR . The con or β-AR ,13,14. I or β-AR . Further or β-AR ,17,18. T or β-AR ,20. DespIncreased renal sympathetic nerve activity leads to increased renin secretion, increased renal tubular sodium reabsorption, and decreased renal blood flow ,10,21. R2-ARs, promotes renal inflammation and interstitial fibrosis [Norepinephrine (NE) is an organic chemical in the catecholamine family that functions in the body as the main neurotransmitter of sympathetic nerve system . NE relefibrosis ,31.1-AR is expressed in arterioles, whereas α2-AR is expressed predominantly in proximal tubules [1-AR and α2-AR are responsible for stimulation of renal vasoconstriction and Na+ reabsorption, respectively [1-AR, β2-AR, and β3-AR [1- and β2-ARs are well known for regulating renal blood flow, glomerular filtration rate (GFR), sodium and water reabsorption, acid-base balance, and secretion of renin in the kidney [3-AR is important for regulating renal water and solute reabsorption [ARs, which are G protein coupled receptors, are well known to participate in various physiological renal functions ,37,38,39 tubules . α1-AR aectively . β-ARs cnd β3-AR  and are nd β3-AR . The rolsorption . The rensorption ,37,39,46Acute kidney injury (AKI) is defined as an abrupt or rapid decline in renal filtration function and is the result of combined acting of multiple factors ,48. PrevRenal sympathetic nervous system and circulating catecholamines including NE are considered to be involved in the development of the progressive renal tissue injury accompanying AKI ,55. In a1-AR antagonist, clonidine, an α2A-AR agonist, and atenolol, a β1-AR antagonist, lowered BP and heart rate in obese dog and human patients [2A-AR. Genetic inhibition of α2A-AR or pharmacological inhibition of α2-AR lowers Ang II-mediated NE release in kidneys with 5/6 nephrectomy [Renal sympathetic nervous system contributes to pathogenesis of not only ischemic AKI, but also of CKD and hypertensive disease ,70,71. Ipatients ,82,83. Ipatients . There ihrectomy .2-AR agonist, and propranolol, β-AR antagonist, are known to have renoprotective actions against post-ischemic AKI [2-ARs prevents interstitial fibrogenesis after IRI, as indicated by reduced TGF-β1 production, Smad3 phosphorylation, α-SMA expression, and collagen deposition [1-AR or β-AR protects the kidney against 5/6 nephrectomy-induced injury, and a combinational inhibition of α1-AR and β-AR is more effective in preventing renal injury than the inhibition of α1-AR or β-AR alone [2-AR blockers and JP-1302, an α2C-AR specific blocker, mitigate I/R-induced renal tubular damage [2-AR inhibition with yohimbine and JP-1302 improves renal blood flow, suppresses increased plasma NE level, and ameliorates cisplatin-induced renal dysfunction and histological damages, by suppressing pro-inflammatory cytokine expression such as TNF- α and MCP-1 [Several studies demonstrated beneficial effects of sympathetic nerve system inhibition on renal function and morphology. Clonidine and moxonidine, αemic AKI ,87,88. Wposition . Other rAR alone . In uninr damage ,90. In tnd MCP-1 . RDNx alnd MCP-1 ,92.In addition, ischemic acute renal failure is ameliorated by RDNx or ganglionic blockade, which blocks the sympathetic nerves, and that effect is accompanied by suppression of elevated renal venous NE level immediately after reperfusion and attenuation of decreased GFR after the I/R . Most stExperimental studies demonstrated that renal denervation slowed or delayed the rise in BP in experimental models of hypertension such as spontaneous hypertension, salt-sensitive hypertension, Nω-nitro-L-arginine methyl ester (L-NAME) induced hypertension, Ang II induced hypertension and deoxycorticosterone acetate (DOCA)-salt hypertension ,104,105.2-AR agonist, exerts notable positive effects in inhibiting sympathetic nerves and plasma NE levels and preventing kidney damage [2-AR and down-regulating TLR4 expression in I/R-induced [2AR agonists have an anti-inflammatory action that targets macrophage activation in diabetic rats, especially in the kidney [2-AR using gene transfer with human β2-AR was effective in preventing endotoxin-induced renal injury with reduced elevation of TNF-α mRNA and leukocyte infiltration into the rat kidney [On the other hand, activation of AR using agonist has protective effect in the kidney. It has been confirmed that dexmedetomidine, a selective αy damage ,109,110.-induced . Dexmedee kidney . Overexpt kidney .2-AR enhances the progression of dysfunction and inflammatory responses in the kidney [2-AR is responsible for upregulation of inflammatory cytokines including TNF-α and IL-6 and enhancement of granulocyte and monocyte recruitment [2-AR agonist UK-14304, significantly elevates LPS-induced TNF production from macrophages via α2-AR located on macrophages, while β-adrenergic agonist, isoproterenol, inhibits TNF release from macrophages [2C-AR with α2c-AR antagonist JP-1302, after I/R, strongly suppressed cytokines, TNF-α, IL-6, IL-1β, and MCP-1 expression [2-AR antagonists has no effect on NE level but decreases cytokine and chemokine expression after UUO, suggesting that norepinephrine may promote leukocyte recruitment and inflammation [The mechanisms underlying AKI to CKD progression involves multiple interactions between injured tubules, immune cells, endothelial cells, and fibroblasts . The impe kidney . Activatruitment . NE or trophages ,120,121.pression . These ipression ,31. In apression , decreaspression . NE has pression ,125. We 2-ARantagonist [2-AR agonist, B-TH933 to the denervated kidney restores both glomerular and tubular responsiveness to NO inhibition [1-AR blockers, prazosin, or doxazosin, and attenuation of L-NAME-induced renal injury [Another potential mechanism for sympathetic nerve system activation in the AKI and CKD model is reduced nitric oxide (NO) availability . In the tagonist ,131. Admhibition . Decreasl injury ,133,134.l injury ,136,137.Kidney tubular cell cycle arrest induced by severe AKI contributes to renal fibrogenesis by releasing profibrotic cytokines such as TGF-β1 and connective tissue growth factor (CTGF) . While c2-AR with dexmedetomidine, α2-AR agonist attenuates renal cell apoptosis and renal tubule impairment by inhibiting mitochondrial apoptosis pathways in the sepsis- or I/R-induced injury model, which may be effective by regulating NE release [The pathophysiology of AKI is generally characterized by a common cascade of cell death-induced inflammation and fibrosis following injury . Inhibit release ,148,149.2-ARs play essential roles in the prevention of heart failure [2A-AR and α2C-AR subtypes increased the susceptibility to develop heart failure following chronic pressure overload in vivo [2A-AR using α2A-AR agonists, clonidine and moxonidine though sympatho-inhibition effect protect the kidney against renal IRI [2-AR accelerates the progression of renal fibrogenesis after renal IRI and UUO, suggesting the activation of α2-AR in this model may trigger inflammatory and fibrotic signaling pathways than sympatho-inhibition to induce fibrogenesis [2-ARs results in increased norepinephrine levels, which elicit typical cardiovascular consequences, including aggressive remodeling of the left ventricle, cardiac hypertrophy, and fibrosis [Heart failure is a disorder in which the heart is unable to pump an adequate supply of blood . SNS ove failure . α2-AR s failure . Deletio in vivo ,162. In enal IRI ,163. Conogenesis ,31. The fibrosis ,164. In fibrosis ,31.1-AR agonist, midodrine administration is associated with a significant improvement in renal function in HRS patients [2-AR agonist, clonidine, reduces portal pressure, arterial BP, and circulating NE in patients with cirrhosis [Hepatorenal syndrome (HRS) is currently defined as the occurrence of AKI, in patients with advanced chronic liver disease ,166. Sinpatients . Other αirrhosis . On the irrhosis ,174. β-birrhosis . For thiirrhosis . As mentirrhosis . RAS actirrhosis ,177. ThuThe role of the sympathetic nervous system in AKI and CKD are discussed through a literature review investigating sympathetic nervous mechanisms ,39,58,96


The field of augmented reality (AR) is rapidly growing with many new potential applications in medical education. This systematic review investigated the current state of augmented reality applications (ARAs) and developed an analytical model to guide future research in assessing ARAs as teaching tools in medical education.A literature search was conducted using PubMed, Embase, Web of Science, Cochrane Library, and Google Scholar. This review followed PRISMA guidelines and included publications from January 1, 2000 to June 18, 2018. Inclusion criteria were experimental studies evaluating ARAs implemented in healthcare education published in English. Our review evaluated study quality and determined whether studies assessed ARA validity using criteria established by the GRADE Working Group and Gallagher et al., respectively. These findings were used to formulate an analytical model to assess the readiness of ARAs for implementation in medical education.We identified 100,807 articles in the initial literature search; 36 met inclusion criteria for final review and were categorized into three categories: Surgery (23), Anatomy (9), and Other (4). The overall quality of the studies was poor and no ARA was tested for all five stages of validity. Our analytical model evaluates the importance of research quality, application content, outcomes, and feasibility of an ARA to gauge its readiness for implementation.While AR technology is growing at a rapid rate, the current quality and breadth of AR research in medical training is insufficient to recommend the adoption into educational curricula. We hope our analytical model will help standardize AR assessment methods and define the role of AR technology in medical education. Virtual reality (VR) technology creates entirely artificial environments through headsets that isolate users from their surroundings. In comparison, augmented reality (AR) overlays digital interfaces upon physical surroundings, producing an environment that is both real and digital.4,5 recent advances in visual technology and the development of new augmented reality applications (ARAs) have drawn consumer and professional attention.6 These applications are software and/or hardware developed explicitly with AR functionality in mind, and have already been applied in many educational settings including environmental sciences, chemistry, humanities, and the arts.7,8 Recent studies have shown that there is a growing number of ARAs in medicine and that AR may foreshadow a new paradigm in medical education.8,9 To date, ARAs have been adapted to every stage of medical training as anatomical teaching tools,10 classroom study aids,11 image training simulators,12 and clinical skills interaction simulators.13While the technology and concept of augmented reality have existed for several decades,9 or applications in general education.8,14 The purposes of this study wereto conduct a systematic review of the role of AR in medical education, including evaluating the quality of studies and the prevalence of formal validity assessments,15,16 and to develop an analytical model to assess the feasibility of ARA implementation into medical educational curricula.This study comprehensively described the use of different ARAs in medical education. Prior systematic reviews have not assessed the quality of recent AR research in medical education and have focused primarily on the integration of surgical ARAs in medical training17We conducted a systematic literature search using PubMed, Cochrane Library, Embase, Web of Science, and Google Scholar from January 1, 2000 through June 18, 2018. The Boolean search terms used were “augmented reality” AND . A university librarian assisted with keyword and database selection to ensure broad coverage that would encompass all existing relevant literature. Search results were recorded per the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.Included articles a) described ARAs in the context of medicine and medical education, b) carried out experimental studies evaluating specific ARAs, c) were obtained from peer-reviewed journals after the year 2000, and d) were written in English. Excluded articles a) discussed VR or similar technologies but not AR, b) were focused on the technological basis for AR or c) discussed AR outside of medicine. Two independent reviewers  conducted the literature search and gathered data, and a third reviewer (E.M.) resolved any conflicts.Reviewed articles were divided into three categories. “Surgical” applications were designed to train medical novices in procedural tasks such as basic laparoscopic skills, suturing, ventriculostomy, and echocardiography. “Anatomy” applications were designed to assist students with learning human anatomy. “Other” applications were developed for general healthcare education, including clinical skills, forensic medicine, dermatology, and pathology.15Studies were assessed for quality using criteria based on the Grades of Recommendation, Assessment, Development, and Evaluation (GRADE) Working Group scoring protocol. Quality analysis was based on metrics including inconsistency in outcomes between different studies, directness of evidence, possibility of bias, confounders, strength of association, dose response, and data quantity.16,18,19 This evaluation was informed by Gallagher et al.’s five forms of validity: face, content, construct, concurrent, and predictive validity  in medical education as well as the quality criteria described by the GRADE Working Group.We identified 100,807 papers in the initial search. Title screening and removal of duplicates left 439 papers that were evaluated based on abstract. Second-level exclusion removed 347 papers, leaving 93 full-text papers that were reviewed in their entirety.Thirty-six articles met proposed inclusion criteria. These papers were divided into three categories— 23 in Surgical, nine in Anatomy, and four in Other. Twenty-two total ARAs were described: 15 in Surgical, five in Anatomy, and two in Other. Of the 36 included articles, 26 (72%) were published in the last five years and eight (22%) were published between 5-10 years ago. A PRISMA flowchart detailing this literature search is displayed in 23-25 Points were primarily lost for study design, lack of data, and outcome inconsistency - seven (19%) of 36 articles were RCTs and twelve (33%) had sample sizes less than 50. Of the seven RCTs, six were given a Low or Moderate rating due to small sample sizes and inconsistent results. Only the ProMIS simulator, ImmersiveTouch, Microsoft Kinect ARMM test system, AR MagicBook, EyeSI, and mARble were evaluated by more than one study. Many ARAs have only been the subject of a single study  DIANA) and thus remain largely untested.Evaluation of study quality is delineated in Validity assessments were not performed for 11 of the included ARAs (50%) and no application achieved all five stages of validity.In the following sections, we describe in more detail the ARAs that have been evaluated by two or more studies. Surgical applications: ProMIS AR Laparoscopic Simulator Of 36 studies, seven involved use of the ProMIS simulator. Composed of a torso-shaped mannequin connected to a computer, this device trains students in laparoscopic procedures and combines the benefits of haptic feedback with the ability to view simulation feedback videos. Three cameras within the mannequin identify inserted instruments from different angles. Substitution of the peritoneal cavity with plastic trays allows the simulator to be used for multiple tasks.26 suturing,27-30 and other basic laparoscopic tasks.30-32 Overall, the ProMIS trainer was an effective educational tool. It was described as highly realistic and improved task-effectiveness across all studies.27 Studies that measured the difference in skill between novice and experienced participants found a significant correlation between high performance metrics and experience, indicating that the ProMIS simulator is reliable for evaluation of laparoscopic skills.26,28,30,32 It is important to note that the majority of these studies were pilot studies with low numbers of participants (n=7-28) with the exception of one (n=115).31 Additionally, none of the studies were randomized, only one was controlled,26 and most depended on subjective means such as Likert-scale surveys to determine performance.The ProMIS AR simulator was used to train users on sigmoid colectomies,ImmersiveTouch System Another AR training simulator that provides haptic feedback is the ImmersiveTouch system. ImmersiveTouch involves the integration of a head-hand tracking system with a stereoscopic display and is typically used for neurosurgical training.33 and the other for ventriculostomies.34 Use of the ARA slightly lowered failure rate in screw placement and demonstrated a statistically significant improvement of correct catheter placement for ventriculostomies. However, these experiments had small sample sizes of 51 and 16 participants, respectively.Two randomized controlled trials (RCTs) evaluated the ImmersiveTouch system -- one for thoracic screw placementEyeSI AR Binocular Indirect Ophthalmoscopy (BIO) Simulator The EyeSI AR simulator displays virtual retinae on a model head through a lens inspired by traditional BIO lenses. The user physically adjusts the lens to look in different directions while their movements are recorded on a separate monitor.35 The AR group significantly outperformed the control group in both raw score and mean performance and was able to complete the procedure in less time. Leitritz et al. (n=37) randomized 4th year medical students with no prior experience with BIO into control and AR groups using the EyeSI simulator.36 All students performed the procedure the day after training and were assessed through their drawings of the patient’s optic disk and arteries/veins. The AR group sketched more vessels correctly and achieved a higher Ophthalmoscopy Training Score.Two RCTs compared traditional BIO lenses to the EyeSI AR simulator. Rai et al. (n=28) randomized first-year ophthalmology residents to traditional and EyeSI training methods and evaluated their performance in three tasks.Anatomyapplications:AR Magic Book (various)Several studies utilized a system called “MagicBook.”37,38 A number of specific ARAs fit into this category , that it increased motivation and interest (75%), and that their grades would improve if professors utilized the technology (67.3%). Another RCT conducted by Kucuk et al.25 demonstrated similar results: medical students utilizing the “MagicBook” ARA scored significantly higher on an academic test with lower cognitive load compared to control and 100% of respondents reported that AR either greatly or partially facilitated learning.Two large RCTs conducted by Ferrer-Torregrosa et al.Microsoft Kinect The Microsoft Kinect was often used as part of an “AR Magic Mirror” (ARMM) approach. The Kinect contains a high-resolution camera for video reproduction and a low-resolution camera for depth perception, allowing the device to accurately track the user’s body movements. The system is often used for interactive video games but can be adapted to allow overlay of tracked virtual information onto a user’s body.39-41 Responses from all three were positive. Varying majorities of respondents reported that ARMM increased learning motivation (58%), was beneficial in an educational setting (69.1%), stimulated active learning (82.4%), and improved 3-Dimensional understanding of anatomy (93.4%) while remaining easy to use.39,40 A large majority (80.5%) rated the system as excellent or good, and surveyed physicians unanimously recommended that ARMM be used to supplement existing anatomy curriculums.41There were three papers exploring the ARMM application; all were surveys directed at medical students and clinicians.Otherapplications:Mobile AR Blended Learning Environment (mARble) 42,43 and one was a survey.44 All three had small sample sizes, with two recruiting ten or less participants.42,44 Students described the application as pragmatic and enjoyable to use, but the two RCTs yielded conflicting results. Albercht et al. concluded that mARble increased knowledge retention with lower cognitive fatigue when compared with traditional textbook material,42 but Noll et al. found no difference in knowledge gain between mARble and control groups immediately after training, although the AR group retained more knowledge in a follow-up assessment 14 days after training.43The mARble is an application developed for the Apple mobile operating system that stores content separately from the program’s code; this allows for the addition of modules to adapt the application for different purposes without changing its source code. Three studies evaluated the mARble application; two were RCTsquality, application content, outcome, and feasibility , and AR telementoring systems . This diversity reflects the well-documented use of simulation as a surgical training tool.9,47,48 Surgical ARAs were more consistently tested for validity than applications in the other two categories, likely due to the surgical origins of modern validation techniques.16 Several articles aimed to demonstrate specific stages of validity. However, we contend that these tests of validity should be modified and adapted to all uses of AR in medical education. The development of AR hardware by leading technology corporations such as Google, Microsoft, Brother, and Epson also indicate the potential integration of consumer products into medical settings. While recent technological advances have made AR simulation more viable for surgical training, further developments will need to broaden in scope to focus on more than technical skill.9,49 A holistic approach to training effective surgeons will require the integration of knowledge and attitude education50,51 as well as development of standardized assessments of simulation training in the operating room.52,53The state and quality of research varied widely between surgical, anatomical, and other ARAs. Surgical ARAs included a variety of laparoscopic simulators , AR glasses  may improve student outcomes.Studies in the Other category did not offer compelling evidence for AR implementation. There was a lack of consistently positive outcomes and high-quality studies for both mARble22,56,57 but have not proposed a model to evaluate the readiness of educational AR tools for curricular implementation. The four categories introduced in our analytical model encompass the largest factors determining an ARA’s success in the medical classroom or operating room. Although the quality and validity metrics used in our systematic review only covered the criteria in three of four categories , future studies should address all four categories (including feasibility) in order to thoroughly consider the key barriers to AR implementation. While we incorporated many aspects of Gallagher et al.’s validity framework into the application content module, we refrained from specifying which framework to use in validating ARAs as we believe the criteria should be adjusted and distinctively prioritized to reflect each application’s unique educational goals. We hope this model will encourage future studies to incorporate both higher quality study designs and formal validity assessments.The breadth of projects identified in this review highlights both the adaptability of AR technology and the lack of standardized assessment tools. Our analytical model  sought t58 However, bias was minimized by using several independent reviewers and consulting with a science librarian. Given the rapid growth of AR technology in recent years, it is also probable that research involving certain cutting-edge applications have not yet been published or are under patent/copyright restrictions, precluding their inclusion in this review. Finally, many criteria put forth in this paper regarding study quality and training potential are inherently subjective and may not be broadly applicable to every program or student population.Our study has several limitations. An inevitable flaw in systematic reviews is the possibility of reporting bias due to search criteria .The use of AR technology in medical education is in its early stages presently lacks evidence-based support for its widespread implementation. Future research should adopt long-term and large-scale RCT or cohort study designs in keeping with the proposed model to evaluate ARA efficacy. Rigorous and standardized validation of commercially viable applications will allow the technology to be more readily integrated into medical educational curricula.


Many studies have shown an association of childhood respiratory diseases with short-term temperature variability such as diurnal temperature range (DTR) and temperature change between two neighboring days (TCN). However, the impact of temperature variability on allergic rhinitis (AR) has not been investigated so far. This study sought to evaluate the short-term effect of temperature variability  on AR, as well as to identify vulnerable subpopulations.We collected daily data on emergency room visits and outpatients for AR and weather variables in Hefei, China during 2014–2016. A distributed lag non-linear model that controlled for long-term trend and seasonality, mean temperature, relative humidity, day of week was used to fit the associations of AR with DTR and TCN. Stratified analyses by age, sex and occupation were also performed.During the study period, there were a total of 53,538 cases and the average values of DTR and TCN were 8.4 °C (range: 1.0 °C to 21.2 °C) and 0 °C (range: − 12.2 °C to 5.9 °C), respectively. While we did not observe an adverse effect of DTR on AR, TCN was significantly associated with increased risk of AR. Specifically, a large temperature drop between two adjacent days  has a delayed and short-lasting effect on AR, with the estimated relative risk of 1.02  at lag 12. Moreover, boys and children older than 15 years seemed to be more vulnerable to the effect of TCN.This study provided evidence of an adverse effect of large temperature drops between two adjacent days on childhood AR. Attention paid to boys and older children may help prevent AR attacks. Allergic rhinitis (AR) is an inflammatory disease of the nasal mucosa, affecting more than 600 million people worldwide, especially in children , 2. In rEpidemiological studies have revealed that the prevalence of childhood AR has a general upward trend in the past 7 years, ranging from 2.2 to 14.6% among children at the age of 6–7 years, and from 4.5 to 45.5% among adolescents aged 13–14 years all over the world . The sevClimate change has been widely recognized as the biggest health threat in the twenty-first century, and temperature play an important role in affecting human health, including cardiovascular and respiratory diseases . In receTo verify our hypothesis, we collected time-series data of daily childhood AR patients and daily weather records to investigate the relationship between childhood AR occurrence and temperature variability including DTR and TCN. Stratified analyses by age, sex and occupation were also conducted to identify potential vulnerable subgroups.2 . It has a typical temperate climate, with the annual average temperature and precipitation being 15.7 °C and 3.0 mm, respectively.Hefei is the capital city of Anhui province and is located in the east central of China . It has a population density of 688 persons per kmDaily AR outpatients and emergency room visits from January 1st, 2014 to December 31st, 2016 were obtained from Anhui Provincial Children’s Hospital and the diagnosis of AR patients was based on the International Classification of Diseases 10th version . Anhui Provincial Children’s Hospital is the biggest and only specialist children’s hospital in Anhui province, which is located at the center of the Hefei city, with advanced facilities, more than 1200 beds and over 1700 staff. This hospital has a well-managed electronic medical record system, which can provide daily hospitalization data including name, sex, age, address, principal diagnosis and treatment date.In addition, we also obtained data for the same period on daily maximum temperature, mean temperature, minimum temperature, relative humidity, rainfall and wind velocity from Hefei Bureau of Meteorology. In line with previous studies , 16, DTRA Poisson generalized linear regression model combined with a distributed lag non-linear model (DLNM) was used to examine the relationship between TCN (or DTR) and AR occurrence. DLNM has been widely applied in studies to quantify the effects of temperature and air pollution on mortality.df). In all cases, the Akaike Information Criterion (AIC) and an analysis of residuals were used to evaluate the choice of df.We controlled for seasonality and long-term trend using a natural cubic spline with 7 df per year for time. The day of week was also included as an indicator in our analysis. Mean temperature and relative humidity were controlled in the model by using a natural cubic spline with three degrees of freedom  of up to 20 days when analyzing the influence of DTR and TCN on children AR occurrence. We also examined TCN effect on childhood AR at lag 5. To examine which groups are most likely to be affected by TCN, we stratified the regression analyses by sex , age group  and occupation .df for time, mean temperature, relative humidity, respectively.All statistical analyses were conducted using the R statistical software (version 2.15) with the ‘dlnm’ package used to fit the regression model. Sensitivity analyses were performed by varying the During the study period, there were a total of 53,538 AR cases. Table Figure Figure Table Figure Figure df (6–8 per year) for time to control for long-term trend and seasonality. We also varied the df (3–5) for temperature, humidity. We found that the results changed a little (results not shown).Sensitivity analyses were conducted by changing the It has been well documented that temperature variability within a short period  affects human health among children –17. We cThe association between temperature and AR was examined in several recent studies, consistently showing an adverse effect of cold weather on AR in many countries such as Korea, China and Finland –14. HoweAlthough DTR and TCN have been widely used in previous studies, litter is known about the relative effects of these two temperature variability indictors. To our knowledge, only one study has evaluated the health risk of DTR and TCN among children . This stWe also observed that TCN had the largest effect 1 week later, namely at lag 12  were more vulnerable to TCN. This result contradicts with our commonsense that, compared with old children, younger children are more vulnerable to temperature effects, because younger children are relatively under-developed and have less self-care ability . TherefoThis study has some strengths. To be best of our knowledge, this is the first study to date investigating the association of AR with short-term temperature variability. Our findings thus add new evidence to the existing literature of temperature and AR. In the preset study, temperature variability was measured by DTR and TCN; these two temperature indictors were often employed by previous researchers to evaluate the effect of unstable weather on human health , 19. OurIn total, this study provides evidence that exposure to a large temperature drop between two adjacent days is associated with increased risk of AR in children. Vulnerable subgroups who are at higher risk of AR are boys and children older than 15 years. Temperature variability should be taken into account in future research assessing the deleterious effect of temperature on AR among children.