Penerapan Metode Eksperimen untuk Meningkatkan Konsep Dasar Sains pada Anak Didik Kelompok A Tk Pkk Suruhwadang Kecamatan Kademangan Kabupaten Blitar

Tujuan penelitian ini adalah untuk memperoleh tentang kemampuan kognitif anak dalamhal konsep dasar sains dengan menggunakan metode eksperimen pada anak didik kelompokA TK PKK Suruhwadang sebelum dan sesudah dilakukan tindakan. Melakukan tindakanberupa penerapan metode eksperimen untuk meningkatkan kemampuan kognitif dalamkonsep dasar sains pada anak didik kelompok A TK PKK Suruhwadang. Mengetahui adatidaknya perbedaan kemampuan konsep dasar sains dengan menggunakan metodeeksperimen pada anak didik kelompok A TK PKK Suruhwadang antara sebelum dan setelahdilakukan tindakan. Rumusan masalah pada penitilian ini adalah apakah metode eksperimendapat meningkatkan kemampuan pemahaman konsep dasar sains pada anak didik kelompokA TK PKK Suruhwadang Kecamatan Kademangan Kabupaten Blitar. Untuk menjawabrumusan masalah digunakan jenis penelitian tindakan kelas (PTK) dengan model Kemmisdan Taggart melalui empat tahapan yaitu tahap perencanaan , pelaksanaan, observasi danrefleksiyang dilalui dengan dua siklus. Teknik pengumpulan data menggunakan teknikobservasi dan dokumentasi. Adapun instrumen yang digunakan adalah lembar observasikegiatan anak dan lembar observasi pembelajaran oleh guru.Hasil penelitian menunjukanbahwa kemampuan kognitif anak kelompok A pada konsep dasar sain pada pra penelitianmenunjukkan prosentase 56.25%. Setelah pelaksanaan siklus I tentang bidang kemampuankognitif pada konsep dasar sains menunjukkan 59% mengalami peningkatan .Setelahpelaksanaan siklus ke II naik menjadi 83%. Hal ini menunjukkan pelaksanaan siklus ke IItelah mencapai kriteria ketuntasan dan membuktikan bahwa dengan metode eksperimendapat meningkatkan kemampuan kognitif dalam konsep dasar sains

Defective Resensitization in Human Airway Smooth Muscle Cells Evokes β-Adrenergic Receptor Dysfunction in Severe Asthma

<div><p>β₂-adrenergic receptor (β₂AR) agonists (β₂-agonist) are the most commonly used therapy for acute relief in asthma, but chronic use of these bronchodilators paradoxically exacerbates airway hyper-responsiveness. Activation of βARs by β-agonist leads to desensitization (inactivation) by phosphorylation through G-protein coupled receptor kinases (GRKs) which mediate β-arrestin binding and βAR internalization. Resensitization occurs by dephosphorylation of the endosomal βARs which recycle back to the plasma membrane as agonist-ready receptors. To determine whether the loss in β-agonist response in asthma is due to altered βAR desensitization and/or resensitization, we used primary human airway smooth muscle cells (HASMCs) isolated from the lungs of non-asthmatic and fatal-asthmatic subjects. Asthmatic HASMCs have diminished adenylyl cyclase activity and cAMP response to β-agonist as compared to non-asthmatic HASMCs. Confocal microscopy showed significant accumulation of phosphorylated β₂ARs in asthmatic HASMCs. Systematic analysis of desensitization components including GRKs and β-arrestin showed no appreciable differences between asthmatic and non-asthmatic HASMCs. However, asthmatic HASMC showed significant increase in PI3Kγ activity and was associated with reduction in PP2A activity. Since reduction in PP2A activity could alter receptor resensitization, endosomal fractions were isolated to assess the agonist ready β₂ARs as a measure of resensitization. Despite significant accumulation of β₂ARs in the endosomes of asthmatic HASMCs, endosomal β₂ARs cannot robustly activate adenylyl cyclase. Furthermore, endosomes from asthmatic HASMCs are associated with significant increase in PI3Kγ and reduced PP2A activity that inhibits β₂AR resensitization. Our study shows that resensitization, a process considered to be a homeostasis maintaining passive process is inhibited in asthmatic HASMCs contributing to β₂AR dysfunction which may underlie asthma pathophysiology and loss in asthma control.</p></div

βAR function in primary human airway smooth muscle cells (HASMCs) from lungs of non-asthma (Non- asthmatic ASM) and asthma (Asthmatic ASM) patients.

<p><b>a</b>, Non- asthmatic ASM and asthmatic ASM cells were stimulated with β-agonist albuterol (0, 5, 10 & 20 minutes (min)). The cell were lysed and assessed for the ability to generate cAMP. *p<0.005 vs. non-asthmatic ASM or asthmatic ASM 0 min (untreated), #p<0.05 vs. asthmatic ASM 5, 10, 20 min, (n = 6 per group, 6 non-asthmatic ASM and 6 asthmatic ASM). <b>b</b>, Plasma membranes were isolated from non-asthmatic ASM and asthmatic ASM cells following pre-treatment of cells with albuterol for 0, 5, 10, and 20 min. The cell-free membranes were stimulated with albuterol to measure adenylyl cyclase activity by providing radioactive ^32[P]γ-ATP and measuring cAMP generation. *p<0.01 vs. respective <i>in vitro</i> vehicle stimulation, #p<0.05 vs. ASM <i>in vitro</i> albuterol stimulated 0 and 5 min, (n = 6/group). V, Vehicle; Alb, Albuterol (β-agonist).</p

Assessment of βAR desensitization in non-asthmatic ASM and asthmatic ASM cells.

<p><b>a</b>, To determine whether βAR are differentially phosphorylated in non-asthmatic ASM and asthmatic ASM cells, the cells were plated on cover slips and β₂AR phosphorylation was visualized by confocal microscopy using anti-phospho-S-355/356 β₂AR antibody (green) (Scale-100 μM) (n = 5/group). <b>b</b>, Plasma membranes isolated from non-asthmatic ASM and asthmatic ASM cells were subjected to ^[125]I-CYP (cyanopindalol) βAR binding at saturation concentration of 250 pmol. *p<0.005 vs. non-asthmatic ASM, (n = 7/group). <b>c</b>, To investigate changes in desensitization βAR components lysates (100 μg) from non-asthmatic ASM and asthmatic ASM cells were immunoblotted for ubiquitously expressed GRKS, GRK2, 3, 5 or 6. The blots were stripped for each probing. Furthermore, the blots were immunoblotted for β-arrestin 1 and 2. Actin was blotted as loading control (n = 7/group).</p

Measure of βAR resensitization in Non-ASM and ASM HASMCs (measure of resensitization components PI3Kγ and PP2A activity).

<p><b>a</b>, Upper panel, PI3Kγ was immunoprecipitated from plasma membrane and endosomes isolated from non-asthmatic ASM and asthmatic ASM cells (120 μg) and the washed immunoprecipitates were subjected to <i>in vitro</i> lipid kinase assays. Lower panel, densitometry for PI3Kγ (n = 4/group). *p<0.01 vs. plasma membrane (P) non-asthmatic ASM, #p<0.0001 vs. endosomes (E) non-asthmatic ASM. <b>b</b>, PP2A was immunoprecipitated from (100 μg) of non-asthmatic ASM and asthmatic ASM cells using anti-PP2A antibodies and the immunoprecipitates were subjected to <i>in vitro</i> phosphatase assay with malachite green as a read for activity, (n = 4/group). *p<0.001 vs. non-asthmatic ASM,). <b>c</b>, Illustration depicting the loss of resensitization as a underlying cause for βAR dysfunction leading to paradoxical loss in β-agonist response. Left Panel (non-asthmatic ASM conditions), βAR activation by β-agonist leads to desensitization by phosphorylation and resensitization by dephosphorylation via PP2A resulting in normal βARs recycling. Right Panel (asthmatic ASM conditions), desensitization is well understood but, little is known of resensitization mechanisms. Since we have shown that βAR resensitization is regulated by PI3Kγ-PP2A axis, assessment in the endosomes shows marked increase in PI3Kγ activity associated with loss in PP2A activity. Based on this observation, we propose that asthma HASMCs are characterized by loss in βAR resensitization wherein, increased PI3Kγ inhibits PP2A activity blocking receptor resensitization resulting in accumulation of βARs in the endosomes.</p

Measure of βAR resensitization in non-asthmatic ASM and asthmatic ASM cells (βAR function, density and distribution).

<p><b>a</b>, To obtain a measure of resensitization that mediates βAR dephosphorylation leading to generation of agonist ready receptors in the endosomes, endosomes were isolated from non-asthmatic ASM and asthmatic ASM cells, and subjected to various assays. <b>b</b>, Plasma membranes and endosomes isolated from non-asthmatic ASM and asthmatic ASM cells were subjected to ^[125]I-CYP (cyanopindalol) βAR binding at saturation concentration of 250 pmol. *p<0.005 vs. Non-ASM, (n = 4/group). <b>c</b>, Plasma membranes and endosomes isolated from non-asthmatic ASM and asthmatic ASM cells were subjected to cell-free membrane associated β-agonist (isoproterenol, ISO)-stimulated adenylyl cyclase activity. *p<0.01 vs. respective cell-free vehicle (V) stimulation, #p<0.05 vs. asthmatic ASM ISO, (n = 4/group). <b>d</b>, Upper panel, plasma membranes and endosomes isolated from non-asthmatic ASM and asthmatic ASM cells (100 μg) were immunoblotted using anti-phospho-S-355/356 β₂AR antibody. Lower panel, densitometry for phospho- β₂AR (n = 4/group). *p<0.01 vs. plasma membrane (P) non-asthmatic ASM, #p<0.05 vs. endosomes (E) non-asthmatic ASM. V, Vehicle, ISO, isoproterenol (β-agonist), P, Plasma membrane, E, Endosome.</p

A representative network of molecules which are predicted targets of miRNAs 1, 214, 342, and 378.

<p>NF kappa B is the central nodal molecule of this network, but importantly, NF kappa B is not a predicted target to any of the altered miRNAs. The NF kappa B could be involved in pathogenesis as the molecules in the network are modulated by miRNAs, and the network function would change as a consequence. The solid arrow between molecules depicts experimentally proven relationship. A dashed line depicts inferred interaction based on experimental evidence. Solid feed-back circular line with an arrow (Auto-regulation). Representation of the shapes: Vertical Diamond–Enzyme; Horizontal Diamond–Peptidase; Equilateral Triangle–Phosphatase; Inverted Equilateral Triangle–Kinase; Solid line square–Cytokine; Dashed line square–Growth Factor; Circle–Other; Trapezoid–Transporter; Double line circle–Complex (like Beta ark).</p

Expression of potential targets and validated miRNA in human cardiomyopathy.

<p>Expression of potential targets and validated miRNA in human cardiomyopathy.</p

Potential targets of validated miRNA occupying central nodes on networks.

<p>Potential targets of validated miRNA occupying central nodes on networks.</p

Patient demographics.

<p>Patient demographics.</p