Selection of reference genes for normalization of quantitative real-time PCR in organ culture of the rat and rabbit intervertebral disc

<p>Abstract</p> <p>Background</p> <p>The accuracy of quantitative real-time RT-PCR (qRT-PCR) is often influenced by experimental artifacts, resulting in erroneous expression profiles of target genes. The practice of employing normalization using a reference gene significantly improves reliability and its applicability to molecular biology. However, selection of an ideal reference gene(s) is of critical importance to discern meaningful results. The aim of this study was to evaluate the stability of seven potential reference genes (Actb, GAPDH, 18S rRNA, CycA, Hprt1, Ywhaz, and Pgk1) and identify most stable gene(s) for application in tissue culture research using the rat and rabbit intervertebral disc (IVD).</p> <p>Findings</p> <p><it>In vitro</it>, four genes (Hprt1, CycA, GAPDH, and 18S rRNA) in rat IVD tissue and five genes (CycA, Hprt1, Actb, Pgk1, and Ywhaz) in rabbit IVD tissue were determined as most stable for up to 14 days in culture. Pair-wise variation analysis indicated that combination of Hprt1 and CycA in rat and the combination of Hprt1, CycA, and Actb in rabbit may most stable reference gene candidates for IVD tissue culture.</p> <p>Conclusions</p> <p>Our results indicate that Hprt1 and CycA are the most stable reference gene candidates for rat and rabbit IVD culture studies. In rabbit IVD, Actb could be an additional gene employed in conjunction with Hprt1 and CycA. Selection of optimal reference gene candidate(s) should be a pertinent exercise before employment of PCR outcome measures for biomedical research.</p

桃の肥料三要素吸収量の季節的変化

1. 桃(3年生高陽白桃, 7年生箕島白桃)樹上葉の三要素吸収量の季節的変化を1957年4月より9月に亘つて調査し, 時期別吸収量の変異について考察した。2. 1樹全葉中の肥料成分吸収量は枝葉の発育増加に伴つて増大し, 8月頃より減少する傾向がある。時期別吸収量の変異は, N, Pでは5-6月に最高となり, 8月以後減少するが, Nの吸収量変異は大きく, Pはその変化は僅少である。Kは5-6月最高となり以後稍減少するが, 新梢生長哀退の初期に再び増加し, 8月下旬以降減少する傾向がある。3. 葉中の時期別含量はP, Kは4月の初期に高く, Nは6-7月が高いが, 9月以降三要素共減少の傾向がある。1. The purpuse of this investigation was to study the seasonal change of N, P and K absorption in the Peach tree, as a function of time from April to september, 1957. 2. The amount of N, P and K absorbed by the leavls increased with the develoment of new shoot, though the rate of absorption tended to be dawn in after August. In variation of absoorption at each growing season, N and P were highest in May to June, but K was increased between May to June, though after in this growing stage absorption to be slow down temporary, and K content was increased in next stage for a second time, though were suddenly decreased in after August. 3. In the seasonal change of N, P and K content of leaves, N, P content was higher in early growing season, but N content was highest in June or July

Nitrosative and Oxidative Stresses Contribute to Post-Ischemic Liver Injury Following Severe Hemorrhagic Shock: The Role of Hypoxemic Resuscitation

Purpose: Hemorrhagic shock and resuscitation is frequently associated with liver ischemia-reperfusion injury. The aim of the study was to investigate whether hypoxemic resuscitation attenuates liver injury. Methods: Anesthetized, mechanically ventilated New Zealand white rabbits were exsanguinated to a mean arterial pressure of 30 mmHg for 60 minutes. Resuscitation under normoxemia (Normox-Res group, n = 16, PaO2 = 95–105 mmHg) or hypoxemia (Hypox-Res group, n = 15, PaO 2 = 35–40 mmHg) followed, modifying the FiO 2. Animals not subjected to shock constituted the sham group (n = 11, PaO 2 = 95–105 mmHg). Indices of the inflammatory, oxidative and nitrosative response were measured and histopathological and immunohistochemical studies of the liver were performed. Results: Normox-Res group animals exhibited increased serum alanine aminotransferase, tumor necrosis factor- alpha, interleukin (IL)-1b and IL-6 levels compared with Hypox-Res and sham groups. Reactive oxygen species generation, malondialdehyde formation and myeloperoxidase activity were all elevated in Normox-Res rabbits compared with Hypox-Res and sham groups. Similarly, endothelial NO synthase and inducible NO synthase mRNA expression was up-regulated and nitrotyrosine immunostaining increased in animals resuscitated normoxemically, indicating a more intense nitrosative stress. Hypox-Res animals demonstrated a less prominent histopathologic injury which was similar to sham animals. Conclusions: Hypoxemic resuscitation prevents liver reperfusion injury through attenuation of the inflammatory respons

Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049

カキ樹の生理生態学的研究 III : 夜温が新梢生長, 開花ならびに果実肥大に及ぼす影響(農学部門)

カキの成木結果樹を供試し, それぞれ樹体の発育周期に応じて, 夜間に補温処理を行ない, 枝梢の生長, 開花ならびに果実の肥大に及ぼす影響を調査した。その結果, 新梢伸長期(自然夜温12&acd;13.4℃)および果実発育の第1期(自然夜温21&acd;22℃)に3&acd;4℃以内で夜間補温すれば, 新梢の生長, 開花および果実の肥大を促進し, とくにその期間中で処理を打ち切った後もその影響が維持されて収穫期にまでおよぶ。しかし, この時期に自然夜温より6&acd;7℃以上の高夜温になると, 一時的には展葉, 開花ならびに幼果の肥大は促されるが, 枝葉は軟弱徒長し開花後の落果が著しい。開花と萠芽時よりの積算夜温とは密接な関係を示し, 4月始めよりほぼ470℃&acd;500℃で開花期に至る。Some effects of night temperature on the shoot elongation, blooming and enlargement of fruit were studied by Confining fruiting limls during the night (7p.m-7a.m) in heated chambers. By the treatment at the shoot growth stage (the mean natural night temperature is 12-13.4℃ through treatment period) and the first stage of fruit growing (the mean natural night temperature is 21-22℃ throught treatmentp eriod), shoot elongation, blooming and fruit enlargement were much acceler ated, in case of 3&acd;4℃ above the natural night temperature, and this tendency was continued till harvest, even if the treatment discontinued merely at the first stage of fruit growing. However, when the treatment tremperature was 6&acd;7℃ higher than the natural night temperature, all young fruits fell down at about 10&acd;20 days after the treatment. The flowering is closely connected with the accumlatively night temperature before the flowering. In the treatment of 1959 and 1960,we observed that accumlatively night temperature from the beginning of April to flowering stage is 470-500

Human DEF6 deficiency underlies an immunodeficiency syndrome with systemic autoimmunity and aberrant CTLA-4 homeostasis

Immune responses need to be controlled tightly to prevent autoimmune diseases, yet underlying molecular mechanisms remain partially understood. Here, we identify biallelic mutations in three patients from two unrelated families in differentially expressed in FDCP6 homolog (DEF6) as the molecular cause of an inborn error of immunity with systemic autoimmunity. Patient T cells exhibit impaired regulation of CTLA-4 surface trafficking associated with reduced functional CTLA-4 availability, which is replicated in DEF6-knockout Jurkat cells. Mechanistically, we identify the small GTPase RAB11 as an interactor of the guanine nucleotide exchange factor DEF6, and find disrupted binding of mutant DEF6 to RAB11 as well as reduced RAB11+CTLA-4+ vesicles in DEF6-mutated cells. One of the patients has been treated with CTLA-4-Ig and achieved sustained remission. Collectively, we uncover DEF6 as player in immune homeostasis ensuring availability of the checkpoint protein CTLA-4 at T-cell surface, identifying a potential target for autoimmune and/or cancer therapy.Mechanistically, we identify the small GTPase RAB11 as an interactor of the guanine nucleotide exchange factor DEF6, and find disrupted binding of mutant DEF6 to RAB11 as well as reduced RAB11+CTLA-4+ vesicles in DEF6-mutated cells. One of the patients has been treated with CTLA-4-Ig and achieved sustained remission. Collectively, we uncover DEF6 as player in immune homeostasis ensuring availability of the checkpoint protein CTLA-4 at T-cell surface, identifying a potential target for autoimmune and/or cancer therapy