Background: Quantitative real-time reverse transcription PCR (qRT-PCR) is a useful tool for assessing gene expression in different tissues, but the choice of adequate controls is critical to normalise the results, thereby avoiding differences and maximizing sensitivity and accuracy. So far, many genes have been used as a single reference gene, without having previously verified their value as controls. This practice can lead to incorrect conclusions and recent evidence indicates a need to use the geometric mean of data from several control genes. Here, we identified an appropriate set of genes to be used as an endogenous reference for quantifying gene expression in human heart tissue.

BackgroundType 1 diabetes (T1D) is a T cell-mediated autoimmune disease that causes a deficit of pancreatic islet β cells. The complexities of overcoming autoimmunity in T1D have contributed to the challenges the research community faces when devising successful treatments with conventional immune therapies. Overcoming autoimmune T cell memory represents one of the key hurdles.MethodsIn this open-label, phase 1/phase 2 study, Caucasian T1D patients (N = 15) received two treatments with the Stem Cell Educator (SCE) therapy, an approach that uses human multipotent cord blood-derived multipotent stem cells (CB-SCs). SCE therapy involves a closed-loop system that briefly treats the patient's lymphocytes with CB-SCs in vitro and returns the “educated” lymphocytes (but not the CB-SCs) into the patient's blood circulation. This study is registered with ClinicalTrials.gov, NCT01350219.FindingsClinical data demonstrated that SCE therapy was well tolerated in all subjects. The percentage of naïve CD4+ T cells was significantly increased at 26 weeks and maintained through the final follow-up at 56 weeks. The percentage of CD4+ central memory T cells (TCM) was markedly and constantly increased at 18 weeks. Both CD4+ effector memory T cells (TEM) and CD8+ TEM cells were considerably decreased at 18 weeks and 26 weeks respectively. Additional clinical data demonstrated the modulation of C–C chemokine receptor 7 (CCR7) expressions on naïve T, TCM, and TEM cells. Following two treatments with SCE therapy, islet β-cell function was improved and maintained in individuals with residual β-cell function, but not in those without residual β-cell function.InterpretationCurrent clinical data demonstrated the safety and efficacy of SCE therapy in immune modulation. SCE therapy provides lasting reversal of autoimmune memory that could improve islet β-cell function in Caucasian subjects.FundingObra Social “La Caixa”, Instituto de Salud Carlos III, Red de Investigación Renal, European Union FEDER Funds, Principado de Asturias, FICYT, and Hackensack University Medical Center Foundation.

Biological rhythms are driven by endogenous biological clocks; in mammals, the master clock is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. This master pacemaker can synchronize other peripheral oscillators in several tissues such as some involved in endocrine or reproductive functions. The presence of an endogenous placental clock has received little attention. In fact, there are no studies in human full-term placentas. To test the existence of an endogenous pacemaker in this tissue we have studied the expression of circadian locomoter output cycles kaput (Clock), brain and muscle arnt-like (Bmal)1, period (Per)2, and cryptochrome (Cry)1 mRNAs at 00, 04, 08, 12, 16, and 20 hours by qPCR. The four clock genes studied are expressed in full-term human placenta. The results obtained allow us to suggest that a peripheral oscillator exists in human placenta. Data were analyzed using Fourier series where only the Clock and Bmal1 expression shows a circadian rhythm.

Background: Intensive care unit (ICU) environment disrupts the circadian rhythms due to environmental and other nonphotic synchronizers. The main purpose of this article is to establish whether critically patients have desynchronization at the molecular level after 1 week of stay in the ICU. Methods: The rhythm of Clock, Bmal1, Cry1, and Per2 genes in neuro-ICU patients (n = 11) on the first day after admission in the unit (1 day) and 1 week later (1 week) was studied, 4 time points throughout the day, at 6, 12, 18, and 24 hours. Human whole blood samples were obtained from neuro-ICU patients. The total RNA was isolated and each sample was reverse transcribed to complementary DNA and quantitative polymerase chain reaction (PCRq) was performed. The possible rhythm was studied using Fourier Series. Results: After 1 week, the clock gene rhythmicity completely disappeared. Messenger RNA (mRNA) expression for the 4 clock genes was shown rhythmicity at the first day after admission in the ICU. Circadian rhythmicity for none of them was observed but rather, ultradian rhythmicity was found. The expression of Clock, Bmal1, and Per2 mRNA after 1 week was similar in the 4-time point studies without significant fluctuation among the 4 time points analyzed. Discussion: Rhythmic mRNA expression is present at the first day after admission in the ICU. However, ICU stay during 1 week affects the molecular machinery of the biological clock generating chronodisruption. Circadian disruption is associated with the risk of several pathologies, thus, it seems to be clear that ICU stay in constant conditions could adversely affect patient evolution and probably, circadian resynchronization restoring clock gene expression could lead to a better clinical evolution of the patient. Conclusions: Clock genes disruption is observed in neuro-ICU patients. Light therapy as well as melatonin treatment could reduce the impact of ICU stay period in biological clock, thereby improving patients’ recovery.

Our results suggest that classic P receptors (PRs) are not the only kind playing a role in the normal physiology of the endometrium. The human decidualization process could be altered by the overexpression or mislocalization of PGRMC1 in ESC.