Developmentally regulated promoter-switch transcriptionally controls Runx1 function during embryonic hematopoiesis

<p>Abstract</p> <p>Background</p> <p>Alternative promoters usage is an important paradigm in transcriptional control of mammalian gene expression. However, despite the growing interest in alternative promoters and their role in genome diversification, very little is known about how and on what occasions those promoters are differentially regulated. Runx1 transcription factor is a key regulator of early hematopoiesis and a frequent target of chromosomal translocations in acute leukemias. Mice deficient in <it>Runx1 </it>lack definitive hematopoiesis and die in mid-gestation. Expression of <it>Runx1 </it>is regulated by two functionally distinct promoters designated P1 and P2. Differential usage of these two promoters creates diversity in distribution and protein-coding potential of the mRNA transcripts. While the alternative usage of P1 and P2 likely plays an important role in <it>Runx1 </it>biology, very little is known about the function of the P1/P2 switch in mediating tissue and stage specific expression of <it>Runx1 </it>during development.</p> <p>Results</p> <p>We employed mice bearing a hypomorphic <it>Runx1 </it>allele, with a largely diminished P2 activity, to investigate the biological role of alternative P1/P2 usage. Mice homozygous for the hypomorphic allele developed to term, but died within a few days after birth. During embryogenesis the P1/P2 activity is spatially and temporally modulated. P2 activity is required in early hematopoiesis and when attenuated, development of liver hematopoietic progenitor cells (HPC) was impaired. Early thymus development and thymopoiesis were also abrogated as reflected by thymic hypocellularity and loss of corticomedullary demarcation. Differentiation of CD4/CD8 thymocytes was impaired and their apoptosis was enhanced due to altered expression of T-cell receptors.</p> <p>Conclusion</p> <p>The data delineate the activity of P1 and P2 in embryogenesis and describe previously unknown functions of Runx1. The findings show unequivocally that the role of P1/P2 during development is non redundant and underscore the significance of alternative promoter usage in Runx1 biology.</p

Waterbomb Origami Mechanism Pneumatic Manipulator Design Based on DH Parameter Method and Symmetric Folding Hypothesis

As robotics and materials science develop, origami mechanical structures have gradually attracted attention in the research field due to their lightweight, foldable, and multifunctional characteristics. As one of them, the water elastic origami mechanism, with its unique structure and flexibility, provides a new perspective and possibility for the design of pneumatic manipulators. This study proposes a research on the design of a pneumatic manipulator for a water elastic paper folding mechanism by the DH parameter method and the symmetric folding assumption, in response to the issue of insensitivity of the manipulator. Firstly, the dynamic factors of the hydroelastic mechanical mechanism, including its degree of freedom and reaction force, are explored. Secondly, the DH parameter method is used to design and optimize the configuration of the paper folding manipulator, making its operation more accurate and capable of handling more types of tasks. Finally, the symmetrical folding assumption is used to guide the design of the water elastic origami mechanism, in order to create a more stable and reliable pneumatic manipulator. Results showed that the recognition accuracy of cola bottles, dental floss boxes, cubes, cylinders, sponge cubes, and glasses boxes reached 100&#x0025;. From this, it can be seen that these data clearly demonstrate that the object recognition system can maintain high recognition accuracy for various different objects and shapes, proving the effectiveness and reliability of the system

Search for an Endogenous Bombesin-Like Receptor 3 (BRS-3) Ligand Using Parabiotic Mice.

Bombesin-like receptor 3 (BRS-3) is an X-linked G protein-coupled receptor involved in the regulation of energy homeostasis. Brs3 null (Brs3-/y) mice become obese. To date, no high affinity endogenous ligand has been identified. In an effort to detect a circulating endogenous BRS-3 ligand, we generated parabiotic pairs of mice between Brs3-/y and wild type (WT) mice or between WT controls. Successful parabiosis was demonstrated by circulatory dye exchange. The Brs3-/y-WT and WT-WT pairs lost similar weight immediately after surgery. After 9 weeks on a high fat diet, the Brs3-/y-WT pairs weighed more than the WT-WT pairs. Within the Brs3-/y-WT pairs, the Brs3-/y mice had greater adiposity than the WT mice, but comparable lean and liver weights. Compared to WT mice in WT-WT pairs, Brs3-/y and WT mice in Brs3-/y-WT pairs each had greater lean mass, and the Brs3-/y mice also had greater adiposity. These results contrast to those reported for parabiotic pairs of leptin receptor null (Leprdb/db) and WT mice, where high leptin levels in the Leprdb/db mice cause the WT parabiotic partners to lose weight. Our data demonstrate that a circulating endogenous BRS-3 ligand, if present, is not sufficient to reduce adiposity in parabiotic partners of Brs3-/y mice

Effect of intermittent cold exposure on brown fat activation, obesity, and energy homeostasis in mice.

Homeotherms have specific mechanisms to maintain a constant core body temperature despite changes in thermal environment, food supply, and metabolic demand. Brown adipose tissue, the principal thermogenic organ, quickly and efficiently increases heat production by dissipating the mitochondrial proton motive force. It has been suggested that activation of brown fat, via either environmental (i.e. cold exposure) or pharmacologic means, could be used to increase metabolic rate and thus reduce body weight. Here we assess the effects of intermittent cold exposure (4°C for one to eight hours three times a week) on C57BL/6J mice fed a high fat diet. Cold exposure increased metabolic rate approximately two-fold during the challenge and activated brown fat. In response, food intake increased to compensate fully for the increased energy expenditure; thus, the mice showed no reduction in body weight or adiposity. Despite the unchanged adiposity, the cold-treated mice showed transient improvements in glucose homeostasis. Administration of the cannabinoid receptor-1 inverse agonist AM251 caused weight loss and improvements in glucose homeostasis, but showed no further improvements when combined with cold exposure. These data suggest that intermittent cold exposure causes transient, meaningful improvements in glucose homeostasis, but without synergy when combined with AM251. Since energy expenditure is significantly increased during cold exposure, a drug that dissociates food intake from metabolic demand during cold exposure may achieve weight loss and further metabolic improvements

Integration of body temperature into the analysis of energy expenditure in the mouse

Objectives: We quantified the effect of environmental temperature on mouse energy homeostasis and body temperature. Methods: The effect of environmental temperature (4–33 °C) on body temperature, energy expenditure, physical activity, and food intake in various mice (chow diet, high-fat diet, Brs3-/y, lipodystrophic) was measured using continuous monitoring. Results: Body temperature depended most on circadian phase and physical activity, but also on environmental temperature. The amounts of energy expenditure due to basal metabolic rate (calculated via a novel method), thermic effect of food, physical activity, and cold-induced thermogenesis were determined as a function of environmental temperature. The measured resting defended body temperature matched that calculated from the energy expenditure using Fourier's law of heat conduction. Mice defended a higher body temperature during physical activity. The cost of the warmer body temperature during the active phase is 4–16% of total daily energy expenditure. Parameters measured in diet-induced obese and Brs3-/y mice were similar to controls. The high post-mortem heat conductance demonstrates that most insulation in mice is via physiological mechanisms. Conclusions: At 22 °C, cold-induced thermogenesis is ∼120% of basal metabolic rate. The higher body temperature during physical activity is due to a higher set point, not simply increased heat generation during exercise. Most insulation in mice is via physiological mechanisms, with little from fur or fat. Our analysis suggests that the definition of the upper limit of the thermoneutral zone should be re-considered. Measuring body temperature informs interpretation of energy expenditure data and improves the predictiveness and utility of the mouse to model human energy homeostasis

Initial body weight and composition of WT and <i>Brs3</i>^<i>-/y</i> mice.

<p>Body weight (A), lean mass (B), and fat mass (C) of the indicated mice at the time of parabiosis surgery. Data are mean ± SEM; N = 7-14/group. There are no significant differences between the groups.</p

Identification of reference genes in blood before and after entering the plateau for SYBR green RT-qPCR studies

Background Tibetans have lived at high altitudes for thousands of years, and they have unique physiological traits that enable them to tolerate this hypoxic environment. However, the genetic basis of these traits is still unknown. As a sensitive and highly efficient technique, RT-qPCR is widely used in gene expression analyses to provide insight into the molecular mechanisms underlying environmental changes. However, the quantitative analysis of gene expression in blood is limited by a shortage of stable reference genes for the normalization of mRNA levels. Thus, systematic approaches were used to identify potential reference genes. Results The expression levels of eight candidate human reference genes (GAPDH, ACTB, 18S RNA, β2-MG, PPIA, RPL13A, TBP and SDHA) were assessed in blood from hypoxic environments. The expression stability of these selected reference genes was evaluated using the geNorm, NormFinder and BestKeeper programs. Interestingly, RPL13A was identified as the ideal reference gene for normalizing target gene expression in human blood before and after exposure to high-altitude conditions. Conclusion These results indicate that different reference genes should be selected for the normalization of gene expression in blood from different environmental settings

Loss of Otopetrin 1 affects thermoregulation during fasting in mice.

ObjectiveOtopetrin 1 (OTOP1) is a proton channel that is highly expressed in brown adipose tissue. We examined the physiology of Otop1-/- mice, which lack functional OTOP1.MethodsMice were studied by indirect calorimetry and telemetric ambulatory body temperature monitoring. Mitochondrial function was measured as oxygen consumption and extracellular acidification.ResultsOtop1-/- mice had similar body temperatures as control mice at baseline and in response to cold and hot ambient temperatures. However, in response to fasting the Otop1-/- mice exhibited an exaggerated hypothermia and hypometabolism. Similarly, in ex vivo tests of Otop1-/- brown adipose tissue mitochondrial function, there was no change in baseline oxygen consumption, but the oxygen consumption was reduced after maximal uncoupling with FCCP and increased upon stimulation with the β3-adrenergic agonist CL316243. Mast cells also express Otop1, and Otop1-/- mice had intact, possibly greater hypothermia in response to mast cell activation by the adenosine A3 receptor agonist MRS5698. No increase in insulin resistance was observed in the Otop1-/- mice.ConclusionsLoss of OTOP1 does not change basal function of brown adipose tissue but affects stimulated responses

A Seamless Gene Deletion Method and Its Application for Regulation of Higher Alcohols and Ester in Baijiu Saccharomyces cerevisiae

The security of engineering Saccharomyces cerevisiae is becoming more focused on industrial production in consideration of the public concern regarding genetically modified organisms. In this work, a rapid and highly efficient system for seamless gene deletion in S. cerevisiae was developed through two-step integration protocol combined with endonuclease I-SCEI expression. The factors affecting the frequency of the second homologous recombination were optimized, and studies indicated that the mutant strains with 500 bp direct repeats and that have been incubating in galactose (0.5 g/100 mL) medium at 30°C and 180 r/min for 24 h permit high frequency (6.86 × 10−4) of the second homologous recombination. Furthermore, DNA sequence assays showed only self-DNA in native location without any foreign genes after deletion using this method. The seamless gene deletion method was applied to the construction of the engineering strains with BAT2 (encoding aminotransferase) deletion and ATF1 (alcohol acetyltransferases) overexpression. The mutants exhibited significant effects on higher alcohol reduction and ester improvement after Baijiu fermentation. The engineered strains can be used in industrial production in security, thereby meeting the requirements of modern science and technology