Antispacer peptide nucleic acids for sequence-specific CRISPR-Cas9 modulation

Despite the rapid and broad implementation of CRISPR-Cas9-based technologies, convenient tools to modulate dose, timing, and precision remain limited. Building on methods using synthetic peptide nucleic acids (PNAs) to bind RNA with unusually high affinity, we describe guide RNA (gRNA) spacer-targeted, or ‘antispacer’, PNAs as a tool to modulate Cas9 binding and activity in cells in a sequence-specific manner. We demonstrate that PNAs rapidly and efficiently target complexed gRNA spacer sequences at low doses and without design restriction for sequence-selective Cas9 inhibition. We further show that short PAM-proximal antispacer PNAs achieve potent cleavage inhibition (over 2000-fold reduction) and that PAM-distal PNAs modify gRNA affinity to promote on-target specificity. Finally, we apply antispacer PNAs for temporal regulation of two dCas9-fusion systems. These results present a novel rational approach to nucleoprotein engineering and describe a rapidly implementable antisense platform for CRISPR-Cas9 modulation to improve spatiotemporal versatility and safety across applications.     

Endothelin-1 and insulin activate the steady-state voltage dependent R-type Ca2+ channel in aortic smooth muscle cells via a pertussis toxin and cholera toxin sensitive G-protein

In single rabbit aortic smooth muscle cells, and at a concentration known to induce a maximum sustained increase of intracellular Ca²⁺ via activation of the steady-state voltage dependent R-type Ca²⁺ channels, endothelin-1 (10^-7 M) and insulin (80 U/ml) were found to induce a sustained increase in cytosolic free Ca²⁺ ([Ca]_i) levels that was significantly attenuated by pre-treatment with either pertussis toxin (PTX), cholera toxin (CTX) or removal of extracellular Ca²⁺.However, both PTX and CTX failed to inhibit the sustained depolarization-evoked sustained Ca²⁺ influx and [Ca]_i elevation via activation of the R-type Ca²⁺ channels. Moreover, ET-1 and insulin-evoked sustained increases in Ca²⁺ influx were not attenuated by the selective PKC inhibitor, bisindolylmaleimide (BIS), or the specific L-type Ca²⁺ channel blocker, nifedipine, but were completely reversed by the R-type Ca²⁺ channel blocker, (-) PN 200-110 (isradipine). These data suggest that both insulin and ET-1 activate the nifedipine-insensitive but isradipine-sensitive steady state voltage dependent R-type Ca²⁺ channels present on rabbit VSMCs and these channels are directly coupled to PTX and CTX sensitive G protein(s).     

REGULATION OF HAEMOPOIESIS IN ALTERED GRAVITY

(1) The aetiology of one of the most striking physiological changes occurring during space-flight, the loss of red blood cells, remains unknown, and its precise time-pattern in flight has not yet been studied. (2) It is suggested that the changes during space-flight responsible for loss of red blood cells in man are (a) loss of plasma volume resulting from disappearance of hydrostatic pressure in the circulation during weightlessness and (b) reduced energy expended in maintenance of form, posture and locomotion resulting from elimination of the usual gravitational load on the muscles. Quadrupeds, like rats, would be expected to suffer minimal blood shifts in weightlessness and therefore have an unchanged plasma volume. However, since in weightlessness the activity-related energy expenditure by the muscles is reduced, the accompanying reduced oxygen demand by the tissues would cause a reduction in erythropoietin levels and so in the production of red blood cells, and a progressive lowering of the total red blood cell mass toward a new steady-state level. (3) Loss of plasma volume alone does not explain the observed loss of red blood cells in astronauts because, in the three manned Skylab missions, as the duration of the missions increased, loss of red blood cell mass decreased, whereas loss of plasma volume increased. This discrepancy is, however, well accounted for by the above hypothesis by taking into consideration the increased level of exercise of the astronauts as the duration of the mission increased. (4) Though water submersion of human subjects does mimic the effects of weightlessness, such effects were overriden in sea mammals because of adaptation to other factors associated with a life in the sea. (5) From the presented analysis of haemopoietic changes observed in spaceflight, an experiment can be designed for a future flight to uncover the causes and mechanisms of these changes and provide a basis for developing protective measures. Thus, the space environment can be used as an investigative tool to enhance the knowledge of the function of the haemopoietic system, which is a major homeostatic system of man and other vertebrates.     

The largest land mammal

Although a lower limit to size of land mammals, about 2·5 g, was established over 30 years ago, no definite upper limit has been proposed. The largest mammal known to have lived on land was the Baluchitherium, with a body mass of about 20 000 kg. In this paper, I show that gravity imposes an upper limit to size of land mammals. This limit results from the metabolic cost of gravity, which increases with body mass; therefore, the larger the body mass of an animal the smaller is the gravitational field intensity that can be tolerated by the animal. From data on gravitational tolerance of a number of mammalian species, it is shown that the size of the land mammal that can not tolerate more than terrestrial gravity is approximately 20 000 kg.     

Predicting maintenance or achievement of healthy weight in children: the impact of changes in physical fitness

Physical fitness is often inversely associated with adiposity in children cross-sectionally, but the effect of becoming fit or maintaining fitness over time on changes in weight status has not been well studied in children. We investigated the impact of changes in fitness over 1-4 years of follow-up on the maintenance or achievement of healthy weight among 2,793 schoolchildren who were first measured as 1st to 7th graders. Students were classified as "fit" or "underfit" according to age- and gender-specific norms in five fitness domains: endurance, agility, flexibility, upper body strength, and abdominal strength. Weight status was dichotomized by BMI percentile: "healthy weight" (<85th percentile) or "overweight/obese" (≥85th percentile). At baseline, of the 38.3% overweight/obese children, 81.9% (N = 875) were underfit. Underfit overweight students were more likely to achieve healthy weight if they achieved fitness (boys: odds ratio (OR) = 2.68, 95% confidence interval (CI) = 1.24-5.77; girls: OR = 4.67, 95%CI = 2.09-10.45). Initially fit overweight children (N = 194) were more likely to achieve healthy weight if they maintained fitness (boys: OR = 11.99, 95%CI = 2.18-65.89; girls: OR = 2.46, 95%CI = 1.04-5.83). Similarly, initially fit healthy-weight children (N = 717) were more likely to maintain healthy weight if they maintained fitness (boys: OR 3.70, 95%CI = 1.40-9.78; girls: OR = 4.14, 95%CI = 1.95-8.78). Overweight schoolchildren who achieve or maintain physical fitness are more likely to achieve healthy weight, and healthy-weight children who maintain fitness are more likely to maintain healthy weight. School-based policies/practices that support physical fitness may contribute to obesity reduction and maintenance of healthy weight among schoolchildren.