Mutations in MIR396e and MIR396f increase grain size and modulate shoot architecture in rice

Grain size and plant architecture are critical factors determining crop productivity. Here, we performed gene editing of the MIR396 gene family in rice and found that MIR396e and MIR396f are two important regulators of grain size and plant architecture. mir396ef mutations can increase grain yield by increasing grain size. In addition, mir396ef mutations resulted in an altered plant architecture, with lengthened leaves but shortened internodes, especially the uppermost internode. Our research suggests that mir396ef mutations promote leaf elongation by increasing the level of a gibberellin (GA) precursor, mevalonic acid, which subsequently promotes GA biosynthesis. However, internode elongation in mir396ef mutants appears to be suppressed via reduced CYP96B4 expression but not via the GA pathway. This research provides candidate gene‐editing targets to breed elite rice varieties.     

Reduced caloric intake and periodic fasting independently contribute to metabolic effects of caloric restriction

Caloric restriction (CR) has positive effects on health and longevity. CR in mammals implements time‐restricted (TR) feeding, a short period of feeding followed by prolonged fasting. Periodic fasting, in the form of TR or mealtime, improves metabolism without reduction in caloric intake. In order to understand the relative contribution of reduced food intake and periodic fasting to the health benefits of CR, we compared physiological and metabolic changes induced by CR and TR (without reduced food intake) in mice. CR significantly reduced blood glucose and insulin around the clock, improved glucose tolerance, and increased insulin sensitivity (IS). TR reduced blood insulin and increased insulin sensitivity, but in contrast to CR, TR did not improve glucose homeostasis. Liver expression of circadian clock genes was affected by both diets while the mRNA expression of glucose metabolism genes was significantly induced by CR, and not by TR, which is in agreement with the minor effect of TR on glucose metabolism. Thus, periodic fasting contributes to some metabolic benefits of CR, but TR is metabolically different from CR. This difference might contribute to differential effects of CR and TR on longevity.     

Interplay between the RNA binding‐protein Musashi and developmental signaling pathways

Musashi comprises an evolutionarily conserved family of RNA‐binding proteins (RBP) that regulate cell fate decisions during embryonic development and play key roles in the maintenance of self‐renewal and differentiation of stem cells and adult tissues. More recently, several studies have shown that any dysregulation of MSI1 and MSI2 can lead to cellular dysfunctions promoting tissue instability and tumorigenesis. Moreover, several reports have characterized many molecular interactions between members of the Musashi family with ligands and receptors of the signaling pathways responsible for controlling normal embryonic development: Notch, Transforming Growth Factor Beta (TGF‐β), Wingless (Wnt) and Hedgehog Signaling (Hh); all of which, when altered, are strongly associated with cancer onset and progression, especially in pediatric tumors. In this context, the present review aims to compile possible cross‐talks between Musashi proteins and members of the above cited molecular pathways for which dysregulation plays important roles during carcinogenesis and may be modulated by these RBP.     

Gene transfer to skeletal muscle using hydrodynamic limb vein injection: current applications,hurdles and possible optimizations

Hydrodynamic limb vein injection is an in vivo locoregional gene delivery method. It consists of administrating a large volume of solution containing nucleic acid constructs in a limb with both blood inflow and outflow temporarily blocked using a tourniquet. The fast, high pressure delivery allows the musculature of the whole limb to be reached. The skeletal muscle is a tissue of choice for a variety of gene transfer applications, including gene therapy for Duchenne muscular dystrophy or other myopathies, as well as for the production of antibodies or other proteins with broad therapeutic effects. Hydrodynamic limb vein delivery has been evaluated with success in a large range of animal models. It has also proven to be safe and well‐tolerated in muscular dystrophy patients, thus supporting its translation to the clinic. However, some possible limitations may occur at different steps of the delivery process. Here, we have highlighted the interests, bottlenecks and potential improvements that could further optimize non‐viral gene transfer following hydrodynamic limb vein injection.     

An on-lattice agent-based Monte Carlo model simulating the growth kinetics of multicellular tumor spheroids

PurposeTo develop an on-lattice agent-based model describing the growth of multicellular tumor spheroids using simple Monte Carlo tools.MethodsCells are situated on the vertices of a cubic grid. Different cell states (proliferative, hypoxic or dead) and cell evolution rules, driven by 10 parameters, and the effects of the culture medium are included. About twenty spheroids of MCF-7 human breast cancer were cultivated and the experimental data were used for tuning the model parameters.ResultsSimulated spheroids showed adequate sizes of the necrotic nuclei and of the hypoxic and proliferative cell phases as a function of the growth time, mimicking the overall characteristics of the experimental spheroids. The relation between the radii of the necrotic nucleus and the whole spheroid obtained in the simulations was similar to the experimental one and the number of cells, as a function of the spheroid volume, was well reproduced. The statistical variability of the Monte Carlo model described the whole volume range observed for the experimental spheroids. Assuming that the model parameters vary within Gaussian distributions it was obtained a sample of spheroids that reproduced much better the experimental findings.ConclusionsThe model developed allows describing the growth of in vitro multicellular spheroids and the experimental variability can be well reproduced. Its flexibility permits to vary both the agents involved and the rules that govern the spheroid growth. More general situations, such as, e. g., tumor vascularization, radiotherapy effects on solid tumors, or the validity of the tumor growth mathematical models can be studied.     

RS‐1 enhances CRISPR‐mediated targeted knock‐in in bovine embryos

Targeted knock‐in (KI) can be achieved in embryos by clustered regularly interspaced short palindromic repeats (CRISPR)‐assisted homology directed repair (HDR). However, HDR efficiency is constrained by the competition of nonhomologous end joining. The objective of this study was to explore whether CRISPR‐assisted targeted KI rates can be improved in bovine embryos by exposure to the HDR enhancer RS‐1. In vitro produced zygotes were injected with CRISPR components (300 ng/µl Cas9 messenger RNA and 100 ng/µl single guide RNA against a noncoding region) and a single‐stranded DNA (ssDNA) repair template (100 ng/µl). ssDNA template contained a 6 bp XbaI site insert, allowing targeted KI detection by restriction analysis, flanked by 50 bp homology arms. Following microinjection, zygotes were exposed to 0, 3.75, or 7.5 µM RS‐1 for 24 hr. No differences were noted between groups in terms of development or genome edition rates. However, targeted KI rates were doubled in the group exposed to 7.5 µM RS‐1 compared to the others (52.8% vs. 25% and 23.1%, for 7.5, 0, and 3.75 µM, respectively). In conclusion, transient exposure to 7.5 µM RS‐1 enhances targeted KI rates resulting in approximately half of the embryos containing the intended mutation, hence allowing direct KI generation in embryos.