The Relationship between Gene Network Structure and Expression Variation among Individuals and Species

Variation among individuals is a prerequisite of evolution by natural selection. As such, identifying the origins of variation is a fundamental goal of biology. We investigated the link between gene interactions and variation in gene expression among individuals and species using the mammalian limb as a model system. We first built interaction networks for key genes regulating early (outgrowth; E9.5–11) and late (expansion and elongation; E11-13) limb development in mouse. This resulted in an Early (ESN) and Late (LSN) Stage Network. Computational perturbations of these networks suggest that the ESN is more robust. We then quantified levels of the same key genes among mouse individuals and found that they vary less at earlier limb stages and that variation in gene expression is heritable. Finally, we quantified variation in gene expression levels among four mammals with divergent limbs (bat, opossum, mouse and pig) and found that levels vary less among species at earlier limb stages. We also found that variation in gene expression levels among individuals and species are correlated for earlier and later limb development. In conclusion, results are consistent with the robustness of the ESN buffering among-individual variation in gene expression levels early in mammalian limb development, and constraining the evolution of early limb development among mammalian species.     

CONSUMER ACCEPTANCE OF COMMERCIALLY AVAILABLE FRANKFURTERS1

Consumers want meat products that are healthful and affordable. However, consumers will repurchase a product only if it tastes good. Consumers identified reasons for purchasing frankfurters and evaluated overall acceptability of 10 commercially available frankfurters. Consumers listed “taste” as the most important factor when purchasing frankfurters; only 16% of the consumers indicated that nutrition/health was the most important criterion. Nineteen consumer-perceived sensory and liking attributes were measured for the frankfurters in this study. The intensities of the attributes ranged from low to moderately high. All the frankfurters scored in the low to moderate range for overall acceptability. Most of the scores for liking of individual attributes correlated highly with overall liking, indicating that many of the questions relating to attribute liking could be eliminated.     

Biodegradable Monocrystalline Silicon Photovoltaic Microcells as Power Supplies for Transient Biomedical Implants

Bioresorbable electronic materials serve as foundations for implantable devices that provide active diagnostic or therapeutic function over a timeframe matched to a biological process, and then disappear within the body to avoid secondary surgical extraction. Approaches to power supply in these physically transient systems are critically important. This paper describes a fully biodegradable, monocrystalline silicon photovoltaic (PV) platform based on microscale cells (microcells) designed to operate at wavelengths with long penetration depths in biological tissues (red and near infrared wavelengths), such that external illumination can provide realistic levels of power. Systematic characterization and theoretical simulations of operation under porcine skin and fat establish a foundational understanding of these systems and their scalability. In vivo studies of a representative platform capable of generating ≈60 µW of electrical power under 4 mm of porcine skin and fat illustrate an ability to operate blue light‐emitting diodes (LEDs) as subdermal implants in rats for 3 d. Here, the PV system fully resorbs after 4 months. Histological analysis reveals that the degradation process introduces no inflammatory responses in the surrounding tissues. The results suggest the potential for using silicon photovoltaic microcells as bioresorbable power supplies for various transient biomedical implants.     

Comparison and evaluation of RNA quantification methods using viral, prokaryotic, and eukaryotic RNA over a 10 concentration range

Quantification of RNA is essential for various molecular biology studies. In this work, three quantification methods were evaluated: ultraviolet (UV) absorbance, microcapillary electrophoresis (MCE), and fluorescence-based quantification. Viral, bacterial, and eukaryotic RNA were measured in the 500 to 0.05-ng μl⁻¹ range via an ND-1000 spectrophotometer (UV), Agilent RNA 6000 kits (MCE), and Quant-iT RiboGreen assay (fluorescence). The precision and accuracy of each method were assessed and compared with a concentration derived independently using inductively coupled plasma-optical emission spectroscopy (ICP-OES). Cost, operator time and skill, and required sample volumes were also considered in the evaluation. Results indicate an ideal concentration range for each quantification technique to optimize accuracy and precision. The ND-1000 spectrophotometer exhibits high precision and accurately quantifies a 1-μl sample in the 500 to 5-ng μl⁻¹ range. The Quant-iT RiboGreen assay demonstrates high precision in the 1 to 0.05-ng μl⁻¹ range but is limited to lower RNA concentrations and is more costly than the ND-1000 spectrophotometer. The Agilent kits exhibit less precision than the ND-1000 spectrophotometer and Quant-iT RiboGreen assays in the 500 to 0.05-ng μl⁻¹ range. However, the Agilent kits require 1 μl of sample and can determine the integrity of the RNA, a useful feature for verifying whether the isolation process was successful.     

Funnels,gas exchange and cliff jumping: natural history of the cliff dwelling ant Malagidris sofina

The Malagasy endemic ant Malagidris sofina (Bolton and Fisher 2014) nests on cliff faces in natural rock alcoves or clay banks. Colonies have single ergatoid queens and reproduce by fission. Each nest has a funnel-shaped entrance that projects horizontally from the cliff face. We examine three hypotheses for the function of the funnels—water exclusion, gas exchange and defense. Entrance funnels are relatively impermeable and divert water from nests, but simple tubes would achieve the same result. Consistent with the gas exchange hypothesis, projected funnel entrances likely increase gas exchange rates over sixfold compared to simple tubes and may increase air flow within the nest. Gas exchange may explain the recurrent evolution of funnel entrances in several ant lineages, especially among cliff dwelling species. We outline M. sofina defense responses to conspecifics and co-occurring ant species, and find no support for a defense role of entrance funnels. Workers display little aggression but respond to several species with an original form of nest defense––cliff jumping—in which workers drop off the cliff face while clinging to invaders and then return to their nest. M. sofina is a restricted range species under threat of extinction by habitat destruction. Its novel lifestyle underscores the urgency of exploration and conservation in a tropical biodiversity hotspot.     

Planarian Immobilization,Partial Irradiation,and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology^1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts³. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal⁴, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a naïve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.Both tissue transplantation^5,6 and partial irradiation⁷ have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of naïve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals⁸. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.     

Phosphorylation of Synaptic GTPase-activating Protein (synGAP) by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) and Cyclin-dependent Kinase 5 (CDK5) Alters the Ratio of Its GAP Activity toward Ras and Rap GTPases

synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAP''s HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons.