A simple physiologically relevant double-agar-layer method for post-germination treatment of seedlings

Arabidopsis thaliana is frequently grown on semisolid medium in Petri dishes, for various experiments that usually consist of two stages on two distinct growth media. Seedlings are germinated under favorable conditions followed by their transfer to another medium containing the given treatment(s). This often causes secondary effects on seedlings due to root shock, or direct and unavoidable contact of the shoot with the second medium. We have developed a simple and efficient method for the transfer of seedlings grown on semisolid medium with minimal damage. In this double-agar-layer method, seeds are germinated on a thin growth-medium-containing agar layer. Subsequently, medium blocks containing the embedded seedlings are excised and placed on the second semisolid medium supplemented with the treatment agent. Differential agar concentrations allow easy penetration of the roots into the second medium, but do not allow the shoots to come into contact with it. This unique method offers several advantages over others that are in common use, in which the seedlings are individually transferred to the second medium or alternatively grown on transfer-carrier matrices, such as filter paper, mesh and cellophane. In the presented method, the entire root system faces the growth medium, the shoots are surrounded by air at all growth stages and transfer of the seedlings is much easier. In addition, a large number of seedlings can be transferred in a single step, without stressing the plants or damaging the delicate root system. This method can also be applied to other plant species grown on semisolid media.     

Genetic analysis of hypertropic mutants of Phycomyces

A new class of Phycomyces behavioral mutants with enhanced tropic responses has been analyzed genetically to determine the number of genes involved and the nature of their expression. These hypertropic mutants carry pleiotropic nuclear mutations. Besides their effects on sensory behavior, they also affect morphology and meiotic processes. Behavioral analyses of heterokaryons containing hypertropic and wild-type nuclei in varying proportions show that the hypertropic mutations in strains L82, L84, L86, and L88 are strongly dominant. Conversely, the hypertropic mutations carried by the strains L83, L85, and L87 are strongly recessive. We performed recombination analyses between hypertropic mutants and mutants with diminished phototropism, affected in the seven genes madA to madG. We found no evidence of linkage between the hypertropic mutations and any of these mad mutations. From crosses, we isolated double mutants carrying hypertropic mutations together with madC (night blind) and madG (stiff) mutations. The behavioral phenotypes of the double mutants are intermediate between those of the parentals. Complementation analyses show that the three recessive hypertropic mutations affect the same gene, which we call madH. The expression of the recessive hypertropic allele becomes dominant in heterokaryons carrying madC and madH nuclei; the madC gene has been implicated separately with the photoreceptor at the input to the sensory pathway, while the madH gene is associated with the growth control output. This result suggests the physical interaction of both gene products, madH and madC, in a molecular complex for the photosensory transduction chain.     

Emergent Group Dynamics Governed by Regulatory Cells Produce a Robust Primary T Cell Response

The currently accepted paradigm for the primary T cell response is that effector T cells commit to autonomous developmental programs. This concept is based on several experiments that have demonstrated that the dynamics of a T cell response is largely determined shortly after antigen exposure and that T cell dynamics do not depend on the level and duration of antigen stimulation. Another experimental study has also shown that T cell responses are robust to variations in antigen-specific precursor frequency.     

Bubble-Induced Cave Collapse

Conventional wisdom among cave divers is that submerged caves in aquifers, such as in Florida or the Yucatan, are unstable due to their ever-growing size from limestone dissolution in water. Cave divers occasionally noted partial cave collapses occurring while they were in the cave, attributing this to their unintentional (and frowned upon) physical contact with the cave walls or the aforementioned “natural” instability of the cave. Here, we suggest that these cave collapses do not necessarily result from cave instability or contacts with walls, but rather from divers bubbles rising to the ceiling and reducing the buoyancy acting on isolated ceiling rocks. Using familiar theories for the strength of flat and arched (un-cracked) beams, we first show that the flat ceiling of a submerged limestone cave can have a horizontal expanse of 63 meters. This is much broader than that of most submerged Florida caves (~ 10 m). Similarly, we show that an arched cave roof can have a still larger expanse of 240 meters, again implying that Florida caves are structurally stable. Using familiar bubble dynamics, fluid dynamics of bubble-induced flows, and accustomed diving practices, we show that a group of 1-3 divers submerged below a loosely connected ceiling rock will quickly trigger it to fall causing a “collapse”. We then present a set of qualitative laboratory experiments illustrating such a collapse in a circular laboratory cave (i.e., a cave with a circular cross section), with concave and convex ceilings. In these experiments, a metal ball represented the rock (attached to the cave ceiling with a magnet), and the bubbles were produced using a syringe located at the cave floor.     

Haptoglobin Genotype-dependent Differences in Macrophage Lysosomal Oxidative Injury

The major function of the Haptoglobin (Hp) protein is to control trafficking of extracorpuscular hemoglobin (Hb) thru the macrophage CD163 receptor with degradation of the Hb in the lysosome. There is a common copy number polymorphism in the Hp gene (Hp 2 allele) that has been associated with a severalfold increased incidence of atherothrombosis in multiple longitudinal studies. Increased plaque oxidation and apoptotic markers have been observed in Hp 2-2 atherosclerotic plaques, but the mechanism responsible for this finding has not been determined. We proposed that the increased oxidative injury in Hp 2-2 plaques is due to an impaired processing of Hp 2-2-Hb complexes within macrophage lysosomes, thereby resulting in redox active iron accumulation, lysosomal membrane oxidative injury, and macrophage apoptosis. We sought to test this hypothesis in vitro using purified Hp-Hb complex and cells genetically manipulated to express CD163. CD163-mediated endocytosis and lysosomal degradation of Hp-Hb were decreased for Hp 2-2-Hb complexes. Confocal microscopy using lysotropic pH indicator dyes demonstrated that uptake of Hp 2-2-Hb complexes disrupted the lysosomal pH gradient. Cellular fractionation studies of lysosomes isolated from macrophages incubated with Hp 2-2-Hb complexes demonstrated increased lysosomal membrane oxidation and a loss of lysosomal membrane integrity leading to lysosomal enzyme leakage into the cytoplasm. Additionally, markers of apoptosis, DNA fragmentation, and active caspase 3 were increased in macrophages that had endocytosed Hp 2-2-Hb complexes. These data provide novel mechanistic insights into how the Hp genotype regulates lysosomal oxidative stress within macrophages after receptor-mediated endocytosis of Hb.     

Extracellular matrix permits the expression of von willebrand’s factor,uptake of Di-I-acetylated low density lipoprotein and secretion of prostacyclin in cultures of endothelial cells from rat brain microvessels

Summary Microvascular endothelial cells from the adult rat brain were cultured on Matrigel and found to express many differentiated properties including secretion of prostacyclin (PGI₂) and von Willebrand’s factor (vWF). Brain microvascular endothelial cells (BMECs) were purified by dextran and percoll gradients after enzymatic treatment and cultured under various conditions. BMECs that were plated on Matrigel stained positively for factor VIII-related antigen and incorporated Di-I-acetylated low density lipoprotein, whereas BMEC plated on fibronectin, gelatin, or uncoated dishes did not express any of the above properties which are characteristic of endothelial cells. vWF was measured by a sensitive ELISA in the culture media of BMECs plated on different types of matrices. Specificity of the anti-human vWF antibodies for the rat vWF was verified by immunoabsorption on a solid phase, sodium dodecyl sulfate, and Western blot analysis. BMECs also secreted vWF into the culture media only when the cells were plated on Matrigel, and this secretion was augmented after a 6 h incubation with an interleukin-1 tumor necrosis factor-α mixture, but not by lipopolysaccharide. From different matrices tested, only Matrigel permitted the secretion of PGI₂ by BMECs. Cells also proved to be sensitive to mechanical stimulation and became refractory to secretagogue if the mechanical stimulation was serially repeated. Under the best conditions, stimulation of the cells with bradykinin (1μM) substantially increased PGI₂ secretion. These data indicate that growth of BMECs on Matrigel in vitro permits the expression of classical endothelial cell markers in a manner similar to the behavior of these cells in situ. Supported by grant 1 RO1 NS2822501 from National Institutes of Health, Bethesda, MD.     

Direct Transfer of Viral and Cellular Proteins from Varicella-Zoster Virus-Infected Non-Neuronal Cells to Human Axons

Varicella Zoster Virus (VZV), the alphaherpesvirus that causes varicella upon primary infection and Herpes zoster (shingles) following reactivation in latently infected neurons, is known to be fusogenic. It forms polynuclear syncytia in culture, in varicella skin lesions and in infected fetal human ganglia xenografted to mice. After axonal infection using VZV expressing green fluorescent protein (GFP) in compartmentalized microfluidic cultures there is diffuse filling of axons with GFP as well as punctate fluorescence corresponding to capsids. Use of viruses with fluorescent fusions to VZV proteins reveals that both proteins encoded by VZV genes and those of the infecting cell are transferred in bulk from infecting non-neuronal cells to axons. Similar transfer of protein to axons was observed following cell associated HSV1 infection. Fluorescence recovery after photobleaching (FRAP) experiments provide evidence that this transfer is by diffusion of proteins from the infecting cells into axons. Time-lapse movies and immunocytochemical experiments in co-cultures demonstrate that non-neuronal cells fuse with neuronal somata and proteins from both cell types are present in the syncytia formed. The fusogenic nature of VZV therefore may enable not only conventional entry of virions and capsids into axonal endings in the skin by classical entry mechanisms, but also by cytoplasmic fusion that permits viral protein transfer to neurons in bulk.     

Integrated Microfluidics for Protein Modification Discovery

Protein post-translational modifications mediate dynamic cellular processes with broad implications in human disease pathogenesis. There is a large demand for high-throughput technologies supporting post-translational modifications research, and both mass spectrometry and protein arrays have been successfully utilized for this purpose. Protein arrays override the major limitation of target protein abundance inherently associated with MS analysis. This technology, however, is typically restricted to pre-purified proteins spotted in a fixed composition on chips with limited life-time and functionality. In addition, the chips are expensive and designed for a single use, making complex experiments cost-prohibitive. Combining microfluidics with in situ protein expression from a cDNA microarray addressed these limitations. Based on this approach, we introduce a modular integrated microfluidic platform for multiple post-translational modifications analysis of freshly synthesized protein arrays (IMPA). The system''s potency, specificity and flexibility are demonstrated for tyrosine phosphorylation and ubiquitination in quasicellular environments. Unlimited by design and protein composition, and relying on minute amounts of biological material and cost-effective technology, this unique approach is applicable for a broad range of basic, biomedical and biomarker research.Protein post-translational modifications (PTMs)¹ vastly diversify eukaryotic proteomes and are integrated in essentially all cellular processes (1). Proteomic approaches, such as mass spectrometry (MS), have been instrumental in monitoring global molecular dynamics for research and clinical applications (2–5). However, even in this modern era, large-scale analyses of PTMs by MS is challenging because of the limited number of modified peptides derived from proteins that, by themselves, may not be abundant. Moreover, comprehensive PTM analysis by MS often requires significant amounts of biological material that may not be available. PTM analysis using protein arrays can overcome these limitations because of the equimolar amount of the arrayed proteins (6, 7). Large-scale protein arrays have been successfully integrated into PTM research (8, 9). However, this technology relies on pre-purified proteins that are arrayed on a surface and thus, incompatible with biochemically challenging proteins, let alone insoluble proteins. Moreover, the production of recombinant protein arrays is impractical in-house. Therefore, such arrays cannot be used fresh, and they are inherently limited to certain designs, protein compositions, and model organisms of high commercial value. To overcome the abovementioned limitations, we designed a modular integrated microfluidic platform for PTM analysis (IMPA).     

Calibrating the Human Mutation Rate via Ancestral Recombination Density in Diploid Genomes

The human mutation rate is an essential parameter for studying the evolution of our species, interpreting present-day genetic variation, and understanding the incidence of genetic disease. Nevertheless, our current estimates of the rate are uncertain. Most notably, recent approaches based on counting de novo mutations in family pedigrees have yielded significantly smaller values than classical methods based on sequence divergence. Here, we propose a new method that uses the fine-scale human recombination map to calibrate the rate of accumulation of mutations. By comparing local heterozygosity levels in diploid genomes to the genetic distance scale over which these levels change, we are able to estimate a long-term mutation rate averaged over hundreds or thousands of generations. We infer a rate of 1.61 ± 0.13 × 10⁻⁸ mutations per base per generation, which falls in between phylogenetic and pedigree-based estimates, and we suggest possible mechanisms to reconcile our estimate with previous studies. Our results support intermediate-age divergences among human populations and between humans and other great apes.