Phosphoproteomics reveals novel modes of function and inter‐relationships among PIKKs in response to genotoxic stress

The DNA damage response (DDR) is a complex signaling network that relies on cascades of protein phosphorylation, which are initiated by three protein kinases of the family of PI3‐kinase‐related protein kinases (PIKKs): ATM, ATR, and DNA‐PK. ATM is missing or inactivated in the genome instability syndrome, ataxia‐telangiectasia (A‐T). The relative shares of these PIKKs in the response to genotoxic stress and the functional relationships among them are central questions in the genome stability field. We conducted a comprehensive phosphoproteomic analysis in human wild‐type and A‐T cells treated with the double‐strand break‐inducing chemical, neocarzinostatin, and validated the results with the targeted proteomic technique, selected reaction monitoring. We also matched our results with 34 published screens for DDR factors, creating a valuable resource for identifying strong candidates for novel DDR players. We uncovered fine‐tuned dynamics between the PIKKs following genotoxic stress, such as DNA‐PK‐dependent attenuation of ATM. In A‐T cells, partial compensation for ATM absence was provided by ATR and DNA‐PK, with distinct roles and kinetics. The results highlight intricate relationships between these PIKKs in the DDR.     

Stimulus Deprivation Increases Pineal Gsα and Gβ

Denervation and other forms of stimulus deprivation cause an increase in the magnitude of subsequent responses, a phenomenon commonly referred to as denervation supersensitivity. This has been well demonstrated with the cyclic AMP response to norepinephrine in the pineal gland. In the present report, we address the question of whether stimulus deprivation alters alpha and beta subunits of the GTP binding regulatory protein that stimulates adenylyl cyclase activity (Gs). Stimulus deprivation of the pineal gland was produced by denervation (superior cervical ganglionectomy), decentralization of the superior cervical ganglia, or by exposure of the animal to continuous lighting. All increased both the alpha and beta subunits of Gs (Gs alpha and G beta) by up to fourfold, as estimated using semiquantitative western blot technology. These effects were detectable after 1 day of stimulus deprivation and were sustained for 2 weeks. The stimulatory effects of constant light-induced stimulus deprivation were also apparent by measuring cholera toxin-dependent ADP-ribosylation of Gs alpha, which revealed a four-fold increase in the amount of labeled substrate. The results of in vivo studies were confirmed with in vitro studies, which demonstrated a spontaneous increase in both Gs alpha and G beta during 72 h of organ culture. The constant light-induced increases in both Gs alpha and G beta were prevented by continuous administration of isoproterenol (0.3 mg/kg/day), supporting the suggestion that adrenergic stimulation controls the levels of Gs alpha and G beta. These studies indicate that stimulus deprivation increases both Gs alpha and G beta.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dinoflagellate bloom development and collapse in Lake Kinneret: a sediment trap study

Warm monomictic Lake Kinneret, Israel, is characterized by awinter–spring water bloom of the large (~50 µm diameter)dinoflagellate Peridinium gatunense Nygaard. Usually the P.gatunense bloom declines in May–June and a less prominentbloom of smaller dinoflagellates (mostly Peridiniopsis spp.of ~20–30 µm diameter) develops. Water column abundancesand sedimentation losses to those dinoflagellates were followedthroughout 1994 and 1995. The objective was to quantify thevariables that describe population dynamics, that in turn willshed more light on the seasonal patterns of bloom dynamics.Sedimentation losses were measured by means of sediment trapswith and without a preservative (formaldehyde) that were exposedfor 24 h once every 2–3 weeks. Annual sedimentation lossesof Peridinium (hypolimnetic trap catches) were 209 g wet wtm^-2 year^-1 in 1994 and 187 g wet wt m^-2 year^-1 in 1995, whichconstituted 16 and 23% of Peridinium production in those years,respectively. This study revealed that increased death ratespreceded a mass sedimentation flux of Peridinium and causedthe decline of the bloom in Lake Kinneret. Annual sedimentationlosses of Peridiniopsis were 55 g wet wt m^-2 year^-1 in 1994and 34 g wet wt m^-2 year^-1 in 1995. In contrast to live Peridiniumcells, Peridiniopsis cells continued to swim to the lower trapafter the onset of thermal stratification, possibly taking advantageof the higher nutrient concentrations below the thermocline,at a time when the lake is already stratified and the epilimnionis nutrient depleted. This could be an important factor allowingPeridiniopsis spp. to peak after the decline of Peridinium.     

Demonstrating the utility of flexible sequence queries against indexed short reads with FlexTyper

Across the life sciences, processing next generation sequencing data commonly relies upon a computationally expensive process where reads are mapped onto a reference sequence. Prior to such processing, however, there is a vast amount of information that can be ascertained from the reads, potentially obviating the need for processing, or allowing optimized mapping approaches to be deployed. Here, we present a method termed FlexTyper which facilitates a “reverse mapping” approach in which high throughput sequence queries, in the form of k-mer searches, are run against indexed short-read datasets in order to extract useful information. This reverse mapping approach enables the rapid counting of target sequences of interest. We demonstrate FlexTyper’s utility for recovering depth of coverage, and accurate genotyping of SNP sites across the human genome. We show that genotyping unmapped reads can correctly inform a sample’s population, sex, and relatedness in a family setting. Detection of pathogen sequences within RNA-seq data was sensitive and accurate, performing comparably to existing methods, but with increased flexibility. We present two examples of ways in which this flexibility allows the analysis of genome features not well-represented in a linear reference. First, we analyze contigs from African genome sequencing studies, showing how they distribute across families from three distinct populations. Second, we show how gene-marking k-mers for the killer immune receptor locus allow allele detection in a region that is challenging for standard read mapping pipelines. The future adoption of the reverse mapping approach represented by FlexTyper will be enabled by more efficient methods for FM-index generation and biology-informed collections of reference queries. In the long-term, selection of population-specific references or weighting of edges in pan-population reference genome graphs will be possible using the FlexTyper approach. FlexTyper is available at https://github.com/wassermanlab/OpenFlexTyper.     

The levels of cyclic GMP and glucose 1,6-diphosphate,and the activity of phosphofructokinase,in muscle from normal and dystrophic mice

A striking reduction in the levels of glucose 1,6-diphosphate and an increase in cyclic GMP were found in muscle from dystrophic mice. Concomitant to these changes, the allosteric activity of phosphofructokinase was found to be markedly reduced. These findings could offer an explanation for the observed reduction in glycolysis in the dystrophic muscle.     

Animal models for Gaucher disease research

Gaucher disease (GD), the most common lysosomal storage disorder (LSD), is caused by the defective activity of the lysosomal hydrolase glucocerebrosidase, which is encoded by the GBA gene. Generation of animal models that faithfully recapitulate the three clinical subtypes of GD has proved to be more of a challenge than first anticipated. The first mouse to be produced died within hours after birth owing to skin permeability problems, and mice with point mutations in Gba did not display symptoms correlating with human disease and also died soon after birth. Recently, conditional knockout mice that mimic some features of the human disease have become available. Here, we review the contribution of all currently available animal models to examining pathological pathways underlying GD and to testing the efficacy of new treatment modalities, and propose a number of criteria for the generation of more appropriate animal models of GD.     

The giant cells produced by Telenomus remus (Hymenoptera: Scelionidae)

The parasitic larva of Telenomus remus is surrounded by giant cells throughout its first instar. These cells arise in the embryonic serosa of the parasite and grow in size, starting with a radius of 5nm and ending with 27nm. Young cells are round and mononuclear, whereas older ones are often polynuclear and have varied, irregular contours. Most cells are profusely vacuolated, the vacuoles being especially large in some of the older cells. Only a few of the giant cells are devoured by the first instar parasite larva, but all disappear at the end of this stage. No giant cells seem to be produced by supernumerary larvae. Once the parasite egg hatches, the host tissue disintegrates almost instantaneously.