Stress response gene family expansions correlate with invasive potential in teleost fish.

The bluegill sunfish Lepomis macrochirus and the closely related redear sunfish Lepomis microlophus have important ecological and recreational value and are widely used for research and aquaculture. While both species have been introduced outside of their native ranges, only the bluegill is considered invasive. Here, we report de novo transcriptome assemblies for these fish as a resource for sunfish biology. Comparative analyses of the transcriptomes revealed an unexpected, bluegill-specific expansion in the HSP70 and HSP90 molecular chaperone gene families. These expansions were not unique to the bluegill as expansions in HSP70s and HSP90s were identified in the genomes of other teleost fish using the NCBI RefSeq database. To determine whether gene family expansions are specific for thermal stress responses, GST and SOD gene families that are associated with oxidative stress responses were also analyzed. Species-specific expansions were also observed for these gene families in distinct fish species. Validating our approach, previously described expansions in the MHC gene family were also identified. Intriguingly, the number of HSP70 paralogs was positively correlated with thermotolerance range for each species, suggesting that these expansions can impact organismal physiology. Furthermore, fish that are considered invasive contained a higher average number of HSP70 paralogs than non-invasive fish. Invasive fish also had higher average numbers of HSP90, MHC and GST paralogs, but not SOD paralogs. Taken together, we propose that expansions in key cellular stress response gene families represent novel genetic signatures that correlate with invasive potential.

Urine Ammonium Predicts Clinical Outcomes in Hypertensive Kidney Disease.

Metabolic acidosis is associated with poor outcomes in CKD. Because impaired renal ammonium excretion is important in the pathogenesis of acidosis, urine ammonium excretion might be a better and perhaps earlier acid-base indicator of risk than serum bicarbonate, particularly in patients without acidosis. We evaluated the association between baseline ammonium excretion and clinical outcomes in African American Study of Kidney Disease and Hypertension participants (n=1044). Median daily ammonium excretion was 19.5 (95% confidence interval [95% CI], 6.5 to 43.2) mEq. In Cox regression models (adjusted for demographics, measured GFR, proteinuria, body mass index, net endogenous acid production, and serum potassium and bicarbonate), hazard ratios of the composite outcome of death or dialysis were 1.46 (95% CI, 1.13 to 1.87) in the low tertile and 1.14 (95% CI, 0.89 to 1.46) in the middle tertile of daily ammonium excretion compared with the high tertile. Among participants without acidosis at baseline, the adjusted hazard ratio for those with ammonium excretion <20 mEq/d was 1.36 (95% CI, 1.09 to 1.71) compared with those with ammonium excretion ≥20 mEq/d. Additionally, compared with participants in the high ammonium tertile, those in the low ammonium tertile had higher adjusted odds of incident acidosis at 1 year (adjusted odds ratio, 2.56; 95% CI, 1.04 to 6.27). In conclusion, low ammonium excretion is associated with death and renal failure in hypertensive kidney disease, even among those without acidosis. Low ammonium excretion could identify patients with CKD and normal bicarbonate levels who might benefit from alkali before acidosis develops.

Visualization of the biphasic calcium wave during fertilization in Caenorhabditis elegans using a genetically encoded calcium indicator.

Fertilization is a critical step in development, yet internal fertilization events are notoriously difficult to visualize. Taking advantage of the calcium response that is a hallmark of sperm-egg fusion, we adapted the genetically encoded calcium indicator jGCaMP7s to visualize the moment of fertilization in Caenorhabditis elegans using fluorescence. We termed this tool the 'CaFE' reporter, for 'calcium during fertilization in C. elegans'. The CaFE reporter produced a robust signal that recapitulated the previously reported, biphasic nature of the calcium wave and had no significant deleterious effects on worm physiology or fecundity. Calcium waves were not observed at the restrictive temperature in the spe-9(hc88) strain, in which sperm can still trigger meiotic maturation but can no longer fuse with the oocyte. Demonstrating the utility of the CaFE reporter, we analyzed polyspermy induced by inhibition of egg-3 via RNAi and observed late calcium waves in the uterus. This finding provides support to the idea that calcium release is not restricted to the first sperm fusion event during polyspermy. Establishment of the CaFE reporter in the genetically tractable and optically transparent worm provides a powerful tool to dissect the oocyte-to-embryo transition inside a living animal.

Integrating experience with databases, bioinformatics, and wet lab exercises for students in an introductory genetics course.

Students often have no exposure to the incredible amount of genomic and proteomic data that is freely and easily accessible online. Becoming familiar with these resources, and seeing how they could be applied to a specific research question, is a prerequisite for students to apply them to their own scientific development. Many students have to "see it" and "do it" before they "get it." This paper describes a teaching laboratory for undergraduate Genetics students that combines exploration of several publicly available databases with some simple bioinformatic exercises and "'real" live experience in a wet lab exercise. The goal is to teach skills in applying genomic data to a real scientific question. In this exercise, students identify a target protein after exploring several protein and signal transduction databases, such as the Kyoto Encyclopedia of Genes and Genomes database. They then search for the encoding RNA in a newly available sea star mature egg transcriptome database and for the DNA in an existing sea star genome database. The students design primers against specific regions or domains in their target RNA and amplify these by reverse transcription PCR against RNA purified from fresh sea star eggs. The PCR reactions are analyzed by agarose gel electrophoresis. It is hoped that the combination of the computational biology exercises with the real lab work will excite the students and stimulate them to explore this exciting new biology further.

Peripheral Joint Radiofrequency Ablation.

This article provides a detailed description of peripheral joint radiofrequency ablation and its contemporary use in the treatment of chronic knee, hip, and shoulder pain. Special attention is given to anatomy and innervation of the joints discussed, technical approach, selection criteria, contraindications, and patient outcomes.

Protein tyrosine kinase signaling during oocyte maturation and fertilization.

The oocyte is a highly specialized cell capable of accumulating and storing energy supplies as well as maternal transcripts and pre-positioned signal transduction components needed for zygotic development, undergoing meiosis under control of paracrine signals from the follicle, fusing with a single sperm during fertilization, and zygotic development. The oocyte accomplishes this diverse series of events by establishing an array of signal transduction pathway components that include a select collection of protein tyrosine kinases (PTKs) that are expressed at levels significantly higher than most other cell types. This array of PTKs includes cytosolic kinases such as SRC-family PTKs (FYN and YES), and FAK kinases, as well as FER. These kinases typically exhibit distinct patterns of localization and in some cases are translocated from one subcellular compartment to another during meiosis. Significant differences exist in the extent to which PTK-mediated pathways are used by oocytes from species that fertilize externally versus internally. The PTK activation profiles as well as calcium signaling pattern seems to correlate with the extent to which a rapid block to polyspermy is required by the biology of each species. Suppression of each of the SRC-family PTKs as well as FER kinase results in failure of meiotic maturation or zygote development, indicating that these PTKs are important for oocyte quality and developmental potential. Future studies will hopefully reveal the extent to which these factors impact clinical assisted reproductive techniques in domestic animals and humans.

Recovery of Sea Star Egg Cell Surface Proteins Released at Fertilization.

To provide a better understanding of the composition of the egg cell membrane, we describe a method in which proteins and peptides that are either naturally released by the egg or cleaved by sperm proteases can be collected, analyzed, and identified. Such molecules are captured and isolated from the surrounding seawater via biotinylation, before being concentrated by an affinity interaction and subsequently analyzed by western blotting and mass spectrometry.

Identification of SH2 Domain-Mediated Protein Interactions that Operate at Fertilization in the Sea Star Patiria miniata.

The signaling mechanisms controlling internal calcium release at fertilization in animals are still largely unknown. Echinoderms, such as the sea star Patiria miniata, produce abundant and easily accessible sperm and eggs. In addition, eggs are naturally synchronized at the same cell cycle stage, collectively making these animals an attractive model to study the signaling proteins controlling fertilization. However, the lack of antibodies to identify proteins in this model system has slowed progress in identifying key signaling molecules. With the advances in mass spectrometry, we present a method for identifying tyrosine phosphorylated proteins binding to GST-tagged SH2 domains in sea star cell lysates for downstream mass spectrometry analysis.

Identification of Prognostic Biomarker Candidates Associated With Melanoma Using High-Dimensional Genomic Data.

Survival of patients with metastatic melanoma varies widely. Melanoma is a highly proliferative, chemo-resistant disease. With the recent availability of immunotherapies such as checkpoint inhibitors, durable response rates have improved but are often still limited to 2-3 years. Response rates to treatment range from 30 to 45% with combination therapy however no improvement in overall survival is frequently observed. Of the available therapies, many have targeted the BRAFV600E mutation that results in abnormal MAPK pathway activation which is important for regulating cell proliferation. Immune checkpoint inhibitors such as anti-PD-1 and anti-PD-L1 offer better success but response rates are still low. Identifying biomarkers to better target those who will respond and identify the right combination of treatment is the best approach. In this study, we utilize data from the Cancer Cell Line Encyclopedia (CCLE), including 62 samples, to examine features of gene expression (19K+) and copy number (20K+) in the melanoma cell lines. We perform a clustering analysis on the feature set to assess genetically similarity among the cell lines. We then discover which specific genes and combinations thereof maximize cluster density. We design a feature selection approach for high-dimensional datasets that integrates multiple disparate machine learning techniques into one cohesive pipeline. Our approach provides a small subset of genes that can accurately distinguish between the clusters of melanoma cell lines across multiple types of classifiers. In particular, we find only the 15 highest ranked genes among the original 19 K are necessary to achieve perfect or near-perfect test split classification performance. Of these 15 genes, some are known to be linked to melanoma or other cancer progressions, while others have not previously been linked to melanoma and are of interest for further examination.

Combining microinjection and immunoblotting to analyze MAP kinase phosphorylation in single starfish oocytes and eggs.

The starfish oocyte has proven useful for studies involving microinjection because it is relatively large (190 mum) and optically clear. These oocytes are easily obtained from the ovary arrested at prophase of meiosis I, making them useful as a model system for the study of cell cycle-related events. In this chapter, a method for combining microinjection with immunoblotting of single cells is described. Individual starfish oocytes are injected, removed from the microinjection chamber, and analyzed by immunoblotting for the dual-phosphorylated form of mitogen-activated protein kinase (MAPK). This method will allow for experiments testing the regulation of MAPK in single cells and for the manipulation of these cells by a quantitative microinjection technique.

Analyzing gene expression in sea star eggs and embryos using bioinformatics.

The explosion in the amount of genomic data has changed how we educate future biologists. Students are inundated with information and need to develop skills in how to observe and understand this data. This chapter describes a teaching laboratory for undergraduate Genetics students that uses a combination of bioinformatics and wet lab exercises to teach skills in applying genomic data to a real scientific question. Students identify a target protein and search for the encoding RNA in newly available sea star transcriptome databases. The students design primers against specific regions or domains in their target RNA and amplify these by reverse transcription PCR. The PCR reactions are analyzed by agarose gel electrophoresis. The goal of this laboratory is to provide an example of how bioinformatics can be used to solve a real biological problem.

Starfish as a Model System for Analyzing Signal Transduction During Fertilization.

The starfish oocyte and egg offer advantages for use as a model system for signal transduction research. Some of these have been recognized for over a century, including the ease of procuring gametes, in vitro fertilization, and culturing the embryos. New advances, particularly in genomics, have also opened up opportunities for the use of these animals. In this chapter, we give a few examples of the historical use of the starfish for research in cell biology and then describe some new areas in which we believe the starfish can contribute to our understanding of signal transduction-particularly in fertilization.

Biotinylation of oocyte cell surface proteins of the starfish Patiria miniata.

Understanding the signal transduction processes that occur during oocyte maturation and fertilization requires knowledge of the constituent proteins from the cell surface to relevant intracellular compartments. To identify starfish oocyte and egg cell surface proteins, a biotinylation method was adapted from prior protocols using B cells, leukocytes, mouse oocytes, and sea urchin eggs (Cole et al. Mol Immunol 24:699-705, 1987; Flaherty and Swann NJ. Mol Reprod Dev 35:285-292, 1993; Haley and Wessel. Dev Biol 272:191-202, 2004; Hurley et al. J Immunol Methods 85:195-202, 1985). This method utilizes the water-soluble Sulfo-NHS-Biotin, which does not cross the egg plasma membrane. The process of biotinylation does not appear to have any effect on the process of oocyte maturation or fertilization. Furthermore, it can be used with either vitelline-intact or vitelline-free oocytes and allows the proteins to be visualized successfully through immunoblotting, immunoprecipitation, or by scanning confocal microscopy.

Roles of Src family kinase signaling during fertilization and the first cell cycle in the marine protostome worm Cerebratulus.

For eggs to generate a calcium response during fertilization, the sperm of many deuterostome animals must first activate a group of egg kinases, called Src family kinases (SFKs). However, whether SFK activation is also required for fertilization-induced calcium signals in eggs of protostomes remains unknown. Thus, in this study, unfertilized oocytes of the marine protostome worm Cerebratulus were treated with either PP2 to inhibit SFKs or with U73122 to block phospholipase C activity downstream of SFK. Compared with control fertilizations, the inhibitors significantly reduced post-insemination levels of polar body formation and cleavage, but apparently did so via different mechanisms, based on the variable effects of these drugs on sperm incorporations and pronuclear differentiation. Moreover, confocal calcium imaging revealed that repetitive calcium waves (=oscillations) were blocked by U73122, but not by PP2, even though immunoblots indicated SFK activity was inhibited by PP2. Such findings fail to support the view that SFKs are required for initiating fertilization-induced calcium oscillations in Cerebratulus, and alternative mechanisms for the observed inhibition of polar body formation and cleavage in drug-treated specimens are discussed.

Identification of a starfish egg PLC-gamma that regulates Ca2+ release at fertilization.

At fertilization, eggs undergo a cytoplasmic free Ca2+ rise, which is necessary for stimulating embryogenesis. In starfish eggs, studies using inhibitors designed against vertebrate proteins have shown that this Ca2+ rise requires an egg Src family kinase (SFK) that directly or indirectly activates phospholipase C-gamma (PLC-gamma) to produce IP3, which triggers Ca2+ release from the egg's endoplasmic reticulum (ER) [reviewed in Semin. Cell Dev. Biol. 12 (2001) 45]. To examine in more detail the endogenous factors in starfish eggs that are required for Ca2+ release at fertilization, an oocyte cDNA encoding PLC-gamma was isolated from the starfish Asterina miniata. This cDNA, designated AmPLC-gamma, encodes a protein with 49% identity to mammalian PLC-gamma1. A 58-kDa Src family kinase interacted with recombinant AmPLC-gamma Src homology 2 (SH2) domains in a specific, fertilization-responsive manner. Immunoprecipitations of sea urchin egg PLC-gamma using an affinity-purified antibody directed against AmPLC-gamma revealed fertilization-dependent phosphorylation of PLC-gamma. Injecting starfish eggs with the tandem SH2 domains of AmPLC-gamma (which inhibits PLC-gamma activation) specifically inhibited Ca2+ release at fertilization. These results indicate that an endogenous starfish egg PLC-gamma interacts with an egg SFK and mediates Ca2+ release at fertilization via a PLC-gamma SH2 domain-mediated mechanism.