Complexities of autonomy in military medical ethics: A brief report.

The first Fallen Surgeons Military Educational Symposium was convened in conjunction with the the American Association for the Surgery of Trauma (AAST) 23 meeting, under the guidance of the AAST Military Liaison Committee. The daylong session included a 1.5-hour segment on military medical ethics in combat and its unique challenges.

JASPer controls interphase histone H3S10 phosphorylation by chromosomal kinase JIL-1 in Drosophila.

In flies, the chromosomal kinase JIL-1 is responsible for most interphase histone H3S10 phosphorylation and has been proposed to protect active chromatin from acquiring heterochromatic marks, such as dimethylated histone H3K9 (H3K9me2) and HP1. Here, we show that JIL-1's targeting to chromatin depends on a PWWP domain-containing protein JASPer (JIL-1 Anchoring and Stabilizing Protein). JASPer-JIL-1 (JJ)-complex is the major form of kinase in vivo and is targeted to active genes and telomeric transposons via binding of the PWWP domain of JASPer to H3K36me3 nucleosomes, to modulate transcriptional output.

Evidence for a role of spindle matrix formation in cell cycle progression by antibody perturbation.

In Drosophila it has recently been demonstrated that a spindle matrix in the form of a membrane-less macromolecular assembly embeds the microtubule-based spindle apparatus. In addition, two of its constituents, Megator and Chromator, were shown to function as spatial regulators of spindle checkpoint proteins. However, whether the spindle matrix plays a wider functional role in spatially regulating cell cycle progression factors was unknown.

H2Av facilitates H3S10 phosphorylation but is not required for heat shock-induced chromatin decondensation or transcriptional elongation.

A model has been proposed in which JIL-1 kinase-mediated H3S10 and H2Av phosphorylation is required for transcriptional elongation and heat shock-induced chromatin decondensation. However, here we show that although H3S10 phosphorylation is indeed compromised in the null mutant, chromatin decondensation at heat shock loci is unaffected in the absence of JIL-1 as well as of H2Av and that there is no discernable decrease in the elongating form of RNA polymerase II in either mutant. Furthermore, mRNA for the major heat shock protein Hsp70 is transcribed at robust levels in both and null mutants.

Digitor/dASCIZ Has Multiple Roles in Drosophila Development.

In this study we provide evidence that the spindle matrix protein Skeletor in Drosophila interacts with the human ASCIZ (also known as ATMIN and ZNF822) ortholog, Digitor/dASCIZ. This interaction was first detected in a yeast two-hybrid screen and subsequently confirmed by pull-down assays. We also confirm a previously documented function of Digitor/dASCIZ as a regulator of Dynein light chain/Cut up expression.

The spindle matrix protein, Chromator, is a novel tubulin binding protein that can interact with both microtubules and free tubulin.

The chromodomain protein, Chromator, is localized to chromosomes during interphase; however, during cell division together with other nuclear proteins Chromator redistributes to form a macro molecular spindle matrix complex that embeds the microtubule spindle apparatus. It has been demonstrated that the CTD of Chromator is sufficient for localization to the spindle matrix and that expression of this domain alone could partially rescue Chro mutant microtubule spindle defects. Furthermore, the presence of frayed and unstable microtubule spindles during mitosis after Chromator RNAi depletion in S2 cells indicated that Chromator may interact with microtubules.

Genome-wide analysis of regulation of gene expression and H3K9me2 distribution by JIL-1 kinase mediated histone H3S10 phosphorylation in Drosophila.

In this study we have determined the genome-wide relationship of JIL-1 kinase mediated H3S10 phosphorylation with gene expression and the distribution of the epigenetic H3K9me2 mark. We show in wild-type salivary gland cells that the H3S10ph mark is predominantly enriched at active genes whereas the H3K9me2 mark is largely associated with inactive genes. Comparison of global transcription profiles in salivary glands from wild-type and JIL-1 null mutant larvae revealed that the expression levels of 1539 genes changed at least 2-fold in the mutant and that a substantial number (49%) of these genes were upregulated whereas 51% were downregulated.

Histone H3S10 phosphorylation by the JIL-1 kinase in pericentric heterochromatin and on the fourth chromosome creates a composite H3S10phK9me2 epigenetic mark.

The JIL-1 kinase mainly localizes to euchromatic interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase in Drosophila. However, recent findings raised the possibility that the binding of some H3S10ph antibodies may be occluded by the H3K9me2 mark obscuring some H3S10 phosphorylation sites. Therefore, we have characterized an antibody to the epigenetic H3S10phK9me2 double mark as well as three commercially available H3S10ph antibodies.

Domain requirements of the JIL-1 tandem kinase for histone H3 serine 10 phosphorylation and chromatin remodeling in vivo.

The JIL-1 kinase localizes to Drosophila polytene chromosome interbands and phosphorylates histone H3 at interphase, counteracting histone H3 lysine 9 dimethylation and gene silencing. JIL-1 can be divided into four main domains, including an NH2-terminal domain, two separate kinase domains, and a COOH-terminal domain. In this study, we characterize the domain requirements of the JIL-1 kinase for histone H3 serine 10 (H3S10) phosphorylation and chromatin remodeling in vivo.

Evidence against a role for the JIL-1 kinase in H3S28 phosphorylation and 14-3-3 recruitment to active genes in Drosophila.

JIL-1 is the major kinase controlling phosphorylation of histone H3S10 and has been demonstrated to function to counteract heterochromatization and gene silencing. However, an alternative model has been proposed in which JIL-1 is required for transcription to occur, additionally phosphorylates H3S28, and recruits 14-3-3 to active genes. Since these findings are incompatible with our previous demonstration that there are robust levels of transcription in the complete absence of JIL-1 and that JIL-1 is not present at developmental or heat shock-induced polytene chromosome puffs, we have reexamined JIL-1's possible role in H3S28 phosphorylation and 14-3-3 recruitment.

The effect of JIL-1 on position-effect variegation is proportional to the total amount of heterochromatin in the genome.

In this study we have taken advantage of recent whole genome sequencing studies that have determined the DNA content in the heterochromatic regions of each Drosophila chromosome to directly correlate the effect on position-effect variegation of a pericentric insertion reporter line, 118E-10 with the total amount of heterochromatic DNA. Heterochromatic DNA levels were manipulated by adding or subtracting a Y chromosome as well as by the difference in the amount of pericentric heterochromatin between the X and Y chromosome. The results showed a direct, linear relationship between the amount of heterochromatic DNA in the genome and the expression of the w marker gene in the 118E-10 pericentric reporter line and that increasing amounts of heterochromatic DNA resulted in increasing amounts of pigment/gene activity.

A nuclear-derived proteinaceous matrix embeds the microtubule spindle apparatus during mitosis.

The concept of a spindle matrix has long been proposed. Whether such a structure exists, however, and what its molecular and structural composition are have remained controversial. In this study, using a live-imaging approach in Drosophila syncytial embryos, we demonstrate that nuclear proteins reorganize during mitosis to form a highly dynamic, viscous spindle matrix that embeds the microtubule spindle apparatus, stretching from pole to pole.

H3S10 phosphorylation by the JIL-1 kinase regulates H3K9 dimethylation and gene expression at the white locus in Drosophila.

The JIL-1 kinase is a multidomain protein that localizes specifically to euchromatin interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase. Genetic interaction assays have suggested that the function of the epigenetic histone H3S10ph mark is to antagonize heterochromatization by participating in a dynamic balance between factors promoting repression and activation of gene expression as measured by position-effect variegation (PEV) assays. Interestingly, JIL-1 loss-of-function alleles can act either as an enhancer or indirectly as a suppressor of w(m4) PEV depending on the precise levels of JIL-1 kinase activity.

Is randomization to placebo safe? Risk in placebo-controlled angina trials: angina risk meta-analysis.

Objective: It was the aim of this study to document the risks of symptomatic patients with angina in placebo-controlled, anti-anginal drug development trials in which symptom-limited exercise testing was used as the primary endpoint.

Patients And Methods: The original case report forms submitted to the United States Food and Drug Administration in support of approval of new or supplemental new drug applications between 1973 and 2001 were identified and subjected to a by-patient meta-analysis, utilizing both a maximum likelihood analysis and classical Mantel-Haenszel methods.

Results: There were 63 placebo-controlled, clinical trials that randomized 10,865 patients, with 1,047 patient-years of observation time.

The epigenetic H3S10 phosphorylation mark is required for counteracting heterochromatic spreading and gene silencing in Drosophila melanogaster.

The JIL-1 kinase localizes specifically to euchromatin interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase. Genetic interaction assays with strong JIL-1 hypomorphic loss-of-function alleles have demonstrated that the JIL-1 protein can counterbalance the effect of the major heterochromatin components on position-effect variegation (PEV) and gene silencing. However, it is unclear whether this was a causative effect of the epigenetic H3S10 phosphorylation mark, or whether the effect of the JIL-1 protein on PEV was in fact caused by other functions or structural features of the protein.

The chromodomain-containing NH(2)-terminus of Chromator interacts with histone H1 and is required for correct targeting to chromatin.

The chromodomain protein, Chromator, can be divided into two main domains, a NH(2)-terminal domain (NTD) containing the chromodomain (ChD) and a COOH-terminal domain (CTD) containing a nuclear localization signal. During interphase Chromator is localized to chromosomes; however, during cell division Chromator redistributes to form a macro molecular spindle matrix complex together with other nuclear proteins that contribute to microtubule spindle dynamics and proper chromosome segregation during mitosis. It has previously been demonstrated that the CTD is sufficient for targeting Chromator to the spindle matrix.

The pleiohomeotic functions as a negative regulator of Drosophila even-skipped gene during embryogenesis.

Polycomb group (PcG) proteins maintain the spatial expression patterns of genes that are involved in cell-fate specification along the anterior-posterior (A/P) axis. This repression requires cis-acting silencers, which are called PcG response elements (PREs). One of the PcG proteins, Pleiohomeotic (Pho), which has a zinc finger DNA binding protein, plays a critical role in recruiting other PcG proteins to bind to PREs.

A balance between euchromatic (JIL-1) and heterochromatic [SU(var)2-5 and SU(var)3-9] factors regulates position-effect variegation in Drosophila.

In this study, we show that the haplo-enhancer effect of JIL-1 has the ability to counterbalance the haplo-suppressor effect of both Su(var)3-9 and Su(var)2-5 on position-effect variegation, providing evidence that a finely tuned balance between the levels of JIL-1 and the major heterochromatin components contributes to the regulation of gene expression.

Do nuclear envelope and intranuclear proteins reorganize during mitosis to form an elastic, hydrogel-like spindle matrix?

The idea of a spindle matrix has long been proposed in order to account for poorly understood features of mitosis. However, its molecular nature and structural composition have remained elusive. Here, we propose that the spindle matrix may be constituted by mainly nuclear-derived proteins that reorganize during the cell cycle to form an elastic gel-like matrix.

Exogenous fms-like tyrosine kinase 3 ligand overrides brain immune privilege and facilitates recognition of a neo-antigen without causing autoimmune neuropathology.

Soluble antigens diffuse out of the brain and can thus stimulate a systemic immune response, whereas particulate antigens (from infectious agents or tumor cells) remain within brain tissue, thus failing to stimulate a systemic immune response. Immune privilege describes how the immune system responds to particulate antigens localized selectively within the brain parenchyma. We believe this immune privilege is caused by the absence of antigen presenting dendritic cells from the brain.

JIL-1 and Su(var)3-7 interact genetically and counteract each other's effect on position-effect variegation in Drosophila.

The essential JIL-1 histone H3S10 kinase is a key regulator of chromatin structure that functions to maintain euchromatic domains while counteracting heterochromatization and gene silencing. In the absence of the JIL-1 kinase, two of the major heterochromatin markers H3K9me2 and HP1a spread in tandem to ectopic locations on the chromosome arms. Here we address the role of the third major heterochromatin component, the zinc-finger protein Su(var)3-7.

Preparation of Drosophila polytene chromosome squashes for antibody labeling.

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae. In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies. The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.

Asator, a tau-tubulin kinase homolog in Drosophila localizes to the mitotic spindle.

We have used a yeast two-hybrid interaction assay to identify Asator, a tau-tubulin kinase homolog in Drosophila that interacts directly with the spindle matrix protein Megator. Using immunocytochemical labeling by an Asator-specific mAb as well as by transgenic expression of a GFP-labeled Asator construct, we show that Asator is localized to the cytoplasm during interphase but redistributes to the spindle region during mitosis. Determination of transcript levels using qRT-PCR suggested that Asator is expressed throughout development but at relatively low levels.

Chromator is required for proper microtubule spindle formation and mitosis in Drosophila.

The chromodomain protein, Chromator, has been shown to have multiple functions that include regulation of chromatin structure as well as coordination of muscle remodeling during metamorphosis depending on the developmental context. In this study we show that mitotic neuroblasts from brain squash preparations from larvae heteroallelic for the two Chromator loss-of-function alleles Chro(71) and Chro(612) have severe microtubule spindle and chromosome segregation defects that were associated with a reduction in brain size. The microtubule spindles formed were incomplete, unfocused, and/or without clear spindle poles and at anaphase chromosomes were lagging and scattered.