EPO improved neurologic outcome in rat pups late after traumatic brain injury.

Unlabelled: In adult rats, erythropoietin improved outcomes early and late after traumatic brain injury, associated with increased levels of Brain Derived Neurotrophic Factor. Using our model of pediatric traumatic brain injury, controlled cortical impact in 17-day old rats, we previously showed that erythropoietin increased hippocampal neuronal fraction in the first two days after injury. Erythropoietin also decreased activation of caspase3, an apoptotic enzyme modulated by Brain Derived Neurotrophic Factor, and improved Novel Object Recognition testing 14 days after injury.

Downmodulation of CCR7 by HIV-1 Vpu results in impaired migration and chemotactic signaling within CD4⁺ T cells.

The chemokine receptor CCR7 plays a crucial role in the homing of central memory and naive T cells to peripheral lymphoid organs. Here, we show that the HIV-1 accessory protein Vpu downregulates CCR7 on the surface of CD4(+) T cells. Vpu and CCR7 were found to specifically interact and colocalize within the trans-Golgi network, where CCR7 is retained.

Erythropoietin improved cognitive function and decreased hippocampal caspase activity in rat pups after traumatic brain injury.

Unlabelled: Traumatic brain injury (TBI) is a leading cause of acquired neurologic disability in children. Erythropoietin (EPO), an anti-apoptotic cytokine, improved cognitive outcome in adult rats after TBI. To our knowledge, EPO has not been studied in a developmental TBI model.

Effects of redox state on the efficient uptake of cell permeable Peptide in Mammalian cells.

We investigated whether a cell-penetrating peptide linked via a disulfide bond to a fluorophore-labeled cargo peptide can be used to interrogate changes in cellular redox state. A fluorescence resonance energy transfer (FRET) pair was constructed so that the cargo peptide was labeled with fluorescein amidite (FAM) and the cell-penetrating peptide was attached to a quencher. Incubation of cells in culture with the FRET construct was visualized using live-cell, time-lapse imaging, which demonstrated earlier cellular uptake of the construct when cells were treated with the reducing agent n-acetylcysteine (NAC).

Real-time fluorescent image analysis of DNA spot hybridization kinetics to assess microarray spot heterogeneity.

Current microarray assay technology predominately uses fluorescence as a detectable signal end point. This study assessed real-time in situ surface hybridization capture kinetics for single printed DNA microspots on solid array surfaces using fluorescence. The influence of the DNA target and probe cyanine dye position on oligo-DNA duplex formation behavior was compared in solution versus surface-hybridized single DNA printed spots using fluorescence resonance energy transfer (FRET) analysis.

Quantification of dystrophin immunofluorescence in dystrophinopathy muscle specimens.

Aims: Duchenne muscular dystrophy (DMD) is usually associated with absent or nearly absent dystrophin expression at the sarcolemmal membrane. Quantification of very low levels of dystrophin signal in immunofluorescent studies of muscle biopsy sections presents a technical challenge. This is particularly true in the setting of proof-of-principle drug trials, in which the detection and quantification of what may be significant changes in levels of expression is important, even if absolute dystrophin levels remain low.

Glutathione-dependent reductive stress triggers mitochondrial oxidation and cytotoxicity.

To investigate the effects of the predominant nonprotein thiol, glutathione (GSH), on redox homeostasis, we employed complementary pharmacological and genetic strategies to determine the consequences of both loss- and gain-of-function GSH content in vitro. We monitored the redox events in the cytosol and mitochondria using reduction-oxidation sensitive green fluorescent protein (roGFP) probes and the level of reduced/oxidized thioredoxins (Trxs). Either H(2)O(2) or the Trx reductase inhibitor 1-chloro-2,4-dinitrobenzene (DNCB), in embryonic rat heart (H9c2) cells, evoked 8 or 50 mV more oxidizing glutathione redox potential, E(hc) (GSSG/2GSH), respectively.

Exploiting antitumor immunity to overcome relapse and improve remission duration.

Cancer survivors often relapse due to evolving drug-resistant clones and repopulating tumor stem cells. Our preclinical study demonstrated that terminal cancer patient's lymphocytes can be converted from tolerant bystanders in vivo into effective cytotoxic T-lymphocytes in vitro killing patient's own tumor cells containing drug-resistant clones and tumor stem cells. We designed a clinical trial combining peginterferon α-2b with imatinib for treatment of stage III/IV gastrointestinal stromal tumor (GIST) with the rational that peginterferon α-2b serves as danger signals to promote antitumor immunity while imatinib's effective tumor killing undermines tumor-induced tolerance and supply tumor-specific antigens in vivo without leukopenia, thus allowing for proper dendritic cell and cytotoxic T-lymphocyte differentiation toward Th1 response.

Human ESCRT-III and VPS4 proteins are required for centrosome and spindle maintenance.

The ESCRT pathway helps mediate the final abscission step of cytokinesis in mammals and archaea. In mammals, two early acting proteins of the ESCRT pathway, ALIX and TSG101, are recruited to the midbody through direct interactions with the phosphoprotein CEP55. CEP55 resides at the centrosome through most of the cell cycle but then migrates to the midbody at the start of cytokinesis, suggesting that the ESCRT pathway may also have centrosomal links.

Time-lapse imaging of mitosis after siRNA transfection.

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered.

Direct toxic effects of aqueous extract of cigarette smoke on cardiac myocytes at clinically relevant concentrations.

Aims: Our goal was to determine if clinically relevant concentrations of aqueous extract of cigarette smoke (CSE) have direct deleterious effects on ventricular myocytes during simulated ischemia, and to investigate the mechanisms involved.

Methods: CSE was prepared with a smoking chamber. Ischemia was simulated by metabolic inhibition (MI) with cyanide (CN) and 0 glucose.

Impaired neutrophil extracellular trap (NET) formation: a novel innate immune deficiency of human neonates.

Neutrophils are highly specialized innate effector cells that have evolved for killing of pathogens. Human neonates have a common multifactorial syndrome of neutrophil dysfunction that is incompletely characterized and contributes to sepsis and other severe infectious complications. We identified a novel defect in the antibacterial defenses of neonates: inability to form neutrophil extracellular traps (NETs).

Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis.

TSG101 and ALIX both function in HIV budding and in vesicle formation at the multivesicular body (MVB), where they interact with other Endosomal Sorting Complex Required for Transport (ESCRT) pathway factors required for release of viruses and vesicles. Proteomic analyses revealed that ALIX and TSG101/ESCRT-I also bind a series of proteins involved in cytokinesis, including CEP55, CD2AP, ROCK1, and IQGAP1. ALIX and TSG101 concentrate at centrosomes and are then recruited to the midbodies of dividing cells through direct interactions between the central CEP55 'hinge' region and GPP-based motifs within TSG101 and ALIX.

An essential Drosophila glutamate receptor subunit that functions in both central neuropil and neuromuscular junction.

A Drosophila forward genetic screen for mutants with defective synaptic development identified bad reception (brec). Homozygous brec mutants are embryonic lethal, paralyzed, and show no detectable synaptic transmission at the glutamatergic neuromuscular junction (NMJ). Genetic mapping, complementation tests, and genomic sequencing show that brec mutations disrupt a previously uncharacterized ionotropic glutamate receptor subunit, named here "GluRIID.

Cellular bases of activity-dependent paralysis in Drosophila stress-sensitive mutants.

Stress-sensitive mutants in Drosophila have been shown to exhibit activity-dependent defects in neurotransmission. Using the neuromuscular junction (NMJ), this study investigates synaptic function more specifically in two stress-sensitive mutants: stress-sensitive B (sesB), which encodes a mitochondrial ADP/ATP translocase (ANT); and Atpalpha(2206), a conditional mutant of the Na+/K+ ATPase alpha-subunit. Mechanical shock induces a period of brief paralysis in both homozygous and double heterozygous mutants, but further analysis revealed distinct activity-dependent neurotransmission lesions in each mutant.

FISHing for chick genes: Triple-label whole-mount fluorescence in situ hybridization detects simultaneous and overlapping gene expression in avian embryos.

Multi-color whole-mount in situ hybridization is a powerful technique for comparing the spatial expression patterns of two or more genes in developing embryos. We have developed an amplified triple-label whole-mount fluorescence in situ hybridization (FISH) protocol that permits detection of three different mRNAs in a single embryo. Our protocol uses simultaneous in situ hybridization to haptenylated riboprobes, followed by sequential antibody detection using anti-hapten antibodies conjugated to horseradish peroxidase, and the tyramide signal amplification (TSA) fluorescence detection system.

The ubiquitin proteasome system acutely regulates presynaptic protein turnover and synaptic efficacy.

Background: The ubiquitin proteasome system (UPS) mediates regulated protein degradation and provides a mechanism for closely controlling protein abundance in spatially restricted domains within cells. We hypothesized that the UPS may acutely determine the local concentration of key regulatory proteins at neuronal synapses as a means for locally modulating synaptic efficacy and the strength of neurotransmission communication.

Results: We investigated this hypothesis at the Drosophila neuromuscular synapse by using an array of genetic and pharmacological tools.

Genetic studies in Drosophila: vesicle pools and cytoskeleton-based regulation of synaptic transmission.

Presynaptic plasticity mechanisms rely on modulation of the synaptic vesicle fusion machinery and the regulated mobilization of synaptic vesicles at the active zone. This review discusses recent evidence suggesting that the relative proportions of synaptic vesicles in the reserve and ready releasable pools is the primary determinant of synaptic transmission strength, and that transport of vesicles between these pools is mediated by cytoskeletal mechanisms. Recent efforts to identify the molecules required for regulation of the presynaptic cytoskeleton suggest that common mechanisms may exist to regulate synaptic vesicle pools in widely divergent neuronal types, ranging from synaptic modulation at the Drosophila neuromuscular junction to the synaptic plasticity required for learning and memory in the mammalian brain.

Drosophila UNC-13 is essential for synaptic transmission.

The UNC-13 protein family has been suggested to be critical for synaptic vesicle dynamics based on its interactions with Syntaxin, Munc-18 and Doc 2alpha. We cloned the Drosophila homolog (Dunc-13) and characterized its function using a combination of electrophysiology and ultrastructural analyses. Dunc-13 contained a C1 lipid-binding motif and two C2 calcium-binding domains, and its expression was restricted to neurons.

The Drosophila ovarian tumor gene is required for the organization of actin filaments during multiple stages in oogenesis.

The ovarian tumor gene is required during both early and late stages of oogenesis. Mutations produce a range of phenotypes, including agametic ovarioles, tumorous egg chambers, and late stage oogenic arrest. We demonstrate that each of these phenotypes is associated with specific aberrations in actin distribution.

[Oocyte donation: ethical aspects related to the donor].

This paper analyses the reasons that brings couples to apprehend very differently the oocyte and sperm donation and highlights the difficulty of donor recruitment in those programs. The various types of donors (occasional, relational, IVF patient and professional) are described with their motivations, resistances, advantages and disadvantages. The contradictory consequences between free or paid donation, the particular risks of eggs donation (in comparison to sperm donation) for the donor as for the recipient are also highlighted.

Oocyte shortage for donation may be overcome in a programme with anonymous permutation of related donors.

Difficulty in recruiting donors plays a crucial role in oocyte donation programmes. We report here an original strategy to overcome this problem, consisting of sharing out anonymous oocytes between recipients, which has markedly improved treatment efficiency by avoiding freezing or destruction of supernumerary embryos. Each patient received part of the supply of oocytes from several donors in four successive cycles, while each donor underwent oocyte retrieval only once.

A P element containing suppressor of hairy-wing binding regions has novel properties for mutagenesis in Drosophila melanogaster.

P elements are widely used as insertional mutagens to tag genes, facilitating molecular cloning and analyses. We modified a P element so that it carried two copies of the suppressor of Hairy-wing [su(Hw)] binding regions isolated from the gypsy transposable element. This transposon was mobilized, and the genetic consequences of its insertion were analyzed.

[Update on ovarian hyperstimulation syndrome].

The ovarian hyperstimulation syndrome (OHSS) is the most important complication of the pharmacological ovulation induction. Exclusively postovulatory, it is clinically characterized by a massive ovarian enlargement associated with an acute third-space fluid shift responsible for the development of ascites, and sometimes pleural and/or pericardial effusion. While mild OHSS has no consequence, severe forms can be life-threatening because of associated hemodynamic troubles.