Drosophila motor neuron retraction during metamorphosis is mediated by inputs from TGF-β/BMP signaling and orphan nuclear receptors

Larval motor neurons remodel during Drosophila neuro-muscular junction dismantling at metamorphosis. In this study, we describe the motor neuron retraction as opposed to degeneration based on the early disappearance of β-Spectrin and the continuing presence of Tubulin. By blocking cell dynamics with a dominant-negative form of Dynamin, we show that phagocytes have a key role in this process. Importantly, we show the presence of peripheral glial cells close to the neuro-muscular junction that retracts before the motor neuron. We show also that in muscle, expression of EcR-B1 encoding the steroid hormone receptor required for postsynaptic dismantling, is under the control of the ftz-f1/Hr39 orphan nuclear receptor pathway but not the TGF-β signaling pathway. In the motor neuron, activation of EcR-B1 expression by the two parallel pathways (TGF-β signaling and nuclear receptor) triggers axon retraction. We propose that a signal from a TGF-β family ligand is produced by the dismantling muscle (postsynapse compartment) and received by the motor neuron (presynaptic compartment) resulting in motor neuron retraction. The requirement of the two pathways in the motor neuron provides a molecular explanation for the instructive role of the postsynapse degradation on motor neuron retraction. This mechanism insures the temporality of the two processes and prevents motor neuron pruning before postsynaptic degradation.     

Short Term Interactions with Long Term Consequences: Modulation of Chimeric Vessels by Neural Progenitors

Vessels are a critical and necessary component of most tissues, and there has been substantial research investigating vessel formation and stabilization. Several groups have investigated coculturing endothelial cells with a second cell type to promote formation and stabilization of vessels. Some have noted that long-term vessels derived from implanted cocultures are often chimeric consisting of both host and donor cells. The questions arise as to whether the coculture cell might impact the chimeric nature of the microvessels and can modulate the density of donor cells over time. If long-term engineered microvessels are primarily of host origin, any impairment of the host''s angiogenic ability has significant implications for the long-term success of the implant. If one can modulate the host versus donor response, one may be able to overcome a host''s angiogenic impairment. Furthermore, if one can modulate the donor contribution, one may be able to engineer microvascular networks to deliver molecules a patient lacks systemically for long times. To investigate the impact of the cocultured cell on the host versus donor contributions of endothelial cells in engineered microvascular networks, we varied the ratio of the neural progenitors to endothelial cells in subcutaneously implanted poly(ethylene glycol)/poly-L-lysine hydrogels. We found that the coculture of neural progenitors with endothelial cells led to the formation of chimeric host-donor vessels, and the ratio of neural progenitors has a significant impact on the long term residence of donor endothelial cells in engineered microvascular networks in vivo even though the neural progenitors are only present transiently in the system. We attribute this to the short term paracrine signaling between the two cell types. This suggests that one can modulate the host versus donor contributions using short-term paracrine signaling which has broad implications for the application of engineered microvascular networks and cellular therapy more broadly.     

Premortem autophagy determines the immunogenicity of chemotherapy-induced cancer cell death

One particular strategy to render anticancer therapies efficient consists of converting the patient's own tumor cells into therapeutic vaccines, via the induction of immunogenic cell death (ICD). One of the hallmarks of ICD dwells in the active release of ATP by cells committed to undergo, but not yet having succumbed to, apoptosis. We observed that the knockdown of essential autophagy-related genes (ATG3, ATG5, ATG7 and BECN1) abolishes the pre-apoptotic secretion of ATP by several human and murine cancer cell lines undergoing ICD. Accordingly, autophagy-competent, but not autophagy-deficient, tumor cells treated with ICD inducers in vitro could induce a tumor-specific immune response in vivo. Cancer cell lines stably depleted of ATG5 or ATG7 normally generate tumors in vivo, and such autophagy-deficient neoplasms, upon systemic treatment with ICD inducers, exhibit the same levels of apoptosis (as monitored by nuclear shrinkage and caspase-3 activation) and necrosis (as determined by following the kinetics of HMGB1 release) as their autophagy-proficient counterparts. However, autophagy-incompetent cancers fail to release ATP, to recruit immune effectors into the tumor bed and to respond to chemotherapy in conditions in which autophagy-competent tumors do so. The intratumoral administration of ecto-ATPase inhibitors increases extracellular ATP concentrations, re-establishes the therapy-induced recruitment of dendritic cells and T cells into the tumor bed, and restores the chemotherapeutic response of autophagy-deficient cancers. Altogether, these results suggest that autophagy-incompetent tumor cells escape from chemotherapy-induced (and perhaps natural?) immunosurveillance because they are unable to release ATP.     

New monolithic enzymatic micro-reactor for the fast production and purification of oligogalacturonides

Fast production and purification of alpha-(1,4)-oligogalacturonides was investigated using a new enzymatic reactor composed of a monolithic matrix. Pectin lyase from Aspergillus japonicus (Sigma) was immobilized on CIM-disk epoxy monolith. Studies were performed on free pectin lyase and immobilized pectin lyase to compare the optimum temperature, optimum pH, and thermal stability. It was determined that optimum temperature for free pectin lyase and immobilized pectin lyase on monolithic support is 30 degrees C, and optimum pH is 5. Monolithic CIM-disk chromatography is one of the fastest liquid chromatographic method used for separation and purification of biomolecules due to high mass transfer rate. In this context, online one step production and purification of oligogalacturonides was investigated associating CIM-disk pectin lyase and CIM-disk DEAE. This efficient enzymatic bioreactor production of uronic oligosaccharides from polygalacturonic acid (PGA) constitutes an original fast process to generate bioactive oligouronides.     

Efficient purification of small unsaturated oligoglucuronides by reversed-phase chromatography

Ion-exchange chromatography has been applied to purification of unsaturated oligoglucuronans. After an isocratic elution on a strong anion-exchange column, the collected fractions were desalted by low pressure size exclusion chromatography. However, this efficient separation was limited by the time required to desalt. So, we developed a reversed-phase chromatography method using back ionization of oligomers. Two C18 columns were tested with trifluoroacetic acid (TFA 0.7%) as eluent. Different selectivities and column stabilities were observed in this acidic condition. The scale up for semi-preparative applications enabled us to recover pure unsaturated oligoglucuronans without desalting step.     

Broad and Gag-biased HIV-1 epitope repertoires are associated with lower viral loads

Background

HLA class-I alleles differ in their ability to control HIV replication through cell-mediated immune responses. No consistent associations have been found between the breadth of Cytotoxic T Lymphocytes (CTL) responses and the control of HIV-1, and it is unknown whether the size or distribution of the viral proteome-wide epitope repertoire, i.e., the intrinsic ability to present fewer, more or specific viral epitopes, could affect clinical markers of disease progression.

Methodology/Principal Findings

We used an epitope prediction model to identify all epitope motifs in a set of 302 HIV-1 full-length proteomes according to each individual''s HLA (Human Leukocyte Antigen) genotype. The epitope repertoire, i.e., the number of predicted epitopes per HIV-1 proteome, varied considerably between HLA alleles and thus among individual proteomes. In a subgroup of 270 chronically infected individuals, we found that lower viral loads and higher CD4 counts were associated with a larger predicted epitope repertoire. Additionally, in Gag and Rev only, more epitopes were restricted by alleles associated with low viral loads than by alleles associated with higher viral loads.

Conclusions/Significance

This comprehensive analysis puts forth the epitope repertoire as a mechanistic component of the multi-faceted HIV-specific CTL response. The favorable impact on markers of disease status of the propensity to present more HLA binding peptides and specific proteins gives impetus to vaccine design strategies that seek to elicit responses to a broad array of HIV-1 epitopes, and suggest a particular focus on Gag.     

The binding of HIV-1 gp41 membrane proximal domain to its mucosal receptor, galactosyl ceramide, is structure-dependent

The peptide of HIV-1 envelope gp41 (a.a 628-683), referred to herein as P5, contains P1, a conserved galactose-specific lectin domain for binding the mucosal HIV-1-receptor, galactosyl ceramide (GalCer), as shown earlier, and a potential calcium-binding site (a.a 628-648). P1 contains contiguous epitopes recognized by the broadly neutralizing antibodies 2F5, 4E10, Z13. However, similar neutralizing antibodies could not be raised in animal model using immunogens based on these epitopes. We now show that the structure of both P5 and P1 peptides, as measured by circular dichroism, differs according to their environment: aqueous or lipidic, and as a function of calcium concentration. P5, but not P1, binds to calcium with a low binding affinity constant in the order of 2.5x10(4). Calcium binding results in a conformational change of P5, leading in turn to a decrease in affinity for GalCer. Hence, the affinity of the gp41-lectin site for the galactose harbored by the mucosal HIV-1 receptor GalCer is modulated by the peptide secondary and tertiary structure and the local environment. Therefore, definition of the conformation of this novel extended gp41 membrane proximal region, containing the conserved peptide P1 and the Ca(2+) binding site, could help designing an immunogen efficient at inducing neutralizing anti-HIV-1 antibodies.     

Life-cycle-generation-specific developmental processes are modified in the immediate upright mutant of the brown alga Ectocarpus siliculosus

Development of the sporophyte and gametophyte generations of the brown alga E. siliculosus involves two different patterns of early development, which begin with either a symmetric or an asymmetric division of the initial cell, respectively. A mutant, immediate upright (imm), was isolated that exhibited several characteristics typical of the gametophyte during the early development of the sporophyte generation. Genetic analyses showed that imm is a recessive, single-locus Mendelian factor and analysis of gene expression in this mutant indicated that the regulation of a number of life-cycle-regulated genes is specifically modified in imm mutant sporophytes. Thus, IMM appears to be a regulatory locus that controls part of the sporophyte-specific developmental programme, the mutant exhibiting partial homeotic conversion of the sporophyte into the gametophyte, a phenomenon that has not been described previously.