Combined treatment with interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha up-regulates the expression of HLA class I determinants in Burkitt lymphoma lines

Cell lines derived from Epstein-Barr virus (EBV)-positive and EBV-negative Burkitt lymphoma (BL) have a low or defective expression of polymorphic HLA class I determinants compared to EBV-transformed lymphoblastoid cell lines (LCL) of normal B cell origin and are resistant to lysis by cytotoxic T lymphocytes (CTL) specific for the corresponding determinants (M. G. Masucci, S. Torsteinsdottir, J. Colombani, C. Brautbar, E. Klein, and G. Klein, Proc. Natl. Acad. Sci. USA 84, 4567, 1987; S. Torsteinsdottir, C. Brautbar, E. Klein, G. Klein, and M. G. Masucci, Int. J. Cancer, 41, 913, 1988). In order to investigate whether this phenotypic trait of the tumor cells can be modulated by agents known to enhance HLA class I antigen expression, pairs of LCL and BL lines were cultured in the presence of recombinant human interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha. Three low HLA A11 expressor EBV-negative BL lines, DG 75, BL 28, and BL 41, reacted significantly stronger with the anti-HLA A11 monoclonal antibody (Mab) AUF 5.13 after combined treatment with 500 U/ml IFN-gamma and 500 U/ml TNF-alpha. Reactivity with the AUF 5.13 and with other anti-polymorphic class I Mab's was up-regulated also in in vitro EBV-converted sublines of BL 28 and BL 41. The increment of antigen expression depended on the baseline expression in untreated cells. It was largest for the low expressor lines and decreased proportionally to the level of up-regulation induced by EBV conversion. Up-regulation of HLA A11 was accompanied by induction of sensitivity to HLA A11-specific CTLs in BL 28 and its converted subline E95A BL28 while BL 41 and E95A BL 41 remained resistant. The treatment did not affect significantly HLA A11 expression of two EBV-carrying, low HLA A11 expressor BL lines, WW-1-BL and WW-2-BL, and of the EBV-carrying BL 72 line that had a high spontaneous expression. The results suggest that the down-regulation of class I antigen expression is reversible in some but not all BL lines.     

Frequent Unanticipated Alleles of lethal giant larvae in Drosophila Second Chromosome Stocks

Forty years ago, a high frequency of lethal giant larvae (lgl) alleles in wild populations of Drosophila melanogaster was reported. This locus has been intensively studied for its roles in epithelial polarity, asymmetric neural divisions, and restriction of tissue proliferation. Here, we identify a high frequency of lgl alleles in the Bloomington second chromosome deficiency kit and the University of California at Los Angeles Bruinfly FRT40A-lethal P collection. These unrecognized aberrations confound the use of these workhorse collections for phenotypic screening or genetic mapping. In addition, we determined that independent alleles of insensitive, reported to affect asymmetric cell divisions during sensory organ development, carry lgl deletions that are responsible for the observed phenotypes. Taken together, these results encourage the routine testing of second chromosome stocks for second-site alleles of lgl.     

Algae sediment dynamics are mediated by herbivorous fishes on a nearshore coral reef

Coral Reefs - Epilithic algae are a ubiquitous component of coral reefs. Components of the epilithic algal matrix (EAM) can have a significant influence on coral settlement and benthic feeding by...     

Cryopreservation of embryonic stem cell‐derived multicellular neural aggregates labeled with micron‐sized particles of iron oxide for magnetic resonance imaging

Magnetic resonance imaging (MRI) provides an effective approach to track labeled pluripotent stem cell (PSC)‐derived neural progenitor cells (NPCs) for neurological disorder treatments after cell labeling with a contrast agent, such as an iron oxide derivative. Cryopreservation of pre‐labeled neural cells, especially in three‐dimensional (3D) structure, can provide a uniform cell population and preserve the stem cell niche for the subsequent applications. In this study, the effects of cryopreservation on PSC‐derived multicellular NPC aggregates labeled with micron‐sized particles of iron oxide (MPIO) were investigated. These NPC aggregates were labeled prior to cryopreservation because labeling thawed cells can be limited by inefficient intracellular uptake, variations in labeling efficiency, and increased culture time before use, minimizing their translation to clinical settings. The results indicated that intracellular MPIO incorporation was retained after cryopreservation (70–80% labeling efficiency), and MPIO labeling had little adverse effects on cell recovery, proliferation, cytotoxicity and neural lineage commitment post‐cryopreservation. MRI analysis showed comparable detectability for the MPIO‐labeled cells before and after cryopreservation indicated by T₂ and T₂* relaxation rates. Cryopreserving MPIO‐labeled 3D multicellular NPC aggregates can be applied in in vivo cell tracking studies and lead to more rapid translation from preservation to clinical implementation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:510–521, 2015     

Mapping coral reefs using consumer-grade drones and structure from motion photogrammetry techniques

We propose a novel technique to measure the small-scale three-dimensional features of a shallow-water coral reef using a small drone equipped with a consumer-grade camera, a handheld GPS and structure from motion (SfM) algorithms. We used a GoPro HERO4 with a modified lens mounted on a DJI Phantom 2 drone (maximum total take-off weight <2 kg) to perform a 10 min flight and collect 306 aerial images with an overlap equal or greater than 90%. We mapped an area of 8380 m², obtaining as output an ortho-rectified aerial photomosaic and a bathymetric digital elevation model (DEM) with a resolution of 0.78 and 1.56 cm pixel⁻¹, respectively. Through comparison with airborne LiDAR data for the same area, we verified that the location of the ortho-rectified aerial photomosaic is accurate within ~1.4 m. The bathymetric difference between our DEM and the LiDAR dataset is −0.016 ± 0.45 m (1σ). Our results show that it is possible, in conditions of calm waters, low winds and minimal sun glint, to deploy consumer-grade drones as a relatively low-cost and rapid survey technique to produce multispectral and bathymetric data on shallow-water coral reefs. We discuss the utility of such data to monitor temporal changes in topographic complexity of reefs and associated biological processes.

     

A new look at computation of the complexity index in mangroves: do disturbed forests have clues to analyze canopy height patchiness?

This paper proposes some guidelines to compute complexity index in those mangroves where either seasonal or strong disturbances have occurred. We surveyed 31 mangrove localities in Buenaventura Bay, Central Pacific Coast of Colombia, where structural parameters were measured within a 0.1 ha plot. Also, most likely disturbances were noted for each plot. Complexity index was calculated in its classical form using the arithmetic mean of the three tallest trees(maximal mean) and alternatively using: a) the total mean: the height average of all trees recorded in each plot; and b) the mode: the most frequent tree height class (10 cm intervals) within each plot. Afterwards, we compared the three computations and discussed there liability of each one according to the current state by plot. In addition, all structural parameters were sorted in two diameter at breast height (dbh) cohorts (2.5–10 cm and ≥ 10 cm) to figure out which contributes more to the forest structure. We conclude the following: (a) The mean of the three tallest trees is not a good estimator of forest development when seasonal or strong disturbances occur since complexity index based in it always overestimates forest structure. (b) Seasonal disturbances and recruitment produce mosaic forests. The best estimator of this condition is the mean height which encompasses both central tendency and variability. (c) The modal height is also helpful to establish the dominant cohort when forests show two or more storied-canopies, or intermediate cohorts are missing. It also applies when a new stock of recruits is entering in a mature forest (the modal or maximal heights can be used interchangeably in this case). (d) Maximal height is the best estimator for uniformly developed forests with closed canopies and/or a single dominant tall-cohort. (e) If one is not confident about which height type to include to compute the complexity index, we recommend to sort out structure data by dbh-cohorts and calculate indices for both of them. This will show which cohort is contributing most to forest complexity. Finally, we suggest to exclude non-mangrove species from the complexity index computation since they mostly do not contribute significantly to forest basal area. This revised version was published online in July 2006 with corrections to the Cover Date.     

The identification of Wos2, a p23 homologue that interacts with Wee1 and Cdc2 in the mitotic control of fission yeasts

The Wee1 kinase inhibits entry into mitosis by phosphorylation of the Cdc2 kinase. Searching for multicopy suppressors that abolish this inhibition in the fission yeast, we have identified a novel gene, here named wos2, encoding a protein with significant homology to human p23, an Hsp90-associated cochaperone. The deletion mutant has a modest phenotype, being heat-shock sensitive. Using antibodies raised against bacterially produced protein, we determined that Wos2 is very abundant, ubiquitously distributed in the yeast cell, and its expression dropped drastically as cells entered into early stationary phase, indicating that its function is associated with cell proliferation. In proliferating cells, the amount of Wos2 protein was not subjected to cell cycle regulation. However, in vitro assays demonstrated that this Hsp90 cochaperone is potentially regulated by phosphorylation. In addition to suppressing Wee1 activity, overproduction of Wos2 displayed synthetic lethality with Cdc2 mutant proteins, indicating that this Hsp90 cochaperone functionally interacts with Cdc2. The level of Cdc2 protein and its associated H1 kinase activity under synthetic lethal conditions suggested a regulatory role for this Wos2-Cdc2 interaction. Hsp90 complexes are required for CDK regulation; the synergy found between the excess of Wos2 and a deficiency in Hsp90 activity suggests that Wos2 could specifically interfere with the Hsp90-dependent regulation of Cdc2. In vitro analysis indicated that the above genetic interactions could take place by physical association of Wos2 with the single CDK complex of the fission yeast. Expression of the budding yeast p23 protein (encoded by the SBA1 gene) in the fission yeast indicated that Wos2 and Sba1 are functionally exchangeable and therefore that properties described here for Wos2 could be of wide significance in understanding the biological function of cochaperone p23 in eukaryotic cells.     

Geminivirus C2 protein might be the key player for geminiviral co-option of SCF-mediated ubiquitination

Viruses are obligate intracellular parasites, and need to create a suitable cell environment for viral propagation to complete their life cycle. In order to achieve this, viruses must usurp or interfere with the cellular machinery. Ubiquitination, a post-translational modification that controls numerous cellular processes, has proven to be a common target for viruses. Recently, geminivirus C2 protein has been shown to interact with the CSN complex and disrupt its activity over CULLIN1, interfering with the function of the CULLIN1-based SCF ubiquitin E3 ligases. Interestingly, over-expression of a given F-box protein may circumvent the general SCF malfunction caused by C2. This result raises the tantalizing idea that geminiviruses might be not only hampering, but also redirecting the activity of SCF complexes, thus co-opting the SCF-mediated ubiquitination pathway. We hypothesize that the mechanism of C2-facilitated co-option of SCF-mediated ubiquitination might not be exclusive for geminiviruses, but rather a common strategy for viruses.Key words: geminivirus, C2 protein, ubiquitination, SCF complex, CSN complex, F-box protein, pathogen co-option     

Autophagy modulates dynamics of connexins at the plasma membrane in a ubiquitin-dependent manner

Different pathways contribute to the turnover of connexins, the main structural components of gap junctions (GJs). The cellular pool of connexins targeted to each pathway and the functional consequences of degradation through these degradative pathways are unknown. In this work, we focused on the contribution of macroautophagy to connexin degradation. Using pharmacological and genetic blockage of macroautophagy both in vitro and in vivo, we found that the cellular pool targeted by this autophagic system is primarily the one organized into GJs. Interruption of connexins' macroautophagy resulted in their retention at the plasma membrane in the form of functional GJs and subsequent increased GJ-mediated intercellular diffusion. Up-regulation of macroautophagy alone is not sufficient to induce connexin internalization and degradation. To better understand what factors determine the autophagic degradation of GJ connexins, we analyzed the changes undergone by the fraction of plasma membrane connexin 43 targeted for macroautophagy and the sequence of events that trigger this process. We found that Nedd4-mediated ubiquitinylation of the connexin molecule is required to recruit the adaptor protein Eps15 to the GJ and to initiate the autophagy-dependent internalization and degradation of connexin 43. This study reveals a novel regulatory role for macroautophagy in GJ function that is directly dependent on the ubiquitinylation of plasma membrane connexins.