Cellular associations of pituitary gonadotrophs in a rodent (Lagostomus maximus maximus) with photoperiod-dependent reproduction

The morphological characteristics and percentage of the cellular associations between gonadotrophs (LH- and FSH-secreting cells) and other cellular types were studied in pituitary pars distalis of adult male viscachas (Lagostomus maximus maximus) by double immunohistochemistry using specific antibodies to LH, FSH, PRL, GH, ACTH, TSH and S-100 protein (by folliculostellate cells; FSC), during long and short photoperiods. Bihormonal gonadotrophs were observed in ventro-medial and dorsal regions, interspersed between monohormonal gonadotrophs, and their number increased in short photoperiod. LH- and FSH-gonadotrophs were found around lactotrophs, enclosed by somatotrophs in the dorsal region, and associated with irregular corticotrophs. Gonadotrophs and thyrotrophs were associated along blood vessels and follicular structures. The cytoplasmic prolongations of FSC were in contact with both gonadotrophs. The percentage of LH–FSH, LH–ACTH, LH–FSC, FSH–LH, FSH–PRL, FSH–GH, FSH–ACTH, FSH–TSH and FSH–FSC associations decreased, whereas LH–PRL increased in short as compared to long photoperiod. The most abundant associations were LH–GH and LH–TSH during long photoperiod, but LH–GH and LH–PRL during short photoperiod. FSH–GH and FSH–PRL were the most numerous associations, and LH–FSC and FSH–FSC were the less abundant ones in both photoperiods. These results provide the morphological evidence for specific cellular associations between gonadotrophs and other cellular types of viscacha pituitary.     

Synthesis and biological evaluation of colchicine C-ring analogues tethered with aliphatic linkers suitable for prodrug derivatisation

Colchicine was modified at the 10-OCH₃ position of the C-ring by reaction with heterocyclic amines or commercially available amines to afford a library of target colchicinoids in high yields (62–99%). Molecular modeling revealed that the incorporation of the linker groups led to a reduction in entropy and therefore binding affinity when compared with colchicine. Some colchicinoids were shown to be equicytotoxic with colchicine when evaluated in the DLD-1 colon cancer cells and retained activity in resistant A2780AD or HeLa cells with mutant Class III β-tubulin. Importantly, unlike colchicine, the analogues in this study are amenable for prodrug derivatisation and with potential for tumor-selective delivery.     

Reduced expression of inflammatory genes in deceased donor kidneys undergoing pulsatile pump preservation

Background

The use of expanded criteria donor kidneys (ECD) had been associated with worse outcomes. Whole gene expression of pre-implantation allograft biopsies from deceased donor kidneys (DDKs) was evaluated to compare the effect of pulsatile pump preservation (PPP) vs. cold storage preservation (CSP) on standard and ECD kidneys.

Methodology/Principal Findings

99 pre-implantation DDK biopsies were studied using gene expression with GeneChips. Kidneys transplant recipients were followed post transplantation for 35.8 months (range = 24–62). The PPP group included 60 biopsies (cold ischemia time (CIT)  = 1,367+/−509 minutes) and the CSP group included 39 biopsies (CIT = 1,022+/−485 minutes) (P<0.001). Donor age (42.0±14.6 vs. 34.1±14.2 years, P = 0.009) and the percentage of ECD kidneys (PPP = 35% vs. CSP = 12.8%, P = 0.012) were significantly different between groups. A two-sample t-test was performed, and probe sets having a P<0.001 were considered significant. Probe set level linear models were fit using cold ischemia time and CSP/PPP as independent variables to determine significant probe sets (P<0.001) between groups after adjusting for cold ischemia time. Thus, 43 significant genes were identified (P<0.001). Over-expression of genes associated with inflammation (CD86, CD209, CLEC4, EGFR2, TFF3, among others) was observed in the CSP group. Cell-to-cell signaling and interaction, and antigen presentation were the most important pathways with genes significantly over-expressed in CSP kidneys. When the analysis was restricted to ECD kidneys, genes involved in inflammation were also differentially up-regulated in ECD kidneys undergoing CSP. However, graft survival at the end of the study was similar between groups (P = 0.2). Moreover, the incidence of delayed graft function was not significant between groups.

Conclusions/Significance

Inflammation was the most important up-regulated pattern associated with pre-implantation biopsies undergoing CSP even when the PPP group has a larger number of ECD kidneys. No significant difference was observed in delayed graft function incidence and graft function post-transplantation. These findings support the use of PPP in ECD donor kidneys.     

Stem cell maintenance in shoot apical meristems

Stem cell homeostasis in shoot apical meristems of higher plants is regulated through a dynamic balance between spatial regulation of gene expression, cell growth patterns and patterns of differentiation. Cell-cell communication mediated by both the local factors and long-range signals have been implicated in stem cell homeostasis. Here we have reviewed recent developments on spatio-temporal regulation of cell-cell communication processes with an emphasis on how ubiquitously utilized signals such as plant hormones function with local factors in mediating stem cell homeostasis. We also provide a brief overview of how the activity of ubiquitously utilized epigenetic regulators are modulated locally to orchestrate gene expression.     

Vinyl sulfone functionalization: a feasible approach for the study of the lectin-carbohydrate interactions

Carbohydrate-mediated molecular recognition is involved in many biological aspects such as cellular adhesion, immune response, blood coagulation, inflammation, and infection. Considering the crucial importance of such biological events in which proteins are normally involved, synthetic saccharide-based systems have emerged as powerful tools for the understanding of protein-carbohydrate interactions. As a new approach to create saccharide-based systems, a set of representative monosaccharides (D-mannose, D-glucose, N-acetyl-D-glucosamine, and L-fucose) and disaccharides (lactose, maltose, and melibiose) were derivatized at their anomeric carbon with a vinyl sulfone group spanned by an ethylthio linker. This vinyl sulfone functionalization is demonstrated to be a general strategy for the covalent linkage of a saccharide in mild conditions via Michael-type additions with the amine and thiol groups from functionalized supports and those naturally present in biomolecules. The introduction of the ethylthio linker between the biorecognizable element (i.e., saccharide) and the reactive group (i.e., vinyl sulfone) was found to preserve the functionality of the former. The capability of the vinyl sulfone saccharides for the study of lectin-carbohydrate interactions was demonstrated by (i) immobilizing them on both amine-functionalized supports (glass slides and microwell plates) and polylysine-coated glass slides to create sugar arrays that selectively bind lectins (ii) coupling to model proteins to yield neoglycoproteins that are recognized by lectins and (iii) using vinyl sulfone saccharides as tags to allow the detection of the labeled biomolecule by HRP-lectins. The above results were further put tothe test with a real case: detection of carbohydrate binding proteins present in rice ( Oryza sativa ).     

Cryo X-ray nano-tomography of vaccinia virus infected cells

We have performed full-field cryo X-ray microscopy in the water window photon energy range on vaccinia virus (VACV) infected cells to produce tomographic reconstructions. PtK2 cells were infected with a GFP-expressing VACV strain and frozen by plunge fast freezing. The infected cells were selected by light fluorescence microscopy of the GFP marker and subsequently imaged in the X-ray microscope under cryogenic conditions. Tomographic tilt series of X-ray images were used to yield three-dimensional reconstructions showing different cell organelles (nuclei, mitochondria, filaments), together with other structures derived from the virus infection. Among them, it was possible to detect viral factories and two types of viral particles related to different maturation steps of VACV (immature and mature particles), which were compared to images obtained by standard electron microscopy of the same type of cells. In addition, the effect of radiation damage during X-ray tomographic acquisition was analyzed. Thin sections studied by electron microscopy revealed that the morphological features of the cells do not present noticeable changes after irradiation. Our findings show that cryo X-ray nano-tomography is a powerful tool for collecting three-dimensional structural information from frozen, unfixed, unstained whole cells with sufficient resolution to detect different virus particles exhibiting distinct maturation levels.     

Phylogeographic patterns of decapod crustaceans at the Atlantic-Mediterranean transition

Comparative multispecies studies allow contrasting the effect of past and present oceanographic processes on phylogeographic patterns. In the present study, a fragment of the COI gene was analyzed in seven decapod crustacean species from five families and with different bathymetric distributions. A total of 769 individuals were sampled along the Atlantic-Mediterranean transition area in order to test the effect of three putative barriers to gene flow: Strait of Gibraltar, Almeria-Oran Front and Ibiza Channel. A significant effect of the Strait of Gibraltar was found in the crabs Liocarcinus depurator and Macropipus tuberculatus. The Ibiza Channel had a significant effect for L. depurator. However, the Almeria-Oran front was not found to have a significant effect on any of the studied species. Higher levels of population structure were found in shallow-water species, although the number of species sampled should be increased to obtain a conclusive pattern. The haplotypes within the different species coalesced at times that could be related with past climatic events occurring before, during and after the last glacial maximum. Given the large diversity of phylogeographic patterns obtained within decapods, it is concluded that both historical and contemporary processes (marine current patterns, bathymetry and life-history traits) shape the phylogeographic patterns of these crustaceans.     

Nothofagus dombeyi regeneration in declining Austrocedrus chilensis forests: Effects of overstory mortality and climatic events

This research examines the regeneration dynamics of Nothofagus dombeyi and Austrocedrus chilensis in A. chilensis-dominated forests growing near the eastern limit of N. dombeyi where precipitation is limiting. In these forests the widespread decline and mortality of overstory A. chilensis trees, known as ‘mal del ciprés’ (cypress sickness), generates large canopy gaps in which new individuals establish. Our objective was to study the population dynamics of N. dombeyi and A. chilensis in these forests to investigate the influences of overstory tree death and climatic variation on establishment. We sampled 6 symptomatic A. chilensis stands and used dendrochronological techniques to reconstruct basal area development and regeneration establishment over time. Bivariate event analysis was performed to examine the temporal relationships between tree establishment and mortality events and climatic variation. Overstory A. chilensis trees established as post-fire cohorts, with subsequent establishment of A. chilensis and N. dombeyi during the past 50–60 years. Regeneration in the past two decades was primarily N. dombeyi. The establishment of both A. chilensis and N. dombeyi was synchronous with overstory tree mortality events, but it was more consistent among stands and prolonged for N. dombeyi. Establishment of A. chilensis was not associated with climatic events but N. dombeyi establishment was synchronous with droughts, possibly related to climate-driven mortality creating canopy gaps or reducing competition within gaps. We have demonstrated that N. dombeyi has the ability to establish in post-fire A. chilensis-dominated forests resulting in mixed-species, uneven-aged forests. The ongoing increase in the abundance of N. dombeyi relative to A. chilensis represents a shift in composition and increased complexity in stand structure driven by ‘mal del ciprés’ and climatic variation.     

High quality long-term CD4+ and CD8+ effector memory populations stimulated by DNA-LACK/MVA-LACK regimen in Leishmania major BALB/c model of infection

Heterologous vaccination based on priming with a plasmid DNA vector and boosting with an attenuated vaccinia virus MVA recombinant, with both vectors expressing the Leishmania infantum LACK antigen (DNA-LACK and MVA-LACK), has shown efficacy conferring protection in murine and canine models against cutaneus and visceral leishmaniasis, but the immune parameters of protection remain ill defined. Here we performed by flow cytometry an in depth analysis of the T cell populations induced in BALB/c mice during the vaccination protocol DNA-LACK/MVA-LACK, as well as after challenge with L. major parasites. In the adaptive response, there is a polyfunctional CD4(+) and CD8(+) T cell activation against LACK antigen. At the memory phase the heterologous vaccination induces high quality LACK-specific long-term CD4(+) and CD8(+) effector memory cells. After parasite challenge, there is a moderate boosting of LACK-specific CD4(+) and CD8(+) T cells. Anti-vector responses were largely CD8(+)-mediated. The immune parameters induced against LACK and triggered by the combined vaccination DNA/MVA protocol, like polyfunctionality of CD4(+) and CD8(+) T cells with an effector phenotype, could be relevant in protection against leishmaniasis.