Recovery of cryopreserved silver birch shoot tips is affected by the pre-freezing age of the cultures and ammonium substitution

The aim of the present study was to improve the cryopreservation protocol for silver birch (Betula pendula Roth) in vitro shoot tips. The recovery of shoot tips derived from young cultures (shoot tips excised from less than 20-month-old cultures) was significantly better than that from old cultures (shoot tips excised from cultures at least 55 months old) when the standard protocol, i.e. the slow-cooling procedure with PGD as cryoprotectant, was used. The recovery of shoot tips of both ages was, however, improved by substituting KNO₃ for ammonium during cold hardening and post-thaw cultivation. The substitution of ammonium with KNO₃ improved recovery in most of the genotypes, indicating the possibility of developing a cryopreservation protocol applicable to several genotypes.     

Radioadaptive response revisited

Radiation-induced adaptive response belongs to the group of non-targeted effects that do not require direct exposure of the cell nucleus by radiation. It is described as the reduced damaging effect of a challenging radiation dose when induced by a previous low priming dose. Adaptive responses have been observed in vitro and in vivo using various indicators of cellular damage, such as cell lethality, chromosomal aberrations, mutation induction, radiosensitivity, and DNA repair. Adaptive response can be divided into three successive biological phenomena, the intracellular response, the extracellular signal, and the maintenance. The intracellular response leading to adaptation of a single cell is a complex biological process including induction or suppression of gene groups. An extracellular signal, the nature of which is unknown, may be sent by the affected cell to neighbouring cells causing them to adapt as well. This occurs either by a release of diffusible signalling molecules or by gap-junction intercellular communication. Adaptive response can be maintained for periods ranging from of a few hours to several months. Constantly increased levels of reactive oxygen species (ROS) or nitric oxide (NO) have been observed in adapted cells and both factors may play a role in the maintenance process. Although adaptive response seems to function by an on/off principle, it is a phenomenon showing a high degree of inter- and intraindividual variability. It remains to be seen to what extent adaptive response is functional in humans at relevant dose and dose-rate exposures. A better understanding of adaptive response and other non-targeted effects is needed before they can be confirmed as risk estimate factors for the human population at low levels of ionising radiation.     

Quantitative proteomic analysis reveals induction of premature senescence in human umbilical vein endothelial cells exposed to chronic low‐dose rate gamma radiation

Chronic low‐dose ionizing radiation induces cardiovascular disease in human populations but the mechanism is largely unknown. We suggested that chronic radiation exposure may induce endothelial cell senescence that is associated with vascular damage in vivo. We investigated whether chronic radiation exposure is causing a change in the onset of senescence in endothelial cells in vitro. Indeed, when exposed to continuous low‐dose rate gamma radiation (4.1 mGy/h), primary human umbilical vein endothelial cells (HUVECs) initiated senescence much earlier than the nonirradiated control cells. We investigated the changes in the protein expression of HUVECs before and during the onset of radiation‐induced senescence. Cellular proteins were quantified using isotope‐coded protein label technology after 1, 3, and 6 weeks of radiation exposure. Several senescence‐related biological pathways were influenced by radiation, including cytoskeletal organization, cell–cell communication and adhesion, and inflammation. Immunoblot analysis showed an activation of the p53/p21 pathway corresponding to the progressing senescence. Our data suggest that chronic radiation‐induced DNA damage and oxidative stress result in induction of p53/p21 pathway that inhibits the replicative potential of HUVECs and leads to premature senescence. This study contributes to the understanding of the increased risk of cardiovascular diseases seen in populations exposed to chronic low‐dose irradiation.     

Mucosal Adhesion Properties of the Probiotic Lactobacillus rhamnosus GG SpaCBA and SpaFED Pilin Subunits

Lactobacillus rhamnosus GG is a well-established Gram-positive probiotic strain, whose health-benefiting properties are dependent in part on prolonged residence in the gastrointestinal tract and are likely dictated by adherence to the intestinal mucosa. Previously, we identified two pilus gene clusters (spaCBA and spaFED) in the genome of this probiotic bacterium, each of which contained the predicted genes for three pilin subunits and a single sortase. We also confirmed the presence of SpaCBA pili on the cell surface and attributed an intestinal mucus-binding capacity to one of the pilin subunits (SpaC). Here, we report cloning of the remaining pilin genes (spaA, spaB, spaD, spaE, and spaF) in Escherichia coli, production and purification of the recombinant proteins, and assessment of the adherence of these proteins to human intestinal mucus. Our findings indicate that the SpaB and SpaF pilin subunits also exhibit substantial binding to mucus, which can be inhibited competitively in a dose-related manner. Moreover, the binding between the SpaB pilin subunit and the mucosal substrate appears to operate through electrostatic contacts and is not related to a recognized mucus-binding domain. We conclude from these results that it is conceivable that two pilin subunits (SpaB and SpaC) in the SpaCBA pilus fiber play a role in binding to intestinal mucus, but for the uncharacterized and putative SpaFED pilus fiber only a single pilin subunit (SpaF) is potentially responsible for adhesion to mucus.The human intestinal microbiota is comprised of more than 1,000 species of commensal and probiotic bacteria, including several members of the Gram-positive genus Lactobacillus (42, 52). Many strains of lactobacilli have a variety of health-promoting effects in humans and consequently have been used commercially as probiotics in foods and nutritional supplements (for a review, see reference 48). Often a necessary precondition for colonization of the human gastrointestinal (GI) tract by probiotic bacteria is preferential adherence to the intestinal mucosa, which in turn prolongs and stabilizes intestinal residence, possibly triggering a variety of defensive host cell immune responses and excluding pathogenic bacteria by competitive inhibition or steric hindrance (48). The outermost layer of the intestinal mucosa, which is a secreted and hydrated mucus gel that acts as a protective barrier and filter, consists primarily of a heterogeneous mixture of highly glycosylated membrane-associated and secreted glycoproteins called mucins (36). Although many studies have demonstrated that various probiotic Lactobacillus spp. adhere initially to the mucus gel layer, relatively few details about the overall molecular mechanism of mucosal adhesion are known (for a review, see reference 23). Nonetheless, several studies have reported that the adherence of Lactobacillus cells to the mucosal barrier is frequently due to a surface protein-mediated interaction. For example, Rojas et al. (44) determined that the ability of Lactobacillus fermentum 104R (reclassified as Lactobacillus reuteri 104R) to bind to porcine small intestinal mucus and gastric mucin was facilitated by a cell surface-localized mucus adhesion-promoting protein (MapA). Similarly, Macías-Rodríguez et al. (25) described two adhesion-associated proteins specific for porcine intestinal mucus-related substrates that are attached noncovalently to the cell surface of L. fermentum BCS87. Also, Roos and Jonsson (45) demonstrated adherence between the surface-associated Mub (mucus binding) protein from L. reuteri 1063 and intestinal mucus components derived from porcine and poultry sources. In addition, Pretzer et al. (38) identified a large multidomain surface protein in Lactobacillus plantarum WCFS1 with binding specificity for the mannose moieties in mucins. Interestingly, Kinoshita et al. (19) discovered that glyceraldehyde 3-phosphate dehydrogenase (GAPDH), an enzyme normally associated with glycolysis, is localized on the surface of L. plantarum LA318 cells and adheres tightly to human colonic mucin.Until quite recently, only indirect or circumstantial evidence suggested that pilus-like structures extend from the surface of probiotic lactobacilli (28, 39). However, in a previous study (18) we demonstrated that Lactobacillus rhamnosus GG, a well-studied and widely used probiotic strain (48), is a piliated microbe. Pili are slender, elongated, heteromeric, proteinaceous surface appendages that are present in numerous other Gram-positive bacteria and often mediate adherence between pathogenic and nonpathogenic species and their host cell targets (for reviews, see references 20, 26, 40, and 49) but have now emerged as possible facilitators of adhesion for probiotic colonization of the GI tract (18). Prototypically, the pilus fiber is composed of one major pilin that forms the pilus backbone and two minor pilin subunits (26, 40, 49), one subunit that has a role in signaling the cessation of pilus polymerization (27, 30) and is deposited at the pilus base and at irregular intervals along the pilus backbone and another subunit with an adhesive property that is often localized at the pilus tip (1, 41). The current model of pilus assembly in Corynebacterium diphtheriae (27) suggests that these pilin subunits are connected covalently to one another through isopeptidyl bonds by a membrane-bound transpeptidase (pilin-specific sortase) to produce polymerized pili, which are then attached covalently to the cell wall by a different transpeptidase (the housekeeping sortase) that is capable of recognizing all C-terminal LPXTG-like substrates. The genes encoding these pilus proteins, as well as the pilin-specific sortase, are clustered at the same locus in the genome (54).In a recent study (18), we discovered that in the L. rhamnosus GG genome the genes encoding two different pilus fibers are in the spaCBA and spaFED gene clusters and, based on a genomic comparison with another L. rhamnosus strain (LC705), that the spaCBA cluster is present in only L. rhamnosus GG. Moreover, in our previous work (18) the predicted genes for the major pilin subunit forming the pilus backbone (SpaA and SpaD), one ancillary minor pilin subunit (SpaB and SpaE) that (based on a model for pilus biogenesis) is likely located at the pilus base and decorates the pilus backbone (27), and another larger adherent minor pilin subunit (SpaC and SpaF) were identified in L. rhamnosus GG on the basis of amino acid identity with pilins from two enterococcal species. In addition, we also detected in the sequences of the predicted spaCBA and spaFED gene products the anticipated consensus motifs and domains characteristic of a pilin primary structure, including the Sec-dependent secretion signal, the sortase recognition site, the YPKN pilin-like motif, and the E box (18). Subsequently, expression and localization of intact SpaCBA pili on the cell surface of L. rhamnosus GG were confirmed by immunoblotting and immunogold-labeled electron microscopy using antiserum specific for the SpaC pilin (18). Adhesion interactions between the L. rhamnosus GG strain and intestinal mucosal surfaces have been reported and characterized in previous studies (15, 31, 33, 46, 55-57). However, in our recent study (18), SpaCBA pilus-mediated binding of L. rhamnosus GG cells to human intestinal mucus was revealed in adhesion experiments performed with both L. rhamnosus GG pretreated with SpaC antiserum and an L. rhamnosus GG spaC insertion mutant. More specifically, we demonstrated that there was significant binding between recombinant SpaC pilin protein and intestinal mucus and thus identified a mucus-binding capacity for one of the minor pilin components localized at the tip and along the backbone of the SpaCBA pilus (18). To expand on these findings, here we describe a study in which each of the remaining predicted pilin subunits (SpaA, SpaB, SpaD, SpaE, and SpaF) encoded by genes in the spaCBA and spaFED gene clusters was overproduced in a recombinant form, purified to apparent homogeneity, and characterized to determine its adherence to human intestinal mucus.     

Cell Survival Following Radiation Exposure Requires miR-525-3p Mediated Suppression of ARRB1 and TXN1

Background

microRNAs (miRNAs) are non-coding RNAs that alter the stability and translation efficiency of messenger RNAs. Ionizing radiation (IR) induces rapid and selective changes in miRNA expression. Depletion of the miRNA processing enzymes Dicer or Ago2 reduces the capacity of cells to survive radiation exposure. Elucidation of critical radiation-regulated miRNAs and their target proteins offers a promising approach to identify new targets to increase the therapeutic effectiveness of the radiation treatment of cancer.

Principal Findings

Expression of miR-525-3p is rapidly up-regulated in response to radiation. Manipulation of miR-525-3p expression in irradiated cells confirmed that this miRNA mediates the radiosensitivity of a variety of non-transformed (RPE, HUVEC) and tumor-derived cell lines (HeLa, U2-Os, EA.hy926) cell lines. Thus, anti-miR-525-3p mediated inhibition of the increase in miR-525-3p elevated radiosensitivity, while overexpression of precursor miR-525-3p conferred radioresistance. Using a proteomic approach we identified 21 radiation-regulated proteins, of which 14 were found to be candidate targets for miR-525-3p-mediated repression. Luciferase reporter assays confirmed that nine of these were indeed direct targets of miR-525-3p repression. Individual analysis of these direct targets by RNAi-mediated knockdown established that ARRB1, TXN1 and HSPA9 are essential miR-525-3p-dependent regulators of radiation sensitivity.

Conclusion

The transient up-regulation of miR-525-3p, and the resultant repression of its direct targets ARRB1, TXN1 and HSPA9, is required for cell survival following irradiation. The conserved function of miR-525-3p across several cell types makes this microRNA pathway a promising target for modifying the efficacy of radiotherapy.