Effect of a Sinorhizobium meliloti strain with a modified putA gene on the rhizosphere microbial community of alfalfa

The success of a rhizobial inoculant in the soil depends to a large extent on its capacity to compete against indigenous strains. M403, a Sinorhizobium meliloti strain with enhanced competitiveness for nodule occupancy, was recently constructed by introducing a plasmid containing an extra copy of a modified putA (proline dehydrogenase) gene. This strain and M401, a control strain carrying the same plasmid without the modified gene, were used as soil inoculants for alfalfa in a contained field release experiment at León, Spain. In this study, we determined the effects of these two strains on the indigenous microbial community. 16S rRNA genes were obtained from the rhizosphere of alfalfa inoculated with strain M403 or strain M401 or from noninoculated plants by amplification of DNA from soil with bacterial group-specific primers. These genes were analyzed and compared by restriction fragment length polymorphism and temperature gradient gel electrophoresis. The results allowed us to differentiate between alterations in the microbial community apparently caused by inoculation and by the rhizosphere effect and seasonal fluctuations induced by the alfalfa plants and by the environment. Only moderate inoculation-dependent effects could be detected, while the alfalfa plants appeared to have a much stronger influence on the microbial community.     

Hippocampus Leads Ventral Striatum in Replay of Place-Reward Information

Associating spatial locations with rewards is fundamental to survival in natural environments and requires the integrity of the hippocampus and ventral striatum. In joint multineuron recordings from these areas, hippocampal–striatal ensembles reactivated together during sleep. This process was especially strong in pairs in which the hippocampal cell processed spatial information and ventral striatal firing correlated to reward. Replay was dominated by cell pairs in which the hippocampal “place” cell fired preferentially before the striatal reward-related neuron. Our results suggest a plausible mechanism for consolidating place-reward associations and are consistent with a central tenet of consolidation theory, showing that the hippocampus leads reactivation in a projection area.     

6-Mercaptopurine reduces cytokine and Muc5ac expression involving inhibition of NFκB activation in airway epithelial cells

Background

Mucus hypersecretion and excessive cytokine synthesis is associated with many of the pathologic features of chronic airway diseases such as asthma. 6-Mercaptopurine (6-MP) is an immunosuppressive drug that is widely used in several inflammatory disorders. Although 6-MP has been used to treat asthma, its function and mechanism of action in airway epithelial cells is unknown.

Methods

Confluent NCI-H292 and MLE-12 epithelial cells were pretreated with 6-MP followed by stimulation with TNFα or PMA. mRNA levels of cytokines and mucins were measured by RT-PCR. Western blot analysis was performed to assess the phosphorylation of IκBα and luciferase assays were performed using an NFκB reporter plasmid to determine NFκB activity. Periodic Acid Schiff staining was used to assess the production of mucus.

Results

6-MP displayed no effect on cell viability up to a concentration of 15 μM. RT-PCR analysis showed that 6-MP significantly reduces TNFα- and PMA-induced expression of several proinflammatory cytokines in NCI-H292 and MLE-12 cells. Consistent with this, we demonstrated that 6-MP strongly inhibits TNFα-induced phosphorylation of IκBα and thus attenuates NFκB luciferase reporter activity. In addition, 6-MP decreases Rac1 activity in MLE-12 cells. 6-MP down-regulates gene expression of the mucin Muc5ac, but not Muc2, through inhibition of activation of the NFκB pathway. Furthermore, PMA- and TNFα-induced mucus production, as visualized by Periodic Acid Schiff (PAS) staining, is decreased by 6-MP.

Conclusions

Our data demonstrate that 6-MP inhibits Muc5ac gene expression and mucus production in airway epithelial cells through inhibition of the NFκB pathway, and 6-MP may represent a novel therapeutic target for mucus hypersecretion in airway diseases.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0236-0) contains supplementary material, which is available to authorized users.     

The effect of drought stress on heterozygosity–fitness correlations in pedunculate oak (Quercus robur)

Background and Aims

The interaction between forest fragmentation and predicted climate change may pose a serious threat to tree populations. In small and spatially isolated forest fragments, increased homozygosity may directly affect individual tree fitness through the expression of deleterious alleles. Climate change-induced drought stress may exacerbate these detrimental genetic consequences of forest fragmentation, as the fitness response to low levels of individual heterozygosity is generally thought to be stronger under environmental stress than under optimal conditions.

Methods

To test this hypothesis, a greenhouse experiment was performed in which various transpiration and growth traits of 6-month-old seedlings of Quercus robur differing in multilocus heterozygosity (MLH) were recorded for 3 months under a well-watered and a drought stress treatment. Heterozygosity–fitness correlations (HFC) were examined by correlating the recorded traits of individual seedlings to their MLH and by studying their response to drought stress.

Key Results

Weak, but significant, effects of MLH on several fitness traits were obtained, which were stronger for transpiration variables than for the recorded growth traits. High atmospheric stress (measured as vapour pressure deficit) influenced the strength of the HFCs of the transpiration variables, whereas only a limited effect of the irrigation treatment on the HFCs was observed.

Conclusions

Under ongoing climate change, increased atmospheric stress in the future may strengthen the negative fitness responses of trees to low MLH. This indicates the necessity to maximize individual multilocus heterozygosity in forest tree breeding programmes.     

Human mesenchymal stem cell-conditioned medium improves cardiac function following myocardial infarction

Recent studies suggest that the therapeutic effects of stem cell transplantation following myocardial infarction (MI) are mediated by paracrine factors. One of the main goals in the treatment of ischemic heart disease is to stimulate vascular repair mechanisms. Here, we sought to explore the therapeutic angiogenic potential of mesenchymal stem cell (MSC) secretions. Human MSC secretions were collected as conditioned medium (MSC-CM) using a clinically compliant protocol. Based on proteomic and pathway analysis of MSC-CM, an in vitro assay of HUVEC spheroids was performed identifying the angiogenic properties of MSC-CM. Subsequently, pigs were subjected to surgical left circumflex coronary artery ligation and randomized to intravenous MSC-CM treatment or non-CM (NCM) treatment for 7 days. Three weeks after MI, myocardial capillary density was higher in pigs treated with MSC-CM (645 ± 114 vs 981 ± 55 capillaries/mm(2); P = 0.021), which was accompanied by reduced myocardial infarct size and preserved systolic and diastolic performance. Intravenous MSC-CM treatment after myocardial infarction increases capillary density and preserves cardiac function, probably by increasing myocardial perfusion.     

MicroRNA‐155 prevents necrotic cell death in human cardiomyocyte progenitor cells via targeting RIP1

To improve regeneration of the injured myocardium, cardiomyocyte progenitor cells (CMPCs) have been put forward as a potential cell source for transplantation therapy. Although cell transplantation therapy displayed promising results, many issues need to be addressed before fully appreciating their impact. One of the hurdles is poor graft‐cell survival upon injection, thereby limiting potential beneficial effects. Here, we attempt to improve CMPCs survival by increasing microRNA‐155 (miR‐155) levels, potentially to improve engraftment upon transplantation. Using quantitative PCR, we observed a 4‐fold increase of miR‐155 when CMPCs were exposed to hydrogen‐peroxide stimulation. Flow cytometric analysis of cell viability, apoptosis and necrosis showed that necrosis is the main cause of cell death. Overexpressing miR‐155 in CMPCs revealed that miR‐155 attenuated necrotic cell death by 40 ± 2.3%via targeting receptor interacting protein 1 (RIP1). In addition, inhibiting RIP1, either by pre‐incubating the cells with a RIP1 specific inhibitor, Necrostatin‐1 or siRNA mediated knockdown, reduced necrosis by 38 ± 2.5% and 33 ± 1.9%, respectively. Interestingly, analysing gene expression using a PCR‐array showed that increased miR‐155 levels did not change cell survival and apoptotic related gene expression. By targeting RIP1, miR‐155 repressed necrotic cell death of CMPCs, independent of activation of Akt pro‐survival pathway. MiR‐155 provides the opportunity to block necrosis, a conventionally thought non‐regulated process, and might be a potential novel approach to improve cell engraftment for cell therapy.     

Distribution of 3-hydroxy oxylipins and acetylsalicylic acid sensitivity in Cryptococcus species

Using a well tested antibody specific for 3-hydroxy oxylipins, we mapped the presence of these oxylipins in selected Cryptococcus (Filobasidiella) species. Immunofluorescence microscopy studies revealed that these compounds are deposited on cell wall surfaces, appendages, and collarettes. In vitro studies revealed that growth of Cryptococcus species was inhibited by acetylsalicylic acid (which is known to inhibit mitochondrial function, including the production of 3-hydroxy oxylipins) at concentrations as low as 1 mmol/L. The results suggest that acetylsalicylic acid is effective in controlling the growth of tested pathogens, probably by targeting their mitochondria. This study further expands the known function of this anti-inflammatory drug as anti-fungal agent.     

Inhibitors of dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Part 2: isoindoline containing inhibitors

To obtain selective and potent inhibitors of dipeptidyl peptidases 8 and 9, we synthesized a series of substituted isoindolines as modified analogs of allo-Ile-isoindoline, the reference DPP8/9 inhibitor. The influence of phenyl substituents and different P2 residues on the inhibitors’ affinity toward other DPPs and more specifically, their potential to discriminate between DPP8 and DPP9 will be discussed. Within this series compound 8j was shown to be a potent and selective inhibitor of DPP8/9 with low activity toward DPP II.     

No evidence for ‘skewed’ inactivation of the X-chromosome as cause of Leber's hereditary optic neuropathy in female carriers

Leber’s hereditary optic neuropathy (LHON) is a maternally inherited disorder of the optic nerves. It has been proposed that the specific mutations in the mitochondrial DNA (mtDNA) that are associated with LHON require and X-chromosomally encoded permissive factor in order to become expressed. This would explain both the preponderance of male patients and the fact that most carriers of specific mtDNA mutations remain unaffected. Although linkage studies have been negative so far, the existence of such a factor has not been ruled out. We investigated the genealogical data of 24 large LHON pedigrees and concluded that the presumed X-linked factor would be recessively inherited and that at least 57% of the affected females would be heterozygous. Therefore, these females must be the victim of nonrandom X-chromosomal inactivation (skewed lyonization). However, analysis of X-chromosomal methylation patterns in 16 LHON-affected females revealed substantial skewing in only 15%–20% of cases, which is not significantly different from the patterns in 49 controls. Moreover, we found the frequency of LHON in daughters of affected heterozygous females to be twice to three times as high as in daughters of unaffected heterozygous females, which cannot be explained by an X-chromosomally inherited factor. We conclude that the results of our investigations do not support the hypothesis that LHON is a digenic disease with an X-linked factor being the main cause of loss of vision in the presence of relevant mtDNA mutations. Received: 1 June 1995 / Revised: 20 September 1995