Heterogenic Final Cell Cycle by Chicken Retinal Lim1 Horizontal Progenitor Cells Leads to Heteroploid Cells with a Remaining Replicated Genome

Retinal progenitor cells undergo apical mitoses during the process of interkinetic nuclear migration and newly generated post-mitotic neurons migrate to their prospective retinal layer. Whereas this is valid for most types of retinal neurons, chicken horizontal cells are generated by delayed non-apical mitoses from dedicated progenitors. The regulation of such final cell cycle is not well understood and we have studied how Lim1 expressing horizontal progenitor cells (HPCs) exit the cell cycle. We have used markers for S- and G2/M-phase in combination with markers for cell cycle regulators Rb1, cyclin B1, cdc25C and p27Kip1 to characterise the final cell cycle of HPCs. The results show that Lim1+ HPCs are heterogenic with regards to when and during what phase they leave the final cell cycle. Not all horizontal cells were generated by a non-apical (basal) mitosis; instead, the HPCs exhibited three different behaviours during the final cell cycle. Thirty-five percent of the Lim1+ horizontal cells was estimated to be generated by non-apical mitoses. The other horizontal cells were either generated by an interkinetic nuclear migration with an apical mitosis or by a cell cycle with an S-phase that was not followed by any mitosis. Such cells remain with replicated DNA and may be regarded as somatic heteroploids. The observed heterogeneity of the final cell cycle was also seen in the expression of Rb1, cyclin B1, cdc25C and p27Kip1. Phosphorylated Rb1-Ser608 was restricted to the Lim1+ cells that entered S-phase while cyclin B1 and cdc25C were exclusively expressed in HPCs having a basal mitosis. Only HPCs that leave the cell cycle after an apical mitosis expressed p27Kip1. We speculate that the cell cycle heterogeneity with formation of heteroploid cells may present a cellular context that contributes to the suggested propensity of these cells to generate cancer when the retinoblastoma gene is mutated.     

Time‐dependent climate impact of a bioenergy system – methodology development and application to Swedish conditions

The area of dedicated energy crops is expected to increase in Sweden. This will result in direct land use changes, which may affect the carbon stocks in soil and biomass, as well as yield levels and the use of inputs. Carbon dioxide (CO₂) fluxes of biomass are often not considered when calculating the climate impact in life cycle assessments (LCA) assuming that the CO₂ released at combustion has recently been captured by the biomass in question. With the extended time lag between capture and release of CO₂ inherent in many perennial bioenergy systems, the relation between carbon neutrality and climate neutrality may be questioned. In this paper, previously published methodologies and models are combined in a methodological framework that can assist LCA practitioners in interpreting the time‐dependent climate impact of a bioenergy system. The treatment of carbon differs from conventional LCA practice in that no distinction is made between fossil and biogenic carbon. A time‐dependent indicator is used to enable a representation of the climate impact that is not dependent on the choice of a specific characterization time horizon or time of evaluation and that does not use characterization factors, such as global warming potential and global temperature potential. The indicator used to aid in the interpretation phase of this paper is global mean surface temperature change (ΔT_s(n)). A theoretical system producing willow for district heating was used to study land use change effects depending on previous land use and variations in the standing biomass carbon stocks. When replacing annual crops with willow this system presented a cooling contribution to ΔT_s(n). However, the first years after establishing the willow plantation it presented a warming contribution to ΔT_s(n). This behavior was due mainly to soil organic carbon (SOC) variation. A rapid initial increase in standing biomass counteracted the initial SOC loss.     

Vitamin D Status,Filaggrin Genotype,and Cardiovascular Risk Factors: A Mendelian Randomization Approach

Background

Vitamin D deficiency is associated with increased cardiovascular disease risk in observational studies. Whether these associations are causal is not clear. Loss-of-function mutations in the filaggrin gene result in up to 10% higher serum vitamin D concentrations, supposedly due to a decreased UV-protection of the keratinocytes. We used a Mendelian randomization approach to estimate the causal effect of vitamin D status on serum lipids, blood pressure, body mass index, waist circumference, and the metabolic syndrome.

Methods

Three population based studies were included, Monica10 (2,656 individuals aged 40–71 years), Inter99 (6,784 individuals aged 30–60 years), and Health2006 (3,471 individuals aged 18–69 years) conducted in 1993–94, 1999–2001, and 2006–2008, respectively. Participants were genotyped for the two most common filaggrin gene mutations in European descendants R501X and 2282del4, in all three studies and further for the R2447X mutation in the Inter99 and Health2006 studies. Filaggrin genotype was used as instrumental variable for vitamin D status. Baseline measurements of serum 25-hydroxyvitamin D were performed in all three studies.

Results

Instrumental variable analyses showed a 23.8% (95% confidence interval, CI 3.0, 48.6) higher HDL cholesterol level and a 30.5% (95% CI: 0.8, 51.3) lower serum level of triglycerides per doubling of vitamin D. These associations were, however, not statistically significant when applying the Bonferroni adjusted significance level. The remaining lipids showed non-significant changes in a favorable direction. Doubling of vitamin D gave a non-significantly lower odds ratio = 0.26 (95% CI: 0.06, 1.17) of the metabolic syndrome. There were no statistically significant causal effects of vitamin D status on blood pressure, body mass index, or waist circumference.

Conclusion

Our results support a causal effect of higher vitamin D status on a more favorable lipid profile, although more studies in other populations are needed to confirm our results.     

Interleukin-1β and Interleukin-1 Receptor Antagonist Appear in Grey Matter Additionally to White Matter Lesions during Experimental Multiple Sclerosis

Background

Multiple sclerosis (MS) has been mainly attributed to white matter (WM) pathology. However, recent evidence indicated the presence of grey matter (GM) lesions. One of the principal mediators of inflammatory processes is interleukin-1β (IL-1β), which is known to play a role in MS pathogenesis. It is unknown whether IL-1β is solely present in WM or also in GM lesions. Using an experimental MS model, we questioned whether IL-1β and the IL-1 receptor antagonist (IL-1ra) are present in GM in addition to affected WM regions.

Methods

The expression of IL-1β and IL-1ra in chronic-relapsing EAE (cr-EAE) rats was examined using in situ hybridization, immunohistochemistry and real-time PCR. Rats were sacrificed at the peak of the first disease phase, the trough of the remission phase, and at the peak of the relapse. Histopathological characteristics of CNS lesions were studied using immunohistochemistry for PLP, CD68 and CD3 and Oil-Red O histochemistry.

Results

IL-1β and IL-ra expression appears to a similar extent in affected GM and WM regions in the brain and spinal cord of cr-EAE rats, particularly in perivascular and periventricular locations. IL-1β and IL-1ra expression was dedicated to macrophages and/or activated microglial cells, at sites of starting demyelination. The time-dependent expression of IL-1β and IL-1ra revealed that within the spinal cord IL-1β and IL-1ra mRNA remained present throughout the disease, whereas in the brain their expression disappeared during the relapse.

Conclusions

The appearance of IL-1β expressing cells in GM within the CNS during cr-EAE may explain the occurrence of several clinical deficits present in EAE and MS which cannot be attributed solely to the presence of IL-1β in WM. Endogenously produced IL-1ra seems not capable to counteract IL-1β-induced effects. We put forward that IL-1β may behold promise as a target to address GM, in addition to WM, related pathology in MS.     

Inhibitors of type III secretion in Yersinia: design, synthesis and multivariate QSAR of 2-arylsulfonylamino-benzanilides

Compound 1, 2-(benzo[1,2,5]thiadiazole-4-sulfonylamino)-5-chloro-N-(3,4-dichloro-phenyl)-benzamide, was identified as a putative type III secretion inhibitor in Yersinia, and the compound thus has a potential to be used to prevent or treat bacterial infections. A set of seven analogues was synthesized and evaluated in a type III secretion dependent reporter-gene assay with viable bacterial to give basic SAR. A second set of 19 compounds was obtained by statistical molecular design in the building block and product space and by subsequent synthesis. Evaluation in the reporter-gene assay showed that the compounds ranged from non-active to compounds more potent than 1. Based on the data multivariate QSAR models were established and the final Hi-PLS model showed good correlation between experimentally determined % inhibition and the calculated % inhibition of the reporter-gene signal.     

Enumeration of Salmonellae in Table Eggs,Pasteurized Egg Products,and Egg-Containing Dishes by Using Quantitative Real-Time PCR

Salmonellae are a major cause of food-borne outbreaks in Europe, with eggs and egg products being identified as major sources. Due to the low levels of salmonellae in eggs and egg products, direct quantification is difficult. In the present study, enrichment quantitative real-time PCR (qPCR) was employed for enumeration of salmonellae in different matrices: table eggs, pasteurized egg products, and egg-containing dishes. Salmonella enterica serovar Enteritidis and S. enterica serovar Tennessee were used to artificially contaminate these matrices. The results showed a linear regression between the numbers of salmonellae and the quantification cycle (C_q) values for all matrices used, with the exception of pasteurized egg white. Standard curves were constructed by using both stationary-phase cells and heat-stressed cells, with similar results. Finally, this method was used to evaluate the fate of salmonellae in two egg-containing dishes, long egg and tiramisu, at abused refrigeration temperatures, and results indicated the growth of bacteria over a 1-week period. In conclusion, enrichment qPCR was shown to be reliable for enumeration of salmonellae in different egg products.     

The heterogenic final cell cycle of chicken retinal Lim1 horizontal cells is not regulated by the DNA damage response pathway

Cells with aberrations in chromosomal ploidy are normally removed by apoptosis. However, aneuploid neurons have been shown to remain functional and active both in the cortex and in the retina. Lim1 horizontal progenitor cells in the chicken retina have a heterogenic final cell cycle, producing some cells that enter S-phase without proceeding into M-phase. The cells become heteroploid but do not undergo developmental cell death. This prompted us to investigate if the final cell cycle of these cells is under the regulation of an active DNA damage response. Our results show that the DNA damage response pathway, including γ-H2AX and Rad51 foci, is not triggered during any phase of the different final cell cycles of horizontal progenitor cells. However, chemically inducing DNA adducts or double-strand breaks in Lim1 horizontal progenitor cells activated the DNA damage response pathway, showing that the cells are capable of a functional response to DNA damage. Moreover, manipulation of the DNA damage response pathway during the final cell cycle using inhibitors of ATM/ATR, Chk1/2, and p38MAPK, neither induced apoptosis nor mitosis in the Lim1 horizontal progenitor cells. We conclude that the DNA damage response pathway is functional in the Lim1 horizontal progenitor cells, but that it is not directly involved in the regulation of the final cell cycle that gives rise to the heteroploid horizontal cell population.     

Synthesis and biological activity of a novel class of pyridazine analogues as non-competitive reversible inhibitors of protein tyrosine phosphatase 1B (PTP1B)

A series of novel pyridazine analogues were prepared and the structure-activity relationship of their behavior as inhibitors of PTP1B was evaluated. Most of the analogues had potencies in the low micromolar range. The in vitro kinetics of this compound series demonstrated that they were reversible non-competitive binders. This indicates that there may exist another site in the enzyme through which enzyme activity can be inhibited, which is not a recognized interaction domain. Some of the analogues exhibited high selectivity for other PTPases, for example, compound 12 mp showed 20-fold selectivity for PTP1B (IC50=5.6 microM) versus both TCPTP and LAR (>100 microM, respectively). In contrast to many tyrosine phosphatase mimetic inhibitors, this compound class lacks negative charge and thus showed high permeability across cell membranes. Selective analogues in the series were analyzed in an in vitro cellular assay, which showed increased insulin-stimulated insulin receptor phosphorylation.