In vivo visualization of Mg-protoporphyrin IX, a coordinator of photosynthetic gene expression in the nucleus and the chloroplast

The photosynthetic apparatus is composed of proteins encoded by genes from both the nucleus and the chloroplast. To ensure that the photosynthetic complexes are assembled stoichiometrically and to enable their rapid reorganization in response to a changing environment, the plastids emit signals that regulate nuclear gene expression to match the status of the plastids. One of the plastid signals, the chlorophyll intermediate Mg-ProtoporphyrinIX (Mg-ProtoIX) accumulates under stress conditions and acts as a negative regulator of photosynthetic gene expression. By taking advantage of the photoreactive property of tetrapyrroles, Mg-ProtoIX could be visualized in the cells using confocal laser scanning spectroscopy. Our results demonstrate that Mg-ProtoIX accumulated both in the chloroplast and in the cytosol during stress conditions. Thus, the signaling metabolite is exported from the chloroplast, transmitting the plastid signal to the cytosol. Our results from the Mg-ProtoIX over- and underaccumulating mutants copper response defect and genome uncoupled5, respectively, demonstrate that the expression of both nuclear- and plastid-encoded photosynthesis genes is regulated by the accumulation of Mg-ProtoIX. Thus, stress-induced accumulation of the signaling metabolite Mg-ProtoIX coordinates nuclear and plastidic photosynthetic gene expression.     

Spatial patterns on the sagebrush steppe/Western juniper ecotone

Analysis of the spatial patterns of woody plants is important to better understand the ecological processes that govern the worldwide expansion of woody plants across semi-arid ecosystems. Second-order characteristics of a marked spatial point pattern of western juniper (Juniperus occidentalis subsp. occidentalis) were analyzed using Ripley’s K-functions and the pair-correlation function g. The marked point process of crown diameters was produced via two-dimensional wavelet analysis of a fine scale aerial photograph at the woodland-steppe ecotone in the Reynolds Creek watershed in the Owyhee Mountains, southwestern Idaho. Colonization of J. occidentalis stems from mature juniper trees growing in rocky, fire resistant areas. Although these areas introduce components of natural heterogeneity within the landscape, the selected study area is situated within a single soil type, and we modeled the expansion of juniper plants into previously juniper-free sagebrush steppe as a homogeneous point process with constant intensity. Through this research we have identified two statistically significant spatial scales characteristic of J. occidentalis on the woodland/steppe ecotone: (1) We observed inhibition between J. occidentalis plants at distances <15 m, resulting in a regular pattern, rather than clumped or random. This short-distance inhibition can be attributed to competition for water and other resources. Recruitment of young J. occidentalis occurs significantly more often in a direction away from older plants, maximizing the utilization of water and light resources, and perpetuating the spread of the species into previously juniper-free shrub-steppe. (2) J. occidentalis on the ecotone exhibits significant clustering within a 30–60 m radius. Bivariate point pattern analyses provide evidence that, within a distance of 50–70 m, there is a spatial dependence in tree size such that medium trees are more likely than small trees to be close to large trees. We attribute these phenomena to the fact that juniper seeds are commonly dispersed by berry-eating birds with small territories (0.3–1 ha). Beyond a distance of 50–70 m, juniper plants are randomly distributed, suggesting that additional long-range seed dispersal processes are at work. We further acknowledge the importance of including a reference to spatial scale when formulating hypotheses in statistical analysis of spatio-temporal point patterns.     

RNA quality in frozen breast cancer samples and the influence on gene expression analysis – a comparison of three evaluation methods using microcapillary electrophoresis traces

Background  

Assessing RNA quality is essential for gene expression analysis, as the inclusion of degraded samples may influence the interpretation of expression levels in relation to biological and/or clinical parameters. RNA quality can be analyzed by agarose gel electrophoresis, UV spectrophotometer, or microcapillary electrophoresis traces, and can furthermore be evaluated using different methods. No generally accepted recommendations exist for which technique or evaluation method is the best choice. The aim of the present study was to use microcapillary electrophoresis traces from the Bioanalyzer to compare three methods for evaluating RNA quality in 24 fresh frozen invasive breast cancer tissues: 1) Manual method = subjective evaluation of the electropherogram, 2) Ratio Method = the ratio between the 28S and 18S peaks, and 3) RNA integrity number (RIN) method = objective evaluation of the electropherogram. The results were also related to gene expression profiling analyses using 27K oligonucleotide microarrays, unsupervised hierarchical clustering analysis and ontological mapping.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Certolizumab pegol plus methotrexate 5-year results from the rheumatoid arthritis prevention of structural damage (RAPID) 2 randomized controlled trial and long-term extension in rheumatoid arthritis patients

IntroductionAs patients with rheumatoid arthritis (RA) receive treatment with anti-tumour necrosis factors over several years, it is important to evaluate their long-term safety and efficacy. The objective of this study was to examine the safety and benefits of certolizumab pegol (CZP)+methotrexate (MTX) treatment for almost 5 years in patients with RA.MethodsPatients who completed the 24-week Rheumatoid Arthritis Prevention of Structural Damage (RAPID) 2 randomized controlled trial (RCT; {"type":"clinical-trial","attrs":{"text":"NCT00160602","term_id":"NCT00160602"}}NCT00160602), or who were American College of Rheumatology (ACR) 20 non-responders at Week 16, entered the open-label extension (OLE; {"type":"clinical-trial","attrs":{"text":"NCT00160641","term_id":"NCT00160641"}}NCT00160641). After ≥6 months treatment with CZP 400 mg every two weeks (Q2W), dose was reduced to 200 mg Q2W, the approved maintenance dose. Safety data are presented from all patients who received ≥1 dose CZP (Safety population, n=612). Efficacy data are presented to Week 232 for the intent-to-treat (ITT, n=492) and Week 24 CZP RCT Completer (n=342) populations, and through 192 weeks of dose-reduction for the Dose-reduction population (patients whose CZP dose was reduced to 200 mg, n=369). Radiographic progression (modified total Sharp score change from RCT baseline >0.5) to Week 128 is reported for the Week 24 CZP Completers.ResultsIn the RCT, 619 patients were randomized to CZP+MTX (n=492) or placebo+MTX (n=127). Overall, 567 patients (91.6%) entered the OLE: 447 CZP and 120 placebo patients. Of all randomized patients, 358 (57.8%) were ongoing at Week 232. Annual drop-out rates during the first four years ranged from 8.4–15.0%. Event rates per 100 patient-years were 163.0 for adverse events (AEs) and 15.7 for serious AEs. Nineteen patients (3.1%) had fatal AEs (incidence rate=0.8). Clinical improvements in the RCT were maintained to Week 232 in the CZP Completers: mean Disease Activity Score 28 (Erythrocyte Sedimentation Rate) change from baseline was −3.4 and ACR20/50/70 responses 68.4%/47.1%/25.1% (non-responder imputation). Similar improvements observed in the ITT were maintained following dose-reduction. 73.2% of CZP Completers had no radiographic progression at Week 128.ConclusionsIn patients with active RA despite MTX therapy, CZP was well tolerated, with no new safety signals identified. CZP provided sustained improvements in clinical outcomes for almost 5 years.

Trial registration

ClinicalTrials.gov, {"type":"clinical-trial","attrs":{"text":"NCT00160602","term_id":"NCT00160602"}}NCT00160602 and {"type":"clinical-trial","attrs":{"text":"NCT00160641","term_id":"NCT00160641"}}NCT00160641. Registered 8 September 2005.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0767-2) contains supplementary material, which is available to authorized users.     

Antiviral, anti-parasitic, and cytotoxic effects of 5,6-dihydroxyindole (DHI), a reactive compound generated by phenoloxidase during insect immune response

Phenoloxidase (PO) and its activation system are implicated in several defense responses of insects. Upon wounding or infection, inactive prophenoloxidase (proPO) is converted to active PO through a cascade of serine proteases and their homologs. PO generates reactive compounds such as 5,6-dihydroxyindole (DHI), which have a broad-spectrum antibacterial and antifungal activity. Here we report that DHI and its spontaneous oxidation products are also active against viruses and parasitic wasps. Preincubation of a baculovirus stock with 1.25 mM DHI for 3 h near fully disabled recombinant protein production. The LC₅₀ for lambda bacteriophage and eggs of the wasp Microplitis demolitor were 5.6 ± 2.2 and 111.0 ± 1.6 ??M, respectively. The toxicity of DHI and related compounds also extended to cells derived from insects that serve as hosts for several of the aforementioned pathogens. Pretreatment of Sf9 cells with 1.0 mM DHI for 4 h resulted in 97% mortality, and LC₅₀ values of 20.3 ± 1.2 ??M in buffer and 131.8 ± 1.1 ??M in a culture medium. Symptoms of DHI toxicity in Sf9 cells included DNA polymerization, protein crosslinking, and lysis. Taken together, these data showed that proPO activation and DHI production is strongly toxic against various pathogens but can also damage host tissues and cells if not properly controlled.     

Discovery of N-(1-adamantyl)-2-(4-alkylpiperazin-1-yl)acetamide derivatives as T-type calcium channel (Cav3.2) inhibitors

Chemical evolution of a HTS-based fragment hit resulted in the identification of N-(1-adamantyl)-2-[4-(2-tetrahydropyran-4-ylethyl)piperazin-1-yl]acetamide, a novel, selective T-type calcium channel (Ca(v)3.2) inhibitor with in vivo antihypertensive effect in rats.     

Allele coding in genomic evaluation

Background

Genomic data are used in animal breeding to assist genetic evaluation. Several models to estimate genomic breeding values have been studied. In general, two approaches have been used. One approach estimates the marker effects first and then, genomic breeding values are obtained by summing marker effects. In the second approach, genomic breeding values are estimated directly using an equivalent model with a genomic relationship matrix. Allele coding is the method chosen to assign values to the regression coefficients in the statistical model. A common allele coding is zero for the homozygous genotype of the first allele, one for the heterozygote, and two for the homozygous genotype for the other allele. Another common allele coding changes these regression coefficients by subtracting a value from each marker such that the mean of regression coefficients is zero within each marker. We call this centered allele coding. This study considered effects of different allele coding methods on inference. Both marker-based and equivalent models were considered, and restricted maximum likelihood and Bayesian methods were used in inference.

Results

Theoretical derivations showed that parameter estimates and estimated marker effects in marker-based models are the same irrespective of the allele coding, provided that the model has a fixed general mean. For the equivalent models, the same results hold, even though different allele coding methods lead to different genomic relationship matrices. Calculated genomic breeding values are independent of allele coding when the estimate of the general mean is included into the values. Reliabilities of estimated genomic breeding values calculated using elements of the inverse of the coefficient matrix depend on the allele coding because different allele coding methods imply different models. Finally, allele coding affects the mixing of Markov chain Monte Carlo algorithms, with the centered coding being the best.

Conclusions

Different allele coding methods lead to the same inference in the marker-based and equivalent models when a fixed general mean is included in the model. However, reliabilities of genomic breeding values are affected by the allele coding method used. The centered coding has some numerical advantages when Markov chain Monte Carlo methods are used.