Artificial endosperm of Cleopatra tangerine zygotic embryos: a model for somatic embryo encapsulation

Synthetic seed technology may be of value in breeding programs and allow the propagation of many elite genotype-derived plants in a short time. In this work, a range of artificial endosperm treatments of Cleopatra tangerine zygotic embryos were evaluated for suitability for encapsulation of somatic embryos. Different complexing ions in the form of alginate capsules, zeolite as an ion exchanger and the relationship between capsule-nutrient gel on germination of zygotic embryos, were evaluated. Artificial endosperm assays showed that abscisic acid (1 μM) and mannitol (0.25 M) delayed germination and conversion of zygotic embryos, whereas amino acid supplements (proline, glutamic acid and arginine) accelerated the conversion process. An artificial endosperm was used to encapsulate somatic and zygotic embryos. After encapsulation, zygotic embryos germinated after four days of culture while somatic embryos germinated asynchronously after 20 days. Somatic embryo-derived plantlets showed greater vigour than zygotic embryo-derived plantlets. Results showed that this artificial endosperm is adequate for Cleopatra tangerine somatic embryo germination and conversion into plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Level of Expression of Thioredoxin is Linked to Fundamental Properties and Applications of Wheat Seeds

Work with cereals （barley and wheat） and a legume （Medicago truncatula） has established thioredoxin h （Trx h） as a central regulatory protein of seeds. Trx h acts by reducing disulfide （S-S） groups of diverse seed proteins （storage proteins, enzymes, and enzyme inhibitors）, thereby facilitating germination. Early in vitro protein studies were complemented with experiments in which barley seeds with Trx h overexpressed in the endosperm showed accelerated germination and early or enhanced expression of associated enzymes （α-amylase and pullulanase）. The current study extends the transgenic work to wheat. Two approaches were followed to alter the expression of Trx h genes in the endosperm： （1） a hordein promoter and its protein body targeting sequence led to overexpression of Trx hS, and （2） an antisense construct of Trx h9 resulted in cytosolic underexpression of that gene （Arabidopsis designation）. Underexpression of Trx h9 led to effects opposite to those observed for overexpression Trx h5 in barley--retardation of germination and delayed or reduced expression of associated enzymes. Similar enzyme changes were observed in developing seeds. The wheat lines with underexpressed Trx showed delayed preharvest sprouting when grown in the greenhouse or field without a decrease in final yield. Wheat with overexpressed Trx h5 showed changes commensurate with earlier in vitro work： increased solubility of disulfide proteins and lower aUergenicity of the gliadin fraction. The results are further evidence that the level of Trx h in cereal endosperm determines fundamental properties as well as potential applications of the seed.     

Detailed imaging and genetic analysis reveal a secondary BRAFL505H resistance mutation and extensive intrapatient heterogeneity in metastatic BRAF mutant melanoma patients treated with vemurafenib

Resistance to treatment is the main problem of targeted treatment for cancer. We followed ten patients during treatment with vemurafenib, by three‐dimensional imaging. In all patients, only a subset of lesions progressed. Next‐generation DNA sequencing was performed on sequential biopsies in four patients to uncover mechanisms of resistance. In two patients, we identified mutations that explained resistance to vemurafenib; one of these patients had a secondary BRAF L505H mutation. This is the first observation of a secondary BRAF mutation in a vemurafenib‐resistant patient‐derived melanoma sample, which confirms the potential importance of the BRAF L505H mutation in the development of therapy resistance. Moreover, this study hints toward an important role for tumor heterogeneity in determining the outcome of targeted treatments.     

The Local Complement Activation on Vascular Bed of Patients with Systemic Sclerosis: A Hypothesis-Generating Study

ObjectiveThe role of complement system in the pathogenesis of systemic sclerosis (SSc) has been debated during the last decade but an evident implication in this disease has never been found. We carried out an explorative study on SSc patients to evaluate the expression of soluble and local C5b-9 complement complex and its relation with a complement regulator, the Membrane Cofactor Protein (MCP, CD46) on skin vascular bed as target distinctive of SSc disease. We also analyzed two polymorphic variants in the complement activation gene cluster involving the MCP region.MethodsC5b-9 plasma levels of SSc patients and healthy subjects were analyzed by ELISA assay. Archival skin biopsies of SSc patients and controls were subjected to immunofluorescence analysis to detect C5b-9 and MCP on vascular endothelial cells. The expression of MCP was validated by immunoblot analysis with specific antibody. Polymorphic variants in the MCP gene promoter were tested by a quantitative PCR technique-based allelic discrimination method.ResultsEven though circulating levels of C5b-9 did not differ between SSc and controls, C5b-9 deposition was detected in skin biopsies of SSc patients but not in healthy subjects. MCP was significantly lower in skin vessels of SSc patients than in healthy controls and was associated with the over-expression of two polymorphic variants in the MCP gene promoter, which has been related to more aggressive phenotypes in other immune-mediated diseases.ConclusionsOur results firsty document the local complement activation with an abnormal expression of MCP in skin vessels of SSc patients, suggesting that a subset of SSc patients might be exposed to more severe organ complications and clinical evolution due to abnormal local complement activation.     

A Human Type 5 Adenovirus-Based Trypanosoma cruzi Therapeutic Vaccine Re-programs Immune Response and Reverses Chronic Cardiomyopathy

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is a prototypical neglected tropical disease. Specific immunity promotes acute phase survival. Nevertheless, one-third of CD patients develop chronic chagasic cardiomyopathy (CCC) associated with parasite persistence and immunological unbalance. Currently, the therapeutic management of patients only mitigates CCC symptoms. Therefore, a vaccine arises as an alternative to stimulate protective immunity and thereby prevent, delay progression and even reverse CCC. We examined this hypothesis by vaccinating mice with replication-defective human Type 5 recombinant adenoviruses (rAd) carrying sequences of amastigote surface protein-2 (rAdASP2) and trans-sialidase (rAdTS) T. cruzi antigens. For prophylactic vaccination, naïve C57BL/6 mice were immunized with rAdASP2+rAdTS (rAdVax) using a homologous prime/boost protocol before challenge with the Colombian strain. For therapeutic vaccination, rAdVax administration was initiated at 120 days post-infection (dpi), when mice were afflicted by CCC. Mice were analyzed for electrical abnormalities, immune response and cardiac parasitism and tissue damage. Prophylactic immunization with rAdVax induced antibodies and H-2K^b-restricted cytotoxic and interferon (IFN)γ-producing CD8⁺ T-cells, reduced acute heart parasitism and electrical abnormalities in the chronic phase. Therapeutic vaccination increased survival and reduced electrical abnormalities after the prime (analysis at 160 dpi) and the boost (analysis at 180 and 230 dpi). Post-therapy mice exhibited less heart injury and electrical abnormalities compared with pre-therapy mice. rAdVax therapeutic vaccination preserved specific IFNγ-mediated immunity but reduced the response to polyclonal stimuli (anti-CD3 plus anti-CD28), CD107a⁺ CD8⁺ T-cell frequency and plasma nitric oxide (NO) levels. Moreover, therapeutic rAdVax reshaped immunity in the heart tissue as reduced the number of perforin+ cells, preserved the number of IFNγ⁺ cells, increased the expression of IFNγ mRNA but reduced inducible NO synthase mRNA. Vaccine-based immunostimulation with rAd might offer a rational alternative for re-programming the immune response to preserve and, moreover, recover tissue injury in Chagas’ heart disease.     

Prevalence of Pulmonary Hypertension in the General Population: The Rotterdam Study

Background

Pulmonary hypertension is characterized by increased pulmonary artery pressure and carries an increased mortality. Population-based studies into pulmonary hypertension are scarce and little is known about its prevalence in the general population. We aimed to describe the distribution of echocardiographically-assessed pulmonary artery systolic pressure (ePASP) in the general population, to estimate the prevalence of pulmonary hypertension, and to identify associated factors.

Methods

Participants (n = 3381, mean age 76.4 years, 59% women) from the Rotterdam Study, a population-based cohort, underwent echocardiography. Echocardiographic pulmonary hypertension was defined as ePASP>40 mmHg.

Results

Mean ePASP was 26.3 mmHg (SD 7.0). Prevalence of echocardiographic pulmonary hypertension was 2.6% (95%CI: 2.0; 3.2). Prevalence was higher in older participants compared to younger ones (8.3% in those over 85 years versus 0.8% in those between 65 and 70), and in those with underlying disorders versus those without (5.9% in subjects with COPD versus 2.3%; 9.2% in those with left ventricular systolic dysfunction versus 2.3%; 23.1% in stages 3 or 4 left ventricular diastolic dysfunction versus 1.9% in normal or stage 1). Factors independently associated with higher ePASP were older age, higher BMI, left ventricular diastolic dysfunction, COPD and systemic hypertension.

Conclusion

In this large population-based study, we show that pulmonary hypertension as measured by echocardiography has a low prevalence in the overall general population in the Netherlands, but estimates may be higher in specific subgroups, especially in those with underlying diseases. Increased pulmonary arterial pressure is likely to gain importance in the near future due to population aging and the accompanying prevalences of underlying disorders.     

Dysregulated Immune Activation in Second-Line HAART HIV+ Patients Is Similar to That of Untreated Patients

Background

Successful highly active antiretroviral therapy (HAART) has changed the outcome of AIDS patients worldwide because the complete suppression of viremia improves health and prolongs life expectancy of HIV-1+ patients. However, little attention has been given to the immunological profile of patients under distinct HAART regimens. This work aimed to investigate the differences in the immunological pattern of HIV-1+ patients under the first- or second-line HAART in Brazil.

Methods

CD4+ T cell counts, Viral load, and plasma concentration of sCD14, sCD163, MCP-1, RANTES, IP-10, IL-1β, IL-6, TNF-α, IL-12, IFN-α, IFN-γ, IL-4, IL-5, and IL-10 were assessed for immunological characterization of the following clinical groups: Non-infected individuals (NI; n = 66), HIV-1+ untreated (HIV; n = 46), HIV-1+ treated with first-line HAART (HAART 1; n = 15); and HIV-1+ treated with second-line HAART (HAART 2; n = 15).

Results

We found that the immunological biosignature pattern of HAART 1 is similar to that of NI individuals, especially in patients presenting slow progression of the disease, while patients under HAART 2 remain in a moderate inflammatory state, which is similar to that of untreated HIV patients pattern. Network correlations revealed that differences in IP-10, TNF-α, IL-6, IFN-α, and IL-10 interactions were primordial in HIV disease and treatment. Heat map and decision tree analysis identified that IP-10>TNF-α>IFN-α were the best respective HAART segregation biomarkers.

Conclusion

HIV patients in different HAART regimens develop distinct immunological biosignature, introducing a novel perspective into disease outcome and potential new therapies that consider HAART patients as a heterogeneous group.