New fluorescence parameters for monitoring photosynthesis in plants - 1. The effect of illumination on the fluorescence parameters of the JIP-test

Chlorophyll fluorescence measurements have a wide range of applications from basic understanding of photosynthesis functioning to plant environmental stress responses and direct assessments of plant health. The measured signal is the fluorescence intensity (expressed in relative units) and the most meaningful data are derived from the time dependent increase in fluorescence intensity achieved upon application of continuous bright light to a previously dark adapted sample. The fluorescence response changes over time and is termed the Kautsky curve or chlorophyll fluorescence transient. Recently, Strasser and Strasser (1995) formulated a group of fluorescence parameters, called the JIP-test, that quantify the stepwise flow of energy through Photosystem II, using input data from the fluorescence transient. The purpose of this study was to establish relationships between the biochemical reactions occurring in PS II and specific JIP-test parameters. This was approached using isolated systems that facilitated the addition of modifying agents, a PS II electron transport inhibitor, an electron acceptor and an uncoupler, whose effects on PS II activity are well documented in the literature. The alteration to PS II activity caused by each of these compounds could then be monitored through the JIP-test parameters and compared and contrasted with the literature. The known alteration in PS II activity of Chenopodium album atrazine resistant and sensitive biotypes was also used to gauge the effectiveness and sensitivity of the JIP-test. The information gained from the in vitro study was successfully applied to an in situ study. This is the first in a series of four papers. It shows that the trapping parameters of the JIP-test were most affected by illumination and that the reduction in trapping had a run-on effect to inhibit electron transport. When irradiance exposure proceeded to photoinhibition, the electron transport probability parameter was greatly reduced and dissipation significantly increased. These results illustrate the advantage of monitoring a number of fluorescence parameters over the use of just one, which is often the case when the F-V/F-M ratio is used.

Induction of iron regulated proteins during normal growth of Neisseria meningitidis in a chemically defined medium

The expression of iron regulated proteins (IRPs) in vitro has been obtained in the past by adding iron chelators to the culture after bacterial growth, in the presence of an organic iron source. We have investigated aspects concerning full expression of the meningococcal IRPs during normal growth, in defined conditions using Catlin medium, Mueller Hinton and Tryptic Soy Broth (TSB). The expression of IRPs varied between different strains with respect to Ethylenediamine Di-ortho-Hidroxy-phenyl-acetic acid (EDDA) concentrations, and according to culture medium, and also between different lots of TSB. For each strain, a specific set of IRPs were expressed and higher EDDA concentrations, or addition of glucose, or use of different culture media did not resulted in a differential expression of IRPs. We were not able to grow N. meningitidis under normal growth conditions using Desferal. We looked for a good yield of outer membrane vesicles (OMVs) expressing IRPs in iron-deficient Catlin medium containing EDDA and Hemin. Culture for 32 h at 30ºC after growing for 16 h at 37ºC supported good bacterial growth. Bacterial lysis was noted after additional 24 h at 30ºC. Approximately 4 times more OMVs was recoverable from a culture supernatant after 24 h at 30ºC than from the cells after 16 h at 37ºC. The IRP were as well expressed in OMVs from culture supernatant obtained after 24 h at 30ºC as from the cells after 16 h at 37ºC.

Differential peptide binding to CD40 evokes counteractive responses.

The antigen-presenting cell-expressed CD40 is implied in the regulation of counteractive immune responses such as induction of pro-inflammatory and anti-inflammatory cytokines interleukin (IL)-12 and IL-10, respectively. The mechanism of this duality in CD40 function remains unknown. Here, we investigated whether such duality depends on ligand binding. Based on CD40 binding, we identifed two dodecameric peptides, peptide-7 and peptide-19, from the phage peptide library. Peptide-7 induces IL-10 and increases Leishmania donovani infection in macrophages, whereas peptide-19 induces IL-12 and reduces L. donovani infection. CD40-peptide interaction analyses by surface plasmon resonance and atomic force microscopy suggest that the functional differences are not associated with the studied interaction parameters. The molecular dynamic simulation of the CD40-peptides interaction suggests that these two peptides bind to two different places on CD40. Thus, we suggest for the first time that differential binding of the ligands imparts functional duality to CD40.

Cured by tonsillectomy: was it really a PFAPA syndrome?

We show how an ultrafast pump-pump excitation induces strong fluorescence depletion in biological samples, such as bacteria-containing droplets, in contrast with fluorescent interferents, such as polycyclic aromatic compounds, despite similar spectroscopic properties. Application to the optical remote discrimination of biotic versus non-biotic particles is proposed. Further improvement is required to allow the discrimination of one pathogenic among other non-pathogenic micro-organisms. This improved selectivity may be reached with optimal coherent control experiments, as discussed in the paper.

Tacrolimus and cyclosporine have differential effects on the risk of development of bronchiolitis obliterans syndrome: results of a prospective, randomized international trial in lung transplantation.

BACKGROUND: Chronic lung allograft dysfunction, which manifests as bronchiolitis obliterans syndrome (BOS), is recognized as the primary cause of morbidity and mortality after lung transplantation. In this study we assessed the efficacy and safety of two de novo immunosuppression protocols to prevent BOS. METHODS: Our study approach was a multicenter, prospective, randomized (1:1) open-label superiority investigation of de novo tacrolimus vs cyclosporine, with both study arms given mycophenolate mofetil and prednisolone after lung transplantation. Cytolytic induction therapy was not employed. Patients were stratified at entry for cystic fibrosis. Primary outcome was incidence of BOS 3 years after transplant (intention-to-treat analysis). Secondary outcomes were survival and incidence of acute rejection, infection and other adverse events. RESULTS: Group demographic data were well matched: 110 of 124 tacrolimus vs 74 of 125 cyclosporine patients were treated per protocol (p < 0.01 by chi-square test). Cumulative incidence of BOS Grade ≥1 at 3 years was 11.6% (tacrolimus) vs 21.3% (cyclosporine) (cumulative incidence curves, p = 0.037 by Gray's test, pooled over strata). Univariate proportional sub-distribution hazards regression confirmed cyclosporine as a risk for BOS (HR 1.97, 95% CI 1.04 to 3.77, p = 0.039). Three-year cumulative incidence of acute rejection was 67.4% (tacrolimus) vs 74.9% (cyclosporine) (p = 0.118 by Gray's test). One- and 3-year survival rates were 84.6% and 78.7% (tacrolimus) vs 88.6% and 82.8% (cyclosporine) (p = 0.382 by log-rank test). Cumulative infection rates were similar (p = 0.91), but there was a trend toward new-onset renal failure with tacrolimus (p = 0.09). CONCLUSIONS: Compared with cyclosporine, de novo tacrolimus use was found to be associated with a significantly reduced risk for BOS Grade ≥1 at 3 years despite a similar rate of acute rejection. However, no survival advantage was detected.

Differential expression of notch signaling-related transcripts accompanies Pro-thymocyte proliferation and phenotype transition induced by epidermal growth factor plus insulin in fetal thymus organ cultures

Thymus regression upon stressing stimuli, such as infectious diseases, is followed by organ reconstitution, paralleling its development in ontogeny. A narrow window of thymus development was here studied, encompassing the pro-T lymphoid precursor expansion during specification stages, by the use of epidermal growth factor plus insulin (INS) in murine fetal thymus organ cultures. Aiming to disclose signaling pathways related to these stages, cultured thymus lobes had their RNA extracted, for the search of transcripts differentially expressed using RNAse protection assays and reverse transcriptase-polymerase chain reactions. We found no difference that could explain INS-driven thymocyte growth, in the pattern of transcripts for death/proliferation mediators, or for a series of growth factor receptors and transcriptional regulators known as essential for thymus development. Thymocyte suspensions from cultured lobes, stained for phenotype analysis by fluorescence activated cell sorting, showed a decreased staining for Notch1 protein at cell surfaces upon INS addition. We analyzed the expression of Notch-related elements, and observed the recruitment of a specific set of transcripts simultaneous and compatible with INS-driven thymocyte growth, namely, transcripts for Notch3, for its ligand Jagged2, and for Deltex1, a mediator of a poorly characterized alternative pathway downstream of the Notch receptor.

Differential lectin labelling of circulating hemocytes from Biomphalaria glabrata and Biomphalaria tenagophila resistant or susceptible to Schistosoma mansoni infection

Lectins/carbohydrate binding can be involved in the Schistosoma mansoni recognition and activation of the Biomphalaria hemocytes. Therefore, expression of lectin ligands on Biomphalaria hemocytes would be associated with snail resistance against S. mansoni infection. To test this hypothesis, circulating hemocytes were isolated from B. glabrata BH (snail strain highy susceptible to S. mansoni), B. tenagophila Cabo Frio (moderate susceptibility), and B. tenagophila Taim (completely resistant strains), labelled with FITC conjugated lectins (ConA, PNA, SBA, and WGA) and analyzed under fluorescence microscopy. The results demonstrated that although lectin-labelled hemocytes were detected in hemolymph of all snail species tested, circulating hemocytes from both strains of B. tenagophila showed a larger number of lectin-labelled cells than B. glabrata. Moreover, most of circulating hemocytes of B. tenagophila were intensively labelled by lectins PNA-FITC and WGA-FITC, while in B. glabrata small hemocytes were labeled mainly by ConA. Upon S. mansoni infection, lectin-labelled hemocytes almost disappeared from the hemolymph of Taim and accumulated in B. glabrata BH. The role of lectins/carbohydrate binding in resistance of B. tengophila infection to S. mansoni is still not fully understood, but the data suggest that there may be a correlation to its presence with susceptibility or resistance to the parasite.

Dengue-2 and yellow fever 17DD viruses infect human dendritic cells, resulting in an induction of activation markers, cytokines and chemokines and secretion of different TNF-α and IFN-α profiles

Flaviviruses cause severe acute febrile and haemorrhagic infections, including dengue and yellow fever and the pathogenesis of these infections is caused by an exacerbated immune response. Dendritic cells (DCs) are targets for dengue virus (DENV) and yellow fever virus (YF) replication and are the first cell population to interact with these viruses during a natural infection, which leads to an induction of protective immunity in humans. We studied the infectivity of DENV2 (strain 16681), a YF vaccine (YF17DD) and a chimeric YF17D/DENV2 vaccine in monocyte-derived DCs in vitro with regard to cell maturation, activation and cytokine production. Higher viral antigen positive cell frequencies were observed for DENV2 when compared with both vaccine viruses. Flavivirus-infected cultures exhibited dendritic cell activation and maturation molecules. CD38 expression on DCs was enhanced for both DENV2 and YF17DD, whereas OX40L expression was decreased as compared to mock-stimulated cells, suggesting that a T helper 1 profile is favoured. Tumor necrosis factor (TNF)-α production in cell cultures was significantly higher in DENV2-infected cultures than in cultures infected with YF17DD or YF17D/DENV. In contrast, the vaccines induced higher IFN-α levels than DENV2. The differential cytokine production indicates that DENV2 results in TNF induction, which discriminates it from vaccine viruses that preferentially stimulate interferon expression. These differential response profiles may influence the pathogenic infection outcome.

Differential in vitro activity of the DNA topoisomerase inhibitor idarubicin against Trypanosoma rangeli and Trypanosoma cruzi

In this study the effect of eight DNA topoisomerase inhibitors on the growth Trypanosoma rangeli epimastigotes in cell culture was investigated. Among the eight compounds tested, idarubicin was the only compound that displayed promising trypanocidal activity with a half-maximal growth inhibition (GI50) value in the sub-micromolar range. Fluorescence-activated cell sorting analysis showed a reduction in DNA content in T. rangeli epimastigotes when treated with idarubicin. In contrast to T. rangeli, against Trypanosoma cruzi epimastigotes idarubicin was much less effective exhibiting a GI50 value in the mid-micromolar range. This result indicates that idarubicin displays differential toxic effects in T. rangeli and T. cruzi. Compared with African trypanosomes, it seems that American trypanosomes are generally less susceptible to DNA topoisomerase inhibitors.

MDM2 and CDK4 immunohistochemistry is a valuable tool in the differential diagnosis of low-grade osteosarcomas and other primary fibro-osseous lesions of the bone.

Low-grade osteosarcoma is a rare malignancy that may be subdivided into two main subgroups on the basis of location in relation to the bone cortex, that is, parosteal osteosarcoma and low-grade central osteosarcoma. Their histological appearance is quite similar and characterized by spindle cell stroma with low-to-moderate cellularity and well-differentiated anastomosing bone trabeculae. Low-grade osteosarcomas have a simple genetic profile with supernumerary ring chromosomes comprising amplification of chromosome 12q13-15, including the cyclin-dependent kinase 4 (CDK4) and murine double-minute type 2 (MDM2) gene region. Low-grade osteosarcoma can be confused with fibrous and fibro-osseous lesions such as fibromatosis and fibrous dysplasia on radiological and histological findings. We investigated MDM2-CDK4 immunohistochemical expression in a series of 72 low-grade osteosarcomas and 107 fibrous or fibro-osseous lesions of the bone or paraosseous soft tissue. The MDM2-CDK4 amplification status of low-grade osteosarcoma was also evaluated by comparative genomic hybridization array in 18 cases, and the MDM2 amplification status was evaluated by fluorescence in situ hybridization or quantitative real-time polymerase chain reaction in 31 cases of benign fibrous and fibro-osseous lesions. MDM2-CDK4 immunostaining and MDM2 amplification by fluorescence in situ hybridization or quantitative real-time polymerase chain reaction were investigated in a control group of 23 cases of primary high-grade bone sarcoma, including 20 conventional high-grade osteosarcomas, two pleomorphic spindle cell sarcomas/malignant fibrous histiocytomas and one leiomyosarcoma. The results showed that MDM2 and/or CDK4 immunoreactivity was present in 89% of low-grade osteosarcoma specimens. All benign fibrous and fibro-osseous lesions and the tumors of the control group were negative for MDM2 and CDK4. These results were consistent with the MDM2 and CDK4 amplification results. In conclusion, immunohistochemical expression of MDM2 and CDK4 is specific and provides sensitive markers for the diagnosis of low-grade osteosarcomas, helping to differentiate them from benign fibrous and fibro-osseous lesions, particularly in cases with atypical radio-clinical presentation and/or limited biopsy samples.

Differential peptide binding to CD40 evokes counteractive responses.

The antigen-presenting cell-expressed CD40 is implied in the regulation of counteractive immune responses such as induction of pro-inflammatory and anti-inflammatory cytokines interleukin (IL)-12 and IL-10, respectively. The mechanism of this duality in CD40 function remains unknown. Here, we investigated whether such duality depends on ligand binding. Based on CD40 binding, we identifed two dodecameric peptides, peptide-7 and peptide-19, from the phage peptide library. Peptide-7 induces IL-10 and increases Leishmania donovani infection in macrophages, whereas peptide-19 induces IL-12 and reduces L. donovani infection. CD40-peptide interaction analyses by surface plasmon resonance and atomic force microscopy suggest that the functional differences are not associated with the studied interaction parameters. The molecular dynamic simulation of the CD40-peptides interaction suggests that these two peptides bind to two different places on CD40. Thus, we suggest for the first time that differential binding of the ligands imparts functional duality to CD40.

Differential stress reaction of human colon cells to oleic-acid-stabilized and unstabilized ultrasmall iron oxide nanoparticles.

Therapeutic engineered nanoparticles (NPs), including ultrasmall superparamagnetic iron oxide (USPIO) NPs, may accumulate in the lower digestive tract following ingestion or injection. In order to evaluate the reaction of human colon cells to USPIO NPs, the effects of non-stabilized USPIO NPs (NS-USPIO NPs), oleic-acid-stabilized USPIO NPs (OA-USPIO NPs), and free oleic acid (OA) were compared in human HT29 and CaCo2 colon epithelial cancer cells. First the biophysical characteristics of NS-USPIO NPs and OA-USPIO NPs in water, in cell culture medium supplemented with fetal calf serum, and in cell culture medium preconditioned by HT29 and CaCo₂ cells were determined. Then, stress responses of the cells were evaluated following exposure to NS-USPIO NPs, OA-USPIO NPs, and free OA. No modification of the cytoskeletal actin network was observed. Cell response to stress, including markers of apoptosis and DNA repair, oxidative stress and degradative/autophagic stress, induction of heat shock protein, or lipid metabolism was determined in cells exposed to the two NPs. Induction of an autophagic response was observed in the two cell lines for both NPs but not free OA, while the other stress responses were cell- and NP-specific. The formation of lipid vacuoles/droplets was demonstrated in HT29 and CaCo₂ cells exposed to OA-USPIO NPs but not to NS-USPIO NPs, and to a much lower level in cells exposed to equimolar concentrations of free OA. Therefore, the induction of lipid vacuoles in colon cells exposed to OA utilized as a stabilizer for USPIO NPs is higly amplified compared to free OA, and is not observed in the absence of this lipid in NS-USPIO NPs.

Gene expression following induction of regeneration in Drosophila wing imaginal discs

Background: Regeneration is the ability of an organism to rebuild a body part that has been damaged or amputated, and can be studied at the molecular level using model organisms. Drosophila imaginal discs, which are the larval primordia of adult cuticular structures, are capable of undergoing regenerative growth after transplantation and in vivo culture into the adult abdomen. Results: Using expression profile analyses, we studied the regenerative behaviour of wing discs at 0, 24 and 72 hours after fragmentation and implantation into adult females. Based on expression level, we generated a catalogue of genes with putative role in wing disc regeneration, identifying four classes: 1) genes with differential expression within the first 24 hours; 2) genes with differential expression between 24 and 72 hours; 3) genes that changed significantly in expression levels between the two time periods; 4) genes with a sustained increase or decrease in their expression levels throughout regeneration. Among these genes, we identified members of the JNK and Notch signalling pathways and chromatin regulators. Through computational analysis, we recognized putative binding sites for transcription factors downstream of these pathways that are conserved in multiple Drosophilids, indicating a potential relationship between members of the different gene classes. Experimental data from genetic mutants provide evidence of a requirement of selected genes in wing disc regeneration. Conclusions: We have been able to distinguish various classes of genes involved in early and late steps of the regeneration process. Our data suggests the integration of signalling pathways in the promoters of regulated genes.

Gene expression following induction of regeneration in Drosophila wing imaginal discs

Background: Regeneration is the ability of an organism to rebuild a body part that has been damaged or amputated, and can be studied at the molecular level using model organisms. Drosophila imaginal discs, which are the larval primordia of adult cuticular structures, are capable of undergoing regenerative growth after transplantation and in vivo culture into the adult abdomen. Results: Using expression profile analyses, we studied the regenerative behaviour of wing discs at 0, 24 and 72 hours after fragmentation and implantation into adult females. Based on expression level, we generated a catalogue of genes with putative role in wing disc regeneration, identifying four classes: 1) genes with differential expression within the first 24 hours; 2) genes with differential expression between 24 and 72 hours; 3) genes that changed significantly in expression levels between the two time periods; 4) genes with a sustained increase or decrease in their expression levels throughout regeneration. Among these genes, we identified members of the JNK and Notch signalling pathways and chromatin regulators. Through computational analysis, we recognized putative binding sites for transcription factors downstream of these pathways that are conserved in multiple Drosophilids, indicating a potential relationship between members of the different gene classes. Experimental data from genetic mutants provide evidence of a requirement of selected genes in wing disc regeneration. Conclusions: We have been able to distinguish various classes of genes involved in early and late steps of the regeneration process. Our data suggests the integration of signalling pathways in the promoters of regulated genes.

Contrasting growth responses in lamina and petiole during neighbor detection depend on differential auxin responsiveness rather than different auxin levels.

Foliar shade triggers rapid growth of specific structures that facilitate access of the plant to direct sunlight. In leaves of many plant species, this growth response is complex because, although shade triggers the elongation of petioles, it reduces the growth of the lamina. How the same external cue leads to these contrasting growth responses in different parts of the leaf is not understood. Using mutant analysis, pharmacological treatment and gene expression analyses, we investigated the role of PHYTOCHROME INTERACTING FACTOR7 (PIF7) and the growth-promoting hormone auxin in these contrasting leaf growth responses. Both petiole elongation and lamina growth reduction are dependent on PIF7. The induction of auxin production is both necessary and sufficient to induce opposite growth responses in petioles vs lamina. However, these contrasting growth responses are not caused by different auxin concentrations in the two leaf parts. Our work suggests that a transient increase in auxin levels triggers tissue-specific growth responses in different leaf parts. We provide evidence suggesting that this may be caused by the different sensitivity to auxin in the petiole vs the blade and by tissue-specific gene expression.