FRAP Analysis on Red Alga Reveals the Fluorescence Recovery Is Ascribed to Intrinsic Photoprocesses of Phycobilisomes than Large-Scale Diffusion

BACKGROUND: Phycobilisomes (PBsomes) are the extrinsic antenna complexes upon the photosynthetic membranes in red algae and most cyanobacteria. The PBsomes in the cyanobacteria has been proposed to present high lateral mobility on the thylakoid membrane surface. In contrast, direct measurement of PBsome motility in red algae has been lacking so far. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we investigated the dynamics of PBsomes in the unicellular red alga Porphyridium cruentum in vivo and in vitro, using fluorescence recovery after photobleaching (FRAP). We found that part of the fluorescence recovery could be detected in both partially- and wholly-bleached wild-type and mutant F11 (UTEX 637) cells. Such partial fluorescence recovery was also observed in glutaraldehyde-treated and betaine-treated cells in which PBsome diffusion should be restricted by cross-linking effect, as well as in isolated PBsomes immobilized on the glass slide. CONCLUSIONS/SIGNIFICANCE: On the basis of our previous structural results showing the PBsome crowding on the native photosynthetic membrane as well as the present FRAP data, we concluded that the fluorescence recovery observed during FRAP experiment in red algae is mainly ascribed to the intrinsic photoprocesses of the bleached PBsomes in situ, rather than the rapid diffusion of PBsomes on thylakoid membranes in vivo. Furthermore, direct observations of the fluorescence dynamics of phycoerythrins using FRAP demonstrated the energetic decoupling of phycoerythrins in PBsomes against strong excitation light in vivo, which is proposed as a photoprotective mechanism in red algae attributed by the PBsomes in response to excess light energy

Cell-free (RNA) and cell-associated (DNA) HIV-1 and postnatal transmission through breastfeeding

<p>Introduction - Transmission through breastfeeding remains important for mother-to-child transmission (MTCT) in resource-limited settings. We quantify the relationship between cell-free (RNA) and cell-associated (DNA) shedding of HIV-1 virus in breastmilk and the risk of postnatal HIV-1 transmission in the first 6 months postpartum.</p> <p>Materials and Methods - Thirty-six HIV-positive mothers who transmitted HIV-1 by breastfeeding were matched to 36 non-transmitting HIV-1 infected mothers in a case-control study nested in a cohort of HIV-infected women. RNA and DNA were quantified in the same breastmilk sample taken at 6 weeks and 6 months. Cox regression analysis assessed the association between cell-free and cell-associated virus levels and risk of postnatal HIV-1 transmission.</p> <p>Results - There were higher median levels of cell-free than cell-associated HIV-1 virus (per ml) in breastmilk at 6 weeks and 6 months. Multivariably, adjusting for antenatal CD4 count and maternal plasma viral load, at 6 weeks, each 10-fold increase in cell-free or cell-associated levels (per ml) was significantly associated with HIV-1 transmission but stronger for cell-associated than cell-free levels [2.47 (95% CI 1.33–4.59) vs. aHR 1.52 (95% CI, 1.17–1.96), respectively]. At 6 months, cell-free and cell-associated levels (per ml) in breastmilk remained significantly associated with HIV-1 transmission but was stronger for cell-free than cell-associated levels [aHR 2.53 (95% CI 1.64–3.92) vs. 1.73 (95% CI 0.94–3.19), respectively].</p> <p>Conclusions - The findings suggest that cell-associated virus level (per ml) is more important for early postpartum HIV-1 transmission (at 6 weeks) than cell-free virus. As cell-associated virus levels have been consistently detected in breastmilk despite antiretroviral therapy, this highlights a potential challenge for resource-limited settings to achieve the UNAIDS goal for 2015 of eliminating vertical transmission. More studies would further knowledge on mechanisms of HIV-1 transmission and help develop more effective drugs during lactation.</p&gt

The influence of allogenic blood transfusion in patients having free-flap primary surgery for oral and oropharyngeal squamous cell carcinoma

The influence of perioperative blood transfusion in oral and oropharyngeal squamous cell carcinoma remains uncertain. It is believed that blood transfusion downregulates the immune system and may have an influence on cancer recurrence and survival. In all, 559 consecutive patients undergoing primary surgery for oral and oropharyngeal squamous cell carcinoma between 1992 and 2002 were included in this study. Known prognostic variables along with transfusion details were obtained from head and neck cancer and blood transfusion service databases, respectively. Adjusting for relevant prognostic factors in Cox regression, the hazard ratio for patients having 3 or more transfused units relative to those not transfused was 1.52 (95% confidence interval (CI) 0.93–2.47) for disease-specific and 1.52 (95% CI 1.05–2.22) for overall mortality. Blood transfusion of 3 or more units might confer a worse prognosis in patients undergoing primary surgery for oral and oropharyngeal squamous cell carcinoma. Therefore, every effort should be made to limit the amount of blood transfused to the minimum requirement

Fifty years of scheduling: a survey of milestones

Scheduling has become a major field within operational research with several hundred publications appearing each year. This paper explores the historical development of the subject since the mid 1950s when the landmark publications started to appear. A discussion of the main topics of scheduling research for the past five decades is provided, highlighting the key contributions that helped shape the subject. The main topics covered in the respective decades are combinatorial analysis, branch and bound, computational complexity and classification, approximate solution algorithms, and enhanced scheduling models

Mixed inflammatory/regulatory cytokine profile marked by simultaneous raise of interferon-γ and interleukin-10 and low frequency of tumour necrosis factor-α(+) monocytes are hallmarks of active human visceral Leishmaniasis due to Leishmania chagasi infection

Considering the complexity of the immunological events triggered during active visceral Leishmaniasis (VL), the relevance of the segregation of the immune response during human VL into type 1 and type 2 still remains unclear. For this purpose, in individuals living in risk areas for VL, we have evaluated especially asymptomatic individuals and patients with active VL, the plasmatic levels of cytokines and reactive nitrogen species under ex vivo conditions. In addition, we have also performed an analysis of intracellular cytokine patterns of circulating leucocytes after short-term culture, particularly in the absence of antigenic-specific stimulation, in order to reflect dynamic events of immune response in vivo during Leishmania chagasi infection. Although asymptomatic individuals and non-infected subjects presented a similar immunological profile, an outstanding inflammatory/regulatory profile, based on higher plasmatic levels of cytokines such as interleukin (IL)-8, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, IL-6 and IL-10, was associated with clinical status observed in active VL. In this context, we hypothesize that IL-10, through its ability to inhibit anti-leishmanial macrophage activation, associated with the lower frequency of TNF-α(+) monocytes and ordinary levels of nitrite and nitrate are the major mechanisms associated with disease onset

Compartmentalized Immune Response in Leishmaniasis: Changing Patterns throughout the Disease

Visceral leishmaniasis (VL) is characterized by loss of T-cell responsiveness and absence of Leishmania-specific IFN-γ production by peripheral blood mononuclear cells. However, the expressions of IFN-γ and TNF-α are up-regulated in the tissues and plasma of VL patients. There is a paucity of information regarding the cytokine profile expressed by different target tissues in the same individual and the changes it undergoes throughout the course of infection. In this work we evaluated IFN-γ, TNF-α, IL-10, and TGF-β mRNA expression using real-time RT-PCR in 5 target tissues at 6 months and 16 months post-infection (PI) in a canine experimental model which mimics many aspects of human VL. The spleen and liver of Leishmania infantum experimentally-infected dogs elicited a pro- and anti- inflammatory response and high parasite density at 6 and 16 months PI. The popliteal lymph node, however, showed an up-regulation of IFN-γ cytokin at commencement of the study and was at the chronic phase when the IL-10 and TGF-β expression appeared. In spite of skin parasite invasion, local cytokine response was absent at 6 months PI. Parasite growth and onset of clinical disease both correlated with dermal up-regulation of all the studied cytokines. Our VL model suggests that central target organs, such as the spleen and liver, present a mixed cytokine immune response early on infection. In contrast, an anti-inflammatory/regulatory immune response in peripheral tissues is activated in the later chronic-patent stages of the disease.This work was funded by Projects AGL2008-00748 and AGL2010-16678-GAN from the Spanish Government, and Grant 2005 FI 01116 from Generalitat de Catalunya Spain.S