PAI-1-dependent endothelial cell death determines severity of radiation-induced intestinal injury

Normal tissue toxicity still remains a dose-limiting factor in clinical radiation therapy. Recently, plasminogen activator inhibitor type 1 (SERPINE1/PAI-1) was reported as an essential mediator of late radiation-induced intestinal injury. However, it is not clear whether PAI-1 plays a role in acute radiation-induced intestinal damage and we hypothesized that PAI-1 may play a role in the endothelium radiosensitivity. In vivo, in a model of radiation enteropathy in PAI-1 -/- mice, apoptosis of radiosensitive compartments, epithelial and microvascular endothelium was quantified. In vitro, the role of PAI-1 in the radiation-induced endothelial cells (ECs) death was investigated. The level of apoptotic ECs is lower in PAI-1 -/- compared with Wt mice after irradiation. This is associated with a conserved microvascular density and consequently with a better mucosal integrity in PAI-1 -/- mice. In vitro, irradiation rapidly stimulates PAI-1 expression in ECs and radiation sensitivity is increased in ECs that stably overexpress PAI-1, whereas PAI-1 knockdown increases EC survival after irradiation. Moreover, ECs prepared from PAI-1 -/- mice are more resistant to radiation-induced cell death than Wt ECs and this is associated with activation of the Akt pathway. This study demonstrates that PAI-1 plays a key role in radiation-induced EC death in the intestine and suggests that this contributes strongly to the progression of radiation-induced intestinal injury.     

Plasma membrane calcium pump (PMCA) isoform 4 is targeted to the apical membrane by the w-splice insert from PMCA2

Local Ca(2+) signaling requires proper targeting of the Ca(2+) signaling toolkit to specific cellular locales. Different isoforms of the plasma membrane Ca(2+) pump (PMCA) are responsible for Ca(2+) extrusion at the apical and basolateral membrane of polarized epithelial cells, but the mechanisms and signals for differential targeting of the PMCAs are not well understood. Recent work demonstrated that the alternatively spliced w-insert in PMCA2 directs this pump to the apical membrane. We now show that inserting the w-insert into the corresponding location of the PMCA4 isoform confers apical targeting to this normally basolateral pump. Mutation of a di-leucine motif in the C-tail thought to be important for basolateral targeting did not enhance apical localization of the chimeric PMCA4(2w)/b. In contrast, replacing the C-terminal Val residue by Leu to optimize the PDZ ligand site for interaction with the scaffolding protein NHERF2 enhanced the apical localization of PMCA4(2w)/b, but not of PMCA4x/b. Functional studies showed that both apical PMCA4(2w)/b and basolateral PMCA4x/b handled ATP-induced Ca(2+) signals with similar kinetics, suggesting that isoform-specific functional characteristics are retained irrespective of membrane targeting. Our results demonstrate that the alternatively spliced w-insert provides autonomous apical targeting information in the PMCA without altering its functional characteristics.     

Human neurocysticercosis: in vivo expansion of peripheral regulatory T cells and their recruitment in the central nervous system

Human neurocysticercosis (NC) is caused by Taenia solium larvae lodged in the central nervous system. Most cases occur with no, or mild, neurological symptoms. However, in some patients, neuroinflammation is exacerbated, leading to severe forms of the disease. Considering the critical role of regulatory T cells (Tregs) in balancing inflammation in chronic diseases, their participation in restraining the inflammatory response in NC was explored in the present study. The frequency of Tregs and their relationship with the level of the proliferative response, the level of activated lymphocytes, and the cytokines expressed were determined in severe NC patients compared with those from healthy donors. Significantly increased peripheral Tregs (CD4(+)CD25(high) and CD4(+)CD25(high)FoxP3(+), CD4(+)CD25(high)CTLA4(+), and CD4(+)CD25(high) IL10(+)) and a significant decrease in activated (CD38(+) and CD69(+)) T cells were observed in 19 NC patients versus 10 healthy subjects. Significantly increased Tregs in NC are accompanied by a depressed specific, and non-specific, lymphocyte proliferative response, and they negatively correlate with activated CD4(+)CD69(+) lymphocytes. Treg frequencies were also determined in cerebral spinal fluid for 8 of the 19 NC patients. A positive significant correlation between peripheral and local Tregs was observed. Here, we report for the first time data that support the possible contribution of local and systemic Tregs in limiting neuroinflammation in NC.     

Extracellular signal-regulated kinase 1/2 signaling promotes oligodendrocyte myelination in vitro

J. Neurochem. (2012) 122, 1167-1180. ABSTRACT: Multiple extracellular factors have been implicated in orchestrating myelination of the CNS; however, less is known about the intracellular signaling cascades that regulate this process. We have previously shown that brain-derived neurotrophic factor (BDNF) promotes oligodendrocyte myelination. Here, we screened for the activation of candidate signaling pathways in in vitro myelination assays and found that extracellular signal-regulated kinase (Erk) signaling positively correlated with basal levels of oligodendrocyte myelination as well as BDNF-induced myelination in vitro. By selectively manipulating Erk1/2 activation in oligodendrocytes in vitro, we found that constitutive activation of Erk1/2 significantly increased myelination, mimicking the promyelinating effect of BDNF, and also caused myelination to occur earlier. Conversely, selective inhibition of Erk1/2 in oligodendrocytes significantly reduced the basal level of myelination and blocked the promyelinating effect of BDNF. Analysis of myelinating spinal cord and corpus callosum white matter tracts revealed that the majority of mature oligodendrocytes are co-labeled with phospho-Erk1/2, whereas phospho-Erk1/2 was rarely observed in oligodendrocyte progenitor cells. Finally, the total level of phospho-Erk1/2 correlated with myelin formation during the early postnatal period. Collectively, these data identify that Erk1/2 signaling within oligodendrocytes exerts an important and direct effect to promote myelination.     

Infant B cell memory differentiation and early gut bacterial colonization

Germ-free animal models have demonstrated that commensal bacterial colonization of the intestine induces B cell differentiation and activation. Whether colonization with particular bacterial species or groups is associated with B cell development during early childhood is not known. In a prospective newborn/infant cohort including 65 Swedish children, we examined the numbers and proportions of CD20(+), CD5(+), and CD27(+) B cells in blood samples obtained at several time points during the first 3 y of life using flow cytometry. Fecal samples were collected and cultured quantitatively for major facultative and anaerobic bacteria at 1, 2, 4, and 8 wk of life. We found that the numbers of CD20(+) B cells and CD5(+)CD20(+) B cells reached their highest levels at 4 mo, whereas CD20(+) B cells expressing the memory marker CD27 were most numerous at 18 and 36 mo of age. Using multivariate analysis, we show that early colonization with Escherichia coli and bifidobacteria were associated with higher numbers of CD20(+) B cells that expressed the memory marker CD27 at 4 and 18 mo of age. In contrast, we were unable to demonstrate any relation between bacterial colonization pattern and numbers of CD20(+) or CD5(+)CD20(+) B cells. These results suggest that the intestinal bacterial colonization pattern may affect the B cell maturation also in humans, and that an early gut microbiota including E. coli and bifidobacteria might promote this maturation.     

Considerations of physicians about the depth of palliative sedation at the end of life

Background:

Although guidelines advise titration of palliative sedation at the end of life, in practice the depth of sedation can range from mild to deep. We investigated physicians’ considerations about the depth of continuous sedation.

Methods:

We performed a qualitative study in which 54 physicians underwent semistructured interviewing about the last patient for whom they had been responsible for providing continuous palliative sedation. We also asked about their practices and general attitudes toward sedation.

Results:

We found two approaches toward the depth of continuous sedation: starting with mild sedation and only increasing the depth if necessary, and deep sedation right from the start. Physicians described similar determinants for both approaches, including titration of sedatives to the relief of refractory symptoms, patient preferences, wishes of relatives, expert advice and esthetic consequences of the sedation. However, physicians who preferred starting with mild sedation emphasized being guided by the patient’s condition and response, and physicians who preferred starting with deep sedation emphasized ensuring that relief of suffering would be maintained. Physicians who preferred each approach also expressed different perspectives about whether patient communication was important and whether waking up after sedation is started was problematic.

Interpretation:

Physicians who choose either mild or deep sedation appear to be guided by the same objective of delivering sedation in proportion to the relief of refractory symptoms, as well as other needs of patients and their families. This suggests that proportionality should be seen as a multidimensional notion that can result in different approaches toward the depth of sedation.Palliative sedation is considered to be an appropriate option when other treatments fail to relieve suffering in dying patients.^1,2 There are important questions associated with this intervention, such as how deep the sedation must be to relieve suffering and how important it is for patients and their families for the patient to maintain a certain level of consciousness.¹ In the national guidelines for the Netherlands, palliative sedation is defined as “the intentional lowering of consciousness of a patient in the last phase of life.”^3,4 Sedatives can be administered intermittently or continuously, and the depth of palliative sedation can range from mild to deep.^1,5Continuous deep sedation until death is considered the most far reaching and controversial type of palliative sedation. Nevertheless, it is used frequently: comparable nationwide studies in Europe show frequencies of 2.5% to 16% of all deaths.^6–8 An important reason for continuous deep sedation being thought of as controversial is the possible association of this practice with the hastening of death,^9–11 although it is also argued that palliative sedation does not shorten life when its use is restricted to the patient’s last days of life.^12,13 Guidelines for palliative sedation often advise physicians to titrate sedatives,^2,3,14 which means that the dosages of sedatives are adjusted to the level needed for proper relief of symptoms. To date, research has predominantly focused on the indications and type of medications used for sedation. In this study, we investigated how physicians decide the depth of continuous palliative sedation and how these decisions relate to guidelines.     

βTrCP controls GH receptor degradation via two different motifs

The physiological roles of GH are broad and include metabolism regulation and promotion of somatic growth. Therefore, the responsiveness of cells to GH must be tightly regulated. This is mainly achieved by a complex and well-controlled mechanism of GH receptor (GHR) endocytosis. GHR endocytosis occurs independently of GH and requires the ubiquitin ligase, SCF (βTrCP) that is recruited to the ubiquitin-dependent endocytosis (UbE) motif in the cytoplasmic tail of the GHR. In this study we report that, in addition to the UbE motif, a downstream degron, DSGRTS, binds to βTrCP. The WD40 residues on βTrCP involved in the interaction with this sequence are identical to the ones necessary for binding the classical motif, DSGxxS, in inhibitor of NFκB signalling, and β-catenin. Previously, we showed that this motif is not involved in GH-induced endocytosis. We show here that the DSGRTS sequence significantly contributes to GHR endocytosis/degradation in basal conditions, whereas the UbE motif is involved both in basal and GH-induced conditions. These findings explain the high rate of GHR degradation under basal conditions, which is important for regulating the responsiveness of cells to GH.     

Vertical niche partitioning between cryptic sibling species of a cosmopolitan marine planktonic protist

A large portion of the surface‐ocean biomass is represented by microscopic unicellular plankton. These organisms are functionally and morphologically diverse, but it remains unclear how their diversity is generated. Species of marine microplankton are widely distributed because of passive transport and lack of barriers in the ocean. How does speciation occur in a system with a seemingly unlimited dispersal potential? Recent studies using planktonic foraminifera as a model showed that even among the cryptic genetic diversity within morphological species, many genetic types are cosmopolitan, lending limited support for speciation by geographical isolation. Here we show that the current two‐dimensional view on the biogeography and potential speciation mechanisms in the microplankton may be misleading. By depth‐stratified sampling, we present evidence that sibling genetic types in a cosmopolitan species of marine microplankton, the planktonic foraminifer Hastigerina pelagica, are consistently separated by depth throughout their global range. Such strong separation between genetically closely related and morphologically inseparable genetic types indicates that niche partitioning in marine heterotrophic microplankton can be maintained in the vertical dimension on a global scale. These observations indicate that speciation along depth (depth‐parapatric speciation) can occur in vertically structured microplankton populations, facilitating diversification without the need for spatial isolation.     

Characterization of the hemoglobin of the backswimmer Anisops deanei (Hemiptera)

While O(2)-binding hemoglobin-like proteins are present in many insects, prominent amounts of hemoglobin have only been found in a few species. Backswimmers of the genera Anisops and Buenoa (Notonectidae) have high concentrations of hemoglobin in the large tracheal cells of the abdomen. Oxygen from the hemoglobin is delivered to a gas bubble and controls the buoyant density, which enables the bugs to maintain their position without swimming and to remain stationary in the mid-water zone where they hunt for prey. We have obtained the cDNA sequences of three Anisops deanei hemoglobin chains by RT-PCR and RACE techniques. The deduced amino acid sequences show an unusual insertion of a single amino acid in the conserved helix E, but this does not affect protein stability or ligand binding kinetics. Recombinant A. deanei hemoglobin has an oxygen affinity of P(50) = 2.4 kPa (18 torr) and reveals the presence of a dimeric fraction or two different conformations. The absorption spectra demonstrate that the Anisops hemoglobin is a typical pentacoordinate globin. Phylogenetic analyses show that the backswimmer hemoglobins evolved within Heteroptera and most likely originated from an intracellular hemoglobin with divergent function.