Stroke in young diver with patent foramen ovale

Patent foramen ovale (PFO) is the most common cause of right-to-left shunt which carries a significant risk for stroke when associated with venous thrombosis, coagulation abnormalities or other conditions. We present a young male in whom diving was associated with stroke in a subject with otherwise clinically silent PFO.     

Peptidomics approach to elucidate the proteolytic regulation of bioactive peptides

Peptide hormones and neuropeptides have important roles in physiology and therefore the regulation of these bioactive peptides is of great interest. In some cases proteolysis controls the concentrations and signaling of bioactive peptides, and the peptidases that mediate this biochemistry have proven to be extremely successful drug targets. Due to the lack of any general method to identify these peptidases, however, the role of proteolysis in the regulation of most neuropeptides and peptide hormones is unknown. This limitation prompted us to develop an advanced peptidomics-based strategy to identify the peptidases responsible for the proteolysis of significant bioactive peptides. The application of this approach to calcitonin gene-related peptide (CGRP), a neuropeptide associated with blood pressure and migraine, revealed the endogenous CGRP cleavage sites. This information was then used to biochemically purify the peptidase capable of proteolysis of CGRP at those cleavage sites, which led to the identification of insulin-degrading enzyme (IDE) as a candidate CGRP-degrading enzyme. CGRP had not been identified as an IDE substrate before and we tested the physiological relevance of this interaction by quantitative measurements of CGRP using IDE null (IDE(-/-)) mice. In the absence of IDE, full-length CGRP levels are elevated in vivo, confirming IDE as an endogenous CGRP-degrading enzyme. By linking CGRP and IDE, this strategy uncovers a previously unknown pathway for CGRP regulation and characterizes an additional role for IDE. More generally, this work suggests that this may be an effective general strategy for characterizing these pathways and peptidases moving forward.     

Calcium and pH-dependent packing and release of the gel-forming MUC2 mucin

MUC2, the major colonic mucin, forms large polymers by N-terminal trimerization and C-terminal dimerization. Although the assembly process for MUC2 is established, it is not known how MUC2 is packed in the regulated secretory granulae of the goblet cell. When the N-terminal VWD1-D2-D'D3 domains (MUC2-N) were expressed in a goblet-like cell line, the protein was stored together with full-length MUC2. By mimicking the pH and calcium conditions of the secretory pathway we analyzed purified MUC2-N by gel filtration, density gradient centrifugation, and transmission electron microscopy. At pH 7.4 the MUC2-N trimer eluted as a single peak by gel filtration. At pH 6.2 with Ca(2+) it formed large aggregates that did not enter the gel filtration column but were made visible after density gradient centrifugation. Electron microscopy studies revealed that the aggregates were composed of rings also observed in secretory granulae of colon tissue sections. The MUC2-N aggregates were dissolved by removing Ca(2+) and raising pH. After release from goblet cells, the unfolded full-length MUC2 formed stratified layers. These findings suggest a model for mucin packing in the granulae and the mechanism for mucin release, unfolding, and expansion.     

Thylakoid potassium channel is required for efficient photosynthesis in cyanobacteria

A potassium channel (SynK) of the cyanobacterium Synechocystis sp. PCC 6803, a photoheterotrophic model organism for the study of photosynthesis, has been recently identified and demonstrated to function as a potassium selective channel when expressed in a heterologous system and to be located predominantly to the thylakoid membrane in cyanobacteria. To study its physiological role, a SynK-less knockout mutant was generated and characterized. Fluorimetric experiments indicated that SynK-less cyanobacteria cannot build up a proton gradient as efficiently as WT organisms, suggesting that SynK might be involved in the regulation of the electric component of the proton motive force. Accordingly, measurements of flash-induced cytochrome b(6)f turnover and respiration pointed to a reduced generation of ΔpH and to an altered linear electron transport in mutant cells. The lack of the channel did not cause an altered membrane organization, but decreased growth and modified the photosystem II/photosystem I ratio at high light intensities because of enhanced photosensitivity. These data shed light on the function of a prokaryotic potassium channel and reports evidence, by means of a genetic approach, on the requirement of a thylakoid ion channel for optimal photosynthesis.     

p63-microRNA feedback in keratinocyte senescence

We investigated the expression of microRNAs (miRNAs) associated with replicative senescence in human primary keratinocytes. A cohort of miRNAs up-regulated in senescence was identified by genome-wide miRNA profiling, and their change in expression was validated in proliferative versus senescent cells. Among these, miRNA (miR)-138, -181a, -181b, and -130b expression increased with serial passages. miR-138, -181a, and -181b, but not miR-130b, overexpression in proliferating cells was sufficient per se to induce senescence, as evaluated by inhibition of BrdU incorporation and quantification of senescence-activated β-galactosidase staining. We identified Sirt1 as a direct target of miR-138, -181a, and -181b, whereas ΔNp63 expression was inhibited by miR-130b. We also found that ΔNp63α inhibits miR-138, -181a, -181b, and -130b expression by binding directly to p63-responsive elements located in close proximity to the genomic loci of these miRNAs in primary keratinocytes. These findings suggest that changes in miRNA expression, by modulating the levels of regulatory proteins such as p63 and Sirt1, strongly contribute to induction of senescence in primary human keratinocytes, thus linking these two proteins. Our data also indicate that suppression of miR-138, -181a, -181b, and -130b expression is part of a growth-promoting strategy of ΔNp63α in epidermal proliferating cells.     

Janus-faced liposomes enhance antimicrobial innate immune response in Mycobacterium tuberculosis infection

We have generated unique asymmetric liposomes with phosphatidylserine (PS) distributed at the outer membrane surface to resemble apoptotic bodies and phosphatidic acid (PA) at the inner layer as a strategy to enhance innate antimycobacterial activity in phagocytes while limiting the inflammatory response. Results show that these apoptotic body-like liposomes carrying PA (ABL/PA) (i) are more efficiently internalized by human macrophages than by nonprofessional phagocytes, (ii) induce cytosolic Ca(2+) influx, (iii) promote Ca(2+)-dependent maturation of phagolysosomes containing Mycobacterium tuberculosis (MTB), (iv) induce Ca(2+)-dependent reactive oxygen species (ROS) production, (v) inhibit intracellular mycobacterial growth in differentiated THP-1 cells as well as in type-1 and -2 human macrophages, and (vi) down-regulate tumor necrosis factor (TNF)-α, interleukin (IL)-12, IL-1β, IL-18, and IL-23 and up-regulate transforming growth factor (TGF)-β without altering IL-10, IL-27, and IL-6 mRNA expression. Also, ABL/PA promoted intracellular killing of M. tuberculosis in bronchoalveolar lavage cells from patients with active pulmonary tuberculosis. Furthermore, the treatment of MTB-infected mice with ABL/PA, in combination or not with isoniazid (INH), dramatically reduced lung and, to a lesser extent, liver and spleen mycobacterial loads, with a concomitant 10-fold reduction of serum TNF-α, IL-1β, and IFN-γ compared with that in untreated mice. Altogether, these results suggest that apoptotic body-like liposomes may be used as a Janus-faced immunotherapeutic platform to deliver polar secondary lipid messengers, such as PA, into phagocytes to improve and recover phagolysosome biogenesis and pathogen killing while limiting the inflammatory response.     

Non-O blood type is the commonest genetic risk factor for VTE: results from a meta-analysis of the literature

It is well known that the ABO blood group exerts a major influence on hemostasis, as O blood group individuals have lower von Willebrand factor and factor VIII levels than non-O blood group subjects. To evaluate the possible clinical implication of the different ABO blood groups on the risk of developing venous thromboembolism (VTE), we conducted a meta-analysis of the existing literature. After an electronic search strategy using Medline and Embase and a manual review of abstract books of the International Society on Thrombosis and Haemostasis and of reference lists of all retrieved articles, we included in the systematic review 38 studies with 10,305 VTE cases. The prevalence of non-O blood group was significantly higher in VTE patients compared with controls with a resulting pooled odds ratio (OR) of 2.09 (95% confidence interval [CI], 1.83, 2.38; p < 0.00001). Similar findings were obtained when the genotypes A1O/BO/A2B (OR 1.73, 95% CI, 1.47, 2.05; p < 0.00001) and A1B/A1A1/BB (OR 1.87, 95% CI, 1.84, 2.44; p < 0.00001) were analyzed. The maximum VTE risk was observed in non-O-factor V Leiden patients (OR 7.60, 95% CI, 3.21, 17.99), while for G20210A prothrombin mutation it was not possible to perform a pooled analysis due to a paucity of published studies. Finally, the association between non-O blood group and VTE was weaker when provoked VTE cases were considered (OR 1.33, 95% CI, 1.18, 1.50), while it was substantially unchanged when unprovoked VTE cases were analyzed (OR 1.88, 95% CI, 1.42, 2.50). In conclusion, considering its prevalence, non-O blood group is a candidate to be one of the most important genetic risk factors for venous thrombosis.