The actin cytoskeleton differentially regulates platelet alpha-granule and dense-granule secretion

Stimulation of platelets with strong agonists results in centralization of cytoplasmic organelles and secretion of granules. These observations have led to the supposition that cytoskeletal contraction facilitates granule release by promoting the interaction of granules with one another and with membranes of the open canalicular system. Yet, the influence of the actin cytoskeleton in controlling the membrane fusion events that mediate granule secretion remains largely unknown. To evaluate the role of the actin cytoskeleton in platelet granule secretion, we have assessed the effects of latrunculin A and cytochalasin E on granule secretion. Exposure of platelets to low concentrations of these reagents resulted in acceleration and augmentation of agonist-induced alpha-granule secretion with comparatively modest effects on dense granule secretion. In contrast, exposure of platelets to high concentrations of latrunculin A inhibited agonist-induced alpha-granule secretion but stimulated dense granule secretion. Incubation of permeabilized platelets with low concentrations of latrunculin A primed platelets for Ca(2+)- or guanosine triphosphate (GTP)-gamma-S-induced alpha-granule secretion. Latrunculin A-dependent alpha-granule secretion was inhibited by antibodies directed at vesicle-associated membrane protein (VAMP), demonstrating that latrunculin A supports soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-dependent membrane fusion. These results indicate that the actin cytoskeleton interferes with platelet exocytosis and differentially regulates alpha-granule and dense granule secretion.     

The distribution pattern of ERα expression,ESR1 genetic variation and expression of growth factor receptors: association with breast cancer prognosis in Russian patients treated with adjuvant tamoxifen

Identification of additional biomarkers associated with ER genomic and nongenomic pathways could be very useful to distinguish patients who will benefit from tamoxifen treatment. The aim of this study was to analyze the prognostic significance of the distribution pattern of ERα expression, ESR1 gene single-nucleotide polymorphisms and expression levels of growth factor receptors in Russian hormone receptor-positive breast cancer patients treated with adjuvant tamoxifen. Formalin-fixed paraffin-embedded tumor tissue samples from 97 patients were examined for the distribution pattern of ERα expression, as well as for EGFR and TGF-βR1 expression by immunohistochemistry. Genotypes for ESR1 +30T>C (rs2077647) and ESR1 2014G>A (rs2228480) were analyzed using a TaqMan assay. Progression-free survival (PFS) was used as an endpoint for the survival analyses. We found that patients with the heterogeneous distribution of ERα expression had poor prognosis on tamoxifen treatment (P = 0.021). We identified a high EGFR expression in patients who developed distant metastasis or recurrence during tamoxifen treatment (a tamoxifen-resistant group—TR) in contrast to the distant metastasis-free patients (a tamoxifen-sensitive group—TS) (80.0 vs. 41.9 %, respectively, P = 0.009). Carriers of the ESR12014A mutant allele were more prevalent among the TR patients compared to the TS patients (26.3 vs. 8.0 %, respectively, P = 0.009). EGFR expression and the distribution pattern of ERα expression were associated with the response to tamoxifen by both univariate and multivariate logistic regression analyses. The presence of these markers either alone or in combination was correlated with the worse PFS for all patients. Analysis of the distribution pattern of ERα expression and the EGFR status in tumor tissue may be valuable for patient selection for tamoxifen adjuvant therapy.     

Preschool Children with Gender Normative and Gender Non-Normative Peer Preferences: Psychosocial and Environmental Correlates

We addressed several issues concerning children who show gender non-normative (GNN) patterns of peer play. First, do young children with GNN peer preferences differ from children with gender normative (GN) peer preferences in problem behaviors? Second, do GNN and GN children differ in sociability and isolation and do they have differential socialization opportunities with externalizing, internalizing, and socially competent peers? We employed a Bayesian approach for classifying children as GNN based on their peer preferences as compared to their peers using a sample of Head Start preschool children from a large Southwestern city (N = 257; 53 % boys; M age = 51 months; 66 % Mexican American). To calculate socialization opportunities, we assessed affiliation to each child in the class and weighted that by each peer’s characteristics to determine the exposure that each child had to different kinds of peers. GN children of both sexes interacted more with same-sex peers, which may limit learning of different styles of interaction. As compared to GN children, GNN children exhibited more engagement in other-sex activities and with other-sex play partners and GNN children experienced somewhat fewer peer interactions, but did not differ on problem behaviors or social competence. Boys with GNN peer preferences had increased exposure to peers with problem behaviors. GNN girls experienced little exposure to peers with problem behaviors, but they also had little exposure to socially competent peers, which may reduce learning social skills from peers. Implications of these findings for future socialization and development will be discussed.     

The role of lung biopsy in children with perinatally acquired AIDS

There is a need for a rapid and efficacious method of diagnosis of pulmonary infiltrates in perinatal HIV infection. However, controversy still exists about which method--open lung biopsy (OLB) versus bronchoscopic techniques--is the best for this population. We present our results with OLB in 24 children with HIV-related lung disease. Over a 6-year period, 27 OLBs were performed on 24 children with diagnosis of HIV infection. The procedures were performed under general anesthesia using a limited anterolateral thoracotomy. Suspicious areas were removed with the autostapler. The specimens were studied for the presence of non-infectious as well as bacterial, viral, fungal, and mycobacterial diseases. There were no operative deaths related to the procedure. Morbidity was limited to prolonged but self-resolving air leaks in two patients (8.3%). Five hospital deaths occurred between 3 and 12 weeks postoperatively and 11 late deaths between 3 months and 6.5 years. All deaths were related to AIDS. Eight patients (33.3%) are still alive 2 to 8 years postoperatively. A total of 43 pathologies were found in 27 specimens. A positive pathologic finding was obtained in all patients, with two patients having nonspecific minimal changes. This resulted in a change of therapy in all but one case. The technique of OLB in children with AIDS is safe and simple. It should be performed early in the course of the disease and, a careful selection of candidates can minimize the incidence of complications.     

An open‐label,long‐term evaluation of the safety,efficacy and tolerability of avanafil in male patients with mild to severe erectile dysfunction

Aim: Determine the long‐term efficacy, safety and tolerability of avanafil, a highly specific, rapidly absorbed phosphodiesterase type 5 inhibitor in male patients with mild to severe erectile dysfunction (ED), with or without diabetes. Methods: This was a 52‐week, open‐label extension of two 12‐week, randomised, placebo‐controlled, phase 3 trials. Patients were assigned to avanafil 100 mg, but could request 200 mg (for increased efficacy; ‘100/200‐mg’ group) or 50 mg (for improved tolerability). Primary end points included percentage of sexual attempts ending in successful vaginal penetration [Sexual Encounter Profile 2 (SEP2)] and intercourse (SEP3) and erectile function domain score per the International Index of Erectile Function (IIEF‐EF). Results: Some 712 patients enrolled; 686 were included in the intent to treat population and contributed to the data. All primary end points showed sustained improvement. SEP2 and SEP3 success rates improved from 44% to 83% and from 13% to 68% (100‐mg group) and from 43% to 79% and from 11% to 66% (100/200‐mg group), respectively. Mean IIEF‐EF domain scores improved from 13.6 to 22.2 (100‐mg group) and from 11.9 to 22.7 (100/200‐mg group). Avanafil was effective in some patients ≤ 15 min and > 6 h postdose. Sixty‐five per cent (112/172) of ‘nonresponders’ to avanafil 100 mg responded to the 200‐mg dose. The most common (≥ 2%) treatment‐emergent adverse events were headache, flushing, nasopharyngitis and nasal congestion; < 3% of patients discontinued therapy because of adverse events. Conclusions: The long‐term tolerability and improvement in sexual function, coupled with rapid onset, suggest that avanafil is well suited for the on‐demand treatment of ED.     

Characterization of TEM-1 β-Lactamase-Producing Kingella kingae Clinical Isolates

Kingella kingae is a human pathogen that causes pediatric osteoarticular infections and infective endocarditis in children and adults. The bacterium is usually susceptible to β-lactam antibiotics, although β-lactam resistance has been reported in rare isolates. This study was conducted to identify β-lactam-resistant strains and to characterize the resistance mechanism. Screening of a set of 90 K. kingae clinical isolates obtained from different geographic locations revealed high-level resistance to penicillins among 25% of the strains isolated from Minnesota and Iceland. These strains produced TEM-1 β-lactamase and were shown to contain additional ≥50-kb plasmids. Ion Torrent sequencing of extrachromosomal DNA from a β-lactamase-producing strain confirmed the plasmid location of the bla_TEM gene. An identical plasmid pattern was demonstrated by multiplex PCR in all β-lactamase producers. The porin gene''s fragments were analyzed to investigate the relatedness of bacterial strains. Phylogenetic analysis revealed 27 single-nucleotide polymorphisms (SNPs) in the por gene fragment, resulting in two major clusters with 11 allele types forming bacterial-strain subclusters. β-Lactamase producers were grouped together based on por genotyping. Our results suggest that the β-lactamase-producing strains likely originate from a single plasmid-bearing K. kingae isolate that traveled from Europe to the United States, or vice versa. This study highlights the prevalence of penicillin resistance among K. kingae strains in some regions and emphasizes the importance of surveillance for antibiotic resistance of the pathogen.