Molecular mechanism and functional implications of thrombin-mediated tyrosine phosphorylation of PKCdelta in platelets

Thrombin has been known to cause tyrosine phosphorylation of protein kinase C delta (PKCdelta) in platelets, but the molecular mechanisms and function of this tyrosine phosphorylation is not known. In this study, we investigated the signaling pathways used by protease-activated receptors (PARs) to cause tyrosine phosphorylation of PKCdelta and the role of this event in platelet function. PKCdelta was tyrosine phosphorylated by either PAR1 or PAR4 in a concentration- and time-dependent manner in human platelets. In particular, the tyrosine 311 residue was phosphorylated downstream of PAR receptors. Also the tyrosine phosphorylation of PKCdelta did not occur in Galpha(q)-deficient mouse platelets and was inhibited in the presence of a phospholipase C (PLC) inhibitor U73122 and calcium chelator BAPTA (5,5'-dimethyl-bis(o-aminophenoxy)ethane-N, N, N ', N '-tetraacetic acid), suggesting a role for Galpha(q) pathways and calcium in this event. Both PAR1 and PAR4 caused a time-dependent activation of Src (pp60c-src) tyrosine kinase and Src tyrosine kinase inhibitors completely blocked the tyrosine phosphorylation of PKCdelta. Inhibition of tyrosine phosphorylation or the kinase activity of PKCdelta dramatically blocked PAR-mediated thromboxane A2 generation. We conclude that thrombin causes tyrosine phosphorylation of PKCdelta in a calcium- and Src-family kinase-dependent manner in platelets, with functional implications in thromboxane A2 generation.     

Neuroleptic malignant syndrome.

Five patients with neuroleptic malignant syndrome (NMS) are described. The syndrome developed earlier and took longer to resolve in patients with schizophrenic disorders. Deterioration in the level of consciousness presented earlier than rigidity and fever in all five patients and was thus considered a major criterion. A significant proportion of the patients showed abnormalities of gaze. In four of the five patients spontaneous recovery occurred without the need for specific drugs.     

Different G protein-coupled signaling pathways are involved in alpha granule release from human platelets.

Alpha granule release plays an important role in propagating a hemostatic response upon platelet activation. We evaluated the ability of various agonists to cause alpha granule release in platelets. Alpha granule release was measured by determining P-selectin surface expression in aspirin-treated washed platelets. ADP-induced P-selectin expression was inhibited both by MRS 2179 (a P2Y1 selective antagonist) and AR-C69931MX (a P2Y12 selective antagonist), suggesting a role for both Galpha(q) and Galpha(i) pathways in ADP-mediated alpha granule release. Consistent with these observations, the combination of serotonin (a Galpha(q) pathway stimulator) and epinephrine (a Galpha(z) pathway stimulator) also caused alpha granule release. Furthermore, U46619-induced P-selectin expression was unaffected by MRS 2179 but was dramatically inhibited by AR-C69931, indicating a dominant role for P2Y12 in U46619-mediated alpha granule release. Additionally, the Galpha(12/13)-stimulating peptide YFLLRNP potentiated alpha granule secretion in combination with either ADP or serotonin/epinephrine costimulation but was unable to induce secretion by itself. Finally, costimulation of the Galpha(i) and Galpha(12/13) pathways resulted in a significant dose-dependent increase in alpha granule release. We conclude that ADP-induced alpha granule release in aspirin-treated platelets occurs through costimulation of Galpha(q) and Galpha(i) signaling pathways. The P2Y12 receptor plays an important role in thromboxane A(2)-mediated alpha granule release, and furthermore activation of Galpha(12/13) and Galpha(q) signaling pathway can cause alpha granule release.     

Karyotype of first clinical miscarriage and prognosis of subsequent pregnancy outcome

Research questionIs the karyotype of the first clinical miscarriage in an infertile patient predictive of the outcome of the subsequent pregnancy?DesignRetrospective cohort study of infertile patients undergoing manual vacuum aspiration with chromosome testing at the time of the first (index) clinical miscarriage with a genetic diagnosis and a subsequent pregnancy. Patients treated at two academic-affiliated fertility centres from 1999 to 2018 were included; those using preimplantation genetic testing for aneuploidy were excluded. Main outcome was live birth in the subsequent pregnancy.ResultsOne hundred patients with euploid clinical miscarriage and 151 patients with aneuploid clinical miscarriage in the index pregnancy were included. Patients with euploid clinical miscarriage in the index pregnancy had a live birth rate of 63% in the subsequent pregnancy compared with 68% among patients with aneuploid clinical miscarriage (adjusted odds ratio [aOR] 0.75, 95% CI 0.47–1.39, P = 0.45, logistic regression model adjusting for age, parity, body mass index and mode of conception). In a multinomial logistic regression model with three outcomes (live birth, clinical miscarriage or biochemical miscarriage), euploid clinical miscarriage for the index pregnancy was associated with similar odds of clinical miscarriage in the subsequent pregnancy compared with aneuploid clinical miscarriage for the index pregnancy (32% versus 24%, respectively, aOR 1.49, 95% CI 0.83–2.70, P = 0.19). Euploid clinical miscarriage for the index pregnancy was not associated with likelihood of biochemical miscarriage in the subsequent pregnancy compared with aneuploid clinical miscarriage (5% versus 8%, respectively, aOR 0.46, 95% CI 0.14–1.55, P = 0.21).ConclusionPrognosis after a first clinical miscarriage among infertile patients is equally favourable among patients with euploid and aneuploid karyotype, and independent of the karyotype of the pregnancy loss.     

Characterization of a novel,papain‐inducible murine model of eosinophilic rhinosinusitis

Background

Eosinophilic chronic rhinosinusitis (ECRS) is a disease characterized by eosinophilic inflammatory infiltrate and a local type 2 cytokine milieu. Current animal models fail to recapitulate many of the innate and adaptive immunologic hallmarks of the disease, thus hindering the development of effective therapeutics. In the present study, mice were exposed intranasally to the cysteine protease papain, which shares functional similarities with parasitic proteases and aeroallergens, to generate a rapidly inducible murine model of eosinophilic rhinosinusitis.

Methods

C57BL/6 mice were intranasally instilled with 20 μg papain or heat‐inactivated papain (HP) on days 0–2 and days 7–10, and then euthanized on day 11. Nasal lavage fluid (NALF) was analyzed to quantify eosinophils and inflammatory cytokine secretion. Sinonasal tissue was sectioned and stained for goblet cells or homogenized to analyze cytokine levels. Serum samples were assayed for immunoglobulin E (IgE) by enzyme‐linked immunoassay. Sinonasal mucosal tissue was dissociated and analyzed by flow cytometry.

Results

Compared with HP treatment, papain induced significant eosinophilia in NALF, goblet cell hyperplasia, innate and adaptive immune cell infiltration, type 2 cytokine production, and IgE responses. Flow cytometric analysis of sinonasal tissues revealed significant inflammatory cell infiltration and interleukin‐13–producing cell populations.

Conclusion

In this study, we demonstrated that the cysteine protease papain induces allergic sinonasal eosinophilic rhinosinusitis and resembles T‐helper 2 cell inflammation and innate immune characteristics of ECRS. This model permits further study into the molecular mechanisms underlying ECRS pathology and provides a model system for the evaluation of potential pharmacologic interventions.

     

The association of early postoperative desaturation in the operating theatre with hospital discharge to a skilled nursing or long-term care facility

It is unclear which criteria should be used to define readiness for tracheal extubation in the operating theatre. We studied the effects of desaturation in the operating theatre immediately after tracheal extubation on long-term outcomes. Performing a pre-specified, retrospective analysis of 71,025 cases involving previously independent adults undergoing non-cardiac surgery, we evaluated the association between desaturation events (oxygen saturation < 90%) within 10 min of tracheal extubation and adverse discharge (to a skilled nursing facility or long-term care facility). A total of 404 (12.3%) cases with, and 5035 (7.4%) cases without, early postoperative desaturation had an adverse discharge. Early postoperative desaturation was associated with higher odds of being discharged to a nursing facility (adjusted odds ratio 1.36 (95%CI 1.20–1.54); p < 0.001). Increased duration of desaturation augmented the effect (p for trend < 0.001). Desaturation was associated with a higher risk of respiratory, renal and cardiovascular complications as well as increased duration of hospital stay, postoperative intensive care unit admission frequency and cost. Several modifiable factors were associated with desaturation including: high intra-operative long-acting opioid administration; high neostigmine dose; high intra-operative inspired oxygen concentration; and low oxygen delivery immediately before tracheal extubation. There was substantial provider variability between anaesthetists in the incidence of postoperative desaturation unexplained by patient- and procedure-related factors. Early postoperative desaturation is a potentially preventable complication associated with a higher risk of adverse discharge disposition. Anaesthetists may consider developing guidelines to define tracheal extubation readiness that contain postoperative desaturation as an adverse outcome after tracheal extubation.     

Size-dependent forced PEG partitioning into channels: VDAC,OmpC, and α-hemolysin

Nonideal polymer mixtures of PEGs of different molecular weights partition differently into nanosize protein channels. Here, we assess the validity of the recently proposed theoretical approach of forced partitioning for three structurally different β-barrel channels: voltage-dependent anion channel from outer mitochondrial membrane VDAC, bacterial porin OmpC (outer membrane protein C), and bacterial channel-forming toxin α-hemolysin. Our interpretation is based on the idea that relatively less-penetrating polymers push the more easily penetrating ones into nanosize channels in excess of their bath concentration. Comparison of the theory with experiments is excellent for VDAC. Polymer partitioning data for the other two channels are consistent with theory if additional assumptions regarding the energy penalty of pore penetration are included. The obtained results demonstrate that the general concept of “polymers pushing polymers” is helpful in understanding and quantification of concrete examples of size-dependent forced partitioning of polymers into protein nanopores.Partitioning of polymers into nanosize cavities has broad relevance (1), generally in biology, where the consequences of molecular crowding are well appreciated but not completely understood (2, 3), and in biotechnology for single-molecule sensing and characterization based on the variation of current through ion-conducting aqueous pores (4–7). The partitioning of nonionic polymers such as PEG into α-hemolysin (aHL) from Staphylococcus aureus has been previously studied and shown to be size-dependent at relatively low salt concentrations (8–13). In a different way, namely, as the amplitude of channel blockage, polymer size dependency has also been observed in single-molecule studies at high salt concentrations (4 M KCl) with aHL (14) and recently with aerolysin from Aeromonas hydrophila (15), and was shown to exhibit pronounced size sensitivity with resolution in the submonomer range.We studied passive size-dependent partitioning and size discrimination that can be manipulated to force polymers into nanosize pores under strong nonideality, when polymer partitioning is qualitatively modified by polymer–polymer repulsion that allows polymers, which are excluded in dilute solutions, to enter the channel pore (13). This concentration-dependent partitioning was rationalized by an argument that the overlap concentration of the polymer in the pore is higher than that in the bath (16, 17). For polymer mixtures, where one component is used to preferentially push another into a cavity, this phenomenon of forced polymer partitioning was referred to as “polymers pushing polymers” (PPP) (18). Using the osmotic pressure of a polymer solution composed of various sizes of the same type of polymers, these theoretical advances quantified the forced preferential entry of polymers into a nanopore depending on their size and the pore penetration energy penalty. The theoretical analysis (18) was formulated specifically for a binary polymer mixture, where only one component is allowed to penetrate the pore, whereas the other polymer is excluded. An equation of state (EOS) of a polymer mixture (osmotic pressure as a function of composition) is first validated with osmotic measurements for a binary polymer mixture. Then this EOS, together with the observed polymer selectivity of the pore, is used to interpret the polymer partitioning coefficient.We apply this approach to different β-barrel pores to assess to what extent it can be useful in understanding concrete examples of size-dependent forced polymer partitioning. We performed partitioning measurements of differently sized PEGs into three pores: mitochondrial voltage-dependent anion channel (VDAC), bacterial porin OmpC (outer membrane protein C), and bacterial toxin aHL. In what follows we limit ourselves exclusively to the case of PEG mixtures, composed of “short” (PEG200) and “long” (PEG3400) polymer components.