Topography of signaling molecules as detected by electron microscopy on plasma membrane sheets isolated from non-adherent mast cells

Immunolabeling of isolated plasma membrane (PM) sheets combined with high-resolution electron microscopy is a powerful technique for understanding the topography of PM-bound signaling molecules. However, this technique has been mostly confined to analysis of membrane sheets from adherent cells. Here we present a rapid, simple and versatile method for isolation of PM sheets from non-adherent cells, and show its use for examination of the topography of Fcepsilon receptor I (FcepsilonRI) and transmembrane adaptors, LAT (linker for activation of T cells) and NTAL (non-T cell activation linker), in murine bone marrow-derived mast cells (BMMC). The data were compared with those obtained from widely used but tumor-derived rat basophilic leukemia (RBL) cells. In non-activated cells, FcepsilonRI was distributed either individually or in small clusters of comparable size in both cell types. In multivalent antigen-activated BMMC as well as RBL cells, FcepsilonRI was internalized to a similar extent, but, strikingly, internalization in BMMC was not preceded by formation of large (~200 nm) aggregates of FcepsilonRI, described previously in activated RBL cells. On the other hand, downstream adaptor proteins, LAT and NTAL, were localized in independent domains in both BMMC and RBL cells before and after FcepsilonRI triggering. The combined data demonstrate unexpected properties of FcepsilonRI signaling assemblies in BMMC and emphasize the importance of studies of PM sheets isolated from non-tumor cells.     

A semi-analytical radiobiological model may assist treatment planning in light ion radiotherapy

A semi-analytical model of light ions' Bragg peaks is presented and used in conjunction with a detailed probabilistic radiobiological module to predict the biological effectiveness of light ion irradiation for hadrontherapy applications. The physical Bragg peak model is based on energy-loss calculations with the SRIM code and phenomenological formulae for the energy-loss straggling. Effects of nuclear reactions are accounted for on the level of reducing the number of primary particles only. Reaction products are not followed at all and their contribution to dose deposition is neglected. Beam widening due to multiple scattering and calculations of spread-out Bragg peaks are briefly discussed. With this simple physical model, integral depth-dose distributions are calculated for protons, carbon, oxygen and neon ions. A good agreement with published experimental data is observed for protons and lower energy ions (with ranges in water up to approximately 15 cm), while less satisfactory results are obtained for higher energy ions due to the increased role of nuclear reaction products, neglected in this model. A detailed probabilistic radiobiological module is used to complement the simple physical model and to estimate biological effectiveness along the penetration depth of Bragg peak irradiation. Excellent agreement is found between model predictions and experimental data for carbon beams, indicating potential applications of the present scheme in treatment planning in light ion hadrontherapy. Due to the semi-analytical character of the model, leading to high computational speed, applications are foreseen in particular in the fully biological optimization of multiple irradiation fields and intensity-modulated beams.     

Rotational Rheology of Bovine Serum Albumin Solutions: Confounding Effects of Impurities,Mechanistic Considerations and Potential Implications on Protein Formulation Development

Purpose

To show and rationalize the confounding effects on the rotational/oscillatory rheology of surface active impurities in commercial protein formulations such as bovine serum albumin, BSA.

Methods

Bulk and interfacial rotational/oscillatory rheology were used to study the viscosity, complex viscosity, storage/elastic modulus, G’ and loss/viscous modulus, G”, as a function of time of aqueous formulations of BSA and their purified components.

Results

Viscosity/time profiles at steady shear for different commercial BSA products and lots showed viscosity increase, decrease and time-independent profiles at low shear rates. All lots showed shear thinning. BSA monomer and dimers/aggregates, in general, showed similar profiles. Addition of low levels of surfactant or high shear rates rendered all solutions to be Newtonian-like. Interfacial viscosity studies paralleled those on the rotational rheometer. G’?>?G” with viscosity increase and G’?<?G” with viscosity decrease over time.

Conclusions

We provide a rational explanation for the time-dependent and source-dependent rheological behavior of aqueous formulations of commercially available BSA proteins based on the migration of protein and surface active impurities to the air/water interface within the rheometer plates leading to the formation and breakdown of protein networks. Highly purified proteins is warranted in rheological studies of protein drug product candidates.

     

Macitentan in Pulmonary Arterial Hypertension: A Focus on Combination Therapy in the SERAPHIN Trial

American Journal of Cardiovascular Drugs - SERAPHIN was a double-blind, placebo-controlled, event-driven phase III trial that evaluated the effects of long-term treatment with macitentan, an oral...     

Reproducibility of sonographic measurement of the substantia nigra

The aim of this study was to evaluate inter-reader, intra-investigator and inter-investigator reproducibility and correlations in the assessment of substantia nigra (SN) echogenicity and area measurement by a physician-sonographer (PS), a sonographic laboratory assistant (SLA) and a physician without sonographic experience (PN). A total of 22 patients with extrapyramidal symptoms were examined using transcranial sonography (TCS). SN images were encoded and evaluated by the three readers. A second TCS examination was performed after 7+/-2 d. A second investigator performed TCS examination 1 mo later. Spearman rank correlation and Pearson's correlation coefficient were used when assessing the agreement between readers. All three readers identified the same 15 patients with SN echogenicity III or more. Inter-reader SN echogenicity and area measurement correlations were r=0.55 to 0.82 and r=0.31 to 0.74 between PS and SLA and r=0.55 to 0.77 and 0.49 to 0.62 between PS and PN, respectively (p<0.05 in all cases). Intra-reader echogenicity and area measurement correlations (r=0.85 to 0.96 and r=0.51 to 0.69) were statistically significant only for PS (p<0.001). All intra- and inter-investigator correlations of SN area measurement (r=0.69 to 0.88 and r=0.5 to 0.61) and SN echogenicity (r=0.64 to 0.92 and r=0.51 to 0.69) were statistically significant (p<0.05). Semiquantitative evaluation of SN echogenicity and area using TCS is highly dependent on the experience of the sonographer. Only an experienced sonographer was able to produce very reproducible results with statistically significant correlations; SLA and PN intra-reader correlations were poor.     

Does status epilepticus modify the effect of ifenprodil on cortical epileptic afterdischarges in immature rats?

Background

Ifenprodil as a specific antagonist of NMDA receptors containing a dominant NR2B subunit exhibits age-dependent anticonvulsant action. Possible changes of this action due to status epilepticus (SE) elicited at early stage of development were studied using cortical epileptic afterdischarges (ADs) as a model.

Muscle transfers in children and adults improve external rotation in cases of obstetrical brachial plexus paralysis: a comparative study

Purpose

Latissimus dorsi and teres major transfers to the lateral side of the humerus with lengthening of the pectoralis major and subscapularis muscles for residual shoulder deformity were compared in children and skeletally mature patients.

Methods

Fifteen patients (nine children, six skeletally mature patients aged three to 30 years, follow-up one to 22 years) were treated for internal shoulder contracture after birth plexus lesions: C5–C6 (seven patients); C5–7 (five patients); C5-C8-T1 (three patients, respectively). Range of movement, Mallet shoulder function score and radiographs were assessed.

Results

Pre-operatively, shoulder function restrictions were comparable in all patients. Postoperatively, external rotation, abduction and Mallet function score improved significantly (p < 0.05) in all patients except one. There were no differences in improvement between children and skeletally mature patients (p = 0.24–1.0).

Conclusions

This technique improves external rotation and abduction of the shoulder for daily living activities in children and young, skeletally mature, patients.     

Thapsigargin Is a Broad-Spectrum Inhibitor of Major Human Respiratory Viruses: Coronavirus,Respiratory Syncytial Virus and Influenza A Virus

The long-term control strategy of SARS-CoV-2 and other major respiratory viruses needs to include antivirals to treat acute infections, in addition to the judicious use of effective vaccines. Whilst COVID-19 vaccines are being rolled out for mass vaccination, the modest number of antivirals in use or development for any disease bears testament to the challenges of antiviral development. We recently showed that non-cytotoxic levels of thapsigargin (TG), an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca²⁺ ATPase pump, induces a potent host innate immune antiviral response that blocks influenza A virus replication. Here we show that TG is also highly effective in blocking the replication of respiratory syncytial virus (RSV), common cold coronavirus OC43, SARS-CoV-2 and influenza A virus in immortalized or primary human cells. TG’s antiviral performance was significantly better than remdesivir and ribavirin in their respective inhibition of OC43 and RSV. Notably, TG was just as inhibitory to coronaviruses (OC43 and SARS-CoV-2) and influenza viruses (USSR H1N1 and pdm 2009 H1N1) in separate infections as in co-infections. Post-infection oral gavage of acid-stable TG protected mice against a lethal influenza virus challenge. Together with its ability to inhibit the different viruses before or during active infection, and with an antiviral duration of at least 48 h post-TG exposure, we propose that TG (or its derivatives) is a promising broad-spectrum inhibitor against SARS-CoV-2, OC43, RSV and influenza virus.