Dynamic FRET-FLIM based screening of signal transduction pathways

Fluorescence Lifetime Imaging (FLIM) is an intrinsically quantitative method to screen for protein–protein interactions and is frequently used to record the outcome of signal transduction events. With new highly sensitive and photon efficient FLIM instrumentation, the technique also becomes attractive to screen, with high temporal resolution, for fast changes in Förster Resonance Energy Transfer (FRET), such as those occurring upon activation of cell signaling. The second messenger cyclic adenosine monophosphate (cAMP) is rapidly formed following activation of certain cell surface receptors. cAMP is subsequently degraded by a set of phosphodiesterases (PDEs) which display cell-type specific expression and may also affect baseline levels of the messenger. To study which specific PDEs contribute most to cAMP regulation, we knocked down individual PDEs and recorded breakdown rates of cAMP levels following transient stimulation in HeLa cells stably expressing the FRET/FLIM sensor, Epac-SH189. Many hundreds of cells were recorded at 5 s intervals for each condition. FLIM time traces were calculated for every cell, and decay kinetics were obtained. cAMP clearance was significantly slower when PDE3A and, to a lesser amount, PDE10A were knocked down, identifying these isoforms as dominant in HeLa cells. However, taking advantage of the quantitative FLIM data, we found that knockdown of individual PDEs has a very limited effect on baseline cAMP levels. By combining photon-efficient FLIM instrumentation with optimized sensors, systematic gene knockdown and an automated open-source analysis pipeline, our study demonstrates that dynamic screening of transient cell signals has become feasible. The quantitative platform described here provides detailed kinetic analysis of cellular signals in individual cells with unprecedented throughput.

On the unusual properties dielectric constant of the electric field of the free atmosphere

On the basis of experimental data vertical distribution electric field strength of the atmosphere, the applied problem of fitting constants in the model of the average self-consistent electric field is solved.The model is based on the nonlinear Poisson equation. Such an approach is not trivial because generally known in meteorology interpolation exponential function describing the empirical distribution of the electric field, space charge density and conductivity with a height not quite correctly reproduce a stable stratification of the electric field. Since aircraft measurements are carried out in a natural environment, the dielectric constant is lost, which leads to underestimated values of the electron-ion concentration.This is due to the fact that the potential in situ is screened and the Gauss theorem does not hold for it, and if it does, then for the radius of the Gaussian sphere it is less than the Debye screening radius. For a large Gaussian sphere, only the near-wall part of the electrometer is experimentally determined, and the shielded (inner) part does not contribute to the field flux through the surface by the dynamic screening of the electron. The magnitude of the screening of electrons in air is very large due to the dynamic polarizability of the medium and consists of two parts — the Debye and ion-plasma screening spheres. This, in turn, requires a redefinition of the dielectric constant for correct reproduction of field measurements. Thus, the verification of the dielectric constant was carried out on different experimental data, and its values lie within the same limits as the values obtained from the classical relations of Penn, Debye, and Landau.

A tertiary care system response to COVID-19 in a resource limited setting: a 6-month experience

Kerala recorded the initial cases of COVID/SARS-CoV-2 in India and managed to successfully contain it initially through its public health system. As the number of cases increased, the private sector also needed to be prepared. Here, we describe the preparedness and response of a tertiary care system in the nonpublic sector. The Health system response and preparedness consisted of establishment of leadership and infrastructural modification; screening-fever clinic, inpatient admission pathway, dynamic screening strategy training; developing protocols for emergencies and review visits of patients requiring dialysis and chemotherapy. Other priorities included maintaining supply chain of PPE, other essentials such as antivirals and establishing tele consultation. A strong leadership spearheaded infrastructural developments, meetings and troubleshooting every day to adapt to the changing epidemiological and clinical scenario. The Fever clinic was the designated area for consultation of patients with positive symptoms complex and epidemiological risk factors. In early March the screening strategy focused on the presence of a combination of epidemiological and clinical risk factors. As the number of contact cases rose, the focus shifted to identifying any of the defined epidemiological risk factors among persons reaching the hospital. The challenges included redesigning a patient movement pathway, deployment of human resources from their regular work to the screening activities, training of the repurposed human resource, procurement of good quality PPE, coordinating among the specialties, ensuring psychological support etc. The implementation of a timely COVID screening strategy aided our tertiary care centre to provide health care to all patients during the pandemic seamlessly.

Optimization of In Vivo Studies by Combining Planar Dynamic and Tomographic Imaging: Workflow Evaluation on a Superparamagnetic Nanoparticles System

Molecular imaging holds great promise in the noninvasive monitoring of several diseases with nanoparticles (NPs) being considered an efficient imaging tool for cancer, central nervous system, and heart- or bone-related diseases and for disorders of the mononuclear phagocytic system (MPS). In the present study, we used an iron-based nanoformulation, already established as an MRI/SPECT probe, as well as to load different biomolecules, to investigate its potential for nuclear planar and tomographic imaging of several target tissues following its distribution via different administration routes. Iron-doped hydroxyapatite NPs (FeHA) were radiolabeled with the single photon γ-emitting imaging agent [99mTc]TcMDP. Administration of the radioactive NPs was performed via the following four delivery methods: (1) standard intravenous (iv) tail vein, (2) iv retro-orbital injection, (3) intratracheal (it) instillation, and (4) intrarectal installation (pr). Real-time, live, fast dynamic screening studies were performed on a dedicated bench top, mouse-sized, planar SPECT system from t = 0 to 1 hour postinjection (p.i.), and consequently, tomographic SPECT/CT imaging was performed, for up to 24 hours p.i. The administration routes that have been studied provide a wide range of possible target tissues, for various diseases. Studies can be optimized following this workflow, as it is possible to quickly assess more parameters in a small number of animals (injection route, dosage, and fasting conditions). Thus, such an imaging protocol combines the strengths of both dynamic planar and tomographic imaging, and by using iron-based NPs of high biocompatibility along with the appropriate administration route, a potential diagnostic or therapeutic effect could be attained.

Hamiltonians and solution techniques

Chapter 1 introduces several of the common principles, techniques and approximations that will be employed throughout the text. Classically, the Hamiltonian is the sum of kinetic energy and potential energy. In quantum mechanics, it is an operator that, by operating on the statefunction, leads to the energy observable. The chapter begins with a preliminary description of the crystal’s Hamiltonian and then introduces approximation techniques that permit useful solutions. Beginning with the simple jellium model, Hartree and Hartree-Fock approaches are developed, exchange interactions and correlation effects are explored, and both time-independent perturbation and time-dependent perturbation techniques discussed. Examples illustrate scattering by perturbation as well as adiabatic evolution. The centrality of fast-and-slow interactions is stressed, the Born-Oppenheimer approximation is illustrated through the configuration coordinate diagram, and interacting electron systems are analyzed. The multi-electron aspects are stressed by discussing static screening, dynamic screening and the meaning of permittivity therein.

SAT0343 NUTRITIONAL STATUS OF SYSTEMIC SCLEROSIS PATIENTS: A PILOT STUDY

Background:Gastrointestinal involvement (GI) in systemic sclerosis (SSc) is one of the major disease burdens. Its consequences on the nutritional status of SSc patients and their quality of life is poorly evaluated during routine check-ups. Since malnutrition is an important cause of morbidity and mortality, addressing this issue seems necessary.Objectives:The aim of this study was to evaluate the risk of malnutrition in SSc patients and to identify potential associations between the risk of malnutrition and clinical features or laboratory parameters.Methods:All patients aged >18 years old with a definite diagnosis of SSc according to the 2013 ACR/EULAR classification criteria from the EUSTAR Center 16 and ERN ReCONNET cohort of the County Emergency Clinical Hospital Cluj-Napoca were included in the study. Patients with localized scleroderma, scleroderma sine scleroderma, overlap syndromes and mixed connective tissue disease were excluded. Clinical and laboratory data was collected from the EUSTAR database and medical charts. A telephone survey was conducted and patients were interviewed using the Malnutrition Universal Screening Tool (MUST) questionnaire.Results:75 patients were eligible for the study. Female to male ratio was 10:1 with an almost equal distribution among limited (57%) and diffuse (43%) SSc subtypes. The most prevalent autoantibodies were anti-TOPO-I and anti-centromere. GI symptoms were reported in 48.6% patients out of which 86% SSc patients underwent further evaluation by upper GI endoscopy. Abnormal endoscopic findings, such as esophagitis, Barret esophagus and gastritis were identified in 80% patients. Most patients had a low risk of malnutrition (93%) with only a minority carrying a medium (6%) or high (1%) risk. No significant association was demonstrated between MUST score and the extend of cutaneous involvement (limited SSc versus diffuse SSc; p=0.39), presence of GI symptoms (p=0.35), presence of abnormal endoscopic findings (p=0.45) or presence of anemia (p=0.83).Conclusion:The majority of SSc patients from this cohort exhibited a low risk of malnutrition. These results are contradictory to previous literature reports. A possible explanation is that the MUST score is a dynamic screening tool and therefore interviewing patients with a stable disease (outpatient care) versus patients with active disease (inpatient care) might lead to different results. Another limitation of this study is the small number of patients included. This is a pilot study. We aim to further extend the study population to the other EUSTAR cohorts and to prospectively evaluate these patients in an inpatient care setting.References:[1]Dupont R et al. Impact of micronutrient deficiency & malnutrition in systemic sclerosis: Cohort study and literature review. Autoimmun Rev. 2018 Nov;17(11):1081-1089;[2]Caimmi C et al. Malnutrition and sarcopenia in a large cohort of patients with systemic sclerosis. Clin Rheumatol. 2018 Apr;37(4):987-997;[3]Türk İ et al. Malnutrition, associated clinical factors, and depression in systemic sclerosis: a cross-sectional study. Clin Rheumatol. 2020 Jan;39(1):57-67.Disclosure of Interests:None declared