Glycine Transporter 2: Mechanism and Allosteric Modulation

Neurotransmitter sodium symporters (NSS) are a subfamily of SLC6 transporters responsible for regulating neurotransmitter signalling. They are a major target for psychoactive substances including antidepressants and drugs of abuse, prompting substantial research into their modulation and structure-function dynamics. Recently, a series of allosteric transport inhibitors have been identified, which may reduce side effect profiles, compared to orthosteric inhibitors. Allosteric inhibitors are also likely to provide different clearance kinetics compared to competitive inhibitors and potentially better clinical outcomes. Crystal structures and homology models have identified several allosteric modulatory sites on NSS including the vestibule allosteric site (VAS), lipid allosteric site (LAS) and cholesterol binding site (CHOL1). Whilst the architecture of eukaryotic NSS is generally well conserved there are differences in regions that form the VAS, LAS, and CHOL1. Here, we describe ligand-protein interactions that stabilize binding in each allosteric site and explore how differences between transporters could be exploited to generate NSS specific compounds with an emphasis on GlyT2 modulation.

Glycemic Control and Metabolic Adaptation in Response to High-Fat versus High-Carbohydrate Diets—Data from a Randomized Cross-Over Study in Healthy Subjects

Granular study of metabolic responses to alterations in the ratio of dietary macro-nutrients can enhance our understanding of how dietary modifications influence patients with impaired glycemic control. In order to study the effect of diets enriched in fat or carbohydrates, fifteen healthy, normal-weight volunteers received, in a cross-over design, and in a randomized unblinded order, two weeks of an iso-caloric high-fat diet (HFD: 60E% from fat) and a high-carbohydrate diet (HCD: 60E% from carbohydrates). A mixed meal test (MMT) was performed at the end of each dietary period to examine glucose clearance kinetics and insulin and incretin hormone levels, as well as plasma metabolomic profiles. The MMT induced almost identical glycemia and insulinemia following the HFD or HCD. GLP-1 levels were higher after the HFD vs. HCD, whereas GIP did not differ. The HFD, compared to the HCD, increased the levels of several metabolomic markers of risk for the development of insulin resistance, e.g., branched-chain amino acid (valine and leucine), creatine and α-hydroxybutyric acid levels. In normal-weight, healthy volunteers, two weeks of the HFD vs. HCD showed similar profiles of meal-induced glycemia and insulinemia. Despite this, the HFD showed a metabolomic pattern implying a risk for a metabolic shift towards impaired insulin sensitivity in the long run.

Resveratrol and related stilbene-derivatives induce Stress Granules with distinct clearance kinetics

Stress Granules are ribonucleoprotein functional condensates that form during stress conditions in all eukaryotic cells. Although their stress-survival function is far from clear, Stress Granules have been implicated in the regulation of many vital cellular pathways. Consequently, SG dysfunction is thought to be a mechanistic point of origin for many neurodegenerative disorders, including Amyotrophic Lateral Sclerosis (ALS). Additionally, SGs are thought to play a role in pathogenic pathways as diverse as viral infection and chemotherapy resistance. There is a growing consensus around the hypothesis that understanding the mechanistic regulation of SG physical properties is essential to understanding their function. Although the internal dynamics and condensation mechanisms of SGs have been broadly investigated, there have been fewer investigations into the timing of SG formation and clearance in live cells. Since the lifetime of SG persistence can be a key factor in their function and tendency towards pathological dysregulation, SG clearance mechanisms deserve particular attention. Here we show that resveratrol and its analogues, piceatannol, pterostilbene, and 3,4,5,4′ tetramethoxystilbene induce G3BP-dependent SG formation with atypically rapid clearance kinetics. Resveratrol binds to G3BP, thereby reducing its protein-protein association valency. We suggest that altering G3BP valency is a pathway for the formation of uniquely transient SGs.

Quantifying dose-, strain-, and tissue-specific kinetics of parainfluenza virus infection

Human parainfluenza viruses (HPIVs) are a leading cause of acute respiratory infection hospitalization in children, yet little is known about how dose, strain, tissue tropism, and individual heterogeneity affects the processes driving growth and clearance kinetics. Longitudinal measurements are possible by using reporter Sendai viruses, the murine counterpart of HPIV 1, that express luciferase, where the insertion location yields a wild-type (rSeV-luc(M-F*)) or attenuated (rSeV-luc(P-M)) phenotype. Bioluminescence from individual animals suggests that there is a rapid increase in expression followed by a peak, biphasic clearance, and resolution. However, these kinetics vary between individuals and with dose, strain, and whether the infection was initiated in the upper and/or lower respiratory tract. To quantify the differences, we translated the bioluminescence measurements from the nasopharynx, trachea, and lung into viral loads and used a mathematical model together a nonlinear mixed effects approach to define the mechanisms distinguishing each scenario. The results confirmed a higher rate of virus production with the rSeV-luc(M-F*) virus compared to its attenuated counterpart, and suggested that low doses result in disproportionately fewer infected cells. The analyses indicated faster infectivity and infected cell clearance rates in the lung and that higher viral doses, and concomitantly higher infected cell numbers, resulted in more rapid clearance. This parameter was also highly variable amongst individuals, which was particularly evident during infection in the lung. These critical differences provide important insight into distinct HPIV dynamics, and show how bioluminescence data can be combined with quantitative analyses to dissect host-, virus-, and dose-dependent effects.

Daily sampling of early SARS-CoV-2 infection reveals substantial heterogeneity in infectiousness

The dynamics of SARS-CoV-2 replication and shedding in humans remain poorly understood. We captured the dynamics of infectious virus and viral RNA shedding during acute infection through daily longitudinal sampling of 60 individuals for up to 14 days. By fitting mechanistic models, we directly estimate viral reproduction and clearance rates, and overall infectiousness for each individual. Significant person-to-person variation in infectious virus shedding suggests that individual-level heterogeneity in viral dynamics contributes to superspreading. Viral genome load often peaked days earlier in saliva than in nasal swabs, indicating strong compartmentalization and suggesting that saliva may serve as a superior sampling site for early detection of infection. Viral loads and clearance kinetics of B.1.1.7 and non-B.1.1.7 viruses were indistinguishable, however B.1.1.7 exhibited a significantly slower pre-peak growth rate in saliva. These results provide a high-resolution portrait of SARS-CoV-2 infection dynamics and implicate individual-level heterogeneity in infectiousness in superspreading.

Multichromatic Near-Infrared Imaging to Assess Interstitial Lymphatic and Venous Uptake In Vivo

SignificanceChanges in interstitial fluid clearance are implicated in many diseases. Using NIR imaging with properly sized tracers could enhance our understanding of how venous and lymphatic drainage are involved in disease progression or enhance drug delivery strategies.AimWe investigated multichromatic NIR imaging with multiple tracers to assess in vivo microvascular clearance kinetics and pathways in different tissue spaces.ApproachWe used a chemically inert IR Dye 800CW (free dye) to target venous capillaries and a purified conjugate of IR Dye 680RD with a 40 kDa PEG (PEG) to target lymphatic capillaries in vivo. Optical imaging settings were validated and tuned in vitro using tissue phantoms. We investigated multichromatic NIR imaging’s utility in two in vivo tissue beds – the mouse tail and rat knee joint. We then tested the ability of the approach to detect interstitial fluid perturbations due to exercise.ResultsIn an in vitro simulated tissue environment, free dye and PEG mixture allowed for simultaneous detection without interference. Co-injected NIR tracers cleared from the interstitial space via distinct routes allowed assessment lymphatic and venous uptake in the mouse tail. We determined that exercise after injection transiently increased lymphatic drainage as measured by lower normalized intensity immediately after exercise, while exercise pre-injection exhibited a transient delay in clearance from the jointConclusionsNIR imaging enables of simultaneous imaging of lymphatic and venous-mediated fluid clearance with great sensitivity and can be used to measure transient changes in clearance rates and pathways.

Inclusion of cycle threshold (CT) values when reporting SARS-CoV-2 RT-PCR results improves clinical Interpretation in suspected and confirmed COVID-19

IntroductionThe Cycle Threshold (CT) value in Real-time Polymerase Chain Reaction (RT-PCR) is where a target specific amplification signal becomes detectable and can infer viral load, risk of transmission and recovery in SARS-CoV-2 infections. Adoption into routine practice is however uncommon.Gap StatementThe lack of inclusion of CT values when reporting SARS-CoV-2 RT-PCR results in routine practice.AimTo use CT values when reporting SARS-CoV-2 RT-PCR results in Qatar to aid clinical interpretation and patient management.MethodologyRoutine CT values across 3 different RT-PCR platforms were reviewed for concordance at presentation and clearance in patients with COVID-19. An Indicative Threshold of CT 30 based on viral clearance kinetics categorized low and high CT values.ResultsThere was very high Correlation and Kappa Score agreement between the different gene targets in each platform (p<0.001). Using the Indicative Threshold it was possible to autoverify and add average CT values and append Interpretive Comments to all RT-PCR reports. The new reporting algorithm impacted immediately and safely on: physician interpretation of SARS-CoV-2 results; patient management; staff surveillance protocols; length of stay in quarantine; a redefinition of patient recovery.ConclusionIncorporation of CT values into routine practice is possible across different RT-PCR platforms and adds useful information for patient management. The use of an Indicative Threshold and interpretive comments improves clinical interpretation of the result and could be a model for reporting other respiratory infections. The current accepted practice of withholding CT values should be reviewed by the profession, accreditation bodies and regulators.

Functional multispectral optoacoustic tomography imaging of hepatic steatosis development in mice

The increasing worldwide prevalence of obesity, fatty liver diseases, as well as the emerging understanding of the importance of lipids in multi-faceted aspects of various other diseases is generating significant interest in lipid research. Lipid visualization in particular can play a critical role in understanding functional relations in lipid metabolism. In this study, we investigate the potential of multispectral optoacoustic tomography (MSOT) as a novel modality to non-invasively visualize lipids in laboratory mice. Using an obesity-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we examined whether MSOT could detect and differentiate different grades of hepatic steatosis and monitor the accumulation of lipids in the liver quantitatively over time, without the use of contrast agents, i.e. in label free mode. Moreover, we demonstrate the efficacy of using the real-time clearance kinetics of indocyanine green (ICG) in the liver as a biomarker to evaluate the organs’ function and assess the severity of NAFLD. This study demonstrates MSOT as an efficient imaging tool for lipid visualization in preclinical studies, particularly for the assessment of NAFLD.