A System for Assessing Dual Action Modulators of Glycine Transporters and Glycine Receptors

Reduced inhibitory glycinergic neurotransmission is implicated in a number of neurological conditions such as neuropathic pain, schizophrenia, epilepsy and hyperekplexia. Restoring glycinergic signalling may be an effective method of treating these pathologies. Glycine transporters (GlyTs) control synaptic and extra-synaptic glycine concentrations and slowing the reuptake of glycine using specific GlyT inhibitors will increase glycine extracellular concentrations and increase glycine receptor (GlyR) activation. Glycinergic neurotransmission can also be improved through positive allosteric modulation (PAM) of GlyRs. Despite efforts to manipulate this synapse, no therapeutics currently target it. We propose that dual action modulators of both GlyTs and GlyRs may show greater therapeutic potential than those targeting individual proteins. To show this, we have characterized a co-expression system in Xenopus laevis oocytes consisting of GlyT1 or GlyT2 co-expressed with GlyRα1. We use two electrode voltage clamp recording techniques to measure the impact of GlyTs on GlyRs and the effects of modulators of these proteins. We show that increases in GlyT density in close proximity to GlyRs diminish receptor currents. Reductions in GlyR mediated currents are not observed when non-transportable GlyR agonists are applied or when Na+ is not available. GlyTs reduce glycine concentrations across different concentration ranges, corresponding with their ion-coupling stoichiometry, and full receptor currents can be restored when GlyTs are blocked with selective inhibitors. We show that partial inhibition of GlyT2 and modest GlyRα1 potentiation using a dual action compound, is as useful in restoring GlyR currents as a full and potent single target GlyT2 inhibitor or single target GlyRα1 PAM. The co-expression system developed in this study will provide a robust means for assessing the likely impact of GlyR PAMs and GlyT inhibitors on glycine neurotransmission.

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A System for Assessing Dual Action Modulators of Glycine Transporters and Glycine Receptors

10.20944/preprints202010.0504.v1 ◽

2020 ◽

Author(s):

Diba Sheipouri ◽

Casey Gallagher ◽

Susan Shimmon ◽

Tristan Rawling ◽

Robert Vandenberg

Keyword(s):

Therapeutic Potential ◽

Glycine Receptor ◽

Expression System ◽

Xenopus Laevis Oocytes ◽

Glycine Transporters ◽

Single Target ◽

Dual Action ◽

Coupling Stoichiometry ◽

The Impact ◽

Glycinergic Neurotransmission

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BOARD INVITED REVIEW: The pig microbiota and the potential for harnessing the power of the microbiome to improve growth and health1

Journal of Animal Science ◽

10.1093/jas/skz208 ◽

2019 ◽

Vol 97 (9) ◽

pp. 3741-3757 ◽

Cited By ~ 8

Author(s):

Nirosh D Aluthge ◽

Dana M Van Sambeek ◽

Erin E Carney-Hinkle ◽

Yanshuo S Li ◽

Samodha C Fernando ◽

...

Keyword(s):

Gastrointestinal Tract ◽

Gut Microbiota ◽

Therapeutic Potential ◽

Animal Health ◽

Microbial Colonization ◽

Critical Importance ◽

Specific Host ◽

Microbiome Research ◽

Health And Disease ◽

The Impact

Abstract A variety of microorganisms inhabit the gastrointestinal tract of animals including bacteria, archaea, fungi, protozoa, and viruses. Pioneers in gut microbiology have stressed the critical importance of diet:microbe interactions and how these interactions may contribute to health status. As scientists have overcome the limitations of culture-based microbiology, the importance of these interactions has become more clear even to the extent that the gut microbiota has emerged as an important immunologic and metabolic organ. Recent advances in metagenomics and metabolomics have helped scientists to demonstrate that interactions among the diet, the gut microbiota, and the host to have profound effects on animal health and disease. However, although scientists have now accumulated a great deal of data with respect to what organisms comprise the gastrointestinal landscape, there is a need to look more closely at causative effects of the microbiome. The objective of this review is intended to provide: 1) a review of what is currently known with respect to the dynamics of microbial colonization of the porcine gastrointestinal tract; 2) a review of the impact of nutrient:microbe effects on growth and health; 3) examples of the therapeutic potential of prebiotics, probiotics, and synbiotics; and 4) a discussion about what the future holds with respect to microbiome research opportunities and challenges. Taken together, by considering what is currently known in the four aforementioned areas, our overarching goal is to set the stage for narrowing the path towards discovering how the porcine gut microbiota (individually and collectively) may affect specific host phenotypes.

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Inhibition of Glycine Re-Uptake: A Potential Approach for Treating Pain by Augmenting Glycine-Mediated Spinal Neurotransmission and Blunting Central Nociceptive Signaling

Biomolecules ◽

10.3390/biom11060864 ◽

2021 ◽

Vol 11 (6) ◽

pp. 864

Author(s):

Christopher L. Cioffi

Keyword(s):

Spinal Cord ◽

Dorsal Horn ◽

Therapeutic Potential ◽

Opioid Analgesic ◽

Critical Role ◽

Analgesic Efficacy ◽

Standard Of Care ◽

Current Standard ◽

Glycine Transporters ◽

Pathological Pain

Among the myriad of cellular and molecular processes identified as contributing to pathological pain, disinhibition of spinal cord nociceptive signaling to higher cortical centers plays a critical role. Importantly, evidence suggests that impaired glycinergic neurotransmission develops in the dorsal horn of the spinal cord in inflammatory and neuropathic pain models and is a key maladaptive mechanism causing mechanical hyperalgesia and allodynia. Thus, it has been hypothesized that pharmacological agents capable of augmenting glycinergic tone within the dorsal horn may be able to blunt or block aberrant nociceptor signaling to the brain and serve as a novel class of analgesics for various pathological pain states. Indeed, drugs that enhance dysfunctional glycinergic transmission, and in particular inhibitors of the glycine transporters (GlyT1 and GlyT2), are generating widespread interest as a potential class of novel analgesics. The GlyTs are Na+/Cl−-dependent transporters of the solute carrier 6 (SLC6) family and it has been proposed that the inhibition of them presents a possible mechanism by which to increase spinal extracellular glycine concentrations and enhance GlyR-mediated inhibitory neurotransmission in the dorsal horn. Various inhibitors of both GlyT1 and GlyT2 have demonstrated broad analgesic efficacy in several preclinical models of acute and chronic pain, providing promise for the approach to deliver a first-in-class non-opioid analgesic with a mechanism of action differentiated from current standard of care. This review will highlight the therapeutic potential of GlyT inhibitors as a novel class of analgesics, present recent advances reported for the field, and discuss the key challenges associated with the development of a GlyT inhibitor into a safe and effective agent to treat pain.

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Virus-Like Particles Produced Using the Brome Mosaic Virus Recombinant Capsid Protein Expressed in a Bacterial System

International Journal of Molecular Sciences ◽

10.3390/ijms22063098 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3098

Author(s):

Aleksander Strugała ◽

Jakub Jagielski ◽

Karol Kamel ◽

Grzegorz Nowaczyk ◽

Marcin Radom ◽

...

Keyword(s):

Ionic Strength ◽

Mosaic Virus ◽

Capsid Protein ◽

Self Assembly ◽

Environmental Changes ◽

Expression System ◽

Brome Mosaic Virus ◽

Virus Like Particles ◽

Assembly Principles ◽

The Impact

Virus-like particles (VLPs), due to their nanoscale dimensions, presence of interior cavities, self-organization abilities and responsiveness to environmental changes, are of interest in the field of nanotechnology. Nevertheless, comprehensive knowledge of VLP self-assembly principles is incomplete. VLP formation is governed by two types of interactions: protein–cargo and protein–protein. These interactions can be modulated by the physicochemical properties of the surroundings. Here, we used brome mosaic virus (BMV) capsid protein produced in an E. coli expression system to study the impact of ionic strength, pH and encapsulated cargo on the assembly of VLPs and their features. We showed that empty VLP assembly strongly depends on pH whereas ionic strength of the buffer plays secondary but significant role. Comparison of VLPs containing tRNA and polystyrene sulfonic acid (PSS) revealed that the structured tRNA profoundly increases VLPs stability. We also designed and produced mutated BMV capsid proteins that formed VLPs showing altered diameters and stability compared to VLPs composed of unmodified proteins. We also observed that VLPs containing unstructured polyelectrolyte (PSS) adopt compact but not necessarily more stable structures. Thus, our methodology of VLP production allows for obtaining different VLP variants and their adjustment to the incorporated cargo.

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COVID-19—The Potential Beneficial Therapeutic Effects of Spironolactone during SARS-CoV-2 Infection

Pharmaceuticals ◽

10.3390/ph14010071 ◽

2021 ◽

Vol 14 (1) ◽

pp. 71

Author(s):

Katarzyna Kotfis ◽

Kacper Lechowicz ◽

Sylwester Drożdżal ◽

Paulina Niedźwiedzka-Rystwej ◽

Tomasz K. Wojdacz ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Therapeutic Potential ◽

Severe Pneumonia ◽

Therapeutic Benefit ◽

World Health ◽

Therapeutic Effects ◽

Severe Acute Respiratory Infection ◽

Dual Action ◽

Health Organization ◽

Transmembrane Serine Protease

In March 2020, coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 was declared a global pandemic by the World Health Organization (WHO). The clinical course of the disease is unpredictable but may lead to severe acute respiratory infection (SARI) and pneumonia leading to acute respiratory distress syndrome (ARDS). It has been shown that pulmonary fibrosis may be one of the major long-term complications of COVID-19. In animal models, the use of spironolactone was proven to be an important drug in the prevention of pulmonary fibrosis. Through its dual action as a mineralocorticoid receptor (MR) antagonist and an androgenic inhibitor, spironolactone can provide significant benefits concerning COVID-19 infection. The primary effect of spironolactone in reducing pulmonary edema may also be beneficial in COVID-19 ARDS. Spironolactone is a well-known, widely used and safe anti-hypertensive and antiandrogenic medication. It has potassium-sparing diuretic action by antagonizing mineralocorticoid receptors (MRs). Spironolactone and potassium canrenoate, exerting combined pleiotropic action, may provide a therapeutic benefit to patients with COVID-19 pneumonia through antiandrogen, MR blocking, antifibrotic and anti-hyperinflammatory action. It has been proposed that spironolactone may prevent acute lung injury in COVID-19 infection due to its pleiotropic effects with favorable renin–angiotensin–aldosterone system (RAAS) and ACE2 expression, reduction in transmembrane serine protease 2 (TMPRSS2) activity and antiandrogenic action, and therefore it may prove to act as additional protection for patients at highest risk of severe pneumonia. Future prospective clinical trials are warranted to evaluate its therapeutic potential.

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Characterization of Transport Activity of SLC11 Transporters in Xenopus laevis Oocytes by Fluorophore Quenching

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/24725552211004123 ◽

2021 ◽

pp. 247255522110041

Author(s):

Raffaella Cinquetti ◽

Francesca Guia Imperiali ◽

Salvatore Bozzaro ◽

Daniele Zanella ◽

Francesca Vacca ◽

...

Keyword(s):

Xenopus Laevis ◽

Voltage Clamp ◽

Expression System ◽

Peptide Transporter ◽

Xenopus Laevis Oocytes ◽

Substrate Uptake ◽

Transport Activity ◽

Experimental Conditions ◽

Metal Transporters ◽

Electrode Voltage

Membrane proteins are involved in different physiological functions and are the target of pharmaceutical and abuse drugs. Xenopus laevis oocytes provide a powerful heterologous expression system for functional studies of these proteins. Typical experiments investigate transport using electrophysiology and radiolabeled uptake. A two-electrode voltage clamp is suitable only for electrogenic proteins, and uptake measurements require the existence of radiolabeled substrates and adequate laboratory facilities. Recently, Dictyostelium discoideum Nramp1 and NrampB were characterized using multidisciplinary approaches. NrampB showed no measurable electrogenic activity, and it was investigated in Xenopus oocytes by acquiring confocal images of the quenching of injected fluorophore calcein. This method is adequate to measure the variation in emitted fluorescence, and thus transporter activity indirectly, but requires long experimental procedures to collect statistically consistent data. Considering that optimal expression of heterologous proteins lasts for 48–72 h, a slow acquiring process requires the use of more than one batch of oocytes to complete the experiments. Here, a novel approach to measure substrate uptake is reported. Upon injection of a fluorophore, oocytes were incubated with the substrate and the transport activity measured, evaluating fluorescence quenching in a microplate reader. The technique permits the testing of tens of oocytes in different experimental conditions simultaneously, and thus the collection of significant statistical data for each batch, saving time and animals. The method was tested with different metal transporters (SLC11), DMT1, DdNramp1, and DdNrampB, and verified with the peptide transporter PepT1 (SLC15). Comparison with traditional methods (uptake, two-electrode voltage clamp) and with quenching images acquired by fluorescence microscopy confirmed its efficacy.

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A Short Note about the Impact Action of a Water Jet Stabilized by a Coaxial Air Stream in the Air and Underwater

Materials ◽

10.3390/ma14175015 ◽

2021 ◽

Vol 14 (17) ◽

pp. 5015

Author(s):

Josef Poláček ◽

Irena Marie Hlaváčová ◽

Martin Tyč

Keyword(s):

Near Field ◽

Pure Water ◽

Air Flow ◽

Relevant Literature ◽

Short Note ◽

Original Method ◽

Water Jet ◽

Impact Action ◽

Dual Action ◽

The Impact

A new original method, applying a coaxial protective airflow, was tested aiming to improve the pure water jet efficiency in surface layer removal or medium hard materials cutting or blasting. The dual action of the air flow is expected: the air co-flowing the water jet with approximately the same velocity should prevent the central jet from breaking up into tiny droplets in the near field, and simultaneously, it should support jet decomposition into big parts with enough destructive potential in the far-field. A brief survey of the relevant literature dealing with the water jet instability is presented, introducing four recognized breakup regimes. An original cutting head designed to generate a waterjet surrounded by protective coaxial air flow is introduced. The submitted device is supposed to operate within the first wind-induced regime. Two types of experiments, consisting of blasting limestone bricks placed either in the air or underwater, were realized. The depths of kerfs produced with different water pressures and air overpressures were evaluated. While no substantial positive effect was recognized in the air performance, the submerged blasting of the same material under similar conditions appeared to be promising.

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The university campus and a sense of belonging: what do students think?

New Directions in the Teaching of Physical Sciences ◽

10.29311/ndtps.v0i15.3590 ◽

2020 ◽

Author(s):

Hilda Mary Mulrooney ◽

Alison Faith Kelly

Keyword(s):

Student Perceptions ◽

Ethnic Diversity ◽

Therapeutic Potential ◽

Emotional Experience ◽

Academic Staff ◽

Physical Space ◽

Student Experiences ◽

University Campus ◽

The University ◽

The Impact

The physical configuration of the university campus impacts upon student learning and experience, and can be used to signal institutional priorities. The extent to which campus, particularly in post 92 institutions, is designed as opposed to evolving as older buildings are repurposed or replaced is variable. Student experiences and perceptions of the campus physical space are also unclear. This study aimed to explore student perceptions using qualitative methods. Data were collected during 8 focus groups from 37 participants. The majority were young and female, with considerable ethnic diversity. Six major themes were identified, many of which did not directly relate to the physical space itself. Rather the impact upon students’ emotional experience and engagement with the institution was emphasised. ‘Belonging’ was the most common theme; indicating that the nature of the physical space on campus is not neutral, but can affect the extent to which students can form attachments with each other and academic staff. ‘Nature’ was the second most commonly mentioned theme. Green space has therapeutic potential in stress management, important given mental health concerns in young people including students. The ideal campus as described by participants would include green non-smoking spaces with an emphasis on health promotion. Opportunities to include nature on campus should be taken, and future proposals to design the campus should canvas student views.

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Effects on Mood and EEG States After Meditation in Augmented Reality With and Without Adjunctive Neurofeedback

Frontiers in Virtual Reality ◽

10.3389/frvir.2021.618381 ◽

2021 ◽

Vol 2 ◽

Author(s):

Jeremy Viczko ◽

Jeff Tarrant ◽

Ray Jackson

Keyword(s):

Augmented Reality ◽

Resting State ◽

Therapeutic Potential ◽

Mood State ◽

Self Regulation ◽

Current Source ◽

The Self ◽

Anterior Cingulate ◽

Open Heart ◽

The Impact

Research and design of virtual reality technologies with mental-health focused applications has increased dramatically in recent years. However, the applications and psychological outcomes of augmented reality (AR) technologies still remain to be widely explored and evaluated. This is particularly true for the use of AR for the self-management of stress, anxiety, and mood. In the current study, we examined the impact of a brief open heart meditation AR experience on participants with moderate levels of anxiety and/or depression. Using a randomized between-group design subjects participated in the AR experience or the AR experience plus frontal gamma asymmetry neurofeedback integrated into the experience. Self-reported mood state and resting-state EEG were recorded before and after the AR intervention for both groups. Participants also reported on engagement and perceived use of the experience as a stress and coping tool. EEG activity was analyzed as a function of the frontal, midline, and parietal scalp regions, and with sLORETA current source density estimates of anterior cingulate and insular cortical regions of interest. Results demonstrated that both versions of the AR meditation significantly reduced negative mood and increased positive mood. The changes in resting state EEG were also comparable between groups, with some trending differences observed, in line with existing research on open heart and other loving-kindness and compassion-based meditations. Engagement was favorable for both versions of the AR experience, with higher levels of engagement reported with the addition of neurofeedback. These results provide early support for the therapeutic potential of AR-integrated meditations as a tool for the self-regulation of mood and emotion, and sets the stage for more research and development into health and wellness-promoting AR applications.

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Characterization of transport mechanisms and determinants critical for Na+-dependent Pisymport of the PiT family paralogs human PiT1 and PiT2

AJP Cell Physiology ◽

10.1152/ajpcell.00015.2006 ◽

2006 ◽

Vol 291 (6) ◽

pp. C1377-C1387 ◽

Cited By ~ 34

Author(s):

Pernille Bøttger ◽

Susanne E. Hede ◽

Morten Grunnet ◽

Boy Høyer ◽

Dan A. Klærke ◽

...

Keyword(s):

Mammalian Cells ◽

Divalent Cations ◽

Maximal Activity ◽

Xenopus Laevis Oocytes ◽

Transport Function ◽

Knockout Mutant ◽

Uptake Medium ◽

The Impact ◽

First Time

The general phosphate need in mammalian cells is accommodated by members of the Pitransport (PiT) family ( SLC20), which use either Na+or H+to mediate inorganic phosphate (Pi) symport. The mammalian PiT paralogs PiT1 and PiT2 are Na+-dependent Pi(NaPi) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with32Pias a traceable Pisource. For PiT1, the Michaelis-Menten constant for Piwas determined as 322.5 ± 124.5 μM. PiT2 was analyzed for the first time and showed positive cooperativity in Piuptake with a half-maximal activity constant for Piof 163.5 ± 39.8 μM. PiT1- and PiT2-mediated Na+-dependent Piuptake functions were not significantly affected by acidic and alkaline pH and displayed similar Na+dependency patterns. However, only PiT2 was capable of Na+-independent Pitransport at acidic pH. Study of the impact of divalent cations Ca2+and Mg2+revealed that Ca2+was important, but not critical, for NaPitransport function of PiT proteins. To gain insight into the NaPicotransport function, we analyzed PiT2 and a PiT2 Pitransport knockout mutant using22Na+as a traceable Na+source. Na+was transported by PiT2 even without Piin the uptake medium and also when Pitransport function was knocked out. This is the first time decoupling of Pifrom Na+transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids E55and E575are responsible for linking Piimport to Na+transport in PiT2.

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