A previously unrecognized superfamily of macro-conotoxins includes an inhibitor of the sensory neuron calcium channel Cav2.3.

Animal venom peptides represent valuable compounds for biomedical exploration. The venoms of marine cone snails constitute a particularly rich source of peptide toxins, known as conotoxins. Here, we identify the sequence of an unusually large conotoxin, Mu8.1, which defines a new class of conotoxins evolutionarily related to the well-known con-ikot-ikots and 2 additional conotoxin classes not previously described. The crystal structure of recombinant Mu8.1 displays a saposin-like fold and shows structural similarity with con-ikot-ikot. Functional studies demonstrate that Mu8.1 curtails calcium influx in defined classes of murine somatosensory dorsal root ganglion (DRG) neurons. When tested on a variety of recombinantly expressed voltage-gated ion channels, Mu8.1 displayed the highest potency against the R-type (Cav2.3) calcium channel. Ca2+ signals from Mu8.1-sensitive DRG neurons were also inhibited by SNX-482, a known spider peptide modulator of Cav2.3 and voltage-gated K+ (Kv4) channels. Our findings highlight the potential of Mu8.1 as a molecular tool to identify and study neuronal subclasses expressing Cav2.3. Importantly, this multidisciplinary study showcases the potential of uncovering novel structures and bioactivities within the largely unexplored group of macro-conotoxins.

Identification and functional evaluation of GRIA1 missense and truncation variants in individuals with ID: An emerging neurodevelopmental syndrome.

GRIA1 encodes the GluA1 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors, which are ligand-gated ion channels that act as excitatory receptors for the neurotransmitter L-glutamate (Glu). AMPA receptors (AMPARs) are homo- or heteromeric protein complexes with four subunits, each encoded by different genes, GRIA1 to GRIA4. Although GluA1-containing AMPARs have a crucial role in brain function, the human phenotype associated with deleterious GRIA1 sequence variants has not been established. Subjects with de novo missense and nonsense GRIA1 variants were identified through international collaboration. Detailed phenotypic and genetic assessments of the subjects were carried out and the pathogenicity of the variants was evaluated in vitro to characterize changes in AMPAR function and expression. In addition, two Xenopus gria1 CRISPR-Cas9 F0 models were established to characterize the in vivo consequences. Seven unrelated individuals with rare GRIA1 variants were identified. One individual carried a homozygous nonsense variant (p.Arg377Ter), and six had heterozygous missense variations (p.Arg345Gln, p.Ala636Thr, p.Ile627Thr, and p.Gly745Asp), of which the p.Ala636Thr variant was recurrent in three individuals. The cohort revealed subjects to have a recurrent neurodevelopmental disorder mostly affecting cognition and speech. Functional evaluation of major GluA1-containing AMPAR subtypes carrying the GRIA1 variant mutations showed that three of the four missense variants profoundly perturb receptor function. The homozygous stop-gain variant completely destroys the expression of GluA1-containing AMPARs. The Xenopus gria1 models show transient motor deficits, an intermittent seizure phenotype, and a significant impairment to working memory in mutants. These data support a developmental disorder caused by both heterozygous and homozygous variants in GRIA1 affecting AMPAR function.

Crystal structure of the GluK1 ligand-binding domain with kainate and the full-spanning positive allosteric modulator BPAM538.

Kainate receptors play an important role in the central nervous system by mediating postsynaptic excitatory neurotransmission and modulating the release of the inhibitory neurotransmitter GABA through a presynaptic mechanism. To date, only three structures of the ligand-binding domain (LBD) of the kainate receptor subunit GluK1 in complex with positive allosteric modulators have been determined by X-ray crystallography, all belonging to class II modulators. Here, we report a high-resolution structure of GluK1-LBD in complex with kainate and BPAM538, which belongs to the full-spanning class III. One BPAM538 molecule binds at the GluK1 dimer interface, thereby occupying two allosteric binding sites simultaneously. BPAM538 stabilizes the active receptor conformation with only minor conformational changes being introduced to the receptor. Using a calcium-sensitive fluorescence-based assay, a 5-fold potentiation of the kainate response (100 µM) was observed in presence of 100 µM BPAM538 at GluK1(Q)b, whereas no potentiation was observed at GluK2(VCQ)a. Using electrophysiology recordings of outside-out patches excised from HEK293 cells, BPAM538 increased the peak response of GluK1(Q)b co-expressed with NETO2 to rapid application of 10 mM L-glutamate with 130 ±â€¯20 %, and decreased desensitization determined as the steady-state/peak response ratio from 23 ±â€¯2 % to 90 ±â€¯4 %. Based on dose-response relationship experiments on GluK1(Q)b the EC50 of BPAM538 was estimated to be 57.5 ±â€¯29.2 µM.

Three-dimensional missense tolerance ratio analysis.

A wealth of genetic information is available describing single-nucleotide variants in the human population that appear to be well-tolerated and in and of themselves do not confer disease. These variant data sets contain signatures about the protein structure-function relationships and provide an unbiased view of various protein functions in the context of human health. This information can be used to determine regional intolerance to variation, defined as the missense tolerance ratio (MTR), which is an indicator of stretches of the polypeptide chain that can tolerate changes without compromising protein function in a manner that impacts human health. This approach circumvents the lack of comprehensive data by averaging the data from adjacent residues on the polypeptide chain. We reasoned that many motifs in proteins consist of nonadjacent residues, but together function as a unit. We therefore developed an approach to analyze nearest neighbors in three-dimensional space as determined by crystallography rather than on the polypeptide chain. We used members of the GRIN gene family that encode subunits of NMDA-type ionotropic glutamate receptors (iGluRs) to exemplify the differences between these methods. Our method, 3DMTR, provides new information about regions of intolerance within iGluRs, allows consideration of protein-protein interfaces in multimeric proteins, and moves this important research tool from one-dimensional analysis to a structurally relevant tool. We validate the improved 3DMTR score by showing that it more accurately classifies the functional consequences of a set of newly measured and published point mutations of Grin family genes than existing methods.

Resonant Laser Printing of Optical Metasurfaces.

One of the challenges for metasurface research is upscaling. The conventional methods for fabrication of metasurfaces, such as electron-beam or focused ion beam lithography, are not scalable. The use of ultraviolet steppers or nanoimprinting still requires large-size masks or stamps, which are costly and challenging in further handling. This work demonstrates a cost-effective and lithography-free method for printing optical metasurfaces. It is based on resonant absorption of laser light in an optical cavity formed by a multilayer structure of ultrathin metal and dielectric coatings. A nearly perfect light absorption is obtained via interferometric control of absorption and operating around a critical coupling condition. Controlled by the laser power, the surface undergoes a structural transition from random, semiperiodic, and periodic to amorphous patterns with nanoscale precision. The reliability, upscaling, and subwavelength resolution of this approach are demonstrated by realizing metasurfaces for structural colors, optical holograms, and diffractive optical elements.

Prolonged loss of force and power following fatiguing contractions in rat soleus muscles. Is low-frequency fatigue an issue during dynamic contractions?

Low-frequency fatigue (LFF) is defined by a relatively larger deficit in isometric force elicited by low-frequency electrical stimulation compared with high-frequency stimulation. However, the effects of LFF on power during dynamic contractions elicited at low and high frequencies have not been thoroughly characterized. In the current study, rat soleus muscles underwent fatiguing either concentric, eccentric, or isometric contractions. Before and 1 h after the fatiguing contractions, a series of brief isometric and dynamic contractions elicited at 20 and 80 Hz stimulation to establish force-velocity relationships. Maximal force (Fmax), velocity (Vmax), and power (Pmax) were assessed for each frequency. Sarcoplasmic reticulum (SR) Ca2+ release and reuptake rates were assessed pre- and postfatigue. Prolonged fatigue was observed as a loss of Fmax and Pmax in muscles fatigued by concentric or eccentric, but not by isometric contractions. When quantified as a decrease in the ratio between 20 Hz and 80 Hz contractile output, LFF was more pronounced for isometric force than for power (-21% vs. -16% for concentrically fatigued muscles, P = 0.003; 29 vs. 13% for eccentrically fatigued muscles, P < 0.001). No changes in SR Ca2+ release or reuptake rates were observed. We conclude that LFF is less pronounced when expressed in terms of power deficits than when expressed in terms of force deficits, and that LFF, therefore, likely affects performance to a lesser degree during fast concentric contractions than during static or slow contractions.

Using state space models to monitor and estimate the effects of interventions on treatment risk and milk yield in dairy farms.

Fast, flexible, and internally valid analytical tools are needed to evaluate the effects of management interventions made on dairy farms to support decisions about which interventions to continue or discontinue. The objective of this observational study was to demonstrate the use of state space models (SSM) to monitor and estimate the effect of interventions on 2 specific outcomes: a dynamic linear model (DLM) evaluating herd-level milk yield and a dynamic generalized linear model evaluating treatment risk in a pragmatic pretest/posttest design under field conditions. This demonstration study is part of a Danish common learning project that ran from March 2020 to May 2021 within the framework of veterinary herd health consultancy in relation to reducing antimicrobial use and improving herd health. Specific interventions for 2 commercial herds were suggested by 4 visiting farmers and were implemented during the project period. The intervention for herd 1 was the application of teat sealers, implemented in August 2020. For herd 2, the intervention was an adjustment of cubicles for cows of parity 2 and above, implemented from November 2020. A shift to an automatic milking system in October 2020 was also modeled as an intervention for herd 1 because the 2 interventions coincided. Data available from the Danish Cattle Database on obligatory registrations for individual cow movements and treatments, as well as test day information on milk yield, were used for model building and testing. Data from a 3-yr period before the project were used to calibrate the SSM to herd conditions, and data from the study period (March 2020 to May 2021) were used for monitoring and intervention testing based on application of the SSM. Herd bulk tank milk recordings were added to the data set during the study period to increase the precision of the estimates in the DLM. The developed SSM monitored herd-level milk yield and the overall probability of treatment throughout the study period in both herds. Furthermore, at the time of intervention, the SSM estimated the effect on herd-level milk yield and treatment risk associated with the implemented intervention in each herd. The SSM were used because they can be calibrated to herd conditions and they take into account herd dynamics and autocorrelation and provide standard deviations of estimates. For herd 1, the intervention effect of applying teat sealers was inconclusive with the current SSM application. For herd 2, no statistically significant changes in cow treatment risk or milk production were identified following the adjustment of cubicles. The use of SSM on observational data under field conditions shows that in this case, the interventions had a nonspecific onset of effect, were implemented during unstable times, and had varying coherence with the measured outcomes, making fully automated SSM analysis difficult. However, similar or expanded SSM with both monitoring and effect estimation functions could, if applied under the right conditions, serve as improved data-based decision support tools for farmers (and veterinarians) to minimize the risk of misinterpreting data due to confounding bias related to dynamics in dairy herds.

Discovery of the First Highly Selective Antagonist of the GluK3 Kainate Receptor Subtype.

Kainate receptors belong to the family of glutamate receptors ion channels, which are responsible for the majority of rapid excitatory synaptic transmission in the central nervous system. The therapeutic potential of kainate receptors is still poorly understood, which is also due to the lack of potent and subunit-selective pharmacological tools. In search of selective ligands for the GluK3 kainate receptor subtype, a series of quinoxaline-2,3-dione analogues was synthesized and pharmacologically characterized at selected recombinant ionotropic glutamate receptors. Among them, compound 28 was found to be a competitive GluK3 antagonist with submicromolar affinity and unprecedented high binding selectivity, showing a 400-fold preference for GluK3 over other homomeric receptors GluK1, GluK2, GluK5 and GluA2. Furthermore, in functional assays performed for selected metabotropic glutamate receptor subtypes, 28 did not show agonist or antagonist activity. The molecular determinants underlying the observed affinity profile of 28 were analyzed using molecular docking and molecular dynamics simulations performed for individual GluK1 and GluK3 ligand-binding domains.

Single-molecule DNA-mapping and whole-genome sequencing of individual cells.

To elucidate cellular diversity and clonal evolution in tissues and tumors, one must resolve genomic heterogeneity in single cells. To this end, we have developed low-cost, mass-producible micro-/nanofluidic chips for DNA extraction from individual cells. These chips have modules that collect genomic DNA for sequencing or map genomic structure directly, on-chip, with denaturation-renaturation (D-R) optical mapping [Marie R, et al. (2013) Proc Natl Acad Sci USA 110:4893-4898]. Processing of single cells from the LS174T colorectal cancer cell line showed that D-R mapping of single molecules can reveal structural variation (SV) in the genome of single cells. In one experiment, we processed 17 fragments covering 19.8 Mb of the cell's genome. One megabase-large fragment aligned well to chromosome 19 with half its length, while the other half showed variable alignment. Paired-end single-cell sequencing supported this finding, revealing a region of complexity and a 50-kb deletion. Sequencing struggled, however, to detect a 20-kb gap that D-R mapping showed clearly in a megabase fragment that otherwise mapped well to the reference at the pericentromeric region of chromosome 4. Pericentromeric regions are complex and show substantial sequence homology between different chromosomes, making mapping of sequence reads ambiguous. Thus, D-R mapping directly, from a single molecule, revealed characteristics of the single-cell genome that were challenging for short-read sequencing.

Is curvature of the force-velocity relationship affected by oxygen availability? Evidence from studies in ex vivo and in situ rat muscles.

The power of shortening contractions in skeletal muscle is determined by the force-velocity relationship. Fatigue has been reported to either increase or decrease the force-velocity curvature depending on experimental circumstances. These discrepant findings may be related to experimental differences in oxygen availability. We therefore investigated how the curvature of the force-velocity relationship in soleus and gastrocnemius rat muscles is affected during fatigue, in both an ex vivo setup without an intact blood perfusion and in an in situ setup with an intact blood perfusion. Furthermore, we investigated the effect of reduced oxygen concentrations and reduced diffusion distance on the curvature of the force-velocity relationship in ex vivo muscles, where muscle oxygen uptake relies on diffusion from the incubation medium. Muscles were electrically stimulated to perform repeated shortening contractions and force-velocity curves were determined in rested and fatigued conditions. The curvature increased during fatigue in the soleus muscles (both in situ and ex vivo), and decreased for the gastrocnemius muscles (in situ) or remained unchanged (ex vivo). Furthermore, under ex vivo conditions, neither reduced oxygen concentrations nor reduced diffusion distance conferred any substantial effect on the force-velocity curvature. In contrast, reduced oxygen availability and increased diffusion distance did increase the loss of maximal power during fatigue, mainly due to additional decreases in isometric force. We conclude that oxygen availability does not influence the fatigue-induced changes in force-velocity curvature. Rather, the observed variable fatigue profiles with regard to changes in curvature seem to be linked to the muscle fiber-type composition.

How good is your metalens? Experimental verification of metalens performance criterion.

A metric for evaluation of overall metalens performance is presented. It is applied to determination of optimal operating spectral range of a metalens, both theoretically and experimentally. This metric is quite general and can be applied to the design and evaluation of future metalenses, particularly achromatic metalenses.

Additional in-series compliance does not affect the length dependence of activation in rat medial gastrocnemius.

NEW FINDINGS: What is the central question of this study? The length dependence of activation (LDA) is typically explained by a length-dependent increase in calcium sensitivity, but recently calcium-independent mechanisms have been suggested: does active muscle shortening provided by a compliant in-series component impact the muscle length at which force output is maximized, thus contributing to LDA? What is the main finding and its importance? Using an in situ rat medial gastrocnemius set-up and varying the magnitude of muscle shortening via an artificial compliant series-elastic component, we were unable to observe any change in optimal length between conditions, contrary to some previous findings. More research is therefore required to explain these discrepancies. ABSTRACT: The force-length relationship dictates the amount of force a muscle can produce as a function of its length, during maximal isometric contractions. When activation is submaximal, it has been shown that the length at which force production is highest (the optimal length) is longer. This is typically explained by a length-dependent increase in Ca2+ sensitivity, known as the 'length dependence of activation'. Recent reports have implicated shortening against in-series compliance to be a potential factor in the observed optimal length (L0 ) of muscle, via the phenomenon of shortening-induced force depression (a phenomenon which describes the relative reduction in muscle force when a muscle is actively shortening to a given length compared to contracting isometrically at that same length). In the current study, rat medial gastrocnemius was stimulated with single and triple pulses (200 Hz) over a range of lengths, both with and without additional in-series compliance provided by a small piece of silicon tubing in series with the muscle, which allowed greater fascicle shortening upon activation. Fascicle length was measured using sonomicrometry crystals, and peak force (Fpeak ) and L0 were estimated by curve-fitting of the force-length data. The additional in-series compliance significantly reduced Fpeak by approximately 14% and 25% for the single and triple pulses, respectively (P = 0.003, P < 0.001), yet L0 remained unchanged (P = 0.405), suggesting that in our model, shortening against in-series compliance does not affect L0 . We offer potential explanations for the discrepancies seen and discuss whether the velocity of shortening may have a role in the length dependence of force.

Conformational Changes in the 5-HT3A Receptor Extracellular Domain Measured by Voltage-Clamp Fluorometry.

The 5-hydroxytryptamine (5-HT) type 3 receptor is a member of the cysteine (Cys)-loop receptor super family of ligand-gated ion channels in the nervous system and is a clinical target in a range of diseases. The 5-HT3 receptor mediates fast serotonergic neurotransmission by undergoing a series of conformational changes initiated by ligand binding that lead to the rapid opening of an intrinsic cation-selective channel. However, despite the availability of high-resolution structures of a mouse 5-HT3 receptor, many important aspects of the mechanistic basis of 5-HT3 receptor function and modulation by drugs remain poorly understood. In particular, there is little direct evidence for the specific conformational changes predicted to occur during ligand-gated channel activation and desensitization. In the present study, we used voltage-clamp fluorometry (VCF) to measure conformational changes in regions surrounding the orthosteric binding site of the human 5-HT3A (h5-HT3A) receptor during binding of 5-HT and different classes of 5-HT3 receptor ligands. VCF utilizes parallel measurements of receptor currents with photon emission from fluorescent reporter groups covalently attached to specific positions in the receptor structure. Reporter groups that are highly sensitive to the local molecular environment can, in real time, report conformational changes as changes in fluorescence that can be correlated with changes in receptor currents reporting the functional states of the channel. Within the loop C, D, and E regions that surround the orthosteric binding site in the h5-HT3A receptor, we identify positions that are amenable to tagging with an environmentally sensitive reporter group that reports robust fluorescence changes upon 5-HT binding and receptor activation. We use these reporter positions to characterize the effect of ligand binding on the local structure of the orthosteric binding site by agonists, competitive antagonists, and allosterically acting channel activators. We observed that loop C appears to show distinct fluorescence changes for ligands of the same class, while loop D reports similar fluorescence changes for all ligands binding at the orthosteric site. In contrast, the loop E reporter position shows distinct changes for agonists, antagonists, and allosteric compounds, suggesting the conformational changes in this region are specific to ligand function. Interpretation of these results within the framework of current models of 5-HT3 and Cys-loop mechanisms are used to expand the understanding of how ligand binding in Cys-loop receptors relates to channel gating. SIGNIFICANCE STATEMENT: The 5-HT3 receptor is an important ligand-gated ion channel and drug target in the central and peripheral nervous system. Determining how ligand binding induced conformational changes in the receptor is central for understanding the structural mechanisms underlying 5-HT3 receptor function. Here, we employ voltage-gated fluorometry to characterize conformational changes in the extracellular domain of the human 5-HT3 receptor to identify intrareceptor motions during binding of a range of 5-HT3 receptor agonists and antagonists.

Mutational Analysis and Modeling of Negative Allosteric Modulator Binding Sites in AMPA Receptors.

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) constitute a subclass of the ionotropic glutamate receptor superfamily, which functions as glutamate-gated cation channels to mediate the majority of excitatory neurotransmission in the central nervous system. AMPARs are therapeutic targets in a range of brain disorders associated with abnormal glutamate hyperactivity. Multiple classes of AMPAR inhibitors have been developed during the past decades, including competitive antagonists, ion channel blockers, and negative allosteric modulators (NAMs). At present, the NAM is the only class of AMPAR ligands that have been developed into safe and useful drugs in humans in the form of perampanel (Fycompa), which was recently approved for treatment of epilepsy. Compared with the detailed understanding of other AMPAR ligand classes, surprisingly little information has been available regarding the molecular mechanism of perampanel and other classes of NAMs at AMPARs; including the location and structure of NAM binding pockets in the receptor complex. However, structures of the AMPAR GluA2 in complex with NAMs were recently reported that unambiguously identified the NAM binding sites. In parallel with this work, our aim with the present study was to identify specific residues involved in the formation of the NAM binding site for three prototypical AMPAR NAMs. Hence, we have performed a mutational analysis of the AMPAR region that links the four extracellular ligand-binding domains to the central ion channel in the transmembrane domain region. Furthermore, we perform computational ligand docking of the NAMs into structural models of the homomeric GluA2 receptor and optimize side chain conformations around the NAMs to model how NAMs bind in this specific site. The new insights provide potentially valuable input for structure-based drug design of new NAMs. SIGNIFICANCE STATEMENT: The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are glutamate-gated ion channels that mediate the majority of excitatory neurotransmission in the brain. Negative allosteric modulators of AMPA receptors are considered to have significant therapeutic potential in diseases linked to glutamate hyperactivity. The present work employs mutational analysis and molecular modeling of the binding site for prototypical NAMs to provide new molecular insight into how NAMs interact with the AMPA receptor, which is of potential use for future design of new types of NAMs.

Hyperspectral spatially offset Raman spectroscopy in a microfluidic channel.

Spatially offset Raman spectroscopy (SORS) enables one to distinguish chemical fingerprints of top and subsurface layers. In this paper, we apply SORS to a microfluidic two-layer system consisting of transparent liquid in a microchannel as the surface layer and microfluidic PDMS chip material as the sublayer. By using an imaging spectrograph connected to a microscope, we perform hyperspectral SORS acquisitions. Furthermore, the focus position z is translated. Thus, we combine the two methods of hyperspectral SORS and defocusing micro-SORS, which leads to an integral characterization of the layered system. The collected top and subsurface layers of Raman scattering at the optical axis (zero spatial offset) largely depends on the focus position z. However, the spatially offset Raman scattered intensity from the subsurface layer is constant for a large range of focus positions z. We claim that there is potential for internal referencing and alignment reproducibility. We demonstrate these findings experimentally in a microfluidic scenario where a 16 µm deep channel is filled with an aqueous hemoglobin solution. Our observation enables consistent concentration measurements in small-volume liquid samples.

Fatiguing stimulation increases curvature of the force-velocity relationship in isolated fast-twitch and slow-twitch rat muscles.

In skeletal muscles, the ability to generate power is reduced during fatigue. In isolated muscles, maximal power can be calculated from the force-velocity relationship. This relationship is well described by the Hill equation, which contains three parameters: (1) maximal isometric force, (2) maximum contraction velocity and (3) curvature. Here, we investigated the hypothesis that a fatigue-induced loss of power is associated with changes in curvature of the force-velocity curve in slow-twitch muscles but not in fast-twitch muscles during the development of fatigue. Isolated rat soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch) muscles were incubated in Krebs-Ringer solution at 30°C and stimulated electrically at 60 Hz (soleus) and 150 Hz (EDL) to perform a series of concentric contractions to fatigue. Force-velocity data were fitted to the Hill equation, and curvature was determined as the ratio of the curve parameters a/F0 (inversely related to curvature). At the end of the fatiguing protocol, maximal power decreased by 58±5% in the soleus and 69±4% in the EDL compared with initial values in non-fatigued muscles. At the end of the fatiguing sequence, curvature increased as judged from the decrease in a/F0 by 81±20% in the soleus and by 31±12% in the EDL. However, during the initial phases of fatiguing stimulation, we observed a small decrease in curvature in the EDL, but not in the soleus, which may be a result of post-activation potentiation. In conclusion, fatigue-induced loss of power is strongly associated with an increased curvature of the force-velocity relationship, particularly in slow-twitch muscles.

In-line whole blood fractionation for Raman analysis of blood plasma.

Blood plasma evaluation has high significance in clinical diagnostics. Current schemes involve the preparation of blood plasma by centrifugation of whole blood followed by electrochemical or spectroscopic analysis. However, centrifugation is often too time-consuming for application in clinical emergency and point-of-care settings. We propose to combine microfluidic, instantaneous plasma fractionation with localized spectroscopic methods for in-line analysis. As an example, we present confocal Raman spectroscopy in fractionated plasma domains at two different Raman excitation wavelengths. Resonance Raman spectroscopy with laser excitation at 408 nm allows the specific detection of free hemoglobin in blood plasma at concentrations above 22 mg dl-1 (level of detection). Consequently, we are able to accurately resolve the range of clinical relevance regarding hemolysis. At near-infrared excitation (785 nm) we furthermore demonstrate the acquisition of characteristic Raman spectra of fractionated blood plasma in the microfluidic setting. These spectra can serve as starting point for a multi-parameter regression analysis to quantify a set of blood plasma parameters from a single Raman spectrum. The combined microfluidics and Raman spectroscopy method is non-destructive and has a whole blood consumption of less than 100 µl per hour. It thus allows for continuous in-line blood plasma monitoring.

Structural rearrangement of the intracellular domains during AMPA receptor activation.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated ion channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. Despite recent advances in structural studies of AMPARs, information about the specific conformational changes that underlie receptor function is lacking. Here, we used single and dual insertion of GFP variants at various positions in AMPAR subunits to enable measurements of conformational changes using fluorescence resonance energy transfer (FRET) in live cells. We produced dual CFP/YFP-tagged GluA2 subunit constructs that had normal activity and displayed intrareceptor FRET. We used fluorescence lifetime imaging microscopy (FLIM) in live HEK293 cells to determine distinct steady-state FRET efficiencies in the presence of different ligands, suggesting a dynamic picture of the resting state. Patch-clamp fluorometry of the double- and single-insert constructs showed that both the intracellular C-terminal domain (CTD) and the loop region between the M1 and M2 helices move during activation and the CTD is detached from the membrane. Our time-resolved measurements revealed unexpectedly complex fluorescence changes within these intracellular domains, providing clues as to how posttranslational modifications and receptor function interact.

Modeling and Mutational Analysis of the Binding Mode for the Multimodal Antidepressant Drug Vortioxetine to the Human 5-HT3A Receptor.

5-Hydroxytryptamine3 (5-HT3) receptors are ligand-gated ion channels that mediate neurotransmission by serotonin in the central nervous system. Pharmacological inhibition of 5-HT3 receptor activity has therapeutic potential in several psychiatric diseases, including depression and anxiety. The recently approved multimodal antidepressant vortioxetine has potent inhibitory activity at 5-HT3 receptors. Vortioxetine has an inhibitory mechanism that differs from classic 5-HT3 receptor competitive antagonists despite being believed to bind in the same binding site. Specifically, vortioxetine shows partial agonist activity followed by persistent and insurmountable inhibition. We have investigated the binding mode of vortioxetine at the human 5-HT3A receptor through computational and in vitro experiments to provide insight into the molecular mechanisms behind the unique pharmacological profile of the drug. We find that vortioxetine binds in a manner different from currently known 5-HT3A orthosteric ligands. Specifically, while the binding pattern of vortioxetine mimics some aspects of both the setron class of competitive antagonists and 5-hydroxytryptamine (5-HT) with regards to interactions with residues of the aromatic box motif in the orthosteric binding site, vortioxetine also forms interactions with residues not previously described to be important for the binding of either setrons or 5-HT such as Val202 on Loop F. Our results expand the framework for understanding how orthosteric ligands drive 5-HT3 receptor function, which is of importance for the potential future development of novel classes of 5-HT3 receptor antagonists.

Placental Sequestration of Plasmodium falciparum Malaria Parasites Is Mediated by the Interaction Between VAR2CSA and Chondroitin Sulfate A on Syndecan-1.

During placental malaria, Plasmodium falciparum infected erythrocytes sequester in the placenta, causing health problems for both the mother and fetus. The specific adherence is mediated by the VAR2CSA protein, which binds to placental chondroitin sulfate (CS) on chondroitin sulfate proteoglycans (CSPGs) in the placental syncytium. However, the identity of the CSPG core protein and the cellular impact of the interaction have remain elusive. In this study we identified the specific CSPG core protein to which the CS is attached, and characterized its exact placental location. VAR2CSA pull-down experiments using placental extracts from whole placenta or syncytiotrophoblast microvillous cell membranes showed three distinct CSPGs available for VAR2CSA adherence. Further examination of these three CSPGs by immunofluorescence and proximity ligation assays showed that syndecan-1 is the main receptor for VAR2CSA mediated placental adherence. We further show that the commonly used placental choriocarcinoma cell line, BeWo, express a different set of proteoglycans than those present on placental syncytiotrophoblast and may not be the most biologically relevant model to study placental malaria. Syncytial fusion of the BeWo cells, triggered by forskolin treatment, caused an increased expression of placental CS-modified syndecan-1. In line with this, we show that rVAR2 binding to placental CS impairs syndecan-1-related Src signaling in forskolin treated BeWo cells, but not in untreated cells.