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 ± 20 %, and decreased desensitization determined as the steady-state/peak response ratio from 23 ± 2 % to 90 ± 4 %. Based on dose-response relationship experiments on GluK1(Q)b the EC50 of BPAM538 was estimated to be 58 ± 29 µM.

Structural determinants for activity of the antidepressant vortioxetine at human and rodent 5-HT3 receptors.

Vortioxetine (VTX) is a recently approved antidepressant that targets a variety of serotonin receptors. Here, we investigate the drug's molecular mechanism of operation at the serotonin 5-HT3 receptor (5-HT3R), which features two properties: VTX acts differently on rodent and human 5-HT3R, and VTX appears to suppress any subsequent response to agonists. Using a combination of cryo-EM, electrophysiology, voltage-clamp fluorometry and molecular dynamics, we show that VTX stabilizes a resting inhibited state of the mouse 5-HT3R and an agonist-bound-like state of human 5-HT3R, in line with the functional profile of the drug. We report four human 5-HT3R structures and show that the human receptor transmembrane domain is intrinsically fragile. We also explain the lack of recovery after VTX administration via a membrane partition mechanism.

The positive allosteric modulator BPAM344 and L-glutamate introduce an active-like structure of the ligand-binding domain of GluK2.

Kainate receptors belong to the family of ionotropic glutamate receptors and contribute to the majority of fast excitatory neurotransmission. Consequently, they also play a role in brain diseases. Therefore, understanding how these receptors can be modulated is of importance. Our study provides a crystal structure of the dimeric ligand-binding domain of the kainate receptor GluK2 in complex with L-glutamate and the small-molecule positive allosteric modulator, BPAM344, in an active-like conformation. The role of Thr535 and Gln786 in modulating GluK2 by BPAM344 was investigated using a calcium-sensitive fluorescence-based assay on transiently transfected cells expressing GluK2 and mutants hereof. This study may aid in the design of compounds targeting kainate receptors, expanding their potential as targets for the treatment of brain diseases.

Small-molecule positive allosteric modulation of homomeric kainate receptors GluK1-3: development of screening assays and insight into GluK3 structure.

The kainate receptors GluK1-3 (glutamate receptor ionotropic, kainate receptors 1-3) belong to the family of ionotropic glutamate receptors and are essential for fast excitatory neurotransmission in the brain, and are associated with neurological and psychiatric diseases. How these receptors can be modulated by small-molecule agents is not well understood, especially for GluK3. We show that the positive allosteric modulator BPAM344 can be used to establish robust calcium-sensitive fluorescence-based assays to test agonists, antagonists, and positive allosteric modulators of GluK1-3. The half-maximal effective concentration (EC50) of BPAM344 for potentiating the response of 100 µm kainate was determined to be 26.3 µm for GluK1, 75.4 µm for GluK2, and 639 µm for GluK3. Domoate was found to be a potent agonist for GluK1 and GluK2, with an EC50 of 0.77 and 1.33 µm, respectively, upon co-application of 150 µm BPAM344. At GluK3, domoate acts as a very weak agonist or antagonist with a half-maximal inhibitory concentration (IC50) of 14.5 µm, in presence of 500 µm BPAM344 and 100 µm kainate for competition binding. Using H523A-mutated GluK3, we determined the first dimeric structure of the ligand-binding domain by X-ray crystallography, allowing location of BPAM344, as well as zinc-, sodium-, and chloride-ion binding sites at the dimer interface. Molecular dynamics simulations support the stability of the ion sites as well as the involvement of Asp761, Asp790, and Glu797 in the binding of zinc ions. Using electron microscopy, we show that, in presence of glutamate and BPAM344, full-length GluK3 adopts a dimer-of-dimers arrangement.

Exploring thienothiadiazine dioxides as isosteric analogues of benzo- and pyridothiadiazine dioxides in the search of new AMPA and kainate receptor positive allosteric modulators.

The synthesis and biological evaluation on AMPA and kainate receptors of new examples of 3,4-dihydro-2H-1,2,4-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxides is described. The introduction of a cyclopropyl chain instead of an ethyl chain at the 4-position of the thiadiazine ring was found to dramatically improve the potentiator activity on AMPA receptors, with compound 32 (BPAM395) expressing in vitro activity on AMPARs (EC2x = 0.24 µM) close to that of the reference 4-cyclopropyl-substituted benzothiadiazine dioxide 10 (BPAM344). Interestingly, the 4-allyl-substituted thienothiadiazine dioxide 27 (BPAM307) emerged as the most promising compound on kainate receptors being a more effective potentiator than the 4-cyclopropyl-substituted thienothiadiazine dioxide 32 and supporting the view that the 4-allyl substitution of the thiadiazine ring could be more favorable than the 4-cyclopropyl substitution to induce marked activity on kainate receptors versus AMPA receptors. The thieno-analogue 36 (BPAM279) of the clinically tested S18986 (11) was selected for in vivo evaluation in mice as a cognitive enhancer due to a safer profile than 32 after massive per os drug administration. Compound 36 was found to increase the cognition performance in mice at low doses (1 mg/kg) per os suggesting that the compound was well absorbed after oral administration and able to reach the central nervous system. Finally, compound 32 was selected for co-crystallization with the GluA2-LBD (L504Y,N775S) and glutamate to examine the binding mode of thienothiadiazine dioxides within the allosteric binding site of the AMPA receptor. At the allosteric site, this compound established similar interactions as the previously reported BTD-type AMPA receptor modulators.

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.

Longitudinal tracking of acute kidney injury reveals injury propagation along the nephron.

Acute kidney injury (AKI) is an important risk factor for chronic kidney disease (CKD), but the underlying mechanisms of failed tubule repair and AKI-CKD transition are incompletely understood. In this study, we aimed for dynamic tracking of tubule injury and remodeling to understand if focal injury upon AKI may spread over time. Here, we present a model of AKI, in which we rendered only half of the kidney ischemic. Using serial intravital 2-photon microscopy and genetic identification of cycling cells, we tracked dynamic tissue remodeling in post- and non-ischemic kidney regions simultaneously and over 3 weeks. Spatial and temporal analysis of cycling cells relative to initial necrotic cell death demonstrated pronounced injury propagation and expansion into non-necrotic tissue regions, which predicted tubule atrophy with epithelial VCAM1 expression. In summary, our longitudinal analyses of tubule injury, remodeling, and fate provide important insights into AKI pathology.

Structure-Activity Relationship and Solubility Studies of N1-Substituted Quinoxaline-2,3-diones as Kainate Receptor Antagonists.

Kainate receptors are a class of ionotropic glutamate receptors that respond to the excitatory neurotransmitter glutamate in the central nervous system and play an important role in the development of neurodegenerative disorders and the regulation of synaptic function. In the current study, we investigated the structure- activity relationship of the series of quinoxaline-2,3-diones substituted at N1, 6, and 7 positions, as ligands of kainate homomeric receptors GluK1-3 and GluK5. Pharmacological characterization showed that all derivatives obtained exhibited micromolar affinity at GluK3 receptors with Ki values in the range 0.1-4.4 µM range. The antagonistic properties of the selected analogues: N-(7-fluoro-6-iodo-2,3-dioxo-3,4-dihydroquinoxalin-1(2H)-yl)-3-sulfamoylbenzamide, N-(7-(1H-imidazol-1-yl)-6-iodo-2,3-dioxo-3,4-dihydroquinoxalin-1(2H)-yl)-3-sulfamoylbenzamide and N-(7-(1H-imidazol-1-yl)-2,3-dioxo-6-(phenylethynyl)-3,4-dihydroquinoxalin-1(2H)-yl)-3-sulfamoylbenzamide at GluK3 receptors, were confirmed by an intracellular calcium imaging assay. To correlate in vitro affinity data with structural features of the synthesized compounds and to understand the impact of the substituent in N1 position on ability to form additional protein-ligand interactions, molecular modeling and docking studies were carried out. Experimental solubility studies using UV spectroscopy detection have shown that 7-imidazolyl-6-iodo analogues with a sulfamoylbenzamide moiety at the N1 position are the best soluble compounds in the series, with molar solubility in TRISS buffer at pH 9 more than 3-fold higher compared to NBQX, a known AMPA/kainate antagonist.

Serial intravital 2-photon microscopy and analysis of the kidney using upright microscopes.

Serial intravital 2-photon microscopy of the kidney and other abdominal organs is a powerful technique to assess tissue function and structure simultaneously and over time. Thus, serial intravital microscopy can capture dynamic tissue changes during health and disease and holds great potential to characterize (patho-) physiological processes with subcellular resolution. However, successful image acquisition and analysis require significant expertise and impose multiple potential challenges. Abdominal organs are rhythmically displaced by breathing movements which hamper high-resolution imaging. Traditionally, kidney intravital imaging is performed on inverted microscopes where breathing movements are partly compensated by the weight of the animal pressing down. Here, we present a custom and easy-to-implement setup for intravital imaging of the kidney and other abdominal organs on upright microscopes. Furthermore, we provide image processing protocols and a new plugin for the free image analysis software FIJI to process multichannel fluorescence microscopy data. The proposed image processing pipelines cover multiple image denoising algorithms, sample drift correction using 2D registration, and alignment of serial imaging data collected over several weeks using landmark-based 3D registration. The provided tools aim to lower the barrier of entry to intravital microscopy of the kidney and are readily applicable by biomedical practitioners.

Systematic Review and Cost-Consequence Analysis of Ambu aScope 5 Broncho Compared with Reusable Flexible Bronchoscopes: Insights from Two US University Hospitals and an Academic Institution.

OBJECTIVE: The aim of this study was to perform a systematic review, meta-analysis and cost-consequence analysis of the single-use bronchoscope, Ambu aScopeTM 5 Broncho, in relation to reusable flexible bronchoscopes (RFB) available within three high procedure volume university hospitals and academic institutions in the USA. METHODS: The primary outcome was incremental cost and the secondary outcome was incremental cross-infection risk of use for both the single-use flexible bronchoscope (SUFB) and RFBs. Cost estimates included capital, repair, and reprocessing costs derived from a prospective observational micro-costing approach within three large university hospitals and academic institutions. All costs were valued in 2022 US dollars (USD). A meta-analysis based on literature covering cross-contamination and infection from 2010 to 2020 investigated cross-infection risk following bronchoscopy procedures with RFBs. Capital costs were discounted at 3% over 5-8 years. All parameters were evaluated using both univariate deterministic and probabilistic sensitivity analyses. RESULTS: In high-volume hospitals, RFBs were cost minimizing compared to SUFBs. Probabilistic sensitivity analysis showed that RFBs were cost saving in 88% of iterations. Univariate analyses illustrated sensitivity of the base-case result to the procedure volume. Data from sensitivity analyses suggest that the two interventions are cost neutral at a break-even point of 756 procedures per year or 46 procedures per bronchoscope per year. CONCLUSION: Assuming equivalent clinical performance, single-use flexible bronchoscopes are not cost minimizing when including the costs associated with cross-infection in high-volume US university hospitals and academic institutions. Overall, the benefits of conversion from RFBs to SUFBs are dependent on the annual procedure volume of individual hospitals, expected cross-infection risk, and purchase price of the aScope 5 Broncho.

Force potentiation during eccentric contractions in rat skeletal muscle.

Postactivation potentiation refers to an acute enhancement of contractile properties following muscle activity. Previously, the effects of prior muscle activation on eccentric force at tetanic activation frequencies have only been sparsely reported. This paper aimed to study acute activity-induced effects on eccentric force of slow and fast-twitch muscles and characterize them in relation to postactivation potentiation. We elicited eccentric contractions in isolated rat extensor digitorum longus and soleus muscles by actively lengthening muscles at a constant velocity. We assessed contractile properties by measuring force over shortly interspaced, identical eccentric, and isometric contractions. We then analyzed stretch force, isometric peak force, rate of force development, and relaxation times. Finally, we compared the time courses for the development and cessation of changes in stretch force to known features of postactivation potentiation. In extensor digitorum longus, muscles stretch force consistently increased in a contraction-to-contraction manner by up to 49% [95% confidence interval (CI): 35-64%] whereas isometric peak force simultaneously showed minor declines (8%, 95% CI: 5-10%). The development and cessation of eccentric force potentiation coincided with the development of twitch potentiation and increases in rate of force development. In soleus muscles we found no consistent eccentric potentiation. Characterization of the increase in eccentric force revealed that force only increased in the very beginning of an active stretch. Eccentric force at tetanic activation frequencies potentiates substantially in extensor digitorum longus muscles over consecutive contractions with a time course coinciding with postactivation potentiation. Such eccentric potentiation may be important in sport performance.NEW & NOTEWORTHY Force during eccentric contractions can increase to a magnitude that may have profound consequences for our understanding of skeletal muscle locomotion. This increase in eccentric force occurs over consecutive, shortly interspaced, tetanic contractions in rat extensor digitorum longus muscles-not in rat soleus muscles-and coincides with well-known traits of postactivation potentiation. Eccentric force potentiation may significantly enhance muscle performance in activities involving stretch-shortening cycles.

The DECIDE project: from surveillance data to decision-support for farmers and veterinarians.

Farmers, veterinarians and other animal health managers in the livestock sector are currently missing sufficient information on prevalence and burden of contagious endemic animal diseases. They need adequate tools for risk assessment and prioritization of control measures for these diseases. The DECIDE project develops data-driven decision-support tools, which present (i) robust and early signals of disease emergence and options for diagnostic confirmation; and (ii) options for controlling the disease along with their implications in terms of disease spread, economic burden and animal welfare. DECIDE focuses on respiratory and gastro-intestinal syndromes in the three most important terrestrial livestock species (pigs, poultry, cattle) and on reduced growth and mortality in two of the most important aquaculture species (salmon and trout). For each of these, we (i) identify the stakeholder needs; (ii) determine the burden of disease and costs of control measures; (iii) develop data sharing frameworks based on federated data access and meta-information sharing; (iv) build multivariate and multi-level models for creating early warning systems; and (v) rank interventions based on multiple criteria. Together, all of this forms decision-support tools to be integrated in existing farm management systems wherever possible and to be evaluated in several pilot implementations in farms across Europe. The results of DECIDE lead to improved use of surveillance data and evidence-based decisions on disease control. Improved disease control is essential for a sustainable food chain in Europe with increased animal health and welfare and that protects human health.

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.

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.

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.

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.

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.

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.

Selective Direct Laser Writing of Pyrolytic Carbon Microelectrodes in Absorber-Modified SU-8.

Pyrolytic carbon microelectrodes (PCMEs) are a promising alternative to their conventional metallic counterparts for various applications. Thus, methods for the simple and inexpensive patterning of PCMEs are highly sought after. Here, we demonstrate the fabrication of PCMEs through the selective pyrolysis of SU-8 photoresist by irradiation with a low-power, 806 nm, continuous wave, semiconductor-diode laser. The SU-8 was modified by adding Pro-Jet 800NP (FujiFilm) in order to ensure absorbance in the 800 nm range. The SU-8 precursor with absorber was successfully converted into pyrolytic carbon upon laser irradiation, which was not possible without an absorber. We demonstrated that the local laser pyrolysis (LLP) process in an inert nitrogen atmosphere with higher laser power and lower scan speed resulted in higher electrical conductance. The maximum conductivity achieved for a laser-pyrolyzed line was 14.2 ± 3.3 S/cm, with a line width and thickness of 28.3 ± 2.9 µm and 6.0 ± 1.0 µm, respectively, while the narrowest conductive line was just 13.5 ± 0.4 µm wide and 4.9 ± 0.5 µm thick. The LLP process seemed to be self-limiting, as multiple repetitive laser scans did not alter the properties of the carbonized lines. The direct laser writing of adjacent lines with an insulating gap down to ≤5 µm was achieved. Finally, multiple lines were seamlessly joined and intersected, enabling the writing of more complex designs with branching electrodes and the porosity of the carbon lines could be controlled by the scan speed.