Single-molecule studies of the neuronal SNARE fusion machinery.

SNAREs are essential components of the machinery for Ca(2+)-triggered fusion of synaptic vesicles with the plasma membrane, resulting in neurotransmitter release into the synaptic cleft. Although much is known about their biophysical and structural properties and their interactions with accessory proteins such as the Ca(2+) sensor synaptotagmin, their precise role in membrane fusion remains an enigma. Ensemble studies of liposomes with reconstituted SNAREs have demonstrated that SNAREs and accessory proteins can trigger lipid mixing/fusion, but the inability to study individual fusion events has precluded molecular insights into the fusion process. Thus, this field is ripe for studies with single-molecule methodology. In this review, we discuss applications of single-molecule approaches to observe reconstituted SNAREs, their complexes, associated proteins, and their effect on biological membranes. Some of the findings are provocative, such as the possibility of parallel and antiparallel SNARE complexes or of vesicle docking with only syntaxin and synaptobrevin, but have been confirmed by other experiments.

Publisher Correction: Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.

Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

Cardiac Therapeutics in Horses.

Many cardiac therapeutics lack significant evidence of benefit in the horse, and in many cases their use is based on extrapolation of evidence from other species. In recent years there has been a push to develop a better understanding of both the pharmacodynamics and pharmacokinetics of these drugs. Recent data have described the use of antiarrhythmic agents including sotalol, flecainide, and amiodarone. Data about the use of ACE inhibitors in the management of congestive heart failure are encouraging and support their use in certain cases, wheras evidence for other medicines, such as pimobendan, remain speculative.

The Comparative Role of the Anterior Cruciate Ligament and Anterolateral Structures in Controlling Passive Internal Rotation of the Knee: A Biomechanical Study.

PURPOSE: To determine the respective functions of the anterior cruciate ligament (ACL) and the anterolateral structures (ALSs) in controlling the tibia's passive internal rotation (IR) with respect to the femur, under uniaxial rotation. METHODS: To test the function of the ACL and the anterolateral ligament (ALL) in IR, we designed a sequential transection study of the ACL and the anterolateral structures (including the ALL) in 24 cadaveric knees divided in 2 groups. Two sequences were conducted successively: group 1 (12 knees) in which the ACL was sectioned first followed by the ALS, and group 2 (12 knees) with reversed transections. Each knee, in neutral rotation position and at flexion angle of 30°, was subjected to a 5 Nm torsion torque of IR. IR was measured using a rotatory laximeter, the Rotam with a gyroscope's measurement accuracy of 0.1°. Laxities were compared using paired t test within each group and using t test between groups. Fisher exact test was used to compare proportions. RESULTS: In group 1, IR increased from 22.1° ± 10.6° to 25.7° ± 10.9° after ACL transection then to 28.1° ± 10.5° after we sectioned the ALS. In group 2, IR increased from 22.5° ± 8.9° to 25.2° ± 8.4° after sectioning the ALS, then to 29.1° ± 8.8° after we sectioned the ACL. Total postsectioning increase in IR was 6.4° ± 2° in group 1, and 6.55° ± 0.9° in group 2. The IR increase after each stage of transection and final IR were statistically significant (P < .001). CONCLUSIONS: In a pure rotational cadaveric test model, the ACL and the ALS contribute to resistance to passive IR of the knee. CLINICAL RELEVANCE: In some specific clinical cases, peripheral lesions may be considered, and injuries to these structures may need to be addressed to improve results controlling postoperative IR.

Do graft diameter or patient age influence the results of ACL reconstruction?

PURPOSE: Hamstring tendons are commonly used as a graft source for ACL reconstruction. This study seeks to determine whether either the diameter of the tendon graft or the age of the patient influences the outcome of the ACL reconstruction when measured using a standard, previously validated laxity measurement device. METHODS: This is a retrospective study of 88 patients who underwent ACL reconstruction with a short, quadrupled tendon technique, using the semitendinosus ± gracilis tendons. Patients included in this study were sequential, unilateral, complete ACL ruptures. The patients were followed for a minimum of 1 year postoperatively, with a mean follow-up of 26 months. Patients were divided into three groups according to the diameter (Ø) of the graft: group 1 (32 patients): 8 mm ≤ Ø ≤ 9 mm; group 2 (28 patients): 9 mm < Ø ≤ 10 mm; and group 3 (28 patients): Ø > 10 mm. Three groups with differential laxity at 134 N (Δ134 = healthy side vs. operated side) measured with the laximeter GNRB(®) were compared. The risk of residual laxity (OR) between the three groups taking age, gender, BMI and meniscus status into account was calculated. A side-to-side laxity >3 mm was considered as a residual laxity. RESULTS: The mean patient age at the time of reconstruction was 29.4 years. The three groups were comparable. Postoperative Δ134 was 1.50 ± 1.3, 1.59 ± 1.5 and 2 ± 1.7 mm for groups 1 through 3, respectively. Δ134 > 3 mm was observed in three patients in group 1, four patients in group 2 and nine patients in group 3. As compared to group 1, OR was 1.46 (95 % CI 0.35-6.05) and 3.31 (95 % CI 0.89-12.34) in groups 2 and 3, respectively. Adjustment for age, gender, BMI and meniscus did not change the estimates [OR 1.44 (95 % CI 0.34-6.16) and 3.92 (95 % CI 1-15.37)] in groups 2 and 3, respectively. Patients younger than 20 had a significantly higher average postoperative laximetry (2.4 ± 1.5 mm) compared to those aged 20 years and over (1.5 ± 1.5 mm) (p = 0.03), regardless of the diameter of the graft. CONCLUSION: The diameter of the graft between 8 and 10 mm does not affect the laximetric results of an ACL reconstruction. Therefore, there does not appear to be a benefit to harvesting and adding further tissue to increase the diameter of the graft above 10 mm. Patients younger than 20 represent a population at risk of graft elongation. In these patients at risk, postoperative management needs to be modified (delayed weight bearing, articulated splinting, slower rehabilitation) in the first months. LEVEL OF EVIDENCE: Retrospective case series, Level IV.

Supertertiary structure of the synaptic MAGuK scaffold proteins is conserved.

Scaffold proteins form a framework to organize signal transduction by binding multiple partners within a signaling pathway. This shapes the output of signal responses as well as providing specificity and localization. The Membrane Associated Guanylate Kinases (MAGuKs) are scaffold proteins at cellular junctions that localize cell surface receptors and link them to downstream signaling enzymes. Scaffold proteins often contain protein-binding domains that are connected in series by disordered linkers. The tertiary structure of the folded domains is well understood, but describing the dynamic inter-domain interactions (the superteritary structure) of such multidomain proteins remains a challenge to structural biology. We used 65 distance restraints from single-molecule fluorescence resonance energy transfer (smFRET) to describe the superteritary structure of the canonical MAGuK scaffold protein PSD-95. By combining multiple fluorescence techniques, the conformational dynamics of PSD-95 could be characterized across the biologically relevant timescales for protein domain motions. Relying only on a qualitative interpretation of FRET data, we were able to distinguish stable interdomain interactions from freely orienting domains. This revealed that the five domains in PSD-95 partitioned into two independent supramodules: PDZ1-PDZ2 and PDZ3-SH3-GuK. We used our smFRET data for hybrid structural refinement to model the PDZ3-SH3-GuK supramodule and include explicit dye simulations to provide complete characterization of potential uncertainties inherent to quantitative interpretation of FRET as distance. Comparative structural analysis of synaptic MAGuK homologues showed a conservation of this supertertiary structure. Our approach represents a general solution to describing the supertertiary structure of multidomain proteins.

A comparison of four tibial-fixation systems in hamstring-graft anterior ligament reconstruction.

The aim of this study was to evaluate at time-zero four tibial fixations on four major criteria: the elongation and cyclic stiffness of the hamstring graft construct under cyclic loading, the yield load and pullout stiffness under load at failure. Four fixation systems were tested: the Delta screw, the WasherLoc, the TightRope Reverse and the tape locking screw on 32 tibiae of adult pigs using 32 pairs of human semitendinosus and gracilis tendons. Two tests were performed: cyclic tests using loads at 70-220 N, to measure the elongation at the end of the cycles, followed by load-to-failure testing to measure the yield load and the cyclic stiffness. The mean elongation was 1.23 mm for the TLS, 3.81 mm for the Delta, 3.59 mm for the WasherLoc and 3.91 mm for the TightRope. The mean yield loads and SD were 1,015 ± 129 N for the TLS, 844 ± 394 N for the Delta, 511 ± 95 N for the WasherLoc and 567 ± 112 N for the TightRope. The results showed the significant superiority of TLS and Delta over WasherLoc and tibial TightRope in regard to yield load. The results showed the significant superiority of TLS over the other fixations in regard to slippage. The TLS system and the Delta screw provide a better quality of primary fixation to the tibia, but further in vitro studies are needed.

Modulating the intrinsic disorder in the cytoplasmic domain alters the biological activity of the N-methyl-D-aspartate-sensitive glutamate receptor.

The NMDA-sensitive glutamate receptor is a ligand-gated ion channel that mediates excitatory synaptic transmission in the nervous system. Extracellular zinc allosterically regulates the NMDA receptor by binding to the extracellular N-terminal domain, which inhibits channel gating. Phosphorylation of the intrinsically disordered intracellular C-terminal domain alleviates inhibition by extracellular zinc. The mechanism for this functional effect is largely unknown. Proline is a hallmark of intrinsic disorder, so we used proline mutagenesis to modulate disorder in the cytoplasmic domain. Proline depletion selectively uncoupled zinc inhibition with little effect on receptor biogenesis, surface trafficking, or ligand-activated gating. Proline depletion also reduced the affinity for a PDZ domain involved in synaptic trafficking and affected small molecule binding. To understand the origin of these phenomena, we used single molecule fluorescence and ensemble biophysical methods to characterize the structural effects of proline mutagenesis. Proline depletion did not eliminate intrinsic disorder, but the underlying conformational dynamics were changed. Thus, we altered the form of intrinsic disorder, which appears sufficient to affect the biological activity. These findings suggest that conformational dynamics within the intrinsically disordered cytoplasmic domain are important for the allosteric regulation of NMDA receptor gating.

Anterior cruciate ligament reconstruction in children with a quadrupled semitendinosus graft: preliminary results with minimum 2 years of follow-up.

BACKGROUND: The management of anterior cruciate ligament (ACL) tears in growing patients must balance activity modification with the risk of secondary (meniscal and cartilaginous) lesions, and surgical intervention, which could adversely affect skeletal growth. Many ACL reconstruction techniques have been developed or modified to decrease the risk of growth disturbance. We have not found any description of ACL reconstruction using a single hamstring, short graft implanted into intraepiphyseal, retroreamed sockets. Our hypothesis was that the technique that we used restored the knee stability and did not cause any growth disturbances. METHODS: We retrospectively studied 28 patients (20 boys, 8 girls) who presented with a unilateral ACL tear and open growth plates. We performed short graft ligament reconstruction with the semitendinosus folded into 4 strands around 2 polyethylene terephthalate tapes. The graft was implanted into sockets that were retroreamed in the femoral and tibial epiphysis and the tapes were fixed remotely by interference screws. After a minimum period of 2 years, we evaluated the comparative knee laxity, the radiographic limb morphology, the appearance of secondary lesions, and the functional outcomes using the Lysholm and Tegner scores. Comparative analyses were performed using the Student t test with subgroups depending on the type of fixation used. RESULTS: The mean age of the patients was 13 years (range, 9 to 15 y). The mean follow-up was 2.8 years (range, 2 to 5 y). The mean difference in laxity at 134 N was 0.3 mm, as determined using a GNRB arthrometer. No patients reported meniscal symptoms or degenerative changes. We found no angular deformity or leg length inequality. Two patients suffered a recurrent ACL tear. CONCLUSIONS: The preliminary results from this series are consistent with prior studies demonstrating that intraepiphyseal ACL reconstruction is a safe reliable alternative for the pediatric population. STUDY DESIGN: Case series; level of evidence 4.

Validation of a novel translumbar ultrasound technique for measuring renal dimensions in horses.

A reliable method for obtaining renal ultrasonographic measurements in the horse is important for diagnosis and monitoring of clinical renal disease. The aims of this prospective study were to develop and validate a novel translumbar ultrasound technique for measuring renal dimensions in horses. Six Thoroughbred or Thoroughbred part bred horses were recruited. All horses were scheduled for euthanasia due to reasons unrelated to the kidneys. Two observers recorded renal length, width, and depth; and dimensions of the cortex, medulla, pyramids, and pelvis for both kidneys in each horse using novel translumbar and conventional transabdominal ultrasound methods. The same measurements were recorded from post-mortem renal specimens. Both kidneys were consistently identified by both methods in the 15-17th intercostal spaces and paralumbar fossa. Using the translumbar technique, maximal dimensions were obtained for the left kidney in the 16th intercostal space (length 16.2 ± 2.0 cm, width 11.8 ± 0.5 cm, depth 6.4 ± 0.9 cm) and for the right kidney in the 15th intercostal space (length 16.1 ± 1.2 cm, width 13.4 ± 1.2 cm, depth 6.7 ± 0.7 cm). Renal dimensions obtained by transabdominal and translumbar projections did not differ (P > 0.05). Good correlations were found between overall renal dimensions and post-mortem measurements for both ultrasound techniques (r(2) > 0.8), but were better for the translumbar method (mean r(2) = 0.92 cf. 0.88). Good-to-excellent reliability was found for all translumbar ultrasound measurements except for the renal cortex. Reproducibility was better for the larger (overall length, width, and depth) than the smaller (cortex, medulla, and pyramids) structures. Findings indicated that translumbar ultrasonography is a valid method for measuring renal dimensions in horses.

Use of recombinant tissue plasminogen activator (rTPA) for treatment of fibrin in the anterior chamber of the horse.

BACKGROUND: Recombinant intracameral tissue plasminogen activator (rTPA) administration can aid clearance of fibrin from the anterior chamber. MATERIALS AND METHODS: In this retrospective multicentre case series, the effect of intracameral rTPA administration to treat fibrin in the anterior chamber resulting from trauma or inflammatory ocular disease was evaluated. Clinical data from 30 treatments in 29 horses were obtained from medical records from 2003 to 2022. Association between time from onset of clinical signs and time for rTPA treatment to effect was studied with regression analysis. RESULTS: Twenty-seven horses (93.1%) had no previous history of ophthalmic disease; one had an iridic cyst, and another had equine recurrent uveitis. The majority of cases were related to trauma (79.3%). Median time from the onset of clinical signs to treatment was 12 h (IQR = 4-48 h). rTPA (72% 20 µg; 24% 25 µg; 3.3% 40 µg) was administered once in all but one eye, which was treated twice. Resolution of fibrin was seen in 96.9% (29/30) of treatments. Fibrin accumulation recurred in one case but resolved 14 days after the second treatment. Complications were seen in four treatments (13.3%): moderate pain for 24 h, intracameral debris and mild intracameral haemorrhage in a horse that received 40 µg of tissue plasminogen activator. Recurrence of fibrin accumulation was absent in 96.7% of cases. Median time to effect was 20 min (IQR = 10-45 min). Time for rTPA treatment to effect was not associated with time from fibrin formation (R2 = 0.09; p = 0.11). CONCLUSION: Intracameral rTPA treatment can be considered at 20-25 µg in 0.1 mL solution to aid resolution of fibrin accumulation.

BEVA primary care clinical guidelines: Equine parasite control.

BACKGROUND: There is a lack of consensus on how best to balance our need to minimise the risk of parasite-associated disease in the individual horse, with the need to limit the use of anthelmintics in the population to preserve their efficacy through delaying further development of resistance. OBJECTIVES: To develop evidence-based guidelines utilising a modified GRADE framework. METHODS: A panel of veterinary scientists with relevant expertise and experience was convened. Relevant research questions were identified and developed with associated search terms being defined. Evidence in the veterinary literature was evaluated using the GRADE evidence-to-decision framework. Literature searches were performed utilising CAB abstracts and PubMed. Where there was insufficient evidence to answer the research question the panel developed practical guidance based on their collective knowledge and experience. RESULTS: Search results are presented, and recommendation or practical guidance were made in response to 37 clinically relevant questions relating to the use of anthelmintics in horses. MAIN LIMITATIONS: There was insufficient evidence to answer many of the questions with any degree of certainty and practical guidance frequently had to be based upon extrapolation of relevant information and the panel members' collective experience and opinions. CONCLUSIONS: Equine parasite control practices and current recommendations have a weak evidence base. These guidelines highlight changes in equine parasite control that should be considered to reduce the threat of parasite-associated disease and delay the development of further anthelmintic resistance.

Reliability and identification of aortic valve prolapse in the horse.

BACKGROUND: The objectives were to determine and assess the reliability of criteria for identification of aortic valve prolapse (AVP) using echocardiography in the horse. RESULTS: Opinion of equine cardiologists indicated that a long-axis view of the aortic valve (AoV) was most commonly used for identification of AVP (46%; n=13). There was consensus that AVP could be mimicked by ultrasound probe malignment. This was confirmed in 7 healthy horses, where the appearance of AVP could be induced by malalignment. In a study of a further 8 healthy horses (5 with AVP) examined daily for 5 days, by two echocardiographers standardized imaging guidelines gave good to excellent agreement for the assessment of AVP (kappa>0.80) and good agreement between days and observers (kappa >0.6). The technique allowed for assessment of the degree of prolapse and measurement of the prolapse distance that provided excellent agreement between echocardiographers, days and observers (kappa/ICC>0.8). Assessments made using real-time zoomed images provided similar measurements to the standard views (ICC=0.9), with agreement for the identification of AVP (kappa>0.8). Short axis views of the AoV were used for identification of AVP by fewer respondents (23%), however provided less agreement for the identification of AVP (kappa>0.6) and only adequate agreement with observations made in long axis (kappa>0.5), with AVP being identified more often in short axis (92%) compared to long axis (76%). Orthogonal views were used by 31% of respondents to identify the presence of AVP, and 85% to identify cusp. Its identification on both views on 4 days was used to categorise horses as having AVP, providing a positive predictive value of 79% and negative predictive value of 18%. Only the non-coronary cusp (NCC) of the AoV was observed to prolapse in these studies. Prolapse of the NCC was confirmed during the optimisation study using four-dimensional echocardiography, which concurred with the findings of two-dimensional echocardiography. CONCLUSIONS: This study has demonstrated reliable diagnostic criteria for the identification and assessment of AVP that can be used for longitudinal research studies to better define the prevalence and natural history of this condition.

The Use of Biologics in NFL Athletes: An Expert Consensus of NFL Team Physicians.

Background: There is a lack of published information outlining the use of biologics in National Football League (NFL) athletes and limited data to guide biologic treatment strategies. Purpose: To develop a consensus on the use of biologics among NFL team physicians. Study Design: Consensus statement. Methods: A working group of 6 experts convened a consensus process involving NFL team physicians using validated Delphi methodology. Physicians from 32 NFL teams as well as NFL London were invited to take part. This iterative process was used to define statements on the use of biologics in NFL athletes. A recent scoping review exploring biologics in professional athletes was used to inform the first of 3 rounds of surveys, with statements considered under 7 headings: biologics in general, challenges of treating NFL athletes, terminology/nomenclature, autologous blood products, cell-based therapies, guidance for NFL team physicians, and biologic research in the NFL. In addition to rating agreement, experts were encouraged to propose further items or modifications. Predefined criteria were used to refine item lists after each survey. For a consensus within the final round, defined a priori, items were included in the final information set if a minimum of 75% of respondents agreed and fewer than 10% disagreed. Results: Physicians from 26 NFL teams and NFL London responded to the initial invitation to participate in the Delphi process; 88.9% of participating team physicians completed the round 1 survey, with response rates of 87.5% in round 2 and 95.2% in round 3. After 3 rounds, 47 statements reached a consensus. A consensus was achieved that platelet-rich plasma has a positive impact on patellar tendinopathy and on symptoms in early osteoarthritis but not for other indications. NFL team physicians agreed that while cell therapies have the potential to improve symptoms, the misrepresentation of uncharacterized preparations as "stem cells" has contributed to the widespread use of unproven therapies. Conclusion: This study established an expert consensus on 47 statements relating to the use of biologics in NFL athletes. In addition to providing clinical guidance for the use of biologics in NFL athletes, this study identified key areas for future focus including the development of athlete education materials.

Effect of Src kinase phosphorylation on disordered C-terminal domain of N-methyl-D-aspartic acid (NMDA) receptor subunit GluN2B protein.

NMDA receptors are ligand-gated ion channels with a regulatory intracellular C-terminal domain (CTD). In GluN2B, the CTD is the largest domain in the protein but is intrinsically disordered. The GluN2B subunit is the major tyrosine-phosphorylated protein in synapses. Src kinase phosphorylates the GluN2B CTD, but it is unknown how this affects channel activity. In disordered proteins, phosphorylation can tip the balance between order and disorder. Transitions can occur in both directions, so it is not currently possible to predict the effects of phosphorylation. We used single molecule fluorescence to characterize the effects of Src phosphorylation on GluN2B. Scanning fluorescent labeling sites throughout the domain showed no positional dependence of the energy transfer. Instead, efficiency only scaled with the separation between labeling sites suggestive of a relatively featureless conformational energy landscape. Src phosphorylation led to a general expansion of the polypeptide, which would result in greater exposure of known protein-binding sites and increase the physical separation between contiguous sites. Phosphorylation makes the CTD more like a random coil leaving open the question of how Src exerts its effects on the NMDA receptor.

Interaction of the M4 segment with other transmembrane segments is required for surface expression of mammalian α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors.

Ionotropic glutamate receptors (GluRs) are ligand-gated ion channels with a modular structure. The ion channel itself shares structural similarity, albeit an inverted membrane topology, with P-loop channels. Like P-loop channels, prokaryotic GluR subunits (e.g. GluR0) have two transmembrane segments. In contrast, eukaryotic GluRs have an additional transmembrane segment (M4), located C-terminal to the ion channel core. However, the structural/functional significance of this additional transmembrane segment is poorly defined. Although topologically similar to GluR0, mammalian AMPA receptor (GluA1) subunits lacking the M4 segment do not display surface expression. This lack of expression is not due to the M4 segment serving as an anchor to the ligand-binding domain because insertion of an artificial polyleucine transmembrane segment does not rescue surface expression. Specific interactions between M4 and the ligand-binding domain are also unlikely because insertion of polyglycines into the linker connecting them has no deleterious effects on function or surface expression. However, tryptophan and cysteine scanning mutagenesis of the M4 segment, as well as recovery of function in the polyleucine background, defined a unique face of the M4 helix that is required for GluR surface expression. In the AMPA receptor structure, this face forms intersubunit contacts with the transmembrane helices of the ion channel core (M1 and M3) from another subunit within the homotetramer. Thus, our experiments show that a highly specific interaction of the M4 segment with an adjacent subunit is required for surface expression of AMPA receptors. This interaction may represent a mechanism for regulating AMPA receptor biogenesis.

An objective study into the effects of an incline on naturally occurring lameness in horses.

BACKGROUND: The clinical examination of lame horses in real world settings often requires the use of sloped surfaces. OBJECTIVES: This pilot study aimed to evaluate the effects of uphill and downhill locomotion on asymmetry in horses with naturally occurring lameness affecting forelimbs and hindlimbs. METHODS: Ten horses (8-19 years) with forelimb lameness and eight horses (7-16 years) with hindlimb lameness were fitted with inertial sensors at the poll, withers, sacrum and both tuber coxae. Data were collected whilst the horses were trotted in hand on a level surface (<0.7%), as well as up and down a minor slope of 2.4%. Data were collected for a minimum of 25 strides at each incline type. Effect of incline was compared using a repeated measures ANOVA and, where significant, a subsequent Bonferroni's multiple comparisons. RESULTS: Of the horses with hindlimb lameness, there were reductions in asymmetry seen during downhill locomotion when compared with trotting on the flat (flat: 6.6 ± 4.4 mm to downhill: 1.9 ± 2.9 mm; p = 0.015) and when compared with uphill locomotion (8.4 ± 4.3 mm; p = 0.007). Horses with forelimb lameness showed no significant difference in asymmetry. However, there were considerable changes in poll asymmetry (>20 mm) among conditions in individual horses. Two horses with hindlimb lameness and two horses with forelimb lameness switched asymmetry between left and right by changing incline. CONCLUSIONS: These results confirm that incline can be an influential factor in the assessment of lame horses. Further work is justified to elucidate the types of pathology associated with the most relevant changes in asymmetry which would allow the use of an incline to prioritise a list of differential diagnoses.

Different Forms of Disorder in NMDA-Sensitive Glutamate Receptor Cytoplasmic Domains Are Associated with Differences in Condensate Formation.

The N-methyl-D-aspartate (NMDA)-sensitive glutamate receptor (NMDAR) helps assemble downstream signaling pathways through protein interactions within the postsynaptic density (PSD), which are mediated by its intracellular C-terminal domain (CTD). The most abundant NMDAR subunits in the brain are GluN2A and GluN2B, which are associated with a developmental switch in NMDAR composition. Previously, we used single molecule fluorescence resonance energy transfer (smFRET) to show that the GluN2B CTD contained an intrinsically disordered region with slow, hop-like conformational dynamics. The CTD from GluN2B also undergoes liquid-liquid phase separation (LLPS) with synaptic proteins. Here, we extend these observations to the GluN2A CTD. Sequence analysis showed that both subunits contain a form of intrinsic disorder classified as weak polyampholytes. However, only GluN2B contained matched patterning of arginine and aromatic residues, which are linked to LLPS. To examine the conformational distribution, we used discrete molecular dynamics (DMD), which revealed that GluN2A favors extended disordered states containing secondary structures while GluN2B favors disordered globular states. In contrast to GluN2B, smFRET measurements found that GluN2A lacked slow conformational dynamics. Thus, simulation and experiments found differences in the form of disorder. To understand how this affects protein interactions, we compared the ability of these two NMDAR isoforms to undergo LLPS. We found that GluN2B readily formed condensates with PSD-95 and SynGAP, while GluN2A failed to support LLPS and instead showed a propensity for colloidal aggregation. That GluN2A fails to support this same condensate formation suggests a developmental switch in LLPS propensity.

Conformational change of Syntaxin-3b in regulating SNARE complex assembly in the ribbon synapses.

Neurotransmitter release of synaptic vesicles relies on the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consisting of syntaxin and SNAP-25 on the plasma membrane and synaptobrevin on the synaptic vesicle. The formation of the SNARE complex progressively zippers towards the membranes, which drives membrane fusion between the plasma membrane and the synaptic vesicle. However, the underlying molecular mechanism of SNARE complex regulation is unclear. In this study, we investigated the syntaxin-3b isoform found in the retinal ribbon synapses using single-molecule fluorescence resonance energy transfer (smFRET) to monitor the conformational changes of syntaxin-3b that modulate the SNARE complex formation. We found that syntaxin-3b is predominantly in a self-inhibiting closed conformation, inefficiently forming the ternary SNARE complex. Conversely, a phosphomimetic mutation (T14E) at the N-terminal region of syntaxin-3b promoted the open conformation, similar to the constitutively open form of syntaxin LE mutant. When syntaxin-3b is bound to Munc18-1, SNARE complex formation is almost completely blocked. Surprisingly, the T14E mutation of syntaxin-3b partially abolishes Munc18-1 regulation, acting as a conformational switch to trigger SNARE complex assembly. Thus, we suggest a model where the conformational change of syntaxin-3b induced by phosphorylation initiates the release of neurotransmitters in the ribbon synapses.