α-Actinin-1 promotes activity of the L-type Ca2+ channel Cav 1.2.

The L-type Ca2+ channel CaV 1.2 governs gene expression, cardiac contraction, and neuronal activity. Binding of α-actinin to the IQ motif of CaV 1.2 supports its surface localization and postsynaptic targeting in neurons. We report a bi-functional mechanism that restricts CaV 1.2 activity to its target sites. We solved separate NMR structures of the IQ motif (residues 1,646-1,664) bound to α-actinin-1 and to apo-calmodulin (apoCaM). The CaV 1.2 K1647A and Y1649A mutations, which impair α-actinin-1 but not apoCaM binding, but not the F1658A and K1662E mutations, which impair apoCaM but not α-actinin-1 binding, decreased single-channel open probability, gating charge movement, and its coupling to channel opening. Thus, α-actinin recruits CaV 1.2 to defined surface regions and simultaneously boosts its open probability so that CaV 1.2 is mostly active when appropriately localized.

Dual Basicity and Nucleophilicity of Organosodium Reagents in Benzylic C-H Additions of Toluenes to Diarylethenes and Ketones.

Profiting from the dual high basicity and nucleophilicity of organosodium complexes, here we report the stepwise lateral metalation of a wide range of alkyl arenes (MeAr), mediated by hydrocarbon-soluble NaCH2SiMe3 ⋅ PMDETA (PMDETA=N,N,N',N'',N''-pentamethyldiethylenetriamine), followed by nucleophilic addition to diarylethenes of the newly generated NaCH2Ar ⋅ PMDETA complexes. This method grants access to a range of functionalised hydrocarbons in excellent yields and can be upgraded to catalytic regimes when using trans-stilbene, a 10 mol% of the alkyl sodium base and toluene as a solvent. Extending this approach to aromatic ketones leads to the formation of stilbenes under mild reaction conditions, resulting from the deprotonative coupling of toluenes with ketones. Combining spectroscopic studies with the trapping and characterisation of key reaction intermediates, mechanistic insights have been gained, advancing the understanding of coordination effects in organosodium chemistry, and shedding light on their special reactivity profiles.

Enhancing prosthesis stability at the cricoid cartilage in equine laryngoplasty using 3-D-printed laryngeal clamps: An ex vivo model study.

OBJECTIVE: To assess a three-dimensional (3-D)-printed laryngeal clamp (LC) designed to enhance the anchoring of laryngeal prostheses at the cricoid cartilage. STUDY DESIGN: Ex vivo biomechanical study. SAMPLE POPULATION: A total of 22 equine larynges. METHODS: Two experimental groups included larynges with standard prosthetic laryngoplasty (PL; n = 10) and larynges with prosthetic laryngoplasty modified with laryngeal clamps (PLLC; n = 10). All constructs underwent 3000 cycles of tension loading and a single tension to failure. Recorded biomechanical parameters included maximum load, actuator displacement, and construct failure. Finite element analysis (FEA) was performed on one PL and one PLLC construct. RESULTS: The maximum load at single tension to failure was 183.7 ± 46.8 N for the PL construct and 292.7 ± 82.3 N for the PLLC construct (p = .003). Actuator displacement at 30 N was 1.7 ± 0.5 mm and 2.7 ± 0.7 mm for the PL and PLLC constructs, respectively (p = .011). The cause of PL constructs failure was mostly tearing through the cartilage whereas the PLLC constructs failed through fracture of the cricoid cartilage (p = .000). FEA revealed an 11-fold reduction in the maximum equivalent plastic strain, a four-fold reduction in maximum compressive stress, and a two-fold increase in the volume of engaged cartilage in PLLC constructs. CONCLUSION: The PLLC constructs demonstrated superior performance in biomechanical testing and FEA compared to standard PL constructs. CLINICAL SIGNIFICANCE: The use of 3-D-printed laryngeal clamps may enhance the outcomes of laryngoplasty in horses. In vivo studies are necessary to determine the feasibility of performing laryngoplasty using the laryngeal clamp in horses.

New Frontiers in Organosodium Chemistry as Sustainable Alternatives to Organolithium Reagents.

With their highly reactive respective C-Na and N-Na bonds, organosodium and sodium amide reagents could be viewed as obvious replacements or even superior reagents to the popular, widely utilised organolithiums. However, they have seen very limited applications in synthesis due mainly to poor solubility in common solvents and their limited stability. That notwithstanding in recent years there has been a surge of interest in bringing these sustainable metal reagents into the forefront of organometallics in synthesis. Showcasing the growth in utilisation of organosodium complexes within several areas of synthetic chemistry, this Minireview discusses promising new methods that have been recently reported with the goal of taming these powerful reagents. Special emphasis is placed on coordination and aggregation effects in these reagents which can impart profound changes in their solubility and reactivity. Differences in observed reactivity between more nucleophilic aryl and alkyl sodium reagents and the less nucleophilic but highly basic sodium amides are discussed along with current mechanistic understanding of their reactivities. Overall, this review aims to inspire growth in this exciting field of research to allow for the integration of organosodium complexes within common important synthetic transformations.

Half-calcified calmodulin promotes basal activity and inactivation of the L-type calcium channel CaV1.2.

The L-type Ca2+ channel CaV1.2 controls gene expression, cardiac contraction, and neuronal activity. Calmodulin (CaM) governs CaV1.2 open probability (Po) and Ca2+-dependent inactivation (CDI) but the mechanisms remain unclear. Here, we present electrophysiological data that identify a half Ca2+-saturated CaM species (Ca2/CaM) with Ca2+ bound solely at the third and fourth EF-hands (EF3 and EF4) under resting Ca2+ concentrations (50-100 nM) that constitutively preassociates with CaV1.2 to promote Po and CDI. We also present an NMR structure of a complex between the CaV1.2 IQ motif (residues 1644-1665) and Ca2/CaM12', a calmodulin mutant in which Ca2+ binding to EF1 and EF2 is completely disabled. We found that the CaM12' N-lobe does not interact with the IQ motif. The CaM12' C-lobe bound two Ca2+ ions and formed close contacts with IQ residues I1654 and Y1657. I1654A and Y1657D mutations impaired CaM binding, CDI, and Po, as did disabling Ca2+ binding to EF3 and EF4 in the CaM34 mutant when compared to WT CaM. Accordingly, a previously unappreciated Ca2/CaM species promotes CaV1.2 Po and CDI, identifying Ca2/CaM as an important mediator of Ca signaling.

Highly Reactive Hydrocarbon Soluble Alkylsodium Reagents for Benzylic Aroylation of Toluenes using Weinreb Amides.

Deaggregating the alkyl sodium NaCH2 SiMe3 with polydentate nitrogen ligands enables the preparation and characterisation of new, hydrocarbon soluble chelated alkylsodium reagents. Equipped with significantly enhanced metalating power over their organolithium counterparts, these systems can promote controlled sodiation of weakly acidic benzylic C-H bonds from a series of toluene derivatives under mild stoichiometric conditions. This has been demonstrated through the benzylic aroylation of toluenes with Weinreb amides, that delivers a wide range of 2-arylacetophenones in good to excellent yields. Success in isolating and determining the structures of key organometallic intermediates has provided useful mechanistic insight into these new sodium-mediated transformations.

A structural basis for lithium and substrate binding of an inositide phosphatase.

Inositol polyphosphate 1-phosphatase (INPP1) is a prototype member of metal-dependent/lithium-inhibited phosphomonoesterase protein family defined by a conserved three-dimensional core structure. Enzymes within this family function in distinct pathways including inositide signaling, gluconeogenesis, and sulfur assimilation. Using structural and biochemical studies, we report the effect of substrate and lithium on a network of metal binding sites within the catalytic center of INPP1. We find that lithium preferentially occupies a key site involved in metal-activation only when substrate or product is added. Mutation of a conserved residue that selectively coordinates the putative lithium-binding site results in a dramatic 100-fold reduction in the inhibitory constant as compared with wild-type. Furthermore, we report the INPP1/inositol 1,4-bisphosphate complex which illuminates key features of the enzyme active site. Our results provide insights into a structural basis for uncompetitive lithium inhibition and substrate recognition and define a sequence motif for metal binding within this family of regulatory phosphatases.

The Crystal Structure of Calmodulin Bound to the Cardiac Ryanodine Receptor (RyR2) at Residues Phe4246-Val4271 Reveals a Fifth Calcium Binding Site.

Calmodulin (CaM) regulates the activity of a Ca2+ channel known as the cardiac ryanodine receptor (RyR2), which facilitates the release of Ca2+ from the sarcoplasmic reticulum during excitation-contraction coupling in cardiomyocytes. Mutations that disrupt this CaM-dependent channel inactivation result in cardiac arrhythmias. RyR2 contains three different CaM binding sites: CaMBD1 (residues 1940-1965), CaMBD2 (residues 3580-3611), and CaMBD3 (residues 4246-4275). Here, we report a crystal structure of Ca2+-bound CaM bound to RyR2 CaMBD3. The structure reveals Ca2+ bound to the four EF-hands of CaM as well as a fifth Ca2+ bound to CaM in the interdomain linker region involving Asp81 and Glu85. The CaM mutant E85A abolishes the binding of the fifth Ca2+ and weakens the binding of CaMBD3 to Ca2+-bound CaM. Thus, the binding of the fifth Ca2+ is important for stabilizing the complex in solution and is not a crystalline artifact. The CaMBD3 peptide in the complex adopts an α-helix (between Phe4246 and Val4271) that interacts with both lobes of CaM. Hydrophobic residues in the CaMBD3 helix (Leu4255 and Leu4259) form intermolecular contacts with the CaM N-lobe, and the CaMBD3 mutations (L4255A and L4259A) each weaken the binding of CaM to RyR2. Aromatic residues on the opposite side of the CaMBD3 helix (Phe4246 and Tyr4250) interact with the CaM C-lobe, but the mutants (F4246A and Y4250A) have no detectable effect on CaM binding in solution. We suggest that the binding of CaM to CaMBD3 and the binding of a fifth Ca2+ to CaM may contribute to the regulation of RyR2 channel function.

Capnometric feedback training decreases 24-h blood pressure in hypertensive postmenopausal women.

BACKGROUND: High normal resting pCO2 is a risk factor for salt sensitivity of blood pressure (BP) in normotensive humans and has been associated with higher resting systolic BP in postmenopausal women. To date, however, no known studies have investigated the effects of regular practice of voluntary mild hypocapnic breathing on BP in hypertensive patients. The objective of the present research was to test the hypothesis that capnometric feedback training can decrease both resting pCO2 and 24-h BP in a series of mildly hypertensive postmenopausal women. METHODS: A small portable end tidal CO2 (etCO2) monitor was constructed and equipped with software that determined the difference between the momentary etCO2 and a pre-programmed criterion range. The monitor enabled auditory feedback for variations in CO2 outside the criterion range. 16 mildly hypertensive postmenopausal women were individually trained to sustain small decreases in etCO2 during six weekly sessions in the clinic and daily sessions at home. 24-h BP monitoring was conducted before and after the intervention, and in 16 prehypertensive postmenopausal women in a control group who did not engage in the capnometric training. RESULTS: Following the intervention, all 16 capnometric training participants showed decreases in resting etCO2 (- 4.3 ± 0.4 mmHg; p < .01) while 15 showed decreases in 24-h systolic BP (- 7.6 ± 2.0 mmHg; p < .01). No significant changes in either measure was observed in the control group. In addition, nighttime (- 9.5 ± 2.6; p < .01) and daytime (- 6.7 ± 0.2 mmHg) systolic BP were both decreased following capnometric training, while no significant changes in nighttime (- 2.8 ± 2.2 mmHg; p = .11) or daytime (- 0.7 ± 1.0 mmHg; p ≤ .247) systolic BP were observed in the control group. CONCLUSIONS: These findings support the hypothesis that regular practice of mild hypocapnic breathing that decreases resting etCO2 reliably decreases 24-h blood pressure in hypertensive postmenopausal women. The extent to which these effects persist beyond the training period or can be observed in other hypertensive subgroups remains to be investigated.

Influence of a novel scaffold composed of polyurethane, hydroxyapatite, and decellularized bone particles on the healing of fourth metacarpal defects in mares.

OBJECTIVE: To determine the effect of a novel scaffold, designed for use in bone regeneration, on healing of splint bone segmental defects in mares. STUDY DESIGN: In vivo experimental study. SAMPLE POPULATION: Five adult mares (4-10 years old; mean weight, 437.7 kg ± 29 kg). METHODS: Bilateral 2-cm full-thickness defects were created in the fourth metacarpal bones (MCIV) of each horse. Each defect was randomly assigned to either a novel scaffold treatment (n = 5) or an untreated control (n = 5). The scaffold was composed of polyurethane, hydroxyapatite, and decellularized bone particles. Bone healing was assessed for a period of 60 days by thermography, ultrasonography, radiography, and computed tomography (CT). Biopsies of each defect were performed 60 days after surgery for histological evaluation. RESULTS: On the basis of radiographic analysis, scaffold-treated defects had greater filling (67.42% ± 26.7%) compared with untreated defects (35.88% ± 32.7%; P = .006). After 60 days, CT revealed that the density of the defects treated with the scaffolds (807.80 ± 129.6 Hounsfield units [HU]) was greater than density of the untreated defects (464.80 ± 81.3 HU; P = .004). Evaluation of histology slides provided evidence of bone formation within an average of 9.43% ± 3.7% of the cross-sectional area of scaffolds in contrast to unfilled defects in which connective tissue was predominant throughout the biopsy specimens. CONCLUSION: The novel scaffold was biocompatible and supported bone formation within the MCIV segmental defects. CLINICAL SIGNIFICANCE: This novel scaffold offers an effective option for filling bone voids in horses when support of bone healing is indicated.

Glaucomatous visual fields and neurocognitive function are independently associated with poor lane maintenance during driving simulation.

BACKGROUND: Driving simulators are a safe alternative to on-road vehicles for studying driving behavior in glaucoma drivers. Visual field (VF) loss severity is associated with higher driving simulator crash risk, though mechanisms explaining this relationship remain unknown. Furthermore, associations between driving behavior and neurocognitive performance in glaucoma are unexplored. Here, we evaluated the hypothesis that VF loss severity and neurocognitive performance interact to influence simulated vehicle control in glaucoma drivers. METHODS: Glaucoma patients (n = 25) and suspects (n = 18) were recruited into the study. All had > 20/40 corrected visual acuity in each eye and were experienced field takers with at least three stable (reliability > 20%) fields over the last 2 years. Diagnosis of neurological disorder or cognitive impairment were exclusion criteria. Binocular VFs were derived from monocular Humphrey VFs to estimate a binocular VF index (OU-VFI). Montreal Cognitive Assessment (MoCA) was administered to assess global and sub-domain neurocognitive performance. National Eye Institute Visual Function Questionnaire (NEI-VFQ) was administered to assess peripheral vision and driving difficulties sub-scores. Driving performance was evaluated using a driving simulator with a 290° panoramic field of view constructed around a full-sized automotive cab. Vehicle control metrics, such as lateral acceleration variability and steering wheel variability, were calculated from vehicle sensor data while patients drove on a straight two-lane rural road. Linear mixed models were constructed to evaluate associations between driving performance and clinical characteristics. RESULTS: Patients were 9.5 years older than suspects (p = 0.015). OU-VFI in the glaucoma group ranged from 24 to 98% (85.6 ± 18.3; M ± SD). OU-VFI (p = .0066) was associated with MoCA total (p = .0066) and visuo-spatial and executive function sub-domain scores (p = .012). During driving simulation, patients showed greater steering wheel variability (p = 0.0001) and lateral acceleration variability (p < .0001) relative to suspects. Greater steering wheel variability was independently associated with OU-VFI (p = .0069), MoCA total scores (p = 0.028), and VFQ driving sub-scores (p = 0.0087), but not age (p = 0.61). CONCLUSIONS: Poor vehicle control was independently associated with greater VF loss and worse neurocognitive performance, suggesting both factors contribute to information processing models of driving performance in glaucoma. Future research must demonstrate the external validity of current findings to on-road performance in glaucoma.

Veterinary Intern Program for Entrustable Professional Activities Skills, a.k.a. Intern Boot Camp.

The University of Tennessee College of Veterinary Medicine (UTCVM) Department of Large Animal Clinical Sciences has developed an intensive training program that all large animal veterinary interns are required to complete at the onset of their internship year. This program was designed to establish a uniform approach to entrustable professional activity (EPA) skills deemed essential by the large animal faculty. These EPA skills emphasize the clinical approaches and skills that interns should understand and demonstrate competency in early in their internship year. The EPA program, completed over 4 consecutive days, was coined the "Intern Boot Camp" and structured to fuse case-based lecture discussions and hands-on wet labs designed to develop or improve skills and techniques. At the conclusion of the boot camp, participants were given an evaluation survey to provide feedback on the program. The results were overwhelmingly positive, with 90% of the participants giving the program a rating of 5 on a scale ranging from 1 (poor opinion or experience) to 5 (excellent opinion or experience). Nearly 95% of participants stated that they felt more prepared for their internship year after attending the boot camp, and 100% of the participants indicated that they would recommend this program to future candidates. Given the positive outcomes over the past 4 years, the implementation of the Veterinary Intern Entrustable Professional Activities program (Intern Boot Camp) is considered a valuable component of the veterinary intern training program and could readily be adapted to other programs.

Visual Pathway Degeneration in Chemotherapy-Related Neurotoxicity: A Review and Directions for Future Research.

Chemotherapy-related neurotoxicity (CRNT) is an emerging public health concern. Visual pathway degeneration may be a symptom of CRNT. We surveyed the current literature for evidence of visual pathway degeneration in cancer patients receiving chemotherapy. A systematic review was conducted in PubMed. Six published articles met our inclusion criteria. The studies showed reduced retinal thickness, primarily in the retinal nerve fibre layer, and impaired inner retinal function in patients receiving chemotherapy. In summary, the current literature suggests chemotherapy may induce visual pathway degeneration. Future research may benefit from improving study design, exploring mechanisms of chemotherapy-related visual pathway degeneration, and incorporating these findings into biomarker development.

Commercially available bone graft substitutes: the impact of origin and processing on graft functionality.

Development of effective and cost-efficient bone tissue engineering grafts has been the key area of research for regenerative medicine, yet an ideal grafting material has remained elusive due in large part to the highly dynamic nature of bone. A wide array of materials, both natural and synthetic, have been implemented as potential candidates for commercially available products, yet the gold standard for grafting material still remains autogenous bone. We review currently commercially available bone graft materials and relevant graft characteristics that impact the effectiveness of tissue repair, emphasizing the advantages and disadvantages of materials based on composition and origin. Examined materials were selected through a web-based search for readily accessible and clinically applicable graft materials. Grafts were then categorized according to material source to examine advantages and disadvantages associated with allogenic, xenogeneic, synthetic materials. Lastly, the application of bioactive molecules onto these basal grafts is explored to illustrate the enhancement and regulative capacity of these additives on traditional osteobiologic materials.

Biomechanical evaluation of peak reverse torque (PRT) in a dynamic compression plate-screw construct used in a goat tibia segmental defect model.

BACKGROUND: Peak reverse torque (PRT) is a valid method to evaluate implants' secondary stability in the healing bone. The secondary stability is achieved by the implant over time and it has been positively correlated with the implants' osseointegration level. In other words, peak reverse torque is the force required to break the bone-implant interface. The purpose of this study was to compare the peak reverse torque for the self-tapping and non-self-tapping screws used in a dynamic compression plate-screw-bone construct after 60 days of loading when used to stabilize 2.5-cm defects in the tibia of goats. The second objective was to compare the peak removal torque of the screws placed in the different positions to evaluate the impact of construct biomechanics on implants osseointegration. RESULTS: In total, 176 non-self-tapping screws and 66 self-tapping screws were used to fix the 8-holes dynamic compression plates to the bones. The screws were placed in the tibiae from proximal (position sites 1,2, 3) to distal (position sites 4,5,6) and were removed 60 days post-implantation. The animals remained weight-bearing throughout the study period. The screws placed in the proximal diaphysis had significantly less peak reverse torque than screws placed in the distal diaphysis in both groups (p < 0.05). The peak reverse torque resistance was also significantly less for the non-self-tapping screws as compared with the self-tapping screws (p < 0.05). The intracortical fractures in the trans-cortex occurred significantly more frequently during the placement of non-self-tapping screws (p < 0.05) as compared with self-tapping screws (p < 0.05). CONCLUSIONS: Based on these results, we concluded that self-tapping screws may be expected to maintain a more stable bone-implant interface during the first 60 days of loading as compared with non-self-tapping screws. This should be a consideration for orthopedic surgeons and scientists using bone plates to stabilize non-load sharing fractures when a stable plate-screw-bone interface is needed to ensure prolonged stability.

Electrophysiological Measure of Impaired Information Processing in Drivers with Hematological Malignancy.

The broad goal of this study is to measure remote effects of cancer on brain physiology and behaviors that underpin instrumental activities of daily living such as automobile driving. Studies of hematological malignancies (HM) have demonstrated impairments in multiple brain functions shown to be critical for safe automobile driving. In the current pilot study, brain physiology during driving simulation was examined in 14 HM patients and 13 healthy comparison drivers. Electroencephalography was used to measure the eye fixation-related potential (EFRP)-a positive amplitude deflection evoked approximately 100 milliseconds after eye movement termination. Previous studies have demonstrated sensitivity of EFRP activity to information-processing capacity. All drivers completed visual search tasks to evaluate the relationship between driving-related changes in performance and EFRP activity. Results showed smaller EFRP amplitudes in drivers who had: (1) greater driving-related changes in visual search performance (p = 0.03, Cohen's d = 0.91); and (2) HM diagnosis (p = 0.18, Cohen's d = 0.54). Extending previous studies, these results provide neural evidence of reduced information-processing capacity associated with cancer diagnosis. Future large-scale studies are needed to confirm these results, given the high level of uncertainty and small sample size. This study provides a novel platform for linking changes in brain physiology and safety-critical driving behaviors.

Vesicopreputial anastomosis for the treatment of obstructive urolithiasis in goats.

OBJECTIVE: To describe a novel surgical technique for the correction of recurrent obstructive urolithiasis in male goats. STUDY DESIGN: Clinical case series. ANIMALS: Castrated male goats (n = 4). METHODS: Medical records of male goats having undergone vesicopreputial anastomosis (VPA) as a treatment for obstructive urolithiasis were reviewed for history, signalment, clinical signs, and intraoperative and postoperative complications. Long-term follow-up (≥12 months) was obtained by telephone interview with owners or by clinical examination. RESULTS: All goats had undergone at least one surgical procedure (median, 2.5, range 2-4) to correct obstructive urolithiasis before undergoing VPA. Postoperative complications included premature removal of the tube from the bladder (1 goat), bacterial cystitis (2), and abscess formation (1). One goat suffered stricture of the anastomosis site 3 months following the original procedure and underwent a second VPA and 1 goat died 7 months after surgery due to severe, acute hydronephrosis and renal failure. Long-term survival ≥12 months was good with 3/4 goats (75%) or 3/5 VPA procedures (60%) having unobstructed urine flow at 12 months. CONCLUSION: Vesicopreputial anastomosis is a feasible surgical procedure for the correction of recurrent obstructive urolithiasis in male goats and one that can result in a favorable clinical outcome. Further investigation in a larger population of goats is warranted for the evaluation of the suitability of VPA in male goats with obstructive urolithiasis.

Induced α rhythms track the content and quality of visual working memory representations with high temporal precision.

Past work has suggested that neuronal oscillations coordinate the cellular assemblies that represent items in working memory (WM). In line with this hypothesis, we show that the spatial distribution of power in the alpha frequency band (8-12 Hz) can be used to decode the content and quality of the representations stored in visual WM. We acquired EEG data during an orientation WM task, and used a forward encoding model of orientation selectivity to reconstruct orientation-specific response profiles (termed channel tuning functions, or CTFs) that tracked the orientation of the memorandum during both encoding and delay periods of the trial. Critically, these EEG-based CTFs were robust predictors of both between- and within-subject differences in mnemonic precision, showing that EEG-based CTFs provide a sensitive measure of the quality of sensory population codes. Experiments 2 and 3 established that these EEG-based CTFs are contingent on the voluntary storage goals of the observer. When observers were given a postsample cue to store or drop the memorandum, the resulting CTF was sustained in the "store" condition and rapidly eliminated following the "drop" cue. When observers were instructed to store one of two simultaneously presented stimuli, only the stored item was represented in a sustained fashion throughout the delay period. These findings suggest that the oscillatory activity in the alpha frequency band plays a central role in the active storage of information in visual WM, and demonstrate a powerful approach for tracking the precision of on-line memories with high temporal resolution.