Twenty-four hour continuous transvenous temporary right ventricular pacing in healthy horses.

BACKGROUND: The ability to perform transvenous temporary cardiac pacing (TV-TP) is critical to stabilize horses with symptomatic bradyarrhythmias. Reports of successful TV-TP in horses are limited, and only briefly describe short-term pacing. OBJECTIVE: To describe temporary, medium-term (24 h) transvenous right ventricular pacing in awake horses using a bipolar torque-directed pacing catheter. ANIMALS: Six healthy adult institutional teaching horses. METHODS: Prospective experimental study with 2 immediately successive TV-TP lead placements in each horse with a target location of the RV apex. One placement was performed primarily with echocardiographic guidance and 1 primarily with fluoroscopic guidance. In all placements, corresponding images were obtained with both imaging modalities. Horses were then paced for 24 h, unrestricted in a stall with continuous telemetric ECG monitoring. Echocardiographically determined lead position, episodes of pacing failure in the preceding 6 h, and pacing thresholds were recorded every 6 h. Pacing failure was defined as a period of loss of capture longer than 20 s. RESULTS: Pacing leads were placed with both guidance methods and maintained for 24 h with no complications. Two horses with leads angled caudally in the right ventricular apex had no pacing failure, the remaining 4 horses had varying degrees of loss of capture. Leads located in the right ventricular apex had longer time to pacing failure and lower capture thresholds P < 0.05. CONCLUSIONS AND CLINICAL IMPORTANCE: Medium-term TV-TP is feasible and has potential for stabilization of horses with symptomatic bradyarrhythmias. Lead position in the right ventricular apex appears optimal. Continuous ECG monitoring is recommended to detect pacing failure.

R-Spondin 3 Regulates Mammalian Dental and Craniofacial Development.

Development of the teeth requires complex signaling interactions between the mesenchyme and the epithelium mediated by multiple pathways. For example, canonical WNT signaling is essential to many aspects of odontogenesis, and inhibiting this pathway blocks tooth development at an early stage. R-spondins (RSPOs) are secreted proteins, and they mostly augment WNT signaling. Although RSPOs have been shown to play important roles in the development of many organs, their role in tooth development is unclear. A previous study reported that mutating Rspo2 in mice led to supernumerary lower molars, while teeth forming at the normal positions showed no significant anomalies. Because multiple Rspo genes are expressed in the orofacial region, it is possible that the relatively mild phenotype of Rspo2 mutants is due to functional compensation by other RSPO proteins. We found that inactivating Rspo3 in the craniofacial mesenchyme caused the loss of lower incisors, which did not progress beyond the bud stage. A simultaneous deletion of Rspo2 and Rspo3 caused severe disruption of craniofacial development from early stages, which was accompanied with impaired development of all teeth. Together, these results indicate that Rspo3 is an important regulator of mammalian dental and craniofacial development.

Pocket-Sized Ultrasound Versus Traditional Ultrasound Images in Equine Imaging: A Pictorial Essay.

This pictorial essay aims to display the image quality of pocket-sized ultrasound devices and hospital-based equipment to provide clinicians visual information about the potential uses of point-of-care ultrasonography (POCUS) in equine practice. Twenty-two paired images were obtained using traditional ultrasound equipment and pocket-sized ultrasound devices from patients evaluated at veterinary teaching hospitals. Images of many common ultrasound windows and miscellaneous sonographic abnormalities were obtained using pocket-sized ultrasound equipment.

Anti-osteogenic function of a LIM-homeodomain transcription factor LMX1B is essential to early patterning of the calvaria.

The calvaria (upper part of the skull) is made of plates of bone and fibrous joints (sutures and fontanelles), and the proper balance and organization of these components are crucial to normal development of the calvaria. In a mouse embryo, the calvaria develops from a layer of head mesenchyme that surrounds the brain from shortly after mid-gestation. The mesenchyme just above the eye (supra-orbital mesenchyme, SOM) generates ossification centers for the bones, which then grow toward the apex gradually. In contrast, the mesenchyme apical to SOM (early migrating mesenchyme, EMM), including the area at the vertex, does not generate an ossification center. As a result, the dorsal midline of the head is occupied by sutures and fontanelles at birth. To date, the molecular basis for this regional difference in developmental programs is unknown. The current study provides vital insights into the genetic regulation of calvarial patterning. First, we showed that osteogenic signals were active in both EMM and SOM during normal development, which suggested the presence of an anti-osteogenic factor in EMM to counter the effect of these signals. Subsequently, we identified Lmx1b as an anti-osteogenic gene that was expressed in EMM but not in SOM. Furthermore, head mesenchyme-specific deletion of Lmx1b resulted in heterotopic ossification from EMM at the vertex, and craniosynostosis affecting multiple sutures. Conversely, forced expression of Lmx1b in SOM was sufficient to inhibit osteogenic specification. Therefore, we conclude that Lmx1b plays a key role as an anti-osteogenic factor in patterning the head mesenchyme into areas with different osteogenic competence. In turn, this patterning event is crucial to generating the proper organization of the bones and soft tissue joints of the calvaria.

Engineering a switch-based biosensor for arginine using a Thermotoga maritima periplasmic binding protein.

The Thermotoga maritima arginine-binding protein (TmArgBP) has been modified to create a reagentless fluorescent protein biosensor. Two design methods for biosensor construction are compared: 1) solvent accessibility of environmentally-sensitive probes and 2) fluorescence deactivation due to photo-induced electron transfer (PET). Nine single cysteine TmArgBP mutants were created and labeled with three different environmentally sensitive fluorescent probes. These mutants demonstrated limited changes in fluorescence emission upon the addition of arginine. In contrast, the PET-based biosensor provides significant enhancements over the traditional approach and provides a fluorescence quenching mechanism that was capable of providing quantitative detection of arginine. Site-directed mutagenesis of TmArgBP was used to create attachment points for the fluorescent probe (K145C) and for an internal aromatic residue (D18X) to serve as the PET quencher. Both tyrosine and tryptophan, but not phenylalanine, were able to quench the emission of the fluorescent probe by more than 80% upon the addition of arginine. The dissociation constant for arginine ranged from 0.87 to 1.5 µM across the different sensors. This PET-based strategy provides a simple and broadly applicable approach for the analytical detection of small molecules that may be applied to any protein that exhibits conformational switching in a ligand dependent manner.

Lhx6 and Lhx8 promote palate development through negative regulation of a cell cycle inhibitor gene, p57Kip2.

Cleft palate is a common birth defect in humans. Therefore, understanding the molecular genetics of palate development is important from both scientific and medical perspectives. Lhx6 and Lhx8 encode LIM homeodomain transcription factors, and inactivation of both genes in mice resulted in profound craniofacial defects including cleft secondary palate. The initial outgrowth of the palate was severely impaired in the mutant embryos, due to decreased cell proliferation. Through genome-wide transcriptional profiling, we discovered that p57(Kip2) (Cdkn1c), encoding a cell cycle inhibitor, was up-regulated in the prospective palate of Lhx6(-/-);Lhx8(-/-) mutants. p57(Kip2) has been linked to Beckwith-Wiedemann syndrome and IMAGe syndrome in humans, which are developmental disorders with increased incidents of palate defects among the patients. To determine the molecular mechanism underlying the regulation of p57(Kip2) by the Lhx genes, we combined chromatin immunoprecipitation, in silico search for transcription factor-binding motifs, and in vitro reporter assays with putative cis-regulatory elements. The results of these experiments indicated that LHX6 and LHX8 regulated p57(Kip2) via both direct and indirect mechanisms, with the latter mediated by Forkhead box (FOX) family transcription factors. Together, our findings uncovered a novel connection between the initiation of palate development and a cell cycle inhibitor via LHX. We propose a model in which Lhx6 and Lhx8 negatively regulate p57(Kip2) expression in the prospective palate area to allow adequate levels of cell proliferation and thereby promote normal palate development. This is the first report elucidating a molecular genetic pathway downstream of Lhx in palate development.

Tryptophan-scanning mutagenesis of the ligand binding pocket in Thermotoga maritima arginine-binding protein.

The Thermotoga maritima arginine binding protein (TmArgBP) is a member of the periplasmic binding protein superfamily. As a highly thermostable protein, TmArgBP has been investigated for the potential to serve as a protein scaffold for the development of fluorescent protein biosensors. To establish a relationship between structural dynamics and ligand binding capabilities, we constructed single tryptophan mutants to probe the arginine binding pocket. Trp residues placed around the binding pocket reveal a strong dependence on fluorescence emission of the protein with arginine for all but one of the mutants. Using these data, we calculated dissociation constants of 1.9-3.3 µM for arginine. Stern-Volmer quenching analysis demonstrated that the protein undergoes a large conformational change upon ligand binding, which is a common feature of this protein superfamily. While still active at room temperature, time-resolved intensity and anisotropy decay data suggest that the protein exists as a highly rigid structure under these conditions. Interestingly, TmArgBP exists as a dimer at room temperature in both the presence and absence of arginine, as determined by asymmetric flow field flow fractionation (AF4) and supported by native gel-electrophoresis and time-resolved anisotropy. Our data on dynamics and stability will contribute to our understanding of hyperthermophilic proteins and their potential biotechnological applications.