James A. Flanders

Low-energy control of electrical turbulence in the heart

Controlling the complex spatio-temporal dynamics underlying life-threatening cardiac arrhythmias such as fibrillation is extremely difficult, because of the nonlinear interaction of excitation waves in a heterogeneous anatomical substrate. In the absence of a better strategy, strong, globally resetting electrical shocks remain the only reliable treatment for cardiac fibrillation. Here we establish the relationship between the response of the tissue to an electric field and the spatial distribution of heterogeneities in the scale-free coronary vascular structure. We show that in response to a pulsed electric field, E, these heterogeneities serve as nucleation sites for the generation of intramural electrical waves with a source density ρ(E) and a characteristic time, τ, for tissue depolarization that obeys the power law τ ∝ Eα. These intramural wave sources permit targeting of electrical turbulence near the cores of the vortices of electrical activity that drive complex fibrillatory dynamics. We show in vitro that simultaneous and direct access to multiple vortex cores results in rapid synchronization of cardiac tissue and therefore, efficient termination of fibrillation. Using this control strategy, we demonstrate low-energy termination of fibrillation in vivo. Our results give new insights into the mechanisms and dynamics underlying the control of spatio-temporal chaos in heterogeneous excitable media and provide new research perspectives towards alternative, life-saving low-energy defibrillation techniques.

The Cbl proteins are binding partners for the Cool/Pix family of p21-activated kinase-binding proteins

Members of the Cool protein family contain SH3, Dbl, and pleckstrin homology domains and are binding partners for the p21‐activated kinase (PAK). Using the yeast two‐hybrid screen, we identified Cbl‐b as a Cool family binding partner. We co‐immunoprecipitated endogenous Cool and Cbl‐b from a variety of breast cancer cell lines. The Cool–Cbl‐b interaction requires the SH3 domain of Cool and competes with the binding of PAK to Cool proteins. Expression of Cbl‐b effectively blocks the ability of Cool‐2 to stimulate PAK, thus providing an additional mechanism, aside from catalyzing receptor ubiquitination, by which Cbl‐b acts as a negative regulator for signaling activities requiring PAK activation.

Multiphoton microscopy and microspectroscopy for diagnostics of inflammatory and neoplastic lung

Limitations of current medical procedures for detecting early lung cancers inspire the need for new diagnostic imaging modalities for the direct microscopic visualization of lung nodules. Multiphoton microscopy (MPM) provides for subcellular resolution imaging of intrinsic fluorescence from unprocessed tissue with minimal optical attenuation and photodamage. We demonstrate that MPM detects morphological and spectral features of lung tissue and differentiates between normal, inflammatory and neoplastic lung. Ex vivo MPM imaging of intrinsic two-photon excited fluorescence was performed on mouse and canine neoplastic, inflammatory and tumor-free lung sites. Results showed that MPM detected microanatomical differences between tumor-free and neoplastic lung tissue similar to standard histopathology but without the need for tissue processing. Furthermore, inflammatory sites displayed a distinct red-shifted fluorescence compared to neoplasms in both mouse and canine lung, and adenocarcinomas displayed a less pronounced fluorescence emission in the 500 to 550 nm region compared to adenomas in mouse models of lung cancer. These spectral distinctions were also confirmed by two-photon excited fluorescence microspectroscopy. We demonstrate the feasibility of applying MPM imaging of intrinsic fluorescence for the differentiation of lung neoplasms, inflammatory and tumor-free lung, which motivates the application of multiphoton endoscopy for the in situ imaging of lung nodules.

Measurement of serum anti-Müllerian hormone concentration in female dogs and cats before and after ovariohysterectomy:

Anti-Müllerian hormone (AMH), or Müllerian inhibitory substance, is a hormone that is best known for its production by fetal testes in mammals and as the inhibitor of Müllerian (paramesonephric) duct development in males. However, following the development of the Müllerian ducts into the oviduct, uterus, and upper vagina in female mammals, the ovaries produce AMH, which can be found in measureable amounts within the peripheral circulation, especially in adults. The ovaries appear to be the sole source of AMH in the circulation; therefore, it may be a useful marker in clinically relevant situations when an assessment of the presence or absence of ovaries or ovarian remnants in dogs and cats is important. To that end, a commercially available, human-based assay was evaluated for the measurement of AMH in dogs and cats. A preliminary assessment involved a single test on a set of serum samples from dogs that were submitted to a diagnostic endocrinology laboratory for other tests. Favorable preliminary results led to a more formal assessment of the assay using serum samples from dogs and cats with the presence or absence of the ovaries known by surgical confirmation. Overall, a single measurement of serum AMH concentration was highly effective at distinguishing ovariohysterectomized from intact adult animals. In addition, the assay also accurately identified several cases of ovarian remnant syndrome.

Identification of a p53-Dependent Pathway in the Induction of Apoptosis of Human Breast Cancer Cells by the Natural Product, Resveratrol

OBJECTIVE Resveratrol, a constituent found in grapes and various other plants, has been shown to have chemo-preventive activity against cancer, and specifically demonstrated to induce apoptosis by p53-dependent pathways in murine cells. The goal of this research was to identify the role of p53-dependent or p53-independent pathways in the induction of apoptosis in human breast cancer cells by this natural product. DESIGN A number of human breast cancer cell lines, as well as a control of a wild-type line (astrocytoma N 1321N1), were investigated for induction of apoptosis by resveratrol using both microscopic evaluation and DNA fragmentation assays. Concurrently, we established the p53 gene status (wild-type or mutant) of each cell line by Western blot using p53-specific antibody. RESULTS Apoptosis induced by resveratrol was found to occur only in breast cancer cells expressing wild-type p53 but not in mutant p53-expressing cells. CONCLUSIONS We therefore conclude that the natural product, resveratrol, induces apoptosis in breast cancer cells via p53-dependent pathways.

Use of transesophageal echocardiography for visualization of the patent ductus arteriosus during transcatheter coil embolization.

Transesophageal echocardiography (TEE) enhances our ability to see the patent ductus arteriosus in the dog. The improved visualization may potentially improve our ability to perform transcatheter coil embolization in patients that are more likely to have a successful outcome. This report uses still and video images to detail the specifics of coil embolization as performed with the assistance of TEE and compares the images with those of angiography, surgery and postmortem examination.

Live Births from Domestic Dog (Canis familiaris) Embryos Produced by In Vitro Fertilization

Development of assisted reproductive technologies (ART) in the dog has resisted progress for decades, due to their unique reproductive physiology. This lack of progress is remarkable given the critical role ART could play in conserving endangered canid species or eradicating heritable disease through gene-editing technologies—an approach that would also advance the dog as a biomedical model. Over 350 heritable disorders/traits in dogs are homologous with human conditions, almost twice the number of any other species. Here we report the first live births from in vitro fertilized embryos in the dog. Adding to the practical significance, these embryos had also been cryopreserved. Changes in handling of both gametes enabled this progress. The medium previously used to capacitate sperm excluded magnesium because it delayed spontaneous acrosome exocytosis. We found that magnesium significantly enhanced sperm hyperactivation and ability to undergo physiologically-induced acrosome exocytosis, two functions essential to fertilize an egg. Unlike other mammals, dogs ovulate a primary oocyte, which reaches metaphase II on Days 4–5 after the luteinizing hormone (LH) surge. We found that only on Day 6 are oocytes consistently able to be fertilized. In vitro fertilization of Day 6 oocytes with sperm capacitated in medium supplemented with magnesium resulted in high rates of embryo development (78.8%, n = 146). Intra-oviductal transfer of nineteen cryopreserved, in vitro fertilization (IVF)-derived embryos resulted in seven live, healthy puppies. Development of IVF enables modern genetic approaches to be applied more efficiently in dogs, and for gamete rescue to conserve endangered canid species.

Surgical Therapy of the Thyroid

The surgical treatment of thyroid cancer in the cat involves excision of one or both thyroid glands while sparing at least one parathyroid gland. The most common postoperative complication of bilateral thyroidectomy is transient hypocalcemia. Most feline thyroid tumors are benign, hyperfunctional thyroid adenomas. Surgical excision is usually curative. Canine thyroid cancer is most commonly malignant and nonfunctional. Successful surgical treatment of canine thyroid cancer depends on histologic and early diagnosis prior to metastasis or invasion of the cancer into adjacent structures within the neck.

Spirocyclic lignans from Guaiacum (Zygophyllaceae) induce apoptosis in human breast cancer cell lines.

We investigated the composition of extracts derived from Guaiacum spp. (Zygophyllaceae), a group of neotropical tree species with varied uses in Central and South American traditional medicine. Activity-guided fractionation of Guaiacum heartwood extracts led to the identification of four new spirocyclic lignans, named ramonanins A-D (1-4). The ramonanins exhibit cytotoxic activity against human breast cancer cell lines with an IC50 value of 18 μM and induce cell death via apoptotic mechanisms. The ramonanins are derived from four units of coniferyl alcohol and feature an unusual spirocyclic ring system.

Cranial vena caval syndrome secondary to central venous obstruction associated with a pacemaker lead in a dog.

This report describes the case of an 11-year-old castrated male Shih Tzu who developed chylothorax three years following implantation of a transvenous pacemaker. Imaging demonstrated one definitive obstruction in the cranial vena cava and 3 additional suspected filling abnormalities within both external jugular veins, brachiocephalic veins and cranial vena cava. A thrombus was visualized in the cranial vena cava via transesophageal echocardiography. Thoracic duct ligation and cisterna chyli ablation were performed, with a resultant change in the nature of the fluid from chylous to a modified transudate. Repeat angiography and computed tomography three months later demonstrated a stenosis within the cranial vena cava. Balloon angioplasty was attempted, however it was unsuccessful in decreasing pleural effusion. Palliative thoracocentesis was continued until the patient developed a lung lobe torsion, at which time euthanasia was elected. Necropsy confirmed cranial vena caval syndrome secondary to transvenous pacemaker implantation induced fibrous proliferation within the vessel lumen.