Retrovirus transduction: segregation of the viral transforming function and the herpes simplex virus tk gene in infectious Friend spleen focus-forming virus thymidine kinase vectors.

A series of deletions and insertions utilizing the herpesvirus thymidine kinase gene (tk) were constructed in the murine retrovirus Friend spleen focus-forming virus (SFFV). In all cases, the coding region for the SFFV-specific glycoprotein (gp55), which is implicated in erythroleukemic transformation, was left intact. These SFFV-TK and SFFV deletion vectors were analyzed for expression of tk and gp55 after DNA-mediated gene transfer. In addition, virus rescued by cotransfection of these vectors with Moloney murine leukemia virus was analyzed for infectious TK-transducing virus, gp55 expression, and erythroleukemia-inducing ability. The experiments demonstrated that deletions or insertions within the intron for the gp55 env gene can interfere with expression of gp55 after both DNA-mediated gene transfer and virus infection. In contrast, the gene transfer efficiency of the tk gene was unaffected in the SFFV-TK vectors, and high-titer infectious TK virus could be recovered. Revertant viruses capable of inducing erythroleukemia and expressing gp55 were generated after cotransfection of the SFFV-TK vectors with murine leukemia virus. The revertant viruses lost both tk sequences and the ability to transduce TK- fibroblasts to a TK+ phenotype. These experiments demonstrate that segregation of the TK and erythroleukemia functions can occur in retrovirus vectors which initially carry both markers.     

Electrical interactions between a real ventricular cell and an anisotropic two-dimensional sheet of model cells

We have extended our "coupling clamp" technique, in which we couple a real cell to a real-time simulation of a model cell, to now incorporate a real cardiac cell as the central element of a two-dimensional sheet of model cells, in which the coupling conductances may be different in the x and y directions and a specific region of lack of coupling conductance may serve as a resistive barrier. We stimulated the real cell in the central location and determined the critical size of the real cell for successful activation of the entire sheet. We found that this critical size was decreased when anisotropy was present compared with the isotropic case and was further decreased when the central site of stimulation was close to the resistive barrier. The heart normally has some degree of anisotropy, and it has been shown that the remodeling that occurs in peri-infarction zones produces a particular loss of lateral connections compared with end-to-end connections among heart cells. We propose that the normal existence of anisotropy and enhancement of the degree of anisotropy both by loss of lateral gap junctions and the development of resistive barriers may play a facilitating role in the development of ectopic foci that may lead to cardiac arrhythmias.     

Avian coronavirus in wild aquatic birds

We detected a high prevalence (12.5%) of novel avian coronaviruses in aquatic wild birds. Phylogenetic analyses of these coronaviruses suggest that there is a diversity of gammacoronaviruses and deltacoronaviruses circulating in birds. Gammacoronaviruses were found predominantly in Anseriformes birds, whereas deltacoronaviruses could be detected in Ciconiiformes, Pelecaniformes, and Anseriformes birds in this study. We observed that there are frequent interspecies transmissions of gammacoronaviruses between duck species. In contrast, deltacoronaviruses may have more stringent host specificities. Our analysis of these avian viral and host mitochondrial DNA sequences also suggests that some, but not all, coronaviruses may have coevolved with birds from the same order.     

A two-phase Bayesian methodology for the analysis of binary phenotypes in genome-wide association studies

Recent advances in sequencing and genotyping technologies are contributing to a data revolution in genome-wide association studies that is characterized by the challenging large p small n problem in statistics. That is, given these advances, many such studies now consider evaluating an extremely large number of genetic markers (p) genotyped on a small number of subjects (n). Given the dimension of the data, a joint analysis of the markers is often fraught with many challenges, while a marginal analysis is not sufficient. To overcome these obstacles, herein, we propose a Bayesian two-phase methodology that can be used to jointly relate genetic markers to binary traits while controlling for confounding. The first phase of our approach makes use of a marginal scan to identify a reduced set of candidate markers that are then evaluated jointly via a hierarchical model in the second phase. Final marker selection is accomplished through identifying a sparse estimator via a novel and computationally efficient maximum a posteriori estimation technique. We evaluate the performance of the proposed approach through extensive numerical studies, and consider a genome-wide application involving colorectal cancer.     

Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor

In many organ systems such as the skin, gastrointestinal tract and hematopoietic system, homeostasis is dependent on the continuous generation of differentiated progeny from stem cells. The rodent incisor, unlike human teeth, grows throughout the life of the animal and provides a prime example of an organ that rapidly deteriorates if newly differentiated cells cease to form from adult stem cells. Hedgehog (Hh) signaling has been proposed to regulate self-renewal, survival, proliferation and/or differentiation of stem cells in several systems, but to date there is little evidence supporting a role for Hh signaling in adult stem cells. We used in vivo genetic lineage tracing to identify Hh-responsive stem cells in the mouse incisor and we show that sonic hedgehog (SHH), which is produced by the differentiating progeny of the stem cells, signals to several regions of the incisor. Using a hedgehog pathway inhibitor (HPI), we demonstrate that Hh signaling is not required for stem cell survival but is essential for the generation of ameloblasts, one of the major differentiated cell types in the tooth, from the stem cells. These results therefore reveal the existence of a positive-feedback loop in which differentiating progeny produce the signal that in turn allows them to be generated from stem cells.     

Sympathetic withdrawal and forearm vasodilation during vasovagal syncope in humans

Dietz, Niki M., John R. Halliwill, John M. Spielmann, LoriA. Lawler, Bettina G. Papouchado, Tamara J. Eickhoff, and Michael J. Joyner. Sympathetic withdrawal and forearm vasodilation duringvasovagal syncope in humans. J. Appl.Physiol. 82(6): 1785-1793, 1997.Our aim was todetermine whether sympathetic withdrawal alone can account for theprofound forearm vasodilation that occurs during syncope in humans. Wealso determined whether either vasodilating ₂-adrenergic receptors ornitric oxide (NO) contributes to this dilation. Forearm blood flow wasmeasured bilaterally in healthy volunteers(n = 10) by using plethysmographyduring two bouts of graded lower body negative pressure (LBNP) tosyncope. In one forearm, drugs were infused via a brachial arterycatheter while the other forearm served as a control. In the controlarm, forearm vascular resistance (FVR) increased from 77 ± 7 unitsat baseline to 191 ± 36 units with 40 mmHg of LBNP(P < 0.05). Mean arterial pressurefell from 94 ± 2 to 47 ± 4 mmHg just before syncope, and allsubjects demonstrated sudden bradycardia at the time of syncope. At theonset of syncope, there was sudden vasodilation and FVR fell to 26 ± 6 units (P < 0.05 vs. baseline). When the experimental forearm was treated withbretylium, phentolamine, and propranolol, baseline FVR fell to 26 ± 2 units, the vasoconstriction during LBNP was absent, and FVR fellfurther to 16 ± 1 units at syncope(P < 0.05 vs. baseline). During thesecond trial of LBNP, mean arterial pressure again fell to 47 ± 4 mmHg and bradycardia was again observed. Treatment of the experimentalforearm with the NO synthase inhibitorN^G-monomethyl-L-arginine in additionto bretylium, phentolamine, and propranolol significantly increasedbaseline FVR to 65 ± 5 units but did not prevent the marked forearmvasodilation during syncope (FVR = 24 ± 4 vs. 29 ± 8 units inthe control forearm). These data suggest that the profound vasodilationobserved in the human forearm during syncope is not mediated solely bysympathetic withdrawal and also suggest that neither₂-adrenergic-receptor-mediated vasodilation nor NO is essential to observe this response.