Bone morphogenic proteins (BMPs) and their inhibitors are increasingly being appreciated for their role in vascular development. Matrix Gla protein (MGP) and crosssveinless 2 (CV2) antagonize BMP-4 and BMP-9, respectively, and the expression of both are stimulated by time-delayed BMP-9/ALK1 signaling, while ALK1 is stimulated by delayed BMP-4 signaling. This double negative feedback system is an important regulator of endothelial cell differentiation, angiogenesis, and maturation. Our collaborators recently discovered oscillatory gene expression of these proteins in human pulmonary artery endothelial cells (HPAECs) undergoing maturation in cell culture. We set out to explore the biological mechanisms guiding this periodic gene expression and to propose a functional role for these oscillations.

We used delay differential equations to mathematically model this network and applied dimensionless analysis to study the impact of relative protein degradation rates on oscillations. Monte Carlo parameter sampling and Hopf bifurcation analysis, while adjusting 1 and 2 parameters, helped identify conditions yielding periodic dynamics. We discovered that the presence of the activator, BMP-4, is critical for oscillations and that its inhibitor, MGP, opposes them, making their mutual regulation important for oscillatory gene expression. We also identified other mechanisms for oscillations including the necessary existence of three time-delays, the presence of BMP-9 as a feedback regulator, and the function of CV2 as an indirect inhibitor of MGP. Findings from the cell culture experiments and the mathematical model led us to postulate that oscillations in this network are important for the maintenance of progenitor HPAECs and the lack of oscillations is associated with HPAEC maturation.

Two recently discovered pictograph sites have similarities indicating common origin to the "Coso" style petroglyphs identified by Grant, Baird and Pringle (1968). These sites were located during field reconnaissance in the Southern Sierra Nevada. The two sites appear unique for two reasons: (1) they date to the late prehistoric and historic periods which previously have not been noted as containing "Coso" style rock art; and (2) "Coso" style pictographs are exceedingly rare and are unknown for these periods.

Ischemic damage from a myocardial infarction can have dramatic negative effects on the heart's ability to adequately perfuse the body, due to the irreversible loss of over a billion cardiomyocytes responsible for ventricular contraction. Regenerative therapies utilizing multipotent cardiac progenitor cells (CPCs) derived from either human embryonic (hESCs) or induced pluripotent stem cells have gained popularity as potential alternatives to someday replace expensive, high-risk medical procedures. Previous studies identified multipotent progenitors localized in the endocardial cushions of the developing mouse heart that co-expressed CD31 and PDGFRα. Additionally, recent data in mice have shown that the loss of a single transcription factor, Scl, enabled robust cardiomyogenic differentiation from endothelium in the heart, resulting in the ectopic appearance of these progenitor cells. However, no studies have shown similar findings in humans.

The purpose of this study is to identify these CD31+/PDGFRα+ cells in the human heart and generate cardiomyocyte-like cells from hESC-derived endothelium. It is hypothesized that human endogenous and embryonic stem cell-derived endothelial cells possess unrealized cardiomyogenic potential. Stained sagittal sections of week 9, 15, and 17 human fetal hearts showed the co-expression of the cell surface markers PDGFRα and CD31 on cells surrounding the ventricular vasculature. In addition, a 5-step protocol was used to differentiate hESCs into cardiomyocyte-like cells through an endothelial cell intermediate. Yield of isolated mesodermal progenitor cells was approximately 10.5%. Under the modified sorting protocol, yield of the mesodermal progenitor cells was approximately 18.6%. The subsequent cell sort yield for CD31+ endothelial cells was approximately 4.8%. Under the modified protocol with and without the addition of lithium chloride, endothelial cell yields were 21.3% and 26.6% respectively. The hESC-derived endothelial cells showed the formation of blood tubes in vitro and, once in cardiac growth medium, expressed Troponin T under fluorescence microscopy. Based on these results, this population of CD31+/PDGFRα+ cells could represent a latent population of cardiac progenitors capable of differentiating into cardiomyocytes.

In a recent issue, Backes (2004) presents an analysis of two pictographs sites in Kern County, California, using ultraviolet fluorescence photography. Two sites, CA-KER-735 and CA-KER-736, were thoroughly studied. Backes' innovative approach is a useful tool in pictograph site research. It has resulted in the identification of new elements that were previously invisible to the 'naked eye' or impossible to document through conventional photographic means. I applaud Backe's efforts. My comments here are aimed at clarifying several minor yet significant contextual, classificatory, and interpretive matters, rather than in disagreeing with his technical methods or results per se.

The ventricle exhibits gradients in repolarization time in both transmural and apicobasal directions. These gradients, caused by the varying strengths of cellular ionic currents in the different regions, affect the morphology of the electrocardiographic T wave. Abnormal gradients, observed clinically by alterations in the ECG, can increase susceptibility to arrhythmias, and therefore the characteristics of both gradients as well as their roles in manifesting the T wave must be understood. The UCLA rabbit ventricular action potential model is based on experimental data from rabbit ventricular myocytes along with reported ion channel properties. To recreate the repolarization gradients in the ventricle, alterations were made to the conduction of repolarizing currents, IKr and Ito,f in the single cell model to simulate the different APDs observed in-situ. The resulting cells were then incorporated into a 3D ventricle model. Simulations were performed with varying presence and direction of repolarization gradients, and an in-silico ECG was calculated. It was found that transmurally, APD epi must be shorter than APD endo and apicobasally, APD base must be shorter than APD apex to give a correct ECG.

Negative feedback loops are well known in physiology. Previous work at UCLA has iden-

tied a negative feedback pathway involving Bone Morphogenetic Protein 4 (BMP-4) and

an inhibitor of BMP-4 called Matrix Gla Protein (MGP) active in vascular development

and pathology.[28] More recently, other inhibitors of BMP-4 have been identied in vascular

tissue, including Crossveinless 2 (CV-2). In addition, experimental works performed by our

collaborators at Dr. Bostrom's lab have identied oscillations in the concentrations of these

morphogens gene expressions.

We developed a mathematical model of the interactions among the new, larger set of

morphogens. In particular, there are at least 2 negative feedback loops, one acting with a

short time delay and the other acting with a longer time delay. In fact, there are 3 distinct

time delays in our mathematical model. The goal was to understand what conditions would give rise to sustained oscillations in this model. With a view to comparing these results to experimental observations in the lab of Dr. Kristina Bostrom at UCLA, we have attempted to draw general conclusions about the behavior of multiple loop feedback systems, a problem of general interest.

Recently obtained radiocarbon analyses of the charred parts of pinyon pine cones and seeds from the Sherwin Grade site. Mono County, California, (CA-Mno-584) are of significance to the controversy concerning the proposition that regular use of pinyon resources initiated in the Owens Valley region between A.D. 600 and A.D. 1000 (Bettinger 1976, 1977a, 19776; McGuire and Garfinkel 1976; Garfinkel and Cook 1979).