Faculty Bibliography

Caspofungin is used for the treatment of acute invasive candidiasis and as salvage treatment for invasive aspergillosis. We report characteristics of isolates of Candida albicans and Aspergillus fumigatus detected in a patient with breakthrough infection complicating severe gastrointestinal surgery and evaluate the capability of susceptibility methods to identify candin resistance. The susceptibility of C. albicans to caspofungin and anidulafungin was investigated by Etest, microdilution (European Committee on Antibiotic Susceptibility Testing [EUCAST] and CLSI), disk diffusion, agar dilution, and FKS1 sequencing and in a mouse model. Tissue was examined by immunohistochemistry, PCR, and sequencing for the presence of A. fumigatus and resistance mutations. The MICs for the C. albicans isolate were as follows: >32 microg/ml caspofungin and 0.5 microg/ml anidulafungin by Etest, 2 microg/ml caspofungin and 0.125 microg/ml anidulafungin by EUCAST methods, and 1 microg/ml caspofungin and 0.5 microg/ml anidulafungin by CLSI methods. Sequencing of the FKS1 gene revealed a mutation leading to an S645P substitution. Caspofungin and anidulafungin failed to reduce kidney CFU counts in animals inoculated with this isolate (P > 0.05 compared to untreated control animals), while both candins completely sterilized the kidneys in animals infected with a control isolate. Disk diffusion and agar dilution methods clearly separated the two isolates. Immunohistochemistry and sequencing confirmed the presence of A. fumigatus without FSK1 resistance mutations in liver and lung tissues. Breakthrough disseminated aspergillosis and candidiasis developed despite an absence of characteristic FKS1 resistance mutations in the Aspergillus isolates. EUCAST and CLSI methodology did not separate the candin-resistant clinical isolate from the sensitive control isolate as well as did the Etest and agar methods.

When asked to relate my experience of anatomy to the first-year medical and physician associate students at Yale before the start of their own first dissection, I found no better words to share than those of my classmates. Why speak with only one tongue, I said, when you can draw on 99 others? Anatomical dissection elicits what our course director, Lawrence Rizzolo, has called a "diversity of experience," which, in turn, engenders a diversity of expressions. For Yale medical and physician associate students, this diversity is captured each year in a ceremony dedicated to those who donated their bodies for dissection. The service is an opportunity to offer thanks, but because only students and faculty are in attendance, it is also a place to share and address the complicated tensions that arise while examining, invading, and ultimately disassembling another's body. It is our pleasure to present selected pieces from the ceremony to the Yale Journal of Biology and Medicine readership.

Atrial natriuretic peptide (ANP) plays a major role in blood pressure and volume regulation. ANP activities are mediated by a cell surface, single-span transmembrane receptor linked to its intrinsic guanylate cyclase activity. The crystal structures of the dimerized ANP receptor extracellular domain (ECD) with and without ANP have revealed a novel hormone-induced rotation mechanism occurring in the juxtamembrane region that appears to mediate signal transduction [Ogawa H, Qiu Y, Ogata CM & Misono KS (2004) J Biol Chem 279, 28625-28631]. However, the ECD crystal packing contains two major intermolecular contacts that suggest two possible dimer pairs: 'head-to-head' (hh) and 'tail-to-tail' (tt) dimers associated via the membrane-distal and membrane-proximal subdomains, respectively. The existence of these two potential dimer forms challenges the proposed signaling mechanism. In this study, we performed single-particle electron microscopy (EM) to determine the ECD dimer structures occurring in the absence of crystal contacts. EM reconstruction yielded the dimer structures with and without ANP in only the hh dimer forms. We further performed steady-state fluorescence spectroscopy of Trp residues, one of which (Trp74) occurs in the hh dimer interface and none of which occurs in the tt dimer interface. ANP binding caused a time-dependent decrease in Trp emission at 350 nm that was attributable to partially buried Trp74 in the unbound hh dimer interface becoming exposed to solvent water upon ANP binding. Thus, the results of single-particle EM and Trp fluorescence studies have provided direct evidence for hh dimer structures for unbound and ANP-bound receptor. The results also support the proposed rotation mechanism for transmembrane signaling by the ANP receptor.

In comparative proteomic studies, it is important to know the variability associated with sample preparation. In this study, we report the strategy of using SILAC (stable isotope labeling by amino acids in cell culture) to evaluate the effect of the variation in sample preparation for quantitative proteomics. Variability can be measured when equal amounts of light and heavy SILAC samples undergo the same sample preparation procedures in parallel, and the two samples are mixed for relative protein quantitation by mass spectrometry. The high quantitative accuracy of SILAC allows for characterization of small variations. First, the reproducibility of immunoprecipitation (IP) and in-gel digestion was evaluated, and the impact of replicate number on quantitative accuracy was characterized. Second, we evaluated the overall variation in a comparative workflow involving three sequential sample preparation steps: IP, SDS-PAGE fractionation, and in-gel digestion. The evaluation of individual sample preparation steps was very valuable for experimental design: the optimal number of replicates for each step could be readily determined and the overall variation of the workflow could be predicted from the variation of the individual steps involved. By using informed experimental design, we demonstrated that the error associated with multiple steps of sample preparation in a comparative experiment can be limited to a reasonably low level

Atherosclerosis is the leading cause of death worldwide. To date, the use of statins to lower LDL levels has been the major intervention used to delay or halt disease progression. These drugs have an incomplete impact on plaque burden and risk, however, as evidenced by the substantial rates of myocardial infarctions that occur in large-scale clinical trials of statins. Thus, it is hoped that by understanding the factors that lead to plaque regression, better approaches to treating atherosclerosis may be developed. A transplantation-based mouse model of atherosclerosis regression has been developed by allowing plaques to form in a model of human atherosclerosis, the apoE-deficient mouse, and then placing these plaques into recipient mice with a normolipidemic plasma environment. Under these conditions, the depletion of foam cells occurs. Interestingly, the disappearance of foam cells was primarily due to migration in a CCR7-dependent manner to regional and systemic lymph nodes after 3 days in the normolipidemic (regression) environment. Further studies using this transplant model demonstrated that liver X receptor and HDL are other factors likely to be involved in plaque regression. In conclusion, through the use of this transplant model, the process of uncovering the pathways regulating atherosclerosis regression has begun, which will ultimately lead to the identification of new therapeutic targets

Classic tissue engineering paradigms are limited by the incorporation of a functional vasculature and a reliable means for reimplantation into the host circulation. We have developed a novel approach to overcome these obstacles using autologous explanted microcirculatory beds (EMBs) as bioscaffolds for engineering complex three-dimensional constructs. In this study, EMBs consisting of an afferent artery, capillary beds, efferent vein, and surrounding parenchymal tissue are explanted and maintained for 24 h ex vivo in a bioreactor that preserves EMB viability and function. Given the rapidly advancing field of stem cell biology, EMBs were subsequently seeded with three distinct stem cell populations, multipotent adult progenitor cells (MAPCs), and bone marrow and adipose tissue-derived mesenchymal stem cells (MSCs). We demonstrate MAPCs, as well as MSCs, are able to egress from the microcirculation into the parenchymal space, forming proliferative clusters. Likewise, human adipose tissue-derived MSCs were also found to egress from the vasculature and seed into the EMBs, suggesting feasibility of this technology for clinical applications. We further demonstrate that MSCs can be transfected to express a luciferase protein and continue to remain viable and maintain luciferase expression in vivo. By using the vascular network of EMBs, EMBs can be perfused ex vivo and seeded with stem cells, which can potentially be directed to differentiate into neo-organs or transfected to replace failing organs and deficient proteins

Today, surgical intervention remains the mainstay of treatment to intervene upon a multitude of skeletal deficits and defects attributable to congenital malformations, oncologic resection, pathologic degenerative bone destruction, and post-traumatic loss. Despite this significant demand, the tools with which surgeons remain equipped are plagued with a surfeit of inadequacies, often resulting in less than ideal patient outcomes. The failings of current techniques largely arise secondary to their inability to produce a regenerate which closely resembles lost tissue. As such, focus has shifted to the potential of mesenchymal stem cell (MSC)-based skeletal tissue engineering. The successful development of such techniques would represent a paradigm shift from current approaches, carrying with it the potential to regenerate tissues which mimic the form and function of endogenous bone. Lessons learned from investigations probing the endogenous regenerative capacity of skeletal tissues have provided direction to early studies investigating the osteogenic potential of MSC. Additionally, increasing attention is being turned to the role of targeted molecular manipulations in augmenting MSC osteogenesis, as well as the development of an ideal scaffold ''vehicle'' with which to deliver progenitor cells. The following discussion presents the authors' current working knowledge regarding these critical aspects of MSC application in cell-based skeletal tissue engineering strategies, as well as provides insight towards what future steps must be taken to make their clinical translation a reality.

Excessive transforming growth factor-beta (TGF-beta) signaling characterizes the progression of aortic aneurysm in mouse models of Marfan syndrome, a systemic disorder of the connective tissue that is caused by mutations in the gene encoding the extracellular matrix protein fibrillin-1. Fibrillin-1 mutations are believed to promote abnormal Smad2/3 signaling by impairing the sequestration of latent TGF-beta complexes into the extracellular matrix. Here we report that promiscuous Smad2/3 signaling is the cell-autonomous phenotype of primary cultures of vascular smooth muscle cells (VSMC) explanted from the thoracic aortas of Fbn1 mutant mice with either neonatal onset or progressively severe aortic aneurysm. This cellular phenotype was characterized in VSMC isolated from Fbn1-null (mgN/mgN) mice, which recapitulate the most severe form of Marfan syndrome. We found that loss of fibrillin-1 deposition promotes the production of intracellular reactive oxygen species and abnormal accumulation of phosphorylated TGF-beta-activated kinase 1 and p38 MAPK, in addition to increasing the levels of endogenous phospho-Smad2. We showed that improper Smad2/3 signaling in Fbn1-null VSMC is in part stimulated by phospho-p38 MAPK, which is in turn activated in response to signals other than those mediated by the kinase activity of the ALK5 receptor. Consistent with these cell culture data, in vivo analyses documented that phospho-p38 MAPK accumulates earlier than phospho-Smad2 in the aortic wall of mgN/mgN mice and that systemic inhibition of phospho-p38 MAPK activity lowers the levels of phospho-Smad2 in this tissue. Collectively, these findings indicate that improper activation of p38 MAPK is a precursor of constitutive Smad2/3 signaling in the aortic wall of a mouse model of neonatal lethal Marfan syndrome