Victoria Scranton

Annexin I is an endogenous ligand that mediates apoptotic cell engulfment

Engulfment of apoptotic cells requires presentation of new cell surface ligands by the dying cells. Using a differential proteomics technology, we identify that annexin I is a caspase-dependent engulfment ligand; it is recruited from the cytosol and exported to the outer plasma membrane leaflet, colocalizes with phosphatidylserine, and is required for efficient clearance of apoptotic cells. Furthermore, phosphatidylserine receptor (PSR) clustering around apoptotic cells indicates a requirement for annexin I. In the nematode Caenorhabditis elegans, downregulation of the annexin homolog prevents efficient engulfment of pharyngeal cell corpses. These results provide novel mechanistic insights into how apoptotic cells are removed and may explain a pathogenic mechanism of chronic inflammatory diseases where annexin I autoantibodies have been described.

The Type I Membrane Protein EFF-1 Is Essential for Developmental Cell Fusion

Multinucleate cells are widespread in nature, yet the mechanism by which cells fuse their plasma membranes is poorly understood. To identify animal fusogens, we performed new screens for mutations that abolish cell fusion within tissues of C. elegans throughout development. We identified the gene eff-1, which is expressed as cells acquire fusion competence and encodes a novel integral membrane protein. EFF-1 sequence motifs suggest physicochemical actions that could cause adjacent bilayers to fuse. Mutations in the extracellular domain of EFF-1 completely block epithelial cell membrane fusion without affecting other perfusion events such as cell generation, patterning, differentiation, and adhesion. Thus, EFF-1 is a key component in the mechanism of cell fusion, a process essential to normal animal development.

Measurement of muscle disease by quantitative second-harmonic generation imaging.

Determining the health of muscle cells by in vivo imaging could impact the diagnosis and monitoring of a large number of congenital and acquired muscular or cardiac disorders. However, currently used technologies are hampered by insufficient resolution, lack of specificity, or invasiveness. We have combined intrinsic optical second-harmonic generation from sarcomeric myosin with a novel mathematical treatment of striation pattern analysis, to obtain measures of muscle contractile integrity that correlate strongly with the neuromuscular health of mice suffering from genetic, acquired, and age-related decline in skeletal muscle function. Analysis of biopsies from a pilot group of human volunteers suggests a similar power in quantifying sarcopenic changes in muscle integrity. These results provide the first strong evidence that quantitative image analysis of sarcomere pattern can be correlated with physiological function, and they invite the application of SHG imaging in clinical practice, either in biopsy samples or via microendoscopy.

Differential effects of aging and sex on stroke induced inflammation across the lifespan

Aging and biological sex are critical determinants of stroke outcome. Post-ischemic inflammatory response strongly contributes to the extent of ischemic brain injury, but how this response changes with age and sex is unknown. We subjected young (5-6 months), middle aged (14-15 months) and aged (20-22 months), C57BL/6 male and female mice to transient middle cerebral artery occlusion (MCAO) and found that a significant age by sex interaction influenced histological stroke outcomes. Acute functional outcomes were worse with aging. Neutrophils, inflammatory macrophages, macrophages, dendritic cells (DCs) and microglia significantly increased in the brain post MCAO. Cycling females had higher Gr1(-) non-inflammatory macrophages and lower T cells in the brain after stroke and these correlated with serum estradiol levels. Estrogen loss in acyclic aged female mice exacerbated stroke induced splenic contraction. Advanced age increased T cells, DCs and microglia at the site of injury, which may be responsible for the exacerbated behavioral deficits in the aged. We conclude that aging and sex have differential effects on the post stroke inflammatory milieu. Putative immunomodulatory therapies need to account for this heterogeneity.

Fusogenic Activity of EFF-1 Is Regulated via Dynamic Localization in Fusing Somatic Cells of C. elegans

BACKGROUND Many animal tissues form via fusion of cells. Yet in all instances of developmental cell fusion, the mechanism underlying fusion of plasma membranes remains poorly understood. EFF-1 is required for most somatic cell fusions in C. elegans, and misexpressed EFF-1 alters the normal pattern of fusing hypodermal cells. However, the autonomous activity of EFF-1, the rules governing its specificity, and the mechanism of its action have not been examined. RESULTS We show that EFF-1 acts as a cellular fusogen, capable of inducing fusion of virtually any somatic cells in C. elegans, yet targeted precisely to fusion-fated contacts during normal development. Misexpression of EFF-1 in early embryos causes fusion among groups of cells composed entirely of nonfusion-fated members. Measurements of cytoplasm diffusion in induced fusion events show that ectopic EFF-1 expression produces fusion pores similar to those in normal fusion events. GFP-labeled EFF-1 is specifically targeted to fusion-competent cell contacts via reciprocal localization to the touching membranes of EFF-1-expressing cells. EFF-1 function is also governed by intercellular barriers that prohibit cell fusion between distinct tissues. Analysis of mutant versions of EFF-1 indicates a novel mode of fusogenicity, employing neither a phospholipase active site nor hydrophobic fusion-peptide acting solely in pore formation. CONCLUSIONS EFF-1 can confer potent fusogenic activity to nonfusing cell types. However, it is normally targeted only to fusion-fated cell borders via mutual interaction between EFF-1-expressing cells and relocalization to the plasma membrane. Because EFF-1 appears evolutionarily unique to nematodes, multiple mechanisms may have evolved for controlled plasma-membrane fusion in development.

ABILITY OF FGFS TO PROMOTE THE OUTGROWTH AND PROLIFERATION OF LIMB MESODERM IS DEPENDENT ON IGF-I ACTIVITY

IGF‐I and members of the FGF family have been implicated in the reciprocal interactions between the apical ectodermal ridge (AER) and underlying subridge mesoderm that are required for outgrowth of the developing limb bud. Several FGFs are expressed by the AER and appear to mediate its outgrowth and patterning effects on the subridge mesoderm. IGF‐I is expressed by the subridge mesoderm that is growing out in response to the AER, and exogenous IGF‐I can promote the outgrowth of the subridge mesoderm in the absence of the AER. Maintenance of IGF‐I expression by the subridge mesoderm is dependent on the AER. Here we report that exogenous FGF‐2 and FGF‐4 can substitute for the AER in maintaining IGF‐I expression by the posterior subridge mesoderm of the chick limb bud. Furthermore, we have found that neutralizing antibodies against IGF‐I inhibit the ability of FGFs to promote the outgrowth and proliferation of the posterior subridge mesoderm cultured in the absence of the AER. These results indicate that FGFs secreted by the AER maintain IGF‐I expression by the subridge mesoderm, and that the ability of FGFs to promote the outgrowth and proliferation of limb mesoderm is dependent on IGF‐I activity. Interestingly, however, the ability of FGFs to maintain the expression of the homeobox containing gene Msx‐1 by the subridge mesoderm is not dependent on IGF‐I activity.

Construction of a laser scanning microscope for multiphoton excited optical fabrication

We present the construction and performance of a simple, programable, laser scanning nonlinear optical microscope for three-dimensional (3D) submicron fabrication. While the primary purpose of the microscope is for optical fabrication this instrument can also perform routine nonlinear optical imaging. The instrument consists of a commercial femtosecond laser system, an upright optical microscope, programable x–y scanning galvo mirrors, axial focus motor with an optical encoder, and single photon counting detection electronics. The mirror scanning and data acquisition are synchronous and are controlled by a single multipurpose I/O board and a graphical user interface written in a LabVIEW framework. The scanner is configured to scan a variety of geometrical patterns with user—controllable speeds, sizes, and aspect ratios, in either filled-in or perimeter patterns. Examples of 3D structures include channel-type structures for potential microfluidic applications and open boxes for encapsulation of cells for t...

The relationship between follicle development and progesterone receptor membrane component-1 expression in women undergoing in vitro fertilization

OBJECTIVE To determine the relationship between progesterone receptor membrane component-1 (PGRMC1) expression and the outcome of IVF treatment. DESIGN A prospective study in which PGRMC1 messenger RNA (mRNA) levels, methylation status of the Pgrmc1 promoter, and the presence of point mutations within Pgrmc1 were obtained from granulosa (GC)/luteal cells of women undergoing controlled ovarian hyperstimulation (COH). SETTING Fertility center/basic science laboratory. PATIENT(S) Eighty-five patients undergoing IVF treatment and 10 women who were undergoing COH for the purpose of oocyte donation were included in this study. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) The PGRMC1 measurements were correlated with clinical outcomes, such as number of follicles, number of retrieved oocytes, and ongoing pregnancy rates (PR). RESULT(S) The PGRMC1 mRNA levels within GC/luteal cells of 18% of IVF patients were >2.25-fold higher than those of oocyte donors. Individuals with elevated PGRMC1 mRNA levels had 30% fewer large follicles and fewer oocytes retrieved. The elevated PGRMC1 mRNA levels were associated with an increase in the methylation of Pgrmc1 promoter. CONCLUSION(S) In patients with elevated PGRMC1 mRNA levels, gonadotropin-induced follicle development is attenuated, although sufficient numbers of follicles develop to allow for ET and subsequent pregnancy.

Quantitative Assays for Cell Fusion

Cell fusion would seem to be obviously recognizable upon visual inspection, and many studies employ a simple microscopic fusion index to quantify the rate and extent of fusion in cell culture. However, when cells are not in monolayers or when there is a large background of multinucleation through failed cytokinesis, cell-cell fusion can only be proven by mixing of cell contents. Furthermore, determination of the microscopic fusion index must generally be carried out manually, creating opportunities for unintended observer bias and limiting the numbers of cells assayed and therefore the statistical power of the assay. Strategies for making assays dependent on fusion and independent of visual observation are critical to increasing the accuracy and throughput of screens for molecules that control cell fusion. A variety of in vitro biochemical and nonbiochemical techniques have been developed to assay and monitor fusion events in cultured cells. In this chapter, we briefly discuss several in vitro fusion assays, nearly all based on systems of two components that interact to create a novel assayable signal only after cells fuse. We provide details for the use of one example of such a system, intracistronic complementation of beta-galactosidase activity by mutants of Escherichia coli lacZ, which allows for either cell-by-cell microscopic assay of cell fusion or quantitative and kinetic detection of cell fusions in whole populations. In addition, we describe a combination of gene knock-down protocols with this assay to study factors required for myoblast fusion.

Implementation of a Computerized Algorithmic Support Tool for Identifying Depression and Anxiety at the Pediatric Well-Child Visit

The high prevalence of unaddressed mental health problems among children and adolescents has received a considerable amount of public attention in recent years with a growing demand for a greater role for pediatric primary care in detecting and managing mental health problems. Indeed, the pediatric primary care setting possesses a number of assets that make it well poised to take on an expanded role. More than 90% of children across all economic and ethnic populations use pediatric primary care services providing a unique opportunity to universally detect mental health problems early and connect children to mental health services. Also, the long-term nature of pediatricians’ relationships with families is conducive to continued mental health surveillance and monitoring across the course of child development. Despite these strengths, evidence indicates that pediatricians are not systematically screening for mental health problems, and when they do, tend to rely on suboptimal procedures that fail to utilize best-practice, evidence-based screening, and assessment strategies. These shortcomings stem from a number of systemic and practice-level barriers including insufficient training, practical resources, time required to administer and score standardized instruments, guidance interpreting results, and options for referring children identified as needing mental health services. The current study examines implementation of a computerized algorithmic support tool that facilitates screening and assessment of depression and anxiety in pediatric primary care and provides referral and treatment guidance. The algorithm incorporates standardized, psychometrically sound screening and next level assessment instruments for detecting clinically significant pediatric depression and anxiety. Pediatricians are prompted to enter demographic data and information indicating the existence of significant risk factors including family history of mental health problems and current family stress or loss. Next, caregivers complete the Pediatric Symptom Checklist (PSC-17). Patients with total scores greater or equal to 15 or who score positive on the depression subscale (sum ≥5 on items 1-5) complete a follow-up assessment—the Patient Health Questionnaire (PHQ-9; ≥5 is clinical threshold) for children older than 12 years and the Center for Epidemiological Studies Depression Scale (CESD; ≥15 is clinical threshold) for children 12 years old or younger. Patients with total scores greater or equal to 15 or who are positive on the anxiety subscale (≥2 on item 4) complete the Self-Report for Childhood Anxiety Related Disorders (SCARED; ≥25 is clinical threshold). The algorithm then summarizes the data from these reports and prompts pediatricians to choose 1 of 11 clinical dispositions, each with clinical recommendations (see Figure 1). The computerized algorithmic support tool was implemented in 4 pediatric primary care practices over a 6-month period for administration at all wellchild visits.