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1.  Pushing the Boundaries: The Development of Super-Resolution Microscopy at Yale and Beyond 
PMCID: PMC3941448  PMID: 24600336
TIRF; microscopy; super-resolution microscopy
4.  Family Presence During Resuscitation: Attitudes of Yale-New Haven Hospital Staff 
Introduction: A novel paradigm of patient- and family-centered care has been promoted and adopted by many parties in the United States. This new attitude emphasizes the role of the family in the care of the patient. One topic that should be affected by the new paradigm is family presence during resuscitation, which continues to be a highly debatable topic with no widespread implementation. The objective of this study was to assess the attitudes of Yale Emergency Department (ED) health care personnel toward Family Presence during Resuscitation (FPDR).
Materials and Methods: In 2012, we surveyed 100 health care professionals in the Yale-New Haven Hospital ED, including physicians, nurses, technicians, social workers, and chaplains. One researcher analyzed the qualitative data, and both researchers reviewed the results to increase internal validity.
Results: Seventy-seven percent of staff members favored allowing the option of FPDR. Seventy-six percent of staff members believed that family members would want to be present during their loved one’s resuscitation.
Conclusion: Given scientific evidence to support FPDR and the staff’s wide acceptance of it, we recommend drafting and implementing a protocol for allowing FPDR. The protocol should be individualized to the Yale-New Haven Hospital ED setting.
PMCID: PMC3941452  PMID: 24600337
family presence during cardiopulmonary resuscitation; family-centered approach, emergency department
6.  The Importance of Student Initiative Both In and Out of The Lab 
In September 2013, graduate students from the Yale Immunobiology Department hosted the second Yale Immunobiology Student Symposium. It was an eclectic and thought-provoking event that encouraged scientists to think outside the box both in their research and in their endeavors outside of the laboratory. The speakers ranged from a government representative to a New York Times science journalist and included four research scientists at the cutting-edge in their field. Speakers discussed their current research, from the role of our gut microbiota in causing colorectal cancers to the biochemical modifications in histone tails that give rise to our unique human biology. The overarching message was to let scientists, especially those of the younger generation, know how to approach, think, and talk about science.
PMCID: PMC3941454  PMID: 24600338
student symposium; science policy; student involvement; government; autoimmunity; microbiome; histone modifications; cancer; science writing
8.  Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination 
Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy.
PMCID: PMC3941456  PMID: 24600334
confocal microscopy; photoactivatable GFP; 2-photon microscopy; superresolution microscopy; selective plane illumination microscopy; light sheet microscopy
10.  What Ticks Do Under Your Skin: Two-Photon Intravital Imaging of Ixodes Scapularis Feeding in the Presence of the Lyme Disease Spirochete 
Lyme disease, due to infection with the Ixodes-tick transmitted spirochete Borrelia burgdorferi, is the most common tick-transmitted disease in the northern hemisphere. Our understanding of the tick-pathogen-vertebrate host interactions that sustain an enzootic cycle for B. burgdorferi is incomplete. In this article, we describe a method for imaging the feeding of Ixodes scapularis nymphs in real-time using two-photon intravital microscopy and show how this technology can be applied to view the response of Lyme borrelia in the skin of an infected host to tick feeding.
PMCID: PMC3941458  PMID: 24600332
Ixodes scapularis ticks; two-photon intravital microscopy; Borrelia burgdorferi; Lyme disease
13.  Comparing the Detection of Iron-Based Pottery Pigment on a Carbon-Coated Sherd by SEM-EDS and by Micro-XRF-SEM 
The same sherd was analyzed using a scanning electron microscope with energy dispersive spectroscopy (SEM-EDS) and a micro X-ray fluorescence tube attached to a scanning electron microscope (Micro-XRF-SEM) to compare the effectiveness of elemental detection of iron-based pigment. To enhance SEM-EDS mapping, the sherd was carbon coated. The carbon coating was not required to produce Micro-XRF-SEM maps but was applied to maintain an unbiased comparison between the systems. The Micro-XRF-SEM analysis was capable of lower limits of detection than that of the SEM-EDS system, and therefore the Micro-XRF-SEM system could produce elemental maps of elements not easily detected by SEM-EDS mapping systems. Because SEM-EDS and Micro-XRF-SEM have been used for imaging and chemical analysis of biological samples, this comparison of the detection systems should be useful to biologists, especially those involved in bone or tooth (hard tissue) analysis.
PMCID: PMC3941461  PMID: 24600333
scanning electron microscopy; pottery pigment; archeology
14.  Science in the Service of Patients: Lessons from the Past in the Moral Battle for the Future of Medical Education 
Medical schools instill a classic moral standoff in which the responsibility for the betterment of the patient stands at odds with the responsibility for the betterment of society. In critical ways, the latter, in the form of a robust research and technology-driven enterprise, has taken precedence over the former, resulting in harm to patients and individual dignity. This tradeoff can be traced to Abraham Flexner, the father of American medical education. In the wake of the Flexner report, American medicine set out on a course of exponential scientific advancement, but the mistreatment of research subjects and the erosion of the doctor-patient relationship in a health care system that is increasingly unaffordable, complex, and impersonal suggest that such progress has come at a price. Recent efforts by medical schools to emphasize humanism in their curricula and admissions processes have shown promise in orienting the values of academic medicine toward the individual patient’s well-being.
PMCID: PMC3941462  PMID: 24600339
bioethics; medical ethics; research ethics; medical education; academic medicine; doctor-patient relationship
15.  How to Design PET Experiments to Study Neurochemistry: Application to Alcoholism  
Positron Emission Tomography (PET) (and the related Single Photon Emission Computed Tomography) is a powerful imaging tool with a molecular specificity and sensitivity that are unique among imaging modalities. PET excels in the study of neurochemistry in three ways: 1) It can detect and quantify neuroreceptor molecules; 2) it can detect and quantify changes in neurotransmitters; and 3) it can detect and quantify exogenous drugs delivered to the brain. To carry out any of these applications, the user must harness the power of kinetic modeling. Further, the quality of the information gained is only as good as the soundness of the experimental design. This article reviews the concepts behind the three main uses of PET, the rationale behind kinetic modeling of PET data, and some of the key considerations when planning a PET experiment. Finally, some examples of PET imaging related to the study of alcoholism are discussed and critiqued.
PMCID: PMC3941463  PMID: 24600335
binding potential; dopamine release; occupancy; alcohol; imaging; tracer kinetics; study design
16.  Introduction 
PMCID: PMC3848097  PMID: 24498669
17.  Hypoxia and DNA Repair 
Hypoxia is a characteristic feature of solid tumors and occurs very early in neoplastic development. Hypoxia transforms cell physiology in multiple ways, with profound changes in cell metabolism, cell growth, susceptibility to apoptosis, induction of angiogenesis, and increased motility. Over the past 20 years, our lab has determined that hypoxia also induces genetic instability. We have conducted a large series of experiments revealing that this instability occurs through the alteration of DNA repair pathways, including nucleotide excision repair, DNA mismatch repair, and homology dependent repair. Our work suggests that hypoxia, as a key component of solid tumors, can drive cancer progression through its impact on genomic integrity. However, the acquired changes in DNA repair that are induced by hypoxia may also render hypoxic cancer cells vulnerable to tailored strategies designed to exploit these changes.
PMCID: PMC3848098  PMID: 24348208
DNA repair; hypoxia; homologous recombination; mismatch repair; BRCA1; MLH1; silencing; epigenetics; microRNAs
18.  Investigations of Homologous Recombination Pathways and Their Regulation 
The DNA double-strand break (DSB), arising from exposure to ionizing radiation or various chemotherapeutic agents or from replication fork collapse, is among the most dangerous of chromosomal lesions. DSBs are highly cytotoxic and can lead to translocations, deletions, duplications, or mutations if mishandled. DSBs are eliminated by either homologous recombination (HR), which uses a homologous template to guide accurate repair, or by nonhomologous end joining (NHEJ), which simply rejoins the two broken ends after damaged nucleotides have been removed. HR generates error-free repair products and is also required for generating chromosome arm crossovers between homologous chromosomes in meiotic cells. The HR reaction includes several distinct steps: resection of DNA ends, homologous DNA pairing, DNA synthesis, and processing of HR intermediates. Each occurs in a highly regulated fashion utilizing multiple protein factors. These steps are being elucidated using a combination of genetic tools, cell-based assays, and in vitro reconstitution with highly purified HR proteins. In this review, we summarize contributions from our laboratory at Yale University in understanding HR mechanisms in eukaryotic cells.
PMCID: PMC3848099  PMID: 24348209
DNA repair; homologous recombination; double-strand breaks; resection; recombinase; presynaptic filament; synaptic complex; double Holliday junction
19.  Cellular Roles of DNA Polymerase Beta 
Since its discovery and purification in 1971, DNA polymerase ß (Pol ß) is one of the most well-studied DNA polymerases. Pol ß is a key enzyme in the base excision repair (BER) pathway that functions in gap filling DNA synthesis subsequent to the excision of damaged DNA bases. A major focus of our studies is on the cellular roles of Pol ß. We have shown that germline and tumor-associated variants of Pol ß catalyze aberrant BER that leads to genomic instability and cellular transformation. Our studies suggest that Pol ß is critical for the maintenance of genomic stability and that it is a tumor suppressor. We have also shown that Pol ß functions during Prophase I of meiosis. Pol ß localizes to the synaptonemal complex and is critical for removal of the Spo11 complex from the 5’ ends of double-strand breaks. Studies with Pol ß mutant mice are currently being undertaken to more clearly understand the function of Pol ß during meiosis. In this review, we will highlight our contributions from our studies of Pol ß germline and cancer-associated variants.
PMCID: PMC3848100  PMID: 24348210
DNA polymerase beta; meiosis; fidelity of DNA synthesis
20.  Triplex-Induced DNA Damage Response 
Cellular DNA damage response is critical to preserving genomic integrity following exposure to genotoxic stress. A complex series of networks and signaling pathways become activated after DNA damage and trigger the appropriate cellular response, including cell cycle arrest, DNA repair, and apoptosis. The response elicited is dependent upon the type and extent of damage sustained, with the ultimate goal of preventing propagation of the damaged DNA. A major focus of our studies is to determine the cellular pathways involved in processing damage induced by altered helical structures, specifically triplexes. Our lab has demonstrated that the TFIIH factor XPD occupies a central role in triggering apoptosis in response to triplex-induced DNA strand breaks. We have shown that XPD co-localizes with γH2AX, and its presence is required for the phosphorylation of H2AX tyrosine142, which stimulates the signaling pathway to recruit pro-apoptotic factors to the damage site. Herein, we examine the cellular pathways activated in response to triplex formation and discuss our finding that suggests that XPD-dependent apoptosis plays a role in preserving genomic integrity in the presence of excessive structurally induced DNA damage.
PMCID: PMC3848101  PMID: 24348211
triplex DNA; H-DNA; DNA repair; apoptosis; XPD; H2AX; genomic instability; nucleotide excision repair; triplex-forming oligonucleotides
21.  BRCA2: One Small Step for DNA Repair, One Giant Protein Purified 
DNA damage, malfunctions in DNA repair, and genomic instability are processes that intersect at the crossroads of carcinogenesis. Underscoring the importance of DNA repair in breast and ovarian tumorigenesis is the familial inherited cancer predisposition gene BRCA2. The role of BRCA2 in DNA double-strand break repair was first revealed based on its interaction with RAD51, a central player in homologous recombination. The RAD51 protein forms a nucleoprotein filament on single-stranded DNA, invades a DNA duplex, and initiates a search for homology. Once a homologous DNA sequence is found, the DNA is used as a template for the high-fidelity repair of the DNA break. Many of the biochemical features that allow BRCA2 to choreograph the activities of RAD51 have been elucidated and include: targeting RAD51 to single-stranded DNA while inhibiting binding to dsDNA, reducing the ATPase activity of RAD51, and facilitating the displacement of the single-strand DNA binding protein, Replication Protein A. These reinforcing activities of BRCA2 culminate in the correct positioning of RAD51 onto a processed DNA double-strand break and initiate its faithful repair by homologous recombination. In this review, I will address current biochemical data concerning the BRCA2 protein and highlight unanswered questions regarding BRCA2 function in homologous recombination and cancer.
PMCID: PMC3848102  PMID: 24348212
BRCA2; DNA repair; homologous recombination; RAD51; breast cancer; ovarian cancer
22.  Fanconi Anemia: A Signal Transduction and DNA Repair Pathway 
Fanconi anemia (FA) is a fascinating, rare genetic disorder marked by congenital defects, bone marrow failure, and cancer susceptibility. Research in recent years has led to the elucidation of FA as a DNA repair disorder and involved multiple pathways as well as having wide applicability to common cancers, including breast, ovarian, and head and neck. This review will describe the clinical aspects of FA as well as the current state of its molecular pathophysiology. In particular, work from the Kupfer laboratory will be described that demonstrates how the FA pathway interacts with multiple DNA repair pathways, including the mismatch repair system and signal transduction pathway of the DNA damage response.
PMCID: PMC3848103  PMID: 24348213
Fanconi anemia; bone marrow failure; DNA repair
23.  Early Days of DNA Repair: Discovery of Nucleotide Excision Repair and Homology-Dependent Recombinational Repair 
The discovery of nucleotide excision repair in 1964 showed that DNA could be repaired by a mechanism that removed the damaged section of a strand and replaced it accurately by using the remaining intact strand as the template. This result showed that DNA could be actively metabolized in a process that had no precedent. In 1968, experiments describing postreplication repair, a process dependent on homologous recombination, were reported. The authors of these papers were either at Yale University or had prior Yale connections. Here we recount some of the events leading to these discoveries and consider the impact on further research at Yale and elsewhere.
PMCID: PMC3848104  PMID: 24348214
DNA repair; homology-dependent repair; leading strand restart; nucleotide excision repair; Yale Radiobiology; recombination; recombinational repair
24.  The Awakening of DNA Repair at Yale 
As a graduate student with Professor Richard Setlow at Yale in the late 1950s, I studied the effects of ultraviolet and visible light on the syntheses of DNA, RNA, and protein in bacteria. I reflect upon my research in the Yale Biophysics Department, my subsequent postdoctoral experiences, and the eventual analyses in the laboratories of Setlow, Paul Howard-Flanders, and myself that constituted the discovery of the ubiquitous pathway of DNA excision repair in the early 1960s. I then offer a brief perspective on a few more recent developments in the burgeoning DNA repair field and their relationships to human disease.
PMCID: PMC3848106  PMID: 24348216
nucleotide excision repair; DNA repair history; repair replication; transcription-coupled repair; ultraviolet light; xeroderma pigmentosum; Cockayne syndrome; UV sensitive syndrome
25.  Introduction 
PMCID: PMC3848107  PMID: 24498668

Results 1-25 (7320)