TIRF; microscopy; super-resolution microscopy
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.
family presence during cardiopulmonary resuscitation; family-centered approach, emergency department
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.
student symposium; science policy; student involvement; government; autoimmunity; microbiome; histone modifications; cancer; science writing
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.
confocal microscopy; photoactivatable GFP; 2-photon microscopy; superresolution microscopy; selective plane illumination microscopy; light sheet microscopy
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.
Ixodes scapularis ticks; two-photon intravital microscopy; Borrelia burgdorferi; Lyme disease
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.
scanning electron microscopy; pottery pigment; archeology
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.
bioethics; medical ethics; research ethics; medical education; academic medicine; doctor-patient relationship
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.
binding potential; dopamine release; occupancy; alcohol; imaging; tracer kinetics; study design
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.
DNA repair; hypoxia; homologous recombination; mismatch repair; BRCA1; MLH1; silencing; epigenetics; microRNAs
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.
DNA repair; homologous recombination; double-strand breaks; resection; recombinase; presynaptic filament; synaptic complex; double Holliday junction
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.
DNA polymerase beta; meiosis; fidelity of DNA synthesis
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
triplex DNA; H-DNA; DNA repair; apoptosis; XPD; H2AX; genomic instability; nucleotide excision repair; triplex-forming oligonucleotides
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
BRCA2; DNA repair; homologous recombination; RAD51; breast cancer; ovarian cancer
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.
Fanconi anemia; bone marrow failure; DNA 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.
DNA repair; homology-dependent repair; leading strand restart; nucleotide excision repair; Yale Radiobiology; recombination; recombinational repair
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.
nucleotide excision repair; DNA repair history; repair replication; transcription-coupled repair; ultraviolet light; xeroderma pigmentosum; Cockayne syndrome; UV sensitive syndrome