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University of, Texas, United States

Choi H.J.,Ohio State University | Ribelayga C.P.,University of Texas Medical School | Mangel S.C.,Ohio State University
Journal of Visualized Experiments | Year: 2012

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue. Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance. Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas, and can be used to study coupling between other retinal neurons, as described here. © 2012 Creative Commons Attribution License.

Imatinib mesylate is a selective tyrosine kinase inhibitor used in the treatment of chronic myelogenous leukemia. Ocular side effects of imatinib include periorbital edema, which may become so severe as to obstruct the visual field. The purpose of this case study is to describe the clinical characteristics of imatinib- induced postoperative periorbital purpura. We retrospectively reviewed the medical literature using PubMed, searching the terms edema, Gleevec, imatinib, periorbital, postoperative and purpura. Patient reports and previous reviews of the subject were critically assessed and the salient features are presented. Three patients have undergone surgery to reduce the imatinib-induced periorbital edema; two of these individuals have developed imatinib-induced postoperative periorbital purpura. We recommend discontinuing imatinib usage one week prior to periorbital surgery and not resuming therapy until the eighth postoperative day.

Abrams J.L.,University of Texas Medical School
Journal of visualized experiments : JoVE | Year: 2013

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences (1). External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases (10). It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme (12). Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 (13), known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system (5), to protein refolding to validate this approach.

News Article
Site: www.nature.com

Twelve years ago, Mary Jane Lapinski had a routine breast-cancer screening mammogram at her local hospital in Baltimore, Maryland. The mammogram showed multiple specks in her left breast. Her physician called it ductal carcinoma in situ (DCIS) — an early-stage, non-invasive cancer of the milk ducts. A surgeon told her he could attempt a lumpectomy to remove the lesions, but he recommended a mastectomy — removal of the entire breast. “I kept thinking, this isn't logical,” says Lapinski, who was 48 at the time. “It was mind-boggling that a non-invasive cancer carried the same or more aggressive treatment than an invasive cancer.” The rogue cells in Lapinski's breast occupied a diagnostic grey area. Some cases of DCIS advance to invasive breast cancer, metastasis and death, but most do not. By current estimates, 20–30% of DCIS tumours will become aggressive within 20 years, says Shelley Hwang, a breast-cancer surgeon and researcher at Duke University School of Medicine in Durham, North Carolina. Still, most oncologists feel that it is best to remove the lesions and offer radiation treatment to stave off their progression. The trouble is that oncologists cannot tell for certain which DCIS lesions will remain idle and which will turn deadly. Identifying breast-cancer biomarkers — molecules that can identify the pre-cancerous cells that are likely to progress to invasive cancer — could lead to better-informed decisions about treatment. Unfortunately, little is known about the natural history of DCIS. It is difficult to track the course of the disease because so many women undergo surgery. “If we can identify a subset of patients that are at risk of developing an invasive cancer and only treat those, we would spare many women unnecessary treatment,” says Eileen Rakovitch, a radiation oncologist at Sunnybrook Health Sciences Center in Toronto, Canada. Before the introduction of widespread screening mammography in the 1980s, DCIS lesions represented about 3% of breast cancers in the United States. They now account for nearly one-third of newly diagnosed breast cancers1. But detecting DCIS does not necessarily add much information about a woman's future or overall health. “It is entirely possible to find cancers that don't matter,” says H. Gilbert Welch, an internist and cancer epidemiologist at the Geisel School of Medicine at Dartmouth in Hanover, New Hampshire. Welch and Archie Bleyer, then at the University of Texas Medical School in Houston, estimated that, in 2008, 70,000 US women received an early-stage breast-cancer diagnosis for lesions that would not have led to clinical symptoms, accounting for 31% of screening-detected breast cancers2. Most women diagnosed with DCIS have a lumpectomy or mastectomy — or a double mastectomy — along with radiation therapy. But the benefits of such treatments are hard to find. A much-discussed observational study of more than 100,000 US women with DCIS found that women who had lumpectomies or mastectomies to treat DCIS had just a 3.3% chance of dying of breast cancer in the next 20 years, not much different than the risk to women in the general population3 (2.7%, according to the American Cancer Society). Ideally, women with DCIS would be tested to assess whether surgery is the best course of action. Although no such test is clinically available, physicians are starting to use biomarkers to predict the future of women who have already had surgery. One test — Oncotype DX DCIS Score, produced by Genomic Health in Redwood City, California — stratifies women who have had breast-conserving surgery for DCIS into low, medium and high risk for future cancer. The test evaluates the expression of seven cancer genes (including those associated with cell proliferation and hormone receptors) in tissue samples taken from breast biopsies. Rakovitch validated DCIS Score in a retrospective study of women diagnosed with DCIS and who'd had breast conserving surgery. In work funded by a research grant from Genomic Health, she and her colleagues applied the test to tissue samples from 718 women4. “Women with an intermediate- or high-risk score had twice the risk of developing local recurrence compared to women with a low-risk score,” says Rakovitch. She says that the assay can pick out some women who are at a high risk of recurrence, but whom doctors might have considered to be low risk based on patient history and tumour characteristics. The search for a reliable measure to prevent surgery in the first place goes on. Any test, Hwang says, would probably involve a large array of markers that could be combined to form a cohesive picture. “We've taken the individual biomarkers as far as they can go and they're not giving us the answers we need,” she says. Thea Tlsty, a molecular pathologist at the University of California, San Francisco, and her colleagues have identified three proteins involved in cell proliferation that are associated with future aggressive breast cancers5. Tlsty's team found that of 1,162 women who had a lumpectomy for DCIS, those whose tissue was positive for all three biomarkers — COX-2, p16 and ki67 — had a 20% risk of developing an invasive cancer within 8 years. If they had none of the proteins, their risk dropped to 4%. “These markers indicate which pre-cancers are the baby, basal-like cancers, which are the most lethal and metastatic,” she says. In unpublished research, Tlsty's group has subsequently identified several other potential biomarkers in proteins that coordinate cell death. Four prospective studies in Australia, the United States and the United Kingdom are further evaluating the trio of markers, Tlsty says. Other biomarkers have also shown promise. Invasive breast cancers often stop the expression of tumour suppressor genes. One of those genes, called SYK, seems to be part of a genetic hub that determines which precancerous cells eventually metastasize. One study found that women who had altered expression of 55 genes that interact with SYK had reduced survival6. The search for circulating markers for early detection has proved frustrating at times, says Jeffrey Marks, a cancer cell biologist at Duke University. Marks and his colleagues selected 90 blood-based biomarkers, but none were useful in distinguishing cases of breast cancer from benign controls7. “They're very difficult to validate in independent populations,” he says. Some researchers are looking for signals that might reveal which DCIS lesions are associated with an increased risk of developing future invasive breast cancer. Andy Beck, a computational biologist at Harvard Medical School in Boston, Massachusetts, and his team are examining patterns of genomic alteration. Using data from DNA profiles of invasive breast cancers catalogued with The Cancer Genome Atlas, the group identified genomic locations that are most frequently copied or deleted in invasive breast cancer lesions. “We're basically saying that if it's not present in invasive cancer then it's not likely to be useful,” says Beck. In this case, the marker proved to be grimly robust. In a study of 271 patients, women with lesions that had extra copies in all three regions had a 17-fold higher risk of having a coincident invasive breast cancer compared with those women who had none8. The group is expanding the study to include about 20 chromosomal regions commonly altered in invasive breast cancer. In collaboration with Stanford University, Washington University and the Nurse's Health Study, the group is launching a study of 1,400 patients to predict the risk of recurrence or a subsequent invasive cancer over time. Researchers have recognized that sheer genetic diversity within precancerous tissues may help to predict cancer formation and progression. As precancerous cells evolve and accumulate genetic and epigenetic alterations, they become more varied. Some studies have shown that diversity can predict progression. Marks is now studying the genetic diversity of the cells within DCIS lesions. In theory, if the DCIS has a more complex mosaic of cells, there is a stronger likelihood that one of them will develop into a more 'fit' cancer cell that can invade the surrounding tissue and metastasize. Until scientists have a fuller understanding of which markers indicate an increased risk of developing invasive cancer, patients with DCIS will lack clarity about their future. For her part, Lapinski never went under the knife. Instead, she tracked down Hwang, who suggested that Lapinski join a three-month clinical study of the oestrogen-blocking drug tamoxifen. Lipinski was supposed to have surgery at the end of the trial, but she opted to forego the operation and continue with the tamoxifen, and, later, raloxifene. She checks in with Hwang twice a year for an examination and a mammogram. Although others see uncertainty in Lapinski's choice, she doesn't see it as a risky move. “Everybody has to make their own decision,” she says. “It has to be comfortable for them.”

News Article
Site: www.cemag.us

Using nanoparticles to engineer a special drug, a team of researchers has demonstrated in mice a new way to both reduce preterm birth and avoid the risks of medication in pregnancy to unborn babies. Jerrie S. Refuerzo, M.D., of the University of Texas Medical School at Houston was frustrated with the limitations of existing tocolytic (anti-contraction or labor-repressant) medications such as indomethacin in treating women experiencing preterm labor. These drugs can cross the placental barrier and cause a heart defect or other problems in the fetus. Refuerzo and Monica Longo, M.D., Ph.D., in collaboration with colleagues from Houston Methodist Research Institute, Biana Godin, PharmD, Ph.D., bioengineered an innovative microscopic nanoparticle of indomethacin aimed at reaching the pregnant uterus but not crossing the placenta to the fetus. This targeted liposomal indomethacin, called LIPINDORA, was coated with an oxytocin receptor antagonist to make it bind to uterine tissue. LIPINDORA was given to near-term pregnant mice and the researchers found that the treated mice were significantly less likely than controls to have preterm uterine contractions or to deliver prematurely. "These findings are exciting because we don't currently have any medication that can reliably stop contractions or prevent preterm birth without also crossing the mom's placenta and causing risks to babies," explains Edward R. B. McCabe, M.D., Ph.D., senior vice president and chief medical officer of the March of Dimes. Preterm birth (birth before 37 weeks of pregnancy) is the number one killer of babies in the U.S. About 380,000 babies are born too soon each year in this country, and those who survive an early birth often face an increased risk of a lifetime of health challenges, such as breathing problems, cerebral palsy, intellectual disabilities and others. Even babies born just a few weeks early have higher rates of hospitalization and illness than full-term infants. McCabe says nanoparticles are a leading-edge technology also being studied for delivery of drugs for other conditions, such as cancer, heart disease, and bacterial infections, in order to target specific cells to reduce toxicity and side effects of those medications and to make them more effective. Nanoparticles range 100-200 nanometers in diameter and a nanometer is one billionth of a meter. To demonstrate scale: a nanometer is to a tennis ball as a tennis ball is to the earth. Refuerzo and her team, F. Leonard, A. E. Ontiveros, N. Buleyeva, Biana Godin, and Monica Longo, are being honored by the March of Dimes at the annual Society for Maternal-Fetal Medicine. Joe Leigh Simpson, M.D., senior vice president for Research and Global Programs, will present Refuerzo with the March of Dimes award for Best Abstract in Prematurity at the SMFM's Annual Meeting. 2016 marks the 13th year that the March of Dimes award has been presented. Refuerzo says the team currently is conducting tests of LIPINDORA's effectiveness in human uterine tissue donated from C-sections. They're looking for biomarkers to indicate that the tissue has an anti-contraction response to the drug. If successful, they hope to begin a Phase I human clinical trial of the drug within the next few years. Release Date: February 1, 2016 Source: The Society for Maternal-Fetal Medicine

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