DNA/RNA Degradation Rate in Long Term Fixed Museum Specimens

In today’s research driven society, it has become commonplace for institutions to rely upon DNA and RNA extraction techniques to help obtain genomic data from old specimens. Generally, specimens were commonly preserved for future gross examination and/or teaching. Using histological examination of specimens from museum jars from the Pathology Department at the Indiana University School of Medicine, the sequential and chronological degradation of DNA and RNA has been studied. We examined gross specimens from nine decades from 1920 until 2000. We evaluated histologic preservation of kidney, liver, heart, lung, spleen, uterus, and brain for nuclear structure in these samples. Nuclear preservation was based on amount of nuclei per 20x microscopic field and the crispness of the nuclear membrane and internal features. The nuclei in high lipid tissues such as the brain were found to degrade at a quicker rate than dense tissues such as the heart and uterus. Our study has shown specimens preserved beyond fifty years were likely to have little to no nuclei left, thus indicating that there was little to no DNA and RNA remaining. This technique of histologic evaluation first is an important finding and a general guideline which may save research institutions from the expensive process of DNA and RNA extraction

Natural Cardiac Deaths in Central Indiana

poster abstractCardiovascular disease is still the major cause of death in the USA for the past 50 to 60 years. Within cardiovascular disease there are many subtypes that cause death including hypertensive heart disease, atherosclerosis, coronary heart disease (CAD), myocardial infarction (MI), dilated cardiomyopathy, hypertrophic cardiomyopathy, cardiomegaly, and misc.). In this review study we examined the Marion County, Indianapolis, Indiana Morgue, Indiana database for the total of deaths that occurred between 2004 through 2012 and evaluated the number of cardiovascular deaths including the various CV subtypes mentioned above. There were approximately 13,000 deaths examined that were sent to the Marion County Morgue during that time frame in Central Indiana. Approximately 2950 deaths were due to CV disease (22.6%). Total ischemia (coronary artery disease) was 1939 made up the majority of the CV related deaths. This was followed by hypertensive heart disease (571) and congestive heart failure (189). Hypertrophic cardiomyopathy (89), cardiomegaly (16), and cardiac tamponade (11) made up the rest. Cardiac arrhythmias and myocarditis made up the remaining CV causes of death (131). In a previous study done at the Marion County Morgue from 1987 to 2003 focused on hypertensive CV disease and hypertrophic cardiomyopathy found 165 deaths and 134 deaths respectively. Compared to the previous local study in the same population the incidence of hypertensive heart disease was moderately increased. There was not much difference between hypertrophic cardiomyopathy between the two studies. Both studies are fairly consistent when compared to national statistics on cardiovascular death in the country

Increased Ischemic Cardiac Deaths in Central Indiana in Summer Months Compared to Winter Months

poster abstractCardiovascular diseases have been the leading cause of death in the United States for several decades. Despite sustained declines in the mortality rates from these diseases, the magnitude of the disease is still staggering. One large recent study, using data on hundreds of heart attacks documented in the National Registry of Myocardial Infarction, found that 53 percent more cases in winter than in summer. The primary culprit, many believe, is temperature. Cold weather narrows coronary arteries and raises blood pressure, stressing the heart. Physical strain and ruptured plaques caused by shoveling snow are also commonly cited. But in a recent study, two researchers, found that the risk increases even in warm climates. Analyzing death certificates in seven regions with different climates, Los Angeles, Texas, Arizona, Pennsylvania, Massachusetts and others found that cardiovascular deaths rose up to 36 percent between summer and winter, regardless of climate and temperatures In this study we evaluated the incidence of ischemic cardiomyopathy in the Central Indiana area in the winter months compared to the summer months for the years 1998 to 2002. Approximately 5325 deaths were seen in the Marion County Morgue in central Indiana in this time period. There were 609 ischemic cardiac deaths seen in the summer (March 15th through October 15th) compared to 434 ischemic cardiac deaths seen in the winter (October 15th through March 15th). The deaths by years in the summer were 129, 131, 92, 127, and 130 and in the winter were 95, 96, 90, 96, and 57 respectively. In conclusion, this study was consistent with the outcome as the previous study done in multiple northern and southern cities in the country

XPC protects against smoking- and carcinogen-induced lung adenocarcinoma

Cigarette smoke (CS) contains hundreds of carcinogens and is a potent inducer of oxidative and bulky DNA damage, which when insufficiently repaired leads to activation of DNA damage response and possibly mutations. The DNA repair protein xeroderma pigmentosum group C (XPC) is primed to play an important role in CS-induced DNA damage because of its function in initiating repair of both bulky oxidative DNA damage. We hypothesized that loss of XPC function will increase susceptibility to developing CS- and carcinogen-induced lung cancer through impaired repair of oxidative DNA damage. Mice deficient in XPC (XPC-/-) exposed to chronic CS developed lung tumors whereas their wild-type littermates (XPC+/+) did not. XPC-/- mice treated with the CS-carcinogen urethane developed lung adenocarcinomas representing progressive stages of tumor development, with lung tumor number increased 17-fold compared with XPC+/+ mice. Mice heterozygous for XPC (XPC+/-) demonstrated a gene-dose effect, developing an intermediate number of lung tumors with urethane treatment. Treatment of XPC-/- mice with the carcinogen 3-methylcholanthrene followed by the proliferative agent butylated hydroxytoluene resulted in a 2-fold increase in lung adenocarcinoma development. Finally, tumor number decreased 7-fold in the lungs of XPC-/- mice by concurrent treatment with the antioxidant, N-acetylcysteine. Altogether, this supports a mechanism by which decreased XPC expression promotes lung adenocarcinoma development in response to CS-carcinogen exposure, due in part to impaired oxidative DNA damage repair

Using TMAs (Tissue MicroArrays) to Evaluate GLS, GLUL, and CAV 1 Immunostaining in Breast Cancer

poster abstractApproximately 1 out of 8 women in the United States will develop invasive breast cancer over the course of their lifetime. Breast cancer has a greater potential of being cured if diagnosed in the earlier phases. We evaluated three well-recognized biomarkers, GLUL, GLS, and Cav 1 (glutamine synthetase, glutaminase, caveolin-1) in 14 TMA (tissue Microarrays). The tissues were normal breast and various subtypes of breast carcinoma by immunohistochemistry (IHC) to determine expression and localization in cancerous tissues in breast carcinoma cases. Approximately 80 to 90 breast biopsies in each of the 14 breast TMA immunostaining were evaluated with the GLUL, GLS, and Cav 1 antibodies. With GLS, immunostaining was seen in most tumor cells (mainly cytoplasm and nucleus) and the stain was clean with no background except in cases that had lymphocytes in the core along with the tumor cells. With GLUL, immunostaining was seen in most tumor cells (mainly cytoplasm and nucleus) and the stain was clean with no background except in cases that had lymphocytes in the core along with the tumor cells. Cav1 was seen only in the endothelial cells in blood vessel walls and some smooth muscle cells in small arterioles in the stroma and surrounding normal ducts, DCIS, and some invasive carcinoma tumor clusters. This information from the immunostains was obtained after analyzing 14 tissue microarrays which is not only time effective but cost effective when analyzing multiple research cases from cancer patients. The data for the three antibodies are currently being analyzed by the biostatistics core group and correlated with the severity of the breast cancer disease with multiple patient demographics

Perfluorooctanoic acid exposure triggers oxidative stress in the mouse pancreas

• PFOA triggers focal ductal hyperplasia following 7 day exposure. • PFOA exposure increases 8-iso-PGF2α levels in the pancreas. • Antioxidant gene expression is upregulated in the pancreas following PFOA exposure. , Perfluorooctanoic acid (PFOA) is used in the manufacture of many industrial and commercial products. PFOA does not readily decompose in the environment, and is biologically persistent. Human epidemiologic and animal studies suggest that PFOA exposure elicits adverse effects on the pancreas. While multiple animal studies have examined PFOA-mediated toxicity in the liver, little is known about the potential adverse effects of PFOA on the pancreas. To address this, we treated C57Bl/6 mice with vehicle, or PFOA at doses of 0.5, 2.5 or 5.0 mg/kg BW/day for 7 days. Significant accumulation of PFOA was found in the serum, liver and pancreas of PFOA-treated animals. Histopathologic examination of the pancreas revealed focal ductal hyperplasia in mice treated with 2.5 and 5.0 mg/kg BW/day PFOA, while inflammation was observed only in the high dose group. Elevated serum levels of amylase and lipase were observed in the 2.5 mg/kg BW/day PFOA treatment group. In addition, PFOA exposure resulted in a dose-dependent increase in the level of the lipid peroxidation product 8-iso-PGF2α and induction of the antioxidant response genes Sod1, Sod2, Gpx2 and Nqo1. Our findings provide additional evidence that the pancreas is a target organ for PFOA-mediated toxicity and suggest that oxidative stress may be a mechanism through which PFOA induces histopathological changes in the pancreas

Use of Aperio Whole Slide Imaging System to Capture and Utilize Digital Virtual Slides for Pathology Education

poster abstractDigital whole slide imaging is the technique of digitizing an entire microscope slide at the highest resolution to produce a “digital virtual microscope slide” with high image quality. This digital image can be viewed in three to four fields, from low to high power, a feature commonly used by pathologists. This digital virtual slide can be used in conjunction with image processing software (both windows-based and browser-based) to view, manipulate, position, and specify the magnification of the image on a screen as if using a regular microscope to view the original glass slide. As the slide is captured in a virtual format, it is possible to use the image for archiving, copying, transferring over networks, distant consultation, as well as integration for educational use on the web and/or DVD. In this study, we captured all C603 and C604 sophomore pathology teaching slides in the general and systemic pathology course for viewing and learning through the Aperio ImageScope viewer. The resulting digital images possessed greater ease of use, were quicker to scan and allowed easier location of pathologic lesions in the slides. The ImageScope viewer allowed students to quickly zoom in and out of the slides at multiple fields of magnification. Instructors that have switched to the Aperio system from the old Bliss system found the Aperio system allowed the instructor to open up to 8 slides at one time, allowing side by side comparison to be completed on the same screen. The system also allows one to measure the size of the cells and to capture detailed images of tumor cells, inflammatory cells, and/or necrosis (cell death). This system is available for use on desktop, laptop, and most digital devices (such as smart phones or tablets). Compared to the old Bliss system, which is unable to perform these functions

Effects of Thrombopoietin (TPO) on Longitudinal Mouse Hind Limb Crush Injury Model

abstractApproximately 645 people suffer from blunt force trauma injury to the femur every day. The recovery time of such injury can last anywhere from 3-6 months. Thrombopoietin (TPO) was used as a growth factor to induce bone and muscle healing. In this study, nine separate mouse groups (10 mice per group) were used: Crush PBS, Crush TPO, Surgery PBS, and Surgery TPO at day 3 and day 17, and controls with no surgery/crush/treatment. Skeletal muscle was harvested from the following sites: experimental impact, experimental adjacent, and normal contralateral skeletal muscle as a control. The muscles were fixed, processed, sectioned, and stained with H&E and Massons Trichrome stains. The slides were reviewed for skeletal muscle injury, muscle necrosis, inflammation, muscle repair, and regeneration. In addition, F4/80, an immunostain for macrophages was performed. On microscopic examination at day 3 the most common histologic changes seen were sporadic muscle fiber vacuolation, focal necrosis of varying sizes, muscle contraction bands, and infiltration of macrophages. On day 17, the skeletal muscle injury was generally healed. The main histologic lesions seen were variable sizes of muscle fibers, early fibroplasia, fat infiltration, some macrophages, satellite cells, and neovascularization. Comparing the TPO treated mice versus the PBS control group, the lesions at both time points were less in the TPO treated mice

Melanoma LAMP-2C Modulates Tumor Growth and Autophagy

Autophagy plays critical but diverse roles in cellular quality control and homeostasis potentially checking tumor development by removing mutated or damaged macromolecules, while conversely fostering tumor survival by supplying essential nutrients during cancer progression. This report documents a novel inhibitory role for a lysosome-associated membrane protein, LAMP-2C in modulating autophagy and melanoma cell growth in vitro and in vivo. Solid tumors such as melanomas encounter a variety of stresses in vivo including inflammatory cytokines produced by infiltrating lymphocytes directed at limiting tumor growth and spread. Here, we report that in response to the anti-tumor, pro-inflammatory cytokine interferon-gamma, melanoma cell expression of LAMP2C mRNA significantly increased. These results prompted an investigation of whether increased melanoma cell expression of LAMP-2C might represent a mechanism to control or limit human melanoma growth and survival. In this study, enhanced expression of human LAMP-2C in melanoma cells perturbed macroautophagy and chaperone-mediated autophagy in several human melanoma lines. In vitro analysis showed increasing LAMP-2C expression in a melanoma cell line, triggered reduced cellular LAMP-2A and LAMP-2B protein expression. Melanoma cells with enhanced LAMP-2C expression displayed increased cell cycle arrest, increased expression of the cell cycle regulators Chk1 and p21, and greater apoptosis and necrosis in several cell lines tested. The increased abundance of Chk1 protein in melanoma cells with increased LAMP-2C expression was not due to higher CHEK1 mRNA levels, but rather an increase in Chk1 protein abundance including Chk1 molecules phosphorylated at Ser345. Human melanoma cell xenografts with increased LAMP-2C expression, displayed reduced growth in immune compromised murine hosts. Melanomas with high LAMP-2C expression showed increased necrosis and reduced cell density upon histological analysis. These results reveal a novel role for LAMP-2C in negatively regulating melanoma growth and survival

Reduced Expression of DNA Repair and Redox Signaling Protein APE1/Ref-1 Impairs Human Pancreatic Cancer Cell Survival, Proliferation, and Cell Cycle Progression

Pancreatic cancer is a deadly disease that is virtually never cured. Understanding the chemoresistance intrinsic to this cancer will aid in developing new regimens. High expression of APE1/Ref-1, a DNA repair and redox signaling protein, is associated with resistance, poor outcome, and angiogenesis; little is known in pancreatic cancer. Immunostaining of adenocarcinoma shows greater APE1/Ref-1 expression than in normal pancreas tissue. A decrease in APE1/Ref-1 protein levels results in pancreatic cancer cell growth inhibition, increased apoptosis, and altered cell cycle progression. Endogenous cell cycle inhibitors increase when APE1/ Ref-1 is reduced, demonstrating its importance to proliferation and growth of pancreatic cancer