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The endothelial glycocalyx is a key component of the glomerular filtration barrier. We have shown that matrix metalloproteinase (MMP)-mediated syndecan 4 shedding is a mechanism of glomerular endothelial glycocalyx damage in vitro, resulting in increased albumin permeability. Here we sought to determine whether this mechanism is important in early diabetic kidney disease, by studying streptozotocin-induced type 1 diabetes in DBA2/J mice. Diabetic mice were albuminuric, had increased glomerular albumin permeability and endothelial glycocalyx damage. Syndecan 4 mRNA expression was found to be upregulated in isolated glomeruli and in flow cytometry-sorted glomerular endothelial cells. In contrast, glomerular endothelial luminal surface syndecan 4 and Marasmium oreades agglutinin lectin labelling measurements were reduced in the diabetic mice. Similarly, syndecan 4 protein expression was significantly decreased in isolated glomeruli but increased in plasma and urine, suggesting syndecan 4 shedding. Mmp-2, 9 and 14 mRNA expression were upregulated in isolated glomeruli, suggesting a possible mechanism of glycocalyx damage and albuminuria. We therefore characterised in detail the activity of MMP-2 and 9 and found significant increases in kidney cortex, plasma and urine. Treatment with MMP-2/9 inhibitor I for 21 days, started six weeks after diabetes induction, restored endothelial glycocalyx depth and coverage and attenuated diabetes-induced albuminuria and reduced glomerular albumin permeability. MMP inhibitor treatment significantly attenuated glomerular endothelial and plasma syndecan 4 shedding and inhibited plasma MMP activity. Thus, our studies confirm the importance of MMPs in endothelial glycocalyx damage and albuminuria in early diabetes and demonstrate that this pathway is amenable to therapeutic intervention. Hence, treatments targeted at glycocalyx protection by MMP inhibition may be of benefit in diabetic kidney disease., Graphical abstract

Lipid Phosphate phosphatase 3 (LPP3), encoded by the Plpp3 gene, is an enzyme that dephosphorylates the bioactive lipid mediator lysophosphatidic acid (LPA). To study the role of LPP3 in the myocardium, we generated a cardiac specific Plpp3 deficient mouse strain. Although these mice were viable at birth in contrast to global Plpp3 knockout mice, they showed increased mortality ~ 8 months. LPP3 deficient mice had enlarged hearts with reduced left ventricular performance as seen by echocardiography. Cardiac specific Plpp3 deficient mice had longer ventricular effective refractory periods compared to their Plpp3 littermates. We observed that lack of Lpp3 enhanced cardiomyocyte hypertrophy based on analysis of cell surface area. We found that lack of Lpp3 signaling was mediated through the activation of Rho and phospho-ERK pathways. There are increased levels of fetal genes Natriuretic Peptide A and B (Nppa and Nppb) expression indicating myocardial dysfunction. These mice also demonstrate mitochondrial dysfunction as evidenced by a significant decrease (P < 0.001) in the basal oxygen consumption rate, mitochondrial ATP production, and spare respiratory capacity as measured through mitochondrial bioenergetics. Histology and transmission electron microscopy of these hearts showed disrupted sarcomere organization and intercalated disc, with a prominent disruption of the cristae and vacuole formation in the mitochondria. Our findings suggest that LPA/LPP3-signaling nexus plays an important role in normal function of cardiomyocytes., Graphical abstract fx1, Highlights • PLPP3 plays a prominent role in the heart compared to other isoforms of PLPP. • Lack of PLPP3 results in deteriorating cardiac function. • PLPP3 regulates LPA signaling in cardiomyocytes. • Presence of PLPP3 is required for optimal mitochondrial function. • Increased free radical production is mitigated with activated PLPP3.

The restoration of dead/degraded oyster reefs is increasingly pursued worldwide to reestablish harvestable populations or renew ecosystem services. Evidence suggests that oysters can improve water quality, but less is known about the role of associated benthic sediments in promoting biogeochemical processes, such as nutrient cycling and burial. There is also limited understanding of if, or how long postrestoration, a site functions like a natural reef. This study investigated key biogeochemical properties (e.g., physiochemical properties, nutrient pools, microbial community size and activity) in the sediments of dead reefs; 1-, 4-, and 7-year-old restored reefs; and natural reference reefs of the eastern oyster, Crassostrea virginica, in Mosquito Lagoon (FL, USA). Results indicated that most of the measured biogeochemical properties (dissolved organic carbon (C), NH4 +, total C, total nitrogen (N), and the activity of major extracellular enzymes involved in C, N, and phosphorus (P) cycling) increased significantly by 1-year postrestoration, relative to dead reefs, and then remained fairly constant as the reefs continued to age. Few differences were observed in biogeochemical properties between restored reefs of any age (1 to 7 years) and natural reference reefs. Variability among reefs of the same treatment category was often correlated with differences in the number of live oysters, reef thickness, and/or the availability of C and N in the sediments. Overall, this study demonstrates the role of live intertidal oyster reefs as biogeochemical hot spots for nutrient cycling and burial and the rapidity (within 1 year) with which biogeochemical properties can be reestablished following successful restoration.

The bioactive lysophosphatidic acid (LPA) plays a well-known role in atherosclerotic disease, whereas, its role in myocardial function remains virtually unexplored. Following acute myocardial infarction, serum LPA concentration rises by six-fold over control human subjects, suggesting LPA may contribute to the pathogenesis of myocardial infarction. LPA production involves hydrolysis of lysophosphatidylcholine by the secreted enzyme autotaxin, whereas lipid phosphate phosphatase-3 (LPP3) catalyzes LPA dephosphorylation to generate lipid products that are not receptor active. We present the first evidence that cardiac ischemia/reperfusion (I/R) injury enhances myocardial autotaxin levels and decreases myocardial LPP3 expression, and this is associated with increased serum LPA levels. Upon reperfusion, reactive oxygen species production arises as a burst of superoxide from mitochondria following I/R injury. The redox-sensitive transcription factor NFAT has been shown to bind to the autotaxin promoter and induce its expression. Therefore, we looked at the autotaxin and LPP3 regulation in mice following I/R injury in the myocardium. After 1h ligation followed by 3h reperfusion in the myocardium, we observed a 3-fold increase in the autotaxin protein levels, whereas LPP3 protein levels were significantly downregulated as observed through Western blot analysis in these myocardial ischemic tissues. Autotaxin and miR-92a mRNA expression levels were significantly upregulated, whereas KLF2 and LPP3 mRNA expressions were significantly downregulated following I/R injury at 24 hours. Western blot analysis showed a 3-fold increase autotaxin protein levels and immunohistochemistry of human infarct tissues at 24 hours showed disruption of the sarcomere with decreased LPP3 staining. We found that I/R injury transactivates miR-92a, and inhibit KLF2, an upstream activator of LPP3. Taken together, us in vivo data, from the myocardial I/R injury and human infarct tissues, suggest that regulation of autotaxin and LPP3 activity might cause the rise in serum LPA levels as reported with acute myocardial infarct patients

Background:The purpose of this study was to examine factors associated with pain after elective ankle and hindfoot reconstruction.Methods:Patients who underwent major ankle or hindfoot reconstruction over a 3-year period were identified. Retrospective chart review determined patient demographics, comorbidities, surgeries, tobacco, alcohol, and narcotic use, chronic pain, and mood disorders. Primary outcomes were cumulative amount of narcotic prescribed (morphine milligram equivalent dose) in the initial 90-day postoperative period, beyond 90 days, and visual analog pain score (VAS) at a minimum of 1-year follow-up. One hundred thirty-two patients (139 operations) met the inclusion criteria.Results:The average narcotic amount prescribed in the initial 90 days after surgery was 1711 mg (morphine equivalent), and narcotic prescriptions were required after 52 surgeries (35%) past 90 days. Preoperative narcotic use ( P < .01), chronic pain disorder ( P = .02), and mood disorder ( P < .01) were significant risk factors for continued narcotic use past 90 days. Tobacco use ( P = .01) and chronic pain disorder ( P < .01) also were significant risk factors for increased initial postoperative narcotic use. The average VAS score in 91 patients at an average of 2.7-year follow-up was 2.1. Mood disorder was a risk factor for increased VAS ( P < .01). No other associations were noted.Conclusion:Patients being treated for chronic pain, diagnosed with a mood disorder, taking any amount of narcotics preoperatively, or using tobacco products had a statistically significant increased risk for pain postoperatively. The presence of risk factors should prompt physicians to discuss pain management strategies before surgery.Level of Evidence:Level III, comparative series.

Understanding aspects of reproduction is important for studies of life history strategy and for resource management. The influences of nine factors (five intrinsic, four extrinsic) on the reproductive attributes egg diameter, egg dry weight, and clutch size (i.e., number of eggs in egg mass) of female stone crabs (genus Menippe (Say)) from Tampa Bay, FL, waters were investigated. Two intrinsic factors influenced reproductive attributes: carapace width was positively correlated with clutch size, and egg stage was positively related to egg size. Three intrinsic factors—genotype class, damaged or missing pleopods, and missing claws or legs—had no influence on any reproductive attribute. The four extrinsic factors each influenced at least two reproductive attributes. Egg diameter and weight were significantly larger and clutch size was significantly smaller (1) in females uninfested by Octolasmis mulleri; (2) during the early part of the spawning season, when water temperature was 27°C or less; and (...

Electrophilic aldehyde (4-hydroxynonenal; 4-HNE), formed after lipid peroxidation, is a mediator of mitochondrial dysfunction and implicated in both the pathogenesis and the progression of cardiovascular disease. Manganese superoxide dismutase (MnSOD), a nuclear-encoded antioxidant enzyme, catalyzes the dismutation of superoxide radicals (O2•-) in mitochondria. To study the role of MnSOD in the myocardium, we generated a cardiomyocyte-specific SOD2 (SOD2Δ) deficient mouse strain. Unlike global SOD2 knockout mice, SOD2Δ mice reached adolescence; however, they die at ~4 months of age due to heart failure. Ultrastructural analysis of SOD2Δ hearts revealed altered mitochondrial architecture, with prominent disruption of the cristae and vacuole formation. Noninvasive echocardiographic measurements in SOD2Δ mice showed dilated cardiomyopathic features such as decreased ejection fraction and fractional shortening along with increased left ventricular internal diameter. An increased incidence of ventricular tachycardia was observed during electrophysiological studies of the heart in SOD2Δ mice. Oxidative phosphorylation (OXPHOS) measurement using a Seahorse XF analyzer in SOD2Δ neonatal cardiomyocytes and adult cardiac mitochondria displayed reduced O2 consumption, particularly during basal conditions and after the addition of FCCP (H+ ionophore/uncoupler), compared to that in SOD2fl hearts. Measurement of extracellular acidification (ECAR) to examine glycolysis in these cells showed a pattern precisely opposite that of the oxygen consumption rate (OCR) among SOD2Δ mice compared to their SOD2fl littermates. Analysis of the activity of the electron transport chain complex identified a reduction in Complex I and Complex V activity in SOD2Δ compared to SOD2fl mice. We demonstrated that a deficiency of SOD2 increases reactive oxygen species (ROS), leading to subsequent overproduction of 4-HNE inside mitochondria. Mechanistically, proteins in the mitochondrial respiratory chain complex and TCA cycle (NDUFS2, SDHA, ATP5B, and DLD) were the target of 4-HNE adduction in SOD2Δ hearts. Our findings suggest that the SOD2 mediated 4-HNE signaling nexus may play an important role in cardiomyopathy., Graphical abstract Image 1, Highlights • SOD2 is essential for cardiomyocyte function; its deficiency causes lethal dilated cardiomyopathy. • SOD2 deficiency disturbs mitochondrial architecture in the heart. • SOD2 is required for optimum mitochondrial respiration. • Deficiency of SOD2 leads to the production of 4-HNE within mitochondria. • 4-HNE targets proteins of the mitochondrial respiratory complex and TCA cycle.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in soils and sediments, particularly in urbanized environments in which the concentrations of 16 (or so) PAHs are regulated. Distinguishing among the numerous PAH sources is of practical and legal concern and thereby is often an objective of environmental forensic chemistry studies. Studies of prospective sources and impacted soils and sediments that rely upon the 16 U.S. EPA Priority Pollutant PAHs are disadvantaged, as these few compounds generally lack the specificity to distinguish among different PAH sources in the environment. Advances in analytical and interpretive methods over several decades have shown that different PAH sources can be more defensibly distinguished using modified EPA Method 8270 that, among other improvements, measure many other polycyclic aromatic compounds (PACs) that co-occur with the Priority Pollutant PAHs in different sources and in the environment. The PACs include variously-alkylated PAHs and polycyclic aromatic sulfu...

Background. The purpose of this study was to examine medical, social, and psychological factors associated with complications and reoperation after foot and ankle reconstruction. Methods. A retrospective chart review was conducted of 132 patients (135 feet; 139 operative cases) who had elective foot and ankle reconstruction. Medical, social, and psychological variables were documented. Primary outcomes included complications and reoperations. Results. The overall complication rate was 28% (39/139), and the reoperation rate was 17% (24/139). Alcohol use (P = .03) and preoperative narcotic use (P = .02) were risk factors for complications, with delayed wound healing more frequent in alcohol users (P = .03) and deep infection (P = .045) and nonunion (P = .046) more frequent preoperative narcotic use. Deep infection also was more frequent in tobacco users (P < .01). Older patients were less likely to undergo reoperation (risk of reoperation increased with age). Other variables were not associated with increased complications. Conclusion. Patients who consumed alcohol or had been prescribed any amount of narcotic within 3 months preoperatively were at increased risk for complications. Patients who smoked were more likely to have a wound infection. Surgeons should be aware of these factors and counsel patients before surgery.Levels of Evidence: Level III: Retrospective comparative study

One of the proteins most frequently found in neuropathological lesions is the ubiquitin binding protein p62 (sequestosome 1). Post-mortem analysis of p62 is a defining diagnostic marker in several neurodegenerative diseases including amyotrophic lateral sclerosis and inclusion body myositis. Since p62 functions in protein degradation pathways including autophagy, the build-up of p62-positive inclusions suggests defects in protein clearance. p62 was expressed unilaterally in the rat substantia nigra with an adeno-associated virus vector (AAV9) in order to study p62 neuropathology. Inclusions formed within neurons from several days to several weeks after gene transfer. By electron microscopy, the inclusions were found to contain packed 10 nm thick filaments, and mitochondria cristae structure was disrupted, resulting in the formation of empty spaces. In corollary cell culture transfections, p62 clearly impaired mitochondrial function. To probe for potential effects on macroautophagy, we co-expressed p62 with a double fluorescent tagged reporter for the autophagosome protein LC3 in the rat. p62 induced a dramatic and specific dissociation of the two tags. By 12 weeks, a rotational behavior phenotype manifested, consistent with a significant loss of dopaminergic neurons analyzed post-mortem. p62 overexpression resulted in a progressive and robust pathology model with neuronal inclusions and neurodegeneration. p62 gene transfer could be a novel methodological probe to disrupt mitochondrial function or autophagy in the brain and other tissues in vivo.

Detailed chemical analysis of petroleum – often referred to as “chemical fingerprinting” – has played a vital role in the identification of oil from accidental spills in the marine and riverine environments (e.g., sediments). The analytical tools used to identify and quantify the spilled oil must provide sufficient chemical resolution to separate the oil from any pre-existing (historical or naturally occurring) background hydrocarbons ubiquitous in many environments. Qualitative chemical fingerprinting analysis is a visual comparison between various spectroscopic or chromatographic fingerprints and is most successfully applied when the oil signature is unweathered and/or not mixed with other hydrocarbon sources (e.g., background). Quantitative chemical fingerprinting is the use of target compound calibration standards during the chemical analysis (e.g., GC/MS) of the sample to quantify the concentrations of source diagnostic hydrocarbons such as polycyclic aromatic hydrocarbons and biomarkers (e.g., triterpanes and steranes). These quantitative results can then be used to develop interpretive tools, such as diagnostic ratios, that allow the forensic scientist to compare spill and source oils and also to develop mixing models capable of allocating hydrocarbons derived from the spilled oil versus any background hydrocarbon signatures. Although the utility of diagnostic ratios has been discussed extensively in regard to oil spill identification in the scientific literature, the same is not true for the development and application hydrocarbon mixing models at oil spill sites. This chapter examines the applications and limitations of qualitative and quantitative oil fingerprinting methods and provides four oil spill case studies where mixing models were used to identify and or allocate liability in complex mixtures of spilled oils and various forms of background hydrocarbons.

Because incidental capture in pelagic longline fishing gear potentially kills or injures thousands of sea turtles annually, solutions to reduce the frequency and mortality rate of these interactions are critical conservation priorities. Understanding factors that affect post-hooking mortality rates remains an important component to evaluating the population-level impact of these interactions. Post-release mortality may be dependent upon the nature of the interaction (hooking location and/or entanglement) and amount of gear remaining at release. Hooking location can impact the ability of the crew to remove gear, as deeply ingested hooks cannot be removed safely. We examined the effects of hook type (circle vs J-hooks), offset (degrees), and other factors on hooking location in leatherback, Dermochelys coriacea (Vandelli, 1761), and loggerhead, Caretta caretta (linnaeus, 1758), sea turtles incidentally captured from 2000 to 2010 using fishery observer data. Significant differences in hooking location in loggerheads were observed between offset J-hooks and non-offset and 10° offset circle hooks; loggerheads were most often mouth/beak hooked with circle hooks, whereas most had swallowed offset J-hooks. Greater offsets appear to increase the frequency of deeply ingested hooks. Leatherback sea turtles were predominately externally hooked regardless of hook type, but mouth hookings occurred significantly more often on non-offset (0°) circle and J-hooks than on 10° offset circle hooks. When combined with outreach and education on careful release protocols, the use of circle hooks may increase post-interaction survival by modifying hooking location and facilitating maximum gear removal.

Background Because α 1 -antitrypsin deficiency (AATD) is underrecognized, strategies to enhance guideline-based diagnostic testing are warranted. Methods We studied the impact of issuing a guideline-based alert within an electronic health record (EHR) to test for AATD on the rate of testing for and detection of AATD. For patients at the Cleveland Clinic whose physician-ordered pulmonary function test results showed airflow obstruction, the rates of testing for and detection of AATD were determined during a baseline (prealert) period and again after implementing the alert. Results During the baseline period, among 979 eligible subjects, 4.7% were tested; 8.9% of those who had phenotype testing performed were found to have AATD (serum levels P P = .041), while carrying a diagnosis of asthma tended to increase the testing frequency ( P = .15). Multivariate analysis showed that younger age and a smoking history were associated with being tested. Conclusions Issuing an alert within an EHR was associated with a severalfold increase in the frequency of testing for α 1 -antitrypsin without a higher rate of diagnosing severe AATD. While the lack of more frequent diagnosis of AATD may reflect a high rate of baseline detection, these results prompt consideration of additional strategies to enhance detection of AATD.

Recessive mutations in the cartilage-associated protein (CRTAP), leucine proline-enriched proteoglycan 1 (LEPRE1) and peptidyl prolyl cis–trans isomerase B (PPIB) genes result in phenotypes that range from lethal in the perinatal period to severe deforming osteogenesis imperfecta (OI). These genes encode CRTAP (encoded by CRTAP), prolyl 3-hydroxylase 1 (P3H1; encoded by LEPRE1) and cyclophilin B (CYPB; encoded by PPIB), which reside in the rough endoplasmic reticulum (RER) and can form a complex involved in prolyl 3-hydroxylation in type I procollagen. CYPB, a prolyl cis–trans isomerase, has been thought to drive the prolyl-containing peptide bonds to the trans configuration needed for triple helix formation. Here, we describe mutations in PPIB identified in cells from three individuals with OI. Cultured dermal fibroblasts from the most severely affected infant make some overmodified type I procollagen molecules. Proα1(I) chains are slow to assemble into trimers, and abnormal procollagen molecules concentrate in the RER, and bind to protein disulfide isomerase (PDI) and prolyl 4-hydroxylase 1 (P4H1). These findings suggest that although CYPB plays a role in helix formation another effect is on folding of the C-terminal propeptide and trimer formation. The extent of procollagen accumulation and PDI/P4H1 binding differs among cells with mutations in PPIB, CRTAP and LEPRE1 with the greatest amount in PPIB-deficient cells and the least in LEPRE1-deficient cells. These findings suggest that prolyl cis–trans isomerase may be required to effectively fold the proline-rich regions of the C-terminal propeptide to allow proα chain association and suggest an order of action for CRTAP, P3H1 and CYPB in procollagen biosynthesis and pathogenesis of OI.

Complex mixtures of volatile organic chemical (VOC) vapors can exist over subsurface accumulations of non-aqueous phase liquids (NAPLs) and contaminated soils. The ability to predict the relative soil gas chemical composition arising from such NAPLs is relevant to studies of the sources and fate of soil gas, and in assessing the possible intrusion of soil gas chemical constituents to indoor air. However, it is difficult to directly compare the chemical ‘fingerprints’ of NAPL and vapor because the differences are significant in liquid-gas partitioning coefficients among the many volatile hydrocarbons that compose the liquid NAPL. In this article, detailed chemical characterization data from analysis of liquid NAPLs are used to calculate equilibrium vapor phase distributions of 66 diagnostic paraffin, isoparaffin, aromatic, naphthene, and olefin (PIANO) compounds. Measured NAPL and predicted vapor phase chemical composition are presented for a diverse group of 10 hydrocarbon products that include crude oil,...

VI (vapor intrusion) is the migration of VOCs (volatile organic compounds) from the subsurface into overlying and nearby buildings. Over the past few years, VI has gained significant attention beca...

Several members of the proteoglycan family are integral components of basement membranes; other proteoglycan family members interact with or bind to molecular residents of the basement membrane. Proteoglycans are polyfunctional molecules, for they derive their inherent bioactivity from the amino acid motifs embedded in the core protein structure as well as the glycosaminoglycan (GAG) chains that are covalently attached to the core protein. The presence of the covalently attached GAG chains significantly expands the "partnering" potential of proteoglycans, permitting them to interact with a broad spectrum of targets, including growth factors, cytokines, chemokines, and morphogens. Thus proteoglycans in the basement membrane are poised to exert diverse effects on the cells intimately associated with basement membranes.

Several members of the proteoglycan family are integral components of basement membranes; other proteoglycan family members interact with or bind to molecular residents of the basement membrane. Proteoglycans are polyfunctional molecules, for they derive their inherent bioactivity from the amino acid motifs embedded in the core protein structure as well as the glycosaminoglycan (GAG) chains that are covalently attached to the core protein. The presence of the covalently attached GAG chains significantly expands the "partnering" potential of proteoglycans, permitting them to interact with a broad spectrum of targets, including growth factors, cytokines, chemokines, and morphogens. Thus proteoglycans in the basement membrane are poised to exert diverse effects on the cells intimately associated with basement membranes.

Virtually all environmental forensics investigations focus on addressing questions pertaining to the nature, source, age, and ownership of site-related contamination. Contamination, particularly at complex historic sites, is usually a multifarious mixture of both organic and inorganic chemicals. Thus, the forensic investigator is typically faced with “unravelling” a complicated mixture of chemicals into component parts in order to better link the chemicals to their historic origins and differentiate them from often similar types and sources of contaminants. This chapter describes advanced methods of chemical analyses that have evolved, and continue to be refined by environmental chemists to address the specific needs of the forensic investigator and focuses on arguably some of the most important organic contaminants commonly encountered in terrestrial and sediment investigations: petroleum hydrocarbons and polycyclic aromatic hydrocarbons (PAH). The details of advanced methods for the measurement of these chemicals in multiple media (water, soils, sediment, air, and biological tissues) are presented. Laboratory techniques, including sample preparation, instrumental analysis, and quality control and quality assurance procedures are presented so that the reader can readily adapt forensic measurement techniques to suit his or her specific site investigation activities. Case studies are presented throughout the text that demonstrate the application of advanced methods of chemical analysis to varying kinds of complicated, real world forensic instigations.

The source(s) of the non-aqueous phase liquid (NAPL) in the downtown area of Mandan, ND, has been investigated through a two-part study in which the detailed chemical compositions of: 1) the NAPLs from the downtown area and remote areas on a nearby rail yard (phase I), and 2) residual petroleum in vadose zone and capillary fringe soils from borings near suspected sources (phase II) have been determined. The NAPL and soils are shown to contain variably weathered middle distillate fuels. The distributions of n-alkylcyclohexanes (CHs) and relative abundance of sulfur-containing aromatics (alkyl-dibenzothiophenes), both of which are stable over the various degrees of weathering exhibited by the NAPLs in the study area, were of particular utility in characterizing these fuels. The data indicate that multiple types of middle distillate fuels exist(ed) in the downtown area, establishing multiple sources and releases to the subsurface.

Petroleum biomarkers are ubiquitous hydrocarbons that occur in crude oils and are derived from formerly living organisms whose organic materials gave rise to the crude oil over geologic time. Biomarkers' specificity and resistance to weathering renders them useful in chemical fingerprinting of petroleum contamination in the environment. The biomarkers most commonly used in forensic issues, viz., tri- and penta-cyclic triterpanes and regular or rearranged steranes, are too high boiling for use in fingerprinting middle distillate petroleum products. The present article describes a suite of “low boiling” biomarkers (i.e., drimane-based bicyclic sesquiterpanes) that boil within the distillate fuel range (n-C13 to n-C16; ∼ 240°C to 285°C). GC/MS analysis of 24 dispensed (fresh) distillate fuels (Jet A, JP-5, and diesel fuel/fuel oil #2) demonstrate the specificity of sesquiterpane patterns in fuels of different origins. Analysis of fresh diesel fuel #2 and its associated, variably weathered NAPLs demonstrate t...

Oil spills of unknown origin, so-called “mystery” spills, occur routinely in rivers, open water, and navigable coastal waterways. The natural resources damage (NRD) liability associated with even a small volume of oil released into the environment warrants that a thorough chemical characterization of the spilled oil be conducted by agencies and potentially responsible parties (PRPs). Chemical fingerprinting methods have played an important role in the identification of mystery oil spills. These methods fall into two categories, viz., qualitative and quantitative. The qualitative approach relies upon visual comparison of various chromatographic fingerprints obtained by GC/FID and GC/MS analysis of spill and candidate source oils and are represented ASTM methods. The quantitative approach relies upon measurements of the concentrations (relative or absolute) of dozens of diagnostic chemicals, typically PAHs and biomarkers, and a subsequent statistical or numerical analysis of various diagnostic parameters ca...

Middle distillate fuels are used in civilian and military jet engines, on-road (truck and bus) and off-road diesel (diesel-electric locomotives, heavy equipment, and farm machinery) engines, marine diesel engines, nonaviation gas turbines, and domestic and commercial furnaces. As such, these fuels are common environmental contaminants that may arise from multiple sources. The considerable liability associated with these fugitive fuels requires that their source(s) be defensibly determined. In this paper we describe the “chemical fingerprinting” features of middle distillate fuels that can be used in assessments of their source(s) in the environment. A case study in which railroad (off-road) diesel fuel #2 could be distinguished from nonrailroad fuels (e.g., on-road diesel fuel #2, fuel oil #2, and/or fuel oil #1) in Mandan, North Dakota, is presented.

The United States Environmental Protection Agency (EPA) estimated that 3,000 to 5,000 manufactured gas plants (MGPs) were constructed in the United States (EPA, 2000). Companies located their MGPs near urban waterways due to the close proximity to cheap condensing liquid (water), improved site drainage, and easy access to delivery channels. However, these site selection criteria were shared by other industries, many of which generated PAHs or handled tar. Contemporary environmental forensic investigations must draw upon a detailed understanding of the relationship between MGP history and tar chemistry in order to differentiate between different types of MGP-derived tars and non-MGP impacts. This article describes the history of MGP operations in the U. S. and how chemical fingerprinting can aid in unraveling liability associated with MGP legacy sites.

The diagnostic imaging of osteomyelitis can require the confluence of multiple imaging technologies. Conventional radiography should always be the first imaging modality. Sonography is most useful in the diagnosis of fluid collections in a joint or in the extra-articular soft tissues but is not useful for evaluating presence of osseous infection. CT scan can be a useful method to detect early osseous erosion and to document the presence of sequestrum, foreign body, or gas formation but generally is less sensitive than other modalities for the detection of bone infection. Nuclear medicine and MRI are the most sensitive and most specific imaging modalities for the detection of osteomyelitis. Nuclear medicine is particularly useful in identifying multifocal involvement, which is common in children. MRI provides more accurate information of the local extent of the soft tissues and possible soft tissue abscess in patients with musculoskeletal infection.

Automotive gasoline is a common petroleum product found at contaminated terrestrial sites. The need to recognize and distinguish different types of gasoline(s) that may be present at a contaminated site and the need to determine the relative proportion of inputs from different sources, are often critical components of environmental 'forensic' investigations. Historically, identification and differentiation of automotive gasolines (particularly when weathered) has been hampered by analytical limitations of existing methods, notably US EPA Method 8260. In this article, we describe a modified EPA Method 8260 that is suitable for environmental investigations involving gasoline (and other light petroleum products). In the modified EPA 8260 method, 109 analytes that can occur in automotive gasoline are quantified in nonaqueous liquid samples (NAPL), water, and soil matrices. The accuracy and precision of the method is demonstrated through comparative analysis using several NIST SRM gasoline standards and replic...

L-2 cells are an immortalized cell line derived from yolk sac parietal endoderm cells, which are responsible for the production of Reichert's membrane, a thick basement membrane produced during rat gestation. Although the L-2 cells secrete all the major components of the basal lamina, they do not assemble a robust matrix in cell culture. We hypothesized that the reason L-2 cells fail to assemble a matrix in cell culture is because the concentrations of matrix components necessary for this matrix assembly do not reach a critical association concentration (CAC) under standard cell culture conditions. To limit the diffusion of secreted molecules while maintaining a nutrient-rich environment for the cells to thrive, we developed a technique that uses a dialysis membrane to limit protein diffusion in a 2-well plate format. This technique permits L-2 cells to assemble a robust matrix in as little as 24 hr that continues to be formed for at least 72 hr. This technique may address some of the physical limitations imposed by cell culture and could be readily applied to other cell types and medium conditions.

Troglitazone suppresses the secretion of type I collagen by mesangial cells in vitro. Background Our laboratory has shown that troglitazone, a thiazolidinedione and peroxisomal proliferator activated receptor γ (PPAR-γ) agonist, prevents mesangial expansion and glomerulosclerosis in diabetic rats. We investigated and compared the action of two PPAR agonists at the level of the mesangial cell. Methods Rat mesangial cells were grown in medium containing 5 mmol/L glucose, 30 mmol/L glucose, or 5 mmol/L glucose plus 25 mmol/L mannitol. The cultures were either left untreated, treated with 10 μmol/L troglitazone (PPAR-γ), or 100 μmol/L clofibrate (PPAR-α). The following parameters were used to assess mesangial cell responses: detection of PPAR-γ and -α mRNA, the degree of PPAR-γ and -α activation, spread cell area, total protein production, and laminin and type I collagen production. Results Reverse transcription-polymerase chain reaction (RT-PCR) showed the presence of PPAR-γ and -α mRNA in rat mesangial cells. PPAR-γ and -α proteins are active in mesangial cells and the extent of activation is affected by different glycemic conditions. Troglitazone and clofibrate treatment corrected in part the increase in spread cell area seen in mesangial cells in hyperglycemic conditions. However, neither agonist corrected the increase in total protein production induced by hyperglycemia. Treatment with troglitazone resulted in a significant, specific decrease in type I collagen along with a slight decrease in laminin production in both medium conditions. Clofibrate had no effect on laminin synthesis in either medium condition but did decrease type I collagen synthesis in cells grown in hyperglycemic conditions. Conclusion PPAR-α and -γ mRNA signaling pathways are in place and active in mesangial cells. Both agonists affect the phenotypic behavior of mesangial cells and ameliorate changes resulting from hyperglycemia. The data indicate that the correction of mesangial cell phenotype by troglitazone may influence production/deposition of a pathological fibrotic connective tissue matrix (that is, type I collagen) by these cells.

Troglitazone is a peroxisome proliferator-activated receptor-γ agonist that has been shown to halt mesangium expansion in experimental models of type 2 diabetes mellitus and to act directly on rat mesangial cells. Because glutamine serves as the precursor for cellular biosynthetic processes, we asked whether troglitazone would inhibit mesangial cell glutamine metabolism under these conditions. Confluent monolayers of rat mesangial cells were incubated in RPMI medium in the presence of troglitazone or vehicle (DMSO). Troglitazone effected a dose-dependent reduction in glutamine utilization and in alanine formation, associated with a decrease in monolayer collagen-glycosaminoglycan content. Despite the reduced glutamine uptake, ammonium formation did not decrease, consistent with increased glutamate flux through the deamination pathway. Assayable activity of the alanine aminotransferase decreased by 63%, whereas assayable glutamate dehydrogenase remained unchanged. In control monolayers, the sum of ammonium plus alanine plus glutamate nitrogen released accounted for

Item does not contain fulltext Heparan sulfate proteoglycans have been shown to modulate podocyte adhesion to--and pedicel organization on--the glomerular basement membrane. Recent studies showed that foot process effacement developed in a mutant mouse model whose podocytes were unable to assemble heparan sulfate glycosaminoglycan chains. This study, a further refinement, explored the role of heparan N-sulfation on podocyte behavior. A novel mutant mouse (Ndst1(-/-)) was developed, having podocyte-specific deletion of Ndst1, the enzyme responsible for N-sulfation of heparan sulfate chains. Podocytes having this mutation had foot process effacement and abnormal adhesion to Bowman's capsule. Although glomerular hypertrophy did develop in the kidneys of mutant animals, mesangial expansion was not seen. The lack of heparan N-sulfation did not affect the expression of agrin or perlecan proteoglycan core proteins. Loss of N-sulfation did not result in significant proteinuria, but the increase in the albumin/creatinine ratio was coincident with the development of the enlarged lysosomes in the proximal tubules. Thus, although the renal phenotype of the Ndst1(-/-) mouse is mild, the data show that heparan chain N-sulfation plays a key role in podocyte organization.

Porous scaffolds made from a biodegradable copolymer of trimethylene carbonate and glycolide were evaluated for tissue-engineered medical products. We examined the scaffold coated with cell adhesion protein and fibronectin and cultured under a dynamic mixing condition to enhance the growth of chondrocytes. Our hypothesis was that the combination of coating and dynamic mixing would be beneficial to the viability of the chondrocytic cells. Fibronectin was selected as the model protein because of its availability and routine assaying methods. Sterile samples of scaffolds of about 1 mm in thickness were coated with fibronectin at 37°C for 1.5 h. Four groups of scaffolds were used: uncoated static or dynamic, and coated static or dynamic. Scaffold samples were placed in either a Petri dish or a spinner flask (static vs. dynamic groups) after inoculation with rat chondrocytes of an initial cell density of 1.29 × 105 cell/mL. After 7, 14, 21, and 28 days, each sample was fixed, embedded, and sectioned at 5 μ thickness. The sections were double-label immunostained using antibodies against cellular fibronectin synthesized by adherent cells as a measure of cell viability. A Hoechst 33258 nuclear stain was used to measure the number of cells attached to the scaffold at each time interval. The slides were examined using a fluorescence microscope to determine the cell ingrowth. At least 25 fields/treatment group (except the 7 day group) were measured. The data showed that cell in-growths into the porous scaffolds were higher at all time periods for the coated dynamic group than those for the other three groups. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res 56: 74–82, 2001

Troglitazone halts diabetic glomerulosclerosis by blockade of mesangial expansion. Background Renal complications of long-term, poorly controlled type 2 diabetes mellitus include glomerulosclerosis and interstitial fibrosis. The onset and progression of these complications are influenced by underlying pathophysiologies such as hyperglycemia, hypertriglyceridemia, and hypercholesterolemia. Troglitazone, a thiazolidinedione, has been shown to ameliorate these metabolic defects. However, it was not known whether therapeutic intervention with troglitazone would prevent the onset and progression of glomerulosclerosis. Methods Sixty male ZDF/Gmi™ rats and 30 age-matched Zucker lean rats were in the study. The ZDF/Gmi™ rats were divided into two groups, one in which blood glucose levels were uncontrolled (30 animals) and another (30) in which blood glucose was controlled via dietary administration of troglitazone. Ten animals from each group were sacrificed at one, three, and six months into the study. The kidneys were harvested and processed for immunostaining with BM-CSPG, a marker for mesangial matrix. Images of 200 glomeruli per animal were captured using digital imaging microscopy, and the index of mesangial expansion (total area mesangium/total area of tuft) per glomerular section was measured. Results The administration of troglitazone ameliorated the metabolic defects associated with type 2 diabetes mellitus. Moreover, the glomeruli from tissue sections of animals given troglitazone showed no mesangial expansion when compared with normoglycemic control animals, whereas the uncontrolled diabetic animals showed significant mesangial expansion at all time intervals. Conclusions Therapeutic intervention with the thiazolidinedione troglitazone halts the early onset and progression of mesangial expansion in the ZDF/Gmi™ rat, preventing the development of glomerulosclerosis in this animal model of type 2 diabetes mellitus.

Our objective was to establish the existence of intercellular communication through gap junctions in synovial lining cells and in primary and passaged cultures of human synovial cells. Communication between cells was assessed using the nystatin perforated-patch method, fluorescent dye transfer, immunochemistry, transmission electron microscopy, and immunoblotting. Functional gap junctions were observed in primary and passaged cultures and were based on measurements of the transient current response to a step voltage. The average resistance between cells in small aggregates was 300 +/- 150 MOmega. Gap junctions were also observed between synovial lining cells in tissue explants; the size of the cell network in synovial tissue was estimated to be greater than 40 cells. Intercellular communication between cultured cells and between synovial lining cells was confirmed by dye injection. Punctate fluorescent regions were seen along intercellular contacts between cultured cells and in synovial membranes in cells and tissue immunostained for connexin43. The presence of the protein was verified in immunoblots. Regular 2-nm intermembrane gap separations characteristic of gap junctions were seen in transmission electron micrographs of synovial biopsies. The results showed that formation of gap-junction channels capable of mediating ionic and molecular communication was a regular feature of synovial cells, both in tissue and in cultured cells. The gap junctions contained connexin43 protein and perhaps other proteins. The physiological purpose of gap junctions in synovial cells is unknown, but it is reasonable to anticipate that intercellular communication serves some presently unrecognized function.

The need for identification, delineation, and differentiation of petroleum-derived contaminants in support of NRDA investigations of chronic or catastrophic release of petroleum is paramount for the equitable settlement of damages related to the incidents. Significant advances have been made over the last decade with regard to these issues for crude oil and heavy residual fuels, but less emphasis has been place on the forensic chemistry of light- and middle-distillates. Historically, identification and differentiation among similar light-distillates by standard EPA methods have been hampered by analytical limitations. In this paper the authors suggest analysis of light-distillate products begin with a chromatographic profile obtained using a modified EPA 8015 GC/FID. Subsequently, the detailed chemical composition of the distillate products is accomplished via a modified version of EPA Method 8260, examining a targeted list of diagnostic petroleum compounds in the C5 to C12 range that represent five important categories of hydrocarbons: Paraffins, Isoparaffins, Aromatics, Naphthenes, and Olefins. Detailed analysis of over 50 2–6 ring PAH and related heteroatomics from a modified EPA 8270 GC/MS analysis can be used to compliment the data set. The analysis of the full suite of hydrocarbons across a broad molecular weight range provides much of the information necessary to identify and distinguish among light- and middle-distillate products.

Backgrounds and Objectives We present the interim findings of our in-house protocol treating the tumor bed alone after lumpectomy with low-dose-rate (LDR) interstitial brachytherapy in selected patients with early-stage breast cancer treated with breast conserving therapy (BCT). Methods From 1 March 1993 through 1 January 1995, 50 women with early-stage breast cancer were entered into a protocol of tumor bed irradiation alone using an interstitial LDR implant. Patients were eligible if their tumor was an infiltrating ductal carcinoma ⩽3 cm in diameter, surgical margins were clear by at least 2 mm, the tumor did not contain an extensive intraductal component, the axilla was surgically staged with ⩽3 nodes involved with cancer, and a postoperative mammogram was performed. Implants were positioned using a template guide delivering 50 Gy over 96 hr to the lumpectomy bed plus a 1–2-cm margin. Local control, cosmetic outcome, and complications were assessed. Results Patients ranged in age from 40 to 84 years (median, 65). The median tumor size was 10 mm (range, 1–25). Seventeen of 50 patients (34%) had well-differentiated tumors, 22 (44%) had moderately differentiated tumors, and in 11 (22%) the tumor was poorly differentiated. Forty-five patients (90%) were node-negative while five (10%) had 1–3 positive nodes. A total of 23 (46%) patients were placed on tamoxifen and 3 (6%) received adjuvant systemic chemotherapy. No patient was lost to follow-up. The median follow-up for surviving patients is 47 months (range, 37–59). No patient has experienced a local, regional, or distant failure. Three patients have died at 19, 33, and 39 months after treatment. All were without clinical evidence of recurrent disease and all deaths were unrelated to treatment. Good-to-excellent cosmetic results have been observed in 49 of 50 patients (98%) (median cosmetic follow-up was 44 months with a range of 19–59). No patient has experienced significant sequelae related to their implant. Conclusions Interim results with treatment of the tumor bed alone with an LDR interstitial implant appear promising. Long-term follow-up of these patients and additional studies will be necessary to establish the equivalence of this treatment approach compared to standard BCT. J. Surg. Oncol. 1999;70:33–40. © 1999 Wiley-Liss, Inc.

The morphogenesis of the glomerular filtration apparatus during pre- and postnatal development in the rodent involves the coordinated assembly of two closely apposed but morphologically different extracellular matrices, the glomerular capillary basement membrane and the mesangial matrix. The cellular origin of these matrices is known to be distinct and complex; however, the mechanisms by which these matrices are assembled during morphogenesis are not entirely understood. It has been shown that in the earliest stages of glomerular morphogenesis the nascent glomerular basement membrane exists as a four-layered structure, the product of both the visceral epithelium and capillary endothelium. During the latter stages of glomerular development, the quadrilaminar structure becomes a trilaminar basement membrane, the event thought to occur by fusion of closely apposed basement membrane layers. In subsequent stages of maturation and throughout the life of the animal, the visceral epithelial cells, which line the periphery of the glomerular capillary, are the primary source of newly synthesized basement membrane material. The mesangial matrix, which lacks the specific organization of a basement membrane, first occurs in the developing glomerulus as a diffuse matrix central to the developing glomerular capillaries. During glomerular maturation the mesangial matrix undergoes a compaction/arborization coincident with the ramification of the vascular histoarchitecture of the glomerular tuft. Recent advances in the cell biology of basement membrane now demonstrate that there is a divergence in isoforms of the molecules that comprise the glomerular capillary basement membrane and mesangial matrices during development, possibly coincidental with functional specialization during the process of glomerular maturation. Microsc. Res. Tech. 39:233–253, 1997. © 1997 Wiley-Liss, Inc.

This paper deals with the development of a human resource planning (HRP) model for hospital laboratory personnel. External and organizational factors impacting the demand and supply of clinical laboratory personnel in an urban academic health center are identified by a Delphi process. These factors are structured into a hierarchy for the application of the Analytic Hierarchy Process (AHP). Computer software, Expert Choice, was used in model hierarchy development and judgmental elicitation process. The model result can be applicable to eliciting the perceptions, insights, and understanding of health-care decision-makers for strategic human resource planning.

Rat mesangial cells in vitro synthesize a spectrum of proteoglycan species including those of the basement membrane and interstitium. Accumulation of extracellular matrix within the mesangium is an important event in the development of glomerular disease. In this report we have used indirect immunofluorescence to positively identify a number of constituents of the mesangial matrix synthesized by rat mesangial cells (RMC) in vitro including laminin, fibronectin, type IV collagen and the basement membrane heparan sulphate proteoglycan (BM-HSPG) known as perlecan. In addition, using Mab 2B5 we demonstrate that RMC synthesize a specific basement membrane chondroitin sulfate (BM-CSPG), a matrix component that in normal animals is localized in the mesangium but is not found in the pericapillary glomerular basement membrane (GBM). Further characterization of the proteoglycans synthesized by RMC in vitro revealed: (i) a second large CSPG, identified as versican; (ii) two small dermatan sulphate proteoglycans identified as biglycan and decorin, which together account for the majority Of the proteoglycans; (iii) a large HSPG-I, probably related to perlecan; and (iv) a small HSPG-II. The cell layer proteoglycans can be sub-divided into a class that are probably free in the membrane, and a class of anchored molecules of the extracellular matrix or stabilized by cytoskeletal elements.