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Freeze Filtration: Description and Evaluation of a Method for Obtaining Samples of Liquid Phase From Frozen SolutionsThe objective of this study was to develop a method for sampling the liquid phase of frozen solutions (i.e. the remaining liquid between ice crystals and other solids) for subsequent chemical analysis in order to determine the extent of ice formation and other physical-chemical effects of freezing upon solutions. Such information could enhance the development of cryoprotective agents (CP As). which would allow the frozen long term preservation of organs for transplantation. The traditional thermometric/ calorimetric methods for the measurement of ice formation are susceptible to bias by composition change of the liquid phase caused by secondary solid phase formations (i.e. solids besides ice). They are not able to determine the nature Df such composition changes, and some changes, like that of the pH, will go unnoticed. The new method uses subatmospheric pressure to aspirate liquid phase from a cooled sample holder into a collection vial. "Freeze Filtration" has been tested on four well-established cryoprotective agents comparing measurements in this study with literature values on these chemicals. Remarkable accuracy and repeatability have been achieved. Deviations from published values depend on the cryoprotectant used, which suggests a CPA-dependent methodical difference that may be rooted in bias within the literature values. Cells and solute precipitates can be isolated along with liquid phase, and the freeze-elevated concentrations of Na+, K+, Ca2+, and H+ of a single sample have been measured. Freeze Filtration differs from the older methods by providing a simple mode of operation, low cost, the ability to determine composition changes, and the ability to serve as a physical-chemical reference during actual cryosurvival studies. It is directly demonstrated that freezing at velocities and sample sizes applicable to organ freezing can result in substantial pH changes in the liquid phase and the precipitation of solutes. The data also indicate that 1,3-butanediol is not a good cryoprotectant.
Mechanisms for Control of Renal Vascular Resistance in Type I Diabetes MellitusGlomerular hyperfiltration and an increase in renal blood flow are hallmark characteristics of Type I_ Diabetes Mellitus in the early stages, and are major risk factors for the development of diabetic nephropathy. Previous studies from our laboratory have implicated an important role for the Nitric Oxide system. in mediating this response, because giving nitric oxide synthase inhibitors· 'prevented the increase in renal plasma flow and glomerular filtration rate during diabetes. However, a limitation of. these studies is that single point measurements were taken and may not reflect the time-dependent role of nitric oxide. Therefore, we have developed a more precise method to measure the role of nitric oxide in the chronic control of renal blood flow during diabetes. We measured renal blood flow continuously, 18 hr/day using a Transonic flow probe in control (C) and diabetic (D) rats. Renal blood flow averaged 8.0±0.1 and 7.8±0 ml/min in the .C and D groups, respectively, during the control period and induction of diabetes caused a marked and progressive increase in renal blood flow in the D rats, averaging 10±6% above control on day 1, and 22±3% and 34±1% above control by the end of diabetes weeks 1 and 2. During the control period, glomerular filtration rate averaged 2.1 ±0.1 and 1.7±0.1 ml/min in the C and D groups, respectively. Glomerular filtration rate did not change during the experiment in the C rats, but increased significantly in the D group, averaging 54±21 and 52±19% above control during diabetic weeks 1 and 2 and renal vascular resistance decreased significantly during the diabetic period. There were no significant changes in filtration fraction in either group. Importantly, chronic blockade of nitric oxide completely prevented the increase in renal blood flow and prevented the diabetes-induced hyperfiltration normally associated with diabetes. These data together suggest that nitric oxide is essential for the renal vasodilation caused by onset of type I diabetes and suggest that the renal vasodilation in diabetes occurs primarily at the afferent arteriole. Autoregulation of the afferent arteriole plays an important role in determining glomerular capillary hydrostatic pressure and glomerular filtration. In diabetes, renal autoregulation may be impaired, but the relative roles of myogenic and tubuloglomerular feedback mechanisms in controlling renal blood flow, and the time course of their involvement, is not known. In addition, there is very little known about autoregulatory mechanisms at the very onset of diabetes, before there has been time for renal structural changes to become manifest. Therefore, we designed experiments to establish the role of the myogenic response and tubuloglomerular feedback mechanism in renal blood flow. control at the onset of diabetes. Coupling continuous measurement of renal blood flow using Transonic flow probes and continuous measurement of arterial pressure, we were able to use transfer function analysis to determine the relationship between arterial pressure and renal blood flow .. This type of analysis examines the dynamic ability of the renal vasculature to attenuate, or autoregulate, the influence of the oscillatory power of blood pressure over the range of frequencies at which the myogenic response and tubuloglomerular feedback mechanism operate. In these studies we demonstrated that transfer function gain was negative, indicating effective· autoregulation, in the frequency range of the myogenic (0.1- 0.3 Hz) and tubuloglomerular feedback (0.03-0.06 Hz) mechanisms during control days. However, at the onset ·of diabetes gain increased to positive values and continued through the 2-week diabetic period. Chronic blockade of nitric oxide in diabetic rats normalized the increase in transfer function gain and possibly enhanced the autoregulatory response. Our model provides a novel method to measure the chronic effects of the nitric oxide on renal blood flow control during diabetes. By using this model we have demonstrated that nitric oxide is required for the immediate increase in renal blood flow in diabetes. Furthermore, these data suggest renal autoregulation is impaired at the onset of diabetes and may play a role in the increase in renal blood flow and glomerular filtration rate early in diabetes. In addition, these data together suggest that nitric oxide contributes to the impaired autoregulatory capacity of the renal vasculature.
microRNA Regulation of Acute Kidney InjuryAcute kidney injury (AKI) is caused by an injury or insult to the kidneys resulting in abrupt loss of renal function. Acute kidney injury is a highly prevalent disease characterized by high rates of morbidity and mortality mainly due to the absence of effective therapeutic options. Dissecting the molecular basis of AKI is vital not only for understanding the mechanisms of disease pathogenesis, but also for designing effective treatments. The small regulatory non-coding RNAs, microRNAs, are vital regulators of normal cellular function and critical modulators of various pathological conditions. An intense focus has recently emerged on the study of microRNA regulation in the maintenance of kidney function and the development of renal diseases. Our laboratory demonstrated the first evidence that microRNAs play a pathogenic role during ischemia-induced AKI by utilizing a conditional Dicer knockout mouse model. The focus of my work was to identify and functionally characterize novel microRNAs that contribute to AKI. Firstly, using a cisplatin-induced nephrotoxicity model of AKI, we showed that miR-34a is up-regulated in a p53 dependent manner and contributes to renal cell survival. Secondly, we identified a novel microRNA, miR-687, as the most significantly upregulated microRNA during ischemia-induced AKI. Mechanistic studies showed that miR-687 is up-regulated in a hypoxia-inducible factor 1 (HIFl)-dependent manner and 3 subsequently negatively regulates PTEN expression under hypoxic conditions. These studies have unearthed an important HIFl-miR-687-PTEN signaling pathway that regulates cell cycle progression during hypoxia. Thirdly, we show that inhibiting miR- 687 significantly ameliorates ischemia-induced AKI. These studies have identified a pivotal signaling mechanism involved in cellular response to hypoxia that may be targeted for renoprotection during ischemic AKI.
The Effect of Filled Adhesive Application Method and Water Storage on the Retentive Features of a Mechanically Retained Ceramic Orthodontic Bracket to Bovine EnamelEase of bracket application, durability of enamel bond, and formation of an intact peripheral seal to maintain color stability are goals of esthetic, ceramic orthodontic bracket use. PURPOSE This research examined the effect of three different types of photo-curable resin applications to a commercial ceramic orthodontic bracket base on bond strength and potential for microleakage on bovine enamel following various periods of water storage. METH 0 D S Resin adhesive was applied to polycrystalline orthodontic brackets (Transcend 6000, 3M Unitek, Monrovia, CA) using three methods: 1) manual application of a light-curable adhesive (Transbond, 3M Unitek) to the bracket base (no resin); 2) with a coat of unfilled resin prior to adhesive placement (resin); 3) or with adhesive paste pre-applied by the manufacturer (APC Brackets, 3M'Unitek). Brackets were applied to flattened, acid-etched bovine enamel surfaces using standardized loading conditions. Bonded brackets were stored in phosphate-buffered saline at 37 degrees C for the following durations prior to testing: 1 day, 1 week, and 14 weeks. Following storage, the brackets (17 per test condition) were debonded in shear or were stored in basic fuchsin dye for 24 h prior to sectioning and examination for microleakage under stereo magnification (12 teeth per condition). RESULTS Two-way ANOVA indicated that type of adhesive resin application (p=0.0001) and duration of water storage (p=0.0063) were both significant factors on strength: resin (13.4 MPa) > APC (10.9 MPa) > no resin (9.3 MPa). Water storage decreased bond strength for all groups after 14 weeks storage. The primary debond location for all groups was the adhesive/bracket interface. APC brackets showed greatest microleakage (42%) after 14 weeks, while the other groups each leaked in 33% of specimens. The method of resin application to a ceramic bracket base is shown to affect both strength and potential for micro leakage.