In vitro Expression of Human Zona Pellucida Protein 3

Human zona pellucida protein 3 (hZP3) is the putative receptor on the zona pellucida of the mature oocyte that recognizes and binds sperm, and therefore plays a critical role in fertilization. A full length cDNA of hZP3 (1278 bp) was amplified from the human ovary mRNA by reverse transcription-polymerase chain reaction (RT-PCR). The hZP3 cDNA was subcloned into PSK and pREP4 expression vectors. The cDNA of hZP3 was further characterized by restriction mapping, PCR, auto-sequencing and Southern blot analysis by using an internal oligonucleotide probe, and found to be identical to the one reported by J. Dean. Using autosequencing, 289 bases were identified and more than 98% homology was found at the 3\u27-end with the reported sequence, whereas, 224 bases were identified and more than 91% homology was found at the 5\sp\prime-end. In vitro translation was performed to ensure the capacity of the cloned cDNA for producing a full length, non-glycosylated hZP3 protein (47 kDa). Stable transfection of a human ovarian tumor cell line, PA-1, was produced by introduction of the hZP3/pREP4 expression vector construct using the calcium phosphate precipitation method. Approximately 200 single colonies of the stable-transfected cells were isolated by the cloning-ring method. Direct PCR, RT-PCR, and ELISA were used to identify and select clones; ten of the isolated clones were found to be positive. Two of the ten selected clones (SKC-P3-12 and SKC-P3-19) were used for further studies. Culture medium was collected from transfected and non-transfected cells (negative control), and the samples were purified by wheat germ agglutinin (WGA)-lectin chromatography and anti-WGA immunoaffinity chromatography; the final eluted fraction was concentrated by ultrafiltration with a limiting molecular weight cut off at 30 kDa. The secreted glycoproteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining, Western blot analysis using a specific anti-ZP3 antibody, and by a metabolic labelling study using 3H-galactose. Several bands including a band at 65 kDa, were observed in both non-transfected and transfected medium by silver staining. Distinct bands were identified by Western blot analysis at 65 kDa and 100 kDa for the recombinant product; solubilized human zonae pellucidae demonstrated a molecular mass of native hZP3 at approximately 65 kDa, and was at the same position identified for the recombinant product. In the metabolic study, several distinct bands including the band at 65 kDa, were identified as biosynthesized glycosylated recombinant proteins. In the hemizona assay (a test for sperm-zona pellucida binding), a significant inhibition (\u3e$40%) of binding was observed in competition studies using a 1:4 dilution of the WGA-purified recombinant glycoprotein solution. This material did not affect sperm viability or motility, and induced a more than 50% increase in the acrosome reaction of sperm after overnight incubation

Assessment of irrigation use on crop yield and water supplies in the Midwestern U.S.

Climate plays an important role in many aspects of hydrological and agricultural systems. Temperature and precipitation are usually the main forcing inputs for hydrological and agricultural simulations, while wind effect has generally been neglected in previous research. Wind speed is, however, an important factor for many physical processes, including evaporation from soil, and transpiration from plants. In order to investigate how climate variability impacts agricultural production, there are three hypotheses addressed in this dissertation. Hypothesis 1 is that the representation of changing wind speed will play an important role in the simulation of hydrological processes, and that the effect of wind speed will directly affect soil hydrology and evapotranspiration. Hypothesis 1 is tested using a factor separation analysis to quantify the contribution of projected future wind speed to hydrologic change under future climate. The result showed that changes in wind speed affected soil hydrology and evapotranspiration directly. ^ Hypothesis 2 is that the application of irrigation will be significantly beneficial for crop productivity in the future as it will mitigate risk associated with water deficits in the growing season, Regional farmers are expected to rely more on irrigation to mitigate risk due to increased climate variability in the future, resulting in a substantial increase in its use. This hypothesis is tested through the analysis of changes in crop (corn and soybean) yield due to climate change between historical and future periods using the VIC-CropSyst model, which incorporates a cropping system model, the CropSyst, model, into a large-scale hydrology model, the VIC model. First the effect of climate change on non-irrigated crop yields is quantified, then the ability of irrigation to mitigate crop yield losses due to changes in climate are quantified. Application of irrigation is found to improve corn yield by up to 5% and soybean yield by 20%, compared to the non-irrigated future scenario. In addition, irrigation is found to have significantly mitigated the impact of climate uncertainty on crop yield, with more benefits for soybean yield than for corn yield. This analysis addressed Hypothesis 2 and showed that irrigation is significantly beneficial for crop productivity and mitigates the impact of future changes in temperature and precipitation, meaning risk associated with water deficits in the growing season can be decreased using irrigation. ^ Hypothesis 3 is that the increased use of irrigation on historically rain-fed crops will affect regional water use significantly, increasing the risk of water supply deficits in the crop growing season. The assessment of water footprint in addition to the previous analysis of irrigation impact on crop yield, found that irrigation leads to significant increases in the blue water footprint. The assessment addressed Hypothesis 3 and suggested that irrigation on traditional rain-fed crops will affect regional water use significantly. However, we cannot assess the risk of water supply deficits because irrigation use is unrestricted in the model

Dual-frequency single-inductor multiple-output (DF-SIMO) power converter topology for SoC applications

Modern mixed-signal SoCs integrate a large number of sub-systems in a single nanometer CMOS chip. Each sub-system typically requires its own independent and well-isolated power supply. However, to build these power supplies requires many large off-chip passive components, and thus the bill of material, the package pin count, and the printed circuit board area and complexity increase dramatically, leading to higher overall cost. Conventional (single-frequency) Single-Inductor Multiple-Output (SIMO) power converter topology can be employed to reduce the burden of off-chip inductors while producing a large number of outputs. However, this strategy requires even larger off-chip output capacitors than single-output converters due to time multiplexing between the multiple outputs, and thus many of them suffer from cross coupling issues that limit the isolation between the outputs. In this thesis, a Dual-Frequency SIMO (DF-SIMO) buck converter topology is proposed. Unlike conventional SIMO topologies, the DF-SIMO decouples the rate of power conversion at the input stage from the rate of power distribution at the output stage. Switching the input stage at low frequency (~2 MHz) simplifies its design in nanometer CMOS, especially with input voltages higher than 1.2 V, while switching the output stage at higher frequency enables faster output dynamic response, better cross-regulation, and smaller output capacitors without the efficiency and design complexity penalty of switching both the input and output stages at high frequency. Moreover, for output switching frequency higher than 100 MHz, the output capacitors can be small enough to be integrated on-chip. A 5-output 2-MHz/120-MHz design in 45-nm CMOS with 1.8-V input targeting low-power microcontrollers is presented as an application. The outputs vary from 0.6 to 1.6 V, with 4 outputs providing up to 15 mA and one output providing up to 50 mA. The design uses single 10-uH off-chip inductor, 2-nF on-chip capacitor for each 15-mA output and 4.5-nF for the 50-mA output. The peak efficiency is 73%, Dynamic Voltage Scaling (DVS) is 0.6 V/80 ns, and settling time is 30 ns for half-to-full load steps with no observable overshoot/undershoot or cross-coupling transients. The DF-SIMO topology enables realizing multiple efficient power supplies with faster dynamic response, better cross-regulation, and lower overall cost compared to conventional SIMO topologies

The Impact of Sleep Apnea on Conventional Doppler Indices

AbstractObjectiveTo prospectively explore the impact of obstructive sleep apnea (OSA) on conventional Doppler indices and to identify possible negative prognostic factors for left ventricular diastolic dysfunction. Materials and Methods: All included subjects had overnight polysomnog-raphy. All subjects underwent a comprehensive echocardiography examination to evaluate systolic and diastolic function of the left ventricle. A multiple logistic regression model was created to identify potential negative prognostic factors for left ventricular dysfunction.ResultsA significant decrease in the ratio of early and atrial mitral flow velocity (E/A ratio) in OSA patients was found. Patients with moderate-to-severe OSA had a significant increase in the odds ratio for development of an abnormal E/A ratio (p=0.014, multivariate logistic regression). There was a significant negative correlation between E/A ratio and apnea-hypopnea index (p = 0.01). Non-obese OSA patients and obese-OSA patients carried significantly increased odds ratios for the development of a reduced E/A ratio (p = 0.02 and 0.038, respectively).ConclusionSubjects with OSA had reduced mitral E/A ratios, which implies possible impaired diastolic heart function. Further study to reverse impaired diastolic function via lifestyle modifications and treatment with nasal continuous positive airway pressure or surgery is warranted