Genetic Association in the Maintenance of the Mitochondrial Microenvironment and Sperm Capacity.

Sperm motility is one of the major determinants of male fertility. Since sperm need a great deal of energy to support their fast movement by active metabolism, they are thus extremely vulnerable to oxidative damage by the reactive oxygen species (ROS) and other free radicals generated as byproducts in the electron transport chain. The present study is aimed at understanding the impact of a mitochondrial oxidizing/reducing microenvironment in the etiopathology of male infertility. We detected the mitochondrial DNA (mtDNA) 4,977 bp deletion in human sperm. We examined the gene mutation of ATP synthase 6 (ATPase6 m.T8993G) in ATP generation, the gene polymorphisms of uncoupling protein 2 (UCP2, G-866A) in the uncoupling of oxidative phosphorylation, the role of genes such as manganese superoxide dismutase (MnSOD, C47T) and catalase (CAT, C-262T) in the scavenging system in neutralizing reactive oxygen species, and the role of human 8-oxoguanine DNA glycosylase (hOGG1, C1245G) in 8-hydroxy-2'-deoxyguanosine (8-OHdG) repair. We found that the sperm with higher motility were found to have a higher mitochondrial membrane potential and mitochondrial bioenergetics. The genotype frequencies of UCP2 G-866A, MnSOD C47T, and CAT C-262T were found to be significantly different among the fertile subjects, the infertile subjects with more than 50% motility, and the infertile subjects with less than 50% motility. A higher prevalence of the mtDNA 4,977 bp deletion was found in the subjects with impaired sperm motility and fertility. Furthermore, we found that there were significant differences between the occurrences of the mtDNA 4,977 bp deletion and MnSOD (C47T) and hOGG1 (C1245G). In conclusion, the maintenance of the mitochondrial redox microenvironment and genome integrity is an important issue in sperm motility and fertility.

High-speed atomic force microscope based on an astigmatic detection system.

High-speed atomic force microscopy (HS-AFM) enables visualizing dynamic behaviors of biological molecules under physiological conditions at a temporal resolution of 1s or shorter. A small cantilever with a high resonance frequency is crucial in increasing the scan speed. However, detecting mechanical resonances of small cantilevers is technically challenging. In this study, we constructed an atomic force microscope using a digital versatile disc (DVD) pickup head to detect cantilever deflections. In addition, a flexure-guided scanner and a sinusoidal scan method were implemented. In this work, we imaged a grating sample in air by using a regular cantilever and a small cantilever with a resonance frequency of 5.5 MHz. Poor tracking was seen at the scan rate of 50 line/s when a cantilever for regular AFM imaging was used. Using a small cantilever at the scan rate of 100 line/s revealed no significant degradation in the topographic images. The results indicate that a smaller cantilever can achieve a higher scan rate and superior force sensitivity. This work shows the potential for using a DVD pickup head in future HS-AFM technology.

Biprism electron interferometry with a single atom tip source.

Experiments with electron or ion matter waves require a coherent, monochromatic and long-term stable source with high brightness. These requirements are best fulfilled by single atom tip (SAT) field emitters. The performance of an iridium covered W(111) SAT is demonstrated and analyzed for electrons in a biprism interferometer. Furthermore we characterize the emission of the SAT in a separate field electron and field ion microscope and compare it with other emitter types. A new method is presented to fabricate the electrostatic charged biprism wire that separates and combines the matter wave. In contrast to other biprism interferometers the source and the biprism size are well defined within a few nanometers. The setup has direct applications in ion interferometry and Aharonov-Bohm physics.

Operation of astigmatic-detection atomic force microscopy in liquid environments.

The astigmatic detection system (ADS) based on commercial optical pickup head was demonstrated to achieve a sub-nanometer sensitivity in detecting the vertical movement of an object surface in air. The detection laser spot of the ADS was sub-µm and the detection bandwidth was over 80 MHz. These advantages allow detection of high-frequency mechanical resonance of very small objects, which would have many important applications in nanotechnology. In this work, we optimized the operation conditions of ADS to achieve good sensitivity in aqueous solutions. We demonstrated good contrast and good spatial resolution of cancer cells in water with the optical profilometry mode. We also built an ADS-AFM (atomic force microscopy) for imaging in water. A novel cantilever holder was designed, and the spurious peaks were suppressed down to 26.0% of the real resonance peak. Most importantly, we demonstrated that the ADS-AFM could resolve single atomic steps on a graphite substrate and image soft DNA molecules on mica in water.

Simultaneous analysis of 17 diuretics in dietary supplements by HPLC and LC-MS/MS.

In order to test health foods for illegally added diuretics for weight loss, we developed simple, rapid, selective, and sensitive methods using HPLC and LC-MS/MS for the simultaneous analysis of 17 diuretics in dietary supplements. HPLC conditions were set with a Capcell-pak C18, using a mobile phase consisting of gradient conditions, UV detection at 254 nm and validated for linearity (r(2)> 0.999), precision (CV ≤ 3%), recoveries (90.4-102.8%) and reproducibility. Identification and quantification of 17 diuretics were accomplished by ion-spray LC-MS/MS using multiple reaction monitoring (MRM). The chromatographic separation was carried out under the reversed-phase mechanism on an HSS-T3 column. The LC-MS/MS method was validated for linearity (r(2)> 0.99) and precision (CV < 13%). Sixteen dietary supplements were tested with the developed methods. Diuretics were not detected in all samples. Extraction recovery was also investigated and the extraction recoveries in different formulations were from 88% to 110% and from 81% to 116% using HPLC and LC-MS/MS, respectively. There was no significant difference in recoveries in the type of dietary supplements. Based on this result, the developed methods to monitor illegal drug adulterations in dietary supplements using HPLC and LC-MS/MS are simple, fast and reliable. Therefore, it is applicable to routine drug-adulteration screening.

Anti-drift and auto-alignment mechanism for an astigmatic atomic force microscope system based on a digital versatile disk optical head.

In this work, an anti-drift and auto-alignment mechanism is applied to an astigmatic detection system (ADS)-based atomic force microscope (AFM) for drift compensation and cantilever alignment. The optical path of the ADS adopts a commercial digital versatile disc (DVD) optical head using the astigmatic focus error signal. The ADS-based astigmatic AFM is lightweight, compact size, low priced, and easy to use. Furthermore, the optical head is capable of measuring sub-atomic displacements of high-frequency AFM probes with a sub-micron laser spot (~570 nm, FWHM) and a high-working bandwidth (80 MHz). Nevertheless, conventional DVD optical heads suffer from signal drift problems. In a previous setup, signal drifts of even thousands of nanometers had been measured. With the anti-drift and auto-alignment mechanism, the signal drift is compensated by actuating a voice coil motor of the DVD optical head. A nearly zero signal drift was achieved. Additional benefits of this mechanism are automatic cantilever alignment and simplified design.

High incidence of multiple aster formation in vitrified-warmed bovine oocytes after in vitro fertilization.

In vitro-matured bovine oocytes do not tolerate vitrification as well as mature murine or human oocytes. Delayed first cleavage in vitrified and in vitro-fertilized bovine oocytes may be responsible for the decreased yield of blastocysts in vitro. Because formation of sperm-aster and the subsequent assembly of microtubule network play an important role for migration and fusion of both pronuclei, aster formation in vitrified-warmed oocytes was analyzed by confocal laser-scanning microscopy. At 10 h post-insemination (hpi), proportions of oocytes fertilized normally were comparable between the vitrified and fresh control groups (67 and 70%, respectively). Proportions of oocytes that exhibited microtubule assembly were similar between the two groups (95% each), but the proportion of oocytes with multiple asters was higher in the vitrified group when compared with the fresh control group (68 vs 29%, P < 0.05). Both migration and development of two pronuclei were adversely affected by multiple aster formation. In the next experiment, multiple asters observed in 5.5 vs 8 hpi pronuclear zygotes were located near the male pronucleus, suggesting that those multiple asters were not the cytoplasmic asters of maternal origin. In conclusion, multiple aster formation frequently observed in vitrified-warmed bovine oocytes may be related to loss of ooplasmic function responsible for normal microtubule assembly from the sperm-aster.

Selenium deficiency contributes to the chronic myocarditis in coxsackievirus-infected mice.

Both coxsackievirus B3 (CVB3) infection and selenium (Se) deficiency play a pivotal role in Keshan disease of the heart. The Se deficiency was known to contribute to the CVB3-induced myocarditis in acute and subacute phase of infection. However, its effect on the myocarditis in chronic phase of infection has not been examined yet. To address this question, we kept mice on a Se-replete or Se-deficient diet for 28 days, infected them intraperitoneally with CVB3 and maintaining previous diets, we examined them for next 90 days for several parameters indicative of the infection or disease. We found out that the mice on the Se-deficient diet exhibited a higher mortality, lower serum glutathione peroxidase (GPx) activity, evident histopathological changes indicative of myocarditis, and a higher level of viral RNA in the heart. Summing up, these data suggest that the Se-deficiency creates a chronic myocarditis-prone condition by fostering the active virus replication.

Time course quantification of spastic hypertonia following spinal hemisection in rats.

Progressive changes in the muscle tone and stretch reflex after spinal cord injury (SCI) provide insight into the time-course development of spasticity. This study quantified the time-course changes of hypertonia for rats following SCI of T8 hemisection. A miniature manual stretching device measured the reactive torque via a pair of pressure sensing balloons; the angular displacement was measured via an optoelectronic device. Various stretching frequencies were tested, specifically 1/3, 1/2, 1, 3/2 and 2 Hz. The reactive torque and angular displacement were used to derive the viscous and elastic components representing the viscosity and stiffness of the rat's ankle joint. The enhanced velocity-dependent properties of spasticity were observed in the SCI hemisection rats (n=9) but not in the controls (n=9). Time-course measurements from pre-surgery to 56 days following SCI showed that the muscle tone of the hemisection rats dropped immediately after spinal shock and then gradually increased to reach a peak around 21 days postinjury (P<0.01). The muscle tone remained at least 75% of the peak value up to the end of an 8 week observation period (P<0.05). The changes of muscle tone can also be verified from the electrophysiological evaluations of electromyography (EMG) (P<0.05). In addition to conventional BBB motor behavior score, our results provided time-course quantification of the biomechanical and electrophysiological properties of muscle tone from the onset of SCI. Such data are useful for investigating progressive recovery of spinal damage in animal model and for future objective assessment of improved treatment for SCI human subjects.

Physiological aspects of MMG and EMG spectra during load-varying isometric dorsiflexion.

The aim of the study was to examine spectral features of the mechanomyogram (MMG) and electromyogram (EMG) during static and load-varying isometric dorsiflexion to characterize force control strategies of the tibialis anterior. Twelve healthy subjects performed two motor tasks including 1) four exertion levels of static isometric dorsiflexion and 2) load-varying isometric dorsiflexion while tracking a target quasi-sinusoidal curve of three different amplitudes. Generally speaking, for both static and load-varying isometric contractions, the mean frequency of MMG-EMG cross spectra (MMG-EMG MF) progressively increased with effort level, whereas the median frequency of EMG auto spectra among higher effort levels remained unchanged. The MMG-EMG MF versus EMG root mean square regression slope was significantly larger for load-varying isometric contraction than for static contraction control measurements. These findings highlight effort-dependent and task-specific rate coding for force regulation of the tibialis anterior.

Adrenomedullin gene expression and peptide levels in the heart and blood vessels of streptozotocin-diabetic rats.

The aim of the present study was to assess the changes in gene expression and peptide adrenomedullin (AM) levels in cardiovascular and other tissues in the streptozotocin-diabetic rats. For this purpose, diabetes was induced by intraperitoneal injection of streptozotocin (STZ, 65 mg/Kg body weight). Half of the diabetic rats were subcutaneously injected with insulin in the afternoon (4 units/day) one week after STZ injection until the day before killing. Control rats received only saline injection. AM mRNA was determined in cardiovascular and other tissues of streptozotocin-diabetic rats using solution-hybridization-RNase protection assay. Circulating AM and peptide AM in cardiovascular and other tissues were estimated using a specific radioimmunoassay. There were increases in preproAM mRNA levels in the left and right ventricles and in the thoracic aorta in both the 2-week and 4-week diabetic rats, but not in the two atria, the mesenteric artery and the lung. In the 2-week diabetic rats, there were decreases in AM contents in the two atria and the lung but an increase in the thoracic aorta. In the 4-week diabetic rats, there were bigger decreases in the atria and also a decrease in the left ventricle. The plasma AM levels were not changed but there was an increase in the excretion of AM in the urine. Our results suggest a possible increase in AM release in the heart and the thoracic aorta in the 2-week and 4-week diabetic rats.

Resource recovery and nitrogen removal from piggery waste using the combined anaerobic processes.

The combined ADEPT (Anaerobic Digestion Elutriated Phased Treatment)- SHARON (Single reactor system High Ammonium Removal Over Nitrite)--ANAMMOX (Anaerobic Ammonium Oxidation) processes were operated for the purpose of resource recovery and nitrogen removal from slurry-type piggery waste. The ADEPT operated at acidogenic loading rates of 3.95 gSCOD/L-day, the SCOD elutriation rate and acid production rate were 5.3 gSCOD/L-day and 3.3 gVFAs(as COD)/L-day, respectively. VS reduction and SCOD reduction by hydrolysis were 13% and 0.19 gSCOD(prod.)/gVS(feeding), respectively. Also, the acid production rate was 0.80 gVFAs/gSCOD(production). In the methanogenic reactor, the gas production rate and methane content were 2.8 L/day (0.3 m3CH4/kgCOD(removal)STP) and 77%, respectively. With these operating condition, the removal of nitrogen and phosphorus were 94.1% as NH4-N (86.5% as TKN) and 87.3% as T-P, respectively.

The effects of weight load and joint immobilization on reorganization of postural tremor.

To investigate change in coordinative strategies due to wrist immobilization and index loading, postural tremors from the index, hand, and forearm were recorded during different postural holding tasks. The wrist joint was immobilized with a thermoplastic splint in the constrained condition, and a copper mass of 100 grams was applied to the index finger in the loaded condition. The structures of the postural tremors of all upper limb segments among the unloaded-unconstrained, unloaded-constrained, loaded-unconstrained, and loaded-constrained conditions were compared. Index loading exaggerated index/forearm postural tremor, while the load-induced tremor enhancement was no longer evident for wrist immobilization. In the unloaded condition, wrist immobilization resulted specifically in enhancement of carpal postural tremor, rather than in the index and forearm. Index loading induced a marked tremor peak and relative power in the range of 5-8 Hz. Wrist immobilization potentiated the carpal tremor peak of 1-4 Hz in association with enhancement of carpal-forearm mechanical coupling. In light of structural changes in postural tremor, our data suggest that (1) a wrist splint is effective to counteract load-induced enhancement of postural tremor, and (2) freezing of the wrist joint might facilitate compensatory strategies to minimize passive fluctuation transmission from the carpal to index.

Full-cyclic control strategy of SBR for nitrogen removal in strong wastewater using common sensors.

A full-cyclic automatic control strategy for sequencing batch reactors (SBR) was proposed using only common sensors such as ORP, DO and pH. The main objective was to develop a generally applicable and robust control strategy. To accomplish this, various control schemes found in the literature or suggested by authors were examined at diverse ammonia loads and SCOD/NH4(+)-N ratios. Advantages and constraints of each scheme were discussed and compared. Ammonia load was estimated with DO lag time during the aerobic stage, and then the influent pump was manipulated to meet the desired load at the next anoxic stage. A partial denitrification scheme was chosen for the anoxic stage period control, to save anoxic time and external carbon. For external carbon dosage control, intermittent feeding at each anoxic stage was concluded to be a suitable scheme. The anoxic stage period could be successfully controlled by the combination of pH increase and DO increase. Every suggested control scheme was incorporated into a full-cyclic control strategy and tested at 0.02, 0.035, 0.08 kg NH4(+)-N/m3/sub-cycle. From the results, it is expected to perform unmanned automatic SBR operation with this strategy.

Monitoring biodegradation of diesel fuel in bioventing processes using in situ respiration rate.

An in situ measuring system of respiration rate was applied for monitoring biodegradation of diesel fuel in a bioventing process for bioremediation of diesel contaminated soil. Two laboratory-scale soil columns were packed with 5 kg of soil that was artificially contaminated by diesel fuel as final TPH (total petroleum hydrocarbon) concentration of 8,000 mg/kg soil. Nutrient was added to make a relative concentration of C:N:P = 100:10:1. One soil column was operated with continuous venting mode, and the other one with intermittent (6 h venting/6 h rest) venting mode. On-line O2 and CO2 gas measuring system was applied to measure O2 utilisation and CO2 production during biodegradation of diesel for 5 months. Biodegradation rate of TPH was calculated from respiration rate measured by the on-line gas measuring system. There were no apparent differences between calculated biodegradation rates from two columns with different venting modes. The variation of biodegradation rates corresponded well with trend of the remaining TPH concentrations comparing other biodegradation indicators, such as C17/pristane and C18/phytane ratio, dehydrogenase activity, and the ratio of hydrocarbon utilising bacteria to total heterotrophic bacteria. These results suggested that the on-line measuring system of respiration rate would be applied to monitoring biodegradation rate and to determine the potential applicability of bioventing process for bioremediation of oil contaminated soil.

Monitoring of pH inhibition on microbial activity in a continuous flow reactor by pseudo toxic concentration (C(PT)) concept and time delay model.

The pseudo toxic concentration (C(PT)) concept was introduced as a quantification method to describe pH as an inhibitor concentration. In this research, the applicability of the C(PT) concept model for the detection of pH inhibitions was expanded for a continuous flow activated sludge reactor. A pilot equipped with an inhibition detection system was installed. Inhibitory wastewater was injected for 1 h and the relative activity was calculated by the maximum respiration rate. At the same time, the coefficients for the C(PT) concept model were estimated. At the dynamic conditions, the estimated relative activity by the C(PT) concept model showed time lag compared to the measured one. However, the time lag problem was successfully resolved by introducing a transfer function into the C(PT) concept model. The C(PT) concept model combined with a transfer function (C(PT) + TF model) successfully tracked the variation of the relative activity under dynamic conditions. The C(PT) + TF model could detect 50% inhibition faster than the respirometry based method by approximately 10 min. Moreover, it had additional advantages such as being inexpensive, easy to install and simple to operate. In conclusion, the C(PT) + TF model was an effective and convenient detection method of pH inhibition.

Ether stress increases adrenomedullin gene expression and levels in the rat adrenal.

To study the contribution of adrenomedullin in the adrenal medulla in the stress response, we measured plasma and adrenal levels of adrenomedullin in sham-operated (intact) rats and in rats without adrenal medulla, with or without exposure to ether vapor for 15 min. Adrenomedullin levels decreased drastically after demedullation. Effect stress resulted in increased adrenomedullin levels in both adrenal and plasma in sham-operated rats, but not in demedullated rats. The responses of plasma adrenocorticotropin to stress were similar, but the elevations in plasma corticosterone levels were significantly less in demedullated rats. In the sham-operated rat, preproadrenomedullin mRNA levels were increased after stress, and this effect was not blocked by pretreatment with hexamethonium. We conclude that stress increases adrenomedullin synthesis and secretion from the adrenal medulla through a hexamethonium-insensitive mechanism, and that adrenomedullin release from the adrenal medulla may play a role in cortical steroidogenesis.

Nitrogen removal from piggery waste using the combined SHARON and ANAMMOX process.

Nitrogen removal in piggery waste was investigated with the combined SHARON-ANAMMOX process. The piggery waste was characterized as strong nitrogenous wastewater with very low C/N ratio. For the preceding SHARON reactor, ammonium nitrogen loading and conversion rates were 0.97 kg NH4-N/m3 reactor/day and 0.73 kg NH4-N/m3 reactor/day, respectively. Alkalinity consumption for ammonium conversion was 8.5 gr bicarbonate utilized per gram ammonium nitrogen converted to NO2-N or NO3-N at steady-states operation. The successive ANAMMOX reactor was fed with the effluent from SHARON reactor. Nitrogen loading and conversion rates were 1.36 kg soluble N/m3 reactor/day and 0.72 kg soluble N/m3 reactor/day, respectively. The average NO2-N/NH4-N removal ratio by ANAMMOX reaction was 2.13. It has been observed that Candidatus "Kuenenia stuttgartiensis" were dominated in the ANAMMOX reactor based on FISH analysis.

ANAMMOX and partial denitritation in anaerobic nitrogen removal from piggery waste.

The anaerobic ammonium removal from a piggery waste with high strength (56 g COD/L and 5 g T-N/L) was investigated using a lab-scale upflow anaerobic sludge bed reactor at a mesophilic condition. Based on the nitrogen and carbon balance in the process, the contribution of autotrophic and heterotrophic organisms was also evaluated in terms of the influent NO2-N/NH4-N ratio (1:0.8 and 1:1.2 for Phase 1 and Phase 2, respectively). The result of this research demonstrates that the anaerobic ammonium removal from the piggery waste, using the UASB reactor, can be performed successfully. Furthermore, it appears that by using granular sludge as the seed biomass, the ANAMMOX reaction can start more quickly. Average nitrogen conversion was 0.59 kg T-N/m3 reactor-day (0.06 kg T-N/kg VSS/day) and 0.66 kg T-N/m3 reactor-day (0.08 kg T-N/kg VSS/day) for Phase 1 and Phase 2. The NO2-N/NH4-N removal ratio by the ANAMMOX was 1.48 and 1.79 for Phase 1 and Phase 2. The higher nitrite contents (about 50%) in the substrate resulted in higher nitrite nitrogen removal by the partial denitritation, as well as the ANAMMOX reaction, implying higher potential of partial denitritation. However, the result reveals that the ANAMMOX reaction was influenced less by the degree of partial denitritation, and the ANAMMOX bacteria did not compete with denitritation bacteria. The colour of the biomass at the bottom of the reactor changed from dark gray to dark red, which was accompanied by an increase in cytochrome content. At the end of the experiment, red-coloured granular sludge with diameter of 1-2 mm at the lower part of the reactor was also observed.